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Biomedical subjects

B H Howard

Publications and source records attributed to B H Howard.

At least 73 records · Page 4Linked to original sources
PubMed1985

Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

Acetyltransferases↗
PubMed1984

Rat growth hormone expression in cell hybrids.

Extinction of rat growth hormone (rGH) gene expression occurs in somatic cell hybrids of GH3 cells (rat pituitary adenoma) and L cells (mouse transformed fibroblast). We have suggested that this is due to transacting factors contributed directly or indirectly by the L cells. To study this in more detail, the cloned rGH gene was introduced into clones of these hybrid cells by microinjection or calcium phosphate transfection. GH peptides were detected 24 hr later in the microinjected cells by immunofluorescence. Production of rGH gene transcripts in the stably transformed hybrid cells was also detected by Northern hybridization. Although the overall patterns of transcripts differed from those present in GH3 cells, normal-sized rGH mRNA species were detected, whereas these had not been observed in previous studies in which the GH gene was transfected into L cells and other mouse fibroblasts. No more than one or two extra GH gene copies per cell were required to restore GH gene expression in the hybrids. Whereas GH gene expression in the hybrid cells transfected with the GH gene might reflect activation of the endogenous GH gene, this was not the case in at least one transfected hybrid cell line which had lost the endogenous gene. These results suggest: (i) the putative transactive repressors of GH gene expression in the hybrid cells do not recognize the transfected genes; and (ii) elements in the hybrid cells, unlike the case with L cells, allow for the production of normal-sized GH mRNA that can also produce GH peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Enhanced gene expression by the poly(dT-dG).poly(dC-dA) sequence.

The sequence poly(dT-dG).poly(dC-dA) (TG-element) is a ubiquitous component of eucaryotic genomes and has the potential to adopt a left-handed DNA conformation (Z-DNA). In this report, we have tested the hypothesis that the TG-element can modulate gene expression. Human genomic DNA fragments (1 to 1.5 kilobases) containing a (dT-dG)n.(dC-dA)n tract (30, 40, or 50 base pairs) or chemically synthesized (dT-dG)n.(dC-dA)n fragments (50 to 130 base pairs) were inserted in the pSV2-cat (simian virus 40 enhancer plus) or pA10-cat (enhancer minus) expression vector plasmid. These constructs were transfected into CV-1 cells or HeLa cells, and their transcription was monitored by assaying chloramphenicol acetyltransferase activity. The results showed that pSV2-cat with the TG-element and pA10-cat with the TG-element synthesized more chloramphenicol acetyltransferase activity (2 to 10 times, depending on the location of the TG-element) than did parental pSV2-cat and pA10-cat DNAs, respectively. Furthermore, the TG-element appeared to have characteristics similar to those of viral enhancers: (i) the TG-element enhanced transcription from a distance, (ii) its closer location to the promoter was more effective, and (iii) its orientation was not crucial. However, its enhancer-like activity was much weaker than that of the simian virus 40 enhancer, and, unlike many viral enhancers, it was equally active in monkey and in human cells. These results suggest that the TG-element may influence the expression of cellular genes.

Acetyltransferases↗
PubMed1983

Expression of recombinant plasmids in mammalian cells is enhanced by sodium butyrate.

We have studied the effects of sodium butyrate on DNA-mediated gene transfer in an effort to investigate interrelationships between chromatin structure and expression of recombinant plasmids. Our results demonstrate that butyrate affects the early stages of gene activity following DNA uptake at least two levels. First, the number of cells able to express foreign DNA increases from 10% to up to 40%. Second, there is an increase in enhancer-dependent transcription, approximately 30 fold in HeLa cells, involving the SV40 early promoter. Stable transformation efficiencies increase to 4% and 10% in HeLa S3 and monkey kidney CV-1 cells, respectively. Finally, expression of integrated recombinant plasmid genes is reinducible by a second treatment five weeks after initial exposure to this agent.

Butyrates↗
PubMed1983

High efficiency DNA-mediated transformation of primate cells.

Tissue culture cells from several mammalian species, including three primate lines, were transfected with recombinant vectors carrying Escherichia coli xanthine-guanine phosphoribosyltransferase or Tn5 aminoglycoside phosphotransferase dominant selectable markers. Human HeLa and SV40-transformed xeroderma pigmentosum cells exhibited stable transformation frequencies of at least 10(-3) (0.1 percent). CV-1, an African green monkey kidney cell line, could be stably transformed with the exceptionally high frequency of 6 X 10(-2) (6 percent).

Animals↗
PubMed1982

Efficient expression of Escherichia coli galactokinase gene in mammalian cells.

The Escherichia coli galactokinase gene (galK) was inserted into a modified early region transcription unit of simian virus 40 (SV40) contained on a bacterial plasmid. Introduction of this pSVK vector into monkey, mouse, and hamster cell lines by transfection resulted in efficient expression of the bacterial galK gene. This expression was shown to be dependent upon fusion of the galK gene to the early promoter of SV40 and did not appear to require SV40 splice signals. Moreover, expression in these cells could be obtained either transiently, 24--72 hr after transfection, or continuously, after stable transformation. In particular, pSVK-dependent galK expression was obtained in a hamster cell line genetically deficient in galactokinase activity. Expression of the bacterial enzyme was shown to complement the galactosemic defect of these cells, thereby allowing their selective survival and growth on galactose as the only carbon source. The ability to readily assay, select for, and potentially select against galK expression from pSVK and its derivatives should prove extremely useful in studying eukaryotic gene regulatory signals.

