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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isolated and sequenced and an active enzyme cloned (F. Cendrin, J. Chroboczek, G. Zaccai, H. Eisenberg and M. Mevarech, unpublished work).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

Budding yeast strains have three CLN genes, which have limited cyclin homology. At least one of the three is required for cell cycle START. Four B cyclins are known in yeast; two have been shown to function in mitosis. We have discovered a fifth B-cyclin gene, called CLB5, which when cloned on a CEN plasmid can rescue strains deleted for all three CLN genes. CLB5 transcript abundance peaks in G1, coincident with the CLN2 transcript but earlier than the CLB2 transcript. CLB5 deletion does not cause lethality, either alone or in combination with other CLN or CLB deletions. However, strains deleted for CLB5 require more time to complete S phase, suggesting that CLB5 promotes some step in DNA synthesis. CLB5 is the only yeast cyclin whose deletion lengthens S phase. CLB5 may also have some role in promoting the G1/S transition, because cln1 cln2 strains require both CLN3 and CLB5 for viability on glycerol media and cln1,2,3- strains require CLB5 for rescue by the Drosophila melanogaster cdc2 gene. In conjunction with cln1,2,3- rescue by CLB5 overexpression and the coincident transcriptional regulation of CLB5 and CLN2, these observations are suggestive of partial functional redundancy between CLB5 and CLN genes. Show less

In Saccharomyces cerevisiae, several of the proteins involved in the Start decision have been identified; these include the Cdc28 protein kinase and three cyclin-like proteins, Cln1, Cln2 and Cln3. We find that Cln3 is a very unstable, low abundance protein. In contrast, the truncated Cln3-1 protein is stable, suggesting that the PEST-rich C-terminal third of Cln3 is necessary for rapid turnover. Cln3 associates with Cdc28 to form an active kinase complex that phosphorylates Cln3 itself and a co-precipitated substrate of 45 kDa. The cdc34-2 allele, which encodes a defective ubiquitin conjugating enzyme, dramatically increases the kinase activity associated with Cln3, but does not affect the half-life of Cln3. The Cln--Cdc28 complex is inactivated by treatment with non-specific phosphatases; prolonged incubation with ATP restores kinase activity to the dephosphorylated kinase complex. It is thus possible that phosphate residues essential for Cln-Cdc28 kinase activity are added autocatalytically. The multiple post-translational controls on Cln3 activity may help Cln3 tether division to growth. Show less

In Saccharomyces cerevisiae, the RNA levels of the G1 cyclins CLN1, CLN2, and HCS26 increase dramatically during the late G1 phase of the cell cycle. The SIT4 gene, which encodes a serine/threonine protein phosphatase, is required for the normal accumulation of CLN1, CLN2, and HCS26 RNAs during late G1. This requirement for SIT4 in normal G1 cyclin RNA accumulation is at least partly via SWI4. Strains containing mutations in SIT4 are sensitive to the loss of either CLN2 or CLN3 function. At the nonpermissive temperature, temperature-sensitive sit4 strains are blocked for both bud emergence and DNA synthesis. Heterologous expression of CLN2 in the absence of SIT4 function results in DNA synthesis, but most of the cells are still blocked for bud emergence. Therefore, SIT4 is required for at least two late G1 or G1/S functions: the normal accumulation of G1 cyclin RNAs (which is required for DNA synthesis) and some additional function that is required for bud emergence or cell cycle progression through late G1 or G1/S. Show less

Following v-Ha-ras transfection of nonmetastatic dimethylbenz(( a ))anthracene-induced rat mammary cancer (RMC1) cells, occasional transfectants were isolated that acquired high metastatic ability. High metastatic ability is not a simple process regulated by v-Ha-ras p21 levels alone in these v-Ha-ras transfectants but involves the development of cytogenetic changes. If such cytogenetic changes involve only gain in gene expression, then all hybrids formed by fusing highly metastatic v-Ha-ras RMC1 transfectants with the parental nonmetastatic RMC1 should be highly metastatic. If loss of a metastatic suppressor gene(s) is also involved, then such hybrids should be nonmetastatic since chromosomes from the nonmetastatic parental cells should supply the suppressor function. To test this possibility, a highly metastatic cloned v-Ha-ras transfectant was fused with the nonmetastatic parental RMC1 cells. Five hybrid clones were isolated that conserved the chromosomes from their parental cells. When these hybrid clones were injected into animals, primary tumors developed with the same tumor-doubling time as that of the highly metastatic parental v-Ha-ras transfectant (i.e., approximately 2 days). High metastatic ability was, however, suppressed in these hybrid clones. All hybrid clones continued to express v-Ha-ras p21. Thus, suppression of metastatic ability in the hybrids can occur even in the presence of an elevated v-Ha-ras p21 level. This suggests that the acquisition of metastatic ability following v-Ha-ras transfection involves loss of metastasis suppressor gene function in rat mammary cancer cells. Show less

