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  • 101
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 8 (1987), S. 135-150 
    ISSN: 0192-253X
    Keywords: mouse ; NADP-isocitrate dehydrogenase ; electrophoresis ; gene regulation ; allele-specific expression ; heart ; kidney ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The murine “housekeeping” enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C. 1.1.1.42) (genetic locus:Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1a allele, but did not vary as a function of sex.Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-hornodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1a allele.While cytosolic NADP-IDH is a “housekeeping” enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.
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  • 102
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 8 (1987), S. 179-185 
    ISSN: 0192-253X
    Keywords: differentiation ; melanogenesis ; tyrosinase ; albino ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Albino mutation in animals blocks pigmentation owing to a deficiency in tyrosinase, although it does not affect the differentiation of colorless melanocytes from the neural crest. In the albino Japanese quail (al, sex-linked), it was demonstrated that morphologically normal melanocytes differentiated from neural crest cells in culture and that these cells contained unmelanized melanosomes as expected for the mutant cells. The mutant melanocytes, however, were shown to exhibit tyrosinase activity in the Golgi-endoplasmic reticulum-lysosome region and in the Golgi vesicles. Our results seem to indicate that the mutation at the al locus affects the transport of tyrosinase from the Golgi area to melanosomes.
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  • 103
    ISSN: 0192-253X
    Keywords: lactate dehydrogenase ; spermatogenesis ; multigene enzyme family ; somatic cell hybrids ; gene mapping ; evolution ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: From the data presented in this report, the human LDHC gene locus is assigned to chromosome 11. Three genes determine lactate dehydrogenase (LDH) in man. LDHA and LDHB are expressed in most somatic tissues, while expression of LDHC is confined to the germinal epithelium of the testes. A human LDHC cDNA clone was used as a probe to analyze genomic DNA from rodent/human somatic cell hybrids. The pattern of bands with LDHC hybridization is easily distinguished from the pattern detected by LDHA hybridization, and the LDHC probe is specific for testis mRNA.The structural gene LDHA has been previously assigned to human chromosome 11, while LDHB maps to chromosome 12. Studies of pigeon LDH have shown tight linkage between LDHB and LDHC leading to the expectation that these genes would be syntenic in man. However, the data presented in this paper show conclusively that LDHC is syntenic with LDHA on human chromosome 11.The terminology for LDH genes LDHA, LDHB, and LDHC is equivalent to Ldhl, Ldh2, and Ldh3, respectively.
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  • 104
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 8 (1987), S. 295-304 
    ISSN: 0192-253X
    Keywords: sequence ; cDNA ; fetal pig ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A cDNA clone of porcine alpha1 acid glycoprotein (α1AGP) has been isolated and sequenced. Sequence homologies between porcine, human, and rat indicate that porcine α1AGP is similar in structure to the rat and human proteins. RNA blots from days 40, 60, 80, and 110 fetal, newborn, and adult livers showed that α1AGP mRNA is relatively abundant throughout fetal development, particularly at the later stages and in the newborn; there is a rapid decline in abundance following birth. From birth to 3 days of age, there is a three- to four-fold decline in abundance, and α1AGP mRNA is approximately 100 times less abundant in the adult liver than in that of perinatal pigs. Southern blots showed that α1AGP is probably a single-copy gene. The isolation of a cloned cDNA for porcine α1AGP provides a tool to investigate the molecular mechanisms involved in the developmental regulation of the gene and to correlate changes in gene expression during development with fetal growth and well being.
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  • 105
    ISSN: 0192-253X
    Keywords: mental retardation ; Down syndrome ; cholinergic neurons ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this study, we examined the neurochemical profiles of selected brain regions (cerebral hemispheres, diencephalon/brainstem) in fetal (day 14 to 18 gestation) trisomy 19 (Ts19) mice. The neurochemical characteristics we observed in Ts19 mice were quite different from those we observed previously in Ts16 mice. Choline acetyltransferase (ChAT) activity was reduced significantly in the cerebral hemispheres, but not in the brainstem/diencephalon, of the fetal Ts19 mouse brain, suggesting a selective vulnerability of telencephalic cholinergic neurons. Additionally, the activity of glutamic acid decarboxylase (GAD) was reduced significantly in both hemispheres and diencephalon/brainstem of late gestation Ts19 fetuses, suggesting a selective vulnerability of GABAergic neurons as well. While the levels of catecholaminergic and dopaminergic markers were reduced significantly at late gestational ages, the relative rate of turnover of dopamine (DA), measured by the ratio of DOPAC/DA, was elevated significantly in Ts19 mice. Neither reduction in the thickness of various cellular zones of the cerebral cortex nor reduced cell density of the cerebral cortex accounts for the alterations in neurochemical parameters observed in Ts19 mice. These results suggest that the effects of the triplication of specific genes on the respective chromosomes, rather than a generalized disruption of developmental homeostasis resulting from extra chromosomal material, may produce selective alterations in neurochemical and neuroanatomical markers observed in these two mouse trisomies.
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  • 106
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 8 (1987), S. 305-320 
    ISSN: 0192-253X
    Keywords: maize ; chlorophyll-deficient mutants ; high-chlorophyll-fluorescent mutants ; albino mutants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Although a wide range of mutations in the nuclear genome also affect chloroplast biogenesis, their pleiotropic nature often limits their use in studying nuclear genes that regulate or facilitate chloroplast development. However, many mutations that cause a high-chlorophyll-fluorescent (hcf) phenotype exhibit limited pleiotrophy, causing the loss of functionally related sets of chloroplast polypeptides. Several hcf mutations are described that result in the loss of one specific protein complex from the thylakoid membrane. Chlorplast and cytosolic mRNAs coding for component polypeptides of the missing complex are unaffected in the mutants, suggesting that each mutation disrupts some process in the synthesis and assembly of the missing complex. Another hcf mutation causes both the loss of three protein complexes and grossly abnormal thylakoid membrane structures. The primary effect of this mutation might be in the assembly of thylakoid membranes or in the stable accumulation of the three protein complexes. Two other hcf mutations are more pleiotropic. Hcf*-38 causes a quantitative reduction of many chloroplast proteins and a reduction of some chloroplast RNAs, including several splicing intermediates. Hcf*-7 causes a major reduction of all chloroplast-encoded proteins examined. The range of pleiotropic effects of hcf mutations indicates that the mutations identify nuclear genes whose products are involved in a number of different steps in chloroplast devclopment. Because some of the mutations described have been generated by transposon insertions, they can be cloned using the transposon to identify the mutant allele.
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  • 107
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 8 (1987), S. 389-403 
    ISSN: 0192-253X
    Keywords: nuclear mutations ; chloroplast assembly ; maize ; light ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The major chlorophyll a/b light harvesting complex (LHCII) of mesophyll chloroplasts is normally assembled late during chloroplast morphogenesis. LHCII occurs at greatly reduced levels in bundle sheath chloroplasts of maize. In order to understand the normal regulatory mechanisms we are examining nuclear maize mutants that alter either (1) the assembly timing or (2) the steady state level of LHCII in mature mesophyll thylakoids. We have found a delayed greening mutant, v24 (on chromosome arm 2L), that unmasks a second unlinked locus, Mof*, that can mediate LHCII assembly timing. The polypeptides of LHCII are encoded by the nuclear multigene cab family. We find that two alleles at Mof* regulate the steady state level of cab mRNA in parallel to their effect on LHCII assembly timing: The genotype Mof*-1 Mof*-1 v24 v24 corresponds to reduced cab mRNA and late LHCII assembly timing, while Mof*-2 Mof*-2 v24 v24 corresponds to reduced cab mRNA and late LHCII assembly timing. A second group of mutations (Oy-700, pg11 and pg12 reduces LHCII levels in mesophyll thylakoids. This is the first report that pg11 and pg12) reduce the LHCII of mesophyll thylakoids. The basis of pg11 and pg12 is unknown. Mutations at the Oy locus block the chlorophyll biosynthetic enzyme, protopor-phyrin IX Mg-chelatase. Heterozygotes of the codominant mutation Oy-700 with the normal allele (Oy) have reduced LHCII. We have defined genetic backgrounds that suppress and those that do not suppress the Oy-700 Oy phenotype under certain conditions: (1) reduced light intensities (200 μE cm-2 sec-1) and/or (2) plant maturity.
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  • 108
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 8 (1987), S. 475-493 
    ISSN: 0192-253X
    Keywords: methylation ; Adh1 ; Zea ; Arabidopsis ; transformed DNA ; CpG-rich islands ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Higher plant DNA is extensively methylated, but the two methylated sequences (CpG and CpNpG) show different characteristics. Using sequence analysis techniques, we demonstrate that while CpG methylation follows the existing models for cytosine methylation in animals, CpNpG methylation does not. Although there is evidence to support the suggestion that the low CpG frequency has arisen from deaminational conversion of 5-methylcytosine to thymidine, there appears to be no comparable conversion of 5-methylcytosine in the CpNpG configuration. It therefore appears that between the evolution of CpG and CpNpG cytosine methylation systems, a mechanism evolved for the correction of C→T conversion, probably using the methylated strand to direct the repair in the correct direction.
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  • 109
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988), S. 13-22 
    ISSN: 0192-253X
    Keywords: ontogeny ; lethality ; gene expression ; mRNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cadmium is a toxic metal that induces the expression of metallothionein genes in many tissues and that binds avidly to metallothionein, a soluble transition metal binding protein. The present study examined the temporal pattern and magnitude of accumulation of metallothionein mRNA in liver of C57BL/6J mice of various ages treated with cadmium. In adult female mice, accumulation was dependent on the dosage level of cadmium and related to the concentration of this metal in liver. The accumulation of metallothionein mRNA in liver depended on age at exposure to cadmium. Intraperitoneal administration of 2 mg of cadmium per kg provoked small increases (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. In contrast, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver with maximum increases occurring 3 to 4 hr after treatment. Because similar patterns for the accumulation of cadmium of liver were found in 7-, 28-, and 56-day-old mice, observed age-dependent differences in induction of metallothionein mRNA in liver were probably not due to differences in the accumulation of cadmium in this organ. Taken together, these data suggest that tissue-specific factors controlling the expression of metallothionein genes may account for developmental variation in the inducibility of these genes by cadmium. Ontogenic variation in accumulation of metallothionein mRNA after cadmium treatment may be a factor in developmental variation in the acute lethality of cadmium in C57BL/6J mice.
