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In Saccharomyces cerevisiae, START has been shown to comprise a series of tightly regulated reactions by which the cellular environment is assessed and under appropriate conditions, cells are commited to a further round of mitotic division. The key effector of START is the product of the CDC28 gene and the mechanisms by which the protein kinase activity of this gene product is regulated at START are well characterized. This is in contrast to the events which follow p34CDC28 activation and the way in which progress to S phase is achieved, which are less clear. We suggest two possible models to describe the regulation of these events. Firstly, it is conceivable that the only post-START targets of the p34CDC28/G1 cyclin kinase complex are components of the SBF and DSC1 transcription factors. This would require that either SBF or DSC1 regulates CDC4 function either directly by activating the transcription of CDC4 itself or else indirectly by activating the transcription of a mediator of CDC4 function in a manner analogous to the way in which the control of CDC7 function may be mediated by transcriptional regulation of DBF4 (Jackson et al., 1993). Potential regulatory effectors of CDC4 function include SCM4, which suppresses cdc4 mutations in an allele-specific manner (Smith et al., 1992) or its homologue HFS1 (J. Hartley & J. Rosamond, unpublished). This possibility is supported by the finding that CDC4 has no upstream SCB or MCB elements, whereas SCM4 and HFS1 have either an exact or close match to the SCB. This model would further require that genes needed for bud emergence and spindle pole body duplication are also subject to transcriptional regulation by DSC1 or SBF. An alternative model is that the p34CDC28/G1 cyclin complexes have several targets post-START, one being DSC1 and the others being as yet unidentified components of the pathways leading to CDC4 function, spindle pole body duplication and bud emergence. This model could account for the functional redundancy observed amongst the G1 cyclins with the various cyclins providing substrate specificity for the kinase complex. We suggest that a complex containing Cln3 protein is primarily responsible for, and acts most efficiently on, the targets containing Swi6 protein (SBF and DSC1), with complexes containing other G1 cyclins (Cln1 and/or Cln2 proteins) principally involved in activating the other pathways. However, there must be overlap in the function of these complexes with each cyclin able to substitute for some or all of the functions when necessary, albeit with differing efficiencies. This hypothesis is supported by several observations.(ABSTRACT TRUNCATED AT 400 WORDS) Show less

Five phosphotyrosine-containing peptides have been synthesized by FMOC solid-phase peptide synthesis. These peptides correspond to the 411-419 sequence of the Xenopus src oncogene, to the 1191-1220 sequence of the human EGF receptor precursor, to the 1146-1158 sequence of the human insulin receptor, to the 856-865 sequence of the human beta-PDGF receptor, and to the 5-16 sequence of the erythrocyte human band 3. The peptides were used as substrates for activity assay of two isoforms (AcP1 and AcP2) of a low molecular weight cytosolic PTPase. The assay, performed in microtiter EIA plates using Malachite green to determine the released phosphate, was rapid, reproducible, and sensitive. Both PTPase isoforms were able to hydrolyze all synthesized peptides, though with different affinity and rate. The main kinetic parameters were compared and discussed with respect to the role of the two enzymes in the cell. Show less

The molecular basis for the photosynthetic defect in four nuclear mutants of maize was investigated. Mutants hcf7, cps1-1, cps1-2, and cps2 contained reduced levels of many chloroplast-encoded proteins without corresponding deficiencies in chloroplast mRNAs. Many chloroplast mRNAs were associated with abnormally few ribosomes, indicating that the protein deficiencies were due to global defects in chloroplast translation. These mutants were used to study the effects of reduced ribosome association on the metabolism of chloroplast RNAs. The level of the rbcL mRNA was reduced fourfold in each mutant, but was unaltered in other nonphotosynthetic mutants with normal chloroplast translation. These results suggest that the rbcL mRNA is destabilized as a consequence of its decreased association with ribosomes. The fact that many other chloroplast mRNAs accumulated to normal levels demonstrated that a decreased association with ribosomes does not significantly alter their stabilities or processing. hcf7 seedlings had a gross defect in the processing of the 16S rRNA: the primary lesion in this mutant may be a defect in 16S rRNA processing itself. Show less