Growth of S. cerevisiae cells by budding gives rise to asymmetric progeny cells: a larger "mother" cell and a smaller "daughter" cell. The mother cell transits a brief G1 phase before forming a new bud and beginning DNA replication. The daughter cell stays in G1 for a longer period, growing in size before initiating a new cell cycle. We show that the timing of cell cycle initiation in mother and daughter cells is governed by different G1 cyclins. In daughter cells, transcription of CLN1 and CLN2 is induced in a size-dependent manner, and these cyclins are necessary for the normal timing of cell cycle initiation. CLN3 is not required in daughter cells, but is crucial for mother cells, in which the G1 phase is much longer in the absence of this cyclin. Show less

The cell cycle in Saccharomyces cerevisiae is controlled by regulation of START in late G1. The CLN1, CLN2 and CLN3 family of cyclin homologues is required for cells to pass START. They probably act by activating the CDC28 protein kinase. Expression of CLN1 or CLN3 under the control of an inducible promoter shows that transcription of either gene is sufficient for cyclin-deficient strains arrested in G1 to traverse START. A model of START regulation involves activation of CDC28 kinase by any CLN protein, leading to activation of CLN1 and CLN2 transcription in a positive feedback loop and passage through START. The cell cycle-dependent transcriptional regulators SWI4 and SWI6 may be components of the feedback loop. Cell cycle commitment entails resistance to the inhibitory action of mating factor, which correlates with peak levels of CLN1 and CLN2 mRNAs. FAR1 encodes an alpha-factor-dependent inhibitor of CLN function whose expression is markedly reduced at the time of START. The interplay of all these factors may sharpen the START transition such that it is close to an all-or-nothing switch event. This may be important for several START-dependent events to be activated at the same time, leading to coordinated cell cycle progression. Show less

Mutants supersensitive to the spindle poison, Isopropyl N-3-chlorophenyl carbamate (CIPC) of the fission yeast Schizosaccharomyces pombe were isolated and characterized genetically. Fourteen different recessive loci were assigned for the mutation (donated as cps1 to cps14) and two, cps1 and cps3, were mapped precisely on the chromosomes. Nine mutant strains were also supersensitive to phenothiazine derivatives, inhibitors of calcium-binding protein calmodulin. Four of nine strains were incapable of growing in the presence of 10 microM calcium ionophore A23187, at which the drug had no effect on cell growth in other strains. Fluorescence microscopy using the DAPI and Calcofluor staining methods showed two strains out of four to be defective in normal cell division; most stationary-phase cells of the cps6 mutant were seen to be bi- or tetra-nucleate, being partitioned with one or three septa, respectively. In the other mutant (cps8), enlarged cells were unequally partitioned with multisepta, and each compartment contained several daughter nuclei. The septa appeared aberrant in position within the cell, and situated diagonally but not vertically along the long cell axis. Show less

Sertoli cell nuclei are characterized by deep invaginations and, in addition, the orientation of the nuclei with respect to the wall of the seminiferous tubules varies during the cycle of the seminiferous epithelium. These events may be the result of cytoplasmic filaments acting at the level of the nuclear capsule and may represent significant changes in Sertoli cell activity. Thus, a study was performed to characterize the nature of the perinuclear filaments of Sertoli cells in vivo and in vitro. In Sertoli cells in vivo, microtubules and microfilaments were often detected in the perinuclear cytoplasm, and these cytoskeletal components were observed to course either parallel to, or abut at, the nuclear capsule. In Sertoli cells in vitro, the nuclear infoldings are retained and the perinuclear cytoskeleton was shown to contain microtubules, f-actin, and intermediate filaments. A fixation-permeabilization protocol employing tannic acid-saponin was used and it significantly enhanced the preservation of cytoskeletal components. The presence of f-actin was demonstrated by using the S1 fragment of muscle myosin to decorate the microfilaments. Treatment of the cultured cells with either microtubule or f-actin depolymerizing agents had no effect on nuclear shape. Thus, at present, the function of the prominent perinuclear cytoskeletal components remains unknown. Show less