Animals↗
PubMed1982

The Rous sarcoma virus long terminal repeat is a strong promoter when introduced into a variety of eukaryotic cells by DNA-mediated transfection.

We characterized the transcriptional activity of the long terminal repeat (LTR) of Rous sarcoma virus by constructing a recombinant plasmid, pRSVcat, in which bacterial chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol 3-O-acetyltransferase, EC 2.3.1.28) coding sequences are placed under LTR control. We find that the LTR directs relatively high levels of CAT synthesis within 48 hr after calcium phosphate-mediated introduction of this plasmid into CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, or mouse NIH/3T3 cells. The level of CAT synthesis is 3-fold higher in CV-1 cells and up to 10-fold higher in HeLa and mouse NIH/3T3 cells than after transfection with a related vector, pSV2cat, carrying CAT sequences under control of the simian virus 40 early promoter. We have shown, by primer extension, that the amounts of CAT-specific mRNAs encoded by pRSVcat and pSV2cat correlate with the levels of CAT enzyme activity. By both S1 nuclease mapping and primer extension, we have demonstrated that the start site for RNA transcription within the LTR of pRSVcat corresponds to previous mapping data. We estimated transfection efficiencies by monitoring immunofluorescence induced by a rhodamine-labeled CAT antibody. Our results indicate that the Rous sarcoma virus LTR can direct synthesis of high levels of functional mRNA and has a wide expression range. The observed high transcriptional activity of the LTR is significant because it has been postulated that this LTR promotes activity of adjacent cellular oncogenes.

Acetyltransferases↗
PubMed1982

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Acetyltransferases↗
PubMed1981

Construction and characterization of SV40 recombinants with beta-globin cDNA substitutions in their early regions.

A cDNA segment coding for rabbit beta-globin has been inserted at different locations in the early region of simian virus 40 (SV40). The inserted sequences in these recombinants are transcribed from the SV40 early region promoter, and the primary transcripts are processed to mature mRNAs using viral intervening sequences and the early region polyadenylation site. After infection with various recombinants, beta-globin polypeptide is synthesized only when the beta-globin translation initiation codon is the first AUG in the messenger RNA sequence. When the early region transcripts contain the beta-globin cDNA sequence 3-proximal to the small t antigen coding sequence, beta-globin synthesis is not detectable. However, these recombinants produce small t antigen and abbreviated forms of large T antigen.

Animals↗
PubMed1979

Synthesis of rabbit beta-globin in cultured monkey kidney cells following infection with a SV40 beta-globin recombinant genome.

Rabbit beta-globin complementary DNA (cDNA) has been inserted into SV40 DNA in place of the gene coding for the virus' major capsid protein, VP1. The recombinant genome, SVGT5-RabetaG, multiplies efficiently in CV1 monkey kidney cell cultures and is transcribed to yield cytoplasmic, polyadenylated hybrid mRNAs containing the beta-globin coding sequence. Cells propagating SVGT5-RabetaG produce substantial quantities of rabbit beta-globin polypeptide.

Animals↗
PubMed1978

Decreased levels of collagen mRNA in rous sarcoma virus-transformed chick embryo fibroblasts.

We have found that double-stranded cDNA synthesized in extended reactions by avian myeloblastosis virus reverse transcriptase is suitable substrate for a variety of restriction endonucleases. Experiments in which rabbit reticulocyte mRNA was reverse-transcribed and restricted to generate beta-globin-specifihe nucleotide sequence of beta-globin mRNA. This method has been applied to study collagen mRNA synthesis in normal and Rous sarcoma virus (RSV)-transformed chick embryo fibroblasts. Characteristic sets of collagen cDNA restriction fragments were produced from RNA fractions rich in collagen message activity. These sets of cDNA fragments, generated by the restriction endonucleases Hae III and Hap II, provided a convenient marker for the presence of collagen mRNA sequences. Equal quantities of high molecular weight mRNA from chick embryo fibroblasts (CEF) and RSV-CEF were reverse-transcribed and the resulting cDNA was restricted. The relative yields of collagen cDNA fragments from such reactions strongly suggest that the decrease in functional collagen RNA following RSV-induced transformation of CEF represents a decrease in the copy number of collagen messenger sequences. The potential of this approach for the study of regulation in other systems is discussed.

Animals↗
PubMed1977

Levels of translatable mRNAs for cell surface protein, collagen precursors, and two membrane proteins are altered in Rous sarcoma virus-transformed chick embryo fibroblasts.

Transformation of chick embryo fibroblasts by Rous sarcoma virus results in decreased amounts of a major cell surface protein and of collagen. To determine the mechanism accounting for the decreased production of these proteins, we have measured the relative amounts of functional mRNAs for these and other transformation-sensitive proteins. Total cellular RNAs extracted from normal cells and from cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus were translated in a cell-free system derived from wheat germ. Analysis of the in vitro translation products of RNAs from normal and transformed chick embryo fibroblasts shows a 5-fold reduction in the translatable mRNA for cell surface protein and a 10-fold reduction in translatable mRNA for two collagen precursors. In addition, increases in functional mRNA are observed for myosin and for two membrane polypeptides with molecular weights of 95,000 and 78,000; the latter two proteins increase on transformation, but the increases are in large part secondary to the depletion of glucose from the medium of transformed cells. Our data suggest that some of the major cellular changes induced by oncogenic viruses are due to changes in the activity of specific cellular genes.

Actins↗
PubMed1977

Transcription in vitro of bacteriophage lambda 4S RNA: studies on termination and