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  • gene expression  (308)
  • Evolution  (260)
  • Springer  (568)
  • American Physical Society
  • MDPI Publishing
  • 1995-1999  (514)
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  • 1
    ISSN: 1433-4909
    Keywords: Key words Thermococcus ; Pyrococcus ; Thermophilic ; Phosphofructokinase ; Evolution ; ADP ; Glycolysis ; ATP
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The ADP-dependent phosphofructokinase (PFK) from Thermococcus zilligii has been purified 950 fold; it had a specific activity of 190 U mg−1. The enzyme required Mg2+ ions for optimal activity and was specific for ADP. The forward reaction kinetics were hyperbolic for both cosubstrates (pH optimum of 6.4), and the apparent K m values for ADP and fructose-6-phosphate were 0.6 mM (apparent V max of 243 U mg−1) and 1.47 mM (apparent V max of 197 U mg−1), respectively. Significantly, the enzyme is indicated to be nonallosteric but was slightly activated by some monovalent cations including Na+ and K+. The protein had a subunit size of 42.2 kDa and an estimated native molecular weight of 66 kDa (gel filtration). Maximal reaction rates for the reverse reaction were attained at pH 7.5–8.0, and the apparent K m values for fructose-1,6-bisphosphate and AMP were 0.56 mM (apparent V max of 2.9 U mg−1) and 12.5 mM, respectively. The biochemical characteristics of this unique ADP-dependent enzymatic activity are compared to ATP and pyrophosphate-dependent phosphofructokinases.
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  • 2
    ISSN: 1433-4909
    Keywords: Key wordsNatronomonas pharaonis ; Natronobacteria ; Archaea ; Serine protease ; Chymotrypsinogen ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A protease of a molecular mass of approximately 30 kDa was isolated and purified from the haloalkaliphilic archaeon Natronomonas (formerly Natronobacterium) pharaonis. The enzyme hydrolyzed synthetic peptides, preferentially at the carboxyl terminus of phenylalanine or leucine, as well as large proteins. Hydrolysis occurred over the range of pH from 6 to 12, with an optimum at pH 10. The temperature optimum was 61°C. The enzyme was nearly equally active over the range of salt concentration from 0.5 to 4 M (NaCl or KCl). A strong cross-reaction with a polyclonal antiserum against human chymotrypsin was observed. Enzymatic activity was inhibited by typical serine protease inhibitors. There was significant homology between N-terminal and internal sequences from autolytic fragments and the sequence of bovine chymotrypsinogen B; the overall amino acid composition was similar to that of vertebrate chymotrypsinogens. Evidence for a zymogen-like processing of the protease was obtained. Cell extracts from other halobacteria exhibited similar proteolytic activity and immunoreactivity. The data suggested a widespread distribution of a chymotrypsinogen B-like protease among halo- and haloalkaliphilic Archaea.
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  • 3
    ISSN: 1432-1211
    Keywords: Key words Antigen processing ; Evolution ; Cell surface molecules ; Mhc ; Class I antigens
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 4
    ISSN: 1432-1211
    Keywords: Key words NRAMP ; Fish ; Carp ; Evolution ; Expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The mouse Lsh/Ity/Bcg locus regulates natural resistance to intracellular pathogens, and the Nramp1 gene was isolated as its candidate. Nramp is part of a small family of at least two genes, Nramp1 and Nramp2. In the present study, a full-length cDNA for carp NRAMP has been isolated and characterized. Nucleotide and predicted amino acid sequence analysis indicate that the carp NRAMP encodes a 548 amino acid membrane protein with 12 putative transmembrane domains, two N-linked glycosylation sites, and an evolutionarily conserved consensus transport motif. The peptide sequence identity among carp and human NRAMP2 is 78%, and 65% with human NRAMP1. Reverse transcription-polymerase chain reaction revealed that carp NRAMP is ubiquitously expressed. Phylogenetic analysis, using neigbor-joining, showed that the carp NRAMP protein clustered together with mammalian NRAMP2 proteins.
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  • 5
    Electronic Resource
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    Springer
    Immunogenetics 50 (1999), S. 301-308 
    ISSN: 1432-1211
    Keywords: Key words T-cell receptors ; Variable region genes ; Evolution ; Phylogeny ; Diversity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The receptor of a T lymphocyte (TCR) recognizes nonself antigens in the company of major histocompatibility complex (MHC) molecules presented to it by the antigen-presenting cell. The variable region of TCR is encoded by either a concatenation of variable region (TCR-V), diversity region (TCR-D), and joining region (TCR-J) genes, or a concatenation of TCR-V and TCR-J genes. The TCR-V genes exist as a multigene family in vertebrate species. Here we study the evolutionary relationships of TCR-V genes from humans, sheep, cattle, rabbits, mice, and chicken. These six species can be classified into two groups according to the frequency of γδ T-cells in their peripheral T-cell populations. The "γδ low" group of species includes humans and mice, in which γδ T-cells constitute very limited portion of the T-cell population. The "γδ high" group includes sheep, cattle, rabbits, and chicken, in which γδ T-cells comprise up to 60% of the T-cell population. Here, we compiled TCR-V sequences from the six species and conducted a phylogenetic analysis. We identified various TCR-V gene subgroups based on the analysis. We found that humans and mice have representatives from nearly all of the subgroups identified, while other species have lost subgroups to different extent. Therefore, the γδ low species have a high degree of diversity of TCR-V genes, while γδ high species all have limited diversity of TCR-V genes. This pattern is similar to that found for immunoglobulin variable region (IGV) genes.
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  • 6
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    Immunogenetics 50 (1999), S. 329-335 
    ISSN: 1432-1211
    Keywords: Key words Marsupials ; Light chains ; Variable regions ; IGK ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  A full-length and several partial cDNAs encoding IGK light chains from the marsupial South American opossum, Monodelphis domestica, were isolated and characterized. Using these clones as a starting point, the expressed IGKV repertoire was sampled by anchored polymerase chain reaction using an IGKC-specific primer. Based on nucleotide sequences of twenty unique, expressed IGKV-J combinations, there are at least four IGKV families and two J segments. Southern blot analysis revealed each IGK-V family contains multiple gene segments totaling at least thirty-five IGKV in the opossum genome. No evidence for particular, recurrent IGKV-J combinations in the opossum IGK repertoire was seen, rather the V-J combinations appeared random and diverse. Each of the four IGKV families appear more closely related to V segments from placental mammals than to each other, suggesting the duplication of the IGKV families prior to the separation of marsupials and placental mammals more than one-hundred-million years ago. Overall, the complexity of opossum light chain V segments appears greater than that found in the heavy chain, and light chains are likely to contribute significantly to Ig diversity in this species.With this report, the homologues encoding all three classes of eutherian Ig chains, IGH, IGL, and IGK, have been described in a non-placental mammal.
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  • 7
    ISSN: 1432-1211
    Keywords: Key words MHC ; MIC ; Nonhuman primates ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 8
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    Immunogenetics 49 (1999), S. 295-302 
    ISSN: 1432-1211
    Keywords: Key words Major histocompatibility complex ; Class II ; Antigen processing ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Proper peptide presentation by major histocompatibility complex (MHC)-encoded class II antigens is dependent on the products of the MHC DM loci. We identified the rabbit orthologues (RLA-DMA and -DMB) of human HLA-DMA and -DMB and found that they have 76.9% and 78.8% identity with HLA-DMA and -DMB, respectively. Like classical class II MHC genes, RLA-DM genes are more closely related to human HLA-DM genes than to mouse H2-DM. Among the DM family, there is a high degree of variability at the amino terminus of the DMa chains, and length variability in the cytoplasmic tails of both DMα and DMβ. The rabbit DM genes are coexpressed with class II genes in lymphoid tissues, as are the DM genes of other mammals. The RLA-DM locus maps to the class II region of the rabbit MHC, and is flanked by the DP and DOB loci. Despite having some similarities to class II genes of bony fishes, the DM family represents a separate branch of the MHC class II family.
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  • 9
    ISSN: 1432-1211
    Keywords: Key words Mhc ; Class II A ; Cichlid ; Fish ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 10
    ISSN: 1432-1211
    Keywords: Key words Beta2-microglobulin ; Evolution ; Sturgeon ; cDNA ; Genomic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 11
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    Journal of comparative physiology 185 (1999), S. 367-372 
    ISSN: 1432-1351
    Keywords: Key words Cortical magnification ; Somatosensory cortex ; Development ; Evolution ; Behavior
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The star-nosed mole (Condylura cristata) has a snout surrounded by 22 fleshy and mobile appendages. This unusual structure is not an olfactory organ, as might be assumed from its location, nor is it used to manipulate objects as might be guessed from its appearance. Rather, the star is devoted to the sense of touch, and for this purpose the appendages are covered with thousands of small mechanoreceptive Eimer's organs. Recent behavioral studies find that the star acts much like a tactile eye, having a small behavioral focus, or “fovea” at the center – used for detailed explorations of objects of interest. The peripheral and central nervous systems of the mole reflect these behavioral specializations, such that the small behavioral focus on the nose is more densely innervated in the periphery, and has a greatly enlarged representation in the somatosensory cortex. This somatosensory representation of the tactile fovea is not correlated with anatomical parameters (innervation density) as found in other species, but rather is highly correlated with patterns of behavior. The many surprising parallels between the somatosensory system of the mole, and the visual systems of other mammals, suggest a convergent and perhaps common organization for highly developed sensory systems.
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  • 12
    Electronic Resource
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    Journal of comparative physiology 184 (1999), S. 543-551 
    ISSN: 1432-1351
    Keywords: Key words Auditory physiology ; Insect ; Frequency discrimination ; Evolution ; Song recognition
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Peripheral auditory frequency tuning in the ensiferan insect Cyphoderris monstrosa (Orthoptera: Haglidae) was examined by comparing tympanal vibrations and primary auditory receptor responses. In this species there is a mis-match between the frequency of maximal auditory sensitivity and the frequency content of the species' acoustic signals. The mis-match is not a function of the mechanical properties of the tympanum, but is evident at the level of primary receptors. There are two classes of primary receptors: low-tuned and broadly tuned. Differences in the absolute sensitivity of the two receptor types at the male song frequency would allow the auditory system to discriminate intraspecific signals from sounds containing lower frequencies. Comparisons of tympanal and receptor tuning indicated that the sensitivity of the broadly tuned receptors did not differ from that of the tympanum, while low-tuned receptors had significantly narrower frequency tuning. The results suggest that the limited specialization for the encoding of intraspecific signals in the auditory system of C. monstrosa is a primitive rather than a degenerate condition. The limited specialization of C. monstrosa may reflect the evolutionary origin of communication-related hearing from a generalized precursor through the addition of peripheral adaptations (tympana, additional receptors) to enhance frequency sensitivity and discrimination.
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  • 13
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    Journal of comparative physiology 185 (1999), S. 199-205 
    ISSN: 1432-1351
    Keywords: Key words Hearing ; Orthoptera ; Phaneropteridae ; Cercal system ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The unusual morphology of a sound-activated plurisegmental ascending interneurone (AN5-AG7) in an insect (Ancistrura nigrovittata, Ensifera, Phaneropteridae) is described. This neurone's soma is located in the penultimate abdominal ganglion. The most prominent arborisations with smooth endings are found in the prothoracic ganglion. The neurone terminates with numerous beaded endings in the brain (protocerebrum). All abdominal ganglia including the penultimate contain only tiny side branches of beaded appearance. The neurone's morphology is compared to the morphology of a `typical' sound-activated plurisegmental neurone of bushcrickets with its soma in the prothorax. In the prothoracic ganglion and in the brain the arborisations of the two cells are very similar. Graded potentials and action potentials are generated in the prothoracic portion of both neurones. Both receive excitation mainly by ultrasound, and inhibition by soma-ipsilateral stimuli. Neither wind, substrate vibration nor touch of the abdomen evoke responses in AN5-AG7. It is assumed that early in evolution this neurone had its dendrites in the ganglion which houses the cell body (like cercal interneurones of this neuromere). Profound evolutionary changes probably have taken place to bring about this neuron's modern morphology.
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  • 14
    ISSN: 1432-1351
    Keywords: Key wordsCydia pomonella ; Adoxophyes orana ; Sex pheromone ; Interspecific interruption ; Evolution ; AbbreviationsZ9-14:Ac (Z9)-tetradecenylacetate Z11-14:Ac (Z11)-tetradecenylacetate ; E8, E10-12:OH (E8, E10)-dodecadienol
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Male moths responding to their species-specific sex pheromone, may cease their upwind flight when pheromone components of sympatric species are added to the mixture. The interspecific interaction between the pheromone response of the tortricid moths Cydia pomonella and Adoxophyes orana was investigated in field-trapping and wind-tunnel studies. Addition of the A. orana pheromone [(Z9)-tetradecenylacetate and (Z11)-tetradecenylacetate] to a source containing the C. pomonella pheromone [(E8, E10)-dodecadienol] resulted in a significant inhibition of attraction by male C. pomonella. It is demonstrated that this behavioural antagonist for C. pomonella must be emitted from the same point source to induce this inhibitory effect. A spatial separation of the two interspecific pheromones (at 14 cm, 5 cm and 0.5 cm crosswind) restored the attraction of the conspecific pheromone for male C. pomonella. In contrast to C. pomonella, male A. orana were not inhibited by point sources releasing both the C. pomonella and A. orana pheromone. We suggest that the discrepancy in the interspecific pheromone interaction between these two tortricids can be explained if we consider the evolutionary ecology of interspecific pheromone communication in C. pomonella and A. orana.
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  • 15
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    Plant molecular biology reporter 17 (1999), S. 371-383 
    ISSN: 1572-9818
    Keywords: epidermal peel ; extraction ; gene expression ; stomata ; tree tobacco
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Stomatal guard cells are critical for maintenance of plant homeostasis and represent an interesting cell type for studies of leaf cell differentiation and patterning. Here we describe techniques for the isolation of guard cell RNA and protein from blended epidermal peels of Nicotiana glauca. The RNA isolation procedure is a modification of the hot borate method, which is particularly well-suited for recalcitrant tissues. Protein was extracted by disrupting guard cell-enriched epidermis with a French® press. This system offers the following advantages: relatively high yield, low or no contamination by other cell types, fresh tissue as a source of RNA and protein rather than protoplasts, and a plant species that is readily transformable. These techniques will allow for cloning and analysis of genes expressed in guard cells, application of traditional biochemical techniques to guard cell proteins, as well as characterization of genetic manipulation of guard cell function in transgenic plants.
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  • 16
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    Economic theory 13 (1999), S. 329-343 
    ISSN: 1432-0479
    Keywords: Keywords and Phrases: Risk ; Evolution ; Entrepreneur. ; JEL Classification Numbers: C72 ; D81.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Economics
    Notes: Summary. I examine a Knightian (1921) model of risk using a general equilibrium model of investment and trade. A population of agents with various preference types can choose between a safe production technology and a risky production technology. In addition, the distribution of types of agents changes through a standard evolutionary dynamic. For a given population distribution, the equilibrium is in general inefficient, however, by allowing the population distribution to change in response to market generated rewards, the population will converge to one where the equilibrium is efficient and where the population as a whole behaves as if all agents were risk neutral.