We have studied the interaction of a human tumor cell line, A253, derived from a submandibular gland carcinoma with a differentiation promoting reconstituted basement membrane extract, Matrigel. When cultured on plastic, these cells maintain a flat, cobblestone, epithelial morphology. On Matrigel, A253 cells initially form a honeycomb network of cords of cells which subsequently thickens. With time, these cords of cells become discontinuous and blunted, whereupon multilobular clusters of cells develop. These clusters possess a lumen with polarized, PAS(+) cells containing numerous desmosomes and an abundance of glycogen. Culture of the cells on laminin, the most abundant protein found in Matrigel, also induces this morphologic differentiation. Using synthetic laminin-derived peptides, the biologically active IKVAV-containing site of laminin was most active in attachment assays, as well as in inhibiting glandular-like morphogenesis when added to the media of cells cultured on Matrigel. Antibodies to the cell surface 67- and 32-kDa laminin binding proteins partially inhibited the glandular-like morphogenesis, suggesting that multiple interactions with laminin are likely required for the differentiation process. Our data demonstrate that A253 cells can undergo glandular-like morphogenesis on basement membrane and that laminin appears to be the major initiating factor. Show less

In this report we present the genomic, cDNA, and predicted protein sequences for mouse apolipoproteins A-I and CIII, as well as sequence comparisons with other species. The genes for these apolipoproteins are within 2.5 kb of each other and convergently transcribed. The almost 9 kb of genomic sequence presented extends from 1298 bp 5' to the apolipoprotein A-I (Apoa-1) gene to 1249 bp 5' to the apolipoprotein CIII (Apoc-3) gene. The mouse Apoa-1 gene is 1.76 kb in length with four exons and three introns. The 5' flanking region contains TATA and CCAAT box sequences, an interferon responsive element homology, and potential binding sites for transcription factors CTF/NF1 and HNF4. Translation of the cDNA predicts that the mouse Apoa-1 primary transcript is 264 amino acids. The mouse Apoc-3 gene is 2.2 kb in length and also consists of four exons and three introns. The 5' flanking region contains TATA and CCAAT box sequences, RXR-1 and ARP-1 binding sites, and potential binding sites for transcription factors HNF4, NFkB, AP-1, and CTF/NF1. Translation of the cDNA predicts that the mouse Apoc-3 primary transcript is 99 amino acids. The clustering and genomic organization of the mouse Apoa-1 and Apoc-3 genes are similar to those of the rat and human genes. Significant sequence homologies between species exist for the proximal promoter and exonic regions of each gene, but not for the intronic or intergenic regions.(ABSTRACT TRUNCATED AT 250 WORDS) Show less

Human pregnancy-specific beta 1-glycoprotein (PSG) is found in high concentrations in the serum of pregnant women, but also has been found in the serum of males and nonpregnant females. Northern slot-blot analysis has demonstrated the presence of PSG mRNA in a variety of tissues in the rat, with the highest levels being found in the testis. Therefore, we have investigated further the expression of PSG in the rat male reproductive tract using in situ hybridization. In testes from immature and adult rats, PSG mRNA was localized in Leydig and peritubular cells, and in the walls of the interstitial blood vessels. PSG transcripts were noted also in the tunica albuginea and in the stromal tissue of the caput and cauda epididymis, prostate, and seminal vesicle from adult rats. The function of PSG is unknown, but it has been speculated that PSG may have immunosuppressive properties or that it may serve as a paracrine regulator of growth and differentiation. It is possible, then, that PSG could contribute to the immunological privilege of the testis or that it plays a role in the cellular interactions which increasingly are being shown to be important in the regulation of male reproductive tract tissues. Show less

