Showing papers by "Cold Spring Harbor Laboratory published in 1991"
TL;DR: A hypothesis is proposed explaining how phosphatases might act synergistically with the kinases to elicit a full physiological response, without regard to the state of phosphorylation of the target proteins.
Abstract: Protein tyrosine phosphatases (PTPs) represent a diverse family of enzymes that exist as integral membrane and nonreceptor forms. The PTPs, with specific activities in vitro 10 to 1000 times greater than those of the protein tyrosine kinases would be expected to effectively control the amount of phosphotyrosine in the cell. They dephosphorylate tyrosyl residues in vivo and take part in signal transduction and cell cycle regulation. Most of the transmembrane forms, such as the leukocyte common antigen (CD45), contain two conserved intracellular catalytic domains; but their external segments are highly variable. The structural features of the transmembrane forms suggest that these receptor-linked PTPs are capable of transducing external signals; however, the ligands remain unidentified. A hypothesis is proposed explaining how phosphatases might act synergistically with the kinases to elicit a full physiological response, without regard to the state of phosphorylation of the target proteins.
942 citations
TL;DR: These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC‐35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils, which may be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.
Abstract: SC-35 is a non-snRNP spliceosome component that is specifically recognized by the anti-spliceosome monoclonal antibody alpha SC-35. In this paper we provide direct evidence that SC-35 is an essential splicing factor and we examine the immunolocalization of SC-35 by confocal laser scanning microscopy and by electron microscopy. We have found that the speckled staining pattern observed by fluorescence microscopy corresponds to structures previously designated as interchromatin granules and perichromatin fibrils. Although snRNP antigens are also concentrated in these nuclear regions, we show that the two types of spliceosome components are localized through different molecular interactions: The distribution of SC-35 was not affected by treatment with DNase I or RNase A, or when the cells were heat shocked. In contrast, snRNP antigens become diffusely distributed after RNase A digestion or heat shock. Examination of cells at different stages of mitosis revealed that the SC-35 speckled staining pattern is lost during prophase and speckles containing SC-35 begin to reform in the cytoplasm of anaphase cells. In contrast, snRNP antigens do not associate with speckled regions until late in telophase. These studies reveal a dynamic pattern of assembly and disassembly of the splicing factor SC-35 into discrete nuclear structures that colocalize with interchromatin granules and perichromatin fibrils. These subnuclear regions may therefore be nuclear organelles involved in the assembly of spliceosomes, or splicing itself.
517 citations
TL;DR: SF2 is a protein factor essential for constitutive pre-mRNA splicing in HeLa cell extracts and also activates proximal alternative 5' splice sites in a concentration-dependent manner, suggesting a role for SF2 in preventing exon skipping, ensuring the accuracy of splicing, and regulating alternative splicing.
484 citations
TL;DR: It is shown that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypePTide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.
Abstract: Bovine papillomavirus (BPV) DNA is maintained as an episome with a constant copy number in transformed cells and is stably inherited. To study BPV replication we have developed a transient replication assay based on a highly efficient electroporation procedure. Using this assay we have determined that in the context of the viral genome two of the viral open reading frames, E1 and E2, are required for replication. Furthermore we show that when produced from expression vectors in the absence of other viral gene products, the full length E2 transactivator polypeptide and a 72 kd polypeptide encoded by the E1 open reading frame in its entirety, are both necessary and sufficient for replication BPV in C127 cells.
479 citations
TL;DR: The results are consistent with CDC28 function being required in both G1 and mitosis, and its mitotic role, the authors believe, involves interaction with a family of at least four G2-specific cyclins.
454 citations
TL;DR: VA RNAs are common to alladenoviruses studied todate, but most work has beencentrated on the groupC adenovirus types2and5(Ad2and AdS), upon which this review will necessarily focus.
