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  • 1
    ISSN: 1432-0428
    Keywords: Genetics ; Type 2 (non-insulin-dependent) ; diabetes mellitus ; insulin receptor ; glucose transporters
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary We have recently examined the exons encoding the insulin receptor tyrosine kinase domain and GLUT 4 in 30 subjects with Type 2 (non-insulin-dependent) diabetes mellitus using a molecular scanning approach. The variant sequences Val-Met985 and Lys-Glu1068 of the insulin receptor and Val-Ile383 of GLUT 4 were each separately found in three different diabetic subjects. In a study of a Welsh population, the GLUT 4383 variant was found in three of 160 diabetic and none of the 80 control subjects. In this study, the same group of Welsh Type 2 diabetic and control subjects was analysed using allele-specific oligonucleotide hybridisation, single nucleotide primer extension and allele-specific restriction digestion to ascertain the frequency of the two insulin receptor mutations. The Val-Met985 mutation was found in none of the 160 Welsh Caucasian Type 2 diabetic subjects and two of 80 control subjects. The Lys-Glu1068 mutation removes a Sty 1 site and digestion of amplified exon 18 with Sty 1 confirmed the presence of this mutation in the heterozygous state in the original subject. None of the Welsh diabetic or control subjects had the Glu1068 mutation. The discovery of a very common silent polymorphism at codon 130 of GLUT 4 allowed examination of the association of this locus with Type 2 diabetes using allele-specific oligonucleotide hybridisation in a subset of the Welsh subjects. The genotypic frequencies (homozygous wild-type and heterzygous polymorphic (poly) sequences) were not significantly different between diabetic and control subjects (Type 2 diabetic subjects: wild-type/wild-type 40%, wild-type/poly 46%, poly/poly 14%; Control subjects: wild-type/wild-type 37%0, wild-type/poly 45 %, poly/poly 18 %;p 〉 0.05). In conclusion, in a British Caucasian population the examined insulin receptor tyrosine kinase domain mutations are uncommon. Also the GLUT 4 locus does not appear to be strongly associated with Type 2 diabetes.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Journal of molecular medicine 70 (1992), S. 377-384 
    ISSN: 1432-1440
    Keywords: Genetics ; Apolipoproteins ; Lipoproteins ; Atherosclerosis ; Transgenic animals
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary In order to elucidate the genetic abnormalities underlying lipoprotein disorders associated with coronary heart disease susceptibility, researchers have looked for candidate genes. The studies have focused particularly on the lipoprotein transport genes. Relatively common as well as rare mutations have already been identified in several of these genes. In addition, further metabolic and genetic studies indicate that some of these loci harbor significant, but as yet undefined, genetic variation. In the next few years, it is not unreasonable to expect that all or most of the significant mutations at these loci will be catalogued. It is too early to know whether this will be sufficient to explain the genetic basis of altered lipoprotein levels or whether new loci will need to be investigated. Additional candidate gene loci might be those coding for genes involved in intracellular cholesterol metabolism, cholesterol absorption, or insulin resistance. New loci may also be revealed by the technique of reverse genetics. A more complete understanding of the genetics of atherosclerosis susceptibility will probably also entail the identification of variants at genetic loci that control both the reaction of the blood vessel wall to atherogenic lipoproteins and the thrombosis system. Investigation of the genetic basis of coronary heart disease susceptibility remains a worthwhile and lively field, with important clinical and public health ramifications.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    European journal of pediatrics 151 (1992), S. 837-841 
    ISSN: 1432-1076
    Keywords: Frontonasal dysplasia ; Craniosynostosis ; Genetics ; X chromosome ; Psychomotor development
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We report on nine patients with craniofrontonasal dysplasia (CFND). Seven classical cases had facial features suggestive of frontonasal dysplasia and coronal craniosynostosis. Extracranial abnormalities such as brittle nails with prominent longitudinal grooves or syndactyly of fingers and toes were observed in individual patients. In two families the father of classical cases showed a milder pattern of abnormalities, consistent with the diagnosis. We present a 2- to 13-year follow-up on our patients. Hypotonia and laxity of joints are common and may necessitate supportive measures. Mild developmental delay was noted in three out of six classical cases studied in detail. Unlike almost all other X-linked disorders, clinical expression in CFND is generally much more severe in females than in males. In contrast to previous reports of this condition, one of our severely affected cases is a male.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Amino acids 3 (1992), S. 229-234 
    ISSN: 1438-2199
    Keywords: Amino acids ; Tyrosine hydroxylase ; Brain ; Genetics ; Mouse
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary L-tyrosine-3-hydroxylase (TH) is the first and rate limiting enzyme in the biosynthetic pathway of catecholamine neurotransmitters (dopamine, noradrenaline, adrenaline). Implication of dopamine (DA) in various psychopathological phenomena, such as schizophrenia, has considerably contributed to the intensity of investigation of basic biochemical regulation of TH by activation and induction. Here we consider a third, constitutional (genotypic) aspect of regulation and present evidence that differences in mesencephalic (TH/SN), striatal (TH/CS), and hypothalamic (TH/HT) TH activity between virtually isogeneic strains of mice can be explained by segregating genetic factors. Biometrical genetic analysis of progenitor strains and their crosses indicated significant additive gene effects for TH/SN, TH/CS, and TH/HT, whereas dominance effects were statistically non-significant. A monogenic model of inheritance for TH/SN and TH/CS could not be rejected, while more than one gene was indicated for TH/HT. Significant positive phenotypic correlations were found in genetically segregating populations among mesencephalic, striatal and hypothalamic TH activities. This would suggest that some common genetic factors (or linked genes) are involved in the genetic variation of all three traits. A genetic selection experiment to elucidate the cellular and biochemical mechanisms underlying these variations is in progress.
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Theoretical and applied genetics 84 (1992), S. 714-719 
    ISSN: 1432-2242
    Keywords: Wheat ; Salinity ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Accessions of Triticum tauschii (Coss.) Schmal. (D genome donor to hexaploid wheat) vary in salt tolerance and in the rate that Na+ accumulates in leaves. The aim of this study was to determine whether these differences in salt tolerance and leaf Na+ concentration would be expressed in hexaploid wheat. Synthetic hexaploids were produced from five T. tauschii accessions varying in salt tolerance and two salt-sensitive T. turgidum cultivars. The degree of salt tolerance of the hexaploids was evaluated as the grain yield per plant in 150 mol m-3 NaCl relative to grain yield in 1 mol m-3 NaCl (control). Sodium concentration in leaf 5 was measured after the leaf was fully expanded. The salt tolerance of the genotypes correlated negatively with the concentration of Na+ in leaf 5. The salt tolerance of the synthetic hexaploids was greater than the tetraploid parents primarily due to the maintenance of kernel weight under saline conditions. Synthetic hexaploids varied in salt tolerance with the source of their D genome which demonstrates that genes for salt tolerance from the diploid are expressed at the hexaploid level.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-5233
    Keywords: Type 2 (non-insulin-dependent) diabetes mellitus ; Genetics ; Polymorphisms ; GLUT 4 ; GLUT 1
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Glucose transporter genes have been proposed as candidate genes for type 2 (non-insulin-dependent) diabetes mellitus. We chose to study the adult skeletal muscle glucose transporter gene (GLUT 4) andGLUT 1 in consideration of previous conflicting results obtained by different authors. We studied 68 patients with type 2 diabetes, and 66 non-diabetic controls matched for age, sex, and body mass index (BMI). Women and men were considered separately, according to BMI (≤24.0 and 〉24.0 for women; ≤25.0 and 〉25.0 for men). Allele and genotype frequencies were not significantly different in controls and in type 2 diabetic patients. ForGLUT 1 allele 1 and genotype x1x1 were more frequent, although not significantly (P=0.064 at χ2,P=0.025 at Fisher exact test) in overweight/obese diabetic women than in overweight/obese non-diabetic women. These data do not support the hypothesis that these genes play a major role in genetic susceptibility to type 2 diabetes mellitus, but suggest a possible association, at least in women, of allele 1 ofGLUT 1 with obese type 2 diabetes mellitus.
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Acta diabetologica 29 (1992), S. 173-177 
    ISSN: 1432-5233
    Keywords: Erythrocyte ; Genetics ; Renal function ; Sodium transport systems
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract Studies of kidney cross-transplantation in the Milan hypertensive strain of rats (MHS) and in its control strain (MNS) have demonstrated that the kidney has a causal role in the development of hypertension in this animal model. The same result was obtained in two other strains of rats with genetic hypertension. Patients receiving a kidney from a donor with hypertensive parents require more antihypertensive therapy than recipients of a kidney from a donor with a normotensive family. When MHS rats and a subset of patients with primary hypertension were compared with their appropriate controls, similar changes in kidney function and Na−K−Cl cotransport were observed. Offspring of hypertensive parents exhibit altered kidney function compared with their controls. Na−K−Cl co-transport in MHS rats is genetically determined and genetically associated with hypertension. In MHS rats the increase in Na−K−Cl co-transport seems to be linked to a cytoskeletal protein, adducin. In conclusion, a consistent sequence of events from a protein abnormality to cell and renal dysfunction may be proposed as being responsible for hypertension.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1437-160X
    Keywords: Systemic lupus erythematosus ; Genetics ; Ro and La antibodies ; Recombinant autoantigens ; MHC ; Multicenter study
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Antibodies against recombinant 52 kD-Ro, recombinant 60 kD-Ro and recombinant La protein were determined by ELISA in over 300 central European patients with systemic lupus erythematosus (SLE). A strong association with HLA-DR3 was found for antibodies against 52 kD-Ro and La, but not for recombinant 60 kD-Ro antibodies in the absence of antibodies against 52 kD-Ro or La. Ro/La negative SLE patients still showed an increased frequency of HLA-DR3 as compared to healthy controls. These results indicated that the preferential formation of Ro and La antibodies was not due to an unspecific stimulatory effect of HLA-DR3 but that the antibody response to certain defined proteins (52 kD-Ro and La) was influenced by MHC genes in SLE. Furthermore, the association of SLE with HLA-DR3 was independent of the effects of DR3 on the formation of 52 kD-Ro and La antibodies.
    Type of Medium: Electronic Resource
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  • 9
    ISSN: 1437-160X
    Keywords: Systemic lupus erythematosus ; Ro and La antibodies ; Multicenter study ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Antibodies against Ro and La, including recombinant La and recombinant 60 kD-Ro, were determined by counter immunoelectrophoresis and ELISA in over 300 central European systemic lupus erythematosus (SLE) patients. The presence of both Ro and La antibodies was strongly associated with the MHC haplotype B8-C4AQ0-DR3-DQ2, the association being stronges for DR3. After exclusion of all B8-DR3 positive patients only DR3 positive patients still showed an increased incidence of Ro and La antibodies, suggesting DR3 as the primary association factor. High titers of La antibody, but not of 60 kD-Ro antibody, were also significantly associated with the presence of DR3. Other DR and DQ antigens or heterozygous DQ combinations were not significantly associated with Ro and La antibodies.
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Reading and writing 4 (1992), S. 307-326 
    ISSN: 1573-0905
    Keywords: Genetics ; Reading disability ; Sex differences ; Twins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Education
    Notes: Abstract The issue of sex differences in reading disability has been of recent interest in relation to sex ratios in families with reading disabled children and to possible sex biases in referred populations. Data from a study of 570 twins are used to develop alternative definitions of reading disability that vary in the manner to which sex effects are taken into account. These definitions include discrepancies between reading quotients and IQ, the use of the regression of reading onto IQ and chronological age/reading age differences. In each case the reading and spelling disability was defined either separately for the sexes or based upon the data for the sexes combined and with and without an IQ〉90 exclusion criterion. The consequences of using the alternative definitions for prevalence, sex ratio and heritability are examined. The results demonstrate that the characteristics of reading disabled children vary with the way disability is defined. The excess of males seems to be a robust finding. Definitions that take into account differences in mean score for males and females reduce but do not eradicate the sex ratio. From the genetic analysis, there is no support for the suggestion that the genetic effect on reading is greater for females than males. It is concluded that the use of regression based procedures for identifying reading disability is desirable but that at present there is insufficient evidence to justify the adoption of separate regression procedures for the two sexes.