An irradiation-reduced somatic cell hybrid mapping panel was constructed of BALB/c mouse Chromosome 1. Nineteen hybrids were selected from a pool of 292 clones to generate a fine structure physical map of the distal 40 cM of the chromosome. The hybrids contain mouse DNA fragments only from Chromosome 1, ranging from approximately 5 cM to approximately 20 cM. Utilizing a viral infectibility assay, a cellular receptor gene, Rmc-1, for the MCF class of murine retroviruses was found to be linked to Lamb2, in the region between the Lamb2 and Bxv-1 loci. In addition, analysis of the hybrid mapping panel resulted in the remapping of three loci, Atpb, Ly-5, and Pmv-24, as compared to the mouse linkage map. Two previously unmapped endogenous proviruses are also putatively assigned positions on the chromosome. Show less

A Saccharomyces cerevisiae genomic DNA encoding vacuolar carboxypeptidase yscS was cloned from a yeast YEp13 library by complementation of the previously characterized mutation cps1-1 [(1981) J. Bacteriol. 147, 418-426], by means of staining carboxypeptidase activity in yeast colonies. The nucleotide sequence of the cloned gene was determined. The open reading frame of CPS1 consists of 576 codons and therefore encodes a protein of 64961 molecular weight. A stretch of 19 residues near the N-terminus of the deduced polypeptide sequence contains characteristics common to known hydrophobic leader sequences. CPS1 was determined by DNA blot analysis to be a single copy gene located on chromosome X. The cloned fragment was used to identify a 2.1 kb mRNA. A transcriptional activation of CPS1 occurs when cells grow on a substrate of carboxy-peptidase yscS as sole nitrogen source. Show less

The neuronal ceroid lipofuscinoses comprise a group of inherited neurodegenerative disorders characterized by the accumulation of autoflourescent lipopigment in neurones and other cell types. Three main childhood sub-types occur: infantile (Haltia-Santavouri disease, locus CLN1), late-infantile (Jansky-Bielschowsky disease, locus CLN2) and juvenile (Spielmeyer-Sjogren-Vogt, Batten disease, locus CLN3). Inheritance is autosomal recessive. The basic biochemical defect remains unknown. The infantile disease Iocus (CLN1) has been mapped to human chromosome 1p32 and the juvenile disease Iocus (CLN3) to human chromosome 16p12 by linkage analysis. Marker loci in strong allelic association with the disease loci have been identified in each case and haplotype analysis suggests a founder mutation for CLN1 and CLN3. Classical late-infantile disease (CLN2) has been shown not to be an allelic variant of either CLN1 or CLN3. Identification of linked markers has provided a new method for pre-natal diagnosis. Work is in progress to clone CLN1 and CLN3 and to map CLN2. This will allow elucidation of the molecular genetic basis of the neuronal ceroid lipofuscinoses. Show less

One hundred fifty-four unrelated French Caucasian subjects were typed for 11 RFLPs at or near the APOA1-C3-A4 gene cluster on the long arm of chromosome 11. All subjects belonged to families having lived in the Toulouse area (in the southwest of France) for over three generations. Allele frequencies for each RFLP were in agreement with previous studies in Caucasian populations for the APOA1/SstI marker. Pairwise linkage disequilibrium was determined. Among the 55 pairs studied, 30 are newly reported. Only three significant nonrandom associations were found: APOA1/MspI-3'APOC3/SstI, APOA1/MspI-3'APOA4/XbaI, and APOA4/DraI-APOA4/XbaI. Extended 11-marker haplotypes were constructed. Haplotype frequencies were estimated by the maximum-likelihood procedure and compared to expected frequencies calculated under the assumption of equilibrium. Among the 37 estimated haplotypes, seven containing at least four nonrandomly associated alleles showed markedly increased frequencies. These results, obtained in a geographically homogeneous population, confirm the existence of disequilibrium in the apolipoprotein cluster, but to a lower extent than previously reported in Caucasian populations, which were geographically more heterogeneous. Show less

