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Showing papers by "Cold Spring Harbor Laboratory published in 1986"


Journal ArticleDOI
05 Sep 1986-Science
TL;DR: The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity, which may represent primary events in the mechanism of action of ras proteins.
Abstract: Expression of the ras oncogene is thought to be one of the contributing events in the initiation of certain types of human cancer. To determine the cellular activities that are directly triggered by ras proteins, the early consequences of microinjection of the human H-ras proteins into quiescent rat embryo fibroblasts were investigated. Within 30 minutes to 1 hour after injection, cells show a marked increase in surface ruffles and fluid-phase pinocytosis. The rapid enhancement of membrane ruffling and pinocytosis is induced by both the proto-oncogenic and the oncogenic forms of the H-ras protein. The effects produced by the oncogenic protein persist for more than 15 hours after injection, whereas the effects of the proto-oncogenic protein are short-lived, being restricted to a 3-hour interval after injection. The stimulatory effect of the ras oncogene protein on ruffling and pinocytosis is dependent on the amount of injected protein and is accompanied by an apparent stimulation of phospholipase A2 activity. These rapid changes in cell membrane activities induced by ras proteins may represent primary events in the mechanism of action of ras proteins.

698 citations


Journal ArticleDOI
11 Apr 1986-Cell
TL;DR: The synthetic peptide VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp 70, S. cerevisiae YG100, and E. coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts that show that uncoating ATPase is a member of the 70 kd heat shock protein family.

568 citations


Journal ArticleDOI
06 Jun 1986-Cell
TL;DR: The cDNA sequence of the mas oncogene reveals a long open reading frame that codes for a 325 amino acid protein that is very hydrophobic and has seven potential transmembrane domains that may reflect a new functional class of oncogenes.

452 citations


Journal ArticleDOI
14 Feb 1986-Cell
TL;DR: A mechanism for VA RNAI action based on its partially double-stranded nature is proposed, which antagonizes the activation of DAI by dsRNA, but it cannot inhibit the activity ofDAI once activated.

246 citations


Journal ArticleDOI
23 May 1986-Cell
TL;DR: The results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.

244 citations


Journal ArticleDOI
09 May 1986-Cell
TL;DR: Evidence is presented that the SV40 enhancer consists of three functional units, A, B, and C, each of which can cooperate with the others or with duplicates of itself to enhance transcription.

242 citations


Journal ArticleDOI
07 Nov 1986-Cell
TL;DR: The designation RAM (RAS protein and a-factor maturation function) for SUPH and STE16 is proposed, which may encode an enzyme responsible for the modification and membrane localization of proteins with this C-terminal sequence.

228 citations


Journal ArticleDOI
TL;DR: The structural gene for cytochrome c(2) (cycA) of the photosynthetic bacterium Rhodopseudomonas capsulata has been cloned, and the nucleotide and deduced polypeptide sequences have been determined.
Abstract: The structural gene for cytochrome c2 (cycA) of the photosynthetic bacterium Rhodopseudomonas capsulata has been cloned, and the nucleotide and deduced polypeptide sequences have been determined. Compared with the known amino acid sequence of the purified cytochrome c2, the nucleotide sequence corresponding to the N-terminal part of the cycA gene product indicates the presence of a putative 21 amino acid signal sequence. Thus, cytochrome c2 may be synthesized as a precursor which is processed during its secretion to the periplasm. Insertion and insertion-deletion mutations were constructed in vitro and the chromosomal cycA+ allele of a wild-type strain was replaced with these mutations by homologous recombination to yield c2- mutants of R. capsulata. The c2- mutants are stable, and they can grow by photosynthesis and by respiration. Since cytochrome c2 is the primary electron donor to the reaction center during photosynthesis, the ability of these mutants to grow photosynthetically indicates that an alternative way(s) of reducing the oxidized reaction center must exist in R. capsulata. One candidate for this role may be the membrane-bound cytochrome c1.

193 citations


Journal ArticleDOI
TL;DR: Anti-tRNA antibody can be absorbed out without depleting antisynthetase activity, showing that the antigens are recognized independently by separable antibodies that coexist in these sera of patients with autoimmune disease.
Abstract: The sera of six patients with autoimmune disease, predominantly myositis with pulmonary fibrosis, contain antibodies of the PL-12 specificity. These autoantibodies react with both protein and RNA components of human cells. The protein has a subunit molecular mass of 110 kD, and the RNA comprises a group of bands in the tRNA size class. Aminoacylation experiments identify the antigens as alanyl-tRNA synthetase and its corresponding tRNAs, tRNAAla. Anti-tRNA antibody can be absorbed out without depleting antisynthetase activity, showing that the antigens are recognized independently by separable antibodies that coexist in these sera. The concurrence of separate antibodies to the two components suggests that the autoimmune response may be mounted against the charging enzyme-tRNA complex. However, the antisynthetase antibody fails to coprecipitate tRNA with the enzyme, suggesting that the antibody reacts with its target only when it is not complexed with tRNA.