Analysis of cell cycle regulation in the budding yeast Saccharomyces cerevisiae has shown that a central regulatory protein kinase, Cdc28, undergoes changes in activity through the cell cycle by associating with distinct groups of cyclins that accumulate at different times. The various cyclin/Cdc28 complexes control different aspects of cell cycle progression, including the commitment step known as START and mitosis. We found that altering the activity of Cdc28 had profound effects on morphogenesis during the yeast cell cycle. Our results suggest that activation of Cdc28 by G1 cyclins (Cln1, Cln2, or Cln3) in unbudded G1 cells triggers polarization of the cortical actin cytoskeleton to a specialized pre-bud site at one end of the cell, while activation of Cdc28 by mitotic cyclins (Clb1 or Clb2) in budded G2 cells causes depolarization of the cortical actin cytoskeleton and secretory apparatus. Inactivation of Cdc28 following cyclin destruction in mitosis triggers redistribution of cortical actin structures to the neck region for cytokinesis. In the case of pre-bud site assembly following START, we found that the actin rearrangement could be triggered by Cln/Cdc28 activation in the absence of de novo protein synthesis, suggesting that the kinase may directly phosphorylate substrates (such as actin-binding proteins) that regulate actin distribution in cells. Show less

The hematopoietic-specific DNA-binding protein B1 binds to the DNA consensus sequence AAAGRGGAARYG located twice in intervening sequence 2 of both of the mouse beta-globin genes (D. L. Galson and D.E. Housman, Mol. Cell. Biol. 8:381-392, 1988). B1 was cloned by expression of a murine erythroleukemia (MEL) cell cDNA library in transfected COS cells and screening by electrophoretic mobility shift analysis. B1 is identical to the proto-oncogene Spi-1/PU.1 (Spi-1), an ets family member. Protein-DNA contacts are shown to resemble those of the helix-turn-helix homeodomain proteins. By Northern (RNA) analysis, we found that Spi-1 mRNA is present at low levels during murine CFU-E maturation and is at least 20-fold higher in uninduced MEL, a transformed proerythroblast-like cell line which contains an activating/transforming insertion of spleen focus-forming virus at the Spi-1 locus. Dimethyl sulfoxide-induced MEL cell differentiation decreases Spi-1 mRNA to approximately 20% of the uninduced level before commitment occurs. In addition to erythroid cells, Spi-1 mRNA is present in B cells, myelomonocytes, and mast cells but not in T cells and nonhematopoietic cell types. In situ hybridization demonstrated Spi-1 mRNA expression in bone marrow, spleen, interstitial nonhepatocytes of the liver, and interstitial nontubular cells of the testis. The Spi-1 locus was mapped on human chromosome 11 to the same interval as ACP2 (lysosomal acid phosphatase), between the anonymous DNA markers D11S33 and D11S14. This region has not yet been found to be associated with a human malignancy. Show less

The functions of the Cdc28 protein kinase in DNA replication and mitosis in Saccharomyces cerevisiae are thought to be determined by the type of cyclin subunit with which it is associated. G1-specific cyclins encoded by CLN1, CLN2, and CLN3 are required for entry into the cell cycle (Start) and thereby for S phase, whereas G2-specific B-type cyclins encoded by CLB1, CLB2, CLB3, and CLB4 are required for mitosis. We describe a new family of B-type cyclin genes, CLB5 and CLB6, whose transcripts appear in late G1 along with those of CLN1, CLN2, and many genes required for DNA replication. Deletion of CLB6 has little or no effect, but deletion of CLB5 greatly extends S phase, and deleting both genes prevents the timely initiation of DNA replication. Transcription of CLB5 and CLB6 is normally dependent on Cln activity, but ectopic CLB5 expression allows cells to proliferate in the absence of Cln cyclins. Thus, the kinase activity associated with Clb5/6 and not with Cln cyclins may be responsible for S-phase entry. Clb5 also has a function, along with Clb3 and Clb4, in the formation of mitotic spindles. Our observation that CLB5 is involved in the initiation of both S phase and mitosis suggests that a single primordial B-type cyclin might have been sufficient for regulating the cell cycle of the common ancestor of many, if not all, eukaryotes. Show less

