Showing papers by "Cold Spring Harbor Laboratory published in 1982"
TL;DR: The H-ras-1 gene cloned from T24 DNA induces transformation in NIH 3T3 cells, while the same gene cloning from normal cellular DNA does not, and the functionally significant difference appears to be a single base mutation.
Abstract: Several different transforming genes have been observed in the DNA of a variety of tumours and tumour cell lines of human and rodent origin by the ability of these genes to induce morphological transformation in NIH 3T3 cells1-5. The transforming gene found in a human bladder carcinoma cell line, T24, is H-ras-1, the human homologue of the Harvey sarcoma virus oncogene (v-H-ras)6-9. In the present study we have compared the H-ras-1 genes cloned from T24 and normal human DNA. The H-ras-1 gene cloned from T24 DNA induces transformation in NIH 3T3 cells, while the same gene cloned from normal cellular DNA does not. The functionally significant difference between the transforming and normal genes appears to be a single base mutation, which produces an amino acid change in the sequence of the proteins that the genes encode.
652 citations
TL;DR: It is found that T24, a cell line derived from a human bladder carcinoma, can induce the morphological transformation of NIH 3T3 cells, and the gene responsible for this transformation is human in origin, <5 kilobase pairs in size and homologous to a 1,100-base polyadenylated RNA species found in T24 and HeLa cells.
Abstract: DNA from T24, a cell line derived from a human bladder carcinoma, can induce the morphological transformation of NIH 3T3 cells. Using techniques of gene rescue to clone the gene responsible for this transformation, we have found that it is human in origin, less than 5 kilobase pairs in size and is homologous to a 1,100-base polyadenylated RNA species found in T24 and HeLa cells. Blot analysis indicates extensive restriction endonuclease polymorphism near this gene, in human DNAs.
446 citations
TL;DR: It is concluded that the cdc 2 and cdc 28 genes perform homologous cell cycle control functions in the two organisms.
Abstract: The cdc 2 (previously called wee 2) cell cycle start gene of Schizosaccharomyces pombe, which is required for start and the control of mitosis, has been isolated from an S. pombe gene bank by complementation of a cdc 2 mutation. A functionally homologous sequence which complements the cdc 2 mutation has also been isolated from a Saccharomyces cerevisiae gene bank and this sequence has been shown to contain the cdc 28 cell cycle start gene of S. cerevisiae. It is concluded that the cdc 2 and cdc 28 genes perform homologous cell cycle control functions in the two organisms.
443 citations
TL;DR: A double-stranded cut at the MAT locus appears to initiate cassette transposition-substitution and defines MAT as the recipient in this process, suggesting that cutting initiates the switching process.
367 citations
TL;DR: The gene encoding a gonococcal pilus protein into Escherichia coli is cloned and it is shown that conversion of the pilus positive to pilus negative state in N. gonorrhoeae involves chromosomal rearrangement.
306 citations
TL;DR: The DNA sequences encoding the amino- terminal signal peptide or the carboxy-terminal hydrophobic anchor have been deleted from a cloned gene coding for the haemagglutinin (HA) of influenza virus.
Abstract: The DNA sequences encoding the amino-terminal signal peptide or the carboxy-terminal hydrophobic anchor have been deleted from a cloned gene coding for the haemagglutinin (HA) of influenza virus. The wild-type gene has previously been shown to be expressed with high efficiency from simian virus 40 (SV40)-HA recombinant vectors into a fully glycosylated protein that is displayed on the infected cell's surface in an antigenically and biologically active form. The anchor-minus HA also is glycosylated but is secreted efficiently into the medium. By contrast, the signal-minus HA is produced only at low levels, is not glycosylated and is located intracellularly.
231 citations
TL;DR: It is found that SIR is necessary for the maintenance of a different chromatin structure at HM loci from their active counterparts at MAT, and provides a simple explanation for one of its biological properties: control of directionality of switching.
224 citations
TL;DR: Cells expressing HA from a cloned copy of the HA gene inserted into a recombinant simian virus 40 (SV40) vector are used to demonstrate that the HA molecule displays fusion in the absence of any other influenza virus-encoded components, opening the possibility of using HA-mediated membrane fusion as a system to deliver foreign molecules into mammalian cells.
