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  • Saccharomyces cerevisiae  (412)
  • Drosophila  (342)
  • AERODYNAMICS
  • Chemistry
  • ddc:330
  • Springer  (763)
  • Berlin : Schmidt
  • 1990-1994  (439)
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  • 1
    ISSN: 1572-8889
    Keywords: host selection ; experience ; learning ; extinction ; reinforcement ; parasitoids ; Drosophila ; Leptopilina heterotoma ; Hymenoptera ; Eucoilidae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The host-foraging behavior of female entomophagous parasitoids is commonly modified by positive associative learning. Typically, a rewarding experience (e.g., successful oviposition in a host) increases a female's foraging effort in a host microhabitat of the type associated with that experience. Less well understood are the effects of unrewarding experiences (i.e., unsuccessful foraging). The influence of unrewarding experience on microhabitat choice and residence time within a microhabitat was examined for the eucoilid parasitoid,Leptopilina heterotoma, in laboratory and greenhouse assays. As determined previously, females which oviposited successfully in either of two microhabitat types (fermenting apple or decaying mushroom) strongly preferred to forage subsequently on that microhabitat type. However, failure to find hosts in the formerly rewarding microhabitat caused females to reverse their preference in favor of a novel microhabitat type. The effect, though striking, was transient: within 1–2 h, the original learned preference was nearly fully restored. Similar effects of unrewarding experiences were observed with respect to the length of time spent foraging in a microhabitat. As determined previously, oviposition experience in a particular microhabitat type increased the time spent foraging in a patch of that microhabitat type. However, failure to find hosts in the patch caused the time a wasp spent in the next unoccupied patch of that type to decrease to almost nothing. In addition, there was a tendency for an unrewarding experience on a formerly rewarding microhabitat type to extend the time spent in a patch of a novel type. The function of the observed effects of unrewarding experiences is discussed.
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  • 2
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    Journal of insect behavior 8 (1994), S. 231-239 
    ISSN: 1572-8889
    Keywords: Drosophila ; sex ratio ; life history ; optimality model
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Based on both previously published literature and results reported here, it appears thatDrosophila melanogaster meet the explicit assumptions of the Trivers and Willard offspring sex allocation model. However, contrary to the model's predictions, offspring sex ratio was not significantly affected when we manipulated factors that influence offspring quality. We suggest that contrary to implicit predictions of offspring sex ratio models,Drosophila may lack the genetic plasticity to readily alter sex ratio.
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  • 3
    ISSN: 1572-8889
    Keywords: parasitoid ; superparasitism ; learning ; motivation ; egg load ; Drosophila
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The influence of egg-laying experience on the response of females of the eucoilid parasitoid,Leptopilina heterotoma, to parasitized and unparasitizedDrosophila melanogaster host larvae was examined under more controlled conditions than those used in past studies. In laboratory assays, we precisely manipulated both the number of eggs laid by females and the kind of larvae (parasitized versus unparasitized) in which the eggs were laid. We found that the tendency to avoid laying eggs in parasitized hosts depended markedly on whether or not eggs had been laid previously, but depended little on whether those eggs had been laid in parasitized or unparasitized hosts. The observed effect of general egg-laying experience on avoidance of parasitized hosts may reflect responses to either changes in the wasp's internal state (perhaps, changes in egg load) or changes in the wasp's neural representation of the external environment (such as those presumed to occur during learning). In light of these results, we offer a tentative reinterpretation of several earlier studies.
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  • 4
    ISSN: 1572-8889
    Keywords: Drosophila ; sexual selection gradients ; courtship success
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Using wild-reared flies, we examined sexual selection on five phenotypic traits (thorax length, wing length, wing width, head width, and face width) inDrosophila buzzatii, by scoring copulatory status in nine mass mating cages. Only male face width was identified as a direct target of sexual selection in an analysis of selection gradient, while indirect selection was present on all other studied traits, as expected from their correlations with face width. In contrast to males, there was no indication of selection in females. Nor was there evidence of assortative mating. The suggested direct selection on face width seems to take place during licking behavior of the courtship and might be related to courtship feeding. This study suggests that courtship success gives rise to indirect selection on body size.
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  • 5
    ISSN: 1572-8773
    Keywords: catalase ; copper resistance ; pH-dependent growth ; Saccharomyces cerevisiae ; superoxide dismutase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract A strain of Saccharomyces cerevisiae has been adapted to increasing concentrations of copper at two different pH values. The growth curve at pH 5.5 is characterized by a time generation increasing with the amount of added copper. A significant decrease of cell volume as compared with the control is also observed. At pH 3 the cells grow faster than at pH 5.5 and resist higher copper concentrations (3.8 against 1.2 mm). Experimental evidence indicates that, after copper treatment, the metal is not bound to the cell wall, but is localized intracellularly. A significant precipitation of copper salts in the medium was observed only at pH 5.5. Increased levels of superoxide dismutase (SOD) activity were observed in copper-treated cells and which persisted after 20 subsequent inocula in a medium without added metal. On the contrary, catalase activity was not stimulated by copper treatment and, hence, not correlated with SOD levels. The mechanism of copper resistance, therefore, probably involves a persistent induction of SOD, but not of catalase, and it is strongly pH-dependent.
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  • 6
    ISSN: 1572-8889
    Keywords: Drosophila ; parasitoid wasp ; behavior ; genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
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  • 7
    ISSN: 1572-8889
    Keywords: temperature preference ; Drosophila ; acclimation ; compensation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The effects of rearing and acclimation on the response of adultDrosophila to temperature were investigated in a gradient.D. melanogaster flies preferred a higher mean temperature and were distributed over a wider range of temperatures thanD. simulans flies. Acclimating adults at different temperatures for a week did not influence the response of either species. Adults reared at 28°C as immatures had a lower mean preference than those reared at cooler temperatures, suggesting that flies compensated for the effects of rearing conditions. Adults from tropical and temperate populations ofD. melanogaster andD. simulans did not differ in the mean temperature they preferred in a gradient, suggesting little genetic divergence for this trait within species. The species differences and environmental responses may be related to changes in optimal physiological conditions for the flies.
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  • 8
    ISSN: 1572-8889
    Keywords: review ; Drosophila ; larva ; phototaxis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this paper we examine theDrosophila melanogaster larval response to light. We survey the morphology of the larval visual and motor systems in relation to larval locomotory behavior and phototaxis. In addition, this paper proposes a model of sensorimotor transformation and examines the reversal in taxis occurring at theD. melanogaster larval wnadering stage.
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  • 9
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    Journal of molecular evolution 39 (1994), S. 478-488 
    ISSN: 1432-1432
    Keywords: Mitochondrial DNA ; Nucleotide sequences ; Drosophila ; Rapid phyletic radiation ; Molecular phylogeny
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Approximately 2 kb corresponding to different regions of the mtDNA of 14 different species of the obscura group of Drosophila have been sequenced. In spite of the uncertainties arising in the phylogenetic reconstruction due to a restrictive selection toward a high mtDNA A+T content, all the phylogenetic analysis carried out clearly indicate that the obscura group is formed by, at least, four well-defined lineages that would have appeared as the consequence of a rapid phyletic radiation. Two of the lineages correspond to monophyletic subgroups (i.e., afftnis and pseudoobscura), whereas the obscura subgroup remains heterogeneous assemblage that could be reasonably subdivided into at least two complexes (i.e., subobscura and obscura).
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  • 10
    ISSN: 1432-1432
    Keywords: Concerted evolution ; Molecular drive ; Drosophila ; rDNA spacers ; PCR length polymorphism ; MVR-PCR mapping
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Polymerase chain reaction (PCR)-amplified, sequenced, and digitally typed intergenic spacers (IGSs) of the ribosomal (r)DNA in D. melanogaster reveal unexpected features of the mechanisms of turnover involved with the concerted evolution of the gene family. Characterization of the structure of three isolated IGS length variants reveals breakage “hot spots” within the 330-base-pair (bp) subrepeat array found in the spacers. Internal mapping of variant repeats within the 240-bp subrepeat array using a novel digital DNA typing procedure (minisatellite variant repeat [MVR]-PCR) shows an unexpected pattern of clustering of variant repeats. Each 240-bp subrepeat array consists of essentially two halves with the repeats in each half identified by specific mutations. This bipartite structure, observed in a cloned IGS unit, in the majority of genomic DNA of laboratory and wild flies and in PCR-amplified products, has been widely homogenized yet is not predicted by a model of unequal crossing over with randomly placed recombination breakpoints. Furthermore, wild populations contain large numbers of length variants in contrast to uniformly shared length variants in laboratory stocks. High numbers of length variants coupled to the observation of a homogenized bipartite structure of the 240-bp subrepeat array suggest that the unit of turnover and homogenization is smaller than the IGS and might involve gene conversion. The use of PCR for the structural analysis of members of the rDNA gene family coupled to digital DNA typing provides powerful new inroads into the mechanisms of DNA turnover affecting the course of molecular evolution in this family.
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  • 11
    ISSN: 1432-1432
    Keywords: Drosophila ; Y chromosome ; Male fertility genes ; Lampbrush loops ; Germ line ; Transposable elements ; Gypsy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract During the evolution of the Y chromosome of Drosophila hydei, retrotransposons became incorporated into the lampbrush loop pairs formed by several of the male fertility genes on this chromosome. Although insertions of retrotransposons are involved in many spontaneous mutations, they do not affect the functions of these genes. We have sequenced gypsy elements that are expressed as constituents of male fertility gene Q in the lampbrush loop pair Nooses. We find that these gypsy elements are all truncated and specifically lost those sequences that may interfere with the continuity of lampbrush loop transcription. Only defective coding regions are found within the loop. Gypsy is not transcribed in loops of many other Drosophila species harboring the family. These results suggest that any contribution of gypsy to the function of male fertility gene Q does not depend on a conserved DNA sequence.
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  • 12
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    Journal of comparative physiology 175 (1994), S. 587-596 
    ISSN: 1432-1351
    Keywords: Wing beat frequency ; Optomotor responses ; Landing response ; Drosophila
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The present study shows that the wing beat frequency of Drosophila is visually controlled and modulated in response to different optomotor stimuli. Whereas rotational large field stimuli do not appear to modulate wing beat frequency, single rotating vertical stripes increase or decrease wing beat frequency when moving back-to-front or front-to-back, respectively. Maximal modulations occur at lateral stripe positions. Expansion stimuli eliciting the landing response cause a marked increase in wing beat frequency. Parameters of this frequency response depend in a graded fashion on certain stimulus properties, and the frequency response co-habituates with the landing response. Several results indicate that the frequency response is an integral component of the landing response, although it can also occur when the characteristic front leg extension is not observed. The complex spatial input integration underlying the frequency response and other motor components of the landing response cannot easily be explained by a system of large field integration units, but might indicate the existence of local expansion detectors.
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  • 13
    ISSN: 1432-1351
    Keywords: Drosophila ; Neuromuscular ; Haemolymph ; Membrane potential ; Synaptic potential
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Neuromuscular preparations from third instar larvae of Drosophila are not well-maintained in commonly used physiological solutions: vacuoles form in the muscle fibers, and membrane potential declines. These problems may result from the Na∶K ratio and total divalent cation content of these physiological solutions being quite different from those of haemolymph. Accordingly haemolymph-like solutions, based upon ion measurements of major cations, were developed and tested. Haemolymph-like solutions maintained the membrane potential at a relatively constant level, and prolonged the physiological life of the preparations. Synaptic transmission was well-maintained in haemolymph-like solutions, but the excitatory synaptic potentials had a slower time course and summated more effectively with repetitive stimulation, than in standard Drosophila solutions. Voltage-clamp experiments suggest that these effects are linked to more pronounced activation of muscle fiber membrane conductances in standard solutions, rather than to differences in passive muscle membrane properties or changes in postsynaptic receptor channel kinetics. Calcium dependence of transmitter release was steep in both standard and haemolymph-like solutions, but higher external calcium concentrations were required for a given level of release in haemolymph-like solutions. Thus, haemolymph-like solutions allow for prolonged, stable recording of synaptic transmission.