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  • 11
    ISSN: 1573-2592
    Keywords: Genetics ; immune deficiency
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract A family with 13 members included 2 subjects with selective IgA deficiency (IgA-D) and 3 subjects with common-variable immune deficiency (CVID), diseases which usually occur sporadically. Reciprocal combinations of B and T cellsin vitro between one normal and two immune-deficient family members and normal subjects revealed that defective Ig synthesis was determined by the B cells, while the patient T cells functioned normally. Normal T helper and suppressor function was demonstrated even in one patient with CVID who developed a T-cell lymphoproliferative disorder associated with elevated IgM; this patient's B cells made only IgMin vitro. Immune deficiencies were inherited in this family in a pattern consistent with an autosomal dominant trait with incomplete penetrance. All the immune-deficient patients in this family possessed at least one copy of an MHC haplotype previously shown to be abnormally frequent in IgA-D and CVID: HLA-DQB1*0201, HLA-DR3, C4B-Sf, C4A-deleted, G11-15, Bf-0.4, C2-a, HSP70-7.5, TNFα-5, HLA-B8, and HLA-A1. The patient who developed the lymphoproliferative disorder was homozygous for this haplotype. Four immunologically normal members, one of whom was 80 years old, also possessed this MHC haplotype, indicating that its presence is not sufficient for disease expression. A small segment of another MHC haplotype associated with Ig deficiency in the population also occurred in this family, but it was not associated with immune deficiency. The presence of neutral amino acids at position 57 of DQβ, previously correlated with IgA-D, was associated with disease in this family approximately to the same degree reported previously in unrelated patients. Thus the expression of immunodeficiency in individuals bearing a disease-associated MHC haplotype appears to require either additional genes or an environmental trigger.
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  • 12
    Electronic Resource
    Electronic Resource
    Springer
    European journal of epidemiology 8 (1992), S. 3-9 
    ISSN: 1573-7284
    Keywords: Atherosclerosis ; Cladistics ; Genetics
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We seek to understand the relative contribution of allelic variations of a particular gene to the determination of an individual's risk of atherosclerosis or hypertension. Work in progress is focusing on the identification and characterization of mutations in candidate genes that are known to be involved in determining the phenotypic expression of intermediate biochemical and physiological traits that are in the pathway of causation between genetic variation and variation in risk of disease. The statistical strategy described in this paper is designed to aid geneticists and molecular biologists in their search to find the DNA sequences responsible for the genetic component of variation in these traits. With this information we will have a more complete understanding of the nature of the organization of the genetic variation responsible for quantitative variation in risk of disease. It will then be possible to fully evaluate the utility of measured genetic information in predicting the risk of common diseases having a complex multifactorial etiology, such as atherosclerosis and hypertension.
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  • 13
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 26-33 
    ISSN: 0192-253X
    Keywords: Tetrahymena thermophila ; cilia ; gly-coprotein ; Con A ; Western blot ; lectin ; mating ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.
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  • 14
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 16-25 
    ISSN: 0192-253X
    Keywords: Nucleotide sequence of macronuclear chromosome ; multiple introns ; ciliate pheromone ; UGA codon translation ; leader peptide ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned and sequenced a 1.7 kb macronuclear chromosome encoding the pheromone 4 gene of Euplotes octocarinatus. The sequence of the secreted pheromone is preceded by a 42 amino acid leader peptide, which ends with a lysine residue. The sequence coding for the leader peptide contains information for a putative signal peptide and is interrupted by a 772 bp intron as shown by comparison with a cDNA clone. A 64 bp intron and a 145 bp intron interrupt the sequence coding for the secreted pheromone. The three introns contain typical 5′ and 3′ splice junctions and a putative branch point site. The small introns have a low GC content. The large intron has a GC content similar to that of the pheromone 4 gene exons. The amino acid sequence of pheromone 4, deduced from both the genomic DNA and the cDNA of pheromone 4, shows that the secreted pheromone consists of 85 amino acids. One of its amino acids is encoded by a UGA codon. Since it has been shown for pheromone 3 of E. octocarinatus that UGA is translated as cysteine, it is assumed that the UGA codon encodes cysteine in pheromone 4 as well. The 164 bp noncoding region upstream of the leader peptide is AT-rich and contains an inverted repeat capable of forming a stem-loop structure with a stem of 11 bp. The 151 bp noncoding region at the 3′ end of the chromosome contains a putative polyadenylation sequence and an inverted repeat. The macro-nuclear molecule is flanked by telomeres and carries the pentanucleotide motif TTGAA, located at a distance of 17 nucleotides from the telomeres. This motif has been suggested to be involved in the formation of macronuclear chromosomes. © 1992 Wiley-Liss, Inc.
    Additional Material: 4 Ill.
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  • 15
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 41-46 
    ISSN: 0192-253X
    Keywords: Analysis of variance ; protists ; mat locus ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The life styles of ciliated protists are particularly suitable for experimental analyses of certain aspects of developmental and genetic biology. The progression from sexual immaturity tomaturity to senescence represents one of the most intriguing aspects of developmental programs. The extent to which progeny clones, their subclones, and testers used in the assay result in different lengths of immaturity has been investigated in Euplotes crassus. Six subclones from each of 12 progeny clones from a cross between stocks EC1 and EC2 were tested for maturity with stocks EC3, EC4, and EC5 on every transfer. Analysis of variance was used to partition thetotal variation in fissions to maturity into parts due to clones, subclones, and testers and the interactions between these levels. The error, interaction of subclones and testers, corresponds to a standard deviation of only 4.1 fissions, while the within clone within tester means range from 15.2 to 46.7 fissions; all levels except testers contribute significantly to the total variation. Most of the variability is attributable to clones (66%), the next most to error (16%), the next most to interaction of clones by testers (13%), and the least to subclones (5%). An a posteriori analysis examined whether the differences among clones were due to the cytoplasm of the clone ancestor (exconjugant), its mat (mating-type) locus genotype, or the mated pair it came from. None of these characteristics was able to interpret simply the large variability among clones. These results provide evidence that the transition from immaturity to maturity is quantitative and complex rather than a jump from one well-defined state to another. © 1992 Wiley-Liss, Inc.
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  • 16
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 47-52 
    ISSN: 0192-253X
    Keywords: Perpetual selfers ; stable progeny from selfers ; Tetrahymena australis ; T. elliotti ; T. shangaiensis ; tetrahymenines ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms.Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing “immature” progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some “immature” progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette. © 1992 Wiley-Liss, Inc.
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  • 17
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 80-86 
    ISSN: 0192-253X
    Keywords: Tetrahymena ; macronuclear development ; chromosome fragmentation ; telomerase ; telomerase RNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Telomeres, the G-rich sequences found at the ends of eukaryotic chromosomes, ensure chromosome stability and prevent sequence loss from chromosome ends during DNA replication. During macronuclear development in Tetrahymena, the chromosomes fragment into pieces ranging from 20 kb to 1,500 kb. Tetrahymena telomerase, a ribonucleoprotein, adds telomeric (TTGGGG)n repeats onto telomeres and onto the newly generated macronuclear DNA ends. We have investigated whether telomerase RNA levels increase during macronuclear development, since such an increase might be expected during chromosomal fragmentation. The steady-state level of the telomerase RNA component was used to estimate the abundance of telomerase present in mating and nonmating Tetrahymena. Northern blot analysis revealed that in vegetatively growing Tetrahymena, there were 18,000-40,000 copies of telomerase RNA per cell. In mating cultures, the levels of RNA increased 2-to 5-fold at 9-15 h, and 1.5- to 3.5-fold in starved nonmating cultures. This increase in telomerase RNA paralleled telomerase activity, which also increased slightly in mating and starved nonmating cells. © 1992 Wiley-Liss, Inc.
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  • 18
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 97-102 
    ISSN: 0192-253X
    Keywords: DNA replication control ; ciliated protozoa ; microinjection ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We show that bacteriophage λ DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. © 1992 Wiley-Liss, Inc.
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  • 19
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 118-125 
    ISSN: 0192-253X
    Keywords: Microinjection ; macronucleoplasm ; transformation ; Paramecium ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA+ or cnrB+) of the donor 9-24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm from P. multimicronucleatum and P. tetraurelia to P. caudatum. resulted in the expression of foreign genes in P. caudatum. In one case, injection of macronucleoplasm of P. tetraurelia produced a stable transformant indicating replication of foreign macronucleoplasm in P. caudatum. This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation among Paramecium species, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley-Liss, Inc.
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  • 20
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 126-132 
    ISSN: 0192-253X
    Keywords: Macronucleus ; macronuclear development ; Rf ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During vegetative, asexual reproduction in heterozygous Tetrahymena thermophila, the macronucleus divides amitotically to produce clonal lineages that express either one or the other allele but not both. Because such phenotypic assortment has been described for every locus studied, its mechanism has important implications concerning the development and structure of the macronucleus. The primary tools to study assortment are Rf/ the rate at which subclones come to express a single allele stably, and the output ratio, the ratio of assortee classes. Because Rf is related to the number of assorting units, a constant Rf for all loci suggests that all genes are maintained at the same copy number. Output ratios reflect the input ratio of assorting units, with a 1:1 output ratio implying equal numbers of alleles at the end of macronuclear development. Because different outcomes would suggest a different macronuclear structure, it is crucial that these parameters be accurately measured. Although published Rf values are similar for all loci measured, there has been no commonly accepted form of presentation and analysis. Here we examine the experimental determination of Rf. First, we use computer simulation to describe how the variability inherent in the assortment process affects experimental determination of Rf. Second, we describe a simple method of plotting assortment data that permits the uniform calculation of Rf, and we describe how to measure Rf accurately in instances when it is possible to score only the recessive allele. Using this method to produce truly comparable Rfs for all published data, we find that most, if not all, loci assort at Rfs consistent with ∼45 assorting units, as has been asserted. © 1992 Wiley-Liss, Inc.
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  • 21
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 151-159 
    ISSN: 0192-253X
    Keywords: Tetrahymena ; mutants ; secretion ; mucocysts ; immunofluorescence ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Dense-core granules represent an adaptation of specialized secretory cell to facilitate stimulus-regulated release of stored proteins. Such granules are a prominent feature of mammalian neuroendocrine and exocrine cells and are also well developed in the ciliates. In Tet-rahymena thermophila, the ability to generate mutants in dense-core granule biosynthesis and fusion presents a versatile system for dissecting steps in regulated exocytosis. We have previously shown that defective granules in such mutants could be characterized by several biochemical criteria, including buoyant density, which increases during maturation, and the degree of proteolytic processing of the content precursors. We have now used indirect immunofluorescence, taking advantage of a monoclonal antibody directed against a granule protein, to visualize the morphology and distribution of both granules and putative granule intermediates in mutant and wild-type cells. The results are consistent with the biochemical analysis and extend our characterization of the mutants, allowing us to distinguish four classes. In addition, the assay represents a powerful technique for diagnosis of new mutants. © 1992 Wiley-Liss, Inc.
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  • 22
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 174-179 
    ISSN: 0192-253X
    Keywords: Conjugation rescue ; Tetrahymena ; nonexcitable mutant ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Tetrahymena nonreversal (TNR) mutants of Tetrahymena thermophila are behavioral mutants with nonexcitable membranes. When cells of the tnrB mutant were mated with wild type, a phenotypic change occurred about l h after pair formation. The pairs began to lose their heterotypic character in stimulation solution containing high potassium and, within 1 1/2h, they were not distinguishable from the wild-type homotypic pairs. On the contrary, although pairs of the tnrA and wild type also lost their heterotypic character about 1 1/2 h after pair formation, they never showed a full response as wild-type homotypic pairs. When tnrA was mated with tnrB more than 50% of pairs expressed a heterotypic pair character 2 h after pair formation, consistent with the tnrB defect having been rescued but not the tnrA defect. Thus, conjugation rescue of the mutant phenotype is locus dependent and probably reflects the nature of the gene products controlling voltage-dependent Ca2+ channels. © 1992 Wiley-Liss, Inc.
