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The expression of most nitrogen catabolic genes in Saccharomyces cerevisiae is regulated at the level of transcription in response to the quality of nitrogen source available. This regulation is accomplished through four GATA-family transcription factors: two positively acting factors capable of transcriptional activation (Gln3p and Gat1p) and two negatively acting factors capable of down-regulating Gln3p- and/or Gat1p-dependent transcription (Dal80p and Deh1p). Current understanding of nitrogen-responsive transcriptional regulation is the result of extensive analysis of genes required for the catabolism of small molecules, e.g., amino acids, allantoin, or ammonia. However, cells contain another, equally important source of nitrogen, intracellular protein, which undergoes rapid turnover during special circumstances such as entry into stationary phase, and during sporulation. Here we show that the expression of some (CPS1, PEP4, PRB1, and LAP4) but not all (PRC1) vacuolar protease genes is nitrogen catabolite repression sensitive and is regulated by the GATA-family proteins Gln3p, Gat1p, and Dal80p. These observations extend the global participation of GATA-family transcription factors to include not only well-studied genes associated with the catabolism of small nitrogenous compounds but also genes whose products are responsible for the turnover of intracellular macromolecules. They also point to the usefulness of considering control of the nitrogen-responsive GATA factors when studying the regulation of the protein turnover machinery. Show less

This report extends the genetic map of the common shrew (Sorex araneus) by use of a clone panel of shrew-Chinese hamster and shrew-mouse hybrid cells (Pack et al., 1995; Matyakhina et al., 1996). This set of hybrid clones made it possible to assign the shrew genes for isocitrate dehydrogenase 2 (IDH2), inorganic pyrophosphatase (PP), glutamicpyruvate transaminase (GPT), adenosine kinase (ADK), glucuronidase 2 (GUSB) and acid phosphatase 2 (ACP2) to chromosome ik; the genes for adenylate kinases 1 and 3 (AK1 and AK3) to chromosome af; the genes for glutamate-oxaloacetate transaminase 2 (GOT2), peptidase D (PEPD) and growth hormone (GH) to chromosome hn; the gene for phosphoglucomutase 2 (PGM2) to chromosome go, the gene for enolase 1 (ENO1) to chromosome ji, the gene for ornithine carbamoyl-transferase (OTC) to chromosome de, the gene for aminoacylase 1 (ACY1) to arm m (chromosome mp), the gene for glutamate-oxaloacetate transaminase 1 (GOT1) to arm q (chromosome qr). Thus, the genetic map of the common shrew now contains 33 genes and it is possible to compare the syntenic associations with other species. Show less

PSD-95 is a component of postsynaptic densities in central synapses. It contains three PDZ domains that localize N-methyl-D-aspartate receptor subunit 2 (NMDA2 receptor) and K+ channels to synapses. In mouse forebrain, PSD-95 bound to the cytoplasmic COOH-termini of neuroligins, which are neuronal cell adhesion molecules that interact with beta-neurexins and form intercellular junctions. Neuroligins bind to the third PDZ domain of PSD-95, whereas NMDA2 receptors and K+ channels interact with the first and second PDZ domains. Thus different PDZ domains of PSD-95 are specialized for distinct functions. PSD-95 may recruit ion channels and neurotransmitter receptors to intercellular junctions formed between neurons by neuroligins and beta-neurexins. Show less

Although the activity of telomerase, an enzyme which synthesizes telomeres de novo and stabilizes telomere length has been demonstrated in the testis, the precise expression of activity in different germ cell types is not known. We examined telomerase activity using a PCR-based telomeric repeat amplification protocol during development of the rat testis from birth to adulthood. Telomerase activity was relatively high from birth to the 4th week of age, and then low between the 5th to 10th week, suggesting that the type A spermatogonial stem cells may be the population which is expressing the highest levels of telomerase activity. To ascertain which germ cells expresses the telomerase activity, purified populations of type A spermatogonia from 9-day old rats, and pachytene spermatocytes, round spermatids and epididymal spermatozoa from adult rats were isolated. While type A spermatogonia expressed very strong telomerase activity, the fractions containing pachytene spermatocytes and round spermatids also expressed telomerase activity, but at comparatively lower levels. Telomerase activity was totally absent in epididymal spermatozoa. Thus, it appears that the telomerase activity is expressed at high levels in the type A spermatogonial stem cells, is down-regulated during spermatogenesis, and is absent in the differentiated spermatozoa. Show less