Abstract: Theadenovirus genome istranscribed bytwoRNA polymerases furnished bythehostcell. RNA polymerase II transcribes bothstrands oftheviral DNA over nearly allof itslength, andtheresultant mRNAs encode more than50 viral proteins. RNA polymerase IIItranscribes lessthan1% oftheviral genome, giving rise to one or two species (depending on thevirus serotype) ofshort, noncoding RNAs (44, 59). ThisRNA was namedvirus-associated (VA)RNA whenits origin fromtheviral genome was still uncertain (45). Eventhough VA RNA was detected ininfected cells asearly as1966, itsrole intranslational control andincounteracting hostantiviral defenses onlybecameapparentinthe1980s andthedetails ofitsaction remain thesubject ofactive investigation. VA RNAs are common toalladenoviruses studied todate, butmostworkhasconcentrated on the groupC adenoviruses, adenovirus types2and5(Ad2and AdS), upon whichthis review will necessarily focus.
397 citations
TL;DR: Results suggest that the sequential binding of RF-C, PCNA, and pol delta to a primer-template junction might directly account for the initiation of leading strand DNA synthesis at a replication origin.
355 citations
TL;DR: The tropomyosins are a family of actin filament binding proteins that exhibit extensive cell type specific isoform diversity and their possible significance to cellular function is discussed.
Abstract: The tropomyosins are a family of actin filament binding proteins. In multicellular animals, they exhibit extensive cell type specific isoform diversity. In this essay we discuss the genetic mechanisms by which this diversity is generated and its possible significance to cellular function.
342 citations
TL;DR: Using tryptic phosphopeptide mapping, it is shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2.
Abstract: The retinoblastoma gene product (pRB) is a nuclear phosphoprotein that is thought to play a key role in the negative regulation of cellular proliferation. pRB is phosphorylated in a cell cycle dependent manner, and studies in both actively dividing and differentiated cells suggest that this modification may be essential for cells to progress through the cell cycle. Using tryptic phosphopeptide mapping we have shown that pRB is phosphorylated on multiple serine and threonine residues in vivo and that many of these phosphorylation events can be mimicked in vitro using purified p34cdc2. Using synthetic peptides corresponding to potential cdc2 phosphorylation sites, we have developed a strategy which has allowed the identification of five sites. S249, T252, T373, S807 and S811 are phosphorylated in vivo, and in each case these sites correspond closely to the consensus sequence for phosphorylation by p34cdc2. This and the observation that pRB forms a specific complex with p34cdc2 in vivo suggests that p34cdc2 or a p34cdc2-related protein is a major pRB kinase.
339 citations
TL;DR: It is suggested that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.
Abstract: To understand the principles of control and selectivity in gene expression, the biochemical mechanisms by which promoter- and enhancer-binding factors regulate transcription by RNA polymerase II were analyzed. A general observed repressor of transcription was purified and identified as histone H1. Since many aspects of H1 binding to naked DNA resemble its interaction with chromatin, purified H1 bound to naked DNA was used as a model for the repressed state of the DNA template. Three sequence-specific transcription factors, Sp1, GAL4-VP16, and GAGA factor, were shown to counteract H1-mediated repression (antirepression). In addition, Sp1 and GAL4-VP16, but not the GAGA factor, activated transcription in the absence of H1. Therefore, true activation and antirepression appear to be distinct activities of sequence-specific factors. Furthermore, transcription antirepression by GAL4-VP16 was sustained for several rounds of transcription. These findings, together with previous studies on H1, suggest that H1 participates in repression of the genome in the ground state and that sequence-specific transcription factors induce selected genes by a combination of true activation and release of basal repression that is mediated at least in part by H1.
332 citations
TL;DR: Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase, and identifies a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of S IT4 and can also partially suppress the phenotypic defectsDue to a null mutation in BCY1.
Abstract: Saccharomyces cerevisiae strains containing temperature-sensitive mutations in the SIT4 protein phosphatase arrest in late G1 at the nonpermissive temperature. Order-of-function analysis shows that SIT4 is required in late G1 for progression into S phase. While the levels of SIT4 do not change in the cell cycle, SIT4 associates with two high-molecular-weight phosphoproteins in a cell-cycle-dependent fashion. In addition, we have identified a polymorphic gene, SSD1, that in some versions can suppress the lethality due to a deletion of SIT4 and can also partially suppress the phenotypic defects due to a null mutation in BCY1. The SSD1 protein is implicated in G1 control and has a region of similarity to the dis3 protein of Schizosaccharomyces pombe. We have also identified a gene, PPH2alpha, that in high copy number can partially suppress the growth defect of sit4 strains. The PPH2 alpha gene encodes a predicted protein that is 80% identical to the catalytic domain of mammalian type 2A protein phosphatases but also has an acidic amino-terminal extension not present in other phosphatases.
