Showing papers on "Gene expression published in 1982"

Activation of the T24 bladder carcinoma transforming gene is linked to a single amino acid change

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.

onc gene amplification in promyelocytic leukaemia cell line HL-60 and primary leukaemic cells of the same patient

Abstract: Cellular onc genes are a group of evolutionarily conserved sequences which are homologous to the transforming genes (v-onc) of oncogenic retroviruses1. Their function in normal cells is not yet known, but the sequence homology between viral and cellular onc genes is consistent with the idea that neoplastic transformation may, in some cases, be due to increased levels of cellular onc gene expression. The cellular homologue, c-myc, of the transforming gene of avian myelocytomatosis virus (MC29)1,2 is involved in the pathogenesis of chicken B-cell lymphomas induced by the non-acute leukosis virus (RAV-2)3–6, and in these tumours, c-myc expression is enhanced by the nearby integration of the RAV-2 terminal repeat region3–6. Transcripts from the c-myc gene are detectable in a variety of human cells7,8, and increased levels of myc mRNA have been occasionally detected in some neoplastic tissues7,8. The highest levels have been detected in the cell line HL-60 (ref. 8) derived from neoplastic cells from a patient with acute promyelocytic leukaemia (APL)9. We have now investigated whether any structural alteration at the genomic level could account for the increased expression of c-myc in HL-60 and report here that the c-myc gene is stably amplified in HL-60 DNA. Amplification was also detected in primary uncultured leukaemic cells from the same individual, suggesting that the c-myc amplification may have been involved in the leukaemic transformation in this case.

Isolation and Preliminary Characterization of a Human Transforming Gene from T24 Bladder-Carcinoma 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.

DNA strand breaks and ADP-ribosyl transferase activation during cell differentiation.

Abstract: The nuclear enzyme ADP-ribosyl transferase (ADPRT) catalyses the formation of poly(ADP-ribose)-modified chromatin proteins from NAD+ (refs 1–5) and is entirely dependent on DNA6 containing nicks7–11. Nuclear ADPRT activity is required for efficient DNA excision repair12,13, probably because it regulates DNA ligase activity14. Indirect evidence has suggested that ADPRT activity may also be involved in control of gene expression and cell differentiation15–21. We report here an obligatory involvement of ADPRT activity in the differentiation of muscle cells. Inhibitors of ADPRT activity reversibly inhibit both fusion of myoblasts to form multi-nucleate muscle fibres and the differentiation-specific increase in creatine phos-phokinase (CPK) activity. These two markers of differentiation can also be reversibly inhibited by depriving the cells of nicotinamide and thus lowering their cellular NAD content. Specific gene expression sometimes requires gene rearrangements or DNA transposition; this implies that DNA strand-breaking and rejoining might be involved in gene expression. We also describe the appearance during cytodifferentiation of single-strand DNA breaks which are not due to a general deficiency in DNA repair.

Gene expression in Streptomyces : Construction and application of promoter-probe plasmid vectors in Streptomyces lividans

Abstract: Promoter-probe plasmid vectors were constructed for Streptomyces lividans using expression of the Escherichia coli chloramphenicol acetyltransferase gene as an indicator of promoter activity. These vectors have been used to isolate and to study the activity of DNA sequences that contain transcriptional control signals from Streptomyces, Bacillus licheniformis, E. coli, and Serratia marcescens. Studies of these promoter regions in heterospecific hosts indicate that genus or species-specific factors may present barriers to the expression of bacterial genetic material in certain heterologous cellular environments. While promoter regions isolated from E. coli, S. marcescens and B. licheniformis all appear to be recognized by the RNA polymerase of S. lividans, the Streptomyces transcriptional control signals isolated do not appear to function normally in E. coli.

Resolving the functions of overlapping viral genes by site-specific mutagenesis at a mRNA splice site.

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.

Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog

Abstract: The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.

Isolation of cloned cDNAs to auxin-responsive poly(A)+RNAs of elongating soybean hypocotyl