We have cloned and sequenced the mouse cDNA homologous to the human Hereditary Multiple Exostoses (EXT1) gene. The mouse homolog shows 94% similarity at the nucleotide level and 99% similarity at the protein level compared to the human gene. The 5' UTRs are unusually conserved for non-coding sequences showing 94% similarity compared to 80% for the 3' UTRs. The high level of evolutionary conservation between the EXT1 proteins as well as the 5' UTR suggests that each plays an important and related role in both species. Show less

The final step in the biosynthesis of testosterone is the reduction of androstenedione to testosterone catalysed by the enzyme 17beta-hydroxysteroid dehydrogenase (17betaHSD). Five isoforms of the enzyme have been identified in the mouse and the type 3 isoform has been shown to be the predominant reductive form present in the adult human and mouse testis. In this study the regulation of 17betaHSD type 3 isoform mRNA levels and the cellular localisation of the enzyme mRNA have been studied in the mouse testis. To examine regulation of 17betaHSD type 3 mRNA expression in the testis, mRNA levels were measured during development in normal mice and in mice lacking circulating gonadotrophins (hpg) or functional androgen receptors (Tfm). In these mutants testicular descent does not occur at the normal time (25 days) and control animals were, therefore, rendered cryptorchid at 19 days. In neonatal mice, it has been shown a peak of type 3 expression occurs around day 5 and this was found to be normal in all groups in the current study. In normal animals there was a marked increase in type 3 isoform expression between 25 and 30 days and this continued into adulthood. In cryptorchid animals the increase in type 3 mRNA levels after 25 days was less marked than in untreated controls and by 90 days was about 15% of normal animals. In Tfm mice, levels of 17betaHSD type 3 mRNA failed to show any increase around puberty (25 days) and in adult Tfm mice, levels were less than 1% of cryptorchid controls. In hpg mice, levels of type 3 mRNA increased slowly after puberty and were about 30% of cryptorchid controls by 90 days. Studies using in situ hybridisation showed that the type 3 isoform was expressed only in the interstitial tissue of the adult normal mouse testis. No specific hybridisation could be determined in adult hpg or Tfm testes. Results show that 17betaHSD type 3 is an interstitial enzyme in the testis and is, probably, localised in the Leydig cells. During neonatal development expression of 17betaHSD type 3 is independent of gonadotrophin action while the increase in type 3 expression at puberty is primarily dependent upon androgen action although testicular descent and gonadotrophins are also required. Show less

Genes for the snRNP proteins U1-70K, U1-A, Sm-B'/B, Sm-D1 and Sm-E have been isolated from various metazoan species. The genes for Sm-D1 and Sm-E, which were isolated from a murine and human source respectively, appear to belong to a multigene family. It has been suggested that also for the mammalian U1-C protein such a multigene family exists. With the human U1-C cDNA as a probe, two genes containing sequences homologous to the probe sequence were isolated from a mouse genomic library. Simultaneously, a murine U1-C cDNA was isolated from a mouse cDNA library. This 0.74 kb cDNA contains an open reading frame (ORF) of 477 bp encoding a polypeptide of 159 amino acids (aa) which differs at only one position (position 65) from the human U1-C protein. One of the isolated U1-C genes contains an ORF as well and shares 92% nucleotide sequence identity with the mouse U1-C cDNA. The features of this gene, in particular the absence of introns, the acquisition of a 3' poly(A) tail and flanking direct repeats, indicate that it represents a processed pseudogene. At the predicted aa sequence level, substitutions of conserved residues at functionally important positions are observed, strongly suggesting that expression of this gene would not lead to a functional polypeptide. The second U1-C gene appeared to be a pseudogene as well because it is also intronless and contains a frameshift mutation compared to the ORF in the mouse U1-C cDNA. The characterization of these two pseudogenes points to the existence of a U1-C multigene family in mice. Furthermore, comparison of aa sequences of the murine, human and Xenopus U1-C shows that the protein is highly conserved through evolution. Since the Xenopus U1-C differs from the two mammalian counterparts solely at a number of positions in the C-terminal region, it can be concluded that aa changes are less well tolerated in the N-terminal region of U1-C than in the rest of the protein. Show less