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  • 23
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 181-186 
    ISSN: 0192-253X
    Keywords: Inheritance ; non-genic ; pattern-formation ; ciliate ; Stentor coeruleus ; Tartar ; Vance ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The one form of cytoplasmic inheritance that has not been assimilated into the Central Dogma is the inheritance of surface structural patterns, a phenomenon most clearly expressed in cilates. Vance Tartar, although he worked with a genetically undomesticated organism (Stentor coeruleus), provided early evidence for the crucial role of clonally propagated features of the cell cortex. He showed that the capacity for development of cortical organelle systems is associated with a particular relational feature, the “locus of stripe contrast” (LSC), and that clonally inherited cortical variants (homopolar doublets) could be created at will by microsurgical operations that duplicated the LSC. Tartar also hoped to demonstrate the existence of what David Nanney called “cellular architects” by provoking stentors to carry out entirely novel types of morphogenetic performances. He eventually acknowledged failure, although the bizarre juxtapositions by which he attempted to elicit such novel performances did bring about specific and illuminating defects in cortical development. Subsequent analyses of similar defects in other ciliates revealed not the unitary “pattern factor” postulated by Tartar, but rather a hierarchy of distinct patterning mechanisms. Nonetheless, by pursuing an embryological approach toward morphogenesis in a highly regulative ciliate, Tartar uncovered relational aspects of pattern-determination; this, in my view, delineates the major problem that we must solve to gain understanding of intracellular patterning. © 1992 Wiley-Liss, Inc.
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  • 24
    ISSN: 0192-253X
    Keywords: Cell division ; microinjection ; division plane ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Understanding the mechanism that determines the cell division plane is one of the most important problems in the fields of cell and developmental biology. Studying the timing and site of formation of contractile ring (CR) micro-filaments provides key information for solving the problem. We tried to create a nonfunctional CR in Tetrahymena by microinjecting rabbit skeletal muscle actin, which can copolymerize with Tetrahymena actin but has properties different from those of Tetrahymena actin. When skeletal muscle actin was injected in a predivision stage, before the onset of furrow constriction, long-term arrest of cell division was observed. Muscle actin did not cause any delay in cell division when the actin was injected at any stage other than the predivision stage. In all cases, muscle actin had little affect on other actin-related functions. Injected skeletal muscle actin polymerized near the equatorial division plane in cases of cell division arrest; it polymerized at other nonspecific locations when cell division was observed. Arrest occurred when the microinjection took place in the 17-min period just before the start of furrowing. This period coincides with the occurrence of equatorial deposits of p85, which is also suggested to be required for the determination of the division plane. The present experimental results are consistent with the idea that p85 is a crucial factor for determining the cell division plane and also functions as a polymerization nucleus for CR microfilaments. © 1992 Wiley-Liss, Inc.
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  • 25
    ISSN: 0192-253X
    Keywords: Surface antigen ; Paramecium primaurelia ; macronuclear DNA ; DNA rearrangement ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Paramecium primaurelia cell surface is covered with a high molecular weight protein called the surface antigen. Several genes encode alternative surface antigens, but only one is expressed at a time. In addition, each of these genes shows a high degree of allelic polymorphism. Paramecium primaurelia strains 156 and 168 have different alleles of the G antigen gene whose respective antigens can be distinguished in vivo using specific antibodies. An interallelic exclusion phenomenon has been previously described: 94% of the 156/168 heterozygotes express only the 156 allele of the G gene; 6% express both the 156 and the 168 alleles. The phenotype of the heterozygotes is determined at the time of macronuclear differentiation. We have investigated the molecular basis for the different heterozygous phenotypes. Both mRNAs are always produced, and the 156 mRNA is always more abundant than the 168 mRNA. The relative amounts of these messages, however, vary greatly between different heterozygotes and parallel their phenotype. Pushing the analysis further, we show that the copy number of each allele in the macronucleus correlates with the relative amounts of the mRNAs. However, allelic dosage alone is not sufficient to explain the variations of the mRNA ratio. The G antigen gene is located near a telomere in the macronucleus. We show that the distance between the 156G gene and the telomere is different in homozygotes and heterozygotes. It also varies among heterozygotes and is correlated with the mRNA ratio. Thus, we have identified two different parameters, both linked to the genome rearrangements occurring during macronuclear differentiation, that correlate with the relative expression of the two alleles. Two hypotheses concerning the influence of the telomere position on the expression of the gene are discussed. © 1992 Wiley-Liss, Inc.
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  • 26
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    Developmental Genetics 13 (1992), S. 277-288 
    ISSN: 0192-253X
    Keywords: Broad-Complex ; gypsy ; eggshell ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Broad-Complex (BR-C) appears to encode factors that mediate ecdysone effects during the larva-adult transition. The main goal of this study was to gain insight into what roles the BR-C might play during oogenesis. The main findings are as follows. First, as determined by heteroallele studies and clonal analysis, de12 is a somatic line mutation that appears to fall into the broad domain of the BR-C. Second, the de12 mutation is associated with the insertion of the gypsy transposon at position 169.5 (Chao and Guild, Embo J, 1986, 5:143-150) in the BR-C domain. In its new context this gypsy element exhibits ovarianspecific activation. Both this gypsy activation and the de12 phenotype are partially suppressible by su(f) and su(Hw). Third, we have identified a set of transcripts that cross-hybridize with BR-C sequence spanning the gypsy insertion site (166-179). There are significant differences in these cross-hybridizing species, both in size and relative abundance, between de12 and its parent strain. Finally we have determined that in de12 there is a premature arrest of chorion gene amplification in the late stages of oogenesis. © 1992 Wiley-Liss, Inc.
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  • 27
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 223-228 
    ISSN: 0192-253X
    Keywords: Micronucleus ; macronucleus ; conjugation ; oral apparatus ; nuclear transplantation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.
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  • 28
    ISSN: 0192-253X
    Keywords: rRNA ; litostomes ; hypotrichs ; hetero-trichs ; karyorelictids ; postciliodesmatophora ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An rRNA phylogeny of 22 species of ciliates belonging to seven of Small and Lynn's eight classes has been obtained by distance and parsimony methods. It displays good congruence with classical systematics at low taxonomic levels and several major surprises at higher levels: (1) The species analyzed group into five major branches, four of which emerge almost simultaneously: hypotrichs, oligohymenophorans, lito-stomes, and nassophoreans corresponding to four of Small and Lynn's classes. The simultaneous emergence of these groups contradicts the long accepted view that litostomes (a group with “simple”, symmetrical, apical oral apparatus) are “primitive,” while hypotrichs are “highly evolved.” (2) Heterotrichs group with a karyorelictid, together forming the first emerging branch. While this supports the view that karyorelictids may be early-emerging ciliates, it completely explodes the traditional “spirotrichs” taxon, which united heterotrichs and hypotrichs. Instead, this reinforces the concept of Postciliodesmatophora and suggests that asymmetric oral apparatuses (i.e., with distinct paroral and adoral ciliatures) may be primitive in ciliates. The global topology of the tree therefore does not fit with the classical views of ciliate evolution, from “simple” oral apparatus and stomatogenesis to “complex” ones. Instead, a rather striking agreement with the strategy adopted to construct the cortical framework was disclosed. We noted that the cytoskeletal elements used to strengthen the cell surface could be subdivided into four main types: epiplasm, filaments, continuous microtu-bules, or basal body derived fibers. These four types fitted quite well with the major evolutionary lines disclosed by the molecular phylogeny. We therefore discuss unorthodox hypotheses assuming an early explosive radiation of ciliates into a small number of major lineages differing essentially in the solution adopted to subtend the cell surface and anchor the infraciliature. © 1992 Wiley-Liss, Inc.
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  • 29
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 30
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 31
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    Developmental Genetics 13 (1992), S. 387-391 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 32
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    Developmental Genetics 13 (1992), S. 87-93 
    ISSN: 0192-253X
    Keywords: Chromosome fragmentation ; ciliated protozoa ; copy number control ; DNA rearrangement ; gene amplification ; mating type determination ; nuclear dimorphism ; polymerase chain reaction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Ciliates possess nuclear dimorphism, i.e., they carry two structurally and functionally differentiated types of nuclei. The micronucleus and macronucleus serve as the germline and somatic nuclei, respectively, of the cell. The macronucleus differentiates from a mitotic sister of the micronucleus once per life cycle. Macronuclear differentiation is accompanied by a developmentally programmed set of DNA rearrangements, including chromosome fragmentation, telomere addition, and amplification. Given the diploidy of the MAC anlage, are both homologous copies of a chromosome processed and amplified equally and simultaneously in an individual differentiating MAC? We have approached this question for the case of the rDNA, exploiting previously identified DNA polymorphisms and the sensitivity of PCR. We determined allelic ratios in individual caryonide cells, i.e., the cells carrying the primary products of MAC differentiation, prior to the first division of the newly differentiated MAC. We observed stochastic variability in allelic ratios among caryonides that start with genetically identical heterozygous MACs. Either rDNA type can be in the majority. Appropriate controls make it unlikely that the ratios observed were significantly affected by variation in the assay itself. The variability may well result from the statistical variation associated with the relative timing of individual biochemical events initiating the processing and/or amplification of a few rDNA precursor molecules, presumably 4-8 at the most, in a MAC anlage. In addition to this stochastic variability, we observed a small but distinct bias in favor of the C3 rDNA. Thus the replication advantage of C3 relative to B rDNA in heterozygous MACs, previously detected during vegetative multiplication, may begin to be expressed during developmental amplification. We discuss the relevance of this stochastic developmental variability to classical genetic observations of Nanney and their collaborators on other T. thermophila loci. © 1992 Wiley-Liss, Inc.
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  • 33
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    Developmental Genetics 13 (1992), S. 216-222 
    ISSN: 0192-253X
    Keywords: Tetrahymena ; partial cytokinesis ; Positioning ; cdaA1 mutant ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During cytokinesis, furrowing creates new boundaries for daughter cells. Following a shift to a restrictive temperature, cells of the temperature-sensitive cell-division-arrest (cdaA1) mutant of Tetrahymena thermophila complete development of the oral apparatus for the prospective posterior daughter cell before becoming arrested in cytokinesis. When maintained under weak restrictive conditions (35°C), some of the chains were arrested prior to the start of fission line formation (D-shaped chains), whereas others manifested rudimentary unilateral furrowing on the ventral side (B-shaped chains). In their second cell cycle following the temperature shift, the D-shaped chains usually formed only one oral primordium, at a position highly correlated with the length of the entire chain. The B-shaped chains always produced two separate oral primordia, located at irregular positions anterior and posterior to the division furrow, often close to the posterior oral apparatus produced during the first cycle. These results suggest that the formation of the fission line sets a reference boundary to assess the number of oral primordia and influence their position, that appear during subsequent morphogenetic episodes. They also indicate that, during cell division cycles, pre-existing oral apparatuses do not strongly inhibit the formation of new oral apparatuses in their close vicinity. © 1992 Wiley-Liss, Inc.
