Showing papers by "Cold Spring Harbor Laboratory published in 1981"
TL;DR: Three transformed lines of simian cells were established and found to contain T antigen; retain complete permissiveness for lytic growth of SV40; support the replication of tsA209 virus at 40 degrees C; and support the replicate of pure populations of SV 40 mutants with deletions in the early region.
2,445 citations
TL;DR: A rapid and simple method for studying the transcription of cloned eucaryotic genes is developed, which involves transfecting SV40-transformed monkey cell lines (COS cells) with derivatives of the plasmid pBR322 that contain the SV40 viral replication origin but lack regions necessary for viral transcription (SV-ORI vectors).
500 citations
TL;DR: The results indicate that overlapping pathways leading to tumorigenesis may arise independently, and that at least three different transforming genes are present in these five lines.
410 citations
TL;DR: Recombinant viruses are constructed which, in infected cells, express large quantities of haemagglutinin, which accumulates at the cell surface and displays haemabsorbing activity.
Abstract: By replacing either the early or the late genes of SV40 with a cloned copy of the influenza virus haemagglutinin gene we have constructed recombinant viruses which, in infected cells, express large quantities of haemagglutinin. This glycoprotein, over 108 molecules of which are produced per cell, is identical in molecular weight to authentic influenza virus haemagglutinin, accumulates at the cell surface and displays haemabsorbing activity.
280 citations
TL;DR: This rat cell line should prove useful for nonselective transfection of genetic information, particularly potentially transforming viral or cellular sequences, through cotransfection with a thymidine kinase gene.
262 citations
TL;DR: It is reported that α-actinin purified from HeLa cells is inhibited from cross-linking actin filaments by micromolar free calcium, concluding that in this molecular weight range there are at least two classes of non-muscle actin-binding protein: one corresponding to non-Muscle α- actinins and the other to proteins such as gelsolin and villin.
Abstract: Many actin-binding proteins have been purified from different cell types, and a number of them share certain features: they are affected by calcium in their interaction with actin and have similar subunit molecular weights (∼100,000) on SDS-poly-acrylamide gels. These proteins have been given various names, including gelsolin1–3, villin4,5, actinogelin6,7, Dictyostelium 95K protein8,9, platelet 95K protein10 and Acanthamoeba 85K protein11. The actin-binding protein α-actinin has received little attention in this analysis, even though it has a subunit molecular weight in this range (∼100,000) and has been shown immunologically to exist in non-muscle cells12,13. Here we report that α-actinin purified from HeLa cells is inhibited from cross-linking actin filaments by micromolar free calcium. In many properties it closely resembles actinogelin, Dictyostelium 95K protein and Acanthamoeba 85K protein, but it appears distinct from proteins such as gelsolin and villin which, in the presence of calcium, fragment actin filaments into short oligomers3,5. We conclude that in this molecular weight range there are at least two classes of non-muscle actin-binding protein: one corresponding to non-muscle α-actinins and the other to proteins such as gelsolin and villin.
253 citations
TL;DR: Twenty of the antibodies which react with specific neurones were studied in greater detail and are described here and include antibodies against identified sensory neurones and motor neurones as well as against numerous unidentified cells.
Abstract: Monoclonal antibodies were isolated by screening 475 hybridomas obtained from mice immunized with whole leech nerve cords. The majority (about 300) reacted with leech nervous tissue, but only about 40 made antibodies that identified single kinds or small sets of cells. Twenty of the antibodies which react with specific neurones were studied in greater detail and are described here. They include antibodies against identified sensory neurones and motor neurones as well as against numerous unidentified cells.
243 citations
TL;DR: Electron microscopic heteroduplex analysis has revealed a family of 1-strand RNAs that probably encode these proteins, which supports a model of adenovirus DNA replication in which the 87K terminal protein precursor is the primary translation product and primes DNA synthesis.
209 citations
TL;DR: Another region of the SV40 genome showed preferential binding to D2T, however, the decay rate of this site in the EcoRII/D fragment was rapid and, under these conditions, could not be distinguished from non-specific binding.
202 citations
TL;DR: Examination of the transcripts by R-looping and filter hybridization indicates that each cassette is capable of producing two divergent transcripts, and transcription of cassettes at the MAT locus is transcribed constitutively.
Abstract: The genes that control the a, α and a/α cell types in Saccharomyces are carried on transposable elements known as a and α cassettes which reside at three different chromosomal loci. Examination of the transcripts by R-looping and filter hybridization indicates that each cassette is capable of producing two divergent transcripts. Cassettes at the MAT locus are transcribed constitutively. Transcription of cassettes at HML and HMR is prevented by trans-acting negative regulators.