Abstract: Auxin-responsive cDNA clones have been isolated from a cDNA library prepared from elongating soybean hypocotyl poly(A)(+)RNA. The expression of two such sequences has been assessed by RNA blot hybridization analyses during normal developmental transitions in the soybean hypocotyl and during incubation of sections excised from the region of cell elongation. The concentrations of these poly(A)(+)RNAs are higher in the elongating zone than in the apical and mature zones of the hypocotyl. Both poly(A)(+)RNAs are depleted during incubation of the sections in the absence of auxin. The loss of one of these sequences (pJCW1) is prevented by the addition of auxin to the incubation medium while the other sequence (pJCW2) increases above the initial level in the presence of auxin. The addition of auxin to auxin-depleted tissue in which the sequences are depleted results in rapid accumulation of these poly(A)(+)RNAs; pJCW1 accumulates to the control level while pJCW2 increases well above the control level. These data along with others [Baulcombe, D. C. & Key, J. L. (1980) J. Biol. Chem. 255, 8907-8913] demonstrate directly a highly selective effect of auxin on the expression of a small number of mRNAs in tissues undergoing both cell elongation and cell division in response to auxin. Although the data are suggestive of a close association betwen auxin action and altered gene expression, a causal relationship is not established. It seems highly unlikely, however, that such specific effects of auxin on gene expression are unimportant in auxin physiology.

Regulatory sites for his3 gene expression in yeast

Abstract: The expression levels of many genes are regulated in response to particular environmental or developmental cues. A simple regulated gene can conceptually be divided into three elements: structural components that encode the gene product, promoter elements that are essential for gene expression, and regulatory elements responsible for changing the level of expression after a specific stimulus1,2. When cells of the yeast Saccharomyces cerevisiae are subjected to amino acid starvation, his3 and many other amino acid biosynthetic genes are expressed threefold above the basal level3,4. Previously, I isolated mutations mapping outside the structural gene that severely reduce or eliminate his3 expression5,6. These mutations define two distinct his3 promoter elements located 115–155 and 32–52 base pairs (bp) from the site of transcriptional initiation. Here I describe 18 small deletion mutations, some of which express his3 at the basal level but are unable to increase the level of expression in the appropriate physiological conditions. These define two regulatory sites located 32–41 and 80–100 bp upstream from the site of transcriptional initiation, and they strongly suggest that these regions are necessary for the positive regulation of his3 expression. I consider the results in terms of models for the general control of amino acid biosynthesis.

Activation of early adenovirus transcription by the herpesvirus immediate early gene: evidence for a common cellular control factor

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.

Differentiation between alpha promoter and regulator regions of herpes simplex virus 1: the functional domains and sequence of a movable alpha regulator

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

Derivation of macrophage-like lines from the pre-B lymphoma ABLS 8.1 using 5-azacytidine

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.

A gene deletion ending at the midpoint of a repetitive DNA sequence in one form of hereditary persistence of fetal haemoglobin

Abstract: The form of hereditary persistence of fetal haemoglobin (HPFH) that commonly occurs in black populations is an inherited disorder of haemoglobin synthesis characterized by a uniformly high level of fetal haemoglobin (HbF) synthesis in all the erythroid cells of affected adult individuals1,2. The precise molecular basis of the HPFH phenotype remains unknown, but is of great interest because of the knowledge that could be gained, through its understanding, of the mechanisms that regulate the expression of globin genes during development. The most common form of HPFH in black populations is associated with an extensive deletion that includes the normal adult (δ and β) globin genes and adjacent flanking DNA3–8. To investigate this disorder in more detail, we have cloned the DNA encompassing the region of the gene deletion in a case of HPFH and have determined the nucleotide sequence across the 5′ end point of the deletion within the non-α-globin gene complex. We report here that this end point maps at the midpoint of a member of the ‘AluI’ family of repetitive sequences located approximately 4 kilobases (kb) to the 5′ side of the δ-globin gene. Such repetitive sequences may be ‘hot spots’ of recombination, and are possibly involved in regulating gene expression.

Control of dna sequence transcription

Abstract: The transcription of DNA sequences in living cells is subjected to external regulation by incorporation of promoter/regulator DNA sequences responsive to metals and/or steroids. More particularly, regulation of the transcription of selected exogenous DNA sequences incorporated into eukaryotic host cells is facilitated by operative association (e.g., fusion) of the selected sequence to a promoter/regulator DNA sequence which is positively or negatively responsive to environmental variation in the concentration of heavy metal ions and/or steroid hormones. As an example, a structural gene for thymidine kinase from herpes simplex virus, fused to the promoter/regulator DNA sequence of a mouse metallothionein-I gene and incorporated on a suitable DNA plasmid vector, is introduced into mouse embryos and stably incorporated therein. Gene expression in differentiated cells of adult mice resulting from the embryos is subsequently regulatable by administration of heavy metals such as cadmium or steroid hormones such as the glucocorticoid, dexamethasone.

Posttranscriptional control of bacteriophage lambda gene expression from a site distal to the gene.

