Showing papers on "Regulation of gene expression published in 1982"
Journal Article•
TL;DR: A bacterial gene conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion and it is shown that cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.
Abstract: A bacterial gene (neo) conferring resistance to neomycin-kanamycin antibiotics has been inserted into SV40 hybrid plasmid vectors and introduced into cultured mammalian cells by DNA transfusion. Whereas normal cells are killed by the antibiotic G418, those that acquire and express neo continue to grow in the presence of G418. In the course of the selection, neo DNA becomes associated with high molecular weight cellular DNA and is retained even when cells are grown in the absence of G418 for extended periods. Since neo provides a marker for dominant selections, cell transformation to G418 resistance is an efficient means for cotransformation of nonselected genes.
2,555 citations
TL;DR: A model that postulates a “dynamic reciprocity” between the extracellular matrix (ECM) on the one hand and the cytoskeleton and the nuclear matrix on the other hand to alter the pattern of gene expression is presented.
1,427 citations
TL;DR: A DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone was microinjected into the pronuclei of fertilized mouse eggs, and seven mice developed that carried the fusion gene and six of these grew significantly larger than their littermates.
Abstract: A DNA fragment containing the promoter of the mouse metallothionein-I gene fused to the structural gene of rat growth hormone was microinjected into the pronuclei of fertilized mouse eggs. Of 21 mice that developed from these eggs, seven carried the fusion gene and six of these grew significantly larger than their littermates. Several of these transgenic mice had extraordinarily high levels of the fusion mRNA in their liver and growth hormone in their serum. This approach has implications for studying the biological effects of growth hormone, as a way to accelerate animal growth, as a model for gigantism, as a means of correcting genetic disease, and as a method of farming valuable gene products.
1,306 citations
TL;DR: A model is proposed to account for the synthesis and regulation of the two forms of inverts: the larger, regulated mRNA contains the initiation codon for the signal sequence required for synthesis of the secreted, glycosylated form of invertase; the smaller, constitutively transcribed mRNA begins within the coding region of the signal sequences, resulting in synthesis ofThe intracellular enzyme.
1,249 citations
TL;DR: Transcriptional control signals of a model eukaryotic protein-coding gene have been identified by a new procedure of in vitro mutagenesis that allows small clusters of nucleotide residues to be substituted in a site-directed manner without causing the addition or deletion of other sequences.
Abstract: Transcriptional control signals of a model eukaryotic protein-coding gene have been identified by a new procedure of in vitro mutagenesis. This method allows small clusters of nucleotide residues to be substituted in a site-directed manner without causing the addition or deletion of other sequences. Transcription assays of a systematic series of these clustered point mutants have led to the identification of three distinct control signals located within the 105-nucleotide residues immediately upstream from the point where transcription begins.
1,131 citations
TL;DR: These studies demonstrate the use ofvaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vacciniairus transcriptional regulatory sequences.
Abstract: Foreign DNA was inserted into two nonessential regions of the vaccinia virus genome by homologous recombination in cells infected with virus and transfected with plasmids containing the foreign DNA elements flanked by vaccinia virus DNA. Thymidine kinase-negative (TK-) recombinants were selected after inserting foreign DNA into the coding region of the TK gene of wild-type vaccinia virus; TK+ recombinants were selected after inserting the herpesvirus TK gene into TK- mutants of vaccinia virus. For TK+ expression, it was necessary to insert a 275-base-pair DNA fragment containing the initiation site and sequences upstream of an early vaccinia virus transcript next to the coding sequences of the herpesvirus gene. The unique ability of the herpesvirus TK to phosphorylate 125I-labeled deoxycytidine provided independent confirmation of gene expression. These studies demonstrate the use of vaccinia virus as a selectable cloning and expression vector, confirm the map location of the vaccinia virus TK gene, and provide initial information regarding the location of vaccinia virus transcriptional regulatory sequences.
719 citations
TL;DR: Results indicate that the transforming genes of these human tumor cell lines are the cellular homologs of two retroviral transforming genes.
Abstract: Blot hybridization analysis indicated that NIH 3T3 mouse bladder transformed by high molecular weight DNAs of a human bladder and a human lung carcinoma cell line contained new sequences homologous, respectively, to the transforming genes of Harvey (rasH) and Kirsten (rasK) sarcoma viruses. The unique ras sequences were present in multiple independent NIH cell lines transformed in both primary and secondary transfection assays and corresponded to ras sequences normally present in human DNAs. The ras gene product was expressed in NIH cells transformed by bladder carcinoma DNAs and in the human bladder carcinoma cell lines at levels 2- to 4-fold greater than the level observed in nontransformed NIH 3T3 cells. These results indicate that the transforming genes of these human tumor cell lines are the cellular homologs of two retroviral transforming genes.