174 citations
TL;DR: The microinjection of highly specific monoclonal antibodies that recognize and alter components of the cell provides an additional approach to determine the in vivo functions of intracellular elements.
TL;DR: Two different genes coding for the hormonally regulated rat liver protein α2u globulin were introduced into mouse Ltk− cells through co-transfection with the HSV-1 thymidine kinase gene, suggesting that the information necessary for hormonal response is contained in the DNA fragment used for transfer.
Abstract: Two different genes coding for the hormonally regulated rat liver protein α2u globulin were introduced into mouse Ltk− cells through co-transfection with the HSV-1 thymidine kinase gene. Three to ten copies of the α2u globulin genes were detected in several tk+ clones, over 50% of which could be induced with dexamethasone, to produce α2u globulin mRNA and protein. This suggests that the information necessary for hormonal response is contained in the DNA fragment used for transfer.
TL;DR: In appropriate strains where the silent loci are also allowed to express, for example in mar mutants, efficient switches of HML and HMR are shown to occur at rates equivalent to those observed for MAT.
TL;DR: It is shown here that recombination occurs at the normal rate in compound female mice containing two different complementing lethal haplotypes (th17/tw12), and it is concluded that cross-over suppression is due to mismatching.
Abstract: We show here that recombination occurs at the normal rate in compound female mice containing two different complementing lethal haplotypes (th17/tw12 where there is a long stretch of homologous t-mutant chromatin. Thus the recombination suppression of a complete t-haplotype cannot be due to an intrinsic factor(s) which suppresses along the length of its own chromosome but is due to ‘mismatching’ of wild-type and mutant chromatin. Naturally occurring t-haplotypes of mouse chromosome 17 have several interesting genetic properties. First, they are always transmitted from males in much higher proportions than mendelian expectation; presumably this accounts for the maintenance of lethal and semilethal t-haplotypes at polymorphic levels in populations of wild mice, t-Haplotypes also show recombination suppression. The conventional map distance between genetic markers T and tf is 7–12 cM, whereas in (t/+) heterozygotes for naturally occurring t-haplotypes, recombination is suppressed and T and tf seem to be separated by only 0.1–0.5 cM (ref. 1). The region of recombination suppression extends to and includes the major histocompatibility complex (H–2)2. Thus t and H–2 effectively travel as a single unit— a ‘super gene’3. Although recombination suppression is known to be accompanied by failure of chiasmata formation4, the mechanism underlying the suppression has remained an enigma. Lyon suggested a disorder of t-heterochromatin5 and more recently a change in ‘intercalary’ middle repetitive DNA6. She proposed that either t-chromatin is intrinsically incapable of participating in crossing-over, or chiasma formation is prevented because of mismatching and mispairing of normal and abnormal chromatin. We have measured recombination between two chromosomes which carried extensive overlapping segments of t-chromatin. We report here that in this configuration, recombination occurs at a normal rate, and thus we conclude that cross-over suppression is due to mismatching.
TL;DR: The nucleotide sequences of IS5 insertions into the B and cim genes of bacteriophage Mu have allowed tentative identification of the protein-coding frames of B andcim, and based on seven examples, this site of insertion appears to be nonrandom.
TL;DR: Biochemical characterization of SV40 T antigens overproduced by the hybrid viruses indicates that they are structurally indistinguishable from wild-type SV40 large T antigen and are functionally equivalent to the D2 protein.
TL;DR: A mechanism by which integrative replication ofacteriophage Mu and many other transposable elements undergo transposition by a process that involves replication of the element is described.
Abstract: Bacteriophage Mu and many other transposable elements undergo transposition by a process that involves replication of the element. We describe here a mechanism by which such integrative replication may take place. We hve examined electron microscopically the DNA structures generated in host cells after Mu induction and have deduced the following steps in the transposition process, (i) Association. A protein-mediated association is brought about between the transposable element and the target DNA. (ii) Attachment. One end of the element is nicked and attached to a site that undergoes a double-stranded cleavage. (iii) Roll-in replication. While one strand of the target DNA is linked to the nicked strand of the element, the complementary strand of the target DNA is used as a primer for replication into the element such that the replicating DNA is threaded through the replication complex. (iv) Roll-in termination. When the distal end of the element arrives at the replication complex, replication is terminated. The roll-in replication mechanism can also explain laying down of tandem repeats--i.e., amplification of circular DNA sequences.