TL;DR: ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression.
Abstract: ABF2 (ARS-binding factor 2), a small, basic DNA-binding protein that binds specifically to the autonomously replicating sequence ARS1, is located primarily in the mitochondria of the yeast Saccharomyces cerevisiae. The abundance of ABF2 and the phenotype of abf2- null mutants argue that this protein plays a key role in the structure, maintenance, and expression of the yeast mitochondrial genome. The predicted amino acid sequence of ABF2 is closely related to the high-mobility group proteins HMG1 and HMG2 from vertebrate cell nuclei and to several other DNA-binding proteins. Additionally, ABF2 and the other HMG-related proteins are related to a globular domain from the heat shock protein hsp70 family. ABF2 interacts with DNA both nonspecifically and in a specific manner within regulatory regions, suggesting a mechanism whereby it may aid in compacting the mitochondrial genome without interfering with expression.
TL;DR: A cell free system that supports replication‐dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication and suggests a level of cellular control for this fundamental process.
Abstract: A cell free system that supports replication-dependent chromatin assembly has been used to determine the mechanism of histone deposition during DNA replication. CAF-I, a human cell nuclear factor, promotes chromatin assembly on replicating SV40 DNA in the presence of a crude cytosol replication extract. Biochemical fractionation of the cytosol extract has allowed separation of the chromatin assembly reaction into two steps. During the first step, CAF-I targets the deposition of newly synthesized histones H3 and H4 to the replicating DNA. This reaction is dependent upon and coupled with DNA replication, and utilizes the newly synthesized forms of histones H3 and H4, which unlike bulk histone found in chromatin, do not bind to DNA by themselves. The H3/H4-replicated DNA complex is a stable intermediate which exhibits a micrococcal nuclease resistant structure and can be isolated by sucrose gradient sedimentation. In the second step, this replicated precursor is converted to mature chromatin by the addition of histones H2A and H2B in a reaction that can occur after DNA replication. The requirement for CAF-I in at least the first step of the reaction suggests a level of cellular control for this fundamental process.
TL;DR: Both the processivity and the banding pattern analysis imply that in the elongation mechanism there must be a translocation step after the 9 nucleotides of internal template RNA have been copied to the extreme 5' end.
Abstract: Telomerase synthesizes tandem repeats of the sequence d(TTGGGG) onto input d(TTGGGG)n primer oligonucleotides (C. W. Greider and E. H. Blackburn, Cell 43:405-413). An intrinsic RNA component of the enzyme provides the template for d(TTGGGG)n repeat synthesis [C. W. Greider and E. H. Blackburn, Nature (London) 337:331-337, 1989; G.-L. Lu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature (London) 344:126-132, 1990]. In a typical reaction, products greater than 2,000 nucleotides were synthesized in 60 min. Dilution and primer challenge experiments showed that these long products were synthesized processively. The apparent processivity was not due to a higher affinity of the enzyme for long d(TTGGGG) products over the shorter competitors. The degree of processivity was quantitated; telomerase synthesized approximately 520 nucleotides before half of the enzyme had dissociated. After dissociating, telomerase reinitiated d(TTGGGG)n synthesis on new primer oligonucleotides. The products from a telomerase reaction have a characteristic 6-nucleotide banding pattern (C. W. Greider and E. H. Blackburn, Cell 51:887-898, 1987). A strong pause in the reaction occurs after the addition of the first G in the sequence d(TTGGGG). Both the processivity and the banding pattern analysis imply that in the elongation mechanism there must be a translocation step after the 9 nucleotides of internal template RNA have been copied to the extreme 5' end.
TL;DR: A minimal origin fragment from BPV is identified that contains all the sequences required in cis for replication of BPV in short term replication assays and shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines.