Abstract: The bacteriophage lambda int gene product, integrase, recombines the phage DNA with the host DNA at specific sites on each to accomplish lysogeny. The int gene is transcribed from two promoters, PL and PI, each regulated positively by lambda proteins. The expression of integrase is also controlled from a site, sib, in the b region of the phage genome. This is a unique regulatory site because it is located distal to the structural gene in relation to the promoters. The expression of int from the PL promoter is inhibited when sib is present. This effect appears to be specific for PL because sib does not cause inhibition of PI-dependent int synthesis. lambda mutants that contain alterations in the site have been isolated. Sequence analyses of the mutations reveal single base changes, spanning 37 base pairs (bp) in the b region, some 240 bp beyond the int gene. Another mutant, hef13, which has a phenotype similar to that of sib, introduces a nucleotide change within the same 37-bp region. The sib and hef mutations cluster within a region of dyad symmetry. Regulation of int synthesis by sib occurs after transcription of the int gene. There is no difference in the rate of PL-promoted int mRNA synthesis in either sib+ or sib- phage infections, yet int mRNA is less stable in the sib+ infection. Because RNase III host mutants are defective in sib regulation, processing of the PL mRNA at sib by this endoribonuclease may cause int mRNA decay and decrease int synthesis.

Transcription of the hepatitis B surface antigen gene in mouse cells transformed with cloned viral DNA.

Abstract: Mouse L cells transformed with recombinant plasmids carrying hepatitis B virus (HBV) DNA fragments were used to study the transcription of the viral surface antigen gene (gene S). An HBV-specific, polyadenylated, 2.3-kilobase RNA was mapped on the HBV genome. This RNA hybridized with approximately 75% of the genome and excluded the region of the HBV core antigen gene (gene C). The 2.3-kilobase RNA species was present only in cell lines that produced hepatitis B surface antigen. An HBV-specific 2.3-kilobase RNA was also detected in human hepatoma cell line PLC/PRF/5 which produced hepatitis B surface antigen. A study of gene S expression in the transformed mouse L cells allowed us to localize the regions of initiation and termination of gene S transcription. Our results strongly suggest that the 2.3-kilobase RNA molecule is the mRNA of the major polypeptide of the envelope, which carries the viral surface antigen determinants.

Calcitonin mRNA polymorphism: peptide switching associated with alternative RNA splicing events.

Abstract: Evidence is presented which supports a model proposing that differential RNA splicing events may be used in expression of genes of the endocrine system to generate alternative polypeptide hormones. We previously reported that variation in the expression of the gene encoding the small polypeptide hormone calcitonin is associated with the production of a new calcitonin-like or pseudo-calcitonin (psi Cal) mRNA. A plasmid containing psi Cal cDNA sequences has been constructed, and calcitonin genomic clones have been isolated. Hybridization analysis reveals that calcitonin and psi Cal sequences are chromosomally linked and are present in the same nuclear RNA transcripts. Both calcitonin and psi Cal mRNAs are functional and encode different polypeptide products. These data are compatible with the proposed model that alternative RNA splicing of the transcript(s) of the calcitonin gene ultimately results in the production of different polypeptide products.

Phytochrome control of plastid mRNA in mustard (Sinapis alba L.).

Abstract: The steady-state levels of plastid RNA sequences in dark-grown and light-grown mustard (Sinapis alba L.) seedlings have been compared. Total cellular RNAs were labeled in vitro with (32)P and hybridized to separated restriction fragments of plastid DNA. Cloned DNA fragments which encode the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxylase (dimerizing), EC 4.1.1.39] and a 35,000 plastid polypeptide were used as probes to assess the levels of these two plastid mRNAs. The 1.22-kilobase-pair mRNA for the 35,000 polypeptide is almost undetectable in dark-grown seedlings, but is a major plastid mRNA in light-grown seedlings. The hybridization analysis of RNA from seedlings which were irradiated with red and far-red light indicates that the level of this mRNA, but not of LS mRNA, is controlled by phytochrome.

Regulatory mutants of the maize Adh1 gene caused by DNA insertions

Abstract: Transposable elements in maize can induce changes in the timing of gene expression and the quantity of gene product1,2. The timing of expression of the bronze locus has been shown genetically to be affected by insertion of the transposable element Ds (dissociation)3. Molecular evidence has demonstrated that insertion of Ds at another locus, shrunken-1, disrupts the DNA sequence as expected of an insertion4,5. Here we report quantitative changes in gene expression resulting from the insertion of a 1.5-kilobase (kb) DNA element at an affected gene. The alcohol dehydrogenase-1 (Adh1) mutants described here, selected via allyl alcohol-resistant pollen grains6, were recovered from a genetic background containing Robertson's mutator (Mu)7. We suggest that the Mu genetic background caused the DNA insertion at Adh1, and that the insertion assumed some regulatory function in the control of Adh1 expression.