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  • 34
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    Developmental Genetics 13 (1992) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 35
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 111-117 
    ISSN: 0192-253X
    Keywords: Tetrahymena ; copy number ; histone H4 ; macronuclear DNA molecules ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In Tetrahymena, the DNA of the macronucleus exists as very large (100 to 4,000-kb) linear molecules that are randomly partitioned to the daughter cells during cell division. This genetic system leads directly to an assortment of alleles such that all loci become homozygous during vegetative growth. Apparently, there is a copy number control mechanism operative that adjusts the number of each macronuclear DNA molecule so that macronuclear DNA molecules (with their loci) are not lost and aneuploid death is a rare event. In comparing Southern analyses of the DNA from various species of Tetrahymena using histone H4 genes as a probe, we find different band intensities in many species. These differences in band intensities primarily reflect differences in the copy number of macronuclear DNA molecules. The variation in copy number of macronuclear DNA molecules in some species is greater than an order of magnitude. These observations are consistent with a developmental control mechanism that operates by increasing the macronuclear copy number of specific DNA molecules (and the genes located on these molecules) to provide the relatively high gene copy number required for highly expressed proteins. © 1992 Wiley-Liss, Inc.
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  • 36
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    Developmental Genetics 13 (1992), S. 143-150 
    ISSN: 0192-253X
    Keywords: Extrachromosomal nucleoli ; in vitro transcription ; chromatin ; ribosomal DNA (rDNA) ; Tetrahymena ; P1 nuclease mapping ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An in vitro transcription system was established using extrachromosomal nucleoli from Tetrahymena pyriformis macronuclei as a template. Ribosomal precursor RNA (pre-rRNA) and nascent pre-rRNA chains were removed from isolated nucleoli by treatment with RNase T1 and Sarkosyl. Nucleoli were then incubated in a RNA synthetic cocktail containing a cellular extract from Tetrahymena thermophila. The transcription product was examined for the presence of transcripts from T. pyriformis ribosomal DNA (rDNA) by P1 nuclease protection mapping using a DNA probe from a T. pyriformis rDNA clone. A sequence difference between T. pyriformis and T. thermophila in the 5′ region of their 35S pre-rRNAs permitted exclusive detection of T. pyriformis transcripts. The results showed that faithful transcription initiation occurred in vitro from the in vivo initiation site of the nucleolar template and that the nucleolar template had a much higher efficiency of transcription than that of the purified rDNA clone. This system may offer unique advantages for future studies of tran-scriptional control during development and differentiation. © 1992 Wiley-Liss, Inc.
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  • 37
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    Developmental Genetics 13 (1992), S. 160-166 
    ISSN: 0192-253X
    Keywords: Deletion mapping ; exocytosis ; genetic complementation ; genomic exclusion ; mucocyst ; nullisomic strains ; regulated secretion ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Under appropriate conditions, Alcian Blue-induced exocytosis of Tetrahymena mucocysts leads to formation of a capsule that surrounds the cell. This phenomenon is an example of regulated secretion, a mechanism of fundamental significance in eukaryotic cells. In order to dissect genetically the mechanism of mucocyst biogenesis and regulated exocytosis, mutants unable to form capsules (Caps-) were isolated. In this paper we report a genetic characterization of Caps- mutants in this collection. The mutations in mutants SB255 and SB281 behave as single recessive Men-delian mutations. The mutation in SB251 is restricted to the macronucleus, and could not be further characterized by the genetic methods we used. Complementation tests suggest the existence of at least 2 genes, named exoA and exoB; additional mutant loci are likely to be included in the mutant collection. Deletion mapping using nulli-somic strains showed that exoA and exoB are located on the left arm of chromosome 4. The exo-3 mutation, which behaves as recessive and complements with exoA1 in SB255 and exoB2 in SB281, maps to chromosome 3. These Caps- mutants may be useful for the elucidation of the developmental pathway of mucocyst biogenesis and the control of regulated secretion in eukaryotic cells. © 1992 Wiley-Liss, Inc.
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  • 38
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    Developmental Genetics 13 (1992), S. 194-202 
    ISSN: 0192-253X
    Keywords: Cellular morphogenesis ; polyphos-photidylinositide cycle ; myo-inositol ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The long-known teratogenic effects (dorsalisation) of lithium on amphibian embryos has recently raised renewed interest. As it is known that lithium blocks the polyphosphoinositide (PI) cycle, causing a depressed level of myo-inositol, and as injections of myo-inostiol have been shown to rescue the effects of Li+, it was postulated that Li+ causes a flattening of gradients of PI cycle activity underlying the developmental polarities. We have studied the effect of Li+ on the morphogenesis of the unicellular organism, Paramecium. We show (1) that exposure to 25 mM Li+ during division yields precise distorsions of the cortical pattern that can be explained by a uniformisation of surface growth i.e. partial suppression of the right/left and antero/posterior asymmetries and (2) that Li+ effects are rescued by injection of myo- inositol. These results suggest that spatially graded activity of the PI cycle (ensuring in turn a spatially graded distribution of secondary messengers directly involved in the morphogenetic processes) appeared early in evolution. © 1992 Wiley-Liss, Inc.
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  • 39
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    Developmental Genetics 13 (1992), S. 229-234 
    ISSN: 0192-253X
    Keywords: Oral morphogenesis ; cc1 mutation ; cell cycle ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This study examines the timing of micronuclear mitosis during the vegetative cell cycle and shows that mitosis begins early in the division process and coincides approximately with the earliest stages of oral morphogenesis (about 0.6 in the cell cycle in synchronous cell samples). The cc1 mutation blocks cell cycle progression prior to the point of commitment to division. Although the cc1 mutation blocks macronuclear DNA synthesis under restrictive conditions, it does not block micronuclear DNA synthesis. However, absence of functional cc1 gene product leads to blockage of micronuclear mitosis prior to completion of anaphase. This point coincides with commitment to division and is also the point at which oral morphogenesis is blocked in cc1 cells. The tim-ings of the transition points for micronuclear mitosis and oral morphogenesis in cc1 cells are closely associated in both synchronous cell samples and in asynchronous cultures. © 1992 Wiley-Liss, Inc.
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  • 40
    ISSN: 0192-253X
    Keywords: Zea mays ; endosperm development ; in situ hybridization ; zein spatial expression ; highlysine mutants ; Opaque-2 transcript localization ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Endosperm development in maize seed involves the multiplication, enlargement, and differentiation of cells with consequent accumulation of storage products. The storage protein genes, encoding zeins, and glutelins (multigene families) are expressed and developmentally regulated by different loci. Wild-type lines and genotypes carrying mutations at loci affecting zein synthesis (o2, o7, fl2, and prol) were characterized at the molecular level and investigated by Northern analysis in order to define the expression of structural and regulatory genes. In situ hybridization in both wild-type and mutant lines was performed to visualize the spatial distribution of transcripts representing each gene family, during endosperm development. The zein and glutelin mRNAs are expressed in all endosperm cells, except for the aleurone layer. However, each mRNA type accumulates at a different level in the various endosperm regions, thus allowing to recognize specific territories of expression for each storage protein mRNA within the tissue. The spatial expression patterns appear early for each gene type and are maintained during the course of endosperm development. Also, the quantitative distribution of the same transcripts in endosperm of mutant lines is specific for each mutant and different from that of the wild-type. Furthermore, the amount of the O2 transcript, present in the nucleus and cytoplasm of wild-type cells, varies substantially in the different o2 mutations considered, in one mutant almost exclusively confined within the nucleus. These data suggest a specific control of the spatial expression of storage protein genes and a heterogeneous molecular composition of protein bodies throughout the endosperm tissue. © 1992 Wiley-Liss, Inc.
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  • 41
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    Developmental Genetics 13 (1992), S. 367-375 
    ISSN: 0192-253X
    Keywords: FLP-recombinase ; FRT ; embryonic mosaics ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An efficient method for generating embryonic mosaics using a yeast site-specific recombinase (FLP), under the control of a heat shock promoter, is described. FLP-recombinase can promote mitotic exchange between homologous chromosomes that contain FRT (FLP Recombination Target) sequences. To demonstrate the efficiency of FLP-recombinase to generate embryonic mosaics, clones of the recessive and cell autonomous mutation armadillo (arm), detected by their ability to differentiate ectopic denticles in the naked cuticle of each abdominal segment, have been induced. We have analyzed the parameters of FLP-recombinase induced embryonic mitotic recombination and have demonstrated that clones can be efficiently induced during the postblastoderm mitotic divisions. We discuss applications of this technique for the analyses of the roles of various mutations during embryonic patterning. © 1992 Wiley-Liss, Inc.
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  • 42
    ISSN: 0192-253X
    Keywords: Vertebrates ; homeodomain ; engrailed ; evolutionary conservation ; gene regulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have isolated and characterized genomic DNA clones for the human and chicken homologues of the mouse En-1 and En-2 genes and determined the genomic structure and predicted protein sequences of both En genes in all three species. Comparison of these vertebrate En sequences with the Xenopus En-2 (Hemmati-Brivanlou et al., 1991) and invertebrate engrailed- like genes showed that the two previously identified highly conserved regions within the En protein [reviewed in Joyner and Hanks, 1991] can be divided into five distinct subregions, designated EH1 to EH5. Sequences 5′ and 3′ to the predicted coding regions of the vertebrate En genes were also analyzed in an attempt to identify cis-acting DNA sequences important for the regulation of En gene expression. Considerable sequence similarity was found between the mouse and human homologues both within the putative 5′ and 3′ untranslated as well as 5′ flanking regions. Between the mouse and Xenopus En-2 genes, shorter stretches of sequence similarity were found within the 3′ untranslated region. The 5′ untranslated regions of the mouse, chicken and Xenopus En-2 genes, however, showed no similarly conserved stretches. In a preliminary analysis of the expression pattern of the human En genes, En-2 protein and RNA were detected in the embryonic and adult cerebellum respectively and not in other tissues tested. These patterns are analogous to those seen in other vertebrates. Taken together these results further strengthen the suggestion that En gene function and regulation has been conserved throughout vertebrate evolution and along with the five highly conserved regions within the En protein, raise an interesting question about the presence of conserved genetic pathways. © 1992 Wiley-Liss, Inc.
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  • 43
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 1-8 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 44
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 58-65 
    ISSN: 0192-253X
    Keywords: Tetrahymena conjugation ; cell separation ; macronuclear gene expression ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A new recessive conjugation lethal mutation was found in Tetrahymena thermophila which was named mra for macronuclear resorption arrest. Other events affected by the mra mutations are separation of pairs, DNA replication in the macronuclear anlagen, and resorption of one of the two micronuclei. In wild-type crosses 50% of the pairs had separated by 12 hr after mixing two mating types and had completed resorption of the old macronucleus 1-2 hr later. In contrast most mra conjugants did not separate even by 24 hr after mixing and the old relic (condensed) macronucleus was seen in over 90% of them.After addition of 10mM calcium to the conjugation medium, the mra conjugants did separate but they still failed to complete resorption of the old macronucleus and to replicate macronuclear anlagen DNA in the exconjugants. The calcium induced separation of the mra conjugants occurred later than the separation of control pairs.During normal conjugation cell separation occurs before the first expression of known macronuclear genes and prior to processing of the macro-nuclear DNA. Therefore, the mra phenotype infers that separation of conjugants requires a signal which is produced by the macronuclear anlagen at an unusually early time. © 1992 Wiley-Liss, Inc.