Cysteine proteinases are hypothesized to be important virulence factors of Entamoeba histolytica, the causative agent of amebic dysentery and liver abscesses. The release of a histolytic cysteine proteinase from E. histolytica correlates with the pathogenicity of both axenic strains and recent clinical isolates as determined by clinical history of invasive disease, zymodeme analysis, and cytopathic effect. We now show that pathogenic isolates have a unique cysteine proteinase gene (ACP1). Two other cysteine proteinase genes (ACP2, ACP3) are 85% identical to each other and are present in both pathogenic and nonpathogenic isolates. ACP1 is only 35 and 45% identical in sequence to the two genes found in all isolates and is present on a distinct chromosome-size DNA fragment. Presence of the ACP1 gene correlates with increased proteinase expression and activity in pathogenic isolates as well as cytopathic effect on a fibroblast monolayer, an in vitro assay of virulence. Analysis of the predicted amino acid sequence of the ACP1 proteinase gene reveals homology with cysteine proteinases released by activated macrophages and invasive cancer cells, suggesting an evolutionarily conserved mechanism of tissue invasion. The observation that a histolytic cysteine proteinase gene is present only in pathogenic isolates of E. histolytica suggests that this aspect of virulence in amebiasis is genetically predetermined. Show less

In the budding yeast Saccharomyces cerevisiae, the G1 cyclins Cln1, Cln2 and Cln3 regulate entry into the cell cycle (Start) by activating the Cdc28 protein kinase. We find that Cln3 is a much rarer protein than Cln1 or Cln2 and has a much weaker associated histone H1 kinase activity. Unlike Cln1 and Cln2, Cln3 is not significantly cell cycle regulated, nor is it down-regulated by mating pheromone-induced G1 arrest. An artificial burst of CLN3 expression early in G1 phase accelerates Start and rapidly induces at least five other cyclin genes (CLN1, CLN2, HCS26, ORFD and CLB5) and the cell cycle-specific transcription factor SWI4. In similar experiments, CLN1 is less efficient than CLN3 at activating Start. Strikingly, expression of HCS26, ORFD and CLB5 is dependent on CLN3 in a cln1 cln2 strain, possibly explaining why CLN3 is essential in the absence of CLN1 and CLN2. To explain the potent ability of Cln3 to activate Start, despite its apparently weak biochemical activity, we propose that Cln3 may be an upstream activator of the G1 cyclins which directly catalyze Start. Given the large number of known cyclins, such cyclin cascades may be a common theme in cell cycle control. Show less

We have recently cloned a cDNA encoding the human phenol-preferring phenol sulfotransferase (P-PST) enzyme. An oligonucleotide primer pair based on the human STP (representing sulfotransferase, phenol-preferring) cDNA sequence was synthesized and was employed in polymerase chain reaction (PCR) amplification of human genomic DNA to identify a 525-bp DNA fragment. The DNA sequence of this portion of the STP gene, near the 5' end of the coding region, was determined. The amplified genomic fragment contained two small introns of 104 and 89 bp. When DNA samples from a human-hamster somatic cell hybrid panel were screened by PCR using these primers, only those hybrids that contained human chromosome 16 were positive for the 525-bp genomic fragment. To identify the specific region on chromosome 16 that contained the STP gene, PCR amplification reactions were performed on a human-mouse somatic cell hybrid panel containing defined portions of human chromosome 16. The results indicated that STP is localized proximal to the gene for protein kinase C, beta 1 polypeptide (PRKCB1), in the region from the distal portion of 16p11.2 to p12.1. The human STP gene maps near the locus for Batten disease (CLN3). Furthermore, we have determined by genotyping of murine interspecific backcross progeny that the homologous gene in mouse (Stp) localizes to the syntenic region of mouse chromosome 7 near the D7Mit8 (at 54 cM) and D7Bir1 markers. Show less