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  • 17
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    Environmental geology 38 (1999), S. 34-46 
    ISSN: 1432-0495
    Keywords: Key words Alkaline/acid/neutral water ; Acid rain ; Geochemical modeling ; Brine ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geosciences
    Notes: Abstract  Analysis and hydrogeochemical modeling of hydrocarbonate groundwater, including its buffering geochemical properties, have demonstrated that this water has undergone a geochemical transformation almost throughout the whole of one industrial region. It is known that hydrocarbonate groundwater possesses a high protective natural geochemical potential, supporting neutralization of acid atmospheric precipitation. Natural hydrocarbonate water undergoes three stages of anthropogenic transformation caused by acid atmospheric precipitation over more than 50 years. The first stage is transformation of neutral hydrocarbonate water into alkaline water, accompanied by calcite precipitation and sodium carbonate dissolution from the unsaturated zone. The second stage is transformation of alkaline water into neutral hydrocarbonate water; the hydrocarbonate content, being less than the lower limit for background concentrations, showed reduced water buffering properties or protective potential relative to acid precipitation. The third stage is transformation of neutral hydrocarbonate water into acidic water, with a complete loss of protective geochemical potential. This unfavorable ecological situation with natural geochemically hydrocarbonate groundwater shows that natural groundwater less protected from the impact of acid atmospheric precipitation is in a worse ecological condition, which is confirmed by observations in northern and Western Europe and other regions of the world.
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  • 18
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    Development genes and evolution 209 (1999), S. 186-197 
    ISSN: 1432-041X
    Keywords: Key words Cnidaria ; Paired class ; Paired-like ; Homeobox gene ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The genes belonging to the Paired class exert primary developmental functions. They are characterized by six invariant amino acid residues in the homeodomain, while the residue at position 50 can be a serine, glutamine or lysine as in the Pax-type, Q50 Paired-like or the K50 Paired-like homeodomains respectively. Genes in this class emerged early in animal evolution: three distinct Pax genes and two Q50 Paired-like genes have recently been characterised from cnidarians. Phylogenetic molecular reconstructions taking into account homeodomain and paired-domain sequences provide some new perspectives on the evolution of the Paired-class genes. Analysis of 146 Paired-class homeodomains from a wide range of metazoan taxa allowed us to identify 18 families among the three sub-classes from which the aristaless family displays the least diverged position. Both Pax-type and K50 families branch within the Q50 Paired-like sequences implying that these are the most ancestral. Consequently, most Pax genes arose from a Paired-like ancestor, via fusion of a Paired-like homebox gene with a gene encoding only a paired domain; the Cnidaria appear to contain genes representing the ’before’ and ’after’ fusion events.
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  • 19
    ISSN: 1432-041X
    Keywords: Key words Notch pathway ; Antagonist ; Hairless ; Orthologue ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Hairless is a member of the Notch signalling pathway, where it acts as antagonist by binding to Suppressor of Hairless [Su(H)], thereby inhibiting Notch target gene activation. The pathway and its members are highly conserved in metazoans from worms to humans. However, a Hairless orthologue from another species has not yet been identified. The identification of Hairless in largely diverged species by cross-hybridization has failed so far probably due to a low degree of conservation. Therefore, we turned to D. hydei where a Hairless mutation has been described before. The D. hydei Hairless orthologue is reasonably well conserved with regard to gene structure and expression. The prospective Hairless protein orthologues share several highly conserved regions which are separated by quite diverged stretches. As to be expected, the largest region of high conservation corresponds to the Su(H) binding domain. This region is also functionally conserved, since this D. hydei protein domain binds very strongly to the D. melanogaster Su(H) protein. The other conserved regions support our earlier structure-function analysis since they nicely correspond to previously defined, functionally important protein domains. Most notably, the very C-terminal domain which is very sensitive to structural alterations, is nearly identical between the two species. In summary, this evolutionary study improves the knowledge on functionally significant domains of the Hairless protein, and may be helpful for the future identification of homologues in other animals, especially in vertebrates.
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  • 20
    ISSN: 1432-041X
    Keywords: Key words Echinoid ; Oogenesis ; Development ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Evolutionary change in developmental mode in sea urchins is closely tied to an increase in maternal provisioning. We examined the oogenic modifications involved in production of a large egg by comparison of oogenesis in congeneric sea urchins with markedly different sized oocytes and divergent modes of development. Heliocidaris tuberculata has small eggs (95 µm diameter) and the ancestral mode of development through feeding larvae, whereas H. erythrogramma has large eggs (430 µm diameter) and highly modified non-feeding lecithotrophic larvae. Production of a large egg in H. erythrogramma involved both conserved and divergent mechanisms. The pattern and level of vitellogenin gene expression is similar in the two species. Vitellogenin processing is also similar with the gonads of both species incorporating yolk protein from coelomic and hemal stores into nutritive cells with subsequent transfer of this protein into yolk granules in the developing vitellogenic oocyte. Immunocytology of the eggs of both Heliocidaris species indicates they incorporate similar levels of yolk protein. However, H. erythrogramma has evolved a highly divergent second phase of oogenesis characterised by massive deposition of non-vitellogenic material including additional maternal protein and lipid. Maternal provisioning in H. erythrogramma exhibits recapitulation of the ancestral vitellogenic program followed by a novel oogenic phase with hypertrophy of the lipogenic program being a major contributor to the increase in egg size.
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  • 21
    ISSN: 1432-1211
    Keywords: Key words MHC ; Transporter ; Evolution ; PCR cloning ; Allelic lineage
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The amphibian Xenopus laevis is one non-mammalian vertebrate in which the major histocompatibility complex (MHC) has been analyzed extensively. Class IIβ, class Ia, LMP2, LMP7, HSP70, C4, Factor B, and Ring3 genes have been identified and mapped to the MHC. Here, we report the isolation of a transporter associated with antigen processing (TAP) gene, TAP2, and demonstrate its linkage to the MHC. While the ATP-binding region of Xenopus TAP2 is highly conserved in evolution, amino acid identity to other vertebrate TAP proteins was not detected in the N-terminal region. Segregation analysis of 34 individuals from two families showed exact restriction fragment length polymorphism matching between the MHC class Ia gene and the one TAP2 gene demonstrating linkage conservation since the mammalian/amphibian divergence ∼350 million years ago. In addition, one non-MHC-linked TAP2–hybridizing fragment was detected in approximately half of the individuals tested. Interestingly, TAP2 allelic lineages appear to match those of LMP7 and classical class I, which previously were categorized into two highly divergent groups that emerged at least 60 million years ago. Similar to LMP7 and class Ia,TAP2 is expressed ubiquitously with highest levels in intestine and spleen.
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  • 22
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    Immunogenetics 49 (1999), S. 438-445 
    ISSN: 1432-1211
    Keywords: Key words PA28 ; Proteasome ; Gene structure ; Evolution ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Two proteasome activators PA28α and β, which have been implicated in antigen processing for loading class I MHC molecules, are synthesized in response to Ifn-γ. The human genes encoding these activators (PSME1 and PSME2, respectively) were analyzed by sequencing. Each gene comprised 11 exons, consistent with gene duplication during vertebrate evolution. The intron/exon organization of both genes was highly conserved, the major difference being the absence of the exon encoding the lysine and glutamic acid-rich 'KEKE' motif in PA28β. Two other genes of relevance to the immune system were located close to those for PA28 at 14q11.2 including ISGF3G, a protein involved in transcription after IFNα signalling. These sequences were also characterized.
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  • 23
    ISSN: 1432-1211
    Keywords: Key words Cattle ; MHC class I ; Haplotypes ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Analysis of cattle major histocompatibility complex (MHC) (BoLA) class I gene expression using serological and biochemical methods has demonstrated a high level of polymorphism. However, analysis of class I cDNA sequences has failed to produce conclusive evidence concerning the number and nature of expressed genes. Such information is essential for detailed studies of cattle immune responses, and to increase our understanding of the mechanisms of MHC evolution. In this study a selective breeding programme has been used to generate a number of MHC homozygous cattle expressing common serologically defined class I specificities. Detailed analysis of five class I haplotypes was carried out, with transcribed class I genes identified and characterized by cDNA cloning, sequence analysis, and transfection/expression studies. Surface expression of the gene products (on lymphocytes) was confirmed using monoclonal antibodies of defined BoLA specificity. Phylogenetic analysis of available transcribed cattle MHC class I sequences revealed complex evolutionary relationships including possible evidence for recombination. The study of individual haplotypes suggests that certain groupings of related sequences may correlate with loci, but overall it was not possible to define the origin of individual alleles using this approach. The most striking finding of this study is that none of the cattle class I genes is consistently expressed, and that in contrast to human, haplotypes differ from one another in both the number and composition of expressed classical class I genes.
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  • 24
    ISSN: 1432-1211
    Keywords: Key words Comparative immunology ; Evolution ; MHC class I ; Molecular biology ; Salmonids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Complementary DNA for two class I genes of the rainbow trout, Oncorhynchus mykiss, were characterized. MhcOnmy-UBA*01 is similar to Onmy-UA-C32 and the classical major histocompatibility complex class I genes of other fish species, whereas Onmy-UAA*01 is divergent from all class I genes so far characterized. Onmy-UAA*01 is expressed at lower levels than Onmy-UBA*01. Although Onmy-UAA*01 exhibits restriction fragment length polymorphism on Southern blotting, the encoded protein is highly conserved. Two allotypes, which differ only by substitution at amino acid position 223 of the α3 domain, have been defined. Onmy-UAA*01 has an exon-intron organization like other class I genes and contains a Tc1-like transposon element in intron III. Orthologues of Onmy-UAA*01 have been characterized in four other species of salmonid. Between four species of Oncorhynchus, UAA*01 proteins differ by only 2–6 amino acids, whereas comparison of Oncorhynchus with Salmo trutta (brown trout) reveals 14–16 amino acid differences. The Onmy-UAA*01 gene has properties indicative of a particularly divergent non-classical class I gene.
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  • 25
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    Immunogenetics 49 (1999), S. 362-367 
    ISSN: 1432-1211
    Keywords: Key words Major histocompatibility complex ; Class I region ; Evolution ; Orthology ; Olfactory receptor genes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  A comparison of the major histocompatibility complex (Mhc) region between human and mouse highlights both stability and differences. The class II and class III regions are orthologous; they probably existed in the ancestor in a similar organization and were not subjected to major rearrangement. The class I genes, by contrast, are definitely paralogous, having been reorganized several times. As long as only class I genes were identified, the class I regions of human and mouse were difficult to compare directly. The identification of non-class I genes has allowed a comparative map to be drawn, which shows that the class I region is orthologous between human and mouse as well. The lack of orthology specifically applies to the class I sequences. However, the comparative map shows that the non-orthologous class I sequences occupy homologous locations with regard to the conserved genes. I propose a model to explain this paradox. The conserved genes may represent samples of a dense "framework" of genes whose alterations are deleterious. The homologous positions occupied by class I genes would thus represent the few permissive places allowing major perturbations. The evolution of the class I sequences, by duplication and deletion, independently in the two species, has taken place within the scope defined by the framework: insertion at the permissive places, and expansion by creation of class I-related DNA by duplication, thus pushing back the boundaries of the framework.
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  • 26
    ISSN: 1432-1211
    Keywords: Key words Antigen processing ; Evolution ; Cell surface ; molecules ; Mhc ; Transporters
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)–2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized.
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  • 27
    ISSN: 1432-1211
    Keywords: Key words New world primates ; MHC ; Evolution ; Gene duplication
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Lymphocytes of a New World primate, the cotton-top tamarin (Saguinus oedipus), express classical G–related major histocompatibility complex (MHC) class I molecules with unusually limited polymorphism and variability. Three G-related loci, an F locus, an E locus, and two pseudogenes (So-N1 and So-N3) have been identified by cDNA library screening and extensive PCR analysis of both cDNA and genomic DNA from the cotton-top tamarin. Furthermore, each genus of the subfamily Callitrichinae (tamarins and marmosets) appears to express its own unique set of MHC class I genes, likely due to a rapid turnover of loci. The rapid emergence of unique MHC class I genes in the Callitrichinae genera, resulting from an active process of duplication and inactivation of loci, may account for the limited diversity of the MHC class I genes in the cotton-top tamarin. To determine the nature of the entire complement of MHC class I genes in the cotton-top tamarin, we synthesized a genomic DNA library and screened it with MHC class I-specific probes. We isolated nine new MHC class I pseudogenes from this library. These newly isolated tamarin G–related MHC class I pseudogenes are not closely related to any of their functional counterparts in the tamarin, suggesting that they do not share a recent common ancestral gene with the tamarin's currently expressed MHC class I loci. In addition, these tamarin sequences display a high rate of nonsynonymous substitutions in their putative peptide binding region. This indicates that the genes from which they have derived were likely subject to positive selection and, therefore, were once functional. Our data support the notion that an extremely high rate of loci turnover is largely responsible for the limited diversity of the MHC class I genes in the cotton-top tamarin.
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  • 28
    ISSN: 1432-1211
    Keywords: Key words MHC ; Primates ; Reproduction ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons.
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  • 29
    ISSN: 1432-1211
    Keywords: Key words Duck ; Immunoglobulin genes ; IgH locus ; Class switching ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
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  • 30
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    Immunogenetics 49 (1999), S. 15-25 
    ISSN: 1432-1211
    Keywords: Key words Rh blood group system ; Phylogenesis ; RH genes ; Evolution ; Nonhuman primates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The presence of Rh30-like polypeptides with an apparent relative molecular mass of 33 000 in the erythrocyte membranes from nonhuman primates and nonprimate mammals (mouse, rat, and dog) was demonstrated by immunoblotting. Nonhuman primates (orangutan, baboon, New World monkeys, lemur) and mouse Rh-like transcripts were amplified and sequenced. Analysis of the deduced amino acids sequences allowed us to determine the amino acid variability of Rh-like polypeptides which correlated with the hydrophylicity indexes. Hence, the putative transmembrane domains exhibited low indexes of variability, while the highest indexes were observed on extramembrane loops with a maximum on the sixth external loop. The cDNA sequences were compared with those previously reported in human, nonhuman primates, and cattle. The time of coalescence of mammalian Rh cDNA sequences was estimated by phylogenetic analysis to be 100 million years.
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  • 31
    ISSN: 1432-0983
    Keywords: Key wordsChlamydomonas ; Chloroplast ; Evolution ; Group-I introns ; psbA gene ; Self-splicing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1–Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3′ end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11–38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas.
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  • 32
    ISSN: 1432-1327
    Keywords: Key words Iron-sulfur ; Nitrogen fixation ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract  The [2Fe-2S] protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster. Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one [2Fe-2S] cluster. The sequence of this protein is very similar to that of the [2Fe-2S] ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the [2Fe-2S] cluster occur in the same positions. The A. vinelandii protein differs from the C. pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues. The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical. These data show that the A. vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the [2Fe-2S] ferredoxin from C. pasteurianum. The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles.