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  • 45
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 103-110 
    ISSN: 0192-253X
    Keywords: Tetrahymena ; macronuclear organization ; DNA elimination ; unequal division ; copy number control ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The macronucleus of Tetrahymena contains a large number of DNA molecules of subchromosomal size. They belong to about 270 species each one occurring at an average number of 45 copies Macronuclei divide unequally and nothing is known of segregation control. This and the elimination and degradation of DNA during macronuclear amitosis make the clonal stability of macronuclei a problem of qualitative and quantitative control on a subchromosomal level.We studied the contribution of DNA elimination to the quantitative composition of the macronucleus cytophotometrically in single cells of different strains. This was done under standard conditions and under conditions known to influence the amount of macronuclear DNA. The following results were found: Elimination of DNA occurs at almost every division. The size of the elimination body is highly variable but still positively correlated with the macronuclear DNA content. In T. thermophila the amount of eliminated DNA is 2.5% of the G2 content and is not dependent on the growth state. It varies with species, amounting to as much as 8% in T pigmentosa. During conditions which increase the macronuclear DNA content, very little DNA is eliminate. On the other hand, large amounts are eliminated under other conditions causing the macronuclear DNA content to decrease. DNA to be eliminated at division is synthesized at the same time as bulk DNA.We developed a computer program which helps us study the effects of DNA elimination and unequal divisions upon the copy numbers of subchromosomal DNA classes.The result indicates that in a given cell line at least one of the DNA molecules becoms extinct after 60 generations which we expect would cause the cell's extinction and restrict a clone's life to 60 generations. As this does not happen in nature, there must be some control of the copy numbers preventing their extinction during vegetative multiplication. Whether elimination increases or decreases the imbalance of genes remains to be investigated. © 1992 Wiley-Liss, Inc.
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  • 46
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 133-142 
    ISSN: 0192-253X
    Keywords: Group I introns ; intron homing ; rDNA inheritance in Tetrahymena ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have previously argued from phylogenetic sequence data that the group I intron in the rRNA genes of Tetrahymena was acquired by different Tetrahymena species at different times during evolution. We have now approached the question of intron mobility experimentally by crossing intron+ and intron- strains looking for a strong polarity in the inheritance of the intron (intron homing). Based on the genetic analysis we find that the intron in T. pigmentosa is inherited as a neutral character and that intron+ and intron- alleles segregate in a Mendelian fashion with no sign of intron homing. In an analysis of vegetatively growing cells containing intron+ and intron- rDNA, initially in the same macronucleus, we similarly find no evidence of intron homing.During the course of this work, we observed to our surprise that progeny clones from some crosses contained three types of rDNA. One possible explanation is that T. pigmentosa has two rdn loci in contrast to the single locus found in T. thermophila. Some of the progeny clones from the genetic analysis were expanded for several hundred generations, and allelic assortment of the rDNA was demonstrated by subcloning analysis. © 1992 Wiley-Liss, Inc.
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  • 47
    ISSN: 0192-253X
    Keywords: Acid hydrolases ; sec allele ; phenotype ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The genetics and phenotypic features (light and electron microscopy) of a secretory mutant, MS-1, of Tetrahymena thermophila blocked in secretion of lysosomal acid hydrolases have been analyzed. Although blocked constitu-tively in secretion, MS-1 contains active lysosomal hydrolases in amounts equivalent to the wild type. The 3:1 segregation in F-2 in sib crosses and the 1:1 segregation in test crosses indicate that the block in secretion of lysosomal hydrolases is controlled by a recessive single gene locus termed sec. The sec allele of MS-1 proved also to be responsible for the highly vacuolarized phenotype the mutant developed when it was transferred from nutrient medium into buffers of low ionic strength. Deletion mapping by crossing MS-1 with nullisomic strains, all secreting lysosomal hydrolases at wild-type rates, was performed. The sec phenotype was expressed in monosomic-4 progeny only, indicating that the sec allele is located on chromosome 4 of T. thermophila. © 1992 Wiley-Liss, Inc.
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  • 48
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 49
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    Developmental Genetics 13 (1992), S. 203-209 
    ISSN: 0192-253X
    Keywords: Ciliate ; basal body ; Stentor ; size dependent regulation ; ultrastructure ; adoral zone of membranelles ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The number and length of oral membranelles were determined for both large and small Stentor from well-fed, growing cultures and nutrient-deprived cultures, respectively. Small cells possess both significantly fewer and shorter membranelles than do large cells. For both large and small cells, each membranelle is composed of three rows of basal bodies. The membranelles closest to the gullet have a third row that is only slightly shorter than the other two. The third row becomes rapidly shorter as membranelles become increasingly distant from the gullet. A short distance from the gullet, and for the remainder of the band, the third row is composed ofonly one to four basal bodies. The first two rows consist of approximately 35 basal bodies each in large cells and approximately 26 basal bodies each in small cells. This indicates that Stentor regulates the number of basal bodies per row, but not the number of rows, in response to changes in cell size. © 1992 Wiley-Liss, Inc.
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  • 50
    ISSN: 0192-253X
    Keywords: Ciliate ; cortex ; basal body ; morphogenesis ; pattern formation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The present study reveals a deficiency in the number of ciliated basal bodies along 180° rotated ciliary rows (IRs) in Tetrahymena. This feature is common to IRs recently generated in young clones with stable corticotypes (total number of ciliary rows per cell), irrespective of the number of IRs present per cell or their cellular location, and is found before the cell loses any of the IRs. In cells bearing three IRs, the IRs on the two sides of the inversion immediately next to normal ciliary rows (junctures) exhibit an even greater deficiency in ciliated basal bodies, compared to the IR located internally between two other IRs; the normal ciliary rows flanking the inversion are also somewhat deficient. These observations show that the IRs of Tetrahymena are structurally deficient, hence developmentally defective, and suggest that they are intrinsically unstable. We propose that basal body development along IRs tends to be truncated before the stage of ciliation; such basal bodies would fail to acquire the potential to serve as nucleating centers for new basal body development in the next round of basal body proliferation, leading to the eventual loss of the IRs. © 1992 Wiley-Liss, Inc.
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  • 51
    ISSN: 0192-253X
    Keywords: Thyroid hormone ; carbamyl phosphate synthetase ; Rana catesbeiana ; metamorphosis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During both spontaneous and thyroid hormone (TH)-induced metamorphosis, the Rana catesbeiana tadpole undergoes postembryonic developmental changes in its liver which are necessary for its transition from an ammonotelic larva to a ureotelic adult. Although this transition ultimately results from marked increases in the activities and/or de novo synthesis of the urea cycle enzymes, the precise molecular means by which TH exerts this tissue-specific response are presently unknown. Recent reports, using RNA from whole Xenopus laevis tadpole homogenates and indirect means of measuring TH receptor (TR) mRNAs, suggest a correlation between the up-regulation of TRβ-mRNAs and the general morphological changes occurring during amphibian metamorphosis. To assess whether or not this same relationship exists in a TH-responsive tissue, such as liver, we isolated and characterized a cDNA clone containing the complete nucleotide sequence for a R. catesbeiana urea cycle enzyme, ornithine transcarbamylase (OTC), as well as a genomic clone containing a portion of the hormone-binding domain of a R. catesbeiana TRβ gene. Through use of these homologous sequences and a heterologous cDNA fragment encoding rat carbamyl phosphate synthetase (CPS), we directly determined the relative levels of the TRβ, OTC, and CPS mRNAs in liver from spontaneous and TH-induced tadpoles. Our results establish that TH affects an up-regulation of mRNAs for its own receptor prior to up-regulating CPS and OTC mRNAs. Moreover, results with cultured tadpole liver demonstrate that TH, in the absence of any other hormonal influence, can affect an up-regulation of both the TRβ and OTC mRNAs. © 1992 Wiley-Liss, Inc.
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  • 52
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    Developmental Genetics 13 (1992), S. 326-330 
    ISSN: 0192-253X
    Keywords: Homeotic ; segmentation ; Pc-group genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: polyhomeotic is a member of a group of genes, the Pc-group responsible for the maintenance of gene expression during development. In particular, the Pc-group of genes is involved in the correct expression of homeotic genes of the bithorax and Antennapedia complexes.Molecular analysis reveals that the Pc-group genes function relatively late in development, once homeotic gene expression has been correctly initiated. This initiation of homeotic gene expression depends on interaction between genes in the segmentation gene hierarchy, the gap and pair-rule genes.The in situ analysis presented here indicates that polyhomeotic transcripts are expressed in a spatially restricted fashion early in development. This blastoderm expression is under the control of genes in the segmentation hierarchy. Given these results, and the role of polyhomeotic in the correct maintenance of homeotic gene expression, I propose that polyhome otic expression may relay the initiation of homeotic gene expression with other mechanisms involved in the maintenance of this expression, involving the other Pc-group genes. © 1992 Wiley-Liss, Inc.
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  • 53
    ISSN: 0192-253X
    Keywords: Meiosis ; heterochromatin ; homology ; pairing ; segregation ; Drosophila ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle. © 1993 Wiley-Liss, Inc.
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  • 54
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    Developmental Genetics 13 (1992), S. 75-79 
    ISSN: 0192-253X
    Keywords: Nuclear development ; sequence stability ; germ-line limited sequence ; transposable element ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: During mocronuclear development in the ciliated protozoan Tetrahymena thermophila, sequence reorganization including sequence loss occurs. Addressing questions about the organization and nucleotide sequence of micronucleus limited regions can lead to insights about mechanisms of DNA rearrangements during macronuclear development as well as mechanisms for the maintenance of the stability of micronucleus-limited sequence families. We have previously identified a moderately repetitive micronu-cleus-limited sequence family called X-H (family members hybridize to an approximately 450 bp Xbal-HindIII restriction fragment), completely absent from macronuclear DNA. The first member of this family which we isolated is associated with terminal sequences characteristic of a Tel-1 element, a putative micronuclear transposable element. Two additional family members have been isolated which are not closely associated with Tel-1 terminal sequences. We have nucleotide sequence data for three cloned members of the X-H family. This analysis has demonstrated that the longest cloned members of the X-H family share a region of homology of approximately 2,400 bp and are highly conserved, differing only by small insertions or deletions of 100 bp or less. The sequences from one of the sequenced family members flanking the region of homology are themselves mostly micronucleus-limited. © 1992 Wiley-Liss, Inc.
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  • 55
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 56
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 9-15 
    ISSN: 0192-253X
    Keywords: Pheromone secretion ; ciliate immaturity ; pheromone gene expression ; autocrine binding sites ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of 125 I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10-9 M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 106 per cell of 5-7 fissions of age, to about 16 × 106 at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from “immature” to “adult,” that is competent to respond as well to pheromones of conspecific, genetically different cells. © 1992 Wiley-Liss, Inc.
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  • 57
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    Developmental Genetics 13 (1992), S. 34-40 
    ISSN: 0192-253X
    Keywords: Deletion mapping ; genomic exclusion ; nullisomic clones ; monosomic clones ; inbred strains ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Tetrahymena thermophila has a multiple mating type system. While a sexually mature cell usually expresses only one mating type, its germline (micronucleus) carries the genetic potential for 5 to 7 mating types. The set of allowed mating types is specified by the mat locus. The choice of which particular mating type is expressed by a cell reflects a somatically inherited, developmentally programmed differentiation of the somatic nucleus (macronucleus). In this work we report that the mat locus maps to the left arm of chromosome 2, as determined by nullisomic deletion mapping. We also report a distance of 29 cM between the mat locus and the ribosomal RNA gene, previously mapped to chromosome 2L. This represents another (rare) case of meiotic linkage in Tetrahymena. © 1992 Wiley-Liss, Inc.
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  • 58
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    Developmental Genetics 13 (1992), S. 53-57 
    ISSN: 0192-253X
    Keywords: Nuclear protein ; macronuclear differentiation ; monoclonal antibody ; Paramecium caudatum ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudotum and P. dubosqui. Immunoblotting showed that the antigen was a poly-peptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macro-nuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus. © 1992 Wiley-Liss, Inc.
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  • 59
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    Developmental Genetics 13 (1992), S. 66-74 
    ISSN: 0192-253X
    Keywords: Gene scrambling ; macronuclear development ; DNA processing ; gene-sized molecules ; recombination ; hypotrich ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the hypotrichous ciliate Oxytricha nova the cloned precursor gene from the micronuclear genome that encodes actin I is composed of highly disordered blocks of deoxynucle-otide sequences. We present and illustrate in detail a recombination model that explains how the actin I gene may be unscrambled during macronuclear development after cell mating. The model was described in a previous publication (Greslin et al.: Proc Natl Acad Sci USA 86:6264-6268, 1989). Here we show the data, described in the earlier publication, that support the model. The data show that scrambling is not an artifact of cloning. They rule against the presence of an unscrambled copy of the actin I gene in the micronucleus, which means that unscrambling must be a part of macro-nuclear development. Finally, the data prove that the actin I gene in O. trifallax is scrambled in a pattern that resembles the pattern in O. nova. © 1992 Wiley-Liss, Inc.