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  • 110
    ISSN: 0192-253X
    Keywords: Daucus carota ; auxin ; gene expression ; mutant ; filtration-enrichment procedure ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cell cultures of the carrot Daucus carota are a useful experimental system for studying the genetic regulation of plant embryogenesis. A modified filtration-enrichment procedure was used to isolate 21 temperature-sensitive variants in somatic embryogenesis; the variants display normal embryo development at the permissive temperature (24°C) and altered development at the restrictive temperature (33°C). Temperature-shift experiments were performed on these variants to determine the timing of gene action for the putative temperature-sensitive alleles. According to their phenotypes at the restrictive temperature, these variants can be divided into six classes: No Growth, Callus Proliferation, Globularstage Block, Oblong-stage Block, Lateral Growth, and Root Formation. Although many variants exhibit lengthy temperature-sensitive periods, the temperature sensitivity of some variants is restricted to one or two embryonic stages. These results plus those in the literature are incorporated into a preliminary model concerning the genetic regulation of carrot embryogenesis.
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  • 111
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 112
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988), S. 155-165 
    ISSN: 0192-253X
    Keywords: embryonic lethal ; agouti locus ; trophectoderm ; inner cell mass ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The tissue specificity of the lethal yellow mutant was investigated by separation of blastocyst tissues. Embryos from experimental (Ay/ae × Ay/ae) and control (ae/ae × Ay/ae) crosses of the AG/CamPa inbred strain were recovered at 3.5 days post coitum, cultured for 24 hours, and then mechanically dissected into the component tissues of the blastocyst, the inner cell mass (ICM), and trophectoderm. These fragments were then cultured separately, with or without a feeder layer of inactivated fibroblasts, for an additional 3-5 days. Comparisons between experimental and control crosses indicated that the lethal Ay/Ay embryos were among the blastocysts successfully dissected but that both the ICM and trophectoderm from lethal embryos failed to develop further in vitro, eithal with or without feeders.With retrospective identification of the lethal embryos, it was found that at 4.5 days, after 1 day of culture, they had formed morphologically normal blastocysts but were frequently more fragile upon dissection and had smaller ICMs. Although none had hatched from the zona pellucida, some had ruptured it and were halfway out. With culture, lethal ICMs showed no development, and lethal trophectoderm usually attached but showed very limited outgrowth. Thus, no rescue of lethal tissue was shown with dissection and in vitro culture, and results are consistent with the gene affecting both tissues of the late blastocyst.
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  • 113
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 114
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988), S. 715-732 
    ISSN: 0192-253X
    Keywords: Regulator of postbithorax ; homeosis ; pattern formation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Genetic analysis has shown that the gap segmentation gene hunchback (hb) is a member of the genetic hierarchy involved in pattern formation in Drosophila. To identify the hb gene, we have mapped the position of hb mutant breakpoints within a chromosomal walk of the 85A region by genomic Southern blots and determined the transcription pattern of DNA from the walk. We detect a single gene within the domain defined by breakpoint mapping. We conclude that we have identified the hunchback gene because three mutations that inactivate hb physically interrupt or delete this gene. Northern analysis shows that the hb gene gives rise to at least five overlapping transcripts ranging in length from 2.6 to 3.5 kilobases. S1 nuclease and primer extension experiments demonstrate that the gene employs two promoters and three polyadenylation sites. The two hb promoters have different temporal specificities. Transcripts arising from the upstream promoter are detected from 0-12 hours of embryogenesis as well as in adult female and male RNA preparations. Transcripts arising from the downstream promoter accumulate only from 0-6 hours of embryogenesis. During the syncytial blastoderm stage, transcripts from the hb gene accumulate over a broad anterior and a narrow posterior domain. This pattern sharpens during the late blastoderm/early gastrula stage to produce an embryo with two stripes of hybridization anterior and one stripe posterior. Later, hb transcripts are detected within the ventral hypoderm in extended germ band stage embryos.
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  • 115
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988), S. 733-741 
    ISSN: 0192-253X
    Keywords: Phycomyces blakesleeanus ; developmental mutants ; phorogenesis ; sexual reproduction ; light ; carotene ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The mycelium of the fungus Phycomyces. essentially a giant multinucleate cell, produces two kinds of asexual reproductive structures, called macrophores and microphores, and a succession of structures for sexual reproduction. Following the treatment of spores with N-methyl-N′ -nitro-N-nitrosoguanidine, conditional imb mutants have been isolated that form no macrophores at 26°C, but do at 14°C. At the restrictive temperature, few imb mutants (2 of 13) develop microphores, and none is able to complete the sexual cycle. This suggests that genes responsible for macrophorogenesis are involved in microphorogenesis and in sexual development as well. Light reduces macrophorogenesis and totally abolishes microphorogenesis in the wild type under the conditions of our experiments. These photomorphogenetic effects require the normal function of genes madA and madB, which are responsible for phototropism. Light inhibits microphorogenesis in the two imb mutants that form microphores at the restrictive temperature. Genetic alterations of carotenogenesis lead to an excess of microphores and a scarcity of macrophores in the dark, but they have little influence on vegetative reproduction in the light.
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  • 116
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 9 (1988), S. 215-226 
    ISSN: 0192-253X
    Keywords: cAMP ; cGMP ; chemotaxis ; mutant ; desensitization ; receptor ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The functional interaction of surface cAMP receptors with effector enzymes via G-proteins was investigated in Dictyostelium discoideum. Several experimental conditions were used to investigate signal transduction, such as reduced temperatures, use of down-regulated cells and of mutants. The results are presented as a model describing the complex interaction between multiple forms of the surface cAMP receptor and different G-proteins that are responsible for the generation of the second messengers, cAMP, cGMP, InsP3 and Ca2+.
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  • 117
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    Developmental Genetics 9 (1988), S. 227-235 
    ISSN: 0192-253X
    Keywords: receptors ; transmembrane signalling ; Dictyostelium ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Using antibodies specific for the 3′, 5′-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened γgtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: (1) β-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, (2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, (3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, (4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and (5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA.The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.
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  • 118
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    Developmental Genetics 9 (1988), S. 247-258 
    ISSN: 0192-253X
    Keywords: protein evolution ; lower eukaryote ; differentiation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The cAMP-dependent protein kinase (cAK) from Dictyostelium discoideum is an enzyme composed of one catalytic and one regulatory subunit. Upon binding of cAMP, the holoenzyme dissociates to liberate free active catalytic subunits. The cAK is developmentally regulated, ranging from very little activity in vegetative cells to maximal expression in postaggregative cells. Although there is no immunological cross-reaction between the subunits of cAKs from Dictyostelium and from other organisms, they share several biochemical properties. A complete cDNA for the regulatory subunit has been cloned and sequenced. Only one copy of the gene for the regulatory subunit is present per haploid genome. On the basis of the comparison of the structure of the cAK from Dictyostelium with its counterparts in yeast and higher eukaryotes, we propose a model for the evolution of cyclic-nucleotide-binding proteins.
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  • 119
    ISSN: 0192-253X
    Keywords: gene regulation ; immunoblotting ; rapid-developing variants ; molecular cloning ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Several lines of evidence indicate that cAMP modulates developmental gene activity via cell-surface receptors. We describe here a novel cAMP receptor, CABP1, whose properties are consistent with the idea that this protein is involved in gene regulation. Firstly, immunological techniques using anti-CABP1 antibodies as probes showed that this cAMP receptor can be detected on the surface of developing cells. Secondly, there is a steady migration of CABP1 to the nucleus during development. Thirdly, some genetic variants exhibiting an altered pattern of development are found to possess modified CABP1. We also showed that CABP1 co-purifies with at least seven other polypeptides which share common epitopes with CABP1. Interestingly, four of the CABP1-related polypeptides can be detected on the cell surface as well as in the nucleus.
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  • 120
    ISSN: 0192-253X
    Keywords: transformation ; gene structure ; cAMP ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The cyclic nucleotide phosphodiesterase (phosphodiesterase) of Dictyostelium discoideum plays an essential role in development by hydrolyzing the cAMP used as a chemoattractant by aggregating cells. We have studied the biochemistry of the phosphodiesterase and a functionally related protein, the phosphodiesterase inhibitor protein, and have cloned the cognate genes. A 1.8-kb and a 2.2-kb mRNA are transcribed from the singlephosphodiesterase gene. The 2.2-kb mRNA comprises the majority of the phosphodiesterase mRNA found in differentiating cells and is transcribed only during development from a promoter at least 2.5 kb upstream of the translational start site. The 1.8-kb phosphodiesterase mRNA is detected at all stages of growth and development, is present at lower levels than the developmentally induced mRNA, and is transcribed from a site proximal to the protein-coding region. The phosphodiesterase gene contains a minimum of three exons, and a 2.3-kb intron, the longest yet reported for this organism. We have shown that the pds A. gene and fourfgd genes affect, the accumulation of the phosphodiesterase mRNAs, and we believe that these loci represent a significant portion of the genes regulating expression of the phosphodiesterase. The phosphodiesterase gene was introduced into cells by transformation and used as a tool to explore the effects of cAMP on the terminal stages of development. In cells expressing high levels of phosphodiesterase activity, final morphogenesis cannot be completed, and differentiated spore and stalk cells do not form. We interpret these results to support the hypothesis that cAMP plays an essential role in organizing cell movements in late development as well as in controlling the aggregation of cells in the initial phase of the developmental program.
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  • 121
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    Developmental Genetics 9 (1988), S. 259-265 
    ISSN: 0192-253X
    Keywords: G-proteins ; gene expression ; developmental regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have identified a two-member gene family in the Dictyostelium genome and have isolated corresponding cDNA or genomic DNA recombinant clones. Analyses of these DNA sequences predicted encoded proteins of ∼200 amino acids with ∼90% sequence identity to each other. These Dictyostelium proteins also share amino acids identity within the GTP-binding domains in the family of G-regulatory proteins involved in cellular regulation and transmembrane signalling. Additional structural similarities are seen with members of the ras supergene family, such as ras, ral, and rho. They are similar in size (usually ∼200 amino acids), possess four conserved domains involved in GTP interaction and are believed to be anchored in the membrane by fatty acid modification of a cysteine residue near the carboxy terminus. More extensive identity is observed with YPT1 and SEC4, two other members of this family of genes that are essential in yeast. The amino-terminal half of both Dictyostelium proteins is 70% identical in amino acid sequence to the YPT1 and SEC4 yeast proteins with less identity continuing through the remainder of the proteins. In addition these proteins terminate in two cysteine residues that are thought to be required for membrane anchorage.The two genes within this Dictyostelium family are organized differently in the genome and are differentially regulated during development. One gene is colinear in sequence with its mRNA in the protein coding region, whereas the other gene encodes a spliced mRNA. The intron-containing gene is associated with a developmentally regulated (AAC)-repeat sequence. Finally, we have shown that the expression of one of the genes is induced during development with kinetics similar to that of other (AAC)n-associated genes; conversely, the expression of the second gene is repressed at a similar developmental stage.
