Showing papers by "Cold Spring Harbor Laboratory published in 1983"
TL;DR: Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmid molecules.
11,144 citations
TL;DR: The topic of this report is rap,d m,croscale methods for,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI, which is of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s.
Abstract: The topic of this report is rap,d m,croscale methods for ,solat,on of plant D N A without tile use of ul tracentr ,fugatlon wEth CsCI. The D N A produced ,s of moderately high molecular weight and serves as a satisfactory substrate for most restrlctum cndonucleases and is statable for genom,c blot analys,s. In addi t ion to the rapidi ty and convenience of mlmpreps which permit a large number of samples to be processed in just a few hours, the small amount of tissue reqmred (less than 1.0 grams) allows tbr molecular analysis of plants at a very young stage Mm,prep D N A y,elds from leaf tissue of most species tested to date are typ,cally 30-100 big per gram tissue, greater than 50 kb, and remarkably uniform from sample to sample. The first mmlprep procedure we reported fi3r maize D N A isolation (Dellaporta et al , ;'*l,;tze Geneta3 Cr162162 Neu'_~letlrt. 1983) was adapted from a procedure commonly used for }'east D N A preparatmn (Dav,s et al. , 1980) Since th,s report, numerous personal commun,cat ,ons have demonstrated that the mm,prep procedure or a modification thereof, can be apphed to most plant species tested. For example, the method has been successfully used on Ntcottana hlgl~um. N. plumklgmgidtum. N. 3)/t'eJtrt~. L)s~opertcum sp.. Amar,mthm sp . Gl)~me max. Petuma h.~hra&. Several modifications have been apphed by these ,nvestlgators and in our own laboratory m order to extend the appl ,catmn of ram,prep procedures to other plant species. The select,on of a part icular protocol depends to a large degree on the plant spec,es used. However, the procedure reported here was selected to be statable for most situations.
7,263 citations
TL;DR: The polyoma virus middle-T and the T24 Harvey ras1 genes are individually unable to transform primary baby rat kidney cells and separate establishment and transforming functions are required for oncogenic transformation of primary cells in culture.
Abstract: The polyoma virus middle-T and the T24 Harvey ras1 genes are individually unable to transform primary baby rat kidney cells. Adenovirus early region 1A provides functions required by these genes to transform primary cells following DNA-mediated gene transfer. These results suggest that separate establishment and transforming functions are required for oncogenic transformation of primary cells in culture.
1,245 citations
TL;DR: It is concluded that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.
474 citations
TL;DR: The identity of the Jo-1 antigen, the first of the RNA-associated antigens familiar in autoimmune disease to be characterized as a specific enzyme, suggests a model for virus involvement in autoantibody generation.
Abstract: In autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus (SLE), autoantibodies are generated against a variety of macromolecules. Myositis is a human autoimmune disease characterized by weakness and wasting of muscle. In American studies, antibodies directed against soluble cellular constituents were detected by immunodiffusion in about 60% of cases; the commonest of these, found in 25% of patients, was antibody to the Jo-1 antigen. An antibody system referred to as PL-1 was recognized at a similar frequency in a series of patients studied at Hammersmith Hospital, London. We show here that this system is identical with the Jo-1 system and demonstrate that the antigen is a polypeptide of molecular weight (Mr) 50,000. The protein is immunoprecipitated with tRNA His and appears to be histidyl-tRNA synthetase. The identity of the Jo-1 antigen, the first of the RNA-associated antigens familiar in autoimmune disease to be characterized as a specific enzyme, suggests a model for virus involvement in autoantibody generation.
369 citations
TL;DR: A common modular structure for ras gene products in which the variable region forms a physiologically important combining site is proposed.
Abstract: The homologue of the viral Kirsten ras (v-Ki-ras) gene found in the human lung carcinoma cell line, Calu-1, has an intron-exon structure similar to that of the human homologue of the viral Harvey ras (v-Ha-ras) gene. A second, potential fourth coding exon is present in the human Ki-ras gene and similar sequences are found in the Kirsten murine sarcoma virus. Cysteine is encoded at the twelfth amino acid position, suggesting that the Calu-1 Ki-ras gene has undergone a mutational activation at the same position as the human Ha-ras gene of the bladder carcinoma cell line, T24. A comparison of their predicted amino acid sequences suggests that ras proteins have a 'constant' region and a 'variable' region. Here we propose a common modular structure for ras gene products in which the variable region forms a physiologically important combining site.
331 citations
TL;DR: The nucleotide sequence of the chick beta-actin gene was determined and it was found that the gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein.
