Showing papers by "Cold Spring Harbor Laboratory published in 1985"
TL;DR: Following the transfer into fertilized mouse eggs of recombinant genes composed of the upstream region of the rat insulin II gene linked to sequences coding for the large-T antigen of simian virus 40, large- T antigen is detected exclusively in the β-Cells of the endocrine pancreas of transgenic mice.
Abstract: Following the transfer into fertilized mouse eggs of recombinant genes composed of the upstream region of the rat insulin II gene linked to sequences coding for the large-T antigen of simian virus 40, large-T antigen is detected exclusively in the beta-cells of the endocrine pancreas of transgenic mice. The alpha- and delta-cells normally found in the islets of Langerhans are rare and disordered. Well-vascularized beta-cell tumours arise in mice harbouring and inheriting these hybrid oncogenes.
1,383 citations
TL;DR: S. cerevisiae strains containing RAS2val19, a Ras2 gene with a missense mutation analogous to one that activates the transforming potential of mammalian ras genes, have growth and biochemical properties strikingly similar to yeast strains carrying IAC or bcy1.
995 citations
TL;DR: Microinjection of the proto-oncogenic and oncogenic forms of the human H-ras protein into living rat pheochromocytoma cells promoted the morphological differentiation of PC12 cells into neuron-like cells.
711 citations
TL;DR: Gene disruption and biochemical experiments indicate that the catalytic domain of adenylate cyclase resides in the carboxyl terminal 400 amino acids, which indicates that elevated expression ofadenylates cyclase suppresses the lethality that otherwise results from loss of RAS gene function in yeast.
381 citations
TL;DR: It is demonstrated that purified RAS proteins, whether derived from the yeast RAS1 or RAS2 or the human H-ras genes, activate yeast adenylate cyclase in the presence of guanine nucleotides, providing a complete biochemical assay for RAS protein function.
307 citations
TL;DR: The results suggest that not all, if any, of the effects of H-rasval12 protein in this system are mediated by adenylate cyclase.
289 citations
TL;DR: The results indicate that the biochemical function of RAS proteins is essential for vegetative haploid yeast and that this function has been conserved in evolution since the progenitors of yeast and mammals diverged.
274 citations
TL;DR: It is proposed that the ran1+ gene encodes a highly controlled protein kinase which shares key substrates with cAMP-dependentprotein kinase and is normally essential for vegetative cell reproduction but is inessential in cells which have abnormally high levels of cAMP.
Abstract: We have investigated the genetic and physiological control of meiosis in fission yeast. Nutritionally depleted h+/h− diploid cells become irreversibly commited to meiosis immediately prior to the initiation of premeiotic S phase. Premeiotic DNA synthesis requires matP+, matM+, mei2+ and mei3+ but not the mitotic cell cycle control gene, cdc2+, ran1+ is an essential gene, loss of which provokes sexual conjugation, premeiotic DNA synthesis, pseudo-meiosis and the sporulation of haploid cells. Our experiments suggest that sexual differentiation is achieved physiologically by the inhibition of ran1+ activity in a two-step process. In the first step, partial inhibition of ran1+ in starved haploid cells, leads to cell cycle arrest in G1 followed by sexual conjugation. In the second step, a pathway requiring the matP+, matM+ and mei3+ genes of the newly-formed zygote, further inhibits ran1+ and thereby commits the cell to meiosis. mei2+ is required for meiotic commitment after full inhibition of ran1+. ran1+ is normally essential for vegetative cell reproduction but is inessential in cells which have abnormally high levels of cAMP-dependent protein kinase. We propose that the ran1+ gene encodes a highly controlled protein kinase which shares key substrates with cAMP-dependent protein kinase.
190 citations
TL;DR: The Antennapedia, Ultrabithorax, and fushi tarazu genes of Drosophila melanogaster each contain a very similar protein coding sequence, the homeo box, and the tissue distribution of transcripts encoded by the two rightmostHomeo box genes of the BithOrax complex are compared with the iab-2 and iab -7 phenotypes.
181 citations
TL;DR: It is reported that microinjection of antibodies specific for amino acid 12 of the oncogenic v-Ki-ras protein into cells transformed by this protein causes a transient reversion of the cells to a normal phenotype, supporting the notion that GTP binding is essential to the transforming function of this oncogene product.