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  • 14
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    Journal of comparative physiology 175 (1994), S. 267-278 
    ISSN: 1432-1351
    Keywords: Drosophila ; Bang sensitivity ; Mechanotransduction ; Adaptation ; Sensory coding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Bang-sensitive mutants of Drosophila melano gaster (bas 1, bssMW1, eas2, tko25t) display seizure followed by paralysis when subjected to mechanical shock. However, no physiological or biochemical defect has been found to be common to all of these mutants. In order to observe the effects of bang-sensitive mutations upon an identified neuron, and to study the nature of mechanically induced paralysis, we examined the response of a mechanosensory neuron in these mutants. In each single mutant and the double mutant bas 1 bssMW1, the frequency of action potentials in response to a bristle displacement was reduced. This is the first demonstration of a physiological defect common to several of the bang-sensitive mutations. Adaptation of spike frequency, cumulative adaptation to repeated stimulation (fatigue) and the time course of recovery from adaptation were also examined. Recovery from adaptation to a conditioning stimulus was examined in two mutants (bas 1 and bss MW1), and initial recovery from adaptation was greater in both mutants. Quantification of receptor potentials was complicated by variability inherent in extracellular recording conditions, but examination of the waveform and range of amplitudes did not indicate clear mutant defects. Therefore the differences observed in the spike response may be due to an alteration of the transfer from receptor potentials to action potential production. DNA sequence analysis of tko and eas has indicated that they encode apparently unrelated biochemical products. Our results suggest that these biochemical lesions lead to a common physiological defect in mechanoreceptors. Although this defect does not provide a straightforward explanation for bang sensitivity, the altered cellular process may lead to bang sensitivity through its action in different parts of the nervous system.
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  • 15
    ISSN: 1432-1432
    Keywords: Artemia franciscana ; Artemia salina ; Artemia parthenogenetica ; Mitochondrial DNA evolution ; Cytochrome c oxidase I ; Cytochrome b ; Drosophila ; Arthropods ; Parthenogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the C↔T substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.
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  • 16
    ISSN: 1432-1432
    Keywords: Drosophila ; Muscle-myosin heavy-chain gene ; Alternative exons ; Synonymous substitutions ; Amino acid substitutions ; Evolution ; Testis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The muscle-myosin heavy-chain (mMHC) gene of Drosophila hydei has been sequenced completely (size 23.3 kb). The sequence comparison with the D. melanogaster mMHC gene revealed that the exonintron pattern is identical. The protein coding regions show a high degree of conservation (97%). The alternatively spliced exons (3a-b, 7a-d, 9a-c, 11a-e, and 15a-b) display more variations in the number of nonsynonymous and synonymous substitutions than the common exons (2, 4, 5, 6, 8, 10, 12, 13, 14, 16, 17, and 19). The base composition at synonymous sites of fourfold degenerate codons (third position) is not biased in the alternative exons. In the common exons there exists a bias for C and against A. These findings imply that the alternative exons of the Drosophila mMHC gene evolve at a different, in several cases higher, rate than the common ones. The 5′ splice junctions and 5′ and 3′ untranslated regions show a high level of similarity, indicating a functional constraint on these sequences. The intron regions vary considerably in length within one species, but the corresponding introns are very similar in length between the two species and all contain stretches of sequence similarity. A particular example is the first intron, which contains multiple regions of similarity. In the conserved regions of intron 12 (head-tail border) sequences were found which have the potential to direct another smaller mMHC transcript.
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  • 17
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    Journal of molecular evolution 38 (1994), S. 363-368 
    ISSN: 1432-1432
    Keywords: Saccharomyces cerevisiae ; 2-μm circle ; DNA sequencing ; Horizontal transmission ; Site-specific recombination ; Selfish DNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We compared the nucleotide substitution pattern over the entire genome of two unique variants of the 6,300-bp selfish DNA (2 μm) plasmid in Saccharomyces cerevisiae. The DNA sequence of the left-unique region is identical among 2-μm variants, while the right-unique region shows substantial divergence. This chimeric pattern cannot be explained by neutral or Darwinian selection models. We propose that horizontal transmission of the 2-μm plasmid coupled with a directed, polarized gene conversion maintains the DNA sequence of the left-unique region, whereas the right-unique region is subject to random drift and Darwinian selection.
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  • 18
    ISSN: 1432-1432
    Keywords: Alcohol dehydrogenase ; Drosophila ; Drosophila lebanonensis ; Gene expression ; Codon usage ; Phylogenetic relationships
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The region of the genome of D. lebanonensis that contains the Adh gene and the downstream Adh-dup gene was sequenced. The structure of the two genes is the same as has been described for D. melanogaster. Adh has two promoters and Adh-dup has only one putative promoter. The levels of expression of the two genes in this species are dramatically different. Hybridizing the same Northern blots with a specific probe for Adh-dup, we did not find transcripts for this gene in D. lebanonensis. The level of Adh distal transcript in adults of D. lebanonensis is five times greater than that of D. melanogaster adults. The maximum levels of proximal transcript are attained at different larval stages in the two species, being three times higher in D. melanogaster late-second-instar larvae than in D. lebanonensis first-instar larvae. The level of Adh transcripts allowed us to determine distal and proximal initiation transcription sites, the position of the first intron, the use of two polyadenylation signals, and the heterogeneity of polyadenylation sites. Temporal and spatial expression profiles of the Adh gene of D. lebanonensis show qualitative differences compared with D. melanogaster. Adh and Adh-dup evolve differently as shown by the synonymous and nonsynonymous substitution rates for the coding region of both genes when compared across two species of the melanogaster group, two of the obscura group of the subgenus Sophophora and D. lebanonensis of the victoria group of the subgenus Scaptodrsophila. Synonymous rates for Adh are approximately half those for Adh-dup, while nonsynonymous rates for Adh are generally higher than those for Adh-dup. Adh shows 76.8% identities at the protein level and 70.2% identities at the nucleotide level while Adh-dup shows 83.7% identities at the protein level and 67.5% identities at the nucleotide level. Codon usage for Adh-dup is shown to be less biased than for Adh, which could explain the higher synonymous rates and the generally lower nonsynonymous substitution rates in Adh-dup compared with Adh. Phylogenetic trees reconstructed by distance matrix and parsimony methods show that Sophophora and Scaptodrosophila subgenera diverged shortly after the separation from the Drosophila subgenus.
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  • 19
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    Journal of molecular evolution 38 (1994), S. 637-641 
    ISSN: 1432-1432
    Keywords: mastermind ; Drosophila ; Homopolymer ; Repeat length variation ; Molecular drive ; Natural selection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Interspecific sequence comparison of the highly repetitive Drosophila gene mastermind (mam) reveals extensive length variation in homopolymer domains. The length variation in homopolymers is due to nucleotide misalignment in the underlying triplet repeats, which can lead to slippage mutations during DNA replication or repair. In mam, the length variation in repetitive regions appears to be balanced by natural selection acting to maintain the distance between two highly conserved charge clusters. Here we report a statistical test of the null hypothesis that the similarity in the amino acid distance between the charge clusters of each species arose by chance. The results suggest that at mam there is a juxtaposition of length variability due to molecular drive and length conservation maintained by natural selection. The analysis of mam allows the extension of current theories of drive-selection interaction to encompass homopolymers. Our model of drive-selection equilibrium suggests that the physical flexibility, length variability, and abundance of homopolymer domains provide an important source of genetic variation for natural populations.
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  • 20
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    Journal of comparative physiology 175 (1994), S. 687-693 
    ISSN: 1432-1351
    Keywords: rdgB ; Maxillary palp ; Drosophila ; Electrophysiology ; Olfaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We describe the kinetics of odorant response in the maxillary palp of Drosophila, and show that the rate of recovery from odorant stimulation is affected by mutation of the rdgB (retinal degeneration B) gene. We use immunocytochemistry to confirm that the rdgB gene product is expressed in the maxillary palp. rdgB has recently been shown to encode a protein with Ca2+-binding sites and sequence similarity to rat brain phosphatidylinositol transfer protein; it is located near the rhabdomeric membranes in photoreceptor cells, where it has been suggested to play a role in membrane transport. The delay in recovery kinetics that we observe in olfactory tissue may reflect a defect in membrane restoration at the conclusion of the olfactory transduction cascade. The use of common molecules in the physiology of two olfactory organs, and in both visual and olfactory physiology, is discussed.
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  • 21
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    Journal of comparative physiology 175 (1994), S. 761-766 
    ISSN: 1432-1351
    Keywords: Olfactory behavior ; Antenna ; Maxillary palp ; Olfaction ; Drosophila
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Maxillary palps have been proposed as secondary olfactory organs, after the antennae, in Drosophila melanogaster. Our study tries to establish the quantitative importance of both organs as olfactory information mediators. Dose-response curves for three odorants: ethyl acetate, propionaldehyde and benzaldehyde were carried out for comparing olfaction in either complete animals or flies surgically deprived of antennae. Antennaless flies tested in our behavioral assay showed indifferent, attractant and repellent responses depending on concentration, similarly as normal flies do. However, they clearly displayed less sensitivity than normal flies. The range of concentrations they were able to perceive was correlated to antennal sensitivity approximately by a factor 1∶10 for ethyl acetate and benzaldehyde, and between 1∶10 and 1∶100 at high concentrations of propionaldehyde. A complementary experiment was performed to test changes in olfactory behavior produced by removing maxillary palps in the presence of antennae. At high concentrations of odorant, responses to ethyl acetate and propionaldehyde experienced small changes when both palps were removed. Results are compatible with a summation model of all olfactory information reaching the brain either through antennae or palps.
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  • 22
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    Development genes and evolution 204 (1994), S. 54-61 
    ISSN: 1432-041X
    Keywords: CNS ; Glia ; Drosophila ; BrdU
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) ofDrosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two otherDrosophila species,D. virilis andD. hydei.
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  • 23
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    Development genes and evolution 204 (1994), S. 118-125 
    ISSN: 1432-041X
    Keywords: Drosophila ; glia ; Proneural ; Neurogenic
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Drosophila proneural genes specify neuronal determination among cells within the ectoderm. Here we address the question of whether proneural genes also affect the specification of glia, the most abundant cell type in the nervous system. We provide evidence that the proneural gene daughterless is essential for the formation of two major classes of PNS glia. In contrast, the proneural genes in the achaete-scute complex have no detectable effect on the specification and differentiation of these PNS glia and certain CNS glia. We also show that, as with neuronal development, glial determination is restricted by the neurogenic genes neuralized, Delta, and the genes of the Enhancer of split complex. Finally, we demonstrate that prospero, a gene involved in neuronal differentiation, also affects glial development. These results demonstrate extensive overlap in the genetic control of glial and neuronal development.
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  • 24
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    Development genes and evolution 203 (1994), S. 266-280 
    ISSN: 1432-041X
    Keywords: Heart ; Drosophila ; Morphogenesis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have followed the normal development of the different cell types associated with the Drosophila dorsal vessel, i.e. cardioblasts, pericardial cells, alary muscles, lymph gland and ring gland, by using several tissue-specific markers and transmission electron microscopy. Precursors of pericardial cells and cardioblasts split as two longitudinal rows of cells from the lateral mesoderm of segments T2-A7 (“cardiogenic region”) during stage 12. The lymph gland and dorsal part of the ring gland (corpus allatum) originate from clusters of lateral mesodermal cells located in T3 and T1/dorsal ridge, respectively. Cardioblast precursors are strictly segmentally organized; each of T2-A6 gives rise to six cardioblasts. While moving dorsally during the stages leading up to dorsal closure, cardioblast precursors become flattened, polarized cells aligned in a regular longitudinal row. At dorsal closure, the leading edges of the cardioblast precursors meet their contralateral counterparts. The lumen of the dorsal vessel is formed when the trailing edges of the cardioblast precursors of either side bend around and contact each other. The amnioserosa invaginates during dorsal closure and is transiently attached to the cardioblasts; however, it does not contribute to the cells associated with the dorsal vessel and degenerates during late embryogenesis. We describe ultrastructural characteristics of cardioblast differentiation and discuss similarities between cardioblast development and capillary differentiation in vertebrates.
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  • 25
    ISSN: 1432-041X
    Keywords: Gut ; Drosophila ; Compartment ; Regional differentiation ; P-element enhancer detectors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We analysed spatial patterns of expression of a lacZ reporter gene in the gut of Drosophila larvae that had been transformed with a P-element-lacZ vector to identify regional differences in gene expression. lacZ-positive epithelial cells formed distinct domains with discrete transverse and longitudinal boundaries along the gut tube. Boundaries were often found at sites at which morphological boundaries were not obvious. The gut epithelium was subdivided into 36 compartments by the boundaries. We refer to these novel compartments as “tissue compartments”. The lacZ-positive domain of each strain appeared as a single tissue compartment or as a combination of several tissue compartments. The tissue compartment is considered to be a unit of regional differentiation. The spatial organization of the tissue compartments may represent the “floor plan”, determined by genes that control the regional differentiation of this nonsegmental organ.