Abstract: Expression of the viral polypeptides E1 and E2 is necessary and sufficient for replication of BPV in mouse C127 cells. By providing these factors from heterologous expression vectors we have identified a minimal origin fragment from BPV that contains all the sequences required in cis for replication of BPV in short term replication assays. This same sequence is also required for stable replication in the context of the entire viral genome. The identified region is highly conserved between different papillomaviruses, and is unrelated to the previously identified plasmid maintenance sequences. The minimal ori sequence contains a binding site for the viral polypeptide E1, which we identify as a sequence specific DNA binding protein, but surprisingly, an intact binding site for the viral transactivator E2 at the ori is not required. The isolated origin shows an extended host region for replication and replicates efficiently in both rodent and primate cell lines.
TL;DR: It is suggested that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin, with consequences for the relative levels of leading and lagging strands.
TL;DR: Transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E 1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved.
Abstract: Infection with adenovirus mutants carrying either point mutations or deletions in the coding region for the 19-kDa E1B gene product (19K protein) causes degradation of host cell and viral DNAs (deg phenotype) and enhanced cytopathic effect (cyt phenotype). Therefore, one function of the E1B 19K protein is to protect nuclear DNA integrity and preserve cytoplasmic architecture during productive adenovirus infection. When placed in the background of a virus incapable of expressing a functional E1A gene product, however, E1B 19K gene mutations do not result in the appearance of the cyt and deg phenotypes. This demonstrated that expression of the E1A proteins was responsible for inducing the appearance of the cyt and deg phenotypes. By constructing a panel of viruses possessing E1A mutations spanning each of the three E1A conserved regions in conjunction with E1B 19K gene mutations, we mapped the induction of the cyt and deg phenotypes to the amino-terminal region of E1A. Viruses that fail to express conserved region 3 (amino acids 140 to 185) and/or 2, (amino acids 121 to 185) or nonconserved sequences between conserved regions 2 and 1 of E1A (amino acids 86 to 120) were still capable of inducing cyt and deg. This indicated that activities associated with these regions, such as transactivation and binding to the product of the retinoblastoma susceptibility gene, were dispensable for induction of E1A-dependent cytotoxic effects. In contrast, deletion of sequences in the amino terminus of E1A (amino acids 22 to 107) resulted in extragenic suppression of the cyt and deg phenotypes. Therefore, a function affected by deletion of amino acids 22 to 86 of E1A is responsible for exerting cytotoxic effects in virally infected cells. Furthermore, transient high-level expression of the E1A region using a cytomegalovirus promoter plasmid expression vector was sufficient to induce the cyt and deg phenotypes, demonstrating that E1A expression alone is sufficient to exert these cytotoxic effects and that other viral gene products are not involved. Finally, placing E1A expression under the control of a strong promoter did not alter the requirement for E1B in the transformation of primary cells. One possibility is that the E1B 19K protein is required to overcome the cytotoxic effects of E1A protein expression and thereby enable primary cells to become transformed.
TL;DR: It is shown that P is required for accumulation in the pericarp of transcripts of two genes (A1 and C2) encoding enzymes for flavonoid biosynthesis--genes also regulated by C1 in the aleurone.
Abstract: The Zea mays P gene has been postulated to regulate the biosynthetic pathway of a flavonoid-derived pigment in certain floral tissues [Styles, E D & Ceska, O (1977) Can J Genet Cytol 19, 289-302] We have characterized two P transcripts that are alternatively spliced at their 3' ends One message of 1802 nucleotides encodes a 437-kDa protein with an N-terminal region showing approximately 40% homology to the DNA-binding domain of several members of the myb family of protooncogene proteins A second message of 945 nucleotides encodes a 173-kDa protein that contains most of the myb-homologous domain but differs from the first protein at the C terminus The deduced P-encoded proteins show an even higher homology (70%) in the myb-homologous domain to the maize regulatory gene C1 Additionally, the P and C1 genes are structurally similar in the sizes and positions of the first and second exons and first intron We show that P is required for accumulation in the pericarp of transcripts of two genes (A1 and C2) encoding enzymes for flavonoid biosynthesis--genes also regulated by C1 in the aleurone
TL;DR: Direct observations of three-dimensional rotations have revealed a close association between the c-fos transcripts and the nuclear speckles that are enriched in pre-mRNA splicing factors.