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function. Show less

The identification of the genetic defect in CLN1 as a palmitoyl-protein thioesterase deficiency initiated a search for the lysosomal storage material. Pulse-chase labelling of fibroblasts and lymphoblastoid cell lines with [35S]cysteine revealed the presence of lipid [35S]cysteine material in CLN1 fibroblasts and not in controls, CLN2 or CLN3 patients or other patients with lipidosis. A single band comigrated with the acylcysteine standard and labelling with [3H]palmitate showed a band of material which eluted from the silicic acid column with the phospholipid fraction and which co-migrated with the lipid-[35S]cysteine band. The storage material is tentatively identified as palmitoylcysteine. Show less

U1C is one of the three human U1 small nuclear ribonucleoprotein (snRNP)-specific proteins and is important for efficient complex formation between U1 snRNP and the pre-mRNA 5' splice site. We identified a hypothetical open reading frame in Saccharomyces cerevisiae as the yeast homolog of the human U1C protein. The gene is essential, and its product, YU1C, is associated with U1 snRNP. YU1C depletion gives rise to normal levels of U1 snRNP and does not have any detectable effect on U1 snRNP assembly. YU1C depletion and YU1C ts mutants affect pre-mRNA splicing in vivo, and extracts from these strains form low levels of commitment complexes and spliceosomes in vitro. These experiments indicate a role for YU1C in snRNP function. Structure probing with RNases shows that only the U1 snRNA 5' arm is hypersensitive to RNase I digestion when YU1C is depleted. Similar results were obtained with YU1C ts mutants, indicating that U1C contributes to a proper 5' arm structure prior to its base pairing interaction with the pre-mRNA 5' splice site. Show less

In budding yeast, entry into the mitotic cell cycle, or Start, requires the Cdc28 cyclin-dependent kinase (Cdk) and one of its three associated G1 cyclins, Cln1, Cln2, or Cln3. In addition, two other G1 cyclins, Pcl1 and Pcl2, associate with a second Cdk, Pho85, to contribute to Start. Although Pho85 is not essential for viability, Pcl1,2-Pho85 kinase complexes become essential for Start in the absence of Cln1,2-Cdc28 kinases. In addition, Pho85 interacts with a third cyclin, Pho80, to regulate acid phosphatase gene expression. Other cellular roles for Pho85 cyclin-Cdk complexes are suggested by the multiple phenotypes associated with deletion of PHO85, in addition to Start defects and deregulated acid phosphatase gene expression. Strains with pho80, pcl1, and pcl2 deletions show only a subset of the pho85 mutant phenotypes, suggesting the existence of additional Pho85 cyclins (Pcls). We used two-hybrid screening and database searching to identify seven additional cyclin-related genes that may interact with Pho85. We found that all of the new genes encode proteins that interacted with Pho85 in an affinity chromatography assay. One of these genes, CLG1, was previously suggested to encode a cyclin, based on the protein's sequence homology to Pcl1 and Pcl2. We have named the other genes PCL5, PCL6, PCL7, PCL8, PCL9, and PCL10. On the basis of sequence similarities, the PCLs can be divided into two subfamilies: the Pcl1,2-like subfamily and the Pho80-like subfamily. We found that deletion of members of the Pcl1,2 class of genes resulted in pronounced morphological abnormalities. In addition, we found that expression of one member of the Pcl1,2 subfamily, PCL9, is cell cycle regulated and is decreased in cells arrested in G1 by pheromone treatment. Our studies suggest that Pho85 associates with multiple cyclins and that subsets of cyclins may direct Pho85 to perform distinct roles in cell growth and division. Show less