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  • 26
    ISSN: 1432-041X
    Keywords: Drosophila ; achaete ; scute ; Taste bristles
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    Topics: Biology
    Notes: Abstract The sensory precursors for labellar taste bristles develop from the labial disc in three distinct temporal waves occurring at 0 h, 8 h and 14 h of pupal development. In each temporal wave, transcripts for the achaete (ac) and scute (sc) genes are expressed in overlapping patterns in cells of the disc epithelium prior to the appearance of sensory mother cells (SMCs). No bristles form in mutant flies in which the ac and sc genes are absent. When the sc gene alone is deleted, a set of seven bristles fail to form. Pulses of ubiquitous sc + expression during pupal development, in a strain mutant for both ac and sc, can result in flies with all the labellar bristles at their correct positions. sc + pulses at times corresponding to the initiation of each of the waves of SMC specification in the disc was sufficient to restore bristle pattern. Bristles were not induced at ectopic positions and times as a result of the ubiquitous expression of sc +. These results suggest that the proneural genes ac and sc do not themselves set the pattern of the labellar bristles. Instead, they are required for the elaboration of the pattern set by other gene products. We also show that the formation and positioning of the later waves of bristles can take place even in the absence of bristles normally specified earlier.
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  • 27
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    Development genes and evolution 203 (1994), S. 367-373 
    ISSN: 1432-041X
    Keywords: Drosophila ; Embryogenesis ; Morphogenetic movements ; Brain ; HRP
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    Topics: Biology
    Notes: Abstract Using intracellular horseradish peroxidase injection we traced the developmental fate of early gastrula cells of the procephalic region in the stage 16/17 embryo. Morphogenetic movements in the developing brain are described in three dimensions. The results are related to head segmentation, and an early gastrula fate map of pregnathal head segments is proposed.
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  • 28
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    Development genes and evolution 204 (1994), S. 54-61 
    ISSN: 1432-041X
    Keywords: CNS ; Glia ; Drosophila ; BrdU
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    Topics: Biology
    Notes: Abstract Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) of Drosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two other Drosophila species, D. virilis and D. hydei.
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  • 29
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    Current genetics 26 (1994), S. 95-99 
    ISSN: 1432-0983
    Keywords: Translational fidelity ; Paromomycin ; Stuttering ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract Missense errors in the translation of mRNAs in Saccharomyces cerevisiae were screened by looking for charge heterogeneity of proteins on two-dimensional gels resulting from the substitution of charged and neutral amino acids. No such mistranslation was detected in wild-type yeast strains grown in the presence of the translational error-inducing antibiotic paromomycin. However, paromomycin-induced mistranslation of a heterologous mRNA, encoding human phosphoglycerate kinase expressed in yeast, was seen. We suggest that the combination of error-prone translation of a heterologous mRNA, and growth in the presence of paromomycin, leads to an accumulation of mistranslated proteins that can be detected by two-dimensional gel electrophoresis.
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  • 30
    ISSN: 1432-0983
    Keywords: ABC superfamily ; Multidrug resistance ; Saccharomyces cerevisiae ; YDR1 gene
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    Topics: Biology
    Notes: Abstract A multidrug resistance gene, YDR1, of Saccharomyces cerevisiae, which encodes a 170-kDa protein of a member of the ABC superfamily, was identified. Disruption of YDR1 resulted in hypersensitivity to cycloheximide, cerulenin, compactin, staurosporine and fluphenazine, indicating that YDR1 is an important determinant of cross resistance to apparently-unrelated drugs. The Ydr1 protein bears the highest similarity to the S. cerevisiae Snq2 protein required for resistance to the mutagen 4-NQO. The drug-specificity analysis of YDR1 and SNQ2 by gene disruption, and its phenotypic suppression by the overexpressed genes, revealed overlapping, yet distinct, specificities. YDR1 was responsible for cycloheximide, cerulenin and compactin resistance, whereas, SNQ2 was responsible for 4-NQO resistance. The two genes had overlapping specificities toward staurosporine and fluphenazine. The transcription of YDR1 and SNQ2 was induced by various drugs, both relevant and irrelevant to the resistance caused by the gene, suggesting that drug specificity can be mainly attributed to the functional difference of the putative transporters. The transcription of these genes was also increased by heat shock. The yeast drug-resistance system provides a novel model for mammalian multidrug resistance.
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  • 31
    ISSN: 1432-0983
    Keywords: Psoralen ; DNA repair mutants ; Gene conversion ; Recombination ; Saccharomyces cerevisiae
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    Notes: Abstract The influence of the DNA repair genePSO3 on photoactivated psoralen-induced meiotic recombination, gene conversion, reverse mutation, and on survival, was assayed in diploid strains ofSaccharomyces cerevisiae homozygous for the wild-type or thepso3-1 mutant allele. Sporulation was normal in thepso3-1 diploid. Wild-type and mutant strains had the same sensitivity to photoactivated monofunctional psoralen (3-CPs+UVA) in meiosis-uncommitted and meiosis-committed stages. The mutant showed higher sensitivity to photoactivated bifunctional psoralen (8-MOP+UVA) during all stages of the meiotic cycle. Mutation induction by 3-CPs+UVA or 8-MOP+UVA in meiosis-committed cells revealed no significant differences between wild-type and thepso3-1 mutant. The status of thePSO3 gene has no influence on the kinetics of induction of gene conversion and crossing-over after 3-CPs+UVA treatment in meiosis-committed cells: gene conversion was blocked while recombination was induced. After treatment with 8-MOP+UVA gene conversion was also blocked in both strains while crossing-over could only be observed in meiosis-committed wild-type cells.
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  • 32
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    Current genetics 25 (1994), S. 180-183 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; In-vivo cloning ; Non-replicative vectors ; Homologous recombination
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    Topics: Biology
    Notes: Abstract We have devised a new strategy to clone DNA sequences from an yeast autonomously-propagating plasmid into a non-autonomous integrative vector by in-vivo recombination. The method consists of a first step in which the replicative plasmid carrying the DNA fragment of interest forms a co-integrate with the non-replicative plasmid by an induced in-vivo reciprocal exchange accompanied by gene conversion. The dimeric plasmid obtained is then purified and cut with an appropriate restriction enzyme and ligated independently to obtain the two intact monomeric plasmids, the original autonomous plasmid plus the new non-autonomous plasmid carrying the subcloned DNA fragment. The dimeric co-integrate can also serve as substrate for a second in-vivo reciprocal exchange that produces new autonomous plasmids carrying the desired DNA fragment. The technique considerably expands the applications of in-vivo cloning in yeast by complementing three important characteristics of previously published methods: (1) it can be used to clone into non-propagating vectors; (2) co-transformation experiments are not required; and (3) the intermediate co-integrate can be used to generate new types of autonomously-propagating plasmids directly. These characteristics are independent of whether the DNA insert is flanked by appropriate restriction sites or whether it does, or does not, express a detectable phenotype in yeast. The method is particularly useful for the cloning of large DNA fragments and can be used for plasmids from organisms other than yeasts.
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  • 33
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    Current genetics 25 (1994), S. 291-298 
    ISSN: 1432-0983
    Keywords: Cytochrome c 1 ; Cytochrome c 1 heme lyase ; GRF2p ; Glucose repression ; HAPp ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract In this paper we examine the expression of the Saccharomyces cerevisiae CYT2 gene, which encodes cytochrome c 1 heme lyase. This enzyme is required for covalent attachment of heme to apocytochrome c 1, a subunit of the mitochondrial respiratory chain. Transcription of the 1-kb CYT2 mRNA initiates at four prominent sites at a distance of 52–225 bp in front of the AUG start codon. The level of CYT2 mRNA is not influenced by the presence or absence of oxygen or of heme, but it is subject to carbonsource control. The concentration of the CYT2 mRNA is significantly reduced in glucose-grown cells as compared to cells grown under non-repressing conditions. Neither the HAPp activator proteins nor MIG1p, a repressor protein involved in glucose repression, seem to mediate this effect.
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  • 34
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; recA gene expression ; UV radiation ; Mitotic gene conversion
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    Notes: Abstract The effect of the Escherichia coli RecA protein on mitotic recombination in the diploid D7 strain of Saccharomyces cerevisiae damaged by UV radiation was investigated. The D7 strain was transformed by two modified versions of the pNF2 plasmid: one, containing the ADH-1 promoter, and the other containing the recA gene tandemly arranged behind the ADH-1 promoter region. Immunological analysis proved the presence of the 38-kDa RecA protein in D7/pNF2ADHrecA transformants. We observed a positive effect of recA gene expression on mitotic gene conversion, mainly at higher doses of UV radiation. The results indicate that a RecA-like activity could participate in steps preceeding mitotic conversion events in yeast.
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  • 35
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    Current genetics 26 (1994), S. 15-20 
    ISSN: 1432-0983
    Keywords: Cell-division cycle ; Mitochondrial genome ; Nuclear mutation ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract In former studies it was found that the ERV1 gene is essential for cell viability and for the biogenesis of functional mitochondria. A temperature-sensitive nuclear mutant exhibits a severe reduction in all the mitochondrial transcripts. Elimination of the gene leads to growth arrest after a few cell divisions. The putative gene product bears the characteristics of a regulatory factor since it has low expression rate and a high content of charged amino acids. In this study it is further verified that the ERV1 gene alone is responsible for the observed cellular and mitochondrial defects. The 5′ region of the gene is analysed by DNA deletions and complementation studies. Expression of the gene under the control of the GAL1-10 promoter in a disruption strain of ERV1 allows a more detailed specification of its influence on mitochondrial and cellular functions. Immediate and complete loss of mitochondrial genomes is observed after the promoter has been shut off, whereas the yeast cells are still able to grow for a limited time under these conditions. Analysis of the cells by in-vivo DNA flurorescence demonstrates a specific arrest in the cell-division cycle as the terminal phenotype. To further characterize the temperature-sensitive allele of ERV1 the mutated gene has been isolated and sequenced. A single point mutation which leads to the exchange of a single amino acid is found in the reading frame.
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  • 36
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Nuclear gene ; Mitochondria ; Mitochondrial ribosomal protein
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    Notes: Abstract The nuclear gene MRP-L13 of Saccharomyces cerevisiae, which codes for the mitochondrial ribosomal protein YmL13, has been cloned and characterized. It is a single-copy gene residing on chromosome XI. Its nucleotide sequence was found to be identical to that of the previously reported ORF YK105. A comparison of the predicted protein sequence of the MRP-L13 gene product and the actual N-terminal amino-acid sequence of the isolated YmL13 protein indicated that the mature protein is preceded by a mitochondrial signal peptide of 86 amino-acid residues, which is the longest among all known mitochondrial ribosomal proteins of S. cerevisiae. No sequence similarity was found to any other ribosomal protein in the current databases. The transcription of MRP-L13 was found to be repressed in the presence of glucose. Its protein product is not strictly essential for mitochondrial functions, but disruption of the gene by insertion of LEU2 noticeably affected cellular growth on non-fermentable carbon sources.
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  • 37
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    Current genetics 25 (1994), S. 289-289 
    ISSN: 1432-0983
    Keywords: Saccharomyces cerevisiae ; Inducible antisense gene ; Acetolactate synthase ; Bradytrophic phenocopy
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    Topics: Biology
    Notes: Abstract A previous report of the use of antisense RNA to regulate gene expression in yeast is incorrect.
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  • 38
    ISSN: 1432-0983
    Keywords: Psoralen sensitivity ; Saccharomyces cerevisiae ; DNA repair ; Oxidative stress
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    Notes: Abstract The complementation and genetical analysis of yeast mutants sensitive to photoactivated 3-carbethoxy-psoralen define three novel recessive mutant alleles pso-5-1, pso6-1, and pso7-1. Their cross-sensitivity to UV254nm, radiomimetic mutagens, and to chemicals enhancing oxidative stress suggest that these mutants are either impaired in metabolic steps protecting from oxidative stress or in mechanisms of the repair of oxygen-dependent DNA lesions. None of the three novel mutant alleles block the induction of reverse mutation by photoactivated mono- and bi-functional psoralens, nitrogen mustards, or UV254nm.
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  • 39
    ISSN: 1432-0983
    Keywords: tRNA processing ; Saccharomyces cerevisiae ; Mitochondria
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    Notes: Abstract We used a genetic approach to study the nuclear factors involved in the biogenesis of mitochondrial tRNAs. A point mutation in the mitochondrial tRNAAsp gene of Saccharomyces cerevisiae had previously been shown to result in a temperature-sensitive respiratory-deficient phenotype as a result of the absence of 3′ end-processing of the tRNAAsp. Analysis of mitochondrial revertants has shown that all revertants sequenced have a G-A compensatory change at position 53, which restores the hydrogen-bond with the mutated nucleotide. We then searched for nuclear suppressors to identify the nuclear gene(s) involved in mitochondrial tRNA 3′ end-processing. One such suppressor mutation was further characterized: it restores tRNAAsp maturation and growth at 36°C on glycerol medium in heterozygous diploids, but leads to a defective growth phenotype in haploids.