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  • 33
    ISSN: 1573-4919
    Keywords: microbodies ; diabetes mellitus ; steroid hormone receptor ; β-oxidation ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract To determine whether the increased fatty acid β-oxidation in the peroxisomes of diabetic rat liver is mediated by a common peroxisome proliferation mechanism, we measured the activation of long-chain (LC) and very long chain (VLC) fatty acids catalyzed by palmitoyl CoA ligase (PAL) and lignoceryl CoA ligase and oxidation of LC (palmitic acid) and VLC (lignoceric acid) fatty acids by isotopic methods. Immunoblot analysis of acyl-CoA oxidase (ACO), and Northern blot analysis of peroxisome proliferator-activated receptor (PPAR-α), ACO, and PAL were also performed. The PAL activity increased in peroxisomes and mitochondria from the liver of diabetic rats by 2.6-fold and 2.1-fold, respectively. The lignoceroyl-CoA ligase activity increased by 2.6-fold in diabetic peroxisomes. Palmitic acid oxidation increased in the diabetic peroxisomes and mitochondria by 2.5-fold and 2.7-fold, respectively, while lignoceric acid oxidation increased by 2.0-fold in the peroxisomes. Immunoreactive ACO protein increased by 2-fold in the diabetic group. The mRNA levels for PPAR-α, ACO and PAL increased 2.9-, 2.8- and 1.6-fold, respectively, in the diabetic group. These results suggest that the increased supply of fatty acids to liver in diabetic state stimulates the expression of PPAR-α and its target genes responsible for the metabolism of fatty acids.
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  • 34
    ISSN: 1573-4919
    Keywords: regucalcin ; Ca2+-binding protein ; protein kinase C ; Ca2+signaling ; gene expression ; H4-II-E hepatoma cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of hepatic Ca2+-binding protein regucalcin in the cloned rat hepatoma cells (H4-II-E) was investigated. The change in regucalcin mRNA levels was analyzed by Northern blotting using rat liver regucalcin complementary DNA (0.9 kb of open reading frame). Regucalcin mRNA was expressed in H4-II-E hepatoma cells. This expression was clearly stimulated in the presence of serum (10% fetal bovine serum). Bay K 8644 (2. 5 × 10-6 M), a Ca2+ channel agonist, significantly stimulated regucalcin mRNA expression in the absence or presence of 10% serum. Dibutyryl cyclic AMP (10-3 M) did not have a stimulatory effect on the regucalcin mRNA expression. The presence of phorbol 12-myristate 13-acetate (PMA; 10-6 M) or estrogen (10-8 M) caused a significant increase in regucalcin mRNA levels in the hepatoma cells cultured in serum-free medium, while insulin (5 × 10-9 M) or dexamethasone (10-6 M) had no effect. Bay K 8644-stimulated regucalcin mRNA expression in the hepatoma cells was completely blocked in the presence of trifluoperazine (10-5 M), an antagonist of calmodulin, or staurosporine (10-7 M), an inhibitor of protein kinase C. The stimulatory effect of PMA was clearly inhibited in the presence of stauroporine. The present study demonstrates that regucalcin mRNA is expressed in the transformed H4-II-E hepatoma cells, and that the expression is stimulated through Ca2+-dependent signaling factors.
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  • 35
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    Molecular and cellular biochemistry 199 (1999), S. 189-200 
    ISSN: 1573-4919
    Keywords: lung ; cancer ; urokinase ; receptor ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The urokinase-type plasminogen activator (uPA) interacts with its receptor (uPAR) to promote proteolysis as well as cell proliferation and migration. These functions contribute to the pathogenesis of neoplastic growth and invasiveness. Expression of uPAR in tumor extracts also inversely correlates with prognosis in many forms of cancer. In this study, we sought to determine if differences in uPAR expression were distinguishable between cultured human lung carcinoma and malignant mesothelioma subtypes. We also sought to determine if, as in malignant mesothelioma cells, uPAR expression is regulated at the posttranscriptional level in cultured malignant lung carcinoma cells. Using 125I-uPA binding and ligand blotting techniques, uPAR was expressed by phenotypically diverse lung carcinoma cell lines, including the H460, H157 and H1395 non-small cell lines and the H146 small cell lung carcinoma line. Increased uPAR expression was also detected in spindle-shaped (M33K) and epithelioid (M9K and MS-1) malignant mesothelioma cells. Selected mediators, including TGF-β, TNF-α, LPS and PMA, uniformly enhanced uPAR expression in each of the tumor cell lines. Steady state uPAR mRNA expression was determined by RNase protection assay and correlated directly with the changes in cell surface uPAR expression. By gel mobility shift and UV-cross linking assays, a uPAR mRNA binding protein (uPAR mRNABp) implicated in the posttranscriptional control of message stability, was identified in each of the cell lines. Expression of uPAR and its message in cultured lung carcinoma and malignant mesothelioma cells is similarly influenced by effectors present in the tumor microenvironment. Regulation of the uPAR message occurs at the posttranscriptional level in cultured small and non-small cell lung carcinoma cells as well as spindle-shaped and fibrous malignant mesothelioma cell lines. Posttranscriptional regulation of uPAR in all these cells involves the interaction of the uPAR mRNABp with uPAR mRNA, which promotes uPAR mRNA destabilization.
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  • 36
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    Molecular and cellular biochemistry 201 (1999), S. 111-123 
    ISSN: 1573-4919
    Keywords: complement factor I ; TPA ; protein kinase C ; gene expression ; Hep G2 cells
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This study examined the role of the protein kinase C (PKC) signalling pathway in the regulation of expression of human complement factor I (CFI) gene. The production of CFI by Hep G2 cells was enhanced in a dose- and time-dependent fashion by 12-O-tetradecanoyl-1,2-phorbol 13-acetate (TPA), a potent PKC activator. 4α-phorbol didecanoate, an inactive phorbol ester, had no effect on CFI synthesis. The TPA-dependent increase in CFI secretion was correlated with an increase in CFI mRNA levels. Forskolin, a cAMP-inducing agent, augmented the TPA response. W7, an inhibitor of protein kinase A and genistein, an inhibitor of protein tyrosine kinase(s) both did not prevent the increase in CFI expression mediated by TPA. However, calphostin C, a specific inhibitor of PKC, abolished the TPA-induced increase in CFI mRNA levels. Down regulation of intracellular PKC levels by prior exposure of Hep G2 cells to a high concentration of TPA also blocked the increase in CFI mRNA levels induced by TPA suggesting that the TPA effects were mediated via activation of PKC. mRNA decay studies indicated that the half-life of CFI mRNA in TPA-induced cells was not significantly different from control. Nuclear run-on transcriptional assays on the other hand demonstrated that whereas the CFI gene is transcribed under basal conditions in Hep G2 cells, TPA induced a 3-4 fold increase in the transcription rate of CFI gene in 24 h. The transcription rate of GAPDH gene did not change, indicating that the effects were not general on gene transcription. Transient transfections of Hep G2 cells with chloramphenicol acetyltransferase reporter gene (CAT) constructs containing a series of sequential 5′ deletions of the CFI promoter and CAT assays showed that the sequence between -136 and -130, containing an AP-1 consensus sequence (TGAGTCA) was required for the TPA response. This observation was substantiated by the finding that mutation of this AP-1 site to TttaTCA or TtAtcCA abolished the TPA responsiveness. The enhancement of the activity of transfected chimeric CAT constructs by TPA was abrogated by calphostin C and by pyrrolidine dithiocarbamate (an inhibitor of NF-κB and AP-1 transactivation). These results indicate that TPA regulation of CFI gene requires PKC signalling and is mediated by via a TPA response element (TRE) in the CFI promoter region located at -136/-130 and involves the transactivation of AP-1 and NF-κB transcription factors. We suggest that PKC may be one of the intracellular pathways that control CFI gene expression and that cellular processes (involving growth factors, hormones, cytokines etc.) that activate PKC may upregulate the expression of the CFI gene.
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  • 37
    ISSN: 1573-4919
    Keywords: regucalcin ; calcium-binding protein ; gene expression ; Ca2+-ATPase ; brain microsomes ; aging
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The expression of calcium-binding protein regucalcin and its effect on the microsomal Ca2+-ATPase activity in rat brain tissues was investigated. The expression of regucalcin mRNA was demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) analysis in brain tissues using rat regucalcin-specific primers. Regucalcin concentration in the brain tissues was about 5 × 10-9 M as measured using enzyme-linked immunoadsorbent assay (ELISA), and this level was lowered with increasing age (50 weeks old). The presence of regucalcin (10-9 to 10-7 M) in the enzyme reaction mixture caused a significant decrease in Ca2+-ATPase activity in the brain microsomes of young rats (5 weeks old). Meanwhile, the enzyme activity was not significantly altered by the addition of calmodulin (1 or 50 μg/ml), calbindin (1 or 10 μg/ml), and S-100 A protein (5 or 25 μg/ml), which are other Ca2+-binding proteins in rat brain. The effect of regucalcin to inhibit microsomal Ca2+-ATPase activity was weakened in the brain of rats with increasing age (50 weeks old). The present study demonstrates that regucalcin is expressed in the brain, and that it can uniquely inhibit Ca2+-ATPase activity in the brain microsomes of rats. The findings suggest that regucalcin plays a role in the regulation of microsomal Ca2+-ATPase activity in rat brain tissues.
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  • 38
    ISSN: 1573-4919
    Keywords: mechanical stretch ; smooth muscle cells ; differential display ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.
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  • 39
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    Journal of evolutionary economics 9 (1999), S. 109-133 
    ISSN: 1432-1386
    Keywords: Key words: Discontinuity ; Evolution ; Logistic diffusion ; Non-linearity ; Non-stationarity ; Self-organisation ; Spectral methods ; JEL-classification: C4; C5; N1; N2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Economics
    Notes: Abstract. This paper offers an econometric methodology for the detection of self-organisational change (defined in terms of the presence of time irreversibility, structural change and fundamental uncertainty) in economic processes that follow logistic diffusion growth paths in historical time. The approach we adopted is built upon recent developments in `moving window' spectral methods which are applied to the scaled residuals generated by estimated logistic diffusion models. We illustrate the use of such methods by examining the case of a financial instrument, namely, the Australian Building Society Deposit, which experienced logistic growth in its market share until bank deregulation was enacted in the 1980s. We show that there is clear evidence that self-organisational change is present over the historical period considered.
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  • 40
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    Journal of evolutionary economics 9 (1999), S. 367-371 
    ISSN: 1432-1386
    Keywords: Key words: Bertrand ; Oligopoly ; Evolution ; Evolutionary stability ; JEL-classification: D43 ; L13 ; C72
    Source: Springer Online Journal Archives 1860-2000
    Topics: Economics
    Notes: Abstract. It is shown that the equilibrium notion of an evolutionary stable strategy (ESS) does have predictive power for standard models of Bertrand competition. This is in contrast to a recent claim by Qin and Stuart (1997). The claim is based on the observation that the solution concept ESS behaves discontinuously when finite (discrete) action games approach an infinite (continuous) action game in the limit. Furthermore, it is argued that from a model-theoretic point of view evolutionary stability in prices (i.e. in the Bertrand model) is quite different from evolutionary stability in quantities (i.e. in the Cournot model).
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  • 41
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    Theoretical and applied genetics 99 (1999), S. 203-209 
    ISSN: 1432-2242
    Keywords: Key words Homeobox gene ; KNOTTED1 ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Homeobox genes encode a family of DNA-binding regulatory proteins which are crucial for development. The first plant homeobox gene identified was knotted1 which plays a major role in leaf development. The knotted1 gene has a homeobox which encodes a homeodomain (HD) and HD proteins have been shown to function as transcription factors. A phylogenetic classification of the KNOTTED1 HD is presented. Here, we report six kn1 HDs from the cereals oat, barley, wheat, rye and rice. The KN1 class-I and -II genes can be divided into two distinct clades. Further, we hypothesize that KN1 and BELL1/MEIS HDs, (the closest non-KN1 class HDs) evolved from a common ancestor after divergence from the common precursor of all the homeobox genes. Our analysis clearly shows the presence of an ancestral KN1 HD from which all the known plant kn1 class of genes evolved.
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  • 42
    ISSN: 1432-2242
    Keywords: Key words Common wild rice ; Cultivated rice ; Evolution ; Genetic analysis ; Molecular marker
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Domesticated rice differs from the wild progenitor in large arrays of morphological and physiological traits. The present study was conducted to identify the genetic factors controlling the differences between cultivated rice and its wild progenitor, with the intention to assess the genetic basis of the changes associated with the processes of rice domestication. A total of 19 traits, including seven qualitative and 12 quantitative traits, that are related to domestication were scored in an F2 population from a cross between a variety of the Asian cultivated rice (Oryza sativa) and an accession of the common wild rice (O. rufipogon). Loci controlling the inheritance of these traits were determined by making use of a molecular linkage map consisting of 348 molecular-marker loci (313 RFLPs, 12 SSRs and 23 AFLPs) based on this F2 population. All seven qualitative traits were each controlled by a single Mendelian locus. Analysis of the 12 quantitative traits resolved a total of 44 putative QTLs with an average of 3.7 QTLs per trait. The amount of variation explained by individual QTLs ranged from a low of 6.9% to a high of 59.8%, and many of the QTLs accounted for more than 20% of the variation. Thus, genes of both major and minor effect were involved in the differences between wild and cultivated rice. The results also showed that most of the genetic factors (qualitative or QTLs) controlling the domestication-related traits were concentrated in a few chromosomal blocks. Such a clustered distribution of the genes may provide explanations for the genetic basis of the “domestication syndrome” observed in evolutionary studies and also for the “linkage drag” that occurs in many breeding programs. The information on the genetic basis of some desirable traits possessed by the wild parent may also be useful for facilitating the utilization of these traits in rice-breeding programs.
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  • 43
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    Theoretical and applied genetics 98 (1999), S. 744-750 
    ISSN: 1432-2242
    Keywords: Key words Chromosome pairing ; Translocations ; T. timopheevii ; T. turgidum ; T. aestivum ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract.  Chromosome pairing at metaphase-I was analyzed in F1 hybrids among T. turgidum (AABB), T. aestivum (AABBDD), and T. timopheevii (AtAtGG) to study the chromosome structure of T. timopheevii relative to durum (T. turgidum) and bread (T. aestivum) wheats. Individual chromosomes and their arms were identified by means of C-banding. Homologous pairing between the A-genome chromosomes was similar in the three hybrid types AAtBG, AAtBGD, and AABBD. However, associations of B-G were less frequent than B-B. Homoeologous associations were also observed, especially in the AAtBGD hybrids. T. timopheevii chromosomes 1At, 2At, 5At, 7At, 2G, 3G, 5G, and 6G do not differ structurally from their counterpart in the A and B genomes. Thus, these three polyploid species inherited translocation 5AL/4AL from the diploid A-genome donor. Chromosome rearrangements that occurred at the tetraploid level were different in T. turgidum and T. timopheevii. Translocation 4AL/7BS and a pericentric inversion of chromosome 4A originated only in the T. turgidum lineage. The two lines of T. timophevii studied carry four different translocations, 6AtS/1GS, 1GS/4GS, 4GS/4AtL, and 4AtL/3AtL, which most likely arose in that sequence. These structural differences support a diphyletic origin of polyploid wheats.