187 citations


Journal ArticleDOI
14 Feb 1986-Cell
TL;DR: The existence of a large inversion of genetic material, encompassing the loci of T and qk, within the proximal portion of t haplotypes, provides an explanation for the suppression of recombination observed along the length of t Haplotype DNA in heterozygous mice.

177 citations


Journal ArticleDOI
TL;DR: The data demonstrate that in fission yeast the transition from G1 to S phase does not mark a point of commitment to the completion of the cell cycle, and suggest that cell cycle arrest in stationary phase is regulated by the activity of the same G1 and G2 controls.
Abstract: The cell cycle of Schizosaccharomyces pombe in continuous culture is controlled at two steps, one which limits the transition from G1 to S phase and the other which determines the timing of cell division. We have investigated, by means of flow-cytofluorometry, the cell cycle characteristics of nutritionally starved cells in stationary phase. Cells were shown to become arrested in either G1 or G2, in ratios which depended on the composition of the growth medium. G1 and G2 stationary phase cells share certain properties. (1) They become relatively resistant to heat shock. (2) They can reenter the cell cycle after subculture into fresh medium. (3) The G1 and G2 arrested populations have equal long-term viability in stationary phase. (4) Both populations require the activity of the cdc2+ gene for reentry into the cell cycle. We suggest that cell cycle arrest in stationary phase is regulated by the activity of the same G1 and G2 controls which limit the rate of cell cycle progression in continuous culture. The data demonstrate that in fission yeast the transition from G1 to S phase does not mark a point of commitment to the completion of the cell cycle.

Journal ArticleDOI
14 Feb 1986-Cell
TL;DR: Results indicate that E 1A contributes complementing biochemical activities that enable ras genes to transform REF52 and suggest that the role of E1A in primary cell transformation may extend beyond facilitating in vitro establishment.

Journal ArticleDOI
TL;DR: Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death.
Abstract: To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.

Journal ArticleDOI
01 Aug 1986-Nature
TL;DR: Transmission of the bovine papillomavirus-1 genome through the mouse germ line results in the heritable formation of fibropapillomas of the skin, a tissue-specific phenotype analogous to that observed in natural BPV-1 infection of cattle.
Abstract: Transmission of the bovine papillomavirus-1 (BPV-1) genome through the mouse germ line results in the heritable formation of fibropapillomas of the skin, a tissue-specific phenotype analogous to that observed in natural BPV-1 infection of cattle. Oncogenesis is slow, with tumours first arising at 8-9 months of age, usually in areas prone to wounding. Extrachromosomal BPV-1 DNA is detected in all tumours, whereas normal tissues show only integrated DNA.

Journal ArticleDOI
TL;DR: The function of the ras+ gene of Schizosaccharomyces pombe has been studied by constructing null and activated alleles of this gene as discussed by the authors, which supports the hypothesis that ras of fission yeast does not modulate adenylate cyclase in a manner analogous to S. cerevisiae RAS.
Abstract: The function of the ras+ gene of Schizosaccharomyces pombe has been studied by constructing null and activated alleles of this gene. An activated allele (rasVal 12) inhibits conjugation but has no effect on cell growth, entry into stationary phase or sporulation. The phenotype of rasVal 12 is distinct from that caused by elevating the intracellular level of cAMP. This supports the hypothesis that ras of fission yeast does not modulate adenylate cyclase in a manner analogous to S. cerevisiae RAS. Introduction of a human ras sequence into fission yeast cells containing a non-functional null allele of ras restored the sexual differentiation process thus indicating that the human sequence can complement S. pombe ras. Our data suggest that although ras genes are highly conserved across a considerable evolutionary divide, the cellular function of the ras gene product varies in different organisms.