Abstract: The nucleotide sequence of the chick beta-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5' untranslated region. The gene has a 97 nucleotide 5'-untranslated region and a 594 nucleotide 3'-untranslated region. A slight heterogeneity in the position of the poly A addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chick skeletal muscle actin gene the beta-actin gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. In the 5' flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous to the sequence found in the same region of the rat beta-actin gene.
313 citations
TL;DR: In vitro analysis of a number of altered recognition sites has delimited the sequences required for cleavage for YZ endo, and the molecular genetics of mating-type interconversion is discussed in the context of this endonucleolytic activity.
292 citations
TL;DR: The co-expression of cytokeratins and vimentin in situ, in the parietal endoderm of the mouse embryo 8.5–13.5 days old, supports the idea that vimentsin expression is specifically related to reduced cell-to-cell contact, and to the independent existence of a cell following detachment from an epithelial sheet.
Abstract: Of the five classes of intermediate filaments found in vertebrate tissues, the cytokeratins are considered unique to epithelial tissues, while vimentin is expressed by endothelial and mesenchymal cells1. In neither case is the precise function of the filament system known. Epithelial cells in culture often express vimentin as well as cytokeratins1–3, but co-expression in vivo, as reported for pleomorphic adenomas of the parotid gland4,5 and metastatic carcinoma cells in ascites or pleural fluid6, is still controversial. Here we report the co-expression of cytokeratins and vimentin in situ, in the parietal endoderm of the mouse embryo 8.5–13.5 days old. This population of individual, motile cells seems to be derived from a conventional epithelium by migration and differentiation. Our results support the idea that vimentin expression is specifically related to reduced cell-to-cell contact, and to the independent existence of a cell following detachment from an epithelial sheet.
256 citations
TL;DR: It is suggested that alterations in the pattern of tropomyosin expression are involved in, or cause, rearrangement of stress fibers and that this may be responsible (in part) for morphological transformation.
191 citations
TL;DR: The nucleotide sequence of the dam gene was determined, and analysis revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam, which corresponds closely to the amino acid composition of the purified dam protein.
Abstract: The E. coli dam (DNA adenine methylase) enzyme is known to methylate the sequence GATC. A general method for cloning sequence-specific DNA methylase genes was used to isolate the dam gene on a 1.14 kb fragment, inserted in the plasmid vector pBR322. Subsequent restriction mapping and subcloning experiments established a set of approximate boundaries of the gene. The nucleotide sequence of the dam gene was determined, and analysis of that sequence revealed a unique open reading frame which corresponded in length to that necessary to code for a protein the size of dam. Amino acid composition derived from this sequence corresponds closely to the amino acid composition of the purified dam protein. Enzymatic and DNA:DNA hybridization methods were used to investigate the possible presence of dam genes in a variety of prokaryotic organisms.
TL;DR: The isolated microfilaments have the ability to activate the Mg2+-ATPase activity of skeletal muscle myosin to almost the same extent as skeletal muscle F-actin (filamentous actin) and this microfilament isolation method is applicable to a variety of cell types.
TL;DR: The mating-type genes of Schizosaccharomyces pombe have been isolated and Restriction and heteroduplex mapping have demonstrated that cell type switching occurs by copy transposition of a 1.1 kilobase sequence from one of two silent storage cassettes to an expression locus at mat1.
Abstract: The mating-type genes of Schizosaccharomyces pombe have been isolated. Restriction and heteroduplex mapping have demonstrated that cell type switching occurs by copy transposition of a 1.1 kilobase sequence from one of two silent storage cassettes to an expression locus at mat1. The switching event is initiated by a long-lived double-strand DNA break at mat1. The properties of this cut are discussed in relation to the known pattern of cell type switching.
TL;DR: Primary cell cultures of African Green monkey kidney contain polarized epithelial cells in which influenza virus matures predominantly at the apical surfaces above tight junctions, and influenza virus glycoproteins were found to be localized at the same membrane domain from which the virus budded.
TL;DR: A galvanotropic response of cultured Xenopus epithelial cells is described, where the anodal side of the cell retracts and both the ends and cathodal edge become active in the extension of ruffling lamellipodia.