Abstract: We have used in situ hybridization and immunocytochemistry to compare the nuclear localization of a specific nascent pre-mRNA and the essential non-snRNP splicing factor SC-35 Nascent c-fos transcripts were detected in serum-induced mouse fibroblasts by in situ hybridization with genomic c-fos probes Prior to serum induction no c-fos RNA is detected, but these transcripts localize to two dots in the interphase nucleus after induction The time course of appearance of the dots correlates with the previously determined time course of transcriptional activation of the gene Upon further analysis by confocal laser scanning microscopy, we have determined that the dots extend through the depth of the nucleus, forming paths By using high-voltage electron microscopy, we have found that the c-fos path extends out and comes into direct contact with the nuclear envelope We have also compared the localization of c-fos transcripts with the speckled nuclear regions that are enriched in snRNPs and the non-snRNP splicing factor SC-35 Direct observations of three-dimensional rotations have revealed a close association between the c-fos transcripts and the nuclear speckles This study demonstrates a direct link between specific nascent RNA transcripts and nuclear speckles that are enriched in pre-mRNA splicing factors
TL;DR: Cl cloning and characterization of the genes encoding RF-A from S. cerevisiae are reported, indicating that the human p34 subunit is 29% identical to the corresponding RFA2 gene product, however, expression of the human protein fails to rescue the rfa2::TRP1 disruption.
Abstract: Replication factor-A (RF-A) is a three-subunit protein complex originally purified from human cells as an essential component for SV40 DNA replication in vitro. We have previously identified a functionally homologous three-subunit protein complex from the yeast Saccharomyces cerevisiae. Here we report the cloning and characterization of the genes encoding RF-A from S. cerevisiae. Each of the three subunits is encoded by a single essential gene. Cells carrying null mutations in any of the three genes arrest as budded and multiply budded cells. All three genes are expressed in a cell-cycle-dependent manner; the mRNA for each subunit peaks at the G1/S-phase boundary. A comparison of protein sequences indicates that the human p34 subunit is 29% identical to the corresponding RFA2 gene product. However, expression of the human protein fails to rescue the rfa2::TRP1 disruption.
TL;DR: Two different signaling pathways act through discrete nuclear targets at the SRE by recruitment of a pathway-specific accessory factor (p62TCF), offering a molecular mechanism to account for the biological specificity of signals that appear to act through common DNA sequence elements.
Abstract: The c-fos serum response element (SRE) is a primary nuclear target for intracellular signal transduction pathways triggered by growth factors. It is the target for both protein kinase C (PKC)-dependent and -independent signals. Function of the SRE requires binding of a cellular protein, termed serum response factor (SRF). A second protein, p62TCF, recognizes the SRE-SRF complex to form a ternary complex. A mutated SRE that bound SRF but failed to form the ternary complex selectively lost response to PKC activators, but retained response to PKC-independent signals. Thus, two different signaling pathways act through discrete nuclear targets at the SRE. At least one of these pathways functions by recruitment of a pathway-specific accessory factor (p62TCF). These results offer a molecular mechanism to account for the biological specificity of signals that appear to act through common DNA sequence elements.
TL;DR: Analysis of cDNA clones encoding a p34cdc2 homologue from a higher plant, Zea mays (maize), and a full-length cDNA clone isolated, sequenced, and shown to complement a cdc28 mutation in Saccharomyces cerevisiae indicates that maize has multiple cdc2 genes.
Abstract: We describe the isolation of cDNA clones encoding a p34cdc2 homologue from a higher plant, Zea mays (maize). A full-length cDNA clone, cdc2ZmA, was isolated, sequenced, and shown to complement a cdc28 mutation in Saccharomyces cerevisiae. Comparison of the deduced amino acid sequence of the maize p34cdc2 protein with other homologues showed that it was 64% identical to human p34cdc2 and 63% identical to Schizosaccharomyces pombe and S. cerevisiae p34cdc2 proteins. Studies of expression of the maize cdc2 gene(s) by Northern blot analysis indicated a correlation between the abundance of cdc2 mRNA and the proliferative state of the tissue. Southern blot analysis, as well as isolation of another cDNA clone, cdc2ZmB, which is 96% identical to cdc2ZmA, indicates that maize has multiple cdc2 genes.