Multiple exostoses is a polygenic disease of bone formation and development characterized by the presence of cartilage-capped osseous projections emanating from the end of the long bones. Two members of a recently defined multigene family of proteins (EXT1 and 2) were shown to be involved in this disease. To investigate the evolutionary relatedness of EXT genes across species we isolated the mouse EXT2 cDNA. As in the human counterpart, the mouse EXT2 cDNA contains an open reading frame of 2154 bp encoding a predicted protein of 718 amino acids. The nucleic acid sequence is 87% identical to the human EXT2 transcript, resulting in an amino acid sequence which is 95% identical to the human protein. The mouse EXT2 gene also shows significant sequence similarity to the mouse and human EXT1 gene. Northern blot analysis shows that this gene is expressed in early stages of embryonic development, and in situ hybridizations suggest that EXT2 plays a role in limb development. The identification of the mouse EXT2 gene will allow functional analysis through insertional inactivation and reverse genetics in mice in order to better understand the formation of exostoses during bone formation. Show less

Mutations at the mouse Fused locus have pleiotropic developmental effects, including the formation of axial duplications in homozygous embryos. The product of the Fused locus, Axin, displays similarities to RGS (Regulators of G-Protein Signaling) and Dishevelled proteins. Mutant Fused alleles that cause axial duplications disrupt the major mRNA, suggesting that Axin negatively regulates the response to an axis-inducing signal. Injection of Axin mRNA into Xenopus embryos inhibits dorsal axis formation by interfering with signaling through the Wnt pathway. Furthermore, ventral injection of an Axin mRNA lacking the RGS domain induces an ectopic axis, apparently through a dominant-negative mechanism. Thus, Axin is a novel inhibitor of Wnt signaling and regulates an early step in embryonic axis formation in mammals and amphibians. Show less

To describe the variation of the phenotype within families with several individuals with Bardet-Biedl syndrome. The phenotypes of affected siblings in 11 Scandinavian families with two or more members who had at least three of the features: retinal dystrophy, polydactyly, obesity, hypogenitalism, and mental retardation, were compared [corrected]. Individuals without retinal dystrophy were excluded. Intrafamilial variation of expressivity of the features obesity, polydactyly, abnormal radiograms of the extremities, hypogenitalism, short stature, paraplegia, and dental abnormalities was found. The retinal dystrophy varied with respect to both the onset of symptoms and the course of the disease. The morphology of the fundus, however, was consistent within the families. The disorder showed statistically significant genetic linkage to the BBS4 locus on chromosome 15 in the affected siblings in two of the families, but the clinical features in these patients did not differ from the other cases of Bardet-Biedl syndrome. Comparison of siblings with the Bardet-Biedl syndrome showed variation of the typical features. In addition, the course of retinal dystrophy varied. No distinctive clinical features were found to separate the BBS4 phenotype from the remaining patients. Show less