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  • 60
    ISSN: 0192-253X
    Keywords: Lembadion-factor ; cell-transformation ; Euplotes octocarinatus ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A morphogenetically active substance released by the predatory ciliate Lem-badion bullinum is recognized by ciliates of the genus Euplotes, which are potential prey organisms of Lembadion. The substance (L-factor) induces cells of the genus Euplotes to become less compact, which reduces their likelihood of becoming engulfed. Under the influence of this Lembadion- derived signal, E. octocarinatus develops extended wings and dorsal and ventral ridges and transforms within a few hours from its typical ovoid morph into an enlarged circular morph. This takes place without cell division. We have isolated the L-factor and report that it is a protein with a mass of 31,500 Da. The factor has been purified to chromatographic and electrophoretic homogeneity and was found to be active at concentrations as low as 10-12 mol/L. © 1992 Wiley-Liss, Inc.
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  • 61
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    Developmental Genetics 13 (1992), S. 255-255 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 62
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 256-263 
    ISSN: 0192-253X
    Keywords: Minute mutations ; oogenesis ; Drosophila ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Antisense RNAs have been used for gene interference experiments in many cell types and organisms. However, relatively few experiments have been conducted with antisense genes integrated into the germ line. In Drosophila reduced ribosomal protein (r-protein) gene function has been hypothesized to result in a Minute phenotype. In this report we examine the effects of antisense r-protein 49 expression, a gene known to correspond to a Minute mutation An antisense rp49 gene driven by a strong and inducible promoter was transformed into the Drosophila germ line. Induction of this gene led to the development of flies with weak Minute phenotypes and to the transient arrest of oogenesis. Parameters that may affect the success of antisense gene inactivation are discussed. © 1992 Wiley-Liss, Inc.
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  • 63
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    Developmental Genetics 13 (1992), S. 302-305 
    ISSN: 0192-253X
    Keywords: Arginine kinase ; developmental regulation ; Drosophila ; ecdysone ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Arginine kinase displays a distinctive rise and fall in specific activity and specific protein levels during the prepupal stage of Drosophila development with maximal activity occurring at morphological stage P3. This developmentally regulated peak is under the influence of ecdysone. Altered doses of the major ecdysone-inducible “early” genes at cytological regions 75B and 2B5 alter this pattern of expression while altered doses of another major “early” gene at 74EF have no effect. We hypothesize that a product of the 2B5 locus and a product of the 75B locus interact to effect this developmental pattern of expression of Drosophila arginine kinase. © 1992 Wiley-Liss, Inc.
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  • 64
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    Developmental Genetics 13 (1992) 
    ISSN: 0192-253X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
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  • 65
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    Developmental Genetics 13 (1992), S. 235-240 
    ISSN: 0192-253X
    Keywords: Micronuclei ; laser tweezers ; micro-manipulation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have constructed a laser optical force trap (“laser tweezers”) by coupling an Nd:YAG laser to an optical microscope with a high numerical aperture objective. The laser beam (approximately 0.1 W power) is focused to a diffraction-limited spot at the specimen plane of the objective: the wavelength chosen (1,064 nm) is not strongly absorbed by most biological materials and is thus not ablative. Because the intensity of the laser beam increases towards the center of the focal spot, small particles brought near the spot will be attracted to the center and held there. Movement of the laser beam will tend to move any trapped particles with it. The laser tweezers can permit precise, nondestructive repositioning of small structures inside a living cell, without recourse to micromanipulators. Initial work has involved the use of laser tweezers on cells of Paramecium tet-raurelia held by a rotocompressor. We have been able to trap and reposition small organelles, especially the highly refractile structures known as crystals. Using a trapped crystal as a “tool”, we have been able to push micronuclei and other structures for many micrometers to virtually any desired location in a cell. In spite of extended exposure of specific structures and of individual cells to the laser beam, no damage has been detectible. Exposed cells, which were removed from the rotocompres-sor and cultured, showed complete viabilty. The laser tweezers technique shows tremendous potential for applications to the study of many fundamental cellular and developmental phenomena in paramecia and other ciliates. For example, we intend to use this technique to investigate temporal and spatial characteristics of nuclear determining regions during sexual reorganization in Paramecium. © 1992 Wiley-Liss, Inc.
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  • 66
    ISSN: 0192-253X
    Keywords: Drosophila melanogaster ; imaginal disc ; epithelial morphogenesis ; ecdysone ; steroid hormone secondary response ; pupariation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Drosophila imaginal discs are induced by the steroid hormone 20-hydroxy-ecdysone to initiate morphogenesis leading to formation of the adult appendages and thoracic epidermis at the end of the third larval instar. Ecdysone-dependent transcriptional activation of a set of genes that encode imaginal disc transcripts found on membrane-bound polysomes precedes and may be responsible for some aspects of the cellular changes that mediate epithelial morpho-genesis in this system. A 1.35 kb transcript from one of these genes, IMP-L1, is first observed in vivo at or just prior to pupariation, as ecdysone titers are peaking and beginning to decline. Expression is initiated in proximal areas of the antennal disc, later spreading to a more widespread but nonuniform distribution throughout other thoracic imaginal discs. IMP-L1 is not, however, expressed in other ecdysone target tissues such as salivary glands or fat body. The IMP-L1 gene encodes a novel protein product containing a signal peptide, a possible transmembrane domain, two highly charged domains and a proline rich C-terminal domain. We suggest that the delayed timing of expression of this secondary response gene is necessary for proper ordering of cellular events associated with disc morphogenesis. © 1992 Wiley-Liss, Inc.
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  • 67
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    Developmental Genetics 13 (1992), S. 376-386 
    ISSN: 0192-253X
    Keywords: Bronze-1 ; transposable element ; germinal reversion ; somatic excision ; microsporogenesis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Three characteristics of standard Mutator lines reflect developmental regulation: new mutants usually involve single gametes, somatic excision is restricted to terminal cell divisions during tissue development, and germinal excision is rare. By selection for earlier (larger) somatic sectors in the aleurone, a Mutator line was identified that exhibits a dramatic elevation in somatic excision frequency during the first three nuclear divisions of the endosperm and more than a 10-fold increase in germinal reversion from the bzl::Mul reporter gene. The programming of early sectoring is dominant in crosses with Mutator lines containing diverse reporter alleles. Germinal reversion is biased 5- to 10-fold for events through the pollen compared to the ear. The timing of germinal excision in the tassel is late because somatic excision sectors in the anthers are small; however, 98% of the germinal revertants are concordant. These observations indicate that in the early sectoring line Mu excision usually occurs before the mitotic divisions that separate gametic nuclei and may be restricted to the early stages of microsporogenesis. © 1992 Wiley-Liss, Inc.
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  • 68
    ISSN: 0192-253X
    Keywords: Chromosome spreads ; meiotic mutants ; synaptonemal complex ; three-dimensional reconstruction ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have utilized spreading methods as well as serial sectioning three-dimensional reconstruction to examine meiotic chromosome behavior in cells homozygous for the rad3-1 mutation in Coprinus cinereus. Comparison of 42 wild-type nuclei that had been spread, stained with silver, and viewed by electron microscopy with 30 mutant nuclei treated in the same manner revealed several defects in the mutant. Axial core formation was defective in the mutant, although limited side-by-side association of axial cores was observed. To detect any differences in three-dimensional architecture between the wild-type and mutant nuclei, we reconstructed three of the former and six of the latter after serial sectioning. It was not possible to trace the expected number of axial cores from section to section in the mutant, although some tripartite synaptonemal complex was observed. Many axial core ends failed to terminate in the nuclear envelope in the mutant. This spectrum of defects (incomplete axial core assembly with some tripartite synaptonemal complex formation) had not been observed previously in either C. cinereus or other systems. We conclude that this combination of spreading and sectioning methods is very useful for analysis of meiotic mutants. © 1993 Wiley-Liss, Inc.
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  • 69
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    Developmental Genetics 13 (1992), S. 319-325 
    ISSN: 0192-253X
    Keywords: Mammalianembryos ; compaction ; cavitation ; blastocoel expansion ; gene transcription ; mRNA ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: This review summarizes information on accumulation profiles of individual gene transcripts in preimplantation development. Most of the information is from the mouse, but some data from other species are reviewed as well. The principal finding is that the transcription of most genes is not temporally linked with any of the three morphogenetic transitions (compaction, cavitation, and blastocoel expansion) that characterize this period. Most genes that are expressed during pre-implantation development of the mouse are already being transcribed in the 4-cell stage, and some clearly begin as early as the 2-cell stage. Once activated, a gene continues to be transcribed at least into the blastocyst stage, resulting in continuous mRNA accumulation. Thus the pattern of gene transcription established at the time of genomic activation in the 2-cell stage is perpetuated into the blastocyst, with a few additions along the way. This information is interpreted in light of previous findings concerning the sensitivity of morphogenetic transitions to inhibition of gene expression. The lack of a clear relationship between the timing of expression of most genes and the schedule of morphogenesis leads one to conclude that temporal regulation is imposed downstream of transcription and translation. This conclusion is substantiated by a consideration of factors controlling the events of compaction. © 1992 Wiley-Liss, Inc.
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  • 70
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 359-366 
    ISSN: 0192-253X
    Keywords: Glutamine synthetase I ; femalesterile mutations ; D. melanogaster ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A female-sterile mutation (fs(2) PM11-19) was recovered in a screen for P-M hybrid dysgenesis induced mutations uncovered by a deletion of region 21B and was identified as an allele of the gene encoding the Drosophila glutamine synthetase I (GSI) mitochondrial isozyme.Molecular analysis has shown that fs(2)PM11-19 contains a 5 kb insert within 500 bp upstream of the transcriptional start site of the gsI gene. Mutant flies have extremely low levels of gsl transcription and GSI activity. A pre-existing deficiency (Df(2L) netpm1) with a breakpoint near the transcription start site was also found to be a female-sterile allele of gsl.All eggs laid by PM11-19 homozygous females, as well as by females heterozygous for this mutation and a deletion or any of several recessive lethal alleles of the gsl gene, fail to hatch. We conclude that an adequate level of maternally supplied GSI activity is necessary in the early stages of Drosophila embryonic development. © 1992 Wiley-Liss, Inc.
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  • 71
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    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 13 (1992), S. 392-402 
    ISSN: 0192-253X
    Keywords: Meiotic recombination in yeast ; spo13 ; prototrophy ; mitotic cells ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The REC104 gene was initially defined by mutations that rescued the inviability of a rad52 spo13 haploid strain in meiosis. We have observed that rec104 mutant strains undergo essentially no induction of meiotic gene conversion, and we have not been able to detect any meiotic crossing over in such strains. The REC104 gene has no apparent role in mitosis, since mutations have no observable effect on growth, mitotic recombination, or DNA repair. The DNA sequence of REC104 reveals that it is a previously unknown gene with a coding region of 549-bp, and genetic mapping has localized the gene to chromosome VIll near FUR1. Expression of the REC104 gene is induced in meiosis, and it appears that the gene is not transcribed in mitotic cells. Possible roles for the REC104 gene product in meiosis are discussed. © 1993 Wiley-Liss, Inc.
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  • 72
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    Developmental Genetics 13 (1992), S. 435-439 
    ISSN: 0192-253X
    Keywords: Synaptonemal complex ; cDNA ; immunocytology ; hamster ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: As an alternative to the production and use of monoclonal antisynaptonemal complex (SC) antibodies to isolate SC genes, we have explored the use of polyclonal anti-SC antibodies to identify SC genes from a cDNA expression library. The method proved relatively simple, reliable, and fast and has yielded two SC genes. A homologue of one of these genes from a different species has previously been isolated in another laboratory. © 1993 Wiley-Liss, Inc.