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  • 40
    ISSN: 1432-0983
    Keywords: Overexpression ; Peroxisomes ; Saccharomyces cerevisiae ; Stabilization
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    Notes: Abstract We have constructed a gene coding for the 12-kDa intermediate form of the 2s methionine-rich protein from Bertholletia excelsa seeds. This protein, expressed intracellularly in yeast, is characterised by a 20-min balf-life. By adding 11 amino acids corresponding to the peroxisome-targeting sequence (PTSc) of luciferase, we have significantly increased its half-life. This stabilization allowed accumulation of the BZN protein into the peroxisome as judged by cell fractionation. Accumulation of the 12-kDa protein results in a significant increase of the total methionine content in yeast cells (30%) indicating that such a microorganism could represent a practicable protected shuttl for an animal-feed additive.
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  • 41
    ISSN: 1432-0983
    Keywords: Cytochrome oxidase ; Revertant ; Mitochondria ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract Three respiratory-deficient mutants of cytochrome oxidase subunit I in the yeast mitochondrion have been sequenced. They are located in, or near, transmembrane segment VI, the catalytic core of the enzyme. Respiratory-competent revertants have been selected and studied. The mutant V244M was found to revert at the same site in valine (wild-type), isoleucine or threonine. The revertants of the mutant G251R were of three types: glycine (wild-type), serine and threonine at position 251. A search for second-site mutations was carried out but none were found. Among 60 revertants tested, the mutant K265M was found to revert only to the wild-type allele.
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  • 42
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    Archives of microbiology 162 (1994), S. 211-214 
    ISSN: 1432-072X
    Keywords: Killer toxin ; Saccharomyces cerevisiae ; Toxin binding ; Cell wall receptor
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    Topics: Biology
    Notes: Abstract A recently described new method for determination of killer toxin activity was used for kinetic measurenments of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K T1=2.6x109 L.U./ml, V max1=0.19 s-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K T2=3.2x107 L.U./ml, V max2=0.03 s-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.
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  • 43
    ISSN: 1432-072X
    Keywords: Rylux BSU ; Fluorescent brightener ; Cell walls ; Chitin synthase ; Glucan synthase ; Yeast ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract Rylux BSU, a new fluorescent brightener from the family of 4,4′-diaminostilbene-2,2′disulfonic acid derivatives, inhibited growth and cytokinesis of the yeast Saccharomyces cerevisiae. In the presence of 0.1–1 mg/ml Rylux BSU the cells grew in clumps, had irregular shape and were larger than controls. They formed apparently normal primary septa but their secondary septa and lateral cell walls, especially those in older cells, were abnormally thick with large deposits of amorphous wall material in the periplasmic spaces all over the cell surface. Chitin content in the cell walls of cells grown in the presence of Rylux BSU was increased 2 to 5 times in comparison to that of the controls and glucan content was reduced by up to 30%. In the in vitro assays with particulate membrane fractions, Rylux BSU acted as a non-competitive inhibitor of β-1,3-glucan synthase with inhibitory constant K i=1.75 mg/ml whereas the chitin synthase was inhibited to a much lesser extent. From the difference of the effects of Rylux BSU on the synthesis of chitin in vivo and in vitro it is concluded that the brightener interacts with chitin synthase only indirectly, possibly by influencing the properties of integral plasma membrane.
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  • 44
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    Archives of microbiology 162 (1994), S. 211-214 
    ISSN: 1432-072X
    Keywords: Key words     Killer toxin ; Saccharomyces cerevisiae ; Toxin binding ; Cell wall receptor
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    Topics: Biology
    Notes: Abstract      A recently described new method for determination of killer toxin activity was used for kinetic measurements of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K T1 = 2.6 × 109 L.U./ml, V max1 = 0.19 s– 1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K T2 = 3.2 × 107 L.U./ml, V max2 = 0.03 s– 1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.
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  • 45
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    Journal of industrial microbiology and biotechnology 13 (1994), S. 30-34 
    ISSN: 1476-5535
    Keywords: Phytate ; Saccharomyces cerevisiae ; Polyacrylamide gel ; Inositol phosphates
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary Saccharomyces cerevisiae in the form of baker's yeast, cells cultivated on a yeast extract-peptone-glucose medium, as well as cells immobilized in 18% (w/v) polyacrylamide gel showed the ability to hydrolyze 1.727 mM sodium phytate solution at 45°C, pH 4.6, in a stirred tank reactor. Seventy percent yield of dephosphorylation was observed after 2 h using a baker's yeast concentration of 5.8 g dry matter per 100 ml. Hydrolytic activity at 1.8–2.0 μM Pi min−1 was observed between 1st and 3rd h of the reaction in cells cultured 24 or 48 h. No inhibition by the substrate was found at sodium phytate concentrations of 0.587–1.727 mM. After 1.5 h of hydrolysis a single, well distinguished peak ofmyo-inositol-triphosphate was the main product found. By means of immobilization the stability of the biocatalyst was enhanced 3.3-fold and reached its half-life at 64 ninety-minute runs.
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  • 46
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    Journal of industrial microbiology and biotechnology 13 (1994), S. 269-272 
    ISSN: 1476-5535
    Keywords: Wine ; Yeasts ; Fatty acids ; Ethyl esters ; Saccharomyces cerevisiae
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    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Summary The evolution of the cell and must contents of three short-chain fatty acids (C6, C8 and C10) and their ethyl esters during fermentations withSaccharomyces cerevisiae racescerevisiae, bayanus andcapensis were studied. The former is a fermentative yeast and the last two are ‘flor’ film yeasts. The acid concentrations in the musts increased throughout the alcoholic fermentations, and maximum cell concentrations of the fatty acids were reached after 48 h of fermentation. Maximum ester concentrations in the cells were attained after 48–72 h of fermentation. In the musts, ethyl octanoate and ethyl decanoate reached a peak also at this point, and ethyl hexanoate after 10 days. After 134 days,S. cerevisiae racecapensis formed a thick ‘flor’ film whileS. cerevisiae racebayanus developed a thin film andS. cerevisiae racecerevisiae formed no film. At this point, acid contents remained constant in the wines produced byS. cerevisiae racescerevisiae andbayanus, and decreased in those obtained with racecapensis. The ethyl ester contents tended to decrease with the exception of ethyl decanoate in the fermentations carried out byS. cerevisiae racescerevisiae andbayanus.
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  • 47
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    Astrophysics and space science 216 (1994), S. 153-154 
    ISSN: 1572-946X
    Keywords: Chemistry ; Molecular Hydrogen ; Cloud Models
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    Topics: Physics
    Notes: Abstract Observations of the CO J=1-0 line are commonly used as a tracer for molecular material in clouds. The ratio of the H2 column density to the integrated intensity of this line,X, is often taken to be constant, despite theoretical and observational uncertainty. We have tried to identify how this ratio depends on cloud parameters, testing a simple theoretical argument suggesting its invariance with respect to density. The apparent constancy can be understood if clouds are clumpy on scales of Av ≈ 1-2 mag.
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  • 48
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    Journal of chemical ecology 20 (1994), S. 1893-1906 
    ISSN: 1573-1561
    Keywords: Drosophila ; Diptera ; Drosophilidae ; cytochrome P-450 ; poly-substrate monooxygenase ; cactus ; alkaloids ; resistance
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    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract The cytochrome P-450 monooxygenase system has been implicated in plant utilization by at least three species ofDrosophila (D. nigrospiracula, D. mettleri, andD. mojavensis) that are endemic to the Sonoran Desert of the southwestern United States and northwestern Mexico. Basal and induced levels of total cytochrome P-450 were determined for third-instar and decapitated 2- to 5-day post eclosion adults of the three desert species. Total P-450 levels, both basal and induced for all species assayed, were significantly higher for adults than for larvae by up to 20-fold. On a per organism basis, the levels of in vitro metabolism of the cactus alkaloid, carnegine, and patterns of response to induction by cactus tissue for adult desertDrosophila approximated those of larvae. Induction by phenobarbital, however, resulted in levels of in vitro carnegine metabolism that were up to 5.6-fold higher in adults than in larvae.
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  • 49
    ISSN: 1573-4927
    Keywords: flightin ; Drosophila ; insect flight muscle ; phosphoprotein ; actin ; myosin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Flightin is a 20-kD myofibrillar protein found in the stretch-activated flight muscles ofDrosophila melanogaster. Nine of the eleven isoelectric variants of flightin are generatedin vivo by multiple phosphorylations. The accumulation of these isoelectric variants is affected differently by mutations that eliminate thick filaments or thin filaments. Mutations in the myosin heavy-chain gene that prevent thick filament assembly block accumulation of all flightin variants except N1, the unphosphorylated precursor, which is present at much reduced levels. Mutations in the flight muscle-specific actin gene that block actin synthesis and prevent thin filament assembly disrupt the temporal regulation of flightin phosphorylation, resulting in premature phosphorylation and premature accumulation of flightin phosphovariants. Cellular fractionation of fibers that are devoid of thin filaments show that flightin remains associated with the thick filamentrich cytomatrix. These results suggest that flightin is a structural component of the thick filaments whose regulated phosphorylation is dependent upon the presence of thin filaments.
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  • 50
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    Molecular biology reports 20 (1994), S. 135-141 
    ISSN: 1573-4978
    Keywords: mitochondria ; multienzyme complex ; replication ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract A 40 S multienzyme complex containing mtDNA polymerase was isolated from mitochondria ofS. cerevisiae by density gradient centrifugation and by gel filtration chromatography. Besides DNA polymerase, RNA polymerase, primase, 3′→5′ exonuclease and an ATPase activities were found to be associated with it. The presence of some of these enzymes were confirmed by Western blot. This high molecular weight multienzyme complex containing DNA has most of the attributes of a putative replisome.
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  • 51
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; cDNA ; complementation ; erg20-2 yeast mutant ; farnesyl diphosphate synthase ; Saccharomyces cerevisiae
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    Topics: Biology
    Notes: Abstract A cDNA encoding farnesyl diphosphate synthase, an enzyme that synthesizes C15 isoprenoid diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate, was cloned from an Arabidopsis thaliana cDNA library by complementation of a mutant of Saccharomyces cerevisiae deficient in this enzyme. The A. thaliana cDNA was also able to complement the lethal phenotype of the erg20 deletion yeast mutant. As deduced from the full-length 1.22 kb cDNA nucleotide sequence, the polypeptide contains 343 amino acids and has a relative molecular mass of 39689. The predicted amino acid sequence presents about 50% identity with the yeast, rat and human FPP synthases. Southern blot analyses indicate that A. thaliana probably contains a single gene for farnesyl diphosphate synthase.
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  • 52
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    Molecular biology reports 19 (1994), S. 211-220 
    ISSN: 1573-4978
    Keywords: Drosophila ; eIF-2 ; eIF-4F ; heat shock ; mRNA translation regulation ; phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract All organisms from bacteria to man respond to an exposure to higher than physiological temperatures by reprogramming their gene expression, leading to the increased synthesis of a unique set of proteins termed heat shock proteins (hsps). The hsps function as molecular chaperones in both normal and stressed cells. The rapid and efficient synthesis of hsps is achieved as a result of changes occurring at gene transcription, RNA processing and degradation, and mRNA translation. With regard to the translational regulation, the emerging picture is that the two key steps of polypeptide chain initiation, namely mRNA binding and Met-tRNA i binding to ribosomes, are regulated in heat-shocked mammalian cells. InDrosophila, mRNA binding is regulated by a structural feature of the leader of heat shock mRNAs and by the inactivation of eukaryotic initiation factor- (eIF-) 4F. No clear evidence for changes in Met-tRNA i binding has been obtained yet.
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  • 53
    ISSN: 1617-4623
    Keywords: Cerulenin ; Saccharomyces cerevisiae ; Fatty acid synthase ; β-Ketoacyl synthase ; Drug resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cerulenin, an antifungal antibiotic produced by Cephalosporium caerulens, is a potent inhibitor of fatty acid synthase in various organisms, including Saccharomyces cerevisiae. The antibiotic inhibits the enzyme by binding covalently to the active center cysteine of the condensing enzyme domain. We isolated 12 cerulenin-resistant mutants of S. cerevisiae following treatment with ethyl methanesulfonate. The mechanism of cerulenin resistance in one of the mutants, KNCR-1, was studied. Growth of the mutant was over 20 times more resistant to cerulenin than that of the wild-type strain. Tetrad analysis suggested that all mutants mapped at the same locus, FAS2, the gene encoding the α subunit of the fatty acid synthase. The isolated fatty acid synthase, purified from the mutant KNCR-1, was highly resistant to cerulenin. The cerulenin concentration causing 50% inhibition (IC50) of the enzyme activity was measured to be 400 μM, whereas the IC50 value was 15 μM for the enzyme isolated from the wild-type strain, indicating a 30-fold increase in resistance to cerulenin. The FAS2 gene was cloned from the mutant. Sequence replacement experiments suggested that an 0.8 kb EcoRV-HindIII fragment closely correlated with cerulenin resistance. Sequence analysis of this region revealed that the GGT codon encoding Gly-1257 of the FAS2 gene was altered to AGT in the mutant, resulting in the codon for Ser. Furthermore, a recombinant FAS2 gene, in which the 0.8 Kb EcoRV-HindIII fragment of the wild-type FAS2 gene was replaced with the same region from the mutant, when introduced into FAS2-defective S. cerevisiae complemented the FAS2 pheno-type and showed cerulenin resistance. These data indicate that one amino acid substitution (Gly → Ser) in the α subunit of fatty acid synthase is responsible for the cerulenin resistance of the mutant KNCR-1.