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  • 44
    ISSN: 1432-2242
    Keywords: Key words Catalase ; Oryza ; Rice ; Evolution ; p-SINE1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  Intron-2 of the Oryza sativa CatA catalase gene is similar in nucleotide sequence to p-SINE1, a retroposon, and seems to have been added to the ancestral genome of rice. To examine when the p-SINE1-like intron was inserted into CatA during the evolutionary divergence of Oryza species, and to elucidate the evolutionary relationships among Oryza species using the sequence of the intron as a marker, we performed polymerase chain reaction (PCR) analyses of 32 accessions of 17 Oryza species with various genome types. Agarose-gel electrophoresis of the PCR products revealed that all the Oryza species with an AA genome have the CatA homolog with the intron, whereas other Oryza species have the CatA homolog without the intron. These results indicate that intron-2 of CatA is a good marker for distinguishing species with an AA genome among Oryza species. Sequencing of the PCR products showed that all the introns are similar to p-SINE1, though with slight variations in length. We also performed PCR analyses using four accessions of three species in genera related to Oryza, and found that there is an intron in the CatA homolog of Leersia perrieri. On the other hand, the CatA homolog of Porteresia coarctata has no intron. Sequence data showed that the L. perrieri homolog has a p-SINE1-like intron similar to that in Oryza species with an AA genome. These results suggest that the p-SINE1-like intron was already present in the common ancestor of Oryza and L. perrieri and was then lost in the ancestors of P. coarctata and of the Oryza species other than those with an AA genome. The phylogenetic tree of Oryza species with an AA genome based on the nucleotide sequences of the introns leads us to propose that Oryza species with an AA genome evolved from an ancestor of Oryza longistaminata.
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  • 45
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    Theoretical and applied genetics 98 (1999), S. 478-484 
    ISSN: 1432-2242
    Keywords: Key words Endosperm development ; Evolution ; 2n gametes ; Breeding ; Potato
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The Endosperm Balance Number (EBN) hypothesis was developed in the early ’80s to explain the basis for normal seed development after intra- and inter-specific crosses, first in the potato and then in several other crop species. According to this hypothesis, each species has a genome-specific effective ploidy, the EBN, which must be in a 2 : 1 maternal to paternal ratio in the hybrid endosperm for normal development of the endosperm itself. This paper reviews how the EBN may act as a powerful isolating mechanism in sexual reproduction, maintaining the genome integrity of the species and playing an important role in the speciation of polyploids from diploids. We also provide further evidence that EBN is more important than chromosome ploidy in determining the success or failure of interspecific crosses. In fact, results from inter-ploidy and inter-EBN crosses to infuse 1EBN Solanum commersonii into 4EBN S. tuberosum demonstrated that the knowledge and manipulation of EBN is a useful tool in designing breeding schemes and in predicting the offspring ploidy and EBN. In this paper we also discuss the exceptions to the 2 : 1 EBN ratio, and report the evidence for endomitosis in the polar nuclei to explain exceptions to the EBN model in the potato.
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  • 46
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    Behavioral ecology and sociobiology 45 (1999), S. 269-275 
    ISSN: 1432-0762
    Keywords: Key words Polyandry ; Evolution ; Social insects ; Apis mellifera ; Task allocation ; Division of labor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A number of hypotheses have been proposed to explain the evolution of multiple mating in the honeybee queen. In particular, the consequences of reduced intracolonial relatedness provide plausible explanations for multiple mating with up to ten drones, but fail to account for the much higher mating frequencies observed in nature. In this paper, we propose an alternative mechanism which builds on non-linear relationships between intracolonial frequencies in genotypic worker specialization and colony fitness. If genes for any worker specialization confer an advantage on colony fitness only when they are rare, this would require a stable mix of sperm from a few drones which contribute that trait, and many which do not. To ensure both specific, low within-colony proportions of “rare specialist” genes, and to reduce random variation of these proportions would require mating with high numbers of drones. The quantitative implementation shows that moderate to very high numbers of matings are required to exploit colony advantages from genotypic allocation of workers to rare tasks. Extreme polyandry thus could result from colony selection dependent on the intracolonial frequency of rare genetic specialists.
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  • 47
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    Cell & tissue research 296 (1999), S. 19-25 
    ISSN: 1432-0878
    Keywords: Key wordsHoxA ; HoxD ; Limb ; Development ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Homeobox genes located in the 5’ part of the HoxA and HoxD complexes are required for proliferation of skeletal progenitor cells of the vertebrate limb. Specific combinations of gene products determine the length of the upper arm (genes belonging to groups 9 and 10), the lower arm (groups 10, 11 and 12) and the digits (groups 11, 12 and 13). In these different domains, individual gene products quantitatively contribute to an overall protein dose, with predominant roles for groups 11 and 13. Quantitative reduction in the gene dose in each set results in truncations of the corresponding anatomical regions. The physical order of the genes in the HoxA and HoxD complexes, as well as a unidirectional sequence in gene activation, allow for completion of the process in a precise order, which in turn makes possible the sequential outgrowth of the respective primordia. While the skeletal patterns of upper and lower limb are relatively stable throughout the tetrapods, more variation is seen in the digits. Molecular analysis of the underlying regulatory processes promises further exciting insights into the genetic control of development, pathology and the course of evolution.
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  • 48
    ISSN: 1432-0878
    Keywords: Key words Metabotropic glutamate/GABA-like receptor ; Evolution ; Geodia cydonium (Porifera)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To date, no conclusive evidence has been presented for the existence of neuronal-like elements in Porifera (sponges). In the present study, isolated cells from the marine sponge Geodia cydonium are shown to react to the excitatory amino acid glutamate with an increase in the concentration of intracellular calcium[Ca2+]i. This effect can also be observed when the compounds L-quisqualic acid (L-QA) or L-(+)-2-amino-4-phosphonobutyric acid (L-AP-4) are used. The effect of L-QA and L-AP-4, both agonists for metabotropic glutamate receptors (mGluRs), can be abolished by the antagonist of group I mGluRs, (RS)-α-methyl-4-carboxyphenylglycine. These data suggest that sponge cells contain an mGluR-like protein. A cDNA encoding rat mGluR subtype 1 has been used to identify the complete nucleotide sequence of G. cydonium cDNA coding for a 528-amino-acid-long protein (59 kDa) that displays marked overall similarity to mGluRs and to γ-amino-butyric acid B receptors. The deduced sponge polypeptide, termed putative mGlu/GABA-like receptor, displays the highest similarity to the two families of metabotropic receptors within the transmembrane segment. The N-terminal part of the sponge sequence shows similarity to mGluR4 and mGluR5. These findings suggest that the earliest evolutionary metazoan phylum, the Porifera, possesses a sophisticated intercellular communication and signaling system, as seen in the neuronal network of higher Metazoa.
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  • 49
    ISSN: 1871-4528
    Keywords: Solanum tuberosum L. ; tuberisation ; extensin ; acyl carrier protein thioesterase ; high mobility group protein ; gene expression ; plant development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary In screening to isolate a full-length copy of a previously isolated cDNA clone, a further three cDNAs were also isolated from a library prepared from sub-apical swelling-stolon tissue of potato (Solanum tuberosum L.). Sequence analysis showed these clones to be similar to extensin-like protein genes, acyl carrier protein thioesterase genes and high mobility group protein genes, respectively. A further cDNA, isolated by subtractive hybridisation, was similar to a tomato cDNA previously isolated on the basis of its down-regulation following nematode infection. While all the newly isolated genes were expressed in swelling stolons, for most, maximal expression was seen to be in stem tissue. Possible roles for these genes in the development of potato plants are discussed, as is the significance of gene expression in stems and stolons to the process of tuberisation.
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  • 50
    ISSN: 1573-9368
    Keywords: transgenic mice ; prolactin ; mammary gland ; gene expression ; Stat5 ; β-globin insulator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to establish a possible correlation between in vitro prolactin induction and the transcriptional activity of mammary gene promoters in transgenic mice, a functional Stat5-binding site was created by means of site-directed mutagenesis at position −70 on a 560 bp murine α-lactalbumin promotor linked to a CAT reporter gene. Surprisingly, the wild-type promoter was constitutively active in vitro and could not be induced by prolactin. Introducing the proximal Stat5 site abolished this constitutive activity and resulted in prolactin dependence in both CHO-K1- and HC11-transfected cells. In transgenic mice, both the frequency of lines expressing the transgene and the prevalence of mid to late pregnancy expression were increased.
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  • 51
    ISSN: 1617-4623
    Keywords: Key words Phytoene synthase ; Lycopene cyclase ; Complementation ; Astaxanthin ; Evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The red heterobasidiomycetous yeast Xanthophyllomyces dendrorhous (perfect state of Phaffia rhodozyma) contains a novel type of carotenoid biosynthetic enzyme. Its structural gene, designated crtYB, was isolated by functional complementation in a genetically modified, carotenogenic Escherichia coli strain. Expression studies in different carotenogenic E. coli strains demonstrated that the crtYB gene encodes a bifunctional protein involved both in synthesis of phytoene from geranylgeranyl diphosphate and in cyclisation of lycopene to β-carotene. By sequence comparison with other phytoene synthases and complementation studies in E. coli with various deletion derivatives of the crtYB gene, the regions responsible for phytoene synthesis and lycopene cyclisation were localised within the protein.
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  • 52
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    Plant systematics and evolution 219 (1999), S. 55-64 
    ISSN: 1615-6110
    Keywords: Evolution ; Encyclia cochleata ; inbreeding depression ; isozymes ; Orchidaceae ; outcrossing ; phenotype ; population genetics ; selfing
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To determine whether inbreeding depression accounts for the maintenance of outcrossing in populations of the self-compatible orchidEncyclia cochleata, the estimated selective advantage of selfing was compared to a measure of inbreeding depression. Individuals from three populations ofE. cochleata and some of their progeny were phenotyped using isozyme analysis. The electrophoretic data were used to estimate the outcrossing rate and the theoretical cost of outcrossing. Inbreeding depression was estimated by comparing the fitness of the progeny resulting from both types of pollinations. The seeds from outcrossed and selfed hand-pollinations and naturally pollinated seeds from a population of the triandrous form ofE. cochleata were grown aseptically on culture media, and their development over the next three years recorded. Inbreeding was common, particularly in one population (outcrossing rate 40%). However, the level of inbreeding depression was only 1–2%, considerably less inbreeding depression than expected.
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  • 53
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    Artificial life and robotics 3 (1999), S. 127-132 
    ISSN: 1614-7456
    Keywords: Evolution ; Cooperative behavior ; Communication ; Species fitness ; Reciprocative
    Source: Springer Online Journal Archives 1860-2000
    Topics: Computer Science
    Notes: Abstract This paper considers the evolution of cooperative behaviors as the interaction among agents using a genetic algorithm to improve the performance of the task in a group (group performance). Previous research often usedthe group fitness method, which evaluates group performance for the evolution of multiple groups in parallel. However, this entails large simulation costs and the evolution speed is slow.The individual fitness method that evaluates theindividual performance of the task entails a smaller simulation cost. However, it can not improve the group performance since each agent behaves selfishly. To optimize the group performance, it is important to include bothcompetition andsharing. Therefore, this paper presentsthe species fitness method, which shares the individual performances of agents belonging to the same species in a group that all have the same chromosomes. We show comparative experiments on these three methods on the evolutionary simulation of a foraging task in a group. To test the interaction among the agents, four kinds of species are evolved which show their communication ability by demonstrating whether the agent can send or receive the signal for food. Experimental results show that evaluating the species variance fitness leads the agents into reciprocative actions.
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  • 54
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    Artificial life and robotics 3 (1999), S. 133-138 
    ISSN: 1614-7456
    Keywords: Bipedal walking ; Evolution ; Neuro-musculoskeletal model ; Genetic algorithms
    Source: Springer Online Journal Archives 1860-2000
    Topics: Computer Science
    Notes: Abstract The acquisition process of bipedal walking in humans was simulated using a neuro-musculo-skeletal model and genetic algorithms, based on the assumption that the shape of the body has been adapted for locomotion. The model was constructed as 10 two-dimensional rigid links with 26 muscles and 18 neural oscillators. Bipedal walking was generated as a mutual entrainment between neural oscillations and the pendulous movement of body dynamics. Evolutionary strategies incorporated, for example, as fitness in the genetic algorithms were assumed to decrease energy consumption, muscular fatigue, and load on the skeletal system. An initial population of 50 individuals was created, and an evolutionary simulation of 5000 steps was conducted. As a result, the shape of the body changed from that of a chimpanzee to that of a modern human, and the body size nearly reached the size of a modern human. These simulation results show that improving locomotive efficiency and reducing the load on the musculo-skeletal system are important factors affecting the evolution of the human body shape and bipedal walking. Such computer simulations help us to understand the process of evolution and adaptation for locomotion in humans.
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  • 55
    ISSN: 1573-0778
    Keywords: CHO cells ; gene expression ; kinetic model ; protein secretion ; transcription ; translation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The elevation of expression levels for secreted glycoproteins by gene amplification in mammalian cells shows a saturation behavior at high levels of gene amplification. At high expression levels a drop in the secretion efficiency for the recombinant protein occurs (Schröder and Friedl, 1997), coinciding with the appearance of misfolded protein in the cell. In this communication we investigated whether additional limitations exist at the levels of transcription and translation. Four Chinese hamster ovary (CHO) cell lines expressing different amounts of human antithrombin III (ATIII) were used as a model system. A tenfold increase in the ATIII cDNA copy number from the lowest to the highest producing cell line coincided with a 38-fold increase in ATIII mRNA levels, and an 80-fold increase in the amount of intracellular ATIII levels. The data was analyzed using a simple kinetic model. The following conclusions were derived: I. The transcriptional activity for the recombinant protein is not saturated. II. Translation itself is not saturated either, but may be downregulated as secretion efficiency drops. III. Two explanations for the previously reported drop in secretion efficiency for the recombinant protein with increasing expression level are possible: A. Protein degradation is an alternative fate for translated ATIII and the fraction of ATIII degraded after translation increases as expression level is increased. B. Translation is downregulated as the secretory apparatus becomes exhausted to maintain cell viability.
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  • 56
    ISSN: 1573-0778
    Keywords: cardiogenesis ; cell differentiation ; gene expression ; mouse embryonic stem cells ; myogenesis ; neurogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro.