Abstract: Enveloped animal viruses enter and infect cells by a process involving fusion of the viral membrane with a cellular membrane. In some cases (for example, Sendai virus1), the fusion event occurs at the plasma membrane; for many other viruses including influenza, entry occurs through the membranes of intracellular vesicles such as endosomes or lysosomes, where the fusion is triggered by the endogenous low pH2–6. This pH-dependent fusion activity has been studied in vitro using as targets cultured cells7–9, erythrocytes9,10 and liposomes11–13. Fusion is a function of the viral surface glycoproteins12 and occurs at a threshold pH that is characteristic of each virus species and strain8. In the case of influenza virus, there is strong evidence that the haemagglutinin glycoprotein has a key role in both virus infectivity and fusion activity9,12,14–16. Both processes require a post-translational proteolytic cleavage of the haemagglutinin precursor, HA0, into the active form of the molecule, HA, which consists of two disulphide-bonded subunits, HA1 and HA217,18. A new N-terminus is generated on the HA2 subunit, the C-terminus of which is embedded in the virus membrane19. At the low pH values required for fusion the cleaved HA undergoes a conformational change exposing this previously buried hydrophobic N-terminus of HA220, possibly enabling it to interact with the target membrane. While it has been established that HA is necessary for fusion, it is unclear whether HA alone is sufficient or whether other viral proteins are involved21. Here we use cells expressing HA from a cloned copy of the HA gene inserted into a recombinant simian virus 40 (SV40) vector22 to demonstrate that the HA molecule displays fusion in the absence of any other influenza virus-encoded components. These results open the possibility of using HA-mediated membrane fusion as a system to deliver foreign molecules into mammalian cells.
215 citations
TL;DR: The β-subunit of the human placental glycoprotein hormone, chorionic gonadotropin (HCG), is coded for by at least eight different genes or pseudogenes, four of which are arranged in inverted pairs while the other four are arrangedin tandem pairs.
Abstract: The β-subunit of the human placental glycoprotein hormone, chorionic gonadotropin (HCG), is coded for by at least eight different genes or pseudogenes, four of which are arranged in inverted pairs while the other four are arranged in tandem pairs. The genes are each 1.45 kilobases (kb) long and have two short introns located in identical positions.
161 citations
TL;DR: The small adenovirus-encoded VA RNAs occur as ribonucleoprotein (RNP) particles in association with a cellular protein antigen, La, recognized by the anti-La class of lupus sera, and the La antigen appears not to be required for the transcription ofVA RNAs in vitro.
Abstract: The small adenovirus-encoded VA RNAs occur as ribonucleoprotein (RNP) particles in association with a cellular protein antigen, La, recognized by the anti-La class of lupus sera [Lerner, M. R., Boyle, J. A., Hardin, J. A. & Steitz, J. A. (1981) Science 211, 400-402]. We have tentatively identified the La antigen as a HeLa cell phosphoprotein of Mr approximately equal to 45,000, present in infected and uninfected cells. The antigen appears not to be required for the transcription of VA RNAs in vitro. RNP particles that contain newly synthesized VA RNAs assemble rapidly in transcription extracts making VA RNA and also can be reconstituted from purified VA RNA and a source of La antigen. Variant forms of VA RNAI with sequence deletions and substitutions bind to the La antigen, suggesting that the recognition site includes the RNA termini or the sequences corresponding to the internal control region (promoter), or both. Upon reconstitution with fragments of VA RNAI, oligonucleotides from both the 5' and 3' termini bind to the antigen, but those from the control region do not. The terminal oligonucleotides of wild-type VA RNA can form a basepaired stem, but structures of comparable stability cannot be formed by the chimeric variant molecules. Therefore, the recognition site is probably the terminal nucleotides themselves rather than the stem structure.
131 citations
TL;DR: It is demonstrated that adenovirus encodes a novel DNA polymerase that is required for priming of DNA synthesis at the origin of replication and may also function in the initiation of transformation of cultured cells.
TL;DR: The efficiency of transformation of HF by origin-defective SV40 DNA (SV ori−) with that of other derivatives containing a functional viral origin of replication, using the calcium phosphate co-precipitation technique was compared and the transforming potential of SV ori− mutants was found to be superior.