Journal ArticleDOI
TL;DR: HAG and HAgC are efficiently routed into the endocytic pathway and HA is not, however, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.
Abstract: Chimeric genes were created by fusing DNA sequences encoding the ectodomain of the influenza virus hemagglutinin (HA) to DNA coding for the transmembrane and cytoplasmic domains of either the G glycoprotein of vesicular stomatitis virus or the gC glycoprotein of Herpes simplex virus 1. CV-1 cells infected with SV40 vectors carrying the recombinant genes expressed large amounts of the chimeric proteins, HAG or HAgC on their surfaces. Although the ectodomains of HAG and HAgC differed in their immunological properties from that of HA, the chimeras displayed the biological functions characteristic of the wild-type protein. Both HAG and HAgC bound erythrocytes as efficiently as HA did and, after brief exposure to an acidic environment, induced the fusion of erythrocyte and CV-1 cell membranes. However, the behavior of HAG and HAgC at the cell surface differed from that of HA in several important respects. HAG and HAgC were observed to collect in coated pits whereas wild-type HA was excluded from those structures. In the presence of chloroquine, which inhibits the exit of receptors from endosomes, HAG and HAgC accumulated in intracellular vesicles. By contrast, chloroquine had no effect on the location of wild-type HA. HAG and HAgC labeled at the cell surface exhibited a temperature-dependent acquisition of resistance to extracellular protease at a rate similar to the rates of internalization observed for many cell surface receptors. HA acquired resistance to protease at a rate at least 20-fold slower. We conclude that HAG and HAgC are efficiently routed into the endocytic pathway and HA is not. However, like HA, HAG was degraded slowly, raising the possibility that HAG recycles to the plasma membrane.

Book ChapterDOI
01 Jan 1986
TL;DR: The complete sequence of 35,937 nucleotides for the Adenovirus-2 genome is presented in a fully annotated form and represents a consensus derived by combining sequence data from many different laboratories.
Abstract: The complete sequence of 35,937 nucleotides for the Adenovirus-2 genome is presented in a fully annotated form. This sequence represents a consensus derived by combining sequence data from many different laboratories.

Journal ArticleDOI
29 Aug 1986-Cell
TL;DR: The DSB at mat1 promotes efficient meiotic recombination in fission yeast and is associated with the recombination of flanking markers.

Journal ArticleDOI
29 Aug 1986-Cell
TL;DR: Although double-strand breaks can initiate meiotic recombination in yeast, the data suggest that they do not normally do so.

Journal ArticleDOI
01 Jul 1986-Genetics
TL;DR: The structures determined for twLub2 and TtOrl indicate that rare recombination can occur between nonequivalent genomic sequences within the inverted proximal t region when wild-type and t chromosomes are paired in a linear, nonhomologous configuration.
Abstract: We have investigated the structure and properties of a chromosomal product recovered from a rare recombination event between a t haplotype and a wild-type form of mouse chromosome 17 . Our embryological and molecular studies indicate that this chromosome ( t wLub 2 ) is characterized by both a deletion and duplication of adjacent genetic material. The deletion appears to be responsible for a dominant lethal maternal effect and a recessive embryonic lethality. The duplication provides an explanation for the t wLub 2 suppression of the dominant T locus phenotype. A reanalysis of previously described results with another chromosome 17 variant called Tt Orl indicates a structure for this chromosome that is reciprocal to that observed for t wLub 2 . We have postulated the existence of an inversion over the proximal portion of all complete t haplotypes in order to explain the generation of the partial t haplotypes t wLub 2 and Tt Orl . This proximal inversion and the previously described distal inversion are sufficient to account for all of the recombination properties that are characteristic of complete t haplotypes. The structures determined for t wLub 2 and Tt Orl indicate that rare recombination can occur between nonequivalent genomic sequences within the inverted proximal t region when wild-type and t chromosomes are paired in a linear, nonhomologous configuration.

Journal ArticleDOI
TL;DR: Two overlapping clones that carry the dcm locus are isolated, from a library of E. coli K-12 DNA, and it is shown that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain.
Abstract: The dcm locus of Escherichia coli K-12 has been shown to code for a methylase that methylates the second cytosine within the sequence 5'-CC(A/T)GG-3'. This sequence is also recognized by the EcoRII restriction-modification system coded by the E. coli plasmid N3. The methylase within the EcoRII system methylates the same cytosine as the dcm protein. We have isolated, from a library of E. coli K-12 DNA, two overlapping clones that carry the dcm locus. We show that the two clones carry overlapping sequences that are present in a dcm+ strain, but are absent in a delta dcm strain. We also show that the cloned gene codes for a methylase, that it complements mutations in the EcoRII methylase, and that it protects EcoRII recognition sites from cleavage by the EcoRII endonuclease. We found no phage restriction activity associated with the dcm clones.

Journal ArticleDOI
01 Nov 1986-Plasmid
TL;DR: The modified plasmid, pU29, greatly facilitates in vitro mutagenesis experiments since previously described techniques and screening procedures are more efficient with M13 derivatives carrying smaller inserts.

Journal ArticleDOI
TL;DR: A group of at least nine nuclear polypeptides ranging in MW from about 12 000 to 110 000 are recognized by immunoblotting with anti-PCNA sera, indicating either that additional antigenic sites are produced on denaturation of native proteins or that additional autoantibodies are present in these sera.

Journal ArticleDOI
01 Jan 1986-Gene
TL;DR: Evidence is presented that suggests that the 4.7-kb HindIII fragment contains a gene coding for 16S rRNA, and that although homology between nif and this fragment can be observed in filter hybridization experiments, a second copy of the nif structural genes seems not to be present in this region.

Book ChapterDOI
01 Jan 1986
TL;DR: An overview of the biological implications of the complete DNA sequence of the Ad2 genome is provided.
Abstract: As a result of work in several laboratories the complete DNA sequence of the 35,937 base pairs long Ad2 genome has been established. In this paper we provide an overview of the biological implications of the sequence.

Journal ArticleDOI
TL;DR: Experiments indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.
Abstract: One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

Journal ArticleDOI
TL;DR: The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop, and it is proposed that Mop may be a regulatory protein binding the anabolic source of molybdenum.
Abstract: mop is the structural gene for the molybdenum-pterin binding protein, which is the major molybdenum binding protein in Clostridium pastuerianum. The mop gene was detected by immunoscreening genomic libraries of C. pastuerianum and identified by determining the nucleotide sequence of the cloned insert of clostridial DNA. The deduced amino acid sequence of an open reading frame proved to be identical to the first twelve residues of purified Mop. The DNA sequence flanking the mop gene contains promoter-like consensus sequences which are probably responsible for the expression of Mop in Escherichia coli. The deduced amino acid composition shows that the protein is hydrophobic, lacks aromatic and cysteine residues and has a calculated molecular weight of 7,038. The N-terminal amino acid sequence of Mop has sequence homology with DNA binding proteins. The pattern and type of residues in the N-terminal region suggest it forms the helix-turn-helix structure observed in DNA binding proteins. We propose that Mop may be a regulatory protein binding the anabolic source of molybdenum.

Journal ArticleDOI
TL;DR: Analysis of the transmission frequency and cosegregation patterns of new proviral loci indicated that viral integration occurs after the first round of DNA replication and before the germ line is set aside during embryogenesis, which is consistent with the hypothesis that virus from the mother infects the egg or the early embryo.
Abstract: Germ line acquisition of ecotropic proviruses occurs at a high frequency in the progeny of SWR/J-RF/J hybrid mice carrying two genetically linked RF/J ecotropic proviral loci, Emv-16 and Emv-17 (N. A. Jenkins and N. G. Copeland, Cell 43:811-819, 1985). To determine if genetic background affects proviral integration frequency, I analyzed a series of crosses in which the two RF/J proviral loci were transferred onto different provirus-negative background strains. Unlike SWR/J-RF/J hybrid progeny, few CBA/CaJ-RF/J hybrid mice were identified that carried new germ line proviral loci. These results indicate that genetic factors other than the linked RF/J proviral loci contribute to the increased frequency of germ line provirus integration seen in the SWR/J-RF/J hybrids. The frequency of proviral acquisition appeared to increase when females carrying Emv-16, Emv-17, and at least one new proviral locus were further backcrossed, suggesting that integration frequency can be increased by genetic manipulation. The breeding data are consistent with the hypothesis that virus from the mother infects the egg or the early embryo. Analysis of the transmission frequency and cosegregation patterns of new proviral loci indicated that viral integration occurs after the first round of DNA replication and before the germ line is set aside during embryogenesis, with a majority of viral integrations occurring at the two-cell stage of development, and independent viral integrations can occur in the same or in different cells of the embryo.

Journal ArticleDOI
TL;DR: Three monoclonal antibodies raised against the leech CNS recognize surface antigens on small sets and subsets of neurons or on glial cells, raising the possibility that different neuronal antigenic determinants are carried on the same protein molecule.

Patent
16 Jul 1986
TL;DR: A method for obtaining E. coli cell lines which carry the deoR mutation is described in this article, and the cell lines themselves are used in cell transfection and transformation, as they transfect transform at much higher frequencies than the previously available cell lines.
Abstract: A method for obtaining E. coli cell lines which carry the deoR mutation is described, as well as the cell lines themselves. These cell lines are useful in cell transfection and transformation, as they transfect transform at much higher frequencies than the previously available cell lines.