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  • 57
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    Cytotechnology 30 (1999), S. 71-83 
    ISSN: 1573-0778
    Keywords: gene expression ; HEK293(EBNA) cells ; serum-free ; transient transfection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In order to establish a simple and scaleable transfection system we have used the cationic polymer polyethylenimine (PEI) to study transient transfection in HEK293 and 293(EBNA) cells grown in serum-free suspension culture. The transfection complexes were made directly within the cell culture by consecutively adding plasmid and PEI (direct method). Alternatively, the DNA-PEI transfection complexes were prepared in fresh medium (1/10 culture volume) and then added to the cells (indirect method). The results of this study clearly show that the ratio of PEI nitrogen to DNA phosphate is very important for high expression levels. The precise ratio is dependent on the DNA concentration. For example, using 1 μg/ml DNA by the indirect method, the ratio of optimal PEI:DNA was about 10–13:1. However, the ratio increases to 33:1 for 0.1–0.2 μg/ml DNA. By testing several different molecular weights of the polycationic polymer we could show that the highest transfection efficiency was obtained with the PEI 25 kDa. Using PEI 25 kDa the indirect method is superior to the direct addition because significantly lower DNA concentrations are needed. The expression levels of the soluble human TNF receptor p55 are even higher at low DNA compared to 1 μg/ml plasmid. The EBV-based pREP vectors gave better transient gene expression when used in 293(EBNA) cells compared to HEK293 cells in suspension culture. No differences in expression levels in the two cell lines were observed when the pC1 (CMV)-TNFR was used. In conclusion, PEI is a low-toxic transfection agent which provides high levels of transient gene expression in 293(EBNA) cells grown in serum-free suspension culture. This system allows highly reproducible, cost-effective production of milligram amounts of recombinant proteins in 2–5 l spinner culture scale within 3–5 days. Fermentor scale experiments, however, are less efficient because the PEI-mediated transient tranfection is inhibited by conditioned medium.
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  • 58
    ISSN: 1573-0778
    Keywords: cell cycle ; CHO ; flow cytometry ; gene expression ; synchronisation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Understanding the relationships between cell cycle and protein expression is critical to the optimisation of media and environmental conditions for successful commercial operation of animal cell culture processes. Using flow cytometry for the analysis of the early phases of synchronised batch cultures, the dependency of product expression on cell cycle related events has been evaluated in a recombinant CHO cell line. Although the production of recombinant protein is initially found to be cell cycle related, the maximum specific protein productivity is only achieved at a later stage of the exponential phase which also sees a maximum in the intracellular protein concentration. Subsequent work suggests that it is the batch phase/medium composition of cultures which is the major determinant of maximum specific productivity in this cell line. Furthermore the effect of the positive association between S phase and specific productivity is subordinate to the effect of batch phase/medium composition on the specific productivity of batch cultures.
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  • 59
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    European journal of plant pathology 105 (1999), S. 519-533 
    ISSN: 1573-8469
    Keywords: genome ; gene expression ; mollicute ; recombination ; transposition ; virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Spiroplasmas are members of the Class Mollicutes, wall-less prokaryotes having a high adenosine–thymidine content in their small genomes. Spiroplasma citri is a plant pathogen that inhabits phloem. Like other phytopathogenic spiroplasmas and the related phytoplasmas, it is transmitted from plant to plant by phloem-feeding leafhoppers that serve as alternate hosts for the spiroplasma as well as vectors. Genetic information in spiroplasmas is carried on a circular chromosome, on plasmids and/or in virus genomes. A picture emerging from recent research on the S. citri genome is one of frequent and often extensive variation, resulting from a number of different mechanisms. Expansion and contraction events must continually be occurring in about equal proportions so that the net genome size varies within defined boundaries. Particularly impressive are large changes in genome size that can occur in only a few generations. As with most organisms, genetic variation in S. citri results from variation in extrachromosomal DNA content, changes due to DNA replication and repair processes and changes due to recombination. The implied flux of genetic information into and out of the S. citri genome should be beneficial to the bacterium, allowing it, with its small genome size, to adapt to new environments.
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  • 60
    ISSN: 1573-4935
    Keywords: Mucin ; lung cancer ; gene expression ; secretion ; lung adenocarcinoma
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Mucins comprise an important class of tumor-associated antigens. The objectives of the present study were (a) to establish an in vitro model system using human non-small cell lung adenocarcinoma cell lines NCIH650 and NCIH2077 (b) provide evidence that these cell lines secrete mucin in culture conditions and (c) investigate the effects of select secretagogues on mucin secretion. The cell lines were established in ACL-4 medium containing several growth factors and retinoic acid and 5% fetal calf serum. The high molecular weight glycoconjugates secreted in the culture medium were purified by ammonium sulfate precipitation and Superose 6 and Superose 12 FPLC chromatography. The purified high molecular weight glycoconjugate fraction and the carcinoma cells were shown to have mucin by dot blot, Western blot and immunohistochemical analysis, respectively, using specific antibodies to purified major mucin, HTM-1. Also, incorporation experiments with mucin precursor 3H-glucosamine demonstrated that the cells indeed synthesize high molecular weight mucins. The effects of secretagogues such as, 8-bromocyclic AMP, ionomycin, phorbol-12-myristate-13-acetate and neutrophil elastase on mucin secretion were also investigated. Only 8-bromocyclic AMP and neutrophil elastase influenced mucin secretion. These studies provided strong evidence that the lung adenocarcinoma cell lines secrete high molecular weight mucins in culture conditions and only two of the four tested secretagogues significantly increased mucin secretion. Thus, this in vitro model system may be useful in determining alterations in mucin structure, if any, in lung adenocarcinomas as well as in studying the regulation of mucin gene expression.
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  • 61
    ISSN: 1573-904X
    Keywords: adriamycin ; doxorubicin ; HPMA copolymer ; apoptosis, multidrug resistance ; gene expression ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To study peculiarities and the mechanism of the anticancer effect of free and HPMA copolymer-bound ADR in sensitive and resistant human ovarian carcinoma cells. Methods. Sensitive A2780 and ADR resistant A2780/AD cells were exposed to different doses of drugs during 12, 24, 36, 48, 60, and 72 hours. Cell viability, drug accumulation, apoptosis, cellular metabolism, lipid peroxidation, DNA content and gene expression were studied. Results. HPMA copolymer-bound ADR (P(GFLG)-ADR) possessed a comparable cytotoxicity to free ADR when comparison was based on intracellular concentrations. While free ADR up-regulated genes encoding ATP driven efflux pumps (MDR1, MRP), P(GFLG)-ADR overcame existing pumps and down regulated the MRP gene. Free ADR also activated cell metabolism and expression of genes responsible for detoxification and DNA repair. P(GFLG)-ADR down-regulated HSP-70, GSr-π, BUDP, Topo-IIα, β, and TK-1 genes. Apoptosis, lipid peroxidation and DNA damage were significantly higher after exposure to P(GFLG)-ADR, as reflected by simultaneous activation of p53, c-fos in A2780 cells) or c-jun (A2780/AD) signaling pathways and inhibition of the bcl-2 gene. Differences between free ADR and P(GFLG)-ADR increased with the time of incubation and drug concentration. Conclusions. P(GFLG)-ADR overcame drug efflux pumps, more significantly induced apoptosis and lipid peroxidation, inhibited DNA repair, replication, and biosynthesis when compared to free ADR.
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  • 62
    ISSN: 1617-4623
    Keywords: Key words Catalase ; Oryza ; Rice ; Evolution ; Tourist
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Tourist-OsaCatA, a transposable element, was found in the 5′-flanking region of the rice gene CatA. The characteristics of this element are similar to those of the other Tourist elements so far found in Oryza sativa. PCR and sequence analyses of 37 accessions of 18 species revealed that all the Oryza species examined, except for one accession, have either a full-length or a partial Tourist element at this locus. Unlike the Tourist elements previously reported, this Tourist element is found in all four Oryza species complexes in the Oryzeae tribe. All AA genome Oryza species, except O. longistaminata, contain the full-length Tourist element. O. longistaminata and the species of the O. officinalis, O. meyeriana and O. ridleyi complexes contain the partial element. A phylogenetic tree of Oryza species based on the nucleotide sequences of these Tourist elements was constructed. The O. longistaminata accessions were placed near the neighboring cluster of the officinalis complex. We propose that the ancestor of O. longistaminata and that of other species with the AA genome diverged, and the ancestor(s) of the O. officinalis, O. ridleyi and O. meyeriana complexes then diverged from the ancestor of O. longistaminata in the course of the evolution of the Oryza species. The Tourist elements associated with CatA and its orthologs thus provide useful tools for examining evolutionary relationships among Oryza species.
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    Molecular genetics and genomics 262 (1999), S. 189-198 
    ISSN: 1617-4623
    Keywords: Key wordsBordetella spp. ; Evolution ; Lipopolysaccharide biosynthesis ; Pulsed-field gel electrophoresis ; Representational difference analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Bordetella pertussis and B. bronchiseptica are genetically very closely related but differ significantly in their virulence properties. Using Representational Difference Analysis (RDA), 11 DNA fragments specific for B. pertussis Tohama I or B. bronchiseptica BB7865 were identified. All B. bronchiseptica BB7865-derived fragments also hybridized with chromosomal DNA from B. parapertussis but not from the B. pertussis strains Tohama I and W28, underlining the close phylogenetic relationship between B. bronchiseptica and B. parapertussis. The B. pertussis type strain BP18323 is a special case, as it contains DNA sequences characteristic for both B. pertussis and B. bronchiseptica. As demonstrated by pulsed-field gel electrophoresis, several of the BB7865-derived fragments are present on a single 30-kb XbaI fragment. Based on the sequences of putative coding regions, four of these fragments may code for proteins involved in carbohydrate metabolism or transport. In agreement with this notion, a mutant for one of these loci synthesizes a significantly altered lipopolysaccharide that lacks the O-specific side chains. The analysis of the corresponding genomic region in various Bordetella species showed that this locus is present in B. bronchiseptica and B. parapertussis but not in B. pertussis. This confirms that the RDA approach has identified a novel strain-specific LPS biosynthesis locus which accounts for the differences between the LPS structures elaborated by different Bordetella species.
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  • 64
    ISSN: 1573-5028
    Keywords: UV-B ; soybean ; chalcone synthase ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract By developing gene-specific RT-PCR and using filters to allow transmission down to 290 nm (UV-B+) or blocking all radiation below 320 nm (UV-B−), the effect of UV-B+ and UV-B− light on expression of each of the presently known seven members of soybean chalcone synthase (CHS) gene family in dark-grown seedlings was analyzed. Dark expression was detectable already in 18 h dark-germinating embryos, with progressive increases on successive days, suggesting that chs belongs to a class of genes expressed very early during germination, and that the expression at this stage is either constitutive or induced by non-light-dependent factors present in the seed or made available following imbibition. Exposure of 18 h dark-germinating embryos to UV-B− or to UV-B+ light did not lead to an increase in chs signal. However, the 24 h dark-germinating embryos showed a distinct effect of UV-B+, interestingly coinciding with the stage when the head of seedlings was in the process of being pushed up above ground by stem elongation, suggesting the possibility of a developmental switch modulating the appearance of UV-B response. The response to UV-B− was most prominent in chs1 and almost silent in chs2, while the up-regulation by UV-B+ was most prominent in chs5 and chs6 and much less so in chs2. Interestingly, chs2 was noted to be the only member of the Gmchs gene family devoid of H-box, raising the possibility that the H-box may be a good indicator of the photo-inducibility of a chs gene.
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  • 65
    ISSN: 1573-5028
    Keywords: amphidiploid genome structure ; gene expression ; glutamine synthetase ; multigene family ; nitrogen assimilation ; oilseed rape
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the amphidiploid genome of oilseed rape (Brassica napus) the diploid ancestral genomes of B. campestris and B. oleracea have been merged. As a result of this crossing event, all gene loci, gene families, or multigene families of the A and C genome types encoding a certain protein are now combined in one plant genome. In the case of the multigene family for glutamine synthetase, the key enzyme of nitrogen assimilation, six different cDNA sequences were isolated from leaf and root specific libraries. One sequence pair (BnGSL1/BnGSL2) was characterized by the presence of amino- terminal transit peptides, a typical feature of all nuclear encoded chloroplast proteins. Two other cDNA pairs (BnGSR1-1/BnGSR1-2 and BnGSR2-1/BnGSR2-2) with very high homology between each other were found in a root specific cDNA library and represent protein subunits for cytosolic glutamine synthetase isoforms. Comparative PCR amplifications of genomic DNA isolated from B. napus, B. campestris and B. oleracea followed by sequence–specific restriction analyses of the PCR products permitted the assignment of the cDNA sequences to either the A genome type (BnGSL1/BnGSR1- 1/BnGSR2-1) or the C genome type (BnGSL2/BnGSR1-2/BnGSR2-2). Consequently, the ancestral GS genes of B. campestris and B. oleracea are expressed simultaneously in oilseed rape. This result was also confirmed by RFLP (restriction fragment length polymorphism) analysis of RT-PCR products. In addition, the different GS genes showed tissue specific expression patterns which are correlated with the state of development of the plant material. Especially for the GS genes encoding the cytosolic GS isoform BnGSR2, a marked increase of expression could be observed after the onset of leaf senescence.
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  • 66
    ISSN: 1573-5028
    Keywords: defense ; gene expression ; leaf senescence ; nitrilase ; pathogen-free ; salicylic acid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To determine the range of gene activities associated with leaf senescence, we have identified genes that show preferential transcript accumulation during this developmental stage. The mRNA levels of a diverse array of gene products increases during leaf senescence, including a protease, a ribosomal protein, two cinnamyl alcohol dehydrogenases, a nitrilase and glyoxalase II. Two of the genes identified are known to be pathogen-induced. The senescence specificity of each gene was determined by characterization of transcript accumulation during leaf development and in different tissues. The increased expression of nitrilase in senescent leaves is paralleled by an increase in free indole-3-acetic acid (IAA) levels. Additionally, we have demonstrated that the induction of defense-related genes during leaf senescence is pathogen-independent and that salicylic acid accumulation is not essential for this induction. Our data indicate that the induction of certain genes involved in plant defense responses is a component of the leaf senescence program.
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  • 67
    ISSN: 1573-5028
    Keywords: gene expression ; gibberellin ; H1 histone ; H2B histones ; leaf ; Lycopersicon esculentum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract After differential screening we isolated cDNA clones encoding a histone H1 (leH1) and three variants of histone H2B (leH2B-1, -2 and -3) from the gibberellin (GA)-deficient mutant of tomato (gib-1). The deduced polypeptide of leH1 is 271 amino acids long and exhibits the typical tripartite structure of histones H1. The full-length cDNA clone leH2B-1 encodes for a protein of 142 amino residues and shows the tripartite organization of histones H2B. The histones leH1 and leH2B, which show no tissue specificity, are developmentally expressed in the leaf. The mRNA accumulation was higher in organs which contain meristematic tissue and/or which have a high proportion of actively cycling cells. In the leaf of the gib-1 mutant we demonstrated GA-enhanced histone leH1 and leH2B expression which was not observed in the wild type. GAs of the early-13-hydroxylated pathway (GA1 and GA3) caused most enhanced transcription compared to GAs of the early-non-hydroxylation pathway (GA4 and GA9). Application of GA to the mutant increased histone expression that could correlate with enhanced DNA replication in leaf tissue. Increased chromosome replication may indicate that there is a higher rate of cell division and/or increase of endopolyploidy which both may be dependent on cell elongation induced by GAs.