Abstract: Transformation of semipermissive human fibroblasts (HF) by wild-type simian virus 40 (SV40) or SV40 DNA is relatively inefficient compared with SV40 transformation of non-permissive rodent cells1. Whereas HF transformed with either SV40 or a subgenomic fragment of SV40 (that is incapable of making virions) containing the early region and the origin of DNA replication produce large amounts of free virus DNA2–5, established human cell lines transformed by SV40 harbour defective virus genomes6 that are incapable of initiating virus DNA replication (M. Botchan, personal communication). We have now investigated whether the low efficiency of transformation is directly related to the ability of SV40 DNA to replicate autonomously in semipermissive HF. We have compared the efficiency of transformation of HF by origin-defective SV40 DNA (SV ori−) with that of other derivatives containing a functional viral origin of replication, using the calcium phosphate co-precipitation technique7. The transforming potential of SV ori− mutants was found to be superior.
TL;DR: A rat DNA fragment is sequenced that contains an unoccupied site of SV40 DNA integration and allowed two conclusions to be reached: first, SV40DNA became linked to rat DNA at a point where the two genomes shared 5 base pairs (bp) of DNA sequence homology; and second, a rearrangement of the rat genome occurred at the site ofsv40 integration.
Abstract: The structure of simian virus 40 (SV40) DNA insertions is different from those of retrovirus proviruses and movable genetic elements. No single DNA sequence in either the cell or the SV40 genome serves as an obligatory site of SV40 integrative recombination1–9, and SV40 DNA insertions are not bordered by the repeat structures characteristic of transposons and retrovirus proviruses10–15. Integration of SV40 could involve the matching of short stretches of homologous sequences present in the otherwise heterologous SV40 and cellular genomes. To explore this possibility, I have now sequenced a rat DNA fragment that contains an unoccupied site of SV40 DNA integration. Comparison of this sequence with that of SV40 and that at the rat-SV40 recombinant junction allowed two conclusions: first, SV40 DNA became linked to rat DNA at a point where the two genomes shared 5 base pairs (bp) of DNA sequence homology; and second, a rearrangement of the rat genome, probably a deletion of at least 3 kilobases (kb), occurred at the site of SV40 integration.
TL;DR: It is reported that strains possessing the reverse HMLa HMR alpha arrangement switch (phenotype) inefficiently at a maximum of about 6%.
TL;DR: The DNA of C6/SV40 mutant does not replicate detectably in permissive, simian cells, but it transforms established rat cells with an efficiency equal to that of wild-type DNA, although the phenotype of the mutant-transformed cells is somewhat different from that of the wild- type transformants.
TL;DR: It is demonstrated that virion-derived DNA-protein complexes from five human adenovirus serological subgroups can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenvirus type 2 (Ad2)-infected HeLa cells.
Abstract: The origin of adenovirus DNA replication lies within an inverted sequence repetition at either end of the linear, double-stranded viral DNA. Initiation of DNA replication is primed by a deoxynucleoside that is covalently linked to a protein, which remains bound to the newly synthesized DNA. We demonstrate that virion-derived DNA-protein complexes from five human adenovirus serological subgroups (A to E) can act as a template for both the initiation and the elongation of DNA replication in vitro, using nuclear extracts from adenovirus type 2 (Ad2)-infected HeLa cells. The heterologous template DNA-protein complexes were not as active as the homologous Ad2 DNA, most probably due to inefficient initiation by Ad2 replication factors. In an attempt to identify common features which may permit this replication, we have also sequenced the inverted terminal repeated DNA from human adenovirus serotypes Ad4 (group E), Ad9 and Ad10 (group D), and Ad31 (group A), and we have compared these to previously determined sequences from Ad2 and Ad5 (group C), Ad7 (group B), and Ad12 and Ad18 (group A) DNA. In all cases, the sequence around the origin of DNA replication can be divided into two structural domains: a proximal A · T-rich region which is partially conserved among these serotypes, and a distal G · C-rich region which is less well conserved. The G · C-rich region contains sequences similar to sequences present in papovavirus replication origins. The two domains may reflect a dual mechanism for initiation of DNA replication: adenovirus-specific protein priming of replication, and subsequent utilization of this primer by host replication factors for completion of DNA synthesis.