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  • 122
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    Developmental Genetics 9 (1988), S. 303-313 
    ISSN: 0192-253X
    Keywords: developmentally regulated cDNAs ; Dictyostelium discoideum gene sequences ; developmentally regulated proteins ; Dictyostelium spore proteins ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Similar to other stages in Dictyostelium development, spore germination is a particularly suitable model for studying the regulation of gene expression, because developmentally regulated changes in both protein and mRNA synthesis occur during the transition from dormant spore to amoeba. Spores are constitutively dormant and must be activated to germinate. Under the proper environmental conditions, spores germinate in a highly synchronous manner to give rise to individual amoebae that can then enter the vegetative growth phase. Protein synthesis is developmentally regulated during this process. Because protein synthesis is transcriptionally controlled during spore germination, the respective genes must be developmentally transcribed, and these can be isolated and analyzed. Three cDNA clones specific for mRNA developmentally regulated during spore germination have been characterized and used as probes to study mRNA accumulation and decay during spore germination. Because we are interested in defining the sequences of developmentally regulated genes that may relate to their regulation of transcription, we have sequenced the cDNAs and have isolated and sequenced their respective genomic clones. The sequences of the three gene families, their genomic organization, and their special structural features are described.
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  • 123
    ISSN: 0192-253X
    Keywords: ions ; developmental regulation ; receptor regulation ; developmental signals ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have analyzed the effects of the cAMP relay inhibitor, caffeine, and the receptor antagonist, adenosine, on the regulation of the cell-surface cAMP receptor in suspensionstarved Dictyostelium discoideum cells by measuring ammonium sulfate-stabilized binding of [3-H]cAMP to intact cells. When cells were starved in fast (230 r.p.m.) shaken suspension in 10 mM Na+/5 mM K+ phosphate buffer, pH 6.5, plus 1 mM CaCl2 and 2.5 mM MgCl2, and assayed for specific cAMP binding, receptor accumulation peaked at approximately 6 hours, reaching a maximum of 1.5 pmol cAMP bound/107 cells (saturation binding). Neither caffeine nor adenosine inhibited the accumulation of cAMP receptors. Similar results were obtained in caffeine-treated, slow shaken (90 r.p.m.) suspension cultures. These results suggest that starvation alone is sufficient stimulus to induce the cAMP receptor. We have also tested the effects of different buffer ionic compositions on the accumulation of cAMP receptors. Elevation of the monovalent ion concentration to 30-40 mM was found to significantly inhibit the induction of cAMP receptors.
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  • 124
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    Developmental Genetics 9 (1988), S. 279-292 
    ISSN: 0192-253X
    Keywords: Dictyostelium ; development ; cAMP ; cell-cell contact ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: cAMP and cell-cell contact are involved in the coordination of differentiation and morphogenesis in Dictyostelium discoideum. The experiments described in this paper establish a relationship between cAMP and cell-cell contact. Contact between Enterobacter aerogenes and aggregation-competent Dictyostelium amoebae and contact between Dictyostelium amoebae themselves results in the transient secretion of cAMP and an alteration in the amount of cAMP secreted in response to subsequent stimulation by cAMP, i.e., an alteration in magnitude of a cAMP relay response. The subsequent cAMP relay response can be enhanced or diminished depending upon the number of contacts formed and the concentration of cAMP present at the time of contact. Latex beads are capable of evoking cAMP secretion. However, the bead/amoebal contact is unable to alter the magnitude of a subsequent response to cAMP. This suggests that a nonspecific interaction via cell-cell contactelicits transient cAMP secretion in aggregation-competent Dictyostelium amoebae.The two responses to cell-cell contact are distinct from each other and distinct from the cAMP relay response. (1) The dose-response curves for the responses to Enterobacter contact are clearly different. (2) Contact with latex beads can elicit cAMP secretion but not alter the magnitude of a subsequent cAMP relay response. (3) The temperature dependences of the contact-induced responses and the cAMP relay response show that only the contact-induced cAMP secretion is inhibited at 12 and 15°C, while only the cAMP relay response is inhibited at 28°C.A 4-second application of cAMP at the time that contact is initiated enhances both contact-induced responses. Whether the relationship between these two developmental regulators is important for the regulation of Dictyostelium development has yet to be established.
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  • 125
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    Developmental Genetics 9 (1988), S. 327-335 
    ISSN: 0192-253X
    Keywords: gene regulation ; initiation of development ; slime mold ; transcription ; cycloheximide ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Several genes that are deactivated upon the initiation of development of Dictyostelium discoideum have been identified by differential screening of various cDNA libraries. These genes have in common a decrease in the steady-state levels of their corresponding mRNAs as development proceeds. When development was carried out in the absence of protein synthesis by inhibition with cycloheximide, the decrease in mRNA levels for most genes (V genes) was normal or slightly accelerated. However, for about 5% of the genes (H genes), cycloheximide caused an apparent induction of expression, as revealed by a slight or dramatic increase in mRNA levels instead of the normal decrease. This effect was due to inhibition of protein synthesis and not to cycloheximide per se. The induction was found to be due to an enhancement of the trascription rate; normal rates of transcription for the H genes were dependent upon continued protein synthesis during vegetative growth and during development. Thus, two general regulatory classes exist for deactivation of gene expression upon initiation of development, one dependent and one independent of protein synthesis. Models concerning the control of expression of these two classes of genes are discussed here. Analysis of expression of these genes in mutant strains that are aggregation-deficient has also been performed, and the results lead to subdivisions of the classes.
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  • 126
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    Developmental Genetics 9 (1988), S. 315-326 
    ISSN: 0192-253X
    Keywords: Dictyostelium ; growth ; development ; regulation ; discoidin I ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have previously shown that growing cells of Dictyostelium discoideum (strains NC4 and AX3) produce a soluble substance that accumulates in the medium in proportion to cell density; this substance regulates the production of certain proteins previously thought to be induced by starvation [Clarke et al., 1987]. We suggest the name PSF (prestarvation factor) for this substance. During growth, Dictyostelium cells monitor the relative concentrations of PSF and food bacteria. When PSF reaches a sufficiently high level relative to the concentration of bacteria, synthesis of PSF-regulated proteins is induced. We propose the name prestarvation response for this induction, which takes place in exponentially growing cells several generations before the food bacteria are depleted. We have explored the mechanism by which the food bacteria inhibit the response of Dictyostelium cells to PSF. We find that the bacteria do not inactivate PSF or inhibit its production; instead, they affect the ability of NC4 cells to detect PSF, possibly by binding to the same cell surface receptor. In the absence of bacteria, as during axenic growth of AX3 cells, the prestarvation response occurs at much lower cell densities, probably accounting for the presence of certain developmentally regulated mRNAs and proteins in axenic cultures.
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  • 127
    ISSN: 0192-253X
    Keywords: cAMP ; Ca2+ ; signal transduction ; cell surface receptor ; gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Elevated levels of cAMP are essential for the expression of many postaggregation prespore and prestalk mRNA species and for the suppression of some growth phase mRNAs. Here we review evidence that this regulation is mediated by cAMP interacting at the cell surface receptor. These effects of cAMP on gene expression can occur under conditions where the receptor-associated adenylate cyclase is inactivated and in concentrations that are consistent with receptor binding. A number of differences are noted in the mechanism by which cAMP regulates prespore and prestalk genes. Finally, evidence is reviewed for the role of a Ca2+-dependent signal transduction system in coupling the expression of some of the prespore mRNAs to the cAMP receptor. This signal transduction system does not appear to be involved in the expression of the cAMP-dependent prestalk gene.
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  • 128
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    Developmental Genetics 9 (1988), S. 337-350 
    ISSN: 0192-253X
    Keywords: Dictyostelium ; cAMP ; receptor ; gene regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have examined the expression of a cAMP pulse-repressed and two cAMP pulse-induced genes in response to cAMP and caffeine under a number of different physiological conditions, and in several classes of developmental mutants altered in cAMP-mediated signal transduction pathways. The data presented help characterize the mutants with regard to early gene expression. Analysis of the data indicates that full induction of the pulse-induced or repression of the pulse-repressed genes requires cycles of activation and adaptation of the cAMP receptor but does not require a rise in intracellular cAMP. Comparison of the results obtained between different mutant classes suggests that repression and activation of the two classes of genes can be uncoupled, implying that different intracellular mechanisms control these processes. In addition, we examined the effects of caffeine and show that it can induce pulse-induced mRNA accumulation in the absence of cAMP.
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  • 129
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    Developmental Genetics 9 (1988), S. 351-358 
    ISSN: 0192-253X
    Keywords: Dictyostelium ; diacylglycerol ; inositol trisphosphate ; developmetal regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In Dictyostelium, extracellular cAMP interacts specifically with cell-surface receptors to promote the accumulation of a variety of intracellular second messengers, such as, 3′-5′ cyclic adenosine monophosphate (cAMP) and 1,4,5 inositol trisphosphate (IP3). We and others have shown that activation of the cell-surface cAMP receptor can also modulate the expression of the Dictyostelium genome during development. In at least one instance, synthesis of intracellular cAMP is required for appropriate gene regulation. However, the induction of most cAMP-dependent gene expression can occur in the absence of receptor-mediated activation of adenylate cyclase and a consequent accumulation of intracellular cAMP. These results suggest that other intracellular second messengers produced in response to receptor activation may potentially act as signal transducers to modulate gene expression during development. In vertebrate cells, IP3 and diacylglycerol (DAG) are intracellular activators of specific protein kinases; they are produced in equimolar amounts by cleavage of phosphoinositol bisphosphate after a receptor-mediated activation of a membrane-bound phosphodiesterase. IP3 and, thus, by inference, diacylglycerol are synthesized in Dictyostelium as a response to cAMP interacting with its cell-surface receptor. Using defined conditions to inhibit the accumulation of extracellular cAMP, we have examined the effects of these compounds on the accumulation of extracellular cAMP, we have examined the effects of these compounds on the expression of genes that require cAMP for their maximal expression. Our results suggest that intracellular IP3 and DAG may in part mediate the action of extracellular cAMP on the expression of the Dictyostelium genome.