The neuronal ceroid lipofuscinoses (NCLs) are a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurons and other cell types. Inheritance is autosomal recessive. Three main childhood subtypes are recognized: infantile (Haltia-Santavuori disease; MIM 256743), late infantile (Jansky-Bielschowsky disease; MIM 204500), and juvenile (Spielmeyer-Sjögren-Vogt, or Batten, disease; MIM 204200). The gene loci for the juvenile (CLN3) and infantile (CLN1) types have been mapped to human chromosomes 16p and 1p, respectively, by linkage analysis. Linkage analysis of 25 families segregating for late-infantile NCL has excluded these regions as the site of this disease locus (CLN2). The three childhood subtypes of NCL therefore arise from mutations at distinct loci. Show less

We analysed the allelic and genotypic frequencies of three restriction fragment length polymorphisms in the region of chromosome 11 encoding apolipoprotein AI and CIII genes in a free-living population from South Italy (Calabria). These markers are located at -2500 and -78 bp from the transcription start site of apolipoprotein AI gene (XmnI and MspI, respectively), and in the 3' untranslated region of apolipoprotein CIII gene (SstI). XmnI and SstI label rare alleles (X2 and S2 indicate the presence of the site), whereas the absence of the MspI site (because of a G to A transition) marks the rare allele, M2. Pairwise linkage disequilibrium analysis was determined. Two significant non-random associations were found: a positive disequilibrium between ApoA1/XmnI and ApoA1/MspI markers (P < 0.0001), and a negative disequilibrium between ApoA1/XmnI and ApoC3/SstI markers (P < 0.05). Statistical analysis showed a significant difference in the S2-M2 haplotype frequency between the group of subjects with serum cholesterol levels in the highest decile (P < 0.005) and the group with serum cholesterol levels below the highest decile. The allelic frequency for each locus showed no significant difference between the two groups for all other metabolic parameters, included total cholesterol serum levels. These haplotypes are a more precise measure of genetic variations in the apolipoprotein cluster and their use should allow the mapping of mutations responsible for high serum cholesterol levels. Show less

The neuronal ceroid lipofuscinoses (NCL) are a group of progressive neurodegenerative disorders characterized by the deposition of autofluorescent proteinaceous fingerprint or curvilinear bodies. We have found that CLN3, the gene underlying the juvenile form of NCL, is very tightly linked to the dinucleotide repeat marker D16S285 on chromosome 16. Integration of D16S285 into the genetic map of chromosome 16 by using the Centre d'Etude du Polymorphisme Humain panel of reference pedigrees yielded a favored marker order in the CLN3 region of qtel-D16S150-.08-D16S285-.04-D16S148-.02-D16S 67-ptel. The most likely location of the disease gene, near D16S285 in the D16S150-D16S148 interval, was favored by odds of greater than 10(4):1 over the adjacent D16S148-D16S67 interval, which was recently reported as the minimum candidate region. Analysis of D16S285 in pedigrees with late-infantile NCL virtually excluded the CLN3 region, suggesting that these two forms of NCL are genetically distinct. Show less

Batten disease, juvenile onset neuronal ceroid lipofuscinosis, is an autosomal recessive neurodegenerative disorder characterized by accumulation of autofluorescent lipopigment in neurons and other cell types. The disease locus (CLN3) has previously been assigned to chromosome 16p. The genetic localization of CLN3 has been refined by analyzing 70 families using a high-resolution map of 15 marker loci encompassing the CLN3 region on 16p. Crossovers in three maternal meioses allowed localization of CLN3 to the interval between D16S297 and D16S57. Within that interval alleles at three highly polymorphic dinucleotide repeat loci (D16S288, D16S298, D16S299) were found to be in strong linkage disequilibrium with CLN3. Analysis of haplotypes suggests that a majority of CLN3 chromosomes have arisen from a single founder mutation. Show less