TL;DR: The structure is discussed in the light of the potential role of PTPH1 in controlling cytoskeletal integrity and the possibility that overexpression of P TPH1 may reverse transformation induced by oncogenic protein-tyrosine kinases, such as the members of the src family.
Abstract: The polymerase chain reaction (PCR), from primers corresponding to conserved sequences within the catalytic domains of the protein-tyrosine phosphatases, was used to amplify protein-tyrosine phosphatase-related cDNAs from a HeLa cell library. After probing the same cDNA library with one of the PCR products, 10 positive clones were identified. The longest of these clones (3984 base pairs) contained 2739 base pairs of open reading frame and, after a stop codon, a 3' nontranslated segment of 1222 base pairs. A 4.3-kilobase transcript was detected by Northern blot analysis of HeLa cell poly(A)+ RNA. The open reading frame predicts a protein of 913 amino acids (approximately 104 kDa), termed PTPH1. The sequence of PTPH1 can be described in terms of three segments. (i) The N-terminal segment displays homology to the domains in the cytoskeletal-associated proteins band 4.1, ezrin, and talin that direct their association with proteins at the interface between the plasma membrane and the cytoskeleton in structures such as focal adhesions. (ii) There is a central segment bearing putative phosphorylation sites for protein-serine/threonine kinases. (iii) A segment that is homologous to the members of the protein-tyrosine phosphatase family is located at the C terminus. The structure is discussed in the light of the potential role of PTPH1 in controlling cytoskeletal integrity and the possibility that overexpression of PTPH1 may reverse transformation induced by oncogenic protein-tyrosine kinases, such as the members of the src family.
Patent•
19 Apr 1991TL;DR: In this paper, the authors present methods for detecting mammalian genes encoding proteins which can function in microorganisms, particularly yeast, to modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism.
Abstract: Disclosed are methods for detecting mammalian genes encoding proteins which can function in microorganisms, particularly yeast, to modify, complement, or suppress a genetic defect associated with an identifiable phenotypic alteration or characteristic in the microorganism. Disclosed also are mammalian DNA sequences cloned by the above method, as well as polypeptide products of the expression of the DNA sequences in procaryotic or eucaryotic host cells and antibody substances which are specifically immunoreactive with said expression products. More specifically, the present invention relates to methods for cloning mammalian genes which encode products which modify, complement or suppress a genetic defect in a biochemical pathway in which cAMP participates or in a biochemical pathway which is controlled, directly or indirectly, by a RAS-related protein, to products (RNA, proteins) encoded by the mammalian genes cloned in this manner, and to antibodies which can bind the encoded proteins.
TL;DR: It is reported that cap- cells bud randomly and are defective in actin distribution, and proposed that CAP and profilin provide a link between growth signals and remodeling of the cellular cytoskeleton.
TL;DR: It is reported here that Tetrahymena telomerase can specifically elongate single-stranded DNA oligonucleotides whose termini are not complementary to the RNA template sequence 5′-CAACCCCAA-3′, suggesting that telomersase may be able to heal chromosomes directly in vivo.
Abstract: Chromosome healing by de novo telomere addition at nontelomeric sites has been well characterized in several organisms. The Tetrahymena telomerase ribonucleoprotein uses an internal RNA template to catalyse d(TTGGGG)n telomere addition to the 3' end of telomeric sequence in vitro and in vivo. Studies of telomerase RNA indicated that hybridization of the RNA template region, 5'-CAACCCCAA-3', to the 3' end of single-stranded telomeric oligonucleotides might be important for primer recognition and utilization. The apparent requirement of telomerase for pre-existing telomeric sequence has raised questions regarding its role in chromosome healing. We report here that Tetrahymena telomerase can specifically elongate single-stranded DNA oligonucleotides whose termini are not complementary to the RNA template sequence 5'-CAACCCCAA-3'. These data suggest that telomerase may be able to heal chromosomes directly in vivo.