Abstract: The development of motility in cultured cells is usually associated with a polarization of the cell shape. In particular, the leading edge of the cell is extended into a lamella which acts as a locus for the elaboration of cell processes and for the formation of cell-substrate contacts and, at the opposite end, retraction fibres often extend beyond the trailing edge of the cell. The alignment of microfilament bundles (stress fibres) along the direction of migration and the presence of a band of actin at the leading edge of the cell suggest an involvement of this protein in the motile process. The direction of growth and orientation of various cell types in tissue culture can be influenced by externally applied d.c. electric fields but the effect of the field on cellular motile activities is unknown. Here we describe a galvanotropic response of cultured Xenopus epithelial cells. At a field strength of 5 V cm-1 these cells elongate perpendicularly with respect to the field. The anodal side of the cell retracts and both the ends and cathodal edge become active in the extension of ruffling lamellipodia. In parallel with the change in the cell axis, stress fibres are oriented perpendicularly to the field, and a band of actin is associated with the lamellae at the cathodal edge and at the ends of the cell.
TL;DR: It is reported here that tubulin synthesis is rapidly and specifically suppressed by injection of an amount of tubulin roughly equivalent to 25–50% of the amount initially present in the cell, thus indicating the presence of an eukaryotic, autoregulatory control mechanism which specifies tubulin content in a cultured mammalian cell line.
Abstract: Most eukaryotic cells rapidly and specifically depress synthesis of alpha- and beta-tubulin polypeptides in response to microtubule inhibitors which cause microtubule depolymerization and presumably increase the intracellular concentration of free subunits. Other drugs which interfere with microtubule function but which lead to a decrease in the subunit pool size have little effect on the rate of new tubulin synthesis. These findings have previously been interpreted to indicate that cultured cells synthesize tubulin constitutively unless the subunit pool rises above a specified level. At this point an autoregulatory control mechanism is triggered which suppresses new tubulin synthesis through specific loss of tubulin mRNAs. That tubulin RNA levels are dramatically lowered by microtubule depolymerizing drugs is unquestionably correct; that fluctuations in the depolymerized tubulin pool size are responsible for altered RNA levels rests, however, entirely on the presumptive effects of different microtubule drugs. This caveat is not trivial, as these drugs induce gross morphological alterations, and the specificities and detailed mechanisms of action of such drugs remain poorly understood. To investigate the effect of altered levels of tubulin subunits on the rate of new tubulin synthesis in mammalian cells, we have microinjected purified tubulin subunits into cells in culture and analysed the synthesized proteins. We report here that tubulin synthesis is rapidly and specifically suppressed by injection of an amount of tubulin roughly equivalent to 25-50% of the amount initially present in the cell, thus indicating the presence of an eukaryotic, autoregulatory control mechanism which specifies tubulin content in a cultured mammalian cell line.
TL;DR: This year marks the 30th anniversary of the discovery of adenoviruses and the publication of the structure of the double helix of DNA, with its implications for the mechanism of DNA replication.
TL;DR: A cell line selected as resistant to a spermidine synthesis inhibitor regenerates to give plants producing flowers with anthers in the place of ovules, a second example of an N. tabacum mutant with altered polyamine metabolism and floral development.
Abstract: We have previously isolated two mutant cell lines of Nicotiana tabacum defective in polyamine metabolism1. One of these two cell lines, a temperature-sensitive mutant ts4, regenerated into light green plantlets with short internodes that could be maintained only in shoot cultures. The other cell line, Rt1, a revertant of the ts4 line, also had short internodes, but was dark green, and produced flowers with a second row of petals in place of anthers2. We describe here a second example of an N. tabacum mutant with altered polyamine metabolism and floral development: in this case a cell line selected as resistant to a spermidine synthesis inhibitor regenerates to give plants producing flowers with anthers in the place of ovules.
TL;DR: It is concluded that each of the four beta tubulin genes is a functional gene which is expressed in a specific program during differentiation, and strongly suggest that fourbeta tubulins are necessary for proper microtubule function in vertebrates.
TL;DR: The results show that groups of growing axons do indeed carry chemically distinct surface molecules and this technique has made possible the production of monoclonal antibodies for identifying and characterizing a family of glycoproteins which are expressed on the surface of specific axon bundles during the development of the leech nervous system.
Abstract: Studies on vertebrate and invertebrate species have established that, during development, axons have the ability to choose particular paths over others. The chemical basis of this pathfinding is not clear but biochemical differences between neurons have long been postulated to account for the specificity of neuronal connections. Such subtle molecular differences between different cells in a single tissue are difficult to study with standard biochemical techniques but hybridoma technology has offered a potential solution to this type of problem. This technique has made possible the production of monoclonal antibodies for identifying and characterizing a family of glycoproteins which are expressed on the surface of specific axon bundles during the development of the leech nervous system. The results show that groups of growing axons do indeed carry chemically distinct surface molecules.
TL;DR: Some form of translational regulation is present in adenovirus-infected cells that is missing from the in vitro translation reaction.
TL;DR: A comparative high-resolution, two-dimensional gel analysis of testicular cell proteins synthesized by inbred mice congenic for a series of t haplotypes on the 129/SvJ background suggests a subset of TCP proteins are most likely to be involved in t haplotype effects on germ cell differentiation and fertility.
TL;DR: The ability of Tn3 to form cointegrates between two plasmids to develop a quantitative assay to detect transposition immunity and it is determined that tnpR is not required for immunity, and only the terminal 38 base pairs of TN3 need be present to confer immunity.
Abstract: A plasmid containing transposon Tn3 is immune to further insertions of Tn3. This phenomenon works in cis and is referred to as transposition immunity. We have used the ability of Tn3 to form cointegrates between two plasmids to develop a quantitative assay to detect transposition immunity. Presence of Tn3 on both the plasmids reduces the cointegration frequency to less than 1/100 of parental. Using this assay, we have determined that (i) tnpR is not required for immunity, (ii) only the terminal 38 base pairs of Tn3 need be present to confer immunity, and (iii) other parts of Tn3 appear not to confer immunity.
TL;DR: Analysis of integrated viral sequences present in the polyoma transformed cell line 82-Rat indicates that multiple recombinant events occur within small regions and that very short homologous stretches participate in joining two non-homologous sequences, which suggests that factors other than sequence homologies drive certain recombination events.
Abstract: Mammalian cells possess mechanisms that allow unrelated sequences to recombine (illegitimate recombination), This is evidenced by the high rate of recombination between largely non-homologous sequences after DNA transfection We have analysed the integrated viral sequences present in the polyoma transformed cell line 82-Rat Within the single insert of integrated viral sequences there are two regions where multiple recombination events have occurred The recombination events are particularly interesting as there was no obvious prior selection for their occurrence, and thus they may accurately reflect a normal mechanism of cellular recombination A total of five recombinant joins have been sequenced Our results, reported here, indicate that multiple recombinant events occur within small regions (about 50 bp) and that very short homologous stretches (3-4 bp) participate in joining two non-homologous sequences This suggests that factors other than sequence homologies drive certain recombination events These results have implications for site-directed recombination following the addition of exogenous DNA
TL;DR: The purified complex is extremely efficient in transfection of E. coli spheroplasts and suggests that this complex might represent the integrative precursor of infecting Mu DNA.
TL;DR: Experiments in which E1B DNA was microinjected together with E1A and E2A suggest that the DNA binding protein may have a stimulatory effect on the synthesis of E1 B products.
TL;DR: Three monoclonal antibodies, originally studied because of their neuron‐specific staining in the leech central nervous system, are characterized further here, both immunocytochemically and biochemically, with Western blot staining using the central nervousSystem and peripheral tissues.
Abstract: Three monoclonal antibodies, originally studied because of their neuron-specific staining in the leech central nervous system, are characterized further here, both immunocytochemically and biochemically, with Western blot staining using the central nervous system and peripheral tissues. The three antibodies react both with neurons and select epithelial tissue in the central nervous system, gut, and penis. Antibody Lan3-8 reacts with neurons in the nerve cord and gut but with epithelial cells in the penis; it binds to a 65K molecule in all three tissues. Lan3-2 and Laz2-369 are considered as a related pair because in the central nervous system the former stains all (four) and the latter generally only half (two) of the neurons in a standard midbody ganglion responding to nociceptive stimulation. In the gut, both antibodies label patches of epithelial cells and Laz2-369 stains a previously unknown type of gut neuron. While a given antibody stains different bands in gut and central nervous system immunoblots, comparing the bands of both antibodies for the same tissue extract makes it apparent that there are similarities in the molecular species that both antibodies recognize.
For each monoclonal antibody, the histologically identified tissue antigens need to be correlated with proteins identified on Western blots. Of particular interest are the broad 130K bands to which Lan3-2 and Laz2-369 bind. The question is raised whether the molecular species in these bands represent a family of proteins that serve a specific nociceptive cell function.
TL;DR: Two MAbs against fixed leech CNS which bind the nociceptive neurons, either the complete set or the lateral subset, yield bands on immunoblots of SDS acrylamide gels that show histological and biochemical properties similar to the original mAbs.
TL;DR: It appears that the interaction of SV40 (a primate virus) with primate cells may be intrinsically less oncogenic than its interaction with rodent cells.