Primary cultures of immature rat Sertoli cells in plastic dishes are highly responsive to follicle stimulating hormone (FSH) and its second messenger, cAMP, in metabolizing testosterone to estradiol, thus indicating the presence of an active, hormone-regulated aromatase cytochrome P450 (P450arom). However, in vivo studies indicated that P450arom is FSH-responsive only in very young animals, where the cells have not yet differentiated, but they lose this ability later on in development. Sertoli cells grown on Matrigel (a reconstituted basement membrane), laminin (a basement membrane component), or in bicameral chambers coated with Matrigel, assume structural and functional characteristics more similar to that of in vivo differentiated Sertoli cells. When the cells were cultured on laminin or Matrigel, the FSH- and cAMP-induced estradiol production was greatly reduced by 30 and 60%, respectively. When Sertoli cells were cultured in bicameral chambers coated with Matrigel, no induction of testosterone aromatization by FSH or cAMP was observed. However, FSH-induced cAMP formation was greater when the cells were cultured on basement membrane or in the chambers than on plastic dishes. These results suggest that culture conditions favoring the assumption by Sertoli cells of a phenotype closer that of the differentiated cells in vivo (tall columnar and highly polarized) suppress the induction of P450arom by FSH and cAMP. We then examined the mechanism(s) by which cell phenotype affects p450arom activity. Northern blot analyses of Sertoli cell RNA revealed one major band of 1.9 Kb and two minor bands of 3.3 and 5.2 Kb. However, there were no changes at the level of the expression of P450arom messenger RNA under the different culture conditions.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

In the yeast Saccharomyces cerevisiae, the Cdc28 protein kinase controls commitment to cell division at Start, but no biologically relevant G1-phase substrates have been identified. We have studied the kinase complexes formed between Cdc28 and each of the G1 cyclins Cln1, Cln2, and Cln3. Each complex has a specific array of coprecipitated in vitro substrates. We identify one of these as Far1, a protein required for pheromone-induced arrest at Start. Treatment with alpha-factor induces a preferential association and/or phosphorylation of Far1 by the Cln1, Cln2, and Cln3 kinase complexes. This induced interaction depends upon the Fus3 protein kinase, a mitogen-activated protein kinase homolog that functions near the bottom of the alpha-factor signal transduction pathway. Thus, we trace a path through which a mitogen-activated protein kinase regulates a Cdc2 kinase. Show less

Batten disease, or neuronal ceroid-lipofuscinosis (CLN) comprises a group of inherited neurodegenerative disorders characterized by the accumulation of autofluorescent lipopigment in neurones. The three main childhood varieties--infantile (CLN1), late-infantile (CLN2) and juvenile (CLN3)--manifest autosomal recessive inheritance. The basic biochemical defect remains unknown. The strategy of positional cloning is being pursued to elucidate the molecular basis of Batten disease. The infantile disease locus (CLN1) has been mapped by linkage analysis to human chromosome 1p32, and the juvenile disease locus (CLN3) to human chromosome 16p12. In each case marker loci in strong linkage disequilibrium with the disease loci have been identified. Locus heterogeneity between classical late-infantile CLN (CLN2) and both CLN1 and CLN3 has been demonstrated. Work is in progress to clone CLN1 and CLN3 and to map CLN2. Identification of linked markers has provided a new approach to prenatal diagnosis. The methodology exists for positional cloning of these genes and elucidation of the molecular genetic basis of the ceroid lipofuscinoses. Show less

Cyclin-dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras-associated GTPase-activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation. Show less

Expression of the vacuolar carboxypeptidase S (CPS1) gene in Saccharomyces cerevisiae is regulated by the availability of nutrients. Enzyme production is sensitive to nitrogen catabolite repression; i.e. the presence of ammonium ions maintains expression of the gene at a low level. Transfer of ammonium-glucose pre-grown cells to a medium deprived of nitrogen causes a drastic increase in CPS1 RNA level provided that a readily usable carbon source, such as glucose or fructose, is available to the cells. Derepression of the gene by nitrogen limitation is cycloheximide-insensitive. Neither glycerol, ethanol, acetate nor galactose support derepression of CPS1 expression under nitrogen starvation conditions. Non-metabolizable sugar analogs (2-deoxyglucose, 6-methyl-glucose or glucosamine) do not allow derepression of CPS1, showing that the process is energy-dependent. Production of carboxypeptidase yscS also increases several-fold when ammonium-pregrown cells are transferred to media containing glucose and a non-readily metabolizable nitrogen source such as proline, leucine, valine or leucyl-glycine. Analysis of CPS1 expression in RAS2+ (high cAMP) and ras2 mutant (low cAMP) strains and in cells grown at low temperature (23 degrees C) and in heat-shocked cells (38 degrees C) shows that steady-state levels of CPS1 mRNA are not controlled by a low cAMP level-signalling pathway. Show less