TL;DR: The phenotype of mutations in this recombination system suggests that transposition proceeds by a mechanism in which cointegrates are intermediates.
Abstract: The bacterial transposon Tn3 encodes a site-specific recombination system. The recombination requires the product of tnpR, a gene previously identified as a repressor of the transposase. This recombination is site specific and takes place somewhere within the sequence C-G-A-A-A-T-A-T-T-A-T-A-A-A-T-T-A-T-C but requires at least one additional sequence outside this. The phenotype of mutations in this recombination system suggests that transposition proceeds by a mechanism in which cointegrates are intermediates.
TL;DR: In contrast to the normal situation, the X90 molecules synthesized in great excess from the plasmid are stable in Su degrees hosts and can be recovered primarily from the 10 000 X g pellets of sonication lysates, suggesting the surprising stability of the overproduced X90 protein may be due to the formation of proteinaceous aggregates.
TL;DR: Evidence is presented to show that mutants of SV40 carrying constructed mutations in the regulatory region13 show reduced binding of D2T protein in vitro, the first demonstration in a eukaryote that a mutation in a regulatory DNA sequence alters the binding of a controllingprotein in vitro.
Abstract: When simian virus 40 (SV40) infects host cells, the viral genes are expressed in two phases Early in infection, the virus genome is transcribed and translated to yield two related proteins, ‘large T’ and ‘small t’ antigens Later the rate of synthesis of the early messenger RNA falls and the viral genes coding for the coat proteins are transcribed and translated Early gene transcription is inhibited only when an active large T antigen is present in the cell (for review see ref 1) These and other observations have led to the hypothesis that T antigen acts as a negative feedback repressor of its own RNA transcript2–4 The properties of SV40 tsA mutants, which produce temperature-sensitive T antigen, provide evidence for another role of T antigen—in the initiation of viral DNA replication5 Mutations around the origin of DNA replication in SV40 lead to a cis-acting defect in DNA replication6,7 These mutations fall outside the region of the A gene which codes for T antigen but within the region known to bind T antigen8 and the closely related and more easily prepared D2T protein9,10 which is synthesized by an adeno–SV40 hybrid virus Ad2D2 and seems to differ from SV40 T antigen only in its amino-terminal segment11 Revertants of origin-defective mutants have been isolated with second site mutations within the DNA A gene coding sequence12 These data support the view that T antigen modulates transcription and replication of SV40 DNA by binding to one or more sites in and around the viral origin of replication We present here evidence to show that mutants of SV40 carrying constructed mutations in the regulatory region13 show reduced binding of D2T protein in vitro This is the first demonstration in a eukaryote that a mutation in a regulatory DNA sequence alters the binding of a controlling protein in vitro
TL;DR: An adenovirus mutant is described which contains a cis dominant defect in the splicing of RNA from one of the early transcriptional units and is used to define sequences required for splicing, but also to investigate the pathways along which a series of related mRNAs is generated.
Abstract: Many primary products of transcription in eukaryotes contain sequences that are removed during maturation of mRNAs, although the structural determinants that direct this highly specific splicing reaction are unknown. Comparative sequence studies of known splice points1–4 have identified a short consensus sequence to which all known splice points conform to varying degrees. However, there is no direct evidence that this consensus sequence is necessary for splicing to occur. In this communication, an adenovirus mutant is described which contains a cis dominant defect in the splicing of RNA from one of the early transcriptional units. The defect is ascribed to changes in two adjacent nucleotides within a splice site consensus sequence. The mutations lie in a region of the adenovirus genome (the transforming region 1A) which normally produces a set of three co-terminal mRNAs differing only by the internal sequences removed during post-transcriptional processing. The mutant has thus been used not only to define sequences required for splicing, but also to investigate the pathways along which a series of related mRNAs is generated.
TL;DR: Comparison of the relationships among the major proteins encoded by early region 1A of Ad2 by comparative analysis of their [ 35 S]methionine-labeled tryptic peptides using reverse phase high-performance liquid chromatography shows that the 50,000, 46, thousands, and 42,000 molecular weight E1A proteins obtained from Ad2-infected HeLa cells are derived from the 1.1 kb region 2A mRNA.
TL;DR: Recombinant plasmids carrying one or both ends of the bacteriophage Mu genome were constructed and conferred full Mu immunity upon the host cells, however, the same mini-Mu's containing kan or lac inserts were defective in immunity.
TL;DR: Biochemical and genetic experiments suggest that the net result of transposition of prophage Mu and internally deleted Mu derivatives (mini-Mu's) during the lytic cycle is replicon fusion, and infer that the process of Mu DNA transposition can occur by either of two alternate pathways which differ with respect to the end-products they generate.