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  • 68
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    Plant molecular biology 39 (1999), S. 933-943 
    ISSN: 1573-5028
    Keywords: cloning ; fruit development ; gene expression ; pea ; polyamine ; spermidine synthase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two cDNAs from young pea fruits coding for functional spermidine synthases (EC 2.5.1.16) were isolated. The corresponding genes were named psSPDSYN1 and psSPDSYN2. Both cDNAs complemented spe3Δ gene when introduced into the Y480 strain of Saccharomyces cerevisiae, which is a null mutant for the spermidine synthase gene. psSPDSYN1 and psSPDSYN2 are regulated differentially. psSPDSYN1 is up-regulated early after fruit set whereas psSPDSYN2 is expressed later. Spermidine synthase activity was detected in pea ovaries, and correlates with the pattern of expression of psSPDSYN1. In the pea plant, psSPDSYN1 is highly expressed in actively growing tissues, whereas the highest level of psSPDSYN2 mRNA was detected in fully elongated stem.
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  • 69
    ISSN: 1573-5028
    Keywords: chitin oligomer ; chitinase ; elicitor ; gene expression ; rice ; signal transduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.
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  • 70
    ISSN: 1573-5028
    Keywords: cytochrome b5 ; fruit and flower development ; gene expression ; in situ hybridisation ; olive
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report the characterisation of two cytochrome b5 genes and their spatial and temporal patterns of expression during development in olive, Olea europaea. A PCR-generated probe, based on a tobacco cytochrome b5 sequence, was used to isolate two full-length cDNA clones (cytochrome b5-15 and cytochrome b5-38) from a library derived from 13 WAF olive fruits. The cDNAs encoded proteins of 17.0 and 17.7 kDa, which contained all the characteristic motifs of cytochromes b5 from other organisms and exhibited 63% identity and 85% similarity with each other. The olive cytochrome b5-15 cDNA was then used as a probe for more detailed analysis. Southern blotting revealed a gene family of at least 4–6 members while northern blotting and in situ hybridisation showed a highly specific pattern of gene expression. Very low levels of cytochrome b5 mRNA were detected in tissues characterised by high rates of lipid accumulation, such as young expanding leaves, maturing seeds and ripening mesocarp. The cytochrome b5 genes were not induced at 6 °C and their response to ABA was relatively slow compared with fatty acid desaturase genes. In contrast, high levels of cytochrome b5 gene expression were found in young fruits at the pattern formation (globular/heart) stage of embryogenesis and in vascular and transmitting tissues of male and female reproductive organs. The data are consistent with a major role for cytochrome b5 in developmental processes related to plant reproduction in addition to being an electron donor to microsomal desaturases.
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  • 71
    ISSN: 1573-5028
    Keywords: Amaranthus hypochondriacus) ; gene expression ; trypsin inhibitor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We previously isolated and sequenced the major trypsin inhibitor from Amaranthus hypochondriacus seeds. This amaranth trypsin inhibitor (AmTI) is a 69 amino acid protein with high homology to members of the potato-1 inhibitor family. This paper describes the cloning and expression of a cDNA encoding this trypsin inhibitor in various vegetative tissues of the amaranth plant during seed development and imbibition, and investigates the possible induction of AmTI expression by wounding. We obtained a 393 bp cDNA sequence with an open reading frame corresponding to a polypeptide with 76 amino acid residues. With the exception of one residue (Ser-41), the polypeptide agrees with the amino acid sequence previously reported, plus 7 more residues at the N-terminus. These N-terminal residues are thought to be part of the signal used for intracellular sorting. The organ specificity of AmTI gene expression was investigated by northern analysis, showing that mRNA corresponding to AmTI genes was present in stems of plants growing under normal conditions. The kinetics of accumulation of the AmTI-mRNA, protein, and inhibitory activity during seed development and imbibition was determined. AmTI-mRNA accumulation reached a maximum at 14 days after anthesis (daa) and then gradually decreased, being barely detectable 36 daa. The AmTI protein accumulation followed the same profile as the inhibitory activity, both were delayed with respect to the mRNA. The maximum level was observed 22 daa, and then gradually decreased until a steady state was reached as seed maturation proceeded. Upon imbibition, a gradual decrease in AmTI protein and inhibitory activity was shown; however, an AmTI transcript was detected 24 h after imbibition. In contrast to representative members of the potato I family, this inhibitor was not inducible by wounding of leaves.
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  • 72
    ISSN: 1573-5028
    Keywords: embryo ; gene expression ; Glycine max ; oxidoreductase ; seed coat ; testa
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The soybean Ep gene encodes an anionic peroxidase enzyme that accumulates in large amounts in seed coat tissues. We have isolated a second peroxidase gene, Prx2, that is also highly expressed in developing seed coat tissues. Sequence analysis of Prx2 cDNA indicates that this transcript encodes a cationic peroxidase isozyme that is far removed from Ep in peroxidase phylogeny. To determine the expression patterns for these two peroxidases in developing seeds, the abundance and localization of the Ep and Prx2 transcripts were compared by in situ hybridization. Results show the expression of Ep begins in a small number of cells flanking the vascular bundle in the seed coat, spreads to encircle the seed, and then migrates to the hourglass cells as they develop. Expression of Prx2 occurs throughout development in all cell layers of the seed coat, and is also evident in the pericarp and embryo. Nonetheless, the Ep-encoded enzyme accounts for virtually all of the peroxidase activity detected in mature seed coats. The Prx2 enzyme is either insoluble in a catalytically inactive form, or is subject to degradation during seed maturation.
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  • 73
    ISSN: 1573-5028
    Keywords: ABRE ; embryogenesis ; G-box ; gene expression ; maize ; protein-DNA interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcription of the rab28 gene from maize is induced in late embryo development and in response to abscisic acid. We have studied the regulation of the activity of the rab28 promoter in embryos. Two abscisic acid-responsive elements (ABREs) were necessary for expression in embryos of transgenic Arabidopsis and in transient transformation in maize embryos. In vivo footprinting showed that there was protein binding to the ABREs and to other cis elements in the promoter in young embryos before expression of rab28. This shows that the rab28 promoter is in an open chromatin structure before developmental activation. The ABREs are important for the induction and have protein binding in young embryos. Nuclear proteins extracted from embryos before activation of rab28 bound to the ABREs in band shift assays. A complex with different mobility was formed between nuclear proteins and the ABREs after induction of rab28 suggesting a modification of the ABRE-binding factor or an exchange of proteins. The footprints on the ABREs were unaltered by induction with abscisic acid or during developmental activation of rab28. These results indicate that constitutive binding of transcription factor(s) on the ABRE is central in embryonic regulation of the rab28 gene.
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  • 74
    ISSN: 1573-5028
    Keywords: alanine aminotransferase ; gene expression ; GUS expression ; promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A cDNA clone encoding alanine aminotransferase (AlaAT) has isolated from randomly sequenced clones derived from a cDNA library of maturing rice seeds by comparison to previously identified genes. The deduced amino acid sequence was 88% and 91% homologous to those of the enzymes from barley and broomcorn millet (Panicum miliaceum), respectively. Using this cDNA as a probe, we isolated and sequenced the corresponding genomic clone. Comparison of the sequences of the cDNA and the genomic gene revealed that the coding region of the gene was interrupted by 14 introns 66 to 1547 bp long. Northern and western blotting analyses showed that the gene was expressed at high levels in developing seeds. When the 5′-flanking region between −930 and +85 from the site of initiation of transcription was fused to a reporter gene for β-glucuronidase (GUS) and then introduced into the rice genome, histochemical staining revealed strong GUS activity in the inner endosperm tissue of developing seeds and weak activity in root tips. Similar tissue-specific expression was also detected by in situ hybridization. These results suggest that AlaAT is involved in nitrogen metabolism during the maturation of rice seed.
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  • 75
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    Plant molecular biology 39 (1999), S. 161-169 
    ISSN: 1573-5028
    Keywords: expansin ; fruit growth ; fruit softening ; gene expression ; Lycopersicon esculentum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA clones encoding homologues of expansins, a class of cell wall proteins involved in cell wall modification, were isolated from various stages of growing and ripening fruit of tomato (Lycopersicon esculentum). cDNAs derived from five unique expansin genes were obtained, termed tomato Exp3 to Exp7, in addition to the previously described ripening-specific tomato Exp1 (Rose et al. (1997) Proc Natl Acad Sci USA 94: 5955–5960). Deduced amino acid sequences of tomato Exp1, Exp4 and Exp6 were highly related, whereas Exp3, Exp5 and Exp7 were more divergent. Each of the five expansin genes showed a different and characteristic pattern of mRNA expression. mRNA of Exp3 was present throughout fruit growth and ripening, with highest accumulation in green expanding and maturing fruit, and lower, declining levels during ripening. Exp4 mRNA was present only in green expanding fruit, whereas Exp5 mRNA was present in expanding fruit but had highest levels in full-size maturing green fruit and declined during the early stages of ripening. mRNAs from each of these genes were also detected in leaves, stems and flowers but not in roots. Exp6 and Exp7 mRNAs were present at much lower levels than mRNAs of the other expansin genes, and were detected only in expanding or mature green fruit. The results indicate the presence of a large and complex expansin gene family in tomato, and suggest that while the expression of several expansin genes may contribute to green fruit development, only Exp1 mRNA is present at high levels during fruit ripening.
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  • 76
    ISSN: 1573-5028
    Keywords: cDNA cloning ; fruit ripening ; gene expression ; non-climacteric fruit ; wild strawberry (Fragaria vesca L.)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Wild strawberry (Fragaria vesca L.) is an attactive model system for studying ripening in non-climacteric fruit, because of its small diploid genome, its short reproductive cycle, and its capacity for transformation. We have isolated eight ripening-induced cDNAs from this species after differential screening of a cDNA library. The predicted polypeptides of seven of the clones exhibit similarity to database protein sequences, including acyl carrier protein, caffeoyl- CoA 3-O-methyltransferase, sesquiterpene cyclase, major latex protein, cystathionine γ-synthase, dehydrin and an auxin- induced gene. A ninth cDNA clone that was constitutively expressed is predicted to encode a metallothionein-like protein. None of these proteins appear to be directly related to events generally associated with ripening such as cell wall metabolism or the accumulation of sugars and pigments, rather, their putative functions are indicative of the wide range of processes upregulated during fruit ripening.
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  • 77
    ISSN: 1573-5028
    Keywords: cowpea (Vigna unguiculata L.) ; drought ; gene expression ; lipid degradation ; phospholipase D
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Phospholipase D, a major lipid-degrading enzyme in plants, was studied in two cultivars of Vigna unguiculata L.Walp, differing in their tolerance to drought (cv. EPACE-1, drought-tolerant, and cv. 1183, drought-susceptible). Enzymatic activities, measured with 14C-PC as substrate, increased when plants were submitted to water stress, the increase being much higher in the drought-sensitive cultivar. A 2911 bp cDNA encoding a putative phospholipase D (VuPLD1) was isolated from a cDNA library prepared from V. unguiculata leaves. The deduced amino acid sequence (809 residues) shows 85.5% identity and 91.3% similarity to that of PLD from Ricinus communis. The expression of the VuPLD1 gene in the leaves is differently modulated by water deficit, depending on the intensity of stress and the tolerance or sensitivity of the plants. In the drought-susceptible V. unguiculata cv. 1183, it readily increased under water stress, reaching maximum values at mild water deficit (−1.5 MPa). In the drought-tolerant cv. EPACE-1, VuPLD1 mRNA remained low throughout the whole drought treatment. Dehydration of leaves led to a dramatic increase in transcript level in both cultivars. Changes in protein amounts semi-quantified by immunoblotting correlated well with variations in transcript steady-state level. Taken together, these results showed that phospholipase D in cowpea plants is essentially regulated at the transcriptional level, and that gene expression is strongly stimulated even by moderate water deficit in the drought-sensitive plant. On the contrary, the drought-tolerant plant presents a remarkable stability of PLD gene expression in conditions of water stress.
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  • 78
    ISSN: 1573-5028
    Keywords: reproductive development ; gene expression ; subtractive hybridization ; cauliflower
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using the meristems of the cauliflower curd as a source of tissue and a series of subtractive hybridizations and amplification reactions, we have constructed a cDNA library highly enriched in cDNAs expressed in reproductive meristems. The analysis of a sample of 250 clones from this library identified 22 cDNA clones corresponding to genes specifically expressed in these cauliflower meristems. Apart from two clones that corresponded to APETALA1, and two other ones showing similarity to different aminoacyl-tRNA synthetases, the remaining clones showed no similarity to any sequence in the databases and may correspond to novel genes. One of these clones, BoREM1, was further characterized and found to correspond to a gene encoding a protein with features of regulatory proteins that follows a expression pattern very similar to the LEAFY transcripts.
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  • 79
    ISSN: 1573-5028
    Keywords: ammonium ; gene expression ; glutamine synthetase ; nodules ; positive element ; promoter
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to identify important promoter elements controlling the ammonium-regulated expression of the soybean gene GS15 encoding cytosolic glutamine synthetase, a series of 5′ promoter deletions were fused to the GUS reporter gene. To allow the detection of positive and negative regulatory elements, a series of 3′ deletions were fused to a −90 CaMV 35S promoter fragment placed upstream of the GUS gene. Both types of construct were introduced into Lotus corniculatus plants and soybean roots via Agrobacterium rhizogenes-mediated transformation. Both spectrophotometric enzymatic analysis and histochemical localization of GUS activity in roots, root nodules and shoots of transgenic plants revealed that a strong constitutive positive element (SCPE) of 400 bp, located in the promoter distal region is indispensable for the ammonium- regulated expression of GS15. Interestingly, this SCPE was able to direct constitutive expression in both a legume and non- legume background to a level similar to that driven by the CaMV 35S full-length promoter. In addition, results showed that separate proximal elements, located in the first 727 bp relative to the transcription start site, are essential for root- and root nodule-specific expression. This proximal region contains an AAAGAT and two TATTTAT consensus sequences characteristic of nodulin or nodule-enhanced gene promoters. A putative silencer region containing the same TATTTAT consensus sequence was identified between the SCPE and the organ-specific elements. The presence of positive, negative and organ-specific elements together with the three TATTTAT consensus sequences within the promoter strongly suggest that these multiple promoter fragments act in a cooperative manner, depending on the spatial conformation of the DNA for trans-acting factor accessibility.
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  • 80
    ISSN: 1573-5028
    Keywords: embryo-abundant cDNAs ; gene expression ; gymnosperm ; Picea glauca ; somatic embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Six somatic embryogenesis-associated cDNAs (PgEMB2, 6, 7, 8, 24 and 34) from white spruce (Picea glauca (Moench) Voss) somatic embryos have been characterized. Transcript accumulation during somatic embryo development and subsequent germination related to these genes, indicated that they were developmentally regulated. The transcripts related to clones PgEMB2, 6, 24 and 34 were also detected during zygotic embryo development, but transcripts of clones PgEMB7 and 8 were not. PgEMB24 had a similar gene expression pattern to spruce Em-like late embryo abundant (lea) gene, but other clones had no similarities in gene expression to either spruce lea-like or storage protein genes. Abscisic acid, a stimulator for spruce somatic embryo maturation, did not obviously affect gene expression corresponding to these cDNAs. The predicted proteins are distinguishable from known LEA proteins based on analyses of hydropathy plots, amino acid compositions and deduced protein structures. The similarities of the spruce cDNAs, and protein sequences predicted from these cDNAs, to other sequence data are described.