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  • 130
    ISSN: 0192-253X
    Keywords: Dictyostelium cell type markers ; PsA ; phosphatidylinosito ; D19 gene ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The nucleotide sequence of D19, a Dictyostelium gene that encodes a prespore-specific mRNA sequence shows it to encode PsA, the cell surface protein detected by the MUD 1 monoclonal antibody. The predicted sequence of the protein reveals a largely hydrophobic C terminus, with chemical similarity to proteins known to be attached to the plasma membrane via a phosphatidylinositol link. The C-terminal region has direct sequence homology to the contact sites A protein and to the phosphatidylinositol-linked form of a chicken N-CAM, suggesting that it might play a role in cell adhesion. Expression of the D19 gene is known to be induced by cAMP and repressed by adenosine. The accumulation of the D19 mRNA is also repressed by DIF, the putative stalk-specific morphogen, and this effect is mediated at the transcriptional level. The pDd56 and pDd63 genes are induced by DIF, and they are specific markers of prestalk and stalk cells. They encode, respectively, ST310 and ST430, two proteins that were first identified by two-dimensional gel electrophoresis. Both proteins are predominantly composed of a highly conserved, 24-amino acid repeat. The two proteins are localized in the slime sheath of the migratory slug and in the stalk tube and stalk cell wall of the mature culminant, where they presumably function as structural components of the extracellular matrix. We have constructed marked derivatives of the pDd56, pDd63, and D19 genes, and these are correctly regulated after transformation into Dictyostelium cells. Thus we have determined the structure, and elucidated possible functions, for one prespore and two prestalk genes. These sequences should be of value, both as markers of the earliest events in cellular differentiation and in identifying the regulatory sequences controlling cell type-specific gene expression.
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  • 131
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    Developmental Genetics 9 (1988), S. 371-382 
    ISSN: 0192-253X
    Keywords: second messenger ; transcription ; DNase I hypersensitive sites ; cis-acting elements ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: cAMP regulates gene expression in Dictyostelium discoideum through the cell surface receptor and is therefore a transmembrane signal transduction event. We have now begun to examine the signal transduction pathway that transmits the cAMP-induced signal to the nucleus. The results presented here indicate that Ca2+ plays a crucial role. A comparison of the accumulation of UDPGP1 mRNA during development with the corresponding transcription rates revealed that this gene is regulated primarily at the level of transcription. To elucidate the factors involved in the regulation of the UDPGP1 gene we characterized its cis acting sequences. We constructed a series of deletions into the 5′ flanking region of the UDPGP1 gene and analyzed the expression of the mutated DNA in transformants. A sequence element essential for the expression of the UDPGP1 gene is located between -500 bp and -288 dp from the transcription start site. This promoter element appears to be a short G + C-rich sequence positioned between -374 to -395 and coincides with a DNase I hypersensitive site.
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  • 132
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    Developmental Genetics 9 (1988), S. 403-419 
    ISSN: 0192-253X
    Keywords: post-transcriptional regulation ; disaggregation ; poly(A)-binding proteins ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This paper reviews our studies of three aspects of post-transcriptional regulation in Dictyostelium discoideum: (1) the determinants of mRNA stability in vegetative amoebae; (2) the effects of disaggregation and cyclic AMP on the decay rates of cell-type-specific mRNAs in late developing cells; and (3) the cytoplasmic function of the 3′ poly(A) tracts present on most mRNAs. We find that: (1) mRNA stability in vegetative amoebae is not dependent on mRNA size, ribosome loading, or poly(A) tract length, but may be determined by specific 3′-untranslated sequences within a given mRNA; (2) mRNA decay rates in late developing cells are heterogeneous, and cyclic AMP does not act directly to stabilize cell-type-specific mRNAs; and (3) poly(A) is most likely involved in the initiation of protein synthesis via an interaction with cytoplasmic poly(A)-binding proteins.
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  • 133
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    Developmental Genetics 9 (1988), S. 421-434 
    ISSN: 0192-253X
    Keywords: translational control ; polyadenylation ; mRNA stability ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated recombinant plasmids that contain cDNA inserts complementary to mRNAs encoding six different r-proteins of Dictyostelium discoideum. Southern and quantitative dot blot analyses have shown that each of the r-protein genes represented in these plasmids is encoded by a single copy gene and that these genes are not tightly linked to each other. We have determined the relative amount of the six r-protein mRNAs present in cells at intervals throughout development and find that for the first 9 hours of development, each of the mRNAs remains present at virtually the same level as in vegetatively growing cells. Between 9 and 11 hours of development, there is a rapid loss of these mRNAs to 15% or less of vegetative levels, and that low level remains, or slightly declines, through the late stages of development. We have shown that two post-transcriptional events contribute to the developmental regulation of the expression of the r-protein genes. The first involves a specific block to translational initiation that is not the result of inactivation of these mRNAs by decapping or deadenylation. The second is a change in the stability of these mRNAs during early development. In order to begin to analyze the role of specific sequences that may act as targets or signals in these events, we have cloned and sequenced a 1.9-kb genomic DNA fragment that encodes one of the r-proteins. We find that transcription of this gene begins in a pyrimidine-rich region that is not preceded by a TATA box, the gene contains a single intron of 350 bp, and there are two alternative 3′ processing sites. In addition, the 5′-untranslated region of the transcript contains an unusually high percentage of G and C residues relative to other Dictyostelium mRNAs.
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  • 134
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    Developmental Genetics 9 (1988), S. 597-605 
    ISSN: 0192-253X
    Keywords: cell differentiation ; differentiation inducing factor ; cyclic AMP ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Stalk cell formation in low-cell-density monolayers of Dictyostelium discoideum, strain V12-M2, occurs following the sequential addition of cyclic AMP and the differentiation-inducing factor (DIF). Both cyclic AMP and DIF are essential for the appearance of the prestalk-specific isozyme alkaline phosphatase-II, which suggests that both factors are necessary for prestalk cell formation. The available evidence suggests that the cyclic AMP requirement for stalk cell formation is mediated through the cell surface cyclic AMP receptor. However, stalk cell formation is inhibited by caffeine and this inhibition is reversed by the cell-permeable analogue 8-Br-cyclic AMP, which suggests in addition a possible involvement for elevated intracellular cyclic AMP concentrations in stalk cell formation.During in vivo development ceils first become independent of cyclic AMP at the tipped aggregate stage, but the acquisition of cyclic AMP independence is advanced by several hours when cells are incubated in the presence of cyclic AMP for 2 hours. Cells do not become independent of DIF until the culmination stage of development, which suggests the possibility that DIF is required for the conversion of prestalk cells to stalk cells.There is an absolute requirement for DIF for stalk cell formation in low-density monolayers of prestalk cells but only part of population exhibits a requirement for cyclic AMP, which suggests that the prestalk cell population consists of two distinct cell types. Stalk cell formation from prespore cells is totally dependent on both cyclic AMP and DIF.When isolated prestalk and prespore cells are plated in high-density monolayers, the former cells accumulate more DIF. which suggests the possibility that DIF is preferentially synthesized by prestalk cells.
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    Developmental Genetics 9 (1988), S. 615-628 
    ISSN: 0192-253X
    Keywords: colony morphology ; yeast cell wall ; aggregation variants ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Recently, high frequency switching systems have been identified in the infectious yeast Candida albicans and the cellular slime mold Dictyostelium discoideum. In C. albicans, cells can switch at spontaneous frequencies as high as 10-2 between seven general colony morphologies in the case of strain 3153A or between two major phenotypes in the white-opaque transition in strain WO-1. In the latter system, dramatic changes occur in cellular phenotype as well. In D. discoideum, cells can switch at spontaneous frequencies of roughly 10-2 between a number of colony phenotypes which include alterations in developmental timing, blocks at particular morphogenetic stages, morphological aberrations, and aggregation-minus. In the C. albicans and D. discoideum switching systems, the following characteristics are shared: (l) a limited number of switch phenotypes; (2) heritability; (3) high frequency reversibility; (4) low and high frequency modes of switching; and (5) ultraviolet (UV) stimulation of switching of cells in a low frequency mode of switching.
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    Developmental Genetics 9 (1988), S. 607-614 
    ISSN: 0192-253X
    Keywords: pattern formation ; cell sorting ; differential chemotaxis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: When cells dissociated from Dictyostelium discoideum slugs were cultured in roller tubes, they formed agglomerates in which prestalk cells were initially dispersed but soon sorted out to the center and then moved to the edge to reconstitute the prestalk/prespore pattern. To examine the mechanism of sorting out, individual prestalk cells were traced by a videotape recorder. The radial component of the rate of movement toward the center of the presumptive prestalk region was calculated. Prestalk cells did not move randomly, but rather directionally toward the center. Thei movement was pulsatile, with a period of ca. 15 min, and accompanied by occasional formation of cell streams, thus resembling the movement observable during cell aggregation. These results favor the idea that prestalk cells sort out to the prestalk region due to differential chemotaxis rather than differential adhesiveness. After formation of the prestalk/prespore pattern, the prestalk region rotated along the circumference of the agglomerates. This appears comparable to migration of slugs on the substratum, the rate of rotation being similar to that of slug migration.To examine the processes of pattern formation during development. washed vegetative cells were cultured in roller tubes. Prespore cells identified by antispore immunoglobulin initially appeared randomly within the agglomerates, but then nonprespore cells accumulated in the center and finally moved to the edge to establish the prestalk/prespore pattern, the processes being similar to those of pattern reconstruction with differentiated prestalk and prespore cells.