TL;DR: An adenovirus-SV40 recombinant has been constructed in which the SV40 early region was substituted for a small portion of the adenvirus late region and all of early region 3, and the RNAs produced lack both the second and third members of the tripartite leader normally found on adenavirus late RNA, yet are translationally active, directing the synthesis of substantial amounts of SV40 T antigen.
TL;DR: In this paper, surface staining of the intact vascular endothelial cell layer lining the lumen of guinea pig thoracic aorta with antibodies to vimentin revealed that at least 70% of the cells contained intact perinuclear rings of 10-nm filaments.
TL;DR: Analysis of the phosphorylation sites on the transformation-specific proteins encoded by RSV and the class III viruses shows that, although there is no appreciable sequence relationship between large portions of these molecules, a tryptic peptide containing the phosphotyrosine seems to be identical in classes I and III.
Abstract: Three classes of avian sarcoma viruses have been defined recently (Table 1)1–5. Transformation-specific proteins of all three viral classes have associated tyrosine-specific protein kinase activity4–12. The protein kinase activity of Rous sarcoma viruses (RSV), representing class I, seems to be a property of the transformation-specific protein, pp60src, itself13 and seems to be essential for cellular transformation, as the activity is labile or absent in transformation mutants6,7,14,15. This connection between transformation-specific protein, enzyme activity and oncogenic cellular changes has yet to be established for the viruses of classes II and III, which code for polyproteins in which gag-derived and transformation-specific sequences are fused. We report here an analysis of the phosphorylation sites on the transformation-specific proteins encoded by RSV and the class III viruses which shows that, although there is no appreciable sequence relationship between large portions of these molecules, a tryptic peptide containing the phosphotyrosine seems to be identical in classes I and III.
TL;DR: A temperature-sensitive mutant of adenovirus type 5 whose alteration maps in the structural gene for the viral DNA binding protein that form plaques at 39° in HeLa cells has been isolated.
01 Jan 1981
TL;DR: It appears that the simplest and smallest device that is compatible with the scrambling influence of thermal fluctuations as are demonstrated by Brownian motion is a pair of cylinders oriented at right angles to each other.
Abstract: The paper suggests several principles of construction of a microscopically small device for locating the directions of signal sources in microscopic dimensions It appears that the simplest and smallest device that is compatible with the scrambling influence of thermal fluctuations as are demonstrated by Brownian motion is a pair of cylinders oriented at right angles to each other Nine equally spaced blades run in a pitched fashion along the mantle of each cylinder The blades have a concave cross-section and bend around the circumference of the cylinder in a certain rotational pattern Considering the striking similarity of this hypothetical device with centrioles, the paper puts forward the conjecture that centrioles locate the direction of hypothetical signals inside cells
TL;DR: The patterns of integration of persisting viral DNA have been examined in 30 to 40 different adenovirus type 2 (Ad2)- and type 12 (Ad12)-transformed hamster cell lines, Ad12-induced hamster and rat tumours and tumour cell lines and so far no evidence has been found for specific sites of virus integration.
Abstract: The patterns of integration of persisting viral DNA have been examined in 30 to 40 different adenovirus type 2 (Ad2)- and type 12 (Ad12)-transformed hamster cell lines, Ad12-induced hamster and rat tumours and tumour cell lines1–5. These patterns have proved to be quite complex, and so far no evidence has been found for specific sites of virus integration. However, the methods of analysis used so far, are not sufficiently sensitive to decide definitively the issue of specificity. Moreover, specific signals directing the interactions between viral and cellular DNA may reside in DNA sequences distant from the site of junction between the two genomes and may be hidden in hitherto undetected structural peculiarities of DNA sequences. From the DNA of an Ad12-induced hamster tumour line (CLAC3) carrying five copies of the Ad12 genome1, the site of junction between the left terminus of Ad12 DNA and cellular DNA has now been recloned in a λ gtWES·λB′ vector. Determination of the nucleotide sequence at this site of junction reveals that the Ad12 DNA sequence is preserved starting with base pair (bp) 46 of the authentic left end of Ad12 DNA. The first 82 bp of the recloned fragment are not viral and contain scattered, patch-like octa- to undecanucleotide pair homologies to distant sequences within the first 2,722 left terminal base pairs of Ad12 DNA. Close to and encompassing the site of junction a stemmed loop can be constructed based on extensive regions of dyad symmetry. The stability of this loop and its biological function are still uncertain.