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  • 81
    ISSN: 1573-5028
    Keywords: gene expression ; GT-1 ; PR-1a ; PR proteins ; salicylic acid-induced ; transcription factors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Infection of Nicotiana tabacum Samsun NN with tobacco mosaic virus (TMV) results in a hypersensitive plant response and leads to systemic acquired resistance (SAR). The induction of SAR is mediated by the plant hormone salicylic acid (SA) and is accompanied by the induced expression of a number of genes including the pathogenesis-related (PR) gene 1a. Previously, it has been found that TMV infection and SA treatment resulted in a reduction of binding of nuclear protein GT-1 to far-upstream regions (−902 to −656) of the PR-1a gene. To test if GT-1 is a negative regulator of PR-1a gene expression, the effects of mutations in the seven putative GT-1 binding sites in this region were studied in vitro using dimethyl sulfate interference footprinting and band shift assays. This showed that at least one of the seven sites is indeed a GT-1 binding site. However, when tested in transgenic plants, the mutations did not result in constitutive expression of the chimeric PR-1a/GUS transgene, while inducible expression after SA treatment was decreased. The results suggest that binding of GT-1-like proteins to far-upstream PR-1a promoter regions indeed influences gene expression. A possible model for GT-1's mode of action in PR-1a gene expression is discussed.
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  • 82
    ISSN: 1573-5028
    Keywords: aquaporin ; gene expression ; growth ; Oryza sativa ; plasma membrane intrinsic protein (PIP) ; rice
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Membrane intrinsic proteins facilitate movement of small molecules often times functioning as water channels. We have identified two genes from rice which encode proteins with characteristic features of plasma membrane intrinsic proteins (PIP). They possess six membrane-spanning domains, an NPA repeat, overall high sequence homologies and characteristic C- and N-terminal hallmark motifs which allowed assignment of OsPIP1a to the PIP1 subfamily and of OsPIP2a to the PIP2 subfamily. OsPIP1a and OsPIP2a showed similar but not identical expression patterns. The two genes were expressed at higher levels in seedlings than in adult plants and expression in the primary root was regulated by light. In internodes of deepwater rice plants which were induced to grow rapidly by submergence, transcript levels were slightly induced in the intercalary meristem (IM) and slightly reduced in the elongation zone (EZ) after 18 h. In internodes of GA-induced excised stem sections transcript levels transiently declined in the IM and EZ after 1 h and subsequently recovered to elevated levels after 18 h. GA also induced OsPIP expression in non-growing tissue after 18 h. In the IM of submergence-induced stem sections transcript levels remained constitutive. The different growth-promoting treatments showed no direct correlation between growth rate and OsPIP gene expression in dividing or expanding cells. In fact, treatment of excised stem sections with ABA or drought stress induced similar changes in OsPIP expression in the growing zone during the first 6 h as GA did. We conclude that regulation of OsPIP1a and OsPIP2a expression is not primarily controlled by growth. GA-induced growth may however change the water status of cells which in turn results in altered PIP abundance.
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  • 83
    ISSN: 1573-5028
    Keywords: ACC synthase ; chilling ; Citrus sinensis ; ethylene ; gene expression ; peel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Diurnal change in the temperature below or above 12.5 °C hastens the degreening of citrus peel and elicits the phytohormone ethylene production in citrus fruit. Ethylene triggers the degradation of chlorophyll and synthesis of carotenoids in citrus peel. To investigate if ethylene is required for the degreening of citrus peel elicited by low temperatures, we studied the chilling-regulated gene expression of ACC synthase, one of the key enzymes catalyzing ethylene biosynthesis. We isolated and characterized a chilling-inducible 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) gene, CS-ACS1, and a chilling-repressible gene, CS-ACS2, from citrus peel. The CS-ACS1 transcript 1.7 kb in length encodes a polypeptide of 483 amino acids (M r 54 115, pI 6.63), whereas the CS-ACS2 transcript of 1.8 kb encodes a polypeptide of 477 amino acids (M r 53 291, pI 6.72). Both genes showed a rapid but transient induction (within 2.4 h) of transcripts upon rewarming after the chilling (4 °C) treatment. After 24 h of incubation at room temperature, CS-ACS1 mRNA diminished to an undetectable level, whereas the CS-ACS2 mRNA regained its basal level of expression attained prior to the chilling treatment. Chilling-induced ethylene production and ACC accumulation were also observed upon rewarming. Both genes were also induced by the wound stress (excision). The protein synthesis inhibitor cycloheximide super-enhances the accumulation of both ACS transcripts at room temperature. Molecular analysis of the 3.3 kb genomic DNA of CS-ACS1 revealed that this gene consists of three introns and four exons. The intron 3 is exceptionally large (1.2 kb) and shares significant homology with mitochondrial DNA, supporting the intron-late theory.
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  • 84
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    Plant molecular biology 41 (1999), S. 351-361 
    ISSN: 1573-5028
    Keywords: chromatin ; gene expression ; high-mobility-group protein HMG1 ; HMGe ; protein stability ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nuclear HMG1 proteins of higher plants are small non-histone proteins that have DNA-bending activity and are considered architectural factors in chromatin. The occurrence of the chromosomal HMG1 proteins, HMGa, HMGc1/2 and HMGd, in various maize tissues was analyzed, and in the course of these studies a novel HMG1 protein, now termed HMGe, was identified. Purification and characterization of HMGe (Mr 13 655) and cloning of the corresponding cDNA revealed that it displays only moderate similarity to other members of the plant HMG1 protein family. The five maize HMG1 proteins could be detected in kernels, leaves, roots and suspension culture cells, indicating that these proteins can be expressed simultaneously and occur relatively ubiquitously. However, the various HMG1 proteins are present in significantly different quantities with HMGa and HMGc1/2 being the most abundant HMG1 proteins in all tissues tested. Furthermore, the relative amounts of the various HMG1 proteins differ among the tissues examined. The HMG1 proteins were found to be relatively stable proteins in vivo, with HMGc1/2, HMGd and HMGe having a half-life of ca. 50 h in cultured cells, while the half-life of the HMGa protein is ca. 65 h. Collectively, these findings are compatible with the concept that the different plant HMG1 proteins might act as general architectural proteins in concert with site-specific factors in the assembly of certain nucleoprotein structures involved in various biological processes.
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  • 85
    ISSN: 1573-5028
    Keywords: alkaloids ; gene expression ; Nicotiana tabacum ; nicotine ; putrescine N-methyltransferase ; tobacco gene evolution
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The structure and nuclear genomic organization of the gene family encoding putrescine N-methyltransferase (PMT), the key enzyme in diverting polyamine metabolism towards the biosynthesis of nicotine and related alkaloids, was examined in Nicotiana tabacum. Five genes encoding PMT are present in the N. tabacum genome and all are expressed. The complete coding region and immediate 5′- and 3′- flanking regions were characterized for four members of the gene family and the Exon 1 region of the fifth member of the family was determined. Comparison of the nucleotide and deduced amino acid sequences of the N. tabacum PMT genes with those of presumed progenitor species, N. sylvestris, N. tomentosiformis and N. otophora, revealed that three members of the N. tabacum PMT gene family were most similar to the three genes present in N. sylvestris, whereas the two remaining PMT genes were similar to PMT genes present in N. tomentosiformis and N. otophora genomes, respectively. These data are consistent with an evolutionary origin of N. tabacum resulting from a cross involving N. sylvestris and an introgressed hybrid between N. tomentosiformis and N. otophora. The five PMT genes present in N. tabacum are expressed in the roots of wild-type plants, but not in other organs. The steady-state level of all five PMT transcripts is transiently increased in roots following topping (removal of the floral meristem), although the maximum level of induction for the individual transcripts varies considerably. In contrast to wild-type plants, no increase in PMT transcript levels was observed in a low-alkaloid (nic1nic2) mutant of Burley 21. These data support a role for nic1 and nic2 in the global regulation of alkaloid formation in tobacco and provide for the first time molecular confirmation of the presumed origin of cultivated tobacco.
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  • 86
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    Plant molecular biology 41 (1999), S. 425-433 
    ISSN: 1573-5028
    Keywords: D1 protein ; gene expression ; psbA genes ; redox regulation ; Synechococcus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Three psbA genes encode two different forms of the photosystem II reaction centre protein D1 in Synechococcus sp. PCC 7942. The psbAI gene encoding D1 protein form I (D1:1) is mainly expressed under low growth light conditions while the psbAII and psbAIII genes, encoding D1 protein form II (D1:2), are induced under stress conditions (e.g. high light or low temperature). In this paper we show that psbAII/III genes can be rapidly induced even under low growth light conditions by adding the thiol reductant (DTTred) to Synechococcus cell culture, at a concentration that does not affect cell growth or photosynthetic activity. Similar induction of psbAII/III genes was obtained by illuminating the cells with photosystem I light. In both instances psbAI gene down-regulation coincided with the up-regulation of psbAII/III genes. DTTred-induced exchange in transcript pools was subsequently followed by an exchange of D1:1 for D1:2 at the protein level. Thiol oxidants, iodosobenzoic acid or diamide, reverted the effects of DTTred on psbA gene expression. Thiol oxidants and the thiol-modifying agent N-ethylmaleimide also totally prevented high-light induction of psbAII/III genes. These data strongly suggest that the up-regulation of psbAII/III genes that occurs under stress conditions is mediated by production of thiol reductants, whereas the expression of the psbAI gene is sustained by the more oxidizing conditions that prevail during the steady-state growth of cells.
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  • 87
    ISSN: 1573-5036
    Keywords: ammonium assimilation ; Amanita muscaria ; carbon allocation ; ectomycorrhiza ; gene expression ; Picea abies ; sugar transport
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract The symbiosis (ectomycorrhiza, ECM) between roots of trees and shrubs of boreal and temperate forest ecosystems and soil fungi is essential for water and nutrient acquisition of the plants. The functionality of ECM is largely dependent on the ability of the host plant to supply photoassimilates to the fungus via the symbiotic interface. Based on sterile in vitro and non-sterile pot experiments, we review data which gives evidence that hexoses are supplied to the fungus by the host plant (mainly glucose and fructose), and that these sugars, at least in part, control development and function of ECM by interfering with fungal gene expression. We further show that any factor which reduces hexose allocation to the host–fungus interface will adversely affect ECM development. As an example, we address the impact of increased supply of nitrogen on the biochemistry of plant–fungus interaction and discuss potential consequences on host performance.
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  • 88
    ISSN: 1573-5028
    Keywords: bundle sheath cell ; C4 photosynthesis ; gene expression ; PEP carboxykinase ; prokaryotic expression ; Zea mays
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We isolated a full-length cDNA that encodes ATP-dependent phosphoenolpyruvate carboxykinase (EC 4.1.1.49, PCK) from leaves of maize, an NADP-malic enzyme type C4 plant. The mRNA was specifically and rather abundantly expressed in bundle sheath cells in accordance with the recent finding of cell-type-specific localization of PCK protein in maize, which has been detected with antibodies against cucumber PCK protein. The predicted protein had an N-terminal extension, which is characteristic of plant PCKs. The transcript level was much higher in the daytime than at night in 14-day old seedlings. However, in 42-day old plants the extent of diurnal change decreased. The maize PCK was expressed in Escherichia coli with the pET32 plasmid and purified to homogeneity. Through digestion with enterokinase, two types of enzyme were prepared; one with an intact N-terminus and the other lacking its N-terminal 77 amino acid residues due to over-digestion. The truncated protein had about 2-fold higher specific activity than the intact one, and was inhibited by 3-phosphoglycerate (3-PGA) with an I0.5 of 17.5 mM. In contrast, the intact protein was almost insensitive to 3-PGA. These results strongly suggest that the intact N-terminal extension may be involved in the regulation of PCK activity in vivo through some modification such as reversible phosphorylation.
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  • 89
    ISSN: 1573-5028
    Keywords: calcium-dependent protein kinase ; gene expression ; immunoassays ; light regulation ; rice ; seed development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We investigated the spatial and temporal expression patterns of two rice calcium-dependent protein kinases (CDPKs), OsCDPK2 and OSCDPK11, using isoform-specific antisera. Bands of the expected molecular sizes for OsCDPK2 (59 kDa) and OsCDPK11 (61 kDa) were detected on western blots. OsCDPK2 and OsCDPK11 mRNA and protein levels increased in unison during flower development. However, at the onset of seed development, the protein expression profiles diverged significantly. OsCDPK2 protein was expressed at low levels during early seed development, but increased to high levels that were maintained in later stages (20 days after fertilisation, DAF). Conversely, OsCDPK11 protein levels were high at the beginning of seed development, but fell rapidly from 10 DAF onwards. This decrease in the level of OsCDPK11 protein was associated with the abundant synthesis of a truncated mRNA species. OsCDPK2 expression was also closely associated with light perception. OsCDPK2 protein was barely detectable in green leaves exposed to light, but levels increased sharply when plants were shifted to darkness. Initially, this increase reflected a rapid elevation in the levels of OsCDPK2 mRNA, which was normally located in the mesophyll. Conversely, OsCDPK11 mRNA and protein levels were unaffected by light. These data strongly indicate that two rice CDPK isoforms have different functions in seed development and in response to light in leaves.
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  • 90
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    Euphytica 106 (1999), S. 117-123 
    ISSN: 1573-5060
    Keywords: differential display ; gene expression ; heterosis ; hybrid wheat ; seedling leaf
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Differential display of mRNA was used to analyze the differences of gene expression in seedling leaves between heterotic hybrid/nonheterotic hybrid and their parental inbreds in order to study the molecular basis of heterosis in wheat. The results indicated that patterns of gene expression in hybrids differ significantly from their parents. Both quantitative and qualitative differences were observed. The quantitative differences include gene over-expression, gene under-expression in hybrid and dominant expression of highly-expressed parental genes in hybrids. The qualitative differences include silencing in hybrids of genes expressed either in male or female parent, and silencing in hybrids of genes expressed in both parents. Expression in hybrid of genes only expressed either in male or female parent was also observed. It was also found that some genes expressed at high level in heterotic hybrid were underexpressed or expressed at low level in nonheterotic hybrid. One differentially expressed cDNA fragment 4B was cloned and sequenced after being confirmed through Northern blot analysis. Homology search in GenBank proved that the cDNA fragment is a new sequence. The selection of primers for differential RNA display in wheat and the relationship between wheat heterosis and alteration of gene expression in hybrids as compared to their parental inbreds were also discussed.