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  • 73
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    Developmental Genetics 13 (1992), S. 498-514 
    ISSN: 0192-253X
    Keywords: Meiosis ; homologous extent ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The minimal length of contiguous homology required for successful completion of meiotic recombination was investigated by using heterologous insertions to delimit homologous segments of chromosome III in the yeast Saccharomyces cerevisiae. Constructs created in vitro by insertion of selectable markers into the LEU2 locus were transplaced into haploid strains, which were then mated to create diploids containing pairs of insertion heterologies at various distances. Analysis of the meiotic products from these diploids revealed a gradient in the frequency of both reciprocal and nonreciprocal recombination declining monotonically from the 5′ end of LEU2. Both types of event were found to be restricted by the presence of the insertion heterologies. The spo 13 single division meiosis was exploited to develop a plating assay in which LEU2 diploid spores containing reciprocally recombinant strands derived from events occurring completely within the interval flanked by the insertion heterologies were selected by random spore methods. Reciprocal recombination frequencies measured with this assay decreased linearly with extent, extrapolating to a minimal homology requirement of 150-250 nucleotides. When homology was most severely restricted, unexpected flanking marker configurations among reciprocal recombinants within LEU2 demonstrated the occurrence of complex recombination events. In addition to detecting reciprocal recombinants, the system is capable of measuring the probability that a non-reciprocal recombination event will have one endpoint between the heterologous inserts and the other lying outside the interval. The minimal length of homology required for this aspect of recombination was found to be 25-60 nucleotides. © 1993 Wiley-Liss, Inc.
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  • 74
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    Developmental Genetics 13 (1992), S. 411-424 
    ISSN: 0192-253X
    Keywords: Maize ; mutations ; female ; meiosis ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A modified enzyme digestion technique of ovary isolation followed by staining and squash preparation has allowed us to observe female meiosis in normal maize meiotically dividing megaspore mother cells (MMCs). The first meiotic division in megasporogenesis of maize is not distinguishable from that in mi-crosporogenesis. The second female meiotic division is characterized as follows: (1) the two products of the first meiotic division do not simultaneously enter into the second meiotic division; as a rule, the chalazal-most cell enters division earlier than the micropylar one, (2) often the second of the two products does not proceed with meiosis, but degenerates, and (3) only a single haploid meiotic product of the tetrad remains alive, and this cell proceeds with three rounds of mitoses without any intervening cell wall formation to produce the eight-nucleate embryo sac. This technique has allowed us to study the effects of five meiotic mutations (aml, aml-pral, afdl, dsy *-9101, and dvl) on female meiosis in maize. The effects of the two alleles of the aml gene (aml and aml-pral) and of the afdl and dsy *-9101mutations are the same in both male and female meiosis. The aml allele prevents the entrance of MMCs into meiosis and meiosis is replaced by mitosis; the aml-pral permits MMCs to enter into meiosis, but their progress is stopped at early prophase I stages. The afdl gene is responsible for substitution of the first meiotic (reductional) division by an equational division including the segregation of sister chromatid centromeres at anaphase I. The dsy * -9101 gene exhibits abnormal chromosome pairing; paired homologous chromosomes are visible at pachytene, but only univalents are observed at diakinesis and metaphase I stages. These mutation specific patterns of abnormal meiosis are responsible for the bisexual sterility of these meiotic mutants.The abnormal divergent shape of the spindle apparatus and the resulting abnormal segregation of homologous chromosomes observed in micro-sporogenesis in plants homozygous for the dv1 mutation have not been found in meiosis of megasporogenesis. Only male sterility is induced by the dv1 gene in the homozygous condition. © 1993 Wiley-Liss, Inc.
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  • 75
    ISSN: 0192-253X
    Keywords: Meiotic pairing ; rDNA ; Drosophila ; disjunction ; sex chromosomes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The Drosophila melanogaster ribosomal DNA (rDNA) functions as an X-Y meiotic pairing site. Deletions encompassing the X chromosomal rDNA block (located in the heterochromatin) disrupt X-Y pairing and disjunction. Insertions of single, complete rRNA genes at ectopic locations on the heterochromatically deficient X partially restore X-Y pairing capacity. This study was undertaken to test fragments of an rDNA repeat for the ability to stimulate X-Y pairing and disjunction and to test for relationships between pairing capacity and two other phenotypes associated with rDNA insertions: transcription and the ability to organize a nucleolus. Insertions of three different fragments, all of which retained the rDNA promoter and upstream spacer sequences and which differed among each other in the length of downstream sequences, were obtained by P-element mediated transformation. One of the fragments is truncated only 140bp downstream from the promoter. Insertions of all three fragments proved capable of stimulating X-Y disjunction. Double insertions were substantially more effective than single insertions. RNA/PCR analysis was used to show that transcripts initiated at the inserted rDNA promoters are present in testis RNA from all insertions. Treatment with an antinucleolar antibody revealed that none of the insertions was associated with a mininucleolus. Thus promoter-containing rDNA fragments are autonomously capable of being transcribed and of functioning as X-Y pairing sites, but not of forming a mini-nucleolus. © 1993 Wiley-Liss, Inc.
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  • 76
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    Developmental Genetics 13 (1992), S. 425-434 
    ISSN: 0192-253X
    Keywords: Chromosome structure ; histone H1 ; histone variants ; immunocytochemistry ; meiosis ; plant development ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Meiotin-1 is a chromatin associated, conserved protein found in meiocytes immediately preceding and during meiosis and is thought to have a role in determining the higher order structure of meiotic chromosomes [Riggs and Hasenkampf: Chromosoma 101:92-98, 1991]. In the studies reported here we utilized immunoblotting and immunocytochemical techniques to examine the temporal and spatial distribution of meiotin-1 in the anthers of Lilium longiflorum. The results with the anti-meiotin-1 immune serum were compared with those obtained using an anti-his-tone Hl immune serum. The anti-histone Hl immune serum gave constant immunostaining in all cell types of the anther at all of the stages tested. In contrast, the anti-meiotin-1 immune serum only gave immunostaining with the microsporocytes and to a lesser extent with the nutritive layer, the tapetum. It did not react with the cells of the anther wall. Meiotin-1 immunostaining was first present in significant quantities in the microsporocytes as they accumulated in the G1 phase before the onset of premeiotic S phase and reached peak levels in the time interval between leptotene and pachytene - the same interval when chromosome synapsis occurs and when reciprocal genetic exchange is thought to occur. Immunostaining for both meiotin-1 and histone H1 uniformly decorates the longitudinal axes of the chromosomes. Our data are consistent with the idea that the role of meiotin-1 may be to tag certain sequences or to limit the degree of chromosome condensation that occurs during meiotic prophase. © 1993 Wiley-Liss, Inc.
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  • 77
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    Developmental Genetics 13 (1992), S. 485-497 
    ISSN: 0192-253X
    Keywords: Double-strand breaks ; meiotic recombination ; pulsed-field gels ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Pulsed-field gel electrophoresis (PFGE) has been used to study the timing, frequency, and distribution of double-strand breaks (DSBs) in chromosomal-sized DNA during meiosis in yeast. It has previously been shown that DSBs are associated with some genetic hotspots during recombination, and it is important to know whether meiotic recombination events routinely initiate via DSBs. Two strains have been studied here - a highsporulating homothallic wild type and a congenic mutant strain carrying a rad50S mutation. This mutant has previously been reported to accumulate broken molecules in meiosis to much higher frequencies than wild type and to abolish the characteristic wild-type processing of DNA that has been observed at the break sites. When whole chromosomes are resolved by PFGE, both strains show some broken molecules starting at the time that cells commit to genetic recombination. Breakage has been assessed primarily on Chromosome III and Chr. XV, using Southern hybridization to identify these chromosomes and their fragments. At any one time, break frequency in wild type is much lower than the cumulative frequency of recombination events that occur during meiosis. However, there is suggestive evidence that each break is short-lived, and it is therefore difficult to estimate the total number of breaks that may occur. In rad50S, chromosome breaks accumulate to much higher levels, which are probably broadly consistent with the estimated number of recombination events in wild type. However, since rad50S is substantially defective in completing recombination, it is not known for certain if it initiates events at wild-type frequencies.A surprising feature of the data is that a strong banding pattern is observed in the fragment distribution from broken chromosomes in both strains, implying that at least much of the breakage occurs at specific sites or within short regions. However, with the exception of the rDNA region on Chr. XII, assessment of the genetic map indicates that recombination can occur almost anywhere in the genome, although some regions are much hotter than others. Possible reasons for this apparent paradox are discussed. It may in part result from breakage levels too low for adequate detection in cold regions but may also imply that recombination events are localized more than previously realized. Alternatively, there may be a more indirect relationship between break sites and the associated recombination events. © 1993 Wiley-Liss, Inc.
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  • 78
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome II ; RAD16 ; DNA helicase ; double-finger motif ; DNA excision repair ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have analysed a region some 30 kb centromere distal form PHO5 on the right arm of yeast chromosome II and determined the nucleotide sequence of a 8.95 kb DNA segment from this region. By this analysis we were able to derive the precise location and the transcriptional orientation of CMD1, ALG1, SSN6 and LYS2. An open reading frame of 2370 bp was locatlized between SSN6 and LYS2, which has recently been identified (Schild et al., 1991) to be the RAD16 gene. The putative gene product, 790 amino acids in length, reveals several interesting freatures. It contains a nuclear target singnature and shares several blocks of similarity with the yeast recombinational repair protein RAD54 and the nuclear factor SNF2 (SW12), which is required for teh transcriptioal activation of a number of yeast genes. The similarity blocks in these three proteins are reminiscent of those found in the helicase superfamily. Furthrmore, RAD16 contains a novel ‘double-finger’ motif, which has been encountered in a variety of proteins from different organisms that are suggested to interact with DNA and are involved in diverse functions including site-specific recombination, DNA repair, and transcriptional regulation. The putative gene product of RAD16 then is the first example of a proteins in which the novel double-finger motif is found to be combined with a poteintial DNA helicase framework.
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  • 79
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 80
    ISSN: 0749-503X
    Keywords: DNA sequencing ; chromosome XI ; centromere CEN11 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A 24·7 kb segment of the cosmid clone pUKG047 containing a Sau3AI-partial fragment from the centromere region of Saccharomyces cerevisiae chromosome XI was sequenced and analysed. A mexed strategy of directed methods including exonuclease III nested deletion, restriction fragment subcloning and oligonucleotide-directed sequences was carried out. Exclusive use waas made of the Applied Biosystems Taq DyeDeoxy™ Terminator Cycle technology and a laser-based ABI373A sequencing system for reactions, gel electrophoresis and automated reading. A total of 12 open reading frames (ORFs) was found. Nine new ORFs (YK102 to YK110) were identified, three of which (YK102, YK107, YK108)showed homologies to proteins of known function from other organisms. In addition, sequence analysis reveled three recently functionally characterized genes (MET14, VPS/SPO15, PAP1), which could be joined to the earlier published CEN11 region.
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  • 81
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome II ; ARS ; ACH1 ; FUS3 ; PEP1 ; vacuolar protein sorting ; carboxypeptidose Y ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We report the nucleotide sequence of an 11.4 kb DNA segment from the left arm of Saccharomyces cerevisiae chromosome II. This sequence contains a typical structure of a functional ARS as well as five open reading frames (ORFs) longer than 300 bp. One is PEP1, a gene encoding a transmembrane protein of 1579 amino acids which transits through the secretory pathway and is involved in vacuolar protein sorting. Two genes were previously sequenced: ACH1 (Lee et al., 1990) and FUS3 (Elion et al., 1990), which encode an acetyl-CoA hydrolase and a protein kinase involved in the cell division cycle, respectively. The last two ORFs localized on the complementary strand of ACH1 are not likely to be expressed.