TL;DR: A gene is reported that can encode a predicted protein kinase and can partially suppress defects in ras1 mutants and is probably identical to ste8, which has been identified previously as ste1.
Abstract: Schizosaccharomyces pombe contains a single gene, ras1, which is a homolog of the mammalian RAS genes. ras1 is required for conjugation, sporulation, and normal cell shape. ras1 has been previously identified as ste5. We report here a gene we call byr2 that can encode a predicted protein kinase and can partially suppress defects in ras1 mutants. ras1 mutant strains expressing high levels of byr2 can sporulate competently but are still defective in conjugation and abnormally round. byr2 mutants are viable and have normal shape but are absolutely defective in conjugation and sporulation. byr2 is probably identical to ste8. In many respects, byr2 resembles the byr1 gene, another suppressor of the ras1 mutation, which has been identified previously as ste1. Our data indicate that if ras1, byr2, and byr1 act along the same pathway, then the site of action for byr2 is between the sites for ras1 and byr1.
TL;DR: The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase.
Abstract: The Saccharomyces cerevisiae SIS1 gene was identified as a high copy number suppressor of the slow growth phenotype of strains containing mutations in the SIT4 gene, which encodes a predicted serine/threonine protein phosphatase. The SIS1 protein is similar to bacterial dnaJ proteins in the amino-terminal third and carboxyl-terminal third of the proteins. In contrast, the middle third of SIS1 is not similar to dnaJ proteins. This region of SIS1 contains a glycine/methionine-rich region which, along with more amino-terminal sequences, is required for SIS1 to associate with a protein of apparent molecular mass of 40 kD. The SIS1 gene is essential. Strains limited for the SIS1 protein accumulate cells that appear blocked for migration of the nucleus from the mother cell into the daughter cell. In addition, many of the cells become very large and contain a large vacuole. The SIS1 protein is localized throughout the cell but is more concentrated at the nucleus. About one-fourth of the SIS1 protein is released from a nuclear fraction upon treatment with RNase. We also show that overexpression of YDJ1, another yeast protein with similarity to bacterial dnaJ proteins, can not substitute for SIS1.
TL;DR: CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins, and is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.
Abstract: CAP, a protein from Saccharomyces cerevisiae that copurifies with adenylyl cyclase, appears to be required for yeast cells to be fully responsive to RAS proteins. CAP also appears to be required for normal cell morphology and responsiveness to nutrient deprivation and excess. We describe here a molecular and phenotypic analysis of the CAP protein. The N-terminal domain is necessary and sufficient for cellular response to activated RAS protein, while the C-terminal domain is necessary and sufficient for normal cellular morphology and responses to nutrient extremes. Thus, CAP is a novel example of a bifunctional component involved in the regulation of diverse signal transduction pathways.
TL;DR: Isolation of a cellular activity can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation, which finds that sequences in conserved region 1 (CR1) and Conserved region 2 (CR2) are important for dissociation of the E 2F complex, whereas amino-terminal sequences are not required.
Abstract: Recent experiments have shown that the cellular E2F transcription factor is found in complexes with cellular proteins and that one such complex contains the cyclin-A protein. Isolation of a cellular activity, which we term E2F-BF, can reconstitute the E2F-cyclin-A complex and has permitted a more detailed analysis of the mechanism of E1A dissociation. Through the analysis of a series of E1A mutants, we find that sequences in conserved region 1 (CR1) and conserved region 2 (CR2) are important for dissociation of the E2F complex, whereas amino-terminal sequences are not required. In contrast to the requirements for dissociation, only the CR1 sequences are required to block formation of the complex if E1A is added when the components are combined. We have also identified an activity, termed E2F-I, that inhibits E2F binding to DNA, again apparently through the formation of a complex with E2F. This inhibitory activity is also blocked by E1A, dependent on the same elements of the E1A protein that disrupt the interaction with E2F-BF. Because the E1A sequences that are important for releasing E2F from these interactions are also sequences necessary for oncogenesis, we suggest that this activity may be a critical component of the transforming activity of E1A.