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  • 54
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Transcriptional regulation ; Chromatin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract GAL11 was first identified as a gene required for full expression of some galactose-inducible genes that are activated by GAL4, and it was subsequently shown to be necessary for full expression of another set of genes activated by RAP1/GRFl/TUF. Genetic analysis suggests that GAL11 functions as a coactivator, mediating the interaction of sequence-specific activators with basal transcription factors. To test this hypothesis, we first tried to identify functional domains by deletion analysis and found that the 866–910 region is indispensable for function. Using reporters bearing various upstream activating sequences (UAS) and different core promoter structures, we show that the involvement of GAL11 in transcriptional activation varies with the target promoter and the particular combination of cis elements. Gel electrophoresis in the presence of chloroquine shows that GAL11 affects the chromatin structure of a circular plasmid. Based on these findings, the role of GAL 11 in regulation of transcription, including an alteration in chromatin structure, is discussed.
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  • 55
    ISSN: 1573-0778
    Keywords: Baculovirus ; cell culture ; Drosophila ; gene expression ; insect cell ; metallothionein promoter ; recombinant protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In this report, we compare two different expression systems: baculovirus/Sf9 and stable recombinantDrosophila Schneider 2 (S2) cell lines. The construction of a recombinant S2 cell line is simple and quick, and in batch fermentations the cells have a doubling time of 20 hours until reaching a plateau density of 20 million cells/ml. Protein expression is driven by theDrosophila Metallothionein promoter which is tightly regulated. When expressed in S2 cells, the extracellular domain of human VCAM, an adhesion molecule, is indistinguishable from the same protein produced by baculovirus-infected Sf9 cells. Additionally, we present data on the expression of a seven trans-membrane protein, the dopamine D4 receptor, which has been successfully expressed in both systems. The receptor integrates correctly in the S2 membrane, binds [3H]spiperone with high affinity and exhibits pharmacological characteristics identical to that of the receptor expressed in Sf9 and mammalian cells. The general implications for large scale production of recombinant proteins are discussed.
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  • 56
    ISSN: 1573-739X
    Keywords: Antimalarials ; Artemisia annua L ; Artemisinin ; Biosynthesis ; Chemistry ; Clinical trials ; Pharmacology ; Phytochemistry ; Sesquiterpenes ; Toxicology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Artemisinin, a sesquiterpene lactone endoperoxide isolated fromArtemisia annua L., and a number of its semisynthetic derivatives have shown to possess antimalarial properties. They are all eflective againstPlasmodium parasites that are resistant to the newest and commonly used antimalarial drugs. This article gives a survey of the literature dealing with artemisinin-relaled antimalarial issues that have appeared from the end of 1989 up to the beginning of 1994. A broad range of medical and pharmaceutical disciplines is covered, including phytochemical aspects like the selection of high-producing plants, analytical procedures, and plant biotechnology. Furthermore, the organic synthesis of artemisinin derivatives is discussed, as well as their mechanism of action and antimalarial activity, metabolism and pharmacokinetics, clinical studies, sideeffects and toxicology, and biological activities other than antimalarial activity.
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  • 57
    ISSN: 1573-4927
    Keywords: Drosophila ; glycerol-3-phosphate ; dehydrogenase ; low activity ; DNA sequence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Northern analyses of two low-activitysn-glycerol-3-phosphate dehydrogenase(Gpdh) alleles extracted from natural populations ofDrosophila melanogaster showed that one of them,Gpdh ACyg22 , produced wild-type levels of a normal sized (1.7-kb) mRNA but the other,Gpdh AMB5 , had very low levels of a 1.7-kb mRNA together with low levels of a transcript 200 bp larger. The two variant genes were cloned and sequenced. Compared with normal activity alleles, there were two nucleotide differences in the DNA sequence ofGpdh ACyg22 which were in first-codon positions and would be expected to give rise to Asn-13 → Tyr and Arg-272 → Cys substitutions. The second of these changes is most likely to account for the altered properties of the enzyme. In contrast, none of the nucleotide differences inGpdh AMB5 would give rise to amino acid substitutions, but a 76-bp deletion in the 5′ region removed the normal TATA box and there was a 20-bp insertion in the same region. One of the two transcripts was derived from the use of a substitute TATA box sequence in the insertion, but the 1.9-kb transcript had heterogeneous 5′ ends that were not associated with substitute TATA box sequences. The two transcripts either are produced at a lower rate or are less stable than the normal mRNA.
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  • 58
    ISSN: 1573-6849
    Keywords: dosage compensation ; Drosophila ; histone acetylation ; nuclear domains
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the fruit flyDrosophila, dosage compensation involves several proteins acting in concert to double the transcriptional activity of genes on the single male X chromosome. Three of these proteins, MLE, MSL-1 and histone H4 acetylated at lysine 16 (H4Ac16), have recently been shown to be located almost exclusively on the male X chromosome in interphase (polytene) cells. We show here that in neuroblasts from third instarDrosophila larvae antisera to H4Ac16, MLE and MSL-1 uniquely label the distal, euchromatic region of the male X chromosome through mitosis. The centromere-proximal, heterochromatic region of the male X is not labelled with these antisera, nor are male autosomes or any chromosomes in female cells. That the association of H4Ac16 with the male X chromosome persists, even when the chromosome is maximally compacted and transcriptionally quiescent, argues that this modified histone is an integral component of the dosage compensation pathway. In the nuclei of interphase neuroblasts from male (but never female) larvae, antibodies to H4Ac16 revealed a small, brightly labelled patch against a background of generally weak nuclear staining. In double-labelling experiments, this patch was also labelled, albeit comparatively weakly, with antibodies to MSL-1. These results strongly suggest that the distal, euchromatic region of the X chromosome in male cells occupies a limited and relatively compact nuclear domain.
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  • 59
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    Molecular genetics and genomics 244 (1994), S. 260-268 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Amino acid permeases ; Transport ; Tryptophan
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm2Δ1-Δ4) cause slow growth, whereas one disrupted allele (scm2Δ5) is lethal. Cells with both the scm2Δ1 and trp1-Δ101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm2Δ1 or trp1-Δ101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.
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  • 60
    ISSN: 1617-4623
    Keywords: Drug sensitivity ; Saccharomyces cerevisiae ; Major facilitator superfamily ; Drug expulsion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).
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  • 61
    ISSN: 1617-4623
    Keywords: Drosophila ; Enhancer of split ; Helix-loop-helix protein ; Neurogenesis ; Transcriptional repressors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract TheEnhancer of split complex [E(SPL)-C] ofDrosophila participates in the control of cell fate choice by uncommitted neuroectodermal cells in the embryo. It encodes seven proteins that belong to the basic helix-loop-helix (bHLH) family, six of which are expressed in very similar patterns in the neuroectoderm. Here we describe experiments aimed at unravelling the molecular basis of their function. We found that two products of the complex, HLH-M5 andEnhancer of split, are capable of binding as homo-and heterodimers to a sequence in the promoters of theEnhancer of split andachaete genes, called the N-box, which differs slightly from the consensus binding site (the E-box) for other bHLH proteins. In transient expression assays in cell culture, both proteins were found to attenuate the transcriptional activation mediated by the proneural bHLH proteinslethal of scute anddaughterless at theEnhancer of split promoter.
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  • 62
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Transcription factor ; Zinc finger ; Multidrug resistance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract TheSaccharomyces cerevisiae PDR3 gene, located near the centromere of chromosome II, has been completely sequenced and characterised. Mutationspdr3-1 andpdr3-2, which confer resistance to several antibiotics can be complemented by a wild-type allele of the PDR3 gene. The sequence of the wild-typePDR3 gene revealed the presence of a long open reading frame capable of encoding a 976-amino acid protein. The protein contains a single Zn(II)2Cys6 binuclear-type zinc finger homologous to the DNA-binding motifs of other transcriptional activators from lower eukaryotes. Evidence that the PDR3 protein is a transcriptional activator was provided by demonstrating that DNA-bound LexA-PDR3 fusion proteins stimulate expression of a nearby promoter containing LexA binding sites. The use of LexA-PDR3 fusions revealed that the protein contains two activation domains, one localised near the N-terminal, cysteine-rich domain and the other localised at the C-terminus. The salient feature of the PDR3 protein is its similarity to the protein coded byPDR1, a gene responsible forpleiotropicdrugresistance. The two proteins show 36% amino acid identity over their entire length and their zinc finger DNA-binding domains are highly conserved. The fact that the absence of both PDR1 and PDR3 (simultaneous disruption of the two genes) enhances multidrug sensitivity strongly suggests that the two transcriptional factors have closely related functions.
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  • 63
    ISSN: 1617-4623
    Keywords: Nuclear suppressor gene ; Mitochondrial functions ; Glucose repression ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We previously isolated a nuclear 5.7 kb genomic fragment carrying the NAM7/UPF1 gene, which is able to suppress mitochondrial splicing deficiency when present in multiple copies. We show here that an immediately adjacent gene ISF1 (Increasing Suppression Factor) increases the efficiency of the NAM7/UPF1 suppressor activity. The ISF1 gene has been independently isolated as the MBR3 gene and comparison of the ISF1 predicted protein sequence with data libraries revealed a significant similarity with the MBRI yeast protein. The ISF1 and NAM7 genes are transcribed in the same direction, and RNase mapping allowed the precise location of their termini within the intergenic region to be determined. The ISF1 gene is not essential for cell viability or respiratory growth. However as for many mitochondrial genes, ISF1 expression is sensitive to fermentative repression; in contrast expression of the NAM7 gene is unaffected by glucose. We propose that ISF1 could influence the NAM7/UPF1 function, possibly at the level of mRNA turnover, thus modulating the expression of nuclear genes involved in mitochondrial biogenesis.
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  • 64
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Mitochondria ; Cytochrome b ; Complex II ; HAP2/3/4
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Computer-assisted structural analysis of the predicted product of the previously described open reading frame (ORF) YKL4 located on the left arm of chromosome XI of Saccharomyces cerevisiae revealed a high degree of similarity (〉50%) to bovine cytochrome b 560, the sdhC polypeptide of the Escherichia coli succinate dehydrogenase (SDH) complex and the protein specified by ORF137 located on the chloroplast DNA of Marchantia polymorpha. Disruption of the yeast gene severely impaired mitochondrial function, while Northern analysis showed it to be subject to catabolite repression. Deletion analysis of the CYB3 promoter identified a single HAP2/3/4-binding element that is necessary and sufficient for carbon source-dependent transcriptional regulation. These experiments also suggested the presence of additional, as yet unidentified, transcriptional control elements, both negative and positive. Taken together, these data lead us to conclude that the CYB3 gene encodes the yeast homolog of the bovine cytochrome b 560 component of complex II of the mitochondrial electron transport chain.
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  • 65
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; CYP1(HAP1) protein ; Electron transport ; Oxygen and heme regulation ; Trans regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract CYP1 determines the expression of several genes whose transcription is heme-dependent in yeast. It exerts regulatory functions even in the absence of heme, usually considered to be its effector. It mediates both positive and negative effects, depending on the target gene and on the redox state of the cell. In the presence of heme, it binds through a cysteine-rich domain in which a histidine residue occupies the position of the sixth and essential cysteine of the otherwise classical zinc cluster DNA-binding domain exemplified by GAL4. We constructed specific missense mutations in the potential CYP1 zinc cluster domain by site-directed mutagenesis and looked for regulatory effects of the mutated proteins under specific physiological conditions. We show that CYP1 does belong to the zinc cluster regulatory family since a sixth essential cysteine residue is indeed present, albeit at a modified position when compared to the consensus sequence. We also show that the amino acid preceding the first cysteine residue of the DNA-binding domain critically affects the efficiency of regulation both in the presence and in the absence of heme: mutations known to affect DNA binding under heme-sufficient conditions also affect regulation under heme-deficient conditions. We therefore surmise that regulation under hemedeficient conditions is dependent upon DNA binding.