By controlled addition of galactose to synchronized galactose-limited Saccharomyces cerevisiae cultures, the growth rate could be regulated while external conditions were kept constant. By using this method, the G1 phase duration was modulated and expression of cell cycle-regulated genes was investigated. The expression of the cyclin genes CLN1 and CLN2 was always induced just before bud emergence, indicating that this event marks the decision to pass Start. Thus, G1 phase elongation was not due to a slower accumulation of the CLN1 and CLN2 mRNA levels. Only small differences in CLN3 expression levels were observed. The maximal SWI4 expression preceded maximal CLN1 and CLN2 expression under all conditions, as expected for a transcriptional activator. But whereas SWI4 was expressed at about 10 to 20 min, before CLN1 and CLN2 expression at high growth rates, this time increased to about 300 min below a particular consumption rate at which the G1 phase strongly elongated. In the slower-growing cultures, also an increase in SWI6 expression was observed in the G1 phase. The increase in G1 phase duration below a particular consumption rate was accompanied by a strong increase in the reserve carbohydrate levels. These carbohydrates were metabolized again before bud emergence, indicating that below this consumption rate, a transient increase in ATP flux is required for progression through the cell cycle. Since Start occurred at different cell sizes under different growth conditions, it is not just a certain cell size that triggers passage through Start. Show less

Wild-type cells of the budding yeast Saccharbmyces cerevisiae arrest in G1 upon nutrient exhaustion. Cell cycle arrest requires the WHI2 gene since whi2 mutants continue to divide and become abnormally small as nutrients are depleted. Here we show that CLN1 and CLN2 transcript levels in a whi2 strain are higher during exponential growth, and persist longer upon starvation, than in an isogenic wild-type strain. In contrast to CLN1 and CLN2, CLN3 levels declined only at very high cell density and were unaffected by the whi2 mutation. Elevated CLN expression is sufficient to explain the whi2 phenotype since ectopic expression of CLN1 in a nutrient-depleted culture caused cells to continue dividing and interfered with the acquisition of heat resistance. These observations show that, either directly or indirectly, Whi2 negatively regulates G1 cyclin expression. Interestingly extremely high levels of Cln1 induced filamentous growth upon nutrient deprivation, suggesting a direct connection between G1 cyclin activity and morphological responses to poor nutrient conditions. Show less

The Saccharomyces cerevisiae gene BTN1, encodes a 408 amino acid putative integral membrane protein, which is 39% identical and 59% similar to the human Cln3p, whose mutant forms are responsible for Batten's disease and for a diminished degradation of mitochondrial ATPase synthase subunit c. Disruption experiments established that Btn1p is not essential for viability, mitochondrial function, or degradation of mitochondrial ATP synthase in yeast. Show less

The plasma lipid response to changes in dietary fat and cholesterol can vary between individuals. The SstI polymorphism, arising from a cytosine to guanosine substitution in the 3' untranslated region of the APOC3 gene distinguishes between two alleles--S1 and S2. The S2 allele has been associated with elevated plasma triacylglycerol, cholesterol, and apolipoprotein (apo) C-III concentrations. In 90 young men we examined the effect of the same mutation on the response of low-density-lipoprotein (LDL) cholesterol to dietary monounsaturated fat. The frequency for the S2 allele was 0.14. Subjects were fed a low-fat diet for 25 d, followed by a diet rich in monounsaturated fatty acid (22% MUFA, 38% total fat) for 28 d; lipoproteins were measured at the end of each diet. There were no significant differences in initial total cholesterol between subjects with the APOC3*S1/APOC3*S1 (S1/S1) and APOC3*S1/APOC3*S2 (S1/S2) genotypes. After consumption of the diet high in MUFA, significant increases in LDL cholesterol (0.13 mmol/L, P < 0.027) were noted in the S1/S1 subjects whereas a significant decrease was observed in the S1/S2 subjects (-0.18 mmol/L, P < 0.046). Significant genotypic effects were seen for diet-induced changes in LDL cholesterol (P < 0.00034), total cholesterol (P < 0.009), and apo B (P < 0.0014). A study of the effect of the interaction between this mutation with that present in position -76 of the APOA1 gene promoter region (G/A) revealed that both mutations had an additive effect on changes in total cholesterol, LDL cholesterol, and apo B induced by diets. Plasma LDL-cholesterol responsiveness to the diet may be explained, at least in part, by variation at the APOC3 gene locus. Show less