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  • 91
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    Plant growth regulation 29 (1999), S. 47-76 
    ISSN: 1573-5087
    Keywords: cold ; chill ; freezing ; gene expression ; signal transduction pathways
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Temperature is expected to affect all plant processes. Consistent with this, expression of a large number of specific mRNAs and proteins is up-regulated during cold-acclimation. Their possible functions are outlined, encompassing a wide range of processes, some possibly related to other winter stresses besides cold. Some of the cold-responsive sequences are associated with constitutive or stress-related metabolism, others are probably protective, some may influence freezing, and many are of as yet unknown function. While many of the sequences code for intracellular proteins, a significant number code for apoplastic proteins with a variety of possible functions. Transient influx of calcium into the cytosol appears to be a key step in the response to cold, and also to many other stresses and signals. Similarly, the cold-responsive promoter element identified so far is also responsive to drought and salt. However, how cold is sensed, and any cold-specific aspects of the cold signal transduction pathway, are, so far, unknown. What is clear is that, at least in the model plant Arabidopsis thaliana, cold-, desiccation-, salt- and ABA-triggered signal transduction pathways, and possibly others, run partly in parallel and partly intersect. This may be partly explained by a need for an integrated winter-response. The total number of genes which are cold-responsive and the quantity of resources which this implies are used in this way, indicate that many must have a positive role in acclimation. Experiments which modify membrane lipid unsaturation or solute accumulation, achieved by transformation of plants to express exotic or heterologous genes or by other means, confirm that these factors affect chill- or freezing-tolerance. A transgenic test has shown that one cold-up-regulated gene of previously unknown function contributes to freezing-tolerance, but the small effect re-emphasises the probably cumulative nature of the contributions of many cold-up-regulated sequences to acclimation. On the other hand, mutant analysis indicates some genes may make a comparatively larger contribution. Transformation of alfalfa to overexpress a superoxide dismutase gene increased cold-tolerance and drought-resistance and demonstrated that improvements in field-survival of stresses is possible by transgenic means. Over-expression of a transcription factor, CBF1, conferred freezing-tolerance on Arabidopsis, showing that manipulation of the signal transduction pathway could be an important method for modifying cold-tolerance.
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  • 92
    ISSN: 1573-5168
    Keywords: fish ; gene expression ; GH ; GtH ; gonad ; growth factors ; RT-PCR ; salmonid
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this study, Northern blot analysis of RNA from trout testis revealed a single transcript of insulin-like growth factor II (IGF-II) around 4.7 kb. The cellular distribution of IGF-II mRNA was studied and quantified in different testicular cells enriched populations by RT-PCR. IGF-II mRNA appears to be expressed in all cellular types tested: spermatogonia A and B, primary spermatocytes, spermatids and secondary spermatocytes and Sertoli cells. A significantly higher expression of IGF-II was found in premeiotic germ cells. The levels of IGF-II mRNA appear to be higher than those of IGF-I in immature trout testis, as judged from the semi-quantitative RT-PCR results. These data suggest that in addition to IGF-I, IGF-II may play a role in testicular physiology in fish. The hormonal regulation of IGF-I and IGF-II gene expression was investigated both in vitro and in vivo using RT-PCR approach. Gonadotropin (GtH) added to testicular explants increased IGF-II mRNA levels but had no effect on IGF-I. No statistically significant effect was observed with androgens. In vivo, GH and pituitary extracts resulted in an 8 fold and 2-3 fold increase in both IGF-I and IGF-II mRNA levels, respectively. Taken together, our study suggests that IGF-I and IGF-II may act as local mediators of GH and GtHs in fish testis. Moreover, our results imply that in fish testicular cells, IGFs are potential paracrine/autocrine regulators inside the spermatogenic compartment and can act directly on germ cells to stimulate their proliferation.
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  • 93
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    Journal of bioenergetics and biomembranes 31 (1999), S. 467-473 
    ISSN: 1573-6881
    Keywords: Uncoupling proteins ; fatty acids ; skeletal muscle ; brown adipose tissue ; obesity ; thermogenesis ; gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The recently discovered uncoupling protein 3 (UCP3) is highly homologous to the mitochondrialinner membrane protein UCP1, which generates heat by uncoupling the respiratory chainfrom oxidative phosphorylation. The thermogenic function of UCP1 protects against cold andregulates the energy balance in rodents. We review in vitro studies investigating the uncouplingactivity of UCP3 and in vivo studies, which address UCP3 gene expression in brown adiposetissue and skeletal muscle under various metabolic conditions. The data presented are, for themost, consistent with an uncoupling role for UCP3 in regulatory thermogenesis. We alsodiscuss mediators of UCP3 regulation and propose a potential role for intracellular fatty acidsin the mechanism of UCP3 modulation. Finally, we hypothesize a role for UCP3 in themetabolic adaptation of the mitochondria to the degradation of fatty acids.
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  • 94
    ISSN: 1573-5028
    Keywords: gene expression ; Gossypium hirsutum ; plant defence response ; Verticillium dahliae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Verticillium wilt is a vascular disease of cotton (Gossypium spp.) caused by the fungal pathogen Verticillium dahliae. To begin to understand the molecular mechanisms of the disease response in cotton cultivars that display superior wilt tolerance, such as Gossypium hirsutum cv. Sicala V-1, a cDNA library was constructed with mRNA isolated from root tissue of Sicala V-1, 24 h after inoculation with V. dahliae. The library was screened by a differential screening technique which was successful in identifying differences in gene expression between uninfected and V. dahliae-infected G. hirsutum root tissue. Among the differentially expressed clones, 51% represented up-regulated genes which had the potential to be involved in the defence response of G. hirsutum. The temporal expression patterns of nine suspected defence response genes were examined by northern blot analysis at several time intervals after inoculation with V. dahliae. The rapid increase in mRNA transcripts corresponding to each of these clones upon infection suggests a role for these genes in the defence response of G. hirsutum. Genes not previously associated with the defence response of the cotton plant, such as those for a 14-3-3-like protein and pathogenesis-related (PR) proteins, have been identified together with presumably novel genes, for which a definite function could not be ascribed.
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  • 95
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    Plant molecular biology 39 (1999), S. 1063-1071 
    ISSN: 1573-5028
    Keywords: cell cycle ; gene expression ; in vitro fertilization ; maize ; zygote ; embryogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Differential screening of cDNA libraries of unfertilized egg cells and in vitro zygotes of maize resulted in the isolation of more than 50 different genes whose expression is up- or down-regulated after in vitro fertilization (IVF). Amoung these genes, we identified a cDNA encoding the eukaryotic translation initiation factor eIF-5A. This highly conserved factor is thought to be necessary for selective mRNA stabilization and translation. It is also the only known protein that contains the unusual amino acid hypusine which is required for biological activity. High transcript amounts are stored in the egg cell, which is, in terms of metabolism, relatively inactive. Upon fertilization transcript amounts decrease, in contrast to metabolically inactive embryos in which the transcript cannot be detected and transcript levels increase upon germination. The expression pattern during the first embryonic cell cycle is also different from that observed during the somatic cell cycle: egg cells in the G0 phase contain high transcript levels, while arrested suspension cells contain few transcripts. In the somatic cell cycle, eif-5A is strongly induced during the G1 phase and transcripts are continuously degraded during the S, G2 and M phases until new induction during the G1 phase of the next cycle. eif-5A, a member of a small gene family in maize, is expressed in most maize tissues investigated. Based on our results, we suggest that the unfertilized egg cell of maize, although relatively inactive regarding its metabolism, is prepared for selective mRNA translation that is quickly triggered after fertilization. We also suggest that the regulation of eif-5A in the first embryonic cell cycle is different from the somatic cell cycle.
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  • 96
    ISSN: 1573-5028
    Keywords: aquaporin ; canola ; gene expression ; germination ; priming ; salt and osmotic stresses
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two aquaporin genes were isolated from a cDNA library of canola (Brassica napus L.). The first aquaporin, BnPIP1 of 1094 bp, encoding a putative polypeptide of 287 amino acids with a predicted molecular mass of 30.4 kDa and a pI of 7.8, belongs to the family of plasma membrane intrinsic protein (PIPs) aquaporins. The B. napus aquaporin showed 85–94% identity to the Arabidopsis thaliana PIPs. ABA priming of seed induced high levels of BnPIP1 transcript which remained after subsequent re-drying of the seed. The second aquaporin, Bnγ-TIP2 of 1020 bp, encoded a putative polypeptide of 253 amino acids with a predicted molecular mass of 25.8 kDa and a pI of 5.8. Bnγ-TIP2 showed 83–90% identity to γ-TIP genes from a variety of plant species. Bnγ-TIP2 was expressed only when radicle protrusion occurred in either untreated or primed seeds. Seeds primed with PEG or ABA germinated earlier and showed a higher final percentage of germination than unprimed seed, particularly under salt and osmotic stresses at low temperature. Transcripts of both BnPIP1 and Bnγ-TIP2 genes were present earlier during germination of primed seeds than non-primed seed. From these results, we conclude that BnPIP1 is related to the water transportation required for enzymatic metabolism of storage nutrients at the early stages of canola seed germination whereas Bnγ-TIP2 expression is related to cell growth associated with radicle protrusion. Priming induced the expression of BnPIP1 but had no effect on Bnγ-TIP2.
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  • 97
    ISSN: 1573-5028
    Keywords: Brassica napus ; cauliflower mosaic virus ; epidermis ; gene expression ; light induction ; lipid transfer protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract cDNA and genomic clones encoding Brassica napus non-specific lipid transfer proteins (LTP) were isolated and sequenced. The encoded amino acid sequences were very similar to those reported previously for LTPs from B. napus and other species. Sequence information indicates that B. napus contains an LTP gene family. The 5′-flanking region of one gene, designated BnLTP, was fused to GUS and the fusion introduced into Arabidopsis. LTP transcripts and BnLTP-Gus expression were present predominantly in the epidermis of leaf and stem, consistent with the hypothesised function of LTPs in the deposition of cuticular or epicuticular waxes. However, GUS activity was detected in other tissues, including lateral root initials, anthers, stigmas and vascular tissues, which may suggest additional functions. LTP transcript levels in B. napus and Arabidopsis and BnLTP-GUS expression in transgenic Arabidopsis were stimulated by blue and red light but not UV-B. BnLTP promoter activity was also stimulated upon viral infection, at a time when the virus had spread systemically. No increase in expression was observed in response to cold or wounding.
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  • 98
    ISSN: 1573-5028
    Keywords: 1-aminocyclopropane-1-carboxylate synthase ; auxin ; brassinosteroid ; ethylene ; gene expression ; hypocotyl ; promoter ; transgenic tobacco ; Vigna radiata
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Indole-3-acetic acid (IAA) markedly increased ethylene production by inducing the expression of three 1-aminocyclopropane-1-carboxylate (ACC) synthase cDNAs (pVR-ACS1, pVR-ACS6 and pVR-ACS7) in mung bean hypocotyls. Results from nuclear run-on transcription assay and RNA gel blot studies revealed that all three genes were transcriptionally active displaying unique patterns of induction by IAA and various hormones in etiolated hypocotyls. Particularly, 24-epibrassinolide (BR), an active brassinosteroid, specifically enhanced the expression of VR-ACS7 by a distinct temporal induction mechanism compared to that of IAA. In addition, BR synergistically increased the IAA-induced VR-ACS6 and VR-ACS7 transcript levels, while it effectively abolished both the IAA- and kinetin-induced accumulation of VR-ACS1 mRNA. In light-grown plants, VR-ACS1 was induced by IAA in roots, and VR-ACS6 in epicotyls. IAA- and BR-treatments were not able to increase the VR-ACS7 transcript in the light-grown tissues. These results indicate that the expression of ACC synthase multigene family is regulated by complex hormonal and developmental networks in a gene- and tissue-specific manner in mung bean plants. The VR-ACS7 gene was isolated, and chimeric fusion between the 2.4 kb 5′-upstream region and the β-glucuronidase (GUS) reporter gene was constructed and introduced into Nicotiana tabacum. Analysis of transgenic tobacco plants revealed the VR-ACS7 promoter-driven GUS activity at a highly localized region of the hypocotyl-root junction of control seedlings, while a marked induction of GUS activity was detected only in the hypocotyl region of the IAA-treated transgenic seedlings where rapid cell elongation occurs. Although there was a modest synergistic effect of BR on the IAA-induced GUS activity, BR alone failed to increase the GUS activity, suggesting that induction of VR-ACS7 occurs via separate signaling pathways in response to IAA and BR. A scheme of the multiple regulatory pathways for the expression of ACC synthase multigene family by auxin and BR is presented.
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  • 99
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    Plant cell, tissue and organ culture 57 (1999), S. 207-210 
    ISSN: 1573-5044
    Keywords: biolistics ; gene expression ; haploid ; transformation ; Triticum aestivum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using the PDS-1000/He Biolistic® Particle Delivery System, the microprojectile travel distance, rupture disk pressure and DNA/gold particle concentrations were assessed in order to optimise short and longer-term β-glucuronidase reporter gene expression in microspore-derived embryos of wheat. The effects were also evaluated of using sterile filter paper to support explants and treatment with a high osmoticum medium (0.2 M mannitol/0.2 M sorbitol or 0.4 M maltose). In the optimised procedure, wheat microspore-derived embryos (MDEs), were placed on filter paper and incubated on medium containing 0.4 M maltose, for 4 h pre- and 45 h post-bombardment. Five μl pAHC25 (0.75 mg ml-1 in TE buffer) was precipitated onto 25 μl gold particles (60 mg ml-1 in sterile water), using 20 μl spermidine (0.1 M) and 50 μl CaCl2 (2.5 M). The particles were centrifuged and resuspended in 75 μl absolute ethanol prior to the preparation of 6 macrocarriers. A microprojectile travel distance of 70 mm, a rupture pressure of 1300 p.s.i., and a vacuum of 29′′ Hg were employed. Maltose at 0.4 M in the support medium was the most important factor influencing GUS activity in bombarded tissues. GUS activity, 1 day post-bombardment, reached 52 ± 17 GUS-positive foci/MDE (mean ± s.e.m, n=3), with 17 ± 4 foci/MDE at 15 days, giving a 3.0-fold increase (p〈0.05) compared to expression in MDEs bombarded on medium without a high osmoticum treatment.
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  • 100
    ISSN: 1573-6822
    Keywords: cytokines ; gene expression ; in vitro human epidermis ; acute and chronic skin irritation ; predictivity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract An in vitro human reconstructed epidermis model (SkinEthic) used for screening acute and chronic skin irritation potential was validated against in vivo data from skin tolerability studies. The irritation potential of sodium lauryl sulfate (SLS), calcipotriol and trans-retinoic acid was investigated. The in vitro epidermis-like model consists of cultures of keratinocytes from human foreskin on a polycarbonate filter. The modulation of cell viability, the release and gene expression of proinflammatory cytokines, interleukins 1α and 8, and morphological changes were evaluated during 3 days as endpoints representative for an inflammatory reaction. The cumulative irritation potential of the topical products was evaluated in a human clinical study by visual scoring and biophysical measurement of inflammatory skin reaction after repeated 24 h applications over 3 weeks under Finn chamber patches. All topical products that were nonirritating in the human study were noncytotoxic and did not induce cytokine expression in the in vitro acute model (day 1 exposure). All irritating controls exhibited specific cell viability and cytokine patterns, which were predictive of the in vivo human data. The ranking of mild to moderate skin irritation potential was based on the lack of cytotoxicity and the presence of cytokine patterns including gene expression specific for each irritant, using the chronic in vitro model (up to 3 days exposure). The human reconstructed epidermis model SkinEthic was shown to be a reliable preclinical tool predicting the irritation potential of topical products. Moreover, it is a useful model in a two-step tiered strategy for screening acute and chronic irritation potential for the selection of vehicles for new topical drugs.
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