TL;DR: A novel sequence-specific endonuclease has been isolated from Streptococcus cremoris F. as mentioned in this paper, which is the first enzyme to recognize this sequence and reported from the lactic streptococci used in dairy fermentations.
Abstract: A novel sequence-specific endonuclease has been isolated from Streptococcus cremoris F. ScrFI recognises the sequence: (formula; see text) and cleaves as indicated by the arrow ( ). It is the first enzyme to recognise this sequence and the first endonuclease reported from the lactic streptococci used in dairy fermentations.
TL;DR: The H-2 complex region associated with each primordial t chromosome has been maintained within at least the five present-day t haplotypes analyzed here and is an integral component of naturally occurring t Haplotypes.
TL;DR: Three embryonic stem cell lines derived from embryos carrying tw5 are established, and it is reported here that one of the cell lines is homozygous for the mutation (tw5/tw5), whereas the other two are heterozygous (+/Tw5).
Abstract: Mouse embryos homozygous for tw5, a recessive lethal mutation in the t complex located on chromosome 17, develop normally until the elongated egg cylinder stage, approximately 6.5 days after fertilization. At this time, the endoderm is morphologically abnormal and the embryonic ectoderm begins to show signs of pyknosis. Death of the embryo usually occurs within the next 2 days (ref. 1). A serious difficulty in the study of lethal t-mutant gene expression during embryogenesis has been to obtain appropriate experimental material, particularly during the period immediately following implantation. Recently, a method involving the use of teratocarcinoma-conditioned medium was devised for establishing pluripotent stem cell cultures directly from the inner cell mass (ICM) of a normal mouse blastocyst2. Using this method, we have established three embryonic stem cell lines derived from embryos carrying tw5. We report here that one of the cell lines is homozygous for the mutation (tw5/tw5), whereas the other two are heterozygous (+/tw5). When injected into athymic mice, each cell line is capable of forming tumours that contain differentiated derivatives of all three primary germ layers.
TL;DR: DNAs were prepared from twenty-six bacterial species and digested with a variety of restriction endonucleases to determine what modifications the DNAs carry.
Abstract: DNAs were prepared from twenty-six bacterial species and digested with a variety of restriction endonucleases to determine what modifications the DNAs carry. Several general conclusions could be made: 1) First, in no instance was the DNA of a restriction enzyme. 2) The specificity of the DNA modification was the same as that of its restriction counterpart; there were no cases of the DNAs being modified against a less specific class of restriction enzymes. 3) In most (but not all) cases, the resistance of a bacterium's DNA to its own restriction enzyme could be generalized to include resistance to all other restriction enzymes with the same specificity (isoschizomers). 4) DNA modified within the central tetramer of a recognition sequence is usually protected against cleavage by all related hexameric enzymes possessing that central tetramer. Only three families of DNA presented in this study disobey this rule. 5) Finally, a significant number of cases emerge where bacterial DNA carries a modification but no corresponding restriction endonuclease activity.
TL;DR: The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1, and the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences were compared.
Abstract: Rous-associated virus 0 (RAV-0), an endogenous chicken virus, does not cause disease when inoculated into susceptible domestic chickens. An infectious unintegrated circular RAV-0 DNA was molecularly cloned, and the sequence of the long terminal repeat (LTR) and adjacent segments was determined. The sequence of the LTR was found to be very similar to that of replication-defective endogenous virus EV-1. Like the EV-1 LTR, the RAV-0 LTR is smaller (278 base pairs instead of 330) than the LTRs of the oncogenic members of the avian sarcoma virus-avian leukosis virus group. There is, however, significant homology. The most striking differences are in the U3 region of the LTR, and in this region there are a series of small segments present in the oncogenic viruses which are absent in RAV-0. These differences in the U3 region of the LTR could account for the differences in the oncogenic potential of RAV-0 and the avian leukosis viruses. I also compared the regions adjacent to the RAV-0 LTR with the available avian sarcoma virus sequences. A segment of approximately 200 bases to the right of the LTR (toward gag) is almost identical in RAV-0 and the Prague C strain of Rous sarcoma virus. The segment of RAV-0 which lies between the end of the env gene and U3 is approximately 190 bases in length. Essentially this entire segment is present between env and src in the Schmidt-Ruppin A strain of Rous sarcoma virus. Most of this segment is also present between env and src in Prague C; however, in Prague C there is an apparent deletion of 40 bases in the region adjacent to env. In Schmidt-Ruppin A, but not in Prague C, about half of this segment is also present between src and the LTR. This arrangement has implications for the mechanism by which src was acquired. The region which encoded the gp37 portion of env appears to be very similar in RAV-0 and the Rous sarcoma viruses. However, differences at the very end of env imply that the carboxy termini of RAV-0, Schmidt-Ruppin A, and Prague C gp37s are significantly different. The implications of these observations are considered.