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  • 137
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    Developmental Genetics 9 (1988), S. 629-638 
    ISSN: 0192-253X
    Keywords: transposon ; cellular slime mold ; development ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: PN6024 is a mutant strain of P. pallidum which appeared on selection for resistance to MDMP, an inhibitor of translation. It was found to be mutant in four other traits, being resistant to tubercidin, incapable of growth at 31.5°C, abnormal in development, and slow growing at 25°C. Genetic crosses using the macrocyst cycle showed that these five traits are controlled by five unlinked genes. The hypothesis is that movement of a transposon to multiple new locations caused these mutations. A difference in restriction fragment pattern between PN6024 and its parent PN600 support the hypothesis. Attempts were made to find conditions generating other strains like PN6024. Selection for growth in the presence of tubercidin yielded clones which resemble PN6024 in being developmentally abnormal as well as tubercidin resistant. Tubercidin treatment also increased the frequency of clones resistant to canavanine. It is suggested that tubercidin is mutagenic because it causes movement of the putative transposon, not because it generates point mutations. Growth under conditions of stress (at 31.5°C, at 8°C, in the presence of 2% ethanol) had at most an erratic effect in generating strains like PN6024.Three substrains appeared spontaneously in cultures of PN6024. These differed in developmental characteristics from each other and from the parent strain. It is suggested that they carry mutations in genes which control the choices between growth and aggregation, and between aggregation and encystment.
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    Developmental Genetics 9 (1988), S. 663-672 
    ISSN: 0192-253X
    Keywords: patterning ; reaction-diffusion ; slime mold ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The formation of secondary sori in whorls of Polysphondylium pallidum provides an attractive model system for the study of symmetry breaking during morphogenesis. Tip-specific antibodies that permit detection of very early stages in this patterning process are available. We have found that the patterns of tip-specific antigen expression vary considerably depending on the size, shape, and developmental stage of the whorl. All of these patterns, however, are well explained by patterning models that rely on short-range autocatalysis and long-range inhibition, as exemplified by reaction-diffusion theories. In the context of reaction-diffusion, we discuss the possible effects of initial conditions, boundary conditions, and nonlinearities on the selection of patterns in P. pallidum whorls.
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    Developmental Genetics 9 (1988), S. 673-681 
    ISSN: 0192-253X
    Keywords: cytokinesis ; phagocytosis ; adhesion ; cytoskeleton ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cellular slime mold amoebae have become a model system for the study of cell motility and the cytoskeleton. A basic problem which all cells face that involves the cytoskeleton is how to control their size. The varied ways in which cellular slime mold amoebae change their cell size-by changing the size at which division occurs, by cell fusion, and by control over cytokinesis-are reviewed. A model is presented which attempts to explain how the mechanisms affected in certain cytokinesis mutants in Dictyostelium discideum known as phg mutants could be involved in control of cell size in the predatory slime mold Dictyostelium caveatum.
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    Developmental Genetics 10 (1989) 
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    Keywords: Life and Medical Sciences ; Genetics
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  • 141
    ISSN: 0192-253X
    Keywords: Oogenesis ; Eggshell ; Gene family ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This study isolated cDNA clones from egg-chamber and adult female Drosophila cDNA libraries using as probe a DNA fragment from a 200-kb “chromosome walk” in region 32E of the second chromosome of D. melanogaster. The present authors believe that these clones correspond to a new vitelline membrane protein (VMP) gene because (1) cDNA clones in Northern blots identify a transcript expressed in a tissue- and stage-specific manner: stage 10 egg-chambers; (2) the sequence of cDNAs and of the genomic subclone shows homology with the other VMP genes that have been identified to date; (3) the amino acid composition of the translational product has the high content of proline and alanine characteristic of VMPs. Two aspects emerging from this study are worth stressing: (1) the presence of a hydrophobic domain that is highly conserved in all the VMP genes; and (2) the particularly narrow period of expression of the isolated gene, which could be related to the mechanism of vitelline membrane assembly.
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    Developmental Genetics 10 (1989) 
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    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 63-69 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 70-86 
    ISSN: 0192-253X
    Keywords: Arrested cleavage ; Centrosome ; contractile ring ; Fusome ; Germarium ; Models of dividing cells ; Oocyte/nurse cell syncytium ; Ovarian tumor mutation ; POlytrohic meroistic ovary ; Ring canal ; Spindle elongation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Three-dimensional models were constructed utilizing the information gained from electron micrographs of serial sections of two clones of cystocytes undergoing their terminal divisions. In each clone a polyfusome connected all eight cystocytes together. Each of the spindles was oriented so that one pole touched the polyfusomes, while the other pointed away from it. This positioning of spindles ensures that one cell of each dividing pair retains all previously formed canals, while the other receives none. The two cells that eventually come to contain the maximum number of canals and fusomal material are the ones that differentiate as pro-oocytes, while the others become nurse cells. The orientation of each spindle suggests that the polyfusome formed at one division determines the placement of the cytoskeletal fibers that anchor the spindles formed at the next division. There is a centripetal gathering together of new canals following each cycle of cystocyte division, which is thought to result from the subsequent contraction of the polyfusomal system. Females homozygous for the otu1 mutation are characterized by ovarian tumors, which result when germarial cystocytes undergo supernumerary divisions and fail to differentiate into either nurse cells or oocytes. An analysis of electron micrographs taken of serially sectioned, mutant germaria showed that most germ cells were single or belonged to clusters of two or three interconnected cells. Therefore otu1 cystocytes are unable to undergo a sustained series of arrested cleavages. These cystocytes contain fusomal material that shows ultrastructural differences from normal polyfusomes. We conclude: (1) that a normal polyfusomal system is a necessary prerequisite for the production of a branched chain of cystocytes and for their subsequent differentiation into pro-oocytes and nurse cells; and (2) that a product encoded by the otu+ gene is essential for the construction of a functional polyfusome.
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    Developmental Genetics 10 (1989) 
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    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 123-123 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
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  • 147
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    Developmental Genetics 10 (1989), S. 124-142 
    ISSN: 0192-253X
    Keywords: Cell determination in Drosophila ; Pair-rule gene expression ; Negative transcription control ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The pair-rule genes hairy, runt, even-skipped, and fushi tarazu express their mRNAs and proteins in striped patterns in the Drosophila embryo at the blastoderm stage. Previous studies have shown that the generation of these patterns depends upon products of the gap genes and upon interactions between the pair-rule genes themselves. Here we show that blocking protein synthesis induces expression of each of the pair-rule mRNAs in virtually all regions of the embryo. Our observations together with genetic studies carried out in other laboratories suggest that negative feedback between the pair-rule genes plays a key role in striped expression of pair-rule genes. We propose that stable proteins, present in all regions of the embryo, first activate transcription ofthese pair-rule genes constitutively. Then, various combinations of unstable proteins repress their transcription in a patterned fashion; each stripe of accumulated products of a given pair-rule gene marks a region where it was not repressed. We develop this idea in mathematical form and demonstrate that a network of mutual repression by pair-rule genes can make each blastoderm nucleus into a genetic switch with two stable states. If preexisting gap gene patterns provide initial bias to the blastoderm nuclei, then the “bistable switch behavior” of the nuclei can refine an initially weak spatial bias into a final pattern of sharp stripes.
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    Developmental Genetics 10 (1989), S. 143-154 
    ISSN: 0192-253X
    Keywords: Alternative splicing ; Drosophila development ; Sex determination ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The transformer gene is one of a set of regulatory genes that form the hierarchy controlling all aspects of somatic sexual differentiation in Drosophila melanogaster. The gene transformer occupies an intermediate position in this hierarchy. Analysis of this gene has allowed us to determine the mechanism by which it is regulated in a sex-specific manner and to examine the way in which the regulatory hierarchy is organized. The female-specific expression of the tra gene, previously inferred from genetic observations, is bused on sex-specific alternative splicing of tra pre-mRNA and is not the result of sex-specific transcriptional activation. The female-specific RNA produced by this alternative splicing is the functional mediator of tra activity. Multiple genetic, molecular, and transformation experiments show that female-specific activation of genes or gene products occurs in the order Sex lethal 〉 transformer 〉 transformer-2 〉 doublesex · intersex 〉 female differentiation. The results do not distinguish the level at which transformer might regulate the downstream gene transformer-2. Neither transformer nor any of the downstream genes feedback on, or participate in, alternative splicing of transformer RNA. The mechanism by which Sex lethal regulates transformer splicing appears to be a repression of the use of one of a pair of splice acceptor sites.
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    Developmental Genetics 10 (1989), S. 287-291 
    ISSN: 0192-253X
    Keywords: Robertsonian translocation chromosomes ; Lens ; Optic cup ; Triplication of chromosomes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Trisomic animals produced from mice doubly heterozygous for Robertsonian translocation chromosomes [Rb(1.3)/Rb(1.10)] consistently show eye defects (e.g., aphakia, micro-phakia, and retention of lens stalk). To determine if changes in distribution or composition of extracellular matrix material may be a factor in development of these defects, eye structures of tnsomy (ts) 1 embryos and normal littermates were studied his-tochemically using the following methods: Alcian blue 8GX, pH 2.5; periodic acid-Schiff (PAS), Alcian blue/PAS combined; high-iron diamine (HID); and HID/Alcian blue combined. Eye development was divided into stages to account for the known delay in ts 1 mouse development.Differences were found in staining patterns as early as stage 1. In later stages, the most consistent difference was an increased period of contact between lens and optic cup due to retardation of interface matrix dissolution between these rudiments in ts 1 embryos. Eyes in which this occurred had abnormally shaped lenses. Overall, the ts 1 optic cup appeared to have fewer staining abnormalities and dysmorphology than did the lens or interface matrix.Triplication of a chromosome may indirectly alter temporal and spatial organization of extracellular matrix through action on cells responsible for the production of this material. Possible mechanisms of action are discussed.
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  • 150
    ISSN: 0192-253X
    Keywords: 5-Azacytidine ; DNA methylation ; Plant tumorogenesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The phenomenon of habituotion is considered in plant tissue cultures to be a real process of chemical tumorogenesis: the cultures acquire the capacity of autonomous growth in a hormone-free medium under the influence of a variety of chemical and physical agents. Treatments with 5-azacytidine (AzaC) of in vitro cultured cells of the Nicotiana glauca × N. langsdorffii nontumorous hybrid (NNT)during the culture cycle led to the induction of a habituated phenotype. The repetitive DNA sequences showed a significant lower level of endogenous methylation in the treated cells in comparison with the normal ones. It is worth noting that it was impossible until now to habituate this strain by conventional methods and that the treatments were effective only in the first 5 days of subculturing; various evidence (cytological and biochemical) pointed out a phenomenon of DNA amplification, occurring in the same period. Moreover, analysis of DNA from control and treated cells shows the induction of variations in the endogenous methylation pattern by AzaC in a critical period of cell culture. These results suggest that demethylation can act as a switch from hormone-dependent to autonomous proliferation by activation of genes coding for or regulating the synthesis of growth factors.