A total of 36 patients with Batten disease (juvenile-onset neuronal ceroid lipofuscinosis), homozygous or heterozygous for the major mutation, a 1.02-kb deletion, in the CLN3 gene, were studied to relate their genotype to their clinical phenotype. The onset of visual failure and epilepsy was highly concordant in both groups. Great inter- and intrafamilial heterogeneity was demonstrated in the development of mental and physical handicap and in magnetic resonance imaging findings among both homozygous and heterozygous patients. The 1.02-kb deletion in homozygous form was always associated with mental and physical handicap, whereas the heterozygous phenotype could be extremely benign without affecting the intellectual level of the patient. Our data suggest that genetic background, modifying genes, and environmental factors all influence the final phenotype of Batten disease. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by multiple bony outgrowths from the juxtaepiphyseal region of long bones. In a small proportion of cases, these exostoses progress to malignant chondrosarcomas. Genetic linkage of this disorder has been described to three independent loci on chromosomes 8q24.1 (EXT1), 11p11-13 (EXT2), and 19p (EXT-3). The EXT1 and EXT2 genes were isolated recently and show extensive sequence homology to each other. These genes are deleted in exostoses-derived tumors, supporting the hypothesis that they encode tumor suppressors. We have identified a third gene that shows striking sequence similarity to both EXT1 and EXT2 at the nucleotide and amino acid sequence levels, and have derived its entire coding sequence. Although the mRNA transcribed from this gene is similar in size to that from EXT1 and EXT2, its pattern of expression is quite different. We have localized this gene by fluorescence in situ hybridization to metaphase chromosomes and by whole genome radiation hybrid mapping to chromosome 1p36.1 between DIS458 and DIS511, region that frequently shows loss of heterozygosity in a variety of tumor types. This gene, EXTL (for EXT-like), is therefore a new member of the EXT gene family and is a potential candidate for several disease phenotypes. Show less

Mitogen-activated protein (MAP) kinase phosphatases constitute a growing family of dual specificity phosphatases thought to play a role in the dephosphorylation and inactivation of MAP kinases and are therefore likely to be important in the regulation of diverse cellular processes such as proliferation, differentiation, and apoptosis. For this reason it has been suggested that MAP kinase phosphatases may be tumor suppressors. We have determined the chromosomal locations of three human dual specificity phosphatase genes by fluorescence in situ hybridization and radiation hybrid mapping. The genes were localized to three different chromosomes, MKP2 (DUSP4) to 8p11-p12, MKP3 (DUSP6) to 12q22-q23, and MKPX (DUSP7) to 3p21. This will allow the potential roles of these genes in disease processes to be evaluated. Show less

We hypothesized that common genomic variants would be associated with variation in lipoprotein phenotypes in young subjects. We determined genotypes of FABP2, PON, APOC3, and APOE in 188 aboriginal Canadians, aged 9 to 17 years. We found that 13 of 32 possible genotype-phenotype associations were significant: (1) the FABP2 codon 54 genotype was associated with variation in plasma triglycerides (P = .045); (2) the PON codon 192 genotype was associated with variation in plasma total and LDL cholesterol and apoB (P = .0099, P = .0088, and P = .016, respectively); (3) the APOC3 insulin-response-element genotype was associated with variation in plasma triglycerides, HDL cholesterol, apoA-I, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .0014, P = .0069, P = .045, P = .0021, and P = .0081, respectively); and (4) the APOE restriction isotype was associated with variation in plasma LDL cholesterol, apoB, the total cholesterol to HDL cholesterol ratio, and the apoB to apoA-I ratio (P = .025, P = .034, P = .045, and P = .047, respectively). The average young age and relative absence of age-dependent secondary environmental factors could have eased the identification of small genetic effects on lipoprotein phenotypes in this study sample. Show less