718 citations
TL;DR: Retrovirus genomes introduced into mouse zygotes by microinjection of cloned DNA, or into morula stage pre-implantation mouse embryos by infection with Moloney murine leukaemia virus, became de novo methylated and were blocked in expression.
Abstract: Retrovirus genomes introduced into mouse zygotes by microinjection of cloned DNA, or into morula stage pre-implantation mouse embryos by infection with Moloney murine leukaemia virus (M-MuLV), became de novo methylated and were blocked in expression. No restriction of virus expression and no de novo methylation were observed when post-implantation mouse embryos were infected with virus. Efficient de novo methylation activity may be an important characteristic of gene regulation in early mouse embryos.
555 citations
TL;DR: The findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.
Abstract: The simian virus (SV40) 72-base pair (bp) tandem repeated sequences have recently been shown to function as activators or enhancers of early viral transcription. A recombinant viral genome was recently constructed by inserting 72-bp tandem repeats from the Moloney murine sarcoma virus (MSV) in place of the 72-bp repeats of SV40. Although this genome replicates in monkey kidney cells, its rate of large tumor antigen expression and replication is considerably slower than that of wild-type SV40. In mouse cells, however, equivalent levels of large tumor antigen appear to be expressed from both wild-type and recombinant genomes, suggesting a relationship between the level of enhancer activity and the host cell. To confirm this observation, we have applied a sensitive quantitative assay for gene expression based on the conversion of chloramphenicol to its acetylated forms. The gene encoding the enzymatic function chloramphenicol acetyltransferase was inserted into two vectors in which the enhancer sequences from SV40 or MSV were placed adjacent to the early SV40 promoter. The SV40 tandem repeats appear to activate gene expression to significantly higher levels in monkey kidney cells, but the MSV repeats are more active in two lines of mouse cells. These findings suggest that the tandem repeat elements may interact with host-specific molecules and, furthermore, may constitute one of the elements determining the host range of these eukaryotic viruses.
471 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 shown that the 200K protein is under separate developmental control during rat brain differentiation and that the time of its expression differs in different regions, suggesting that this protein probably has a more specialized role in neurofilament architecture and function than the other two triplet proteins.
Abstract: Axonal transport studies and biochemical fractionation have led to the concept that the three 'triplet' proteins [approximate molecular weights 200,000 (200K), 145,000 (145K) and 68,000 (68K)] are the essential components of mammalian neurofilaments. Using a correlated biochemical and immunological approach, we have now shown that the 200K protein is under separate developmental control during rat brain differentiation and that the time of its expression differs in different regions. We were unable to detect 200K protein by immunofluorescence or in total brain filament preparations from prenatal rat brain, although the 145K and 68K proteins are both present in an apparently identical distribution. During development, progressively more 145K- and 68K-positive neurofilamentous bundles can be stained with 200K antibodies, paralleling the increasing quantities of this protein detected biochemically in brain filament preparations. We conclude that 200K protein probably has a more specialized role in neurofilament architecture and function than the other two triplet proteins.
TL;DR: A fusion plasmid, pMK, containing the mouse metallothionein-I promoter/regulatory region joined to the structural gene of herpesvirus thymidine kinase was introduced into mice by microinjection into fertilized eggs followed by reinsertion of the eggs into foster mothers.
TL;DR: The results reported herein suggest that the heat-shock polypeptides controlled by the hin gene play an important role in cell growth at high temperature.
Abstract: When Escherichia coli cells grown at 30 degrees C are transferred to 42 degrees C, synthesis of several polypeptides is markedly and transiently induced. A temperature-sensitive nonsense mutant (tsn-K165) of E. coli K-12 is found to be defective in the induction of these proteins. mRNA for one major heat-shock polypeptide (groE protein) tested is induced in the wild type but not in the mutant upon temperature shift. Hence, the mutation defines a (regulatory) gene, designated hin (heat shock induction), whose product is required for active transcription of a set of heat-inducible operons in E. coli. The results reported herein suggest that the heat-shock polypeptides controlled by the hin gene play an important role in cell growth at high temperature. The possible involvement of the hin gene product in protection against thermal killing is also discussed.
TL;DR: The observation that the transcriptionally active ovalbumin gene is preferentially associated with the nuclear matrix may have significant implications for gene expression and the organization of nuclear DNA into supercoiled-loop domains.