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  • 66
    ISSN: 1617-4623
    Keywords: Drosophila ; Y chromosome ; Fertility genes Lampbrush loops ; Repetitive DNA sequences
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The short arm of the Y chromosome of Drosophila hydei carries a single male fertility gene, gene Q, which forms the lampbrush loop pair Nooses. Conflicting observations have been reported concerning the identity of the repetitive DNA sequences that are transcribed in this loop pair. It has been claimed by other investigators that the loop transcripts contain repeats of two distinct, but related families of Y-specific repetitive DNA sequences, ayl and YsI. We reinvestigated this issue, using as probes single ayl and YsI repeats which, under stringent conditions, hybridize only to members of their own family. Under non-stringent conditions, both repeats hybridize in situ to Nooses transcripts. However, if hybridization conditions are stringent, only the ayl probe hybridizes to loop transcripts. Hybridizations to Northern blots of testis RNA confirm these results. Further, YsI repeats are not found the closely related species D. eohydei. We conclude that the YsI repeats are not relevant for the function of fertility gene Q.
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  • 67
    ISSN: 1617-4623
    Keywords: Drosophila ; Glutamic acid decarboxylase ; GAD ; GABA ; Lethal mutations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Drosophila melanogaster Gad gene maps to region 64A3-5 of chromosome 3L and encodes glutamic acid decarboxylase (GAD), the rate-limiting enzyme for the synthesis of the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Because this neurotransmitter has been implicated in developmental functions, we have begun to study the role of GABA synthesis during Drosophila embryogenesis. We show that Gad mRNA is expressed in a widespread pattern within the embryonic nervous system. Similarly, GAD-immunoreactive protein is present during embryogenesis. These results prompted us to screen for embryonic lethal mutations that affect GAD activity. The chromosomal region to which Gad maps, however, has not been subjected to an extensive mutational analysis, even though it contains several genes encoding important neurobiological, developmental, or cellular functions. Therefore, we have initially generated both chromosomal rearangements and point mutations that map to the Drosophila 64AB interval. Altogether, a total of 33 rearrangements and putative point mutations were identified within region 64A3-5 to 64B12. Genetic complementation analysis suggests that this cytogenetic interval contains a minimum of 19 essential genes. Within our collection of lethal mutations are several chromosomal rearrangements, two of which are in the vicinity of the Gad locus. One of these rearrangements, Df(3L)C175, is a small deletion that removes the Gad locus and at least two essential genes; the second, T(2;3)F10, is a reciprocal translocation involving the second and third chromosomes with a break within region 64A3-5. Both of these rearrangements are associated with embryonic lethality and decreased GAD enzymatic activity.
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  • 68
    ISSN: 1617-4623
    Keywords: Multicopy suppressors ; HAP2/3/4 activation complex ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two new yeast genes, named MBR1 and MBR3, were isolated as multicopy suppressors of the growth defect of a strain lacking the HAP2 transcriptional activator. Both genes when overexpressed can also suppress the growth defect of hap3 and hap4 null mutants. However, overexpression of MBRI cannot substitute for the HAP2/3/4 complex in activation of the CYC1 gene. Nucleotide sequencing of MBR1 and MBR3 revealed that these two genes encode serine-rich, hydrophilic proteins with regions of significant homology. The functional importance of one of these conserved regions was shown by mutagenesis. Disruption of MBR1 leads to a partial growth defect on glycerol medium. Disruption of MBR3 has no major effect but the double disruptant shows a synthetic phenotype suggesting that the MBR1 and MBR3 gene products participate in common function.
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  • 69
    ISSN: 1617-4623
    Keywords: Drosophila ; Fertility genes ; Y chromosome Lampbrush loops ; Repetitive DNA sequences
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Y chromosomal lampbrush loop-forming male fertility genes of Drosophila consist mainly of repetitive DNA sequences that do not code for proteins. We investigated whether differences in the transcription of these sequences can be detected in male-sterile alleles of male fertility gene Q, which forms the loop pair Nooses. The loop consists, for approximately two-thirds, of repeats of the Y-specific ay1 family of repetitive DNA sequences. Of the remaining one-third, at least one-half is represented by defective retrotransposons of the gypsy family. Both sequence types are interspersed throughout the loop. Using both ay1 and gypsy sequences as probes for transcript in situ hybridization, we show that, at the level of the light microscope, transcription of neither sequence is detectably affected in the loops formed by a male-sterile allele of gene Q. We conclude that the transcription of ay1 and gypsy is required, but not sufficient for the function of gene Q.
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  • 70
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Meiosis Sporulation ; Divergent promoter ; Developmental regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Promoters that control gene expression in Saccharomyces cerevisiae only in a sporulation-specific manner have previously been isolated from a genomic yeast DNA library fused to a promoterless Escherichia coli lacZ gene. Two novel sporulation-specific genes, SPS18 and SPS19, were isolated using this technique. These genes are divergently controlled by the same promoter but with SPS18 expressed at four times the level of SPS19. Deletion analysis has shown that the promoter elements that exert sporulation control on each of the genes overlap, having a common 25 bp sequence located within the intergenic region. SPS18 encodes a 34-KDa protein of 300 amino acids that contains a putative zinc-binding domain and a region of highly basic residues that could target the protein to the nucleus. SPS19 encodes a 31-KDa protein of 295 amino acids, which has a peroxisomal targeting signal (SKL) at its C terminus; this protein belongs to the family of non-metallo short-chain alcohol dehydrogenases. A null mutation deleting the intergenic promoter prevented expression of both genes, and when homozygous in diploids, reduced the extent of sporulation four-fold; the spores that did form were viable, but failed to become resistant to ether, and were more sensitive to lytic enzymes. This phenotype reflects a defect in spore wall maturation, indicating that the product of at least one of the genes functions during the process of spore wall formation. Therefore these genes belong to the class of late sporulation-specific genes that are sequentially activated during the process of meiosis and spore formation.
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  • 71
    ISSN: 1617-4623
    Keywords: Antibacterial ; Digestive tract ; Drosophila ; Gene family ; Lysozyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles in immunity and digestion, we have now characterized all six lysozyme genes in the cloned part of the lysozyme locus at 61F, and a seventh gene that maps to the same chromosomal location. The expression of the genes follows four different patterns: firstly, four closely related genes, LysB, C, D and E, are all strongly expressed in the midgut of larvae and adults; secondly, LysP is expressed in the adult salivary gland; thirdly, LysS is expressed mainly in the gastric caecae of larvae; and finally, LysX is primarily expressed in the metamorphosing midgut of late larvae and early pupae. The LysD-like genes and LysS are strongly repressed in artificially infected animals, possibly reflecting a malaise reaction in the digestive tract. None of the genes is expressed in the fat body or haemocytes. Thus rather than being a component of the haemolymph, the Drosophila lysozymes are found mainly in the digestive tract where they are expressed at a high level. Furthermore all genes, except LysP, encode acidic proteins, in contrast to the strongly basic “typical” lysozymes. This is highly reminiscent of the situation in ruminants, where the lysozymes have been recruited for the digestion of symbiotic bacteria in the stomach.
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  • 72
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Duplicate genes ; Synthetic lethal mutants ; CTP synthetase ; Pyrimidine biosynthetic pathway
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In the pyrimidine biosynthetic pathway, CTP synthetase catalyses the conversion of uridine 5′-triphosphate (UTP) to cytidine 5′-triphosphate (CTP). In the yeast Saccharomyces cerevisiae, the URA7 gene encoding this enzyme was previously shown to be nonessential for cell viability. The present paper describes the selection of synthetic lethal mutants in the CTP biosynthetic pathway that led us to clone a second gene, named URA8, which also encodes a CTP synthetase. Comparison of the predicted amino acid sequences of the products of URA7 and URA8 shows 78% identity. Deletion of the URA8 gene is viable in a haploid strain but simultaneous presence of null alleles both URA7 and URA8 is lethal. Based on the codon bias values for the two genes and the intracellular concentrations of CTP in strains deleted for one of the two genes, relative to the wild-type level, URA7 appears to be the major gene for CTP biosynthesis. Nevertheless, URA8 alone also allows yeast growth, at least under standard laboratory conditions.
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  • 73
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    Molecular genetics and genomics 242 (1994), S. 517-527 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; HSP82 ; Random in vitro mutagenesis ; Temperature-sensitive mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.
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  • 74
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell wall ; Protein kinase C ; β-Glucanase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To obtain more information about the cell wall organization of Saccharomyces cerevisiae, we have developed a novel screening system to obtain cell wall-defective mutants, using a density gradient centrifugation method. Nine hypo-osmolarity-sensitive mutants were classified into two complementation groups, hpo1 and hpo2. Phase contrast microscopic observation showed that mutant cells bearing lesions at either locus became abnormally large. A gene that complemented the mutant phenotype of hpo2 was cloned and sequenced. This gene turned out to be identical to PKC1, which encodes the yeast homologue of mammalian protein kinase C. Complementation tests with pkc1Δ showed that hpo2 is allelic to pkc1. To study the reason for the fragility of hpo2 cells, cell wall was isolated and the glucan was analyzed. The amount of alkali, acid-insoluble glucan, which is responsible for the rigidity of the cell wall, was reduced to about 30% that of the wild-type cell and this may be the major cause of the fragility of the hpo2 mutant cell. Analysis of total wall proteins in hpo2 mutant cells on SDS-polyacrylamide gels revealed that a 33 kDa protein was overproduced two- to threefold relative to the wild-type level. This 33 kDa protein was identified as a β-glucanase, encoded by BGL2. Disruption of BGL2 in the hpo2 mutant partially rescued the growth rate defect. This suggests that the PKC1 kinase cascade regulates BGL2 expression negatively and overproduction of the β-glucanase is partially responsible for the growth defect. Since the bgl2 disruption did not rescue the hypo-osmolarty-sensitive phenotype of the hpo2 mutant, PKC1 must negatively regulate other enzymes involved in the biosynthesis and metabolism of the cell wall.
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  • 75
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    Molecular genetics and genomics 243 (1994), S. 358-362 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Oxidative stress ; High temperature viability ; Ubiquitin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract UBI4, the polyubiquitin gene of Saccharomyces cerevisiae, is expressed at a low level in vegetative cells, yet induced strongly in response to starvation, cadmium, DNA-damaging agents and heat shock. UBI4 is also expressed at a higher basal level in cells growing by respiration as compared to glucose-repressed cells growing by fermentation. This higher UBI4 expression of respiratory cultures probably helps to counteract the greater oxidative stress of respiratory growth. The effects of inactivating UBI4 on high temperature viability are more marked with respiratory cultures. Also loss of UBI4 leads to a considerably increased rate of killing of respiring cells by hydrogen peroxide, whereas the same gene inactivation has relatively little effect on the peroxide sensitivity of cells in which mitochondrial functions are repressed. This is the first study to reveal that ubiquitin levels in cells can influence their ability to withstand oxidative stress.
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  • 76
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    Molecular genetics and genomics 243 (1994), S. 363-368 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Glycolysis ; Phosphoglucose isomerase ; Antisense ; Double-strand coding
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Open reading frames longer than 300 bases were observed in the antisense strands of the genes coding for the glycolytic enzymes phosphoglucose isomerase, phosphoglycerate mutase, pyruvate kinase and alcohol dehydrogenase I. The open reading frames on both strands are in codon register. It has been suggested that proteins coded in codon register by complementary DNA strands can bind to each other. Consequently, it was interesting to investigate whether the open reading frames in the antisense strands of glycolytic enzyme genes are functional. We used oligonucleotide-directed mutagenesis of the PGI1 phosphoglucose isomerase gene to introduce pairs of closely spaced base substitutions that resulted in stop codons in one strand and only silent replacements in the other. Introduction of the two stop codons into the PGI1 sense strand caused the same physiological defects as already observed for pgi1 deletion mutants. No detectable effects were caused by the two stop codons in the antisense strand. A deletion that removed a section from − 31 by to + 109 by of the PGI1 gene but left 83 bases of the 3′ region beyond the antisense open reading frame had the same phenotype as a deletion removing both reading frames. A similar pair of deletions of the PYK1 gene and its antisense reading frame showed identical defects. Our own Northern experiments and those reported by other authors using double-stranded probes detected only one transcript for each gene. These observations indicate that the antisense reading frames are not functional. On the other hand, evidence is provided to show that the rather long reading frames in the antisense strands of these glycolytic enzyme genes could arise from the strongly selective codon usage in highly expressed yeast genes, which reduces the frequency of stop codons in the antisense strand.
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  • 77
    ISSN: 1617-4623
    Keywords: Bacterio-opsin ; Expression ; Yeast ; Saccharomyces cerevisiae ; Membranes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium The designation H. salinarium instead of the former designation H. halobium is used throughout this paper following the classification of Tindall (1992) . This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.