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    Developmental Genetics 10 (1989), S. 304-310 
    ISSN: 0192-253X
    Keywords: Maize ; Catalase ; Kernel ; Gene expression ; mRNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In maize three isozymic forms of catalase, CAT-1, CAT-2, and CAT-3 are encoded by three distinct and unlinked structural genes (Catl, Cat2, and Cat3). Catalase activity profiles and zymogram analysis were used to examine the spatial and temporal expression of the three genes during kernel maturation. Three developmental stages of catalase expression were observed in the growing kernel. During stage 1 (6-12 days after pollination), both Catl and Cat3 were expressed; during stage 2 (15-18 days after pollination) only Cat1 expression was observed; and during stage 3 (21-30 days after pollination), Cat1 and Cat2 were expressed. The major constituent tissues of the kernel were examined to determine their contribution to total kernel catalase expression. Each of the tissues was found to have a unique pattern of catalase gene expression. RNA blot analysis, using catalase gene-specific nucleic acid probes, suggests that the differential expression of the three catalase genes observed in the kernel is regulated by controlling the distribution of steady-state mRNA species for the three genes.
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  • 152
    ISSN: 0192-253X
    Keywords: Mouse embryos ; Gap junctions ; Connexin43 ; mRNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Gap junctions appear de novo during compaction in the eight-cell stage of mouse development. This is a critical event in the life of the embryo, because gap junctional intercellular communication is an essential requirement for maintaining compaction and, hence, for development of the blastocyst. Recently, a family of genes encoding gap junction proteins (connexins) has been identified and cloned, and we have taken advantage of the availability of antibodies and cDNA probes to investigate the expression of these genes in early development. We found that a protein with antigenic and size similarity to the “liver” gap junction protein, connexin32, is present throughout preimplantation development from the zygote through the late morula. Connexin32 mRNA, however, could not be detected in any preimplantation stage. This, and the presence of connexin32 in zygotes before activation of embryonic transcription, leads us to conclude that this protein is inherited as an oogenetic product that persists well beyond the transition from the oogenetic to embryonic program of gene expression. Furthermore, we found that mRNA for another gap junction protein, connexin43, is fairly abundant in preimplantation embryos. We conclude that it is more likely connexin43, and not connexin32, that is used to assemble new connexons as the level of intercellular coupling increases after compaction.
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    Developmental Genetics 10 (1989), S. 311-317 
    ISSN: 0192-253X
    Keywords: β-globin ; Human erythroleukemia cells ; RNA transcripts ; K562 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Previous studies have indicated that control and hemin-treated human eryth-roleukemia K-562 cells fail to produce adult-type β-globin mRNA transcripts and to translate them into nascent β-globin chains. Expression of the β-globin DNA sequences in K-562 cells can occur, however, under certain conditions. To readdress this issue and to examine the possibility of whether these cells produce immature and untranslatable β-globin RNA transcripts, we prepared total cyto-plasmic RNA from control and inducer-treated cells and performed Northern blot hybridization analysis using 5′ end-labeled fragments of the human β-globin DNA rather than 3′ end fragments as probes. Although hybridization of both cytoplasmic and nuclear K-562 RNA with a32P-labeled 3′ end fragment (1.6kb Bam H1 cut) coding for a large part of the first exon of β-globin failed to detect β-globin RNA transcripts, hybridization with a 5′ end 32P-labeled 2.0kb Bam H1 fragment (coding for the third exon and part of the second) revealed the presence of relatively small (〈7S) RNA molecules both in nuclear and cytoplasmic fraction. S1 nuclease mapping of both cytoplasmic and nuclear RNA with the use of 5′ end-labeled 2.0 kb Bam H1 fragment of human β-globin DNA indicated protection of a small portion located 64bp 5′ upstream from the Bam H1 site of the second exon. The amount of protected portion was relatively higher in K-562 cells undergoing erythroid maturation. These findings suggest that control and differentiating K-562 cells synthesize β-globin-like RNA transcripts that are 3′ end short, immature, and unable to give rise to adult β-globin chains. These results also indicate that K-562 cells may lack factors that are unique for transcription and processing of the human β-globin RNA transcripts.
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    Developmental Genetics 10 (1989), S. 324-332 
    ISSN: 0192-253X
    Keywords: Heat-shock proteins ; Pollen ; Development ; Maize ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In contrast to sporophytic tissues, mature pollen of higher plants does not synthesize the typical set of heat-shock proteins (HSPs) in response to a marked temperature upshift. Immature grains, however, seem able to do so, at least partially. We investigated the characteristics of HSP synthesis throughout the male gametophytic phase in maize and compared gametophytic and sporophytic heat-shock responses. One-dimensional Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis technique (SDS-PAGE) of newly synthesized proteins revealed that immature pollen synthesizes HSPs, some of which are not induced in sporophytic tissues. The heat-shock response appeared to be related to microgametophytic developmental stages. The strongest response was found in uninucleate microspores: at this stage, in addition to the sporophytic 102, 84, 72, and 18 kD HSPs, three other polypeptides of 74, 56, and 46 kD were observed. In the binucleate and trinucleate stages, only a reduced synthesis of few HSPs could be induced, and differences between genotypes were observed. In germinating pollen, HSP synthesis was not induced under a voriety of heat-stress conditions; however, the consti-tutive synthesis of two polypeptides of the same molecular weight, 72 and 64 kD, as two HSPs was observed. The biological significance of these results is discussed.
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    Developmental Genetics 10 (1989), S. 333-338 
    ISSN: 0192-253X
    Keywords: Cell migration ; Aphidicolin ; Blastula-Gastrula ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Inhibition of DNA replication by aphidicolin in the chick morula interferes with its progression to a normal blastula and prevents induction of the first morphogenetic cell movements of primitive streak formation. Embryos in aphidicolin synthesize some polypeptides typical of blastula but do not display all the characteristic features of morula to blastula transition. Inhibition of DNA replication inteferes with the sequential synthesis of maternally coded polypeptides and with the activation of the embryonic genome in the chick embryo.
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    Developmental Genetics 10 (1989), S. 345-345 
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    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 347-347 
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    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989) 
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    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 339-344 
    ISSN: 0192-253X
    Keywords: Delayed processing ; Splicing ; Transcription ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This study deals with the pattern of developmental expression of the catalase gene in mice. We have used a mouse catalase 2 kb cDNA (pMCT-1) and its 1.4 kb 5′ fragment as probes to characterize the transcripts during embryonic development and differentiation. Total RNA was isolated from 8 days postconceptus (p.c.) whole embryos and from livers and carcasses of 13, 15, and 18 day p.c. embryos as well as from the livers of newborn and adult mice of the S.W. strain. The RNA was applied on slot blots, and run on agarose gels to generate northern blots. Blots were hybridized with the 32P-labeled cDNA probe under different stringency conditions. Autoradiograms were scanned with a densitometer to quantify relative hybridization signals of RNA samples obtained from two or three individual mice representing each stage of development.The catalase transcript is detectable as early as 8 days p.c. with the beginning of somite formation. At this stage, it is primarily in the form of a 12.2 kb transcript. One additional band (2.4 kb) is also apparent at this stage although at a very low intensity. The intensity of the two bands increases with development, particularly during 13-18 days p.c. in liver and carcass. The 2.4 kb RNA band increases sharply from day 8 through 13, 15, and 18 days p.c. and is confined primarily to the liver. Interestingly, only the 2.4 kb RNA band is seen at and after birth. The 2.4 kb RNA is the known mature message of the catalase gene in mice. The presence of large catalase-specific RNA species (seen during development in utero only) is interpreted as the primary transcript of this gene. The complete and efficient processing of this primary transcript takes place only after birth and primarily in the liver, which may be related to the physiological role of this enzyme in oxygen metabolism, particularly stressful superoxides, expected with independent respiration. At a lower stringency wash of the northern blots, a 9.5 kb RNA was seen during a narrow window of in utero development. This 9.5 kb band may represent an uncharacterized catalase-related gene with a possible role in development and differentiation.
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    Developmental Genetics 10 (1989), S. 349-355 
    ISSN: 0192-253X
    Keywords: SV40 promoter ; Expression vector ; Drug resistance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have previously demonstrated systemic resistance to methotrexate (MTX) in transgenic mice carrying a foreign, mutant dihydrofolate reductase (DHFR, E.C. 1.5.1.3) gene. The new gene was introduced as a cDNA cloned into an expression vector driven by the simian virus 40 (SV40) early promoter. Previous physiologic studies suggested that transgenic mice tolerated drug doses invariably lethal to controls on the basis of gastrointestinal (GI) resistance to MTX. In the present study we evaluated foreign gene expression at the RNA level in the three major sites of MTX toxicity: intestine, liver, and bone marrow.The transgene was transcriptionally active in small bowel, and levels of expression were high in animals tolerating the largest doses of MTX. The gene was also expressed in the liver in some pedigrees, but was not detected in hemopoietic tissues of any of the pedigrees tested. Our studies correlate the site of expression of a drug resistant dhfr gene with an altered physiologic response to MTX, and demonstrate that transgenic mice can be used as a test system for expression of genes considered for use in somatic gene therapy.
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    Developmental Genetics 10 (1989), S. 356-364 
    ISSN: 0192-253X
    Keywords: Glucose intolerance ; Insulin resistance ; Diabetes mellitus ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We are investigating human insulin gene expression in transgenic mice. An 8.8 kilobase (kb) human genomic DNA fragment, including the insulin gene (1.4 kb) and 2 kb of 5′ human flanking sequences, was introduced into mouse embryos by pronuclear microinjection. Two lines of transgenic mice have been established, both of which carry the intact human gene in multiple copies. Animals from both lines have significantly higher insulin levels than control mice, and the degree of hyperinsulinemia shows a positive correlation with human gene copy number in the two lines. Expression of the human gene is confirmed by the detection of human C-peptide in plasma. Tissue specificity of expression is maintained, with human insulin mRNA detectable only in the pancreas. The transgenics maintain normal fasting blood glucose in spite of their high insulin levels, but preliminary studies show them to be glucose intolerant when given a glucose load. These mice provide a model system for further studies on the regulation of insulin gene expression and on the effects of chronic hyperinsulinemia on glucose homeostasis.