The Cln3 cyclin homolog of Saccharomyces cerevisiae functions to promote cell cycle START for only a short time following its synthesis. Cln3 protein is highly unstable and is stabilized by C-terminal truncation. Cln3 binds to Cdc28, a protein kinase catalytic subunit essential for cell cycle START, and Cln3 instability requires Cdc28 activity. The long functional lifetime and the hyperactivity of C-terminally truncated Cln3 (Cln3-2) relative to those of full-length Cln3 are affected by mutations in CDC28: the functional lifetime of Cln3-2 is drastically reduced by the cdc28-13 mutation at the permissive temperature, and the cdc28-4 mutation at the permissive temperature completely blocks the function of Cln3-2 while only partially reducing the function of full-length Cln3. Thus, sequences in the C-terminal third of Cln3 might help stabilize functional Cdc28-Cln3 association, as well as decreasing the lifetime of the Cln3 protein. These and other results strongly support the idea that Cln proteins function to activate Cdc28 at START. Show less

The expression of guanine nucleotide-binding proteins (G proteins) during the development of rat testes was investigated. Immunohistochemical studies on frozen sections and isolated testicular cells demonstrated that the expression of the GTP-binding proteins was developmentally regulated and specific for different cell types. The alpha subunit of the cholera toxin-sensitive stimulatory G protein (Gs alpha) was first detected in testes from 7-day-old rats; its value reached a maximum at 23 days and then decreased to very low or undetectable amounts in testes of 45-day-old and adult rats (60-90 days of age). The Gs alpha subunit appears to be expressed by Sertoli, peritubular myoid and interstitial cells. The common beta subunit (G beta) was present at all ages during development and was more prominent around the periphery of the tubules in younger animals but then became more evident in the cytoplasm of germ cells with increasing age. The pertussis toxin-sensitive inhibitory G proteins, Gi1/2 and Gi3, showed a similar pattern of expression. Sertoli cells and peritubular cells expressed Gi1/2 and Gi3 at very low levels at all ages, whereas pachytene spermatocytes and round spermatids expressed the inhibitory binding proteins only at later ages of development (45-day-old and adult testis). Northern blot analysis showed that with increasing age the Gs alpha mRNA in the testis decreased and this was confirmed by in situ hybridization. These latter studies showed localization of the transcripts to somatic cells but not to germ cells.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

In budding yeast, a switch between the mutually exclusive pathways of cell cycle progression and conjugation is controlled at Start in late G1 phase. Mating pheromones promote conjugation by arresting cells in G1 phase before Start. Pheromone-induced cell cycle arrest requires a functional FAR1 gene. We have found that FAR1 transcription and protein accumulation are regulated independently during the cell cycle. FAR1 RNA and protein are highly expressed in early G1, but decline sharply at Start. Far1 is phosphorylated just before it disappears at Start, suggesting that modification may target Far1 for degradation. Although FAR1 mRNA levels rise again during late S or G2 phase, reaccumulation of Far1 protein to functional levels is restricted until after nuclear division. Show less

A family with two siblings, 10 and 8 years old, both with clinical and ultrastructural evidence of juvenile neuronal ceroid lipofuscinosis is described. The family was found to be informative for the restriction fragment length polymorphisms (RFLPs) detected by the probes pCJ52-95M1 (locus D16S148) and pCJ52-94T1 (locus D16S159) flanking the juvenile neuronal ceroid lipofuscinosis locus, CLN3. The parents were both heterozygous using these probes, while their two children with juvenile neuronal ceroid lipofuscinosis were both homozygous. Chorionic villi analysis showed that the fetus was heterozygous and had inherited the one allele of the mother which was not found in the two siblings. This suggested that the fetus had derived one healthy allele from the mother, the risk for a double crossing-over being less than 1 per cent. Electron microscopy showed no fingerprint inclusions in chorionic villi. The child was investigated at 6 months of age and found to be healthy, as new fingerprint inclusions were found at electron microscopy and no vacuolated lymphocytes were found in the blood smear. Due to the risk of heterogeneity, both DNA-based analysis and electron microscopy on chorionic villi are recommended for prenatal examination for juvenile neuronal ceroid lipofuscinosis. Show less