TL;DR: Examination of the synthesis and accumulation of cytoskeletal proteins and of their temporal relation to morphological conversion indicates that the biosynthetic changes are very early events in the differentiation, and suggests strongly that they participate in the development of the adipocyte morphology.
TL;DR: Early region IA of human adenoviruses encodes a function required for normal induction of early viral genes and virus-induced cell transformation.
Abstract: Early region IA of human adenoviruses encodes a function required for normal induction of early viral genes and virus-induced cell transformation. The region is expressed at early times as two overlapping spliced mRNAs, 12S and 13S, which encode closely related proteins. To distinguish between the functions of these proteins, a single T → G transversion was constructed which prevents splicing of the 12S mRNA. This transversion, in the second base of the 12S mRNA intron, does not alter the protein encoded by the 13S mRNA due to degeneracy in the genetic code. Studies with this mutant demonstrated that only the 13S mRNA encodes the regulatory protein required for normal early gene expression.
TL;DR: It is reported here that the complete nucleotide sequence of a βE-gene revealed the expected GAG → AAG change in codon 26 but no other mutations, demonstrating a disturbance in the expression of the βE -gene attributable solely to the exon mutation—a novel mechanism for gene dysfunction.
Abstract: As is typical of all beta-thalassaemias, the erythroid cells of individuals with the variant haemoglobin E (alpha 2 beta 2(26Glu leads to Lys)) exhibit a quantitative deficiency in their content of beta-globin (in this case beta E-globin) and its messenger RNA2,3. To determine the molecular basis of this phenotype, we have investigated the structure and expression of cloned beta E-globin genes. We report here that the complete nucleotide sequence of a beta E-gene revealed the expected GAG leads to AAG change in codon 26 but no other mutations. Expression of beta E-globin genes introduced into HeLa cells revealed two abnormalities of RNA processing: slow excision of intervening sequence-1 (IVS-1) and alternative splicing into exon-1 at a cryptic donor sequence within which the codon 26 nucleotide substitution resides. These results demonstrate a disturbance in the expression of the beta E-gene attributable solely to the exon mutation-a novel mechanism for gene dysfunction.
TL;DR: The results show that the expression of certain genes may be inhibited by site-specific methylation of these sequences and suggest that methylation may play a direct role in the regulation of gene expression.
Abstract: The effect of DNA methylation on the expression of the hamster adenine phosphoribosyltransferase (aprt) gene in mouse cells has been examined. This gene was methylated in vitro at all of its C-C-G-G sites by using Hpa II methylase and was inserted into mouse Ltk- aprt- L cells by cotransformation, with the herpes virus thymidine kinase gene as a selectable vector. Whereas clones carrying unmethylated aprt sequences were found to have an aprt+ phenotype as shown by their ability to grow in azaserine-containing medium, almost all clones carrying methylated aprt sequences were shown to be phenotypically aprt-. Blot hybridization analysis demonstrated that both the methylated and unmethylated aprt sequences were integrated into the cellular genome to the same extent and that the in vitro modification was stably maintained in these cells for many generations. When clones containing methylated aprt genes were exposed to conditions that select for the expression of the aprt gene, a low frequency of reversion to the aprt+ phenotype was observed. In all of these clones, this reversion was accompanied by reorganization and undermethylation of the aprt sequences. These results show that the expression of certain genes may be inhibited by site-specific methylation of these sequences and suggest that methylation may play a direct role in the regulation of gene expression.
TL;DR: The results demonstrate that the galK gene is translationally coupled to the gene immediately preceding galK in the gal operon (that is, galT), and that the coupling effect depends primarily on the position at which upstream translation terminates relative to the gal K start site.
TL;DR: This study was able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants by staining with an antiserum raised against membranes of mutant CHO cells.
Abstract: Colchicine-resistant Chinese hamster ovary (CHO) cell mutants whose resistance results from reduced drug permeability have been isolated previously in our laboratories. This reduced permeability affects a wide range of unrelated drugs, resulting in the mutants displaying a multiple drug resistance phenotype. A 170,000-dalton cell surface glycoprotein (P-glycoprotein) was identified, and its expression appears to correlate with the degree of resistance. In this study we were able to confer the multiple drug resistance phenotype on sensitive mouse L cells by DNA-mediated gene transfer of DNA obtained from the colchicine-resistant mutants. P-glycoprotein was detected in plasma membranes of these DNA transformants by staining with an antiserum raised against membranes of mutant CHO cells. These results are consistent with a causal relationship between P-glycoprotein expression and the multiple drug resistance phenotype.