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    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 411-411 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
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    Developmental Genetics 10 (1989), S. 402-410 
    ISSN: 0192-253X
    Keywords: F9 ECC ; Aggregates ; Embryoid bodies ; Endoderm ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To study the relationship between compaction and differentiation in aggregates of F9 embryonal carcinoma cells, a subline was developed which grows mostly uncompacted in monolayer culture in medium containing a low concentration of calcium (about 0.05 mM). When these cells were trypsinized and cultured in suspension in the same medium, they formed loose, open aggregates, which failed to differentiate into embryoid bodies after exposure to 10 nM retinoic acid, confirming the requirement of compaction for differentiation. If, after culture for 3 days, the uncompacted F9 aggregates were exposed to additional calcium (4 mM), all compacted within an hour. The number of days necessary for aggregates to acquire this ability to compact rapidly was reduced if the monolayer of cells from which the aggregates were derived had been exposed to additional calcium to cause compaction for several days prior to trypsinization and aggregation. Next, treatment of the compacted F9 aggregates with 10 nM retinoic acid was followed by differentiation into embryoid bodies. The number of days required for this was also reduced if the aggregates were formed from previously compacted cells, presumably because compaction of the aggregates occured sooner.The acceleration in compaction and differentiation in aggregates formed from previously compacted cells suggests that some of the proteins important for compaction, which are synthesized in a monolayer of compacted cells, persist through trypsinization and are carried over from monolayer to aggregates. Alternatively, an inhibitor of compaction is decreased in the compacted monolayer. Thus, the process of compaction in its entirety, including its relationship to subsequent differentiation, cannot be studied in aggregates formed from F9 cells grown as usual in the compacted state in monolayer culture. This work provides an alternative system in which aggregation, compaction, and differentiation of F9 cells can be made to occur in stepwise fashion and can be examined separately.
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    Yeast 2 (1986), S. S261 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S221 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S281 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S301 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S321 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S341 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S361 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 2 (1986), S. S381 
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    Keywords: Life and Medical Sciences ; Genetics
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    Yeast 3 (1987), S. 5-9 
    ISSN: 0749-503X
    Keywords: Schizosaccharomyces pombe ; sterile mutants ; ste genes ; protoplast fusion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In previous experiments of Girgsdies (1982), eight sterile (ste) mutants of Schizosaccharomyces pombe did not sporulate when fused with h+ or h- protoplasts. We succeeded in achieving sporulation with these mutants. Two hitherto unknown ste genes, ste7 and ste8, were found.
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    ISSN: 0749-503X
    Keywords: Yeast protein map ; carbon metabolism machinery ; Life and Medical Sciences ; Genetics
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    Notes: Using a modification of the basic two-dimensional polyacrylamide gel electrophoresis technique, we have undertaken a systematic identification of the polypeptides of the protein map of Saccharomyces cerevisiae corresponding to components of the carbon metabolism machinery. To the previous location of nine glycolytic enzyme polypeptides on the yeast protein map we add the location of 23 polypeptides. Ten of them were identified as corresponding to cytoplasmic enzymes of the carbon metabolism machinery and 13 were characterized as mitochondrial proteins. The criteria used to establish the identification of these polypeptides spots include migration with purified proteins, immunodetection, overproduction by plasmid-carrying strains and physiological behaviour.
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  • 175
    ISSN: 0749-503X
    Keywords: Zygosaccharomyces ; weak-acid resistance ; intracellular pH ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Weak acids and hydrogen ions in different concentration combinations affect the intracellular pH value (pHi) of Zygosaccharomyces bailii. The lowest pHi value measured was not at the most extreme, but at intermediate conditions of inhibition. Proton and organic-acid ejection, on a cell volume basis, is greater in cells grown under inhibitory conditions and is stimulated by weak acids, whilst in cells not grown under inhibitory conditions acid efflux is lower and is depressed by weak acids; this may be important in the maintenance of tolerable pHi values in the presence of weak acids. The concentration of benzoic acid measured internally is identical to the value expected from its pK, external pH and pHi. Addition of fructose to starved cells causes both a decreased pHi and a concomitant efflux of previously loaded benzoic acid, quantitatively in accord with the shift in equilibrium of the freely permeable undissociated acid. There is no evidence that weak acids are actively extruded. Protoplast volume also varies with hydrogen-ion and weak-acid concentration and this too may play a role in intracellular pH maintenace.
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  • 176
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    Yeast 3 (1987), S. 43-49 
    ISSN: 0749-503X
    Keywords: Heterologous gene expression ; Hepatitis B ; protein estimation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Purified recombinant hepatitis B surface antigen separated on polyacrylamide gels in the presence of sodium dodecyl sulphate has a very low staining index with Coomassie blue relative to a number of standard proteins. In contrast the protein stains better than average with silver nitrate. This property has been used to develop a semi-quantitative method of estimation of recombinant surface antigen in extracts of Saccharomyces cerevisiae producing this protein. The method can be used to follow purification protocols. It is quick, simple and since it measures the surface antigen biochemically, is independent of the aggregation state or conformation of the protein, a factor which can affect enzyme-linked immunoassays which rely on antigen-antibody interactions.
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  • 177
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    Yeast 3 (1987), S. 33-42 
    ISSN: 0749-503X
    Keywords: Yeast ; acid phosphatase ; gene regulation ; upstream activating sequences ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To identify the sequences involved in the regulation of the yeast acid phosphatase gene (PHO5) we constructed a series of hybrid promoters. Increasing lengths of 5′-flanking sequences of the PHO5 gene were placed in front of the TATA-box of constitutively expressed acid phosphatase gene (PHO3).The PHO5/PHO3 promoter constructions were used to replace the entire PHO5, PHO3 gene cluster on chromosome II. Depending on the length of PHO5 5′-flanking sequences present the PHO3 gene driven by the hybrid promoter could now be derepressed in response to inorganic phosphate (low Pi) exactly as the PHO5 wild type gene. A critical regulatory element was located between position -402 to -351 (upstream from ATG) and sequences further downstream (from -351 to -300) could increase transcriptional activation. The transcription levels of PHO3 were determined by northern blot analysis, under repressed (high Pi) and derepressed (low Pi) conditions which was paralleled by an increase in extra-cellular acid phosphatase activity. Fully regulated promoter hybrids showed a 40-fold induction of mRNA levels, comparable to wild type PHO5 promoter. S1-nuclease protection experiments revealed that the PHO5 5′-flanking sequences, placed in front of PHO3, did not change the PHO3 transcription initiation site/s.
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  • 178
    ISSN: 0749-503X
    Keywords: Cyclic AMP ; nitrogen limitation ; resting state ; cell cycle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have identified a mutation called rcal (for rescue by cAMP) which allows adenylate cyclase-deficient mutants to divide in the presence of cAMP. We took advantage of this rcal mutation to study the effect of externally added cAMP on the onset of the resting state when cells are starved for ammonium. We measured the resistance of the cells to zymolyase treatment as a parameter of the resting state. We observed that the onset of the resting state is reversibly blocked by cAMP. This inhibitory effect of cAMP is discussed together with the cAMP control of the start. This leads us to propose a model in which the cAMP level, controlled by the availability of nutrients, should trigger the choice between the entry of the cell into the resting state and the initiation of a new division cycle.
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  • 179
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    Yeast 3 (1987), S. 95-105 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; tryptophan accumulation ; genetic engineering ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Plasmid pME559, carrying all five yeast TRP genes, was constructed. This plasmid is a yeast/Escherichia coli shuttle vector based on pBR322 and 2 μm-DNA sequences derived from plasmid pJDB207. We studied in yeast (i) the stability of the plasmid under selective and non-selective conditions, (ii) expression of all five TRP genes and (iii) tryptophan accumulation in yeast transformants. These studies were conducted in comparison with an earlier construction, pME554, which differs from plasmid pME559 in the expression of the TRP1 gene and which carries the TRP2 wild type instead of the TRP2fbr mutant allele. For stable maintenance of the plasmids in yeast a selection was necessary. Plasmid pME559 displayed normal expression of all TRP genes, and enzyme levels on average 23-fold higher than in the wild type strain were found. In comparison, the maximal tryptophan flux observed in such a plasmid-carrying strain was about ten-fold higher than the maximal flux capacity in the wild type strain.
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  • 180
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    Yeast 3 (1987), S. 107-115 
    ISSN: 0749-503X
    Keywords: DNA replication ; ARS elements ; histone genes ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have previously identified an autonomously replicating segment (ARS) near the 3′ end of the histone H4 gene at the copy-I H3-H4 locus. We have now searched for additional autonomously replicating segments and sequences homologous with the ARS core consensus sequence near the copy-II histone H4 gene and both of the histone H3 genes. No new ARS elements were identified by functional cloning assays. However, several matches to the ARS core consensus element were found within the DNA sequencs of the copy-I and copy-II genes. An exact match to the ARS core consensus was identified in the region downstream from the copy-I histone H3 gene and a set of sequences with weak homology was also locatd within the copy-II region. However, restriction fragments including these sequences did not demonstrate ARS activity on a plasmid in transformed cells.
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  • 181
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    Yeast 3 (1987) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 182
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    Yeast 3 (1987), S. 117-129 
    ISSN: 0749-503X
    Keywords: Killer ; virus-like particles ; nucleotides ; pyrophosphatase ; RNA polymerase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The intracellular killer virions of yeast co-purify with an RNA polymerase activity which catalyzes the synthesis of fulllength transcripts of the two viral genomic double-stranded RNA segments. This polymerase utilizes ribonucleoside diphosphates or triphosphates as substrates. The virions have other associated nucleotide-metabolizing enzyme activities, including nucleoside diphosphate kinase, adenosine monophosphate kinase, and nucleoside triphosphate phosphotransferase, an activity which catalyzes the exchange of gamma-phosphate from any ribonucleoside triphosphate with any ribonucleoside or deoxyribonucleoside triphosphate. The purified virions also contain an inorganic pyrophosphatase activity. These enzymes may allow the virus to utilize nucleotide pools distinct from those utilized in host cell transcription.