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  • 78
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    Molecular genetics and genomics 244 (1994), S. 197-204 
    ISSN: 1617-4623
    Keywords: Drosophila ; Shaker gene complex Dominant lethality ; K+ channel ; Troponin I
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Different mutations belonging to the HLI and HLII complementation groups of the haplolethal (HL) region of the Shaker complex (ShC) are described. The HLI complementation group includes viable (hdp), recessive lethals [l(1)1614], semidominant lethals [l(1)8384] and dominant lethals [l(1)5051,l(1)9916, l(1)13193], lack-of-function alleles that affect nervous system, cuticle and muscle development. The HLI complementation group encodes troponin I. HLII lack-of-function mutations [l(1)174 and l(l)4058] affect nervous system development. The semidominant lethal HLI mutation 1(1)8384 shows differential complementation with other mutations in the ME and HL regions of ShC. Thus, heterozygous combinations of l(1)8384 with ME mutations l(1)162 and l(1)387 are poorly viable. The same phenomenon is observed for heterozygotes of l(1)8384 with HL mutations l(1)1199, l(1)2288 and l(1)3014. These specific interactions indicate the existence of functional relationships among the genetic elements of ShC. The implications for the understanding of the functional organization of ShC are discussed.
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  • 79
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    Molecular genetics and genomics 244 (1994), S. 205-215 
    ISSN: 1617-4623
    Keywords: Drosophila ; tetanic ; Shaker gene complex ; Maternal effect ; K+ channel modulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Different phenotypes associated with the tetanic (tta) mutation such as appendage contraction, maternal effect and low viability and fertility are enhanced by one extra dose of the Shaker gene complex (ShC). The tta mutation is lethal with two extra doses of ShC. In addition, tta embryos have a defective nervous system. In this paper, I analyse the interaction between tta and ShC to gain insight into their relationship. Aneuploid analysis suggests that the lethality is due to an interaction of the tta mutation with the maternal effect (ME) region of this gene complex. Mutations in the ME region of ShC partially suppress this interaction. Trans-heterozygous combinations of MEI[l(1)305] and MEIII [l(1)459] mutations causes dominant lethality in a tta background. Trans-heterozygous combinations of an MEII [l(1)1359] mutation with the cited MEI and MEIII mutations are lethal in a tta background. Double mutant combinations and gene dosage experiments, suggest that tta also interacts with the viable (V) region of ShC. These specific genetic interactions indicate that tta and the ME and V regions of ShC are functionally related. These results, together with the previous electrophysiological, molecular and biochemical studies on these mutants suggest an interaction at the protein level. Thus, in the case of the V region, the tta gene product may modulate the activity of the K+ channels encoded in this region. Furthermore, the extreme dosage sensitivity of the interaction between tta and ShC suggests a stoichiometric requirement for the different gene products involved, which might be physically associated and form heteromultimers.
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  • 80
    ISSN: 1617-4623
    Keywords: Acetylcholinesterase ; Drosophila ; Thermosensitive mutation
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    Topics: Biology
    Notes: Abstract Ace IJ29 and Ac IJ40 are cold- and heat-sensitive variants of the gene coding for acetylcholinesterase in Drosophila melanogaster. In the homozygous condition, these mutations are lethal when animals are raised at restrictive temperatures, i.e., below 23° C for Ace IJ29 or above 25° C for Ace IJ40. The coding regions of the gene in these mutants were sequenced and mutations changing Ser374 to Phe in Ace IJ29 and Pro75 to Leu in Ace IJ40 were found. Acetylcholinesterases bearing these mutations were expressed in Xenopus oocytes and we found that these mutations decrease the secretion rate of the protein most probably by affecting its folding. This phenomenon is exacerbated at restrictive temperatures decreasing the amount of secreted acetylcholinesterase below the lethality threshold. In parallel, the substitution of the conserved Asp248 by an Asn residue completely inhibits the activity of the enzyme and its secretion, preventing the correct folding of the protein in a non-conditional manner.
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  • 81
    ISSN: 1617-4623
    Keywords: Arabidopsis thaliana ; Saccharomyces cerevisiae ; Complementation ; Aspartate transcarbamylase ; Orotate phosphoribosyl transferase ; Orotidine-5′-phosphate decarboxylase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An Arabidopsis thaliana cDNA library was used to complement Saccharomyces cerevisiae pyrimidine auxotrophic mutants. Mutants in all but one (carbamylphosphate synthetase) of the six steps in the de novo pyrimidine biosynthetic pathway could be complemented. We report here the cloning, sequencing and computer analysis of two cDNAs encoding the aspartate transcarbamylase (ATCase; EC 2.1.3.2) and orotate phosphoribosyltransferase-orotidine-5′-phosphate decarboxylase (OPRTase-OMP-decase; EC 2.4.2.10, EC 4.1.1.23) enzymes. These results confirm the presence in A. thaliana of a bifunctional gene whose product catalyses the last two steps of the pyrimidine biosynthetic pathway, as previously suggested by biochemical studies. The ATCase encoding cDNA sequence (PYRB gene) shows an open reading frame (ORF) of 1173 by coding for 390 amino acids. The cDNA encoding OPRTase-OMPdecase (PYRE-F gene) shows an ORF of 1431 by coding for 476 amino acids. Computer analysis of the deduced amino acid sequences of both cDNAs shows the expected high similarity with the ATCase, ornithine transcarbamylase (OTCase; EC 2.1.3.3), OPRTase and OMPdecase families. This heterospecific cloning approach increases our understanding of the genetic organization and interspecific functional conservation of the pyrimidine biosynthetic pathway and underlines its usefulness as a model for evolutionary studies.
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  • 82
    ISSN: 1617-4623
    Keywords: HAP3 ; Saccharomyces cerevisiae ; Kluyveromyces lactis ; Zinc finger ; Carbon source regulation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Kluyveromyces lactis homologue of the Saccharomyces cerevisiae HAP3 gene was isolated by functional complementation of the respiratory-deficient phenotype of the S. cerevisiae hap3::HIS4 strain SHY40. The KlHAP3 gene encodes a protein of 205 amino acids, of which the central B-domain of 90 residues is highly homologous to HAP3 counterparts of S. cerevisiae and higher eukaryotes. The protein contains a novel 4-cysteine zinc-finger motif and we propose by analogy that all other homologous HAP3 proteins contain the same motif, with the position containing the third cysteine being occupied by a serine residue. In contrast to the situation in S. cerevisiae, disruption of the KlHAP3 gene in K. lactis does not result in a respiratory-deficient phenotype and the growth of the null strain is indistinguishable from wild type. There is also no effect on the expression of the carbon source-regulated KlCYC1 gene, suggesting either a different role for the HAP2/3/4 complex, or the existence of a different mechanism of carbon source regulation. Sequence verification of the S. cerevisiae HAP3 locus reveals that, just as in K. lactis, a long open reading frame (ORF) is present upstream of the HAP3 gene. These highly homologous ORFs are predicted to have at least eight membrane-spanning fragments, but do not show significant homology to any known sequence present in databases. The ScORFX gene is transcribed in the opposite direction to ScHAP3, but, in contrast to an earlier report by Hahn et al. (1988), the transcripts of the two genes do not overlap. The model proposed by these authors, in which the ScHAP3 gene is regulated by an anti-sense non-coding mRNA, is therefore not correct.
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  • 83
    ISSN: 1617-4623
    Keywords: Phosphorylase kinase γ gene ; Kinase ; Drosophila ; Maternal effect
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Partial and total loss of function mutant alleles of a putative Drosophila homologue (DPhK-γ) of the vertebrate phosphorylase kinase γ-subunit gene have been isolated. DPhK-γ is required in early embryonic processes, such as gastrulation and mesoderm formation; however, defects in these processes are seen only when both the maternal and zygotic components of DPhK-γ expression are eliminated. Loss of zygotic expression alone does not appear to affect normal embryonic and larval development; some pupal lethality is observed but the majority of mutant animals eclose as adults. Many of these adults show defects in their leg musculature (e.g. missing and degenerating muscles), in addition to exhibiting melanised “tumours” on their leg joints. Loss of only the maternal component has no obvious phenotypic consequences. The DPhKγ gene has been cloned and sequenced. It has an open reading frame (ORF) of 1680 by encoding a 560 amino acid protein. The predicted amino acid sequence of DPhK-γ has two conserved domains, the catalytic kinase and calmodulin-binding domains, separated by a linker sequence. The amino acid sequence of DPhK-γ is homologous to that of mammalian PhK-γ proteins but differs in the length and amino acid composition of its linker sequence. The expression of DPhK-γ mRNA is developmentally regulated. We discuss the implications of these observations.
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  • 84
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    Molecular genetics and genomics 245 (1994), S. 686-693 
    ISSN: 1617-4623
    Keywords: Yeast ; Saccharomyces cerevisiae ; Poly(ADP-ribose) polymerase ; DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The coding sequence for human poly(ADP-ribose) polymerase was expressed inducibly in Saccharomyces cerevisiae from a low-copy-number plasmid vector. Cell free extracts of induced cells had poly(ADPribose) polymerase activity when assayed under standard conditions; activity could not be detected in non-induced cell extracts. Induced cells formed poly(ADP-ribose) in vivo, and levels of these polymers increased when cells were treated with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The cytotoxicity of this agent was increased in induced cells, and in vivo labelling with [3H]adenine further decreased their viability. Increased levels of poly(ADP-ribose) found in cells treated with the alkylating agent were not accompanied by lowering of the NAD concentration.
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  • 85
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; ts mutant ; Recovery ; HTR1 ; MCS1/SSD1
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    Notes: Abstract A new temperature-sensitive mutant of Saccharomyces cerevisiae was isolated. Arrested cells grown at the nonpermissive temperature were of dumb-bell shape and contained large vacuoles. A DNA fragment was cloned based on its ability to complement this temperature sensitivity. The HTR1 gene encodes a putative protein of 93 kDa without significant homology to any known proteins. The gene was mapped between ade5 and lys5 on the left arm of chromosome VII. The phenotype of the gene disruptant appeared to be strain-specific; disruption of the gene in strain W303 caused the cells to become temperature sensitive. The arrested phenotype here was similar to that of the original is mutant and cells in G2/M phase predominated at high temperature. Another disruptant in a strain YPH background grew slowly at high temperature due to slow progression through G2/M phase, and morphologically abnormal (elongated) cells accumulated. A single-copy suppressor that alleviated the temperature-sensitive defects in both strains was identified as MCS1/SSD1. The wild-type strains W303 and YPH are known to carry defective MCS1/SSD1 alleles; hence HTR1 may function redundantly with MCS1/SSD1 to suppress the temperature-sensitive phenotypes. In addition, based on a halo bioassay, the disruptant strains appeared to be defective in recovery from, or adaptive response to G1 arrest mediated by mating pheromone, even at the permissive temperature. Thus the gene has at least two functions and is designated HTR1 (required for high temperature growth and recovery from G1 arrest induced by mating pheromone).
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  • 86
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Cell cycle ; Bud site selection ; Guanine exchange factor ; Ras
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Guanine Exchange Factor (GEF) activity for Ras proteins has been associated with a conserved domain in Cdc25p, Sdc25p in Saccharomyces cerevisiae and several other proteins recently found in other eukaryotes. We have assessed the structure-function relationships between three different members of this family in S. cerevisiae, Cdc25p, Sdc25p and Bud5p. Cdc25p controls the Ras pathway, whereas Bud5p controls bud site localization. We demonstrate that the GEF domain of Sdc25p is closely related to that of Cdc25p. We first constructed a thermosensitive allele of SDC25 by specifically altering amino acid positions known to be changed in the cdc25-1 mutation. Secondly, we constructed three chimeric genes from CDC25 and SDC25, the products of which are as active in the Ras pathway as are the wild-type proteins. In contrast, similar chimeras made between CDC25 and BUD5 lead to proteins that are inactive both in the Ras and budding control pathways. This difference in the ability of chimeric proteins to retain activity allows us to define two subclasses of structurally different GEFs: Cdc25p and Sdc25p are Ras-specific GEFs, and Bud5p is a putative GEF for the Rsr1/Bud1 Rap-like protein.
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  • 87
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    Molecular genetics and genomics 245 (1994), S. 323-333 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; mRNA decay Poly(A) tail ; Ty transposition ; SSM4 gene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Decay rates of mRNAs depend on many elements and among these, the role of the poly(A) tail is now well established. In the yeast Saccharomyces cerevisiae, thermosensitive mutations in two genes, RNA14 and RNA15, result in mRNAs having shorter poly(A) tails and reduced half-life. To identify other components interacting in the same process, we have used a genetic approach to isolate mutations that suppress the thermosensitivity of an rna14 mutant strain. Mutations in a single locus, named SSM4, not only suppress the cell growth phenotype but also the mRNA instability and extend the short mRNA poly(A) tails. The frequency of appearance and the recessive nature of these mutations suggested that the suppressor effect was probably due to a loss of function. We failed to clone the SSM4 gene directly by complementation, owing to its absence from gene banks; it later emerged that the gene is toxic to Escherichia coli, but we have nevertheless been able to clone the SSM4 sequence by Ty element transposition tagging. Disruption of the SSM4 gene does not affect cell viability and suppresses the rna14 mutant phenotypes. The protein encoded by the SSM4 gene has a calculated molecular mass of 151 kDa and does not contain any known motif or show homology with known proteins. The toxicity of the SSM4 gene in E. coli suggests that a direct biochemical activity is associated with the corresponding protein.