We have identified two processes in the G1 phase of the Saccharomyces cerevisiae cell cycle that are required before nutritionally arrested cells are able to return to proliferative growth. The first process requires protein synthesis and is associated with increased expression of the G1 cyclin gene CLN3. This process requires nutrients but is independent of Ras and cyclic AMP (cAMP). The second process requires cAMP. This second process is rapid, is independent of protein synthesis, and produces a rapid induction of START-specific transcripts, including CLN1 and CLN2. The ability of a nutritionally arrested cell to respond to cAMP is dependent on completion of the first process, and this is delayed in cells carrying a CLN3 deletion. Mating pheromone blocks the cAMP response but does not alter the process upstream of Ras-cAMP. These results suggest a model linking the Ras-cAMP pathway with regulation of G1 cyclin expression. Show less

Two genetic polymorphisms of salivary proteins were found by polyacrylamide gel electrophoresis among inbred strains of rats. Both proteins (RSP-1 and RSP-2) were inherited as a single autosomal trait. The loci were designated as Rsp-1 (rat salivary protein-1) and Rsp-2. Rsp-1 had two codominant alleles (Rsp-1a, Rsp-1b), and Rsp-2 had two alleles (Rsp-2a, and Rsp-2b); Rsp-2a was dominant over Rsp-2b. The Rsp-1 locus is not linked with the linkage groups (LGs) I, II, IV, V and the LGs containing Acp-2 and Pg-1. The Rsp-2 is not linked with the LGs I, II, V, X and the LGs containing Amy-1, Es-6 and Pg-1. Show less

The CLN1, CLN2 and CLN3 gene family of G1-acting cyclin homologs of Saccharomyces cerevisiae is functionally redundant: any one of the three Cln proteins is sufficient for activation of Cdc28p protein kinase activity for cell cycle START. The START event leads to multiple processes (including DNA replication and bud emergence); how Cln/Cdc28 activity activates these processes remains unclear. CLN3 is substantially different in structure and regulation from CLN1 and CLN2, so its functional redundancy with CLN1 and CLN2 is also poorly understood. We have isolated mutations that alter this redundancy, making CLN3 insufficient for cell viability in the absence of CLN1 and CLN2 expression. Mutations causing phenotypes specific for the cell division cycle were analyzed in detail. Mutations in one gene result in complete failure of bud formation, leading to depolarized cell growth. This gene was identified as BUD2, previously described as a non-essential gene required for proper bud site selection but not required for budding and viability. Bud2p is probably the GTPase-activating protein for Rsr1p/Bud1p [Park, H., Chant, I. and Herskowitz, I. (1993) Nature, 365, 269-274]; we find that Rsr1p is required for the bud2 lethal phenotype. Mutations in two other genes (ERC10 and ERC19) result in a different morphogenetic defect: failure of cytokinesis resulting in the formation of long multinucleate tubes. These results suggest direct regulation of diverse aspects of bud morphogenesis by Cln/Cdc28p activity. Show less