We have cloned and sequenced the murine homologue of the human EXT1 gene. At the protein level, these genes show almost complete identity as divergence is limited to only 5 amino acid positions that are scattered about the whole sequence. In addition, similarity searches identified a protein from chromosome III of C. elegans that shows significant similarity to the human and murine EXT/Ext genes. Using high resolution backcross mapping, the murine Ext1 was mapped at 26.55 cM between D15Mit143 and D15Mit153 on mouse chromosome 15. Therefore, Ext1 is part of an evolutionarily conserved linkage group including SDC2/Hspg1, TRHR/Trhr, EXT1/Ext1, MYC/Myc, and TG/Tgn. Show less

In budding yeast, stability of the mitotic B-type cyclin Clb2 is tightly cell cycle-regulated. B-type cyclin proteolysis is initiated during anaphase and persists throughout the G1 phase. Cln-Cdc28 kinase activity at START is required to repress B-type cyclin-specific proteolysis. Here, we show that Clb-dependent kinases, when expressed during G1, are also capable of repressing the B-type cyclin proteolysis machinery. Furthermore, we find that inactivation of Cln- and Clb-Cdc28 kinases is sufficient to trigger Clb2 proteolysis and sister-chromatid separation in G2/M phase-arrested cells, where the B-type cyclin-specific proteolysis machinery is normally inactive. Our results suggest that Cln- and Clb-dependent kinases are both capable of repressing B-type cyclin-specific proteolysis and that they are required to maintain the proteolysis machinery in an inactive state in S and G2/M phase-arrested cells. We propose that in yeast, as cells pass through START, Cln-Cdc28-dependent kinases inactivate B-type cyclin proteolysis. As Cln-Cdc28-dependent kinases decline during G2, Clb-Cdc28-dependent kinases take over this role, ensuring that B-type cyclin proteolysis is not activated during S phase and early mitosis. Show less

To determine what factors regulate gonocyte proliferation in newborn rats, we first examined the expression of several signal transduction molecules by immunocytochemistry in 3-day-old rat testis sections. We found that gonocytes specifically expressed the iota and zeta isoforms of protein kinase (PK) C (PKC) and the phosphatidylinositol 3-kinase (PI3-K). Because both the zeta PKC and PI 3-K have been shown to play a role in platelet-derived growth factor (PDGF)-induced cell proliferation, we examined the effects of PDGF on gonocytes. For this, we developed a method to obtain highly purified and viable gonocytes in culture. After enzymatic digestion, differential adhesion, and two successive gradient fractionations, the gonocyte suspension obtained was over 90% pure, as assessed by light microscopy. The viability of cultured gonocytes exceeded 90% after 48 h in the presence of 2.5% FBS used as a survival factor. Immunodetection studies showed that isolated gonocytes expressed zeta PKC, PI 3-K, and the PDGF receptor. Treatment with 10 ng/ml PDGF induced a 4-fold increase of bromodeoxyuridine incorporation into gonocytes (from 5% proliferative gonocytes under basal conditions to 20% in the presence of PDGF). Because neonatal Sertoli cells secrete high levels of the growth promoting steroid, 17 beta-estradiol, we also tested its effect and found that it induced gonocyte proliferation at a level comparable with that of PDGF and that this effect was blocked by the estrogen receptor antagonist, ICI 164384. The combination of PDGF and estradiol, however, was not additive, suggesting that their effects were mediated by common molecular target(s). These results demonstrate that PDGF and estradiol activate gonocyte proliferation in vitro, suggesting that they may act as the physiological regulators of gonocyte development in vivo. Show less

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by the presence of multiple cartilage-capped exostoses in the juxta-epiphyseal regions of the long bones. EXT is heterogeneous with at least three different locations currently having been identified on chromosomes 8, 11 and 19. We have tested a series of 29 EXT families for possible linkage to the three disease loci and estimated the probability of linkage of the disease to each locus in our series, by using an extension of the admixture test, which makes modelling of heterogeneous monogenic disease feasible. The maximum likelihood was obtained for proportions of 44%, 28% and 28% of families being linked to chromosome 8, 11 and 19, respectively. The a posteriori probability of linkage of the disease to EXT1, EXT2 and EXT3 was greater than 80% for 8/29, 5/29 and 3/29 families, respectively, and did not give evidence of a fourth locus for the disease. The present approach can be generalized to the investigation of genetic heterogeneity in other monogenic diseases, as it simultaneously estimates the location of each disease gene and the proportion of families linked to each locus. Show less