TL;DR: Throughout all stages of mitosis, intact 10-nm filaments were observed to form a cage around the mitotic spindle, as found in a variety of other cell types.
TL;DR: During incubation with 1 M sorbitol the shrunken cells appear to store excess surface area in numerous, rapidly forming microvilli and the cytoplasm is altered, and an intriguing aspect of irreversibility was observed when blebbing microplasts switched to ruffling.
TL;DR: Three monoclonal antibodies to tropomyosin allow us, for the first time, to observe directly the distribution of tropomyOSin molecules along the thin filaments of smooth muscle cells.
TL;DR: Evidence is provided that p63/6.9 is closely associated with the external surface of testicular cells but not as an integral membrane component, and the possibility is suggested that primary effects of t haplotypes on sperm differentiation could be exerted through the extracellular matrix.
TL;DR: Analysis of key structures seen after induction of Mu DNA replication shows that key structures contain replicating Mu DNA linked to non-Mu DNA and that replication can begin at either end of Mu.
TL;DR: Results suggest structural and functional relatedness between the src, fps, and yes gene products despite the lack of hybridization between their DNA sequences.
TL;DR: The nucleotide sequences of cloned DNA segments encoding the IVa2 gene from Ad7 and a portion of Ad12 (group B and group A human adenoviruses, respectively) have been determined and it is found that these sequences have been found to be 80% homologous.
TL;DR: Most spontaneous polar mutations in Escherichia coli appear to the insertions, which is the most common insertion; however, certain DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.
Abstract: It can be argued that all organisms exhibit two levels of DNA rearrangements. At a low level they may occur sporadically in cells, perhaps largely because of spontaneous activity of transposable genetic elements. A high level may be induced in special circumstances if functions that cause rearrangements are hyperactive. As an example of low level genetic rearrangements, we have studied the occurrence of spontaneous polar mutations in the early regions of prophage Mu. We isolated 49 independent prophage mutants, which are defective in replication and expression of late genes; 44 were in the B region and 5 were in the A region. In the B region, 68% were IS1 insertions, 9% were IS5 insertions and 9% were IS2 insertions; 14% showed no insertion. In the A region, all 5 were IS5 insertions. Thus most spontaneous polar mutations in Escherichia coli appear to be insertions. IS1 is the most common insertion; however, certain DNA regions may show preference for a specific element. High level DNA rearrangements are exemplified by DNA fusion and DNA dissociation that occur when replication-transposition functions of Mu are induced.
01 Jan 1982
TL;DR: This chapter describes some examples to illustrate the advantages of monoclonal antibodies directed against cytoskeletal components.
Abstract: The lymphocyte hybridoma technique developed by Kohler and Milstein (1975) has been widely and successfully used in the field of immunology and virology (Melchers et al., 1978; Kennett et al., 1980; Milstein and Lennox, 1980). However, there are few reports describing the monoclonal antibodies to cytoskeletal proteins. Monoclonal antibodies against cytoskeletal components have the potential not only for improving the immunofluorescent localization of specific proteins within cells but also for analyzing the functional sites of specific proteins and identifying the previously unidentified proteins. Furthermore, they can be used together with microinjection techniques (Feramisco, 1979; Lin and Feramisco, 1981) to investigate the physiological roles of specific proteins. In this chapter, we describe some examples to illustrate the advantages of monoclonal antibodies directed against cytoskeletal components.