The ceroid-lipofuscinoses are a group of inherited neurodegenerative disorders characterised by the accumulation of autofluorescent lipopigment in neurones and other cell types. The underlying biochemical defect is unknown. Juvenile onset neuronal ceroid lipofuscinosis (Batten disease; Spielmeyer-Vogt disease) is an autosomal recessive trait. Linkage studies were undertaken to determine the location of the Batten disease (CLN3) mutation. Studies were carried out on 205 members of 42 families in which there were 76 affected individuals. Families originated from 7 North European countries and Canada. Serum samples from 23 families, including a total of 48 affected children, were tested for a set of "classical markers." A positive lod score was found with the haptoglobin (Hp) system. The combined male and female maximum lod score was 3.00 at theta = 0.00 and theta = 0.26, respectively. This provided an indication of localisation to the long arm of chromosome 16. Linkage analysis was then carried out in 42 families using DNA markers for loci on human chromosome 16. The maximal lod score between Batten disease and the locus D16S148 calculated for combined sexes was 6.05. No recombinants were observed. Multilocus analysis using 5 loci indicated the most likely order to be HP-D16S151-D16S150-CLN3-D16S148-D16S147. Work is in progress to refine the genetic and physical localisation of the Batten disease gene using additional markers in this region and a panel of somatic cell hybrids. Methods are now available which should allow the gene to be isolated and characterised. Show less

A number of years ago we reported that tight junctions between adjacent Sertoli cells subdivide the seminiferous epithelium into two compartments, basal and adluminal, thus forming the morphological basis of the blood-testis barrier. It is now generally believed that the special milieu created by the Sertoli cells in the adluminal compartment is essential for germ cell differentiation. In order to duplicate the compartmentalization that occurs in vivo, Sertoli cells were cultured in bicameral chambers on Millipore filters impregnated with a reconstituted basement membrane. Confluent monolayers of these cells were tall columnar (40-60 microns in height) and highly polarized. These Sertoli cell monolayers established electrical resistance that peaked when the Sertoli-Sertoli tight junctions developed in culture. In addition, the monolayers formed a permeability barrier to 3H-inulin and lanthanum nitrate. The bicameral chambers were utilized in a number of studies on protein secretion, and it was revealed that numerous proteins are secreted in a polarized manner. In another study, hormone-stimulated aromatase activity was measured in Sertoli cells grown on plastic culture dishes, plastic dishes coated with laminin or Matrigel, and in the bicameral chambers. Cell culture on basement membrane substrates decreased the FSH-dependent estrogen production. No estrogen production was observed when the Sertoli cells were cultured in the bicameral chambers. These results are in accord with the hypothesis that differentiated Sertoli cells lose their ability to metabolize androgen to estrogen in an hormone-dependent manner, whereas undifferentiated cells in culture, or in vivo, have a very active FSH-dependent aromatase activity.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

Halophilic malate dehydrogenase (hMDH) from Haloarcula marismortui has been isolated, purified and characterized by biochemical and biophysical solution studies. A stabilization mechanism at extremely high concentrations of salt, based on the formation of co-operative hydrate bonds between the protein and hydrated salt ions, was suggested from thermodynamic analysis of native enzyme solutions. Recently the gene coding for hMDH was isolated and sequenced and an active enzyme cloned (F. Cendrin, J. Chroboczek, G. Zaccai, H. Eisenberg and M. Mevarech, unpublished work). A study of the crystal structure of hMDH in a high-salt physiological medium is in progress (O. Butbul-Dym & J. Sussman, personal communication). Here we discuss in depth implications of these recent developments on our earlier results. Show less

Two low M(r) phosphotyrosine protein phosphatases have been isolated from rat liver. The enzymes were previously known as low M(r) acid phosphatases, but several recent studies have demonstrated that this family of enzymes possesses specific phosphotyrosine protein phosphatase activity. We determined the complete amino acid sequences of the two isoenzymes and named them AcP1 and AcP2. Both consist of 157 amino acid residues, are acetylated at the NH2-terminus, and have His as the COOH-terminus. The molecular weights calculated from the sequences are 18,062 for AcP1 and 17,848 for AcP2. They are homologous except in the 40-73 zone, where about 50% of residues are different. This fact suggests that the two isoenzymes are produced by an alternative splicing mechanism. There is no homology between these two isoenzymes and the receptor-like phosphotyrosine protein phosphatases LAR, CD45, human placenta PTPase 1B, and rat brain PTPase-1. AcP1 and AcP2 are also distinct from rat liver PTPase-1 and PTPase-2, since these last enzymes have higher molecular weights. AcP1 differs from AcP2 with respect to (1) substrate affinity and (2) its sensitivity to activators and inhibitors, thus suggesting a their different physiological function. Show less