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  • 88
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    Molecular genetics and genomics 242 (1994), S. 257-262 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Protein phosphatase ; Ras-cAMP pathway ; DIS2S1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Saccharomyces cerevisiae DIS2S1/GLC7 gene encodes a type 1 protein phosphatase indispensable for cell proliferation. We found that introduction of a multicopy DIS2S1 plasmid impaired growth of cells with reduced activity of the cAMP-dependent protein kinase. In order to understand further the interaction between the two enzymes, a temperature-sensitive mutation in the DIS2S1 gene was isolated. The mutant accumulated less glycogen than wild type at the permissive temperature, indicating that activity of the Dis2s1 protein phosphatase is attenuated by the mutation. Furthermore, the dis2s1 ts mutation was shown to be suppressed by a multicopy plasmid harboring PDE2, a gene for cAMP phosphodiesterase. These results indicate that the Ras-cAMP pathway interacts genetically with the DIS2S1/GLC7 gene.
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  • 89
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Transcriptional activator ; AP-1 ; Stress response
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The Saccharomyces cerevisiae YAP2 gene encoding an AP-1-like transcriptional activator protein was cloned by selection for genes that confer pleiotropic drug resistance when present in high copy number. The novel YAP2 gene encodes a protein of 45827 daltons and is homologous in part to a known transcriptional activator protein encoded by YAP1/PDR4/SNQ3/PAR1. Homology was found only in both terminal regions. The N-terminal portion contains a region rich in basic amino acids, followed by a “leucine zipper” motif. Overexpression of YAP2 led to the induction of expression of an AP-1 recognition element (ARE)-dependent promoter. The yap1 disruptant has been shown to be sensitive to H2O2. In this study, we demonstrated that the yap1 disruptant is also unable to grow in medium containing 150 μM cadmium, whereas the yap2 disruptant exhibited no significant phenotypes. However, YAP2 in high copy number did suppress cadmium sensitivity, but not H2O2 sensitivity of the yap1 disruptant. YAP1 was able to mediate both cadmium- and H2O2-induced transcriptional activation of an ARE-dependent promoter. A high-copy-number plasmid bearing YAP2 mediated cadmium-induced transcriptional activation of this promoter. The inductions were prevented by the antioxidant N-acetyl-l-cysteine.
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  • 90
    ISSN: 1617-4623
    Keywords: DNA polymerases ε and δ ; 3′ → 5′ Exonuclease ; Replication errors ; Spontaneous mutations ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract DNA polymerases II (ε) and III(δ) are the only nuclear DNA polymerases known to possess an intrinsic 3′ → 5′ exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase δ and ε 3′ → 5′ exonuclease-deficient mutants, pol3-01 and pol2-4, respectively. pol3-01 and pol2-4 increased spontaneous mutation rates by factors of the order of 102 and 101, respectively, measured as URA3 forward mutation and his7-2 reversion. Surprisingly, a double mutant pol2-4 pol3-01 haploid was inviable. This was probably due to accumulation of unedited errors, since a pol2-4/pol2-4 pol3-01/pol3-01 diploid was viable, with the spontaneous his7-2 reversion rate increased by about 2 × 103-fold. Analysis of mutation rates of double mutants indicated that the 3′ → 5′ exonucleases of DNA polymerases δ and ε can act competitively and that, like the 3′ → 5′ exonuclease of DNA polymerase δ the 3′ → 5′ exonuclease of DNA polymerase ε acts in series with the PMS1 mismatch correction system. Mutational spectra at a URA3 gene placed in both orientations near to a defined replication origin provided evidence that the 3′ → 5′ exonucleases of DNA polymerases δ and ε act on opposite DNA strands, but were in sufficient to distinguish conclusively between different models of DNA replication.
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  • 91
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    Molecular genetics and genomics 243 (1994), S. 253-260 
    ISSN: 1617-4623
    Keywords: Recombinant DNA ; Saccharomyces cerevisiae ; Endo-β-glucanase ; Endo-xylanase ; Heterologous expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have developed a method for fast and efficient isolation of enzyme genes from filamentous fungi by combining the ability of Saccharomyces cerevisiae to express heterologous genes with the utilisation of sensitive and reliable enzyme assays. A cDNA library from the fungus Humicola insolens was constructed in a S. cerevisiae/Escherichia coli shuttle vector in E. coli. Sub-pools of the library were subsequently screened for enzyme activity in S. cerevisiae. More than 130 clones were identified as positive in either an endo-β-glucanase or an endo-xylanase assay. Based on a partial characterization of the DNA sequence of the individual clones, they could be grouped into five distinct types of endo-β-glucanases and three types of endo-xylanases. A representative cDNA from each type was sub-cloned in an Aspergillus vector and expressed in A. oryzae. The new cloning method may be an important alternative to traditional cloning methods based on amino acid sequence information.
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  • 92
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    Molecular genetics and genomics 243 (1994), S. 308-314 
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Integrative plasmids ; Recombinational structures ; UV irradiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The nature of UV-induced pre-recombinational structures was studied using transformation of Saccharomyces cerevisiae cells with non-replicative plasmids. Transformation by double-stranded plasmids irradiated with UV was stimulated up to 50-fold, and both plasmid integration and conversion of the mutated chromosomal selective gene were found to be equally increased. The stimulation observed with such ‘totally’ irradiated plasmids was not found with plasmids bearing lesions in only one strand. This effect is attributed to the formation by excision repair of recombinogenic structures consisting of a pyrimidine dimer opposite a gap. When single-stranded integrative plasmids were irradiated, their transforming potential was decreased but the proportion of transformants that arose by gene conversion, rather than by plasmid integration, was increased from 8% to 49% as a function of the UV dose. Possible reasons why single-strand UV lesions favour gene conversion are discussed.
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  • 93
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; Yeast Catabolite repression ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Maltose utilization in yeast requires the presence of any one of the five unlinked, homologous MAL loci. Transcription of the two structural genes MALT (permease) and MALS (maltase) is induced by maltose and catabolite-repressed by glucose. MAL6T and MAL6S share a common 5′ intergenic sequence; deletion studies within this sequence revealed a bi-directionally functioning upstream activation sequence (UASM) consisting of four 11bp homologous sites. Activation of these sites by the MALR protein results in the coordinate expression of MAL6T and MAL6S. The basal promoter activates MALS expression to a greater extent than MALT and is located in a region that overlaps UASM. Deletion of several subsites within the UASM has an asymmetric effect on MAL gene expression, having a greater affect on MALT than on MALS. Catabolite repression of MAL6T and MAL6S by glucose is controlled at several levels. Using disruption mutants, the positively acting MAL1R protein was also found to play a role in catabolite repression of MAL6T and MAL6S.
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  • 94
    ISSN: 1617-4623
    Keywords: Saccharomyces cerevisiae ; RPK1 gene ; Protein kinase ; DNA replication ; Initiation of mitosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report here the sequence of RPK1 (for Regulatory cell Proliferation Kinase), a new Saccharomyces cerevisiae gene coding for a protein with sequence similarities to serine/threonine protein kinases. The protein sequence of 764 amino acids includes an amino-terminal domain (residues 1–410), which may be involved in regulation of the kinase domain (residues 411–764). The catalytic domain of Rpkl is not closely related to other known yeast protein kinases but exhibits strong homology to a newly discovered group of mammalian kinases (PYT, TTK, esk) with serine/threonine/tyrosine kinase activity. Null alleles of RPK1 are lethal and thus this gene belongs to the small group of yeast protein kinase genes that are essential for cell growth. In addition, eliminating the expression of RPK1 gives rise to the accumulation of non-viable cells with less than a 1 N DNA content suggesting that cells proceed into mitosis without completion of DNA synthesis. Therefore, the Rpkt kinase may function in a checkpoint control which couples DNA replication to mitosis. The level of the RPK1 transcript is extremely low and constant throughout the mitotic cycle. However it is regulated during cellular differentiation, being decreased in α-factor-treated a cells and increased late in meiosis in a/α diploids. Taken together, our results suggest that Rpk1 is involved in a pathway that coordinates cell proliferation and differentiation.
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  • 95
    ISSN: 1573-6857
    Keywords: Drosophila ; transposable elements ; hybrid dysgenesis ; transcriptional regulation ; horizontal transfer
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  • 96
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    Genetica 93 (1994), S. 149-160 
    ISSN: 1573-6857
    Keywords: environment ; genome ; stress ; transposable elements ; Drosophila
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The action of stresses on the genome can be considered as responses of cells or organisms to external aggressions. Stress factors are of environmental origin (climatic or trophic) or of genomic nature (introduction of foreign genetic material, for example). In both cases, important perturbations can occur and modify hereditary potentialities, creating new combinations compatible with survival; such a situation may increase the variability of the genome, and allow evolutive processes to take place. The behavior of transposable elements under stress conditions is thus of particular interest, since these sequences are sources of mutations and therefore of genetic variability; they may play an important role in population adaptation. The survey of the available experimental results suggests that, although some examples of mutations and transposable elements movements induced by external factors are clearly described, environmental injuries or introduction of foreign material into a genome are not systematically followed by drastic genomic changes.
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  • 97
    ISSN: 1573-6857
    Keywords: Adh ; Adh-dup ; Drosophila ; molecular clock ; nucleotide substitution rates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The homologous genomic region that contains two paralogous genes,Adh andAdh-dup, was compared in severalDrosophila species. Sequences were analyzed as follows: a) At the nucleotide level, Ka and Ks values were determined for each pair of species. Ka-Adh and Ka-Adh-dup are not significantly different. However, Ks-Adh values are significantly lower than Ks-Adh-dup, which are more variable. In agreement with other reports, lower Ks values forAdh correlate with a high level of gene expression and relatively high percentage of G+C content in the third codon position, while the opposite applies toAdh-dup. b) At the protein level, amino acid comparisons reveal conserved regions shared by ADH and ADH-DUP, which have been assigned to known functional domains. Key residues for dehydrogenasic function are also found in ADH-DUP, thus pointing to a dehydrogenase activity for ADH-DUP, albeit very different from that of ADH.
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  • 98
    ISSN: 1573-6857
    Keywords: Drosophila ; esterase 6 ; reproduction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previous studies have shown that the esterase 6 (EST6) enzyme ofD. melanogaster is mainly produced in the sperm ejaculatory duct of the adult male and comparisons of wild-type males with laboratory null mutants have suggested that the enzyme plays a role in reproductive fitness. In this study we have compared 18 field-derived lines each isoallelic forEst6 for differences in five components of male reproductive fitness. No consistent fitness differences were found among lines differing in respect of the two major allozyme classes EST6-F and EST6-S, despite other evidence that these two classes are not selectively equivalent in the field. However, differences in reproductive fitness were found among lines differing in the minor mobility variants that segregate within EST6-F and EST6-S. A failure to distinguish among these minor forms may explain the discrepancies in previous studies on the effects of the major EST6 allozymes on reproductive fitness. The most significant associations we have found between EST6 and reproductive fitness were due to variation in EST6 activity levels. Male EST6 activity levels were found to be positively correlated with their time to first mating, negatively correlated with the numbers of eggs laid and progeny produced by their mates, and negatively correlated with the frequency with which their mates remate. We conclude that some EST6 variants differ in components of male reproductive fitness operative in laboratory cultures. However, the evidence for fitness differences is stronger for variants affecting the amount, rather than the structure of the enzyme, and the direction of the differences varies between some of the fitness components tested.
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  • 99
    ISSN: 1573-6857
    Keywords: Drosophila ; glutamine synthetase ; isozyme functional specialization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The glutamine synthetase isozymes ofDrosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. InDrosophila melanogaster two GS. isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the twoDrosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.
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  • 100
    Electronic Resource
    Electronic Resource
    Springer
    World journal of microbiology and biotechnology 10 (1994), S. 572-575 
    ISSN: 1573-0972
    Keywords: Growth inhibition ; L-lysine ε-aminotransferase ; nitrogen limitation ; α-oxoadipic acid ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Lysine added to grain mashes under nitrogen-limiting conditions (as in most industrial fermentations) inhibited growth of Saccharomyces cerevisiae. This inhibition was relieved by raising the assimilable nitrogen content. Lysine-induced inhibition is not mediated through accumulation of α-oxoadipic acid, an intermediate of lysine metabolism which accumulates by a back up of intermediates in de novo synthesis. Lysine degradation is regulated by the synthesis of L-lysine ε-aminotransferase, an enzyme that catalyses the first step in one of three possible routes of lysine degradation (not previously reported in S. cerevisiae). Synthesis is repressed under nitrogenlimiting conditions, but derepressed when excess assimilable nitrogen is available. Derepression results in degradation of lysine and decreases inhibitory effects on growth. The toxic compound appears to be lysine itself.
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