We have identified a novel promoter element that confers M/G1-specific transcription in Saccharomyces cerevisiae. This element, which we call an ECB (early cell cycle box), was first identified in the SWI4 promoter, but it is also present in the promoter of a G1 cyclin CLN3, as well as in the promoters of three DNA replication genes: CDC6, CDC47, and CDC46. Transcripts from all five of these genes oscillate during the cell cycle and peak at the M/G1 boundary, as do isolated ECB elements in reporter constructs. The ECB element contains an Mcm1 binding site to which Mcm1 binds in vitro, and an Mcm1-VP16 fusion, which places a constitutive activator on Mcm1-binding sites in vivo, can deregulate ECB-containing promoters. Mcm1 is a transcription factor that is also required for minichromosome maintenance. We provide evidence that the replication defect of mcm1 mutants can be suppressed by ectopic CDC6 transcription. Periodic expression of SWI4 and CLN3 may be important for cell cycle progression, as we find that these genes are both haploinsufficient and rate limiting for G1 progression. We suggest that ECB-regulated gene products play critical roles in promoting the initiation of S-phase, both by regulating CLN1 and CLN2 transcription and as components of the initiation complex on origins of replication. Show less

Within the core histone octamer each histone H4 interacts with each H2A-H2B dimer subunit through two binding surfaces. Tyrosines play a central role in these interactions with H4 tyrosines 72 and 88 contacting one H2A-H2B dimer subunit, and tyrosine 98 contacting the other. To investigate the roles of these interactions in vivo, we made site-directed amino acid substitutions at each of these tyrosine residues. Elimination of either set of interactions is lethal, suggesting that binding of the tetramer to both dimers is essential. Temperature-sensitive mutants were obtained through single amino acid substitutions at each of the tyrosines. The mutants show both strong positive and negative effects on transcription. Positive effects include Spt- and Sin-phenotypes resulting from mutations at each of the three tyrosines. One allele has a strong negative effect on the expression of genes essential for the G1 cell cycle transition. At restrictive temperature, mutant cells fail to express the CLN1, CLN2, SWI4 and SWI6 genes, and have reduced levels of CLN3 mRNA. These results demonstrate the critical role of histone dimer-tetramer interactions in vivo, and define their essential role in the expression of genes regulating G1 cell cycle progression. Show less

Previous reports have suggested that heterozygotes for ataxia-telangiectasia (A-T) have an increased risk of cancer, in particular breast cancer. The ATM gene, responsible for A-T, was recently cloned. Loss of heterozygosity (LOH) in the chromosome band 11q23, where the ATM gene is located, has been reported in several types of tumours including breast carcinomas. Whether the ATM gene is the target, and the sole target, for the LOH seen in this region is not yet known. In this study, 169 primary breast carcinomas and 10 metastases were examined for allelic imbalance (AI) using 10 microsatellite markers mapping to 11q23.1. Nine of the markers reside within a 10 Mb region surrounding the ATM gene, whereas the tenth locus, APOC-3, is located more than 12 Mb telomeric from this region. The highest frequencies of alteration were found for APOC-3 (45%), and for two markers located approximately 200 and 900 kb telomeric from ATM, D11S1294 (44%) and D11S1818 (44%). The marker located within the ATM gene, D11S2179, was altered in 37% of the informative tumours. The present deletion map indicates that three distinct regions at 11q23.1 may be involved in breast cancer development; one between the markers D11S1294 and D11S1818, a second close to APOC-3, and a third that is possibly the ATM-gene itself. Show less