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  • 82
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    Yeast 8 (1992), S. 787-790 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; ATPase ; chromosome V ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have sequenced a gene that encodes a 377 amino acid putative protein with an ATPase motif typical of the protein family including SEC18p (NSF = N-ethyl maleimide-sensitive fusion protein; vesicle-mediated endoplasmic reticulum to Golgi protein transfer), PAS1p (peroxisome assembly), CDC48p (VCP = valosin-containing protein; cell cycle) and TBP1 (Tat-binding protein). This gene, AFG1 for ATPase family, gene, also has substantial homology to these proteins outside the ATPase domain. AFG1 is located on chromosome V imediately centromere-proximal to M AK10.
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  • 83
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    Yeast 8 (1992), S. 801-804 
    ISSN: 0749-503X
    Keywords: Kluyveromyces lactis ; LEU2 gene ; rat ribosomal protein L7 ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A DNA fragment that can complement the leu2 mutation of Saccharomyces cerevisiae was cloned from the genomic library of Kluyveromyces lactis.The nucleotide sequence revealed an open reading frame of 362 codons, 75% homologous to S. cerevisiae LEU2 gene. The upstream region contained a CCGGAACCGG sequence identical to the site of leucine-specific control of LEU2. Further upstream, there is a partial open reading frame homologous to rat ribosmal protien L7.
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  • 84
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 85
    ISSN: 0749-503X
    Keywords: Chromosome III ; Ty insertion ; gene disruption ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We report the 9210 bp sequence from a segment of yeat chromosome III cloned from strain AB972 in λPM3270. Analysis of this sequence and its comparison with the one derived from the corresponding segment of strain XJ24-24A revealed that the AB972 region contains a duplication of about 2 kb and a Ty element, which are not found in XJ24-24A and cause a quite significant rearrangement of the whole region. We performed analysis of YCR28, the largest open reading frame we found in both AB972 and XJ24-24A. YCR28 encodes a putative protein of 512 amino acids with some similarities to yeast allontoate permease. Its disruption does not cause any detectable phenotype on rich medium or on allantoate medium, while we observed a strain-dependent effect on senstivity to amino acid balance and to 3-aminotriazole, when cells were grown in synthetic medium.
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  • 86
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 87
    ISSN: 0749-503X
    Keywords: mitochondria ; immunogold labelling ; HSP70 ; in vitro import ; respiration ; Q pool ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A technique is described for the isolation and purification of intact, respiratory-competent mitochondria from Sachizo-saccharomyces pombe. The purified mitochondria are capabel of oxidizing NADH and succinate as resporatory substrates. indicating the presence of succinate dehydorgenase and an NADH dehydrogenase located on the outer suface of the inner membrane. Mitochondria display good respiratory control with an ADP/O ratio of 〈2. Respiratory activity is linearly dependent upon the redox poise of the quinone pool, suggesting the presence of an unbranched repiratory pathway to molecular oxygen. Immunogold labelling using antisera raised against mitochodria proteins (SSPI, SSCI, and PHSPI) from three different species, namely S. pombe, Saccharomyces cerevisiane and the plant Pisum sativum respectively, has been used to investigate the presence and ultrastructure of the mitochondria isolated by this procedure. The immunocytochemistry was carried out using cells containing wild-type levels of SSPI protein and cells over-expressing the protein. These results also demonstrate the capacity of mitochondria to import increased levels of protein in vivo. In vitro import experiments using COXIV-DHFR indicate that purified S. pombe mitochondria can efficiently import this precursor, and that protein translocation is dependent upon an oxidizable substrate and a membrane potential.
    Additional Material: 5 Ill.
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  • 88
    ISSN: 0749-503X
    Keywords: Yeast ; Hansenula polymorpha ; microbodies ; biogenesis ; PER genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In the course of our studies on the molecular mechanisms involved in peroxisome biogenesis, we have isolated several mutants of the methylotrophic yeast Hansenula polymorpha impaired in the import of peroximal matrix proteins. These mutants are characterized by the presence of small intact peroxisomes, while the bulk of the peroxisomal matrix protein is not imported and resides in the cytosol (Pim- phenotype). Genetic analysis of back-crossed mutants revealed five different complementation groups, which were designated PERI-PER5. Mapping studies to determine the linkage relationships indicated that the observed Pim- phenotypes were determined by single recessive nuclear mutations.The different mutants had comparable phenotypes: (i) they were impaired to utilize methanol as the sole source of carbon and energy but grew well on various other compounds, including nitrogen sources, the metabolism of which is known to be mediated by peroxisome-borne enzymes in wild-type cells; (ii) all peroxisomal enzymes tested were induced, assembled and activated as in wild-type cells although their activities varied between the different representative mutants; (iii) all peroxisomal proteins, whether constitutive or inducible, were found both in the cytosol and in the small peroxisomes. These results suggest that a general, major import mechanism is affected in all mutants.
    Additional Material: 2 Ill.
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  • 89
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome XI ; CCEI ; shotgun sequencing ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We report the wequence of a 9·3 kb DNA segment of chromosome XI of Saccharomyces cerevisiae, located between the MAK11 locus and the centromere. This sequence contains four long open reading frames (ORFs). YKL160, YKL162, YKL164, YKL165 and part of another ORF, YKL166, covering altogether 90% of the entire sequence. One of these ORFs YKL164, corresponds to CCE1. Translation products of two other ORFs, YKL160 and YKL165, exhibit homology with previously known S. cerevisiae proetins: the robosomal protein L10, and the MYO2 gene product, respectively.
    Additional Material: 6 Ill.
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  • 90
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 1007-1014 
    ISSN: 0749-503X
    Keywords: Electroporation ; spheroplasts ; mRNA ; translation ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: An efficient delivery method for introducing in vitro synthesized RNA into yeast into has been developed using electroporation. Spheroplast preparation, electroporation, and subsequent expression analysis can be accomplished within a single day. The use of introduced mRNA constructs avoids any complications due to nuclear regulation and is particularly suited for cytoplasmic regulatory studies. Moreover, this technique is useful for introduicing those RNas that connot be made in vivo, Such as poly (A)-mRNAs or RNAs with base modifications. We demonstrate that the Escherichia coli GUS gene and the firefly Luc gene are both excellent reporter genes for RNA electroporation.
    Additional Material: 4 Ill.
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  • 91
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 1015-1024 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; mating ; conjugation ; sterols ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Sterol auxotrophic strains of Saccharomyces cerevisiae were grown and allowed to conjugate on media supplemented with various sterols.The mating efficiency of the auxotrophs is perturbed by the relacement of the normal yeast sterol, ergsterol, with other sterols. After 4 h of mating, cells grown on ergosterol a 30-fold higher productive mating efficiency than those cells grown in stigmasterol. Aberrant budding by the conjugants was enhanced following incubation on stigmasterol and other non-ergosterol sterols. Using light and electron microscopy, we demonstrated that there is a reduced ability for stigmasterol-grown cells to undergo cytoplasmic fusion during conjugation. Many of the mated pairs remained adherent but Prezygotic even after 12 h of incubation. The addition of ergosterol to cells previously grown on stigmasterol rescued the organisms, allowing for zygote formation and normal budding.
    Additional Material: 3 Ill.
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  • 92
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 1033-1041 
    ISSN: 0749-503X
    Keywords: polyamines ; yeast architecture ; cell wall ; polysaccharides ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Cells of Saccharomyces cerevisiae 179-5, an ornithine decarboxylase mutant (spe-1), showed several ultrastructural abnormalities when cultivated in the absence of polyamines. Besides the appearance of microvacuole-like spaces in the cytoplasm and of deformed nuclei, the most important alterations seemed to be located in the cell wall, which was thicker and of heterogeneous texture, and in the cell membrane, of irregular contour. These modifications could not be evoked by general stress conditions elicited by lack of nutrients. The relative levels of cell wall polysaccharides were altered in polyamine-deprived organisms, giving an envelope with increased mannan and decreased glucan content; this cell wall was incompletely attacked by the lytic enzyme zymolyase. Polyamine depletion led also to some abnormalities in the budding pattern. The above observations suggest the involvement of polyamines in the correct structure and organization of the yeast cell.
    Additional Material: 4 Ill.
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  • 93
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992) 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 94
    ISSN: 0749-503X
    Keywords: Protein kinase ; chromosome III ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have sequenced a gene on chromosome III of Saccharomyces cerevisiae which codes for a putative serine/threonine protein kinase of 726 amino acids (calculated molecular weight 82 kDa). We have called this gene KIN82. The amino acid sequence of KIN82 is most similar to the cyclic nucleotide-dependent protein kinase subfamily and the protein kinase C subfamily. Gene disruption of KIN82 did not produce any phenotype when tested under a variety of conditons. Reduced stringency hybridizations revealed the presence of another genomic sequence with high homology to the carboxy-terminal catalytic domain of KIN82.
    Additional Material: 4 Ill.
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  • 95
    ISSN: 0749-503X
    Keywords: Maltose ; galactose ; raffinose fermentation ; nculeo-mitochondrial interaction ; yeast ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants of grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level.Mutantions in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency.The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.
    Additional Material: 5 Ill.
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  • 96
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 107-115 
    ISSN: 0749-503X
    Keywords: Yeast ; Saccharomyces cerevisiae ; glycolysis ; hexokinase ; phosphofructokinase ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The enzymatic steps involved in the inhibition of glycolysis by 2-deoxygalactose in Saccharomyces cerevisiae have been investigated. Yeast, incubated with 2-deoxygalactose, accumulates up to 8 mM-2-deoxygalactose, 30 mM-2-deoxygalactose-1-phosphate and 0·25 mM-UDP-2-deoxygalactose and UDP-2-dexyglucose. An inverse correlation between 2-deoxygalactose-1-phosphate content and rate of glycolysis has been observed. The intracellular concentration of glycolytic intermediates and related metabolites point to the hexokinase and phosphofructokinase steps as the targets for the inhibition of glycolysis by 2-deoxygalactose and rule out all other mechanisms that have been proposed to explain this inhibition.
    Additional Material: 4 Ill.
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  • 97
    Electronic Resource
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    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 155-155 
    ISSN: 0749-503X
    Keywords: Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
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  • 98
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; chromosome III ; sup61 ; RADI18 ; chromosome sequening ; Zn finger proteins ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A 7965 bp DNA segment from the right arm of chromosome III of Saccharomyces cerevisiae, encompassing the sup61 and RAD18 genes, was sequenced. Four new open reading frames were found in this DNA fragment. One of them YCR103, is 51% homologous with the G10 gene product of Xenopus laevis.
    Additional Material: 5 Ill.
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  • 99
    ISSN: 0749-503X
    Keywords: Yeast ; protein export ; heat shock ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: A new secreted yeast glycoprotein with an Mr of about 400 kDa (gp400) has been found. The glycoprotein is an O-mannosylated oligomer, whose synthesis and export into culture medium are stimulated by heat shock. Intracellular transport of gp400 is carried out by membrane vesicles distinct form the known constitutive scretory vesicles. Immunological analysis revealed gp400 only in Saccharomyces species.
    Additional Material: 12 Ill.
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  • 100
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 183-192 
    ISSN: 0749-503X
    Keywords: CIF1 gene ; catabolite inactivation ; chromosome II ; S. cerevisiae ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The cif1 mutation of Saccharomyces cerevisia (Navon et al., Biochemistry 18, 4487-4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of funcion and licated it in a 2·75 kb SphI-BstEII fragment situated at ca. 18 kb centomere distal of LYS2 and ca. 80 kb centromere proximal of TYRI on chromosome II. Southern analysis demostrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1·8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increse. cAMP concentration increases in response to galactose or 2,4-dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1/, background did not restore growth on glucose, suggesting that the absence of cAMP signal is not primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.
    Additional Material: 5 Ill.
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