The GPA1 gene of S. cerevisiae encodes a G alpha subunit that plays a positive role in the transduction of signals stimulating recovery from pheromone-induced cell cycle arrest. The GPA1Val50 mutation, in which Gly-50 is replaced by valine, causes hyperadaptation to pheromone. However, GPA1Val50 cells do not recover from division arrest in the absence of both CLN1 and CLN3, which encode G1 cyclins, indicating that the recovery-promoting activity of GPA1Val50 requires the function of G1 cyclins. An sgv1 mutation suppresses the hyperadaptive response caused by GPA1Val50 and also confers cold- and temperature-sensitive growth. The SGV1 gene encodes an apparent protein kinase homologous to CDC28/cdc2 kinase: SGV1 is 42% identical to CDC28. The activated mutation, CLN3-2, partially suppresses the growth defect of sgv1, suggesting that the SGV1 and CLN3 proteins may act in the same growth control pathway. Show less

Laminin is a potent stimulator of neurite outgrowth. We have examined the signal transduction events involved in the neuronal cell response to laminin. Cyclic nucleotides, calcium, and sodium-proton exchange do not appear to be required for the transduction of the laminin signal during neurite outgrowth. Direct measurement of cAMP and cGMP levels shows no changes in NG108-15 cells when cultured on laminin. Exogenous cAMP alone had no effect on either the rate of process formation or process length, but did alter the morphology of laminin-induced neurites. A four-fold increase in the number of branches per neurite and a two-to-three-fold increase in the number of neurites per cell were observed in both NG108-15 and PC12 cells cultured on laminin when either 8-BrcAMP or forskolin was added. The cAMP-induced branching was also observed when PC12 cells were cultured on a laminin-derived synthetic peptide (PA22-2), which contains the neurite-promoting amino acid sequence IKVAV. By immunofluorescence analysis with axonal or dendritic markers, the PC12 processes on laminin and PA22-2 were axonal, not dendritic, and the cAMP-induced morphological changes were due to axonal branching. These data demonstrate that changes in cAMP are not involved in laminin-mediated neurite outgrowth, but cAMP can modulate the effects of laminin. Show less

For the budding yeast Saccharomyces cerevisiae the mitotic cell cycle is coordinated with cell mass at the regulatory step "start". The threshold amount of cell mass (reflected as a "critical size") necessary for "start" is proportional to nutrient quality. This relationship leads to a transient accumulation of cells at "start", termed nutrient modulation, upon enrichment of nutrient conditions. Nutrient enrichment abruptly increases the critical size needed for "start", causing the smaller cells, produced in the previous cell cycle, to be delayed at "start" while growing larger. Here we show that, in S. cerevisiae, a second cell-cycle step, at mitosis, also exhibits nutrient modulation, and is, therefore, another point of cell-cycle regulation. At both mitosis and "start", nutrient modulation was found through mutation to be regulated by the activity of the cyclin-related WHI1 (CLN3) gene product. Show less

A panel of 18 rat x mouse somatic cell hybrid clones segregating individual rat chromosomes in different combinations was used to assign 23 biochemical loci to rat chromosomes. The chromosomal locations for these 23 loci were determined as follows: GOT1 on rat chromosome 1; HAGH on 2; ACP2, ADA, GANC, ITPA, and SORD on 3; LDHB on 4; PEPB on 7; GLB1 and HEXA on 8; IDH1 on 9; UMPH2 on 10; GUSB on 12; FH and PEPC on 13; PEPS on 14; ESD and NP on 15; DIA4 on 19; and PP on 20. In addition, ACP1 and GLO1 were reassigned to rat chromosomes 6 and 20, respectively. The chromosomal assignments of these loci extends the known syntenic homologies among rats, mice, and humans. Show less

The CLN1, CLN2, and CLN3 genes of S. cerevisiae form a redundant family essential for the G1-to-S phase transition. CLN1 and CLN2 mRNAs were previously shown to be negatively regulated by mating pheromone and by cell cycle progression out of G1, whereas CLN3 mRNA is not. The CLN3-2 (DAF1-1) allele prevents both cell cycle arrest and the turnoff of CLN1 and CLN2 mRNAs in response to mating pheromone, but only in the presence of an active CDC28 gene. An internally deleted nonfunctional cln2 gene was used as a reporter gene to demonstrate that in the absence of mating pheromone, efficient expression of cln2 mRNA requires both an active CDC28 gene and at least one functional CLN gene. mRNA from a nonfunctional cln1 gene was regulated similarly. Thus, CLN function and CDC28 activity jointly stimulate CLN1 and CLN2 mRNA levels, potentially forming a positive feedback loop for CLN1 and CLN2 expression. Show less