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  • 183
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    Yeast 3 (1987), S. 131-137 
    ISSN: 0749-503X
    Keywords: Transformation ; Saccharomyces ; plasmid ; DNA uptake ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have studied the mechanism of DNA transformation of whole yeast cells in Saccharomyces cerevisiae with particular emphasis on the role of the cell wall complex in DNA uptake. Two new aspects of the process have been investigated in order to evaluated its specificity. Such aspects are: (i) effect of monovalent vs. divalent cations during incubation with the transforming DNA and (ii) timing of DNA adsorption and uptake. We found that the specificity for cation requirement is a strain-dependent characteristic influenced by the presence of transforming DNA in the cell suspension. This finding is supported by reports from several laboratories that some yeast strains show mutually exclusive transformability with monovalent vs. divalent cations. While irreversible adsorption of plasmid DNA molecules is induced by both heat shock and polyethylene-glycol(PEG), DNA uptake seems to occur only after the removal of PEG. In the course of this study we have developed a new, alternative method of whole cell DNA transformation with CaCl2 able to transform strains that do not respond to other methods.
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  • 184
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    Yeast 3 (1987), S. 255-262 
    ISSN: 0749-503X
    Keywords: Yarrowia lipolytica ; isocitrate lyase ; structural gene ; gene map ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The gene ICL1 codes for the tetrameric enzyme isocitrate lyase of Y. lipolytica. Twenty icl1- alleles have been analysed for their reversion frequency, their interallelic complementation pattern, and the position of the corresponding mutation site on the fine structure map of the gene ICL1. One intragenic temperature-sensitive revertant of the allele icl1D-39 was isolated, which expressed a thermolabile enzyme. In spite of the fact that no nonsense mutations have been detected, the direction of transcription of the gene ICL1 was inferred from the localization of a linked cis-dominant regulatory mutation site. The size of the mitotic map of this gene suggests that recombination frequency in Y. lipolytica is lower than in Saccharomyces cerevisiae.
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  • 185
    ISSN: 0749-503X
    Keywords: Pichia pinus ; alcohol oxidase ; catabolite repression ; metabolic regulation ; methanol ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effect of various carbon compounds on the synthesis of alcohol oxidase in a medium with methanol was studied in the wild type strain of Pichia pinus as well as in gcr1 and ecr1 mutants defective in glucose and ethanol repression of methanol metabolic enzymes, respectively. Compounds repressing the synthesis of alcohol oxidase in the wild type strain were divided into four groups. Repression of alcohol oxidase by compounds of the first group (glucose, fructose, mannose, galactose, L-sorbose and xylose) was impaired only in the gcr1 mutant and that by compounds of the second group (ethanol, acetate, 2-oxoglutarate and erythritol) only in the ecr1 mutant. Repression by compounds of the third group (malate, dihydroxyacetone) was not impaired in both these regulatory mutants and that by compounds of the fourth group (succinate, fumarate, L-arabinose, sorbitol, salicin, xylitol and cellobiose) was partially reduced in both gcr1 and ecr1 strains.Mutation gcr1 causes a significant decrease in phosphofructokinase activity. It also led to a six- to seven-fold increase in intracellular pools of glucose-6-phosphate and fructose-6-phosphate and to a two-fold decrase in the intracellular pool of fructose-1,6-bisphosphate. In ecr1 strains, a decrese in 2-oxoglutarate dehydrogenase activity accompanied by an increae in activities of NAD- and NADP-dependent isocitrate dehydrogenases and NAD- and NADP-dependent glutamate dehydrogenases was demonstrated. The intracellular pool of 2-oxoglutarate was increased 2·5-fold in ecr1 strains. Genes GCR1 and ECR1 are not linked.The mechanisms of catabolite repression of alcohol oxidase in methylotrophic yeasts are discussed.
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  • 186
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    Yeast 3 (1987), S. 273-273 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 187
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 188
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    Yeast 4 (1988) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 189
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    Yeast 3 (1987), S. 263-270 
    ISSN: 0749-503X
    Keywords: Lodderomyces elongisporus ; Rhodotorula gracilis ; Saccharomyces cerevisiae ; accumulation ratio ; membrane transport ; suspension density ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The previously described effect of cell suspension density on metabolic and transport phenomena in yeast, apparently caused by inhibition by dissolved carbon dioxide, is also observed with the accumulation ratio of both sugars and amino acids where not only a kinetic but also an energetic factor comes into play. Unlike all previously measured metabolic and transport parameters, the dependence of the accumulation ratio on suspension density is not monotonic but shows a pronounced maximum in the range of 4-8 mg dry wt/ml, depending on yeast species and on cultivation conditions. In Rhodotorula gracilis and in Lodderomyces elongisporus it is not due to CO2 but is semiquantitatively related to the proton-motive force across the plasma membrane as well as to the intracellular ATP content. It is observed both in oxygen and in argon, over a wide range of pH values and of temperatures, but it is suppressed by metabolic inhibitors. It is expressed only in a range of transported solute concentrations between about 0·1 and 10 mM.
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  • 190
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    Yeast 4 (1988), S. 1-15 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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    Topics: Biology
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  • 191
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    Yeast 4 (1988), S. 27-40 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 192
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    Yeast 4 (1988), S. 17-26 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 193
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    Yeast 4 (1988), S. 41-46 
    ISSN: 0749-503X
    Keywords: Isocitrate lyase ; purification ; Catabolite inactivation ; Saccharomyces cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Isocitrate lyase purified to homogeneity from Saccharomyces cerevisiae was composed of four identical subunits with a molecular mass 75 K Da. The enzyme was most active at pH 7.0 in the presence of 5 mM-Mg2+. The Km value for threo-Ds-isocitrate was 1.4 mM. Isocitrate lyase was shown to be thermostable at 50°C for 60 min at a high salt concentration, but rapidly lost activity at -20°C or by dialysis.
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  • 194
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    Yeast 4 (1988), S. 83-83 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 195
    ISSN: 0749-503X
    Keywords: Bovine leukemia virus ; PH05 ; PGK ; tumor virus ; Saccharomyces cerevisiae ; viral antigens ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: DNA sequences of the envelop (env) gene of the bovine leukemia virus (BLV) were expressed in the yeast saccharomyces cerevisiae. Two yeast promoters, the responsible PH05 promoter and the constitutive PGK promoter, were used to construct four expression plasmids either a sequence of the surface antigen gp51 or a (gp51 + gp30) sequence.The expressed hetrologous gene products were characterized by Western blot analysis and competitive radio-immunoassay. By means of Northern blot analysis the steady-state level of env-specific mRNA was analysed.The highest expression rate was obtained from recombinant plasmid YEpSG 94 comprising a gp51 sequence - a 630 base pair fragment containing 70% of the gp51 but lacking the N terminus - as well as the PH05 promoter including PH05 signal sequence and the PH05 terminator. The recombinant gp51 was partially lycosylated but the PH05 signal peptide did not seem to be cleaved off. No immunoreactive material could be found in the periplasm or in the culture medium.By means of monoclonal antibodies directed against eight different epitopes of viral gp51, all for sequential antigenic determinants were detected in the AH 216(YEpSG 94) expression product.
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  • 196
    ISSN: 0749-503X
    Keywords: Yeast ; Ribosomes ; Kluyveromyces ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In an adenine-requiring mutant strain of the yeast, Kluyveromyces lactis the intracellular content of ATP is one-third to one-fifth that in a protophic wild strain under growing conditions. The quantitatives difference becomes rather small in resisting stationary-phase cells. Temporary changes in the two-dimensional protein patterns of mutant ribosomes occur when the ATP content during the transition phase of growth. The transfer of exponentially growing cells to a synthetic complete medium void of adenine induces the same changes in mutant ribosomes within several hours. Identification of robosomal proteins by two-dimensional gel electrophoresis indicated all changeable proteins (at least five proteins) to belong to 40S ribosomal subunits. The mutant ribosomes prepared from the transitio-phase cells have much lower activity (below 60%) for poly(U)-directed polyphenylalanine synthesis than those in exponentially growing or resisting stationary-phase cells. Thus, changes in ribosomal components associated with the differences in ribosomes activity in a cell-free system were noted in the adenylate-deprived cells of K. lactix.
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  • 197
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; killer DNA plasmid ; gene cloning ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The killer system of Kluveromyces lactis is associated with two linear DNA plasmids, pGKL1 and pGKL2. The killer toxin and the immunity determinant are coded for by pGKL1. Mutations which we have named KEX1. The KEX1 gene of K. lactis has been cloned by complementation of kex1 mutations by using a recombinant plasmid pool containing the entire Kluyveromyces lactis genome, on a multicopy plasmid KEp6, which contains the Saccharomyces cerevisiae URA3 gene as a marker. Genetic analyses of strains carrying a distrupted kex1 allele demonstrated that the cloned DNA corresponded to the KEX1 gene. The cloned KEX1 gene of K. lactis has low but significant sequence homology with the KEX2 gene of Saccharomyces cerevisiae. In vivo complementation of the kex1 mutations of K. lactis by the KEX2 gene of S. cerevisiae, and complementation of the kex2 mutations of S. Cerevisiae by the KEX1 gene of K. lactis, demonstrated that KEX1 of K. Lactis is functionally related to the KEX2 gene of S. cerevisiae. K. lactis diploids homozygous for kex1 are deficient for sporulation.
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  • 198
    ISSN: 0749-503X
    Keywords: Alcohol dehydrogenase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.
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  • 199
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    Yeast 4 (1988), S. 155-155 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
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  • 200
    ISSN: 0749-503X
    Keywords: Aminotransferase ; transaminase ; 4-aminobutyrate ; Candida ; putrescine ; spermidine ; cytisol ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: 4-Aminobutyrate aminotransferase (EC 2.6.1.19) was elevated in activity by 20- to 40-fold in cells of the yeast Candida boidinii grown on spermidine, putrescine, 4-acetamidobutyrate and 4-aminobutyrate compared with activities detected in cells grown on ammonium, methylamine or 6-aminohexanoate, confirming previous suggesions that it plays a key role in polyamine breakdown. Other enzymes of the proposed route of polymine breakdown were found to be non-coordinately induced or derepressed during growth on spermidine or its putative breakdown intermediates. The enzyme was not sedimented from spheroplast lysates at 100 000 × g, and it is concluded that it is conclded that it is probably cytosoilc in its subcellular location (although the data do not exclude the possiblity of its being vacuolar).
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