TL;DR: The rat genome contains two segments closely related to a rat α-tubulin mRNA that are a processed α- Tubulin pseudogene and a dispersed repetitive element inserted within it that possibly reflect a common RNA-mediated process of insertion.
Abstract: The rat genome contains two segments closely related to a rat alpha-tubulin mRNA Both have been cloned and complete nucleotide sequences are presented Analysis of the structure and sequence of one of these establishes it as a functional alpha-tubulin gene The second segment is a processed alpha-tubulin pseudogene Comparison of this pseudogene to the mRNA and gene coding for alpha-tubulin strongly suggests that a mature mRNA was involved in its origin Features of the pseudogene and a dispersed repetitive element inserted within it possibly reflect a common RNA-mediated process of insertion
TL;DR: A chlC-lac operon fusion was used to study regulatory mutations which affect nitrate reductase expression in Escherichia coli and a NarL- mutant apparently lacks a nitrate-specific positive regulatory component.
Abstract: I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli. A NarL- mutant apparently lacks a nitrate-specific positive regulatory component. Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions. These data are consistent with a two-step, positive control model for nitrate reductase regulation.
TL;DR: A gene related to the major heat shock-induced (hsp70) gene of Drosophila has been isolated from the sibling species D. melanogaster and D. simulans.
Abstract: A gene related to the major heat shock-induced (hsp70) gene of Drosophila has been isolated from the sibling species D. melanogaster and D. simulans. This heat shock-cognate (hsc70) gene is present at cytological locus 70C. The primary sequence of approximately one-third of the protein-coding region has been compared with that of the hsp70 gene; 72% homology of the base sequence and 74% homology of the deduced amino acid sequence was found. In the codon specifying amino acid 66, the hsc70 gene contains an insertion of 1.7 kilobases; the hsp70 genes contain no intervening sequences. The sequence at the 5' and 3' junctions of the insertion is similar to that found in many intervening sequences. cDNA extension experiments indicate that the hsc70 gene is transcribed at normal temperatures in adult flies and that transcription is not enhanced by heat treatment.
TL;DR: A recombinant plasmid containing the gene for bacterial β-galactosidase, situated close to the simian virus 40 early promoter, has been constructed and transfection of CHO, L, and COS-1 cells led to the expression and appearance of the enzyme.
Abstract: A recombinant plasmid containing the gene for bacterial β-galactosidase, situated close to the simian virus 40 early promoter, has been constructed. Transfection of CHO, L, and COS-1 cells with this plasmid led to the expression and appearance of the enzyme. Using this system, we have developed a series of promoter cloning vehicles capable of accepting promoter signals for animal genes.
TL;DR: The results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.
Abstract: Saccharomyces cerevisiae contains a family of genes related to the major heat shock-induced gene of Drosophila (hsp 70). Two members of the multigene family (YG100 and YG101) were isolated. The primary DNA sequences of more than one-half of the protein-encoding regions of YG100 and YG101 were determined and compared with the Drosophila hsp 70 gene sequence; the predicted amino acid sequences were 72 and 64% homologous to the sequence of the Drosophila hsp 70 protein, respectively. The predicted amino acid sequences of the yeast genes were 65% homologous. Our results demonstrate a striking sequence conservation of hsp 70-related sequences in evolution. Hybridization of the S. cerevisiae genes to total S. cerevisiae DNA indicated that the multigene family consists of approximately 10 members. Hybridization of labeled RNAs from heat-shocked and control cells suggested that, like transcription of the Drosophila hsp 70 gene, transcription of YG100 or a closely related gene is enhanced after heat shock. However, the amount of RNA sequences homologous to YG101 was reduced after heat shock. A multigene family related to the hsp 70 gene exists in Drosophila; transcription of some members is induced by heat shock, whereas transcription of others is not. Our results suggest that S. cerevisiae, like Drosophila, contains a multigene family of hsp 70-related sequences under complex transcriptional regulation and that the differential control, as well as the nucleotide sequence, has been highly conserved in evolution.
TL;DR: Merodiploid studies demonstrated that both the iclR and fadR genes regulate the glyoxylate shunt in a trans-dominant manner.
Abstract: The expression of the glyoxylate shunt enzymes is required for growth of Escherichia coli on acetate or fatty acids as a sole carbon source. The genes for the two unique enzymes of the glyoxylate shunt, aceA and aceB, are located at 90 min on the E. coli K-12 genetic map. Polar mutations in the aceB gene eliminate aceA gene function, suggesting that these genes constitute an operon and the direction of transcription is from aceB to aceA. Mu d (Ap lac) fusions with the aceA gene have been constructed to study the regulation of the ace operon. Expression of the ace operon is under the transcriptional control of two genes: the iclR gene, which maps near the ace operon, and the fadR gene, which maps at 25 min, and is also involved in the regulation of the fatty acid degradation (fad) regulon. Merodiploid studies demonstrated that both the iclR and fadR genes regulate the glyoxylate shunt in a trans-dominant manner.