Abstract: The proteins encoded by the ras oncogene are thought to trigger expression of the transformed phenotype in some types of cancer cells. In human cells, the ras protein family consists of several members including normal (proto-oncogene) and mutant (oncogene) forms. In general, the proto-oncogene forms are thought to be involved in the normal growth control of cells, while the mutant forms (which apparently result from somatic mutation of the normal ras genes) appear to be responsible, in part, for the loss of normal growth control. On microinjection into living normal cells, the purified ras oncogene protein (p21) induces a characteristic loss of growth control in cells within several hours. The mutant forms of the different ras proteins typically contain a single amino-acid change, usually at position 12 or less frequently at position 61. Here we report that microinjection of antibodies specific for amino acid 12 of the oncogenic v-Ki-ras protein into cells transformed by this protein causes a transient reversion of the cells to a normal phenotype. The fact that this antibody inhibits binding of GTP to the v-Ki-ras protein supports the notion that GTP binding is essential to the transforming function of this oncogene product.
147 citations
TL;DR: A small RNA accumulating late in adenovirus infection is required for efficient protein synthesis, although not specifically for the translation of viral proteins.
Abstract: A small RNA accumulating late in adenovirus infection is required for efficient protein synthesis, although not specifically for the translation of viral proteins. This RNA maintains the activity of an initiation factor catalysing the earliest step of polypeptide chain initiation.
TL;DR: It is concluded that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface and absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.
Abstract: Mutations have been introduced into the cloned DNA sequences coding for influenza virus hemagglutinin (HA), and the resulting mutant genes have been expressed in simian cells by the use of SV40-HA recombinant viral vectors. In this study we analyzed the effect of specific alterations in the cytoplasmic domain of the HA molecule on its rate of biosynthesis and transport, cellular localization, and biological activity. Several of the mutants displayed abnormalities in the pathway of transport from the endoplasmic reticulum to the cell surface. One mutant HA remained within the endoplasmic reticulum; others were delayed in reaching the Golgi apparatus after core glycosylation had been completed in the endoplasmic reticulum, but then progressed at a normal rate from the Golgi apparatus to the cell surface; another was delayed in transport from the Golgi apparatus to the plasma membrane. However, two mutants were indistinguishable from wild-type HA in their rate of movement from the endoplasmic reticulum through the Golgi apparatus to the cell surface. We conclude that changes in the cytoplasmic domain can powerfully influence the rate of intracellular transport and the efficiency with which HA reaches the cell surface. Nevertheless, absolute conservation of this region of the molecule is not required for maturation and efficient expression of a biologically active HA on the surface of infected cells.
TL;DR: The ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state is demonstrated, and the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells are found to be correlated.
Abstract: Transformed monkey cell lines (CMT and BMT) that inducible express simian virus 40 (SV40) T antigen from the metallothionein promoter have been isolated and characterized. Immunoprecipitation of pulse-labeled T antigen demonstrates a 5- to 12-fold increase in the rate of synthesis on addition of heavy-metal inducers to the culture medium. Radioimmunoassay of cell extracts indicates the accumulation of three- to fourfold more total T antigen after 2 days of induction by comparison with uninduced controls. A direct correlation was found between the level of T-antigen synthesis and the extent of SV40 DNA replication in inducible cells. Inducible BMT cells expressing a low basal level of T antigen were efficiently transformed by a vector carrying the neomycin resistance marker and an SV40 origin of replication. These vector sequences were maintained in an episomal form in most G418-resistant cell lines examined and persisted even in the absence of biochemical selection. Extensive rearrangements were observed only if the vector contained bacterial plasmid sequences. Expression of a protein product under the control of the SV40 late promoter in such vectors was increased after heavy-metal-dependent amplification of the template. These results demonstrate the ability of BMT cells to maintain a cloned eucaryotic gene in an amplifiable episomal state.
TL;DR: Using in vitro interposon mutagenesis, Rhodopseudomonas capsulata strains have been constructed wherein all or part of the reaction center (RC), light-harvesting I (LHI), and light-Harvesting II (LHII) structural genes have been deleted.
TL;DR: Analysis of the RNA transcripts by S1-nuclease mapping indicates that the restriction and modification genes are transcribed from different promoters, and there are regions of homology between the BsuRI methylase and two other GGCC specific modification enzymes, the BspRI and SPR methylases.
Abstract: The genes of the 5'-GGCC specific BsuRI restriction-modification system of Bacillus subtilis have been cloned and expressed in E. coli and their nucleotide sequence has been determined. The restriction and modification genes code for polypeptides with calculated molecular weights of 66,314 and 49,642, respectively. Both enzymes are coded by the same DNA strand. The restriction gene is upstream of the methylase gene and the coding regions are separated by 780 bp. Analysis of the RNA transcripts by S1-nuclease mapping indicates that the restriction and modification genes are transcribed from different promoters. Comparison of the amino acid sequences revealed no homology between the BsuRI restriction and modification enzymes. There are, however, regions of homology between the BsuRI methylase and two other GGCC specific modification enzymes, the BspRI and SPR methylases.
TL;DR: Identical organization of the beta and alpha structural genes for both LHI and LHII and sequence homologies suggests that both complexes arose by gene duplication from a single ancestral light-harvesting complex and that the putative bacteriochlorophyll binding sequence Ala-X-X -X- X-His has been absolutely conserved.
Abstract: The light-harvesting II (LHII) structural genes coding for the (B800-B850 complex) β- and α-polypeptides have been cloned and the nucleotide and deduced polypeptide sequences have been determined. This completes the sequencing of all seven structural genes coding for the structural polypeptides of the photosynthetic apparatus that bind the pigments and cofactors participating in the primary light reactions of photosynthesis. Unlike the structural genes coding for the reaction center L, M, and H subunits and the light-harvesting I (LHI) (B870 complex) structural polypeptides, the LHII structural genes are not within the 46-kilobase photosynthetic gene cluster carried by the R-prime plasmid pRPS404. Identical organization of the β and α structural genes for both LHI and LHII and sequence homologies between the two β-polypeptides and between the two α-polypeptides suggests that both complexes arose by gene duplication from a single ancestral light-harvesting complex and that the putative bacteriochlorophyll binding sequence Ala-X-X-X-His has been absolutely conserved.
TL;DR: The structure of 18 revertants of this mutant is described and it is suggested that in each of the 18 revertant, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the ‘core’ element.
Abstract: Enhancers are cis-acting control elements which can stimulate at a distance the activity of a variety of eukaryotic promoters. First identified as a repeated 72 base pair (bp) sequence upstream of the simian virus 40 (SV40) early gene promoter, enhancers have since been shown to be associated with numerous other viral and cellular genes. Although there are no strong homologies between the sequences of different enhancers, a number of short and degenerate consensus sequences have been identified, including the 'core' element GTGGA/TA/TA/TG and stretches of alternating purines and pyrimidines which may have the potential to form left-handed Z DNA. To study the functional significance of two alternating purine and pyrimidine sequences in the SV40 enhancer, we have introduced various combinations of point mutations into a modified SV40 enhancer which contained only one copy of the 72 bp element (W.H., Y.G., A. Nordheim and A. Rich, unpublished results); one of these combinations impaired both the activity of the enhancer and growth of SV40. We describe here the structure of 18 revertants of this mutant and suggest that in each of the 18 revertants, the defects of the original mutant have been overcome by simple tandem duplications in the enhancer region, all of which include the 'core' element.
TL;DR: Four genomic clones obtained from microdissected fragments of the proximal portion of mouse chromosome 17 have been used to identify a series of t-haplotype-specific restriction fragments, providing the first molecular evidence that independent partial t haplotypes contain different lengths of t haplotype DNA.
TL;DR: The stress response, in general, appears to be a defensive one in nature since a brief induction of the stress proteins confers a degree of added protection to the cell upon subsequent stress situations.
Abstract: Exposure of mammalian cells to elevated growth temperatures results in specific changes at both the transcriptional and translational levels. Similar to what was first described in Drosophila rnelanogaster cells’sz and subsequently in all cells from simple prokaryotes to higher eukaryotes, heat-shock treatment of mammalian cells is best characterized by the elevated transcription and translation of genes that encode a specific set of proteins, the so-called heat-shock proteins.’ Concomitant with the increased synthesis of the heat-shock proteins, heat-shock-treated cells also show a diminished production of polypeptides that were being synthesized prior to the change in growth temperature. In addition to heat-shock treatment, it is now recognized that a variety of other treatments to cells can give rise to similar changes at both the transcriptional and translational le~els.4’~ For example, exposure of cells to various drugs, heavy metals, amino acid analogs, and even ethanol, to list just a few, all result in an induction in cells of the same proteins induced by heat-shock treatment. Accordingly, this seemingly generalized response of cells to adverse growth conditions is more aptly referred to as “physiological stress” and the proteins induced referred to as the “stress proteins.” While the identity of the stress proteins has been determined with respect to their relative molecular mass and isoelectric point, the function of any one of the proteins remains unknown. For example, in mammalian cells the stress proteins consist of polypeptides of apparent molecular mass of 28, 72, 73, 80, 90, 100, and 1 10 kilodaltons (kD).5s6 The stress proteins are not simply novel components of the stressed cell since most of the proteins are also constitutively expressed at significant levels in cells grown under “normal” tissue culture conditions. The stress response, in general, appears to be a defensive one in nature since a brief induction of the stress proteins confers a degree of added protection to the cell upon subsequent stress situations?-”
TL;DR: It is proposed that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci, and designated the new locus SUM1 (suppressor of mar).
Abstract: The mating-type information residing at the HML and HMR loci in Saccharomyces cerevisiae is kept unexpressed by the action of at least four MAR (or SIR) loci. To determine possible interactions between the MAR/SIR gene products and to find new regulatory loci, we sought extragenic suppressors of the mar1-1 mutation. A strain with the genotype HMLa MATα mar1-1 is unable to mate because of the simultaneous expression of a and α information. A mutant of this strain was isolated that exhibits an α phenotype and, therefore, presumably fails to express the HML and HMR loci. We designate the new locus SUM1 (supressor of mar). The mutation is recessive, centromere unlinked and does not correspond to the MAT, HML, HMR, SIR1, MAR1, MAR2 (SIR3) or SIR4 loci. The sum1 mutation affects expression of both a and α information at the HM loci. Suppression by sum1-1 is neither allele specific nor locus specific as it suppresses a deletion mutation of the MAR1 locus and mutations in SIR3 and SIR4. The sum1-1 mutation has no discernible phenotype in a Mar+ strain. We propose that the MAR/SIR gene products negatively regulate the SUM1 locus, the gene product of which is necessary for expression of the HM loci.
TL;DR: Polymyxin B is a potentially useful inhibitor of C-kinase activity, and that this enzyme may play an important role in mediating B cell responses.
01 Jan 1985
TL;DR: A major contribution to the dynamic flux of the plant genome is the presence of discrete mobile genetic elements that can cause high rates of genetic instability including spontaneous unstable mutations and chromosome rearrangements.
Abstract: A major contribution to the dynamic flux of the plant genome is the presence of discrete mobile genetic elements that can cause high rates of genetic instability including spontaneous unstable mutations and chromosome rearrangements. These mobile elements, first identified in maize, have since been discovered in a wide variety of prokaryotic and eukaryotic organisms. They may well be ubiquitous but simply not yet identified in other organisms.
TL;DR: Using conventional meiotic and mitotic mapping combined with recombinant DNA techniques, this work has mapped three other SIR genes in Saccharomyces cerevisiae.
Abstract: The HML and HMR loci in the yeast Saccharomyces cerevisiae each contain a complete copy of mating-type information. HML and HMR normally are transcriptionally inactive due to four unlinked genes, known as MAR or SIR or CMT. The map position of MAR1 (SIR2) has been reported previously; it is located on the left arm of chromosome IV, 27 cM from the centromere. Using conventional meiotic and mitotic mapping combined with recombinant DNA techniques, we have mapped three other SIR genes. SIR1 maps near the telomere of the right arm of chromosome XI; SIR3 (MAR2) maps to the right arm of chromosome XII, 31 cM distal to URA4; and SIR4 maps to the right arm of chromosome IV, 16 cM proximal to LYS4.
TL;DR: The evidence suggested the possible mechanism of the induction of multiple MTOCs, and implied that the MT-MTOC complex is significant in the cytokinesis of Dictyostelium by determining the cell locomotory unit.
Abstract: The microtubule inhibitors, ethyl-N-phenylcarbamate (EPC) and thiabendazole (TB), which disrupt cytoplasmic microtubules and induce giant cells inDictyostelium (Kitanishiet al. 1984), were found to induce the occurrence of multiple microtubule organizing centers (MTOCs) in these giant cells. Probing was done by indirect immunofluorescence using monoclonal anti-α-tubulin. The nuclear DNA content of the giant cells increased in parallel with an increase in the number of MTOCs, as shown by microspectrophotometory of cells stained with the fluorescent DNA stain DAPI (4′,6-diamidino-2-phenylindole).
TL;DR: The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli, and the presence of theGal+ phenotype, provided by the clostridial galactokerase, was unstable in E. coli, suggesting that galactose is used in C. pasteurIANum through the Leloir pathway via galactOSE 1-phosphate.
TL;DR: Expression in Escherichia coli of a chimaeric gene fusion containing a porcine parvovirus DNA fragment produced more than one size fusion protein, which suggests that the smaller products are the result of independent initiations of translation within PPV sequences rather than proteolysis of the full-length fusion protein.
Abstract: Expression in Escherichia coli of a chimaeric gene fusion containing a porcine parvovirus (PPV) DNA fragment produced more than one size fusion protein. The introduction of a translation termination codon between the initiation codon and the PPV sequences of the gene fusion did not prevent the expression of the smaller fusion proteins. This suggests that the smaller products are the result of independent initiations of translation within PPV sequences rather than proteolysis of the full-length fusion protein. Three translation initiation sites were mapped to different segments of PPV DNA by protein sizing and deletion analysis. The sequences of these segments are presented.
TL;DR: It is shown that specific peripheral neurons associated with the segmental sensilla also are antibody-positive during development, and the diversity of the staining pattern of the monoclonal antibody is discussed.
TL;DR: The appearance of peripheral neuron within the skin during embryonic development of the leech is described and the development of peripheral neurons in the skin of the rear sucker is also described.
TL;DR: The expression of wild-type HA and analysis of the expression of mutant HA proteins are presented, which provides a useful model system for the study of the structure, function, and biosynthesis of eukaryotic membrane proteins.
Abstract: Publisher Summary Hemagglutinin (HA) glycoprotein of influenza virus is considered the best characterized of all integral membrane proteins. HA is regarded as an ideal candidate for site-specific mutagenesis experiments aimed at elucidating the function of the various domains of the molecule. The structure of the HA molecule is characteristic of the major class of cellular integral membrane proteins, having typical amino and carboxy-terminal hydrophobic regions. Essentially, HA provides a useful model system for the study of the structure, function, and biosynthesis of eukaryotic membrane proteins. The HA protein is naturally encoded by an RNA genome that is not amenable to in vitro mutagenesis. When the HA cDNA is introduced into eukaryotic cells using vectors derived from SV40, it is expressed with high efficiency into a fully glycosylated protein that is displayed on the infected cell's surface in an antigenically and biologically active form. The cloned, expressing copies of the HA gene provide the material to analyze the effect of mutations on the structure and function of the protein. This chapter presents the expression of wild-type HA and analysis of the expression of mutant HA proteins.
01 Jan 1985
TL;DR: In higher eukaryotic cells, the lack of simple genetics has hampered progress toward elucidation of the mechanism of DNA synthesis, particularly how DNA replication gets under way and what immediate events control this process.
Abstract: The replication of DNA is a fundamental process in cell metabolism which is controlled throughout the cell growth cycle both in its timing and extent The initiation of a replicative phase in eukaryotic cells is subject to many environmental influences which we do not understand, but prerequisite to understanding the control of DNA replication is knowledge of the way DNA replicates Elegant studies with bacteria and their phages have established basic principles about the mechanism of DNA replication and the enzymes that complete the task and these studies have relied heavily upon the use of genetics as a method of analysis In higher eukaryotic cells, the lack of simple genetics has hampered progress toward elucidation of the mechanism of DNA synthesis, particularly how DNA replication gets under way and what immediate events control this process Many investigators have turned to the replication of DNA viruses in mammalian cells in an attempt to circumvent some of these problems, particularly because these viruses often allow genetic analysis to be employed