TL;DR: It is argued that the activation, either by the E1A protein or the herpesvirus immediately early protein, most likely occurs indirectly through interaction with a cellular protein rather than by a direct recognition of regulatory sequences at the adenovirus promoters.
Abstract: Adenovirus mutants carrying a defective E1A gene, such as dl312, are unable to express any of the early viral genes upon infection of HeLa cells. However, efficient expression of the other early adenovirus genes was obtained when dl312-infected HeLa cells were coinfected with pseudorabies virus, a herpesvirus. By employing a temperature-sensitive pseudorabies mutant (tsG1) it was demonstrated that the herpesvirus function responsible for the induction of adenovirus transcription was the immediate early gene, a gene required for the activation of herpesvirus early gene expression and the maintenance of early and late herpesvirus transcription. Specifically, HeLa cells coinfected with dl312 and tsG1, when shifted to the nonpermissive temperature, lost their capacity to express the early adenovirus genes. Furthermore, activation of early adenovirus gene expression in herpesvirus coinfection occurred earlier and at a higher level than in wild-type adenovirus infection. Therefore, the herpesvirus immediate early protein not only activates the early adenovirus transcription units but apparently does so more efficiently than the adenovirus E1A gene product. Because of this fact, we argue that the activation, either by the E1A protein or the herpesvirus immediately early protein, most likely occurs indirectly through interaction with a cellular protein rather than by a direct recognition of regulatory sequences at the adenovirus promoters.
TL;DR: The herpes simplex virus genome consists of at least three groups of genes--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion.
Abstract: The herpes simplex virus genome consists of at least three groups of genes--alpha, beta, and gamma--whose expression is coordinately regulated and sequentially ordered in a cascade fashion We have established that the elements involved in regulation of alpha genes are a sequence that promotes gene expression and a sequence that confers alpha regulation on the gene by responding to trans-acting regulatory signals The domains of these sequences were mapped by determining the regulation of thymidine kinase (TK) in L cells converted to TK+ phenotype by chimeric TK indicator genes The chimeric genes were constructed from appropriate portions of the TK gene fused to donor sequences derived from the 5' nontranscribed and nontranslated leader portions of the viral alpha gene 4 The results were as follows (i) The natural beta TK indicator extending 5' up to -80 and the chimeric alpha TK extending 5' up to -110 both converted cells to TK+ phenotype but were not regulated (ii) A segment of the regulator region of the alpha gene 4, extending 5' from position -110, confers inducible alpha-type regulation when fused to the nonregulated but expressible beta TK indicator described above (iii) The extent of gene induction appears to hinge on the size of the regulatory region inserted into the chimeric gene and correlates with the presence of repeated consensus sequences and G+C-rich inverted repeats in the regulatory region of the alpha gene 4 and other alpha genes
TL;DR: Compared the function of the human alpha-, beta- and delta-globin genes using various plasmid expression vectors derived from pBR322, the activity of the beta- globin gene was approximately 50 times greater than that of the delta-Globin gene, approximating the ratio of beta and delta mRNA observed in normal human bone marrow cells.
TL;DR: Following exposure of the pre-B lymphoma ABLS 8.1 to 5-AC, derived cloned cell lines which possess macrophage-like characteristics not expressed by ABLS8.1 are derived and remain stable after 4 months of continuous culture.
Abstract: Variation in the degree of methylation of DNA seems to be one mode of regulating gene expression in eukaryotic cells1–7. The relationship between DNA demethylation and gene activation observed in globin4–6 and viral2 genes, together with evidence that alterations in the degree of DNA methylation of a gene are heritable8, although not with 100% fidelity9, have suggested that this may be a mechanism of control of differentiation. Futhermore, exposure to the demethylating drug 5-azacytidine (5-AC) causes differentiation of 3T3 cells into striated muscle cells, chondrocytes and adipocytes7. Subsequent studies have shown that these effects are due to DNA demethylation10. In view of these observations, we have now attempted to modify several continuous B-cell lines with 5-AC. Following exposure of the pre-B lymphoma ABLS 8.1 to 5-AC, we have derived cloned cell lines which possess macrophage-like characteristics not expressed by ABLS 8.1. Similar macrophage-like cell lines were obtained in two independent experiments; they have been re-cloned and remain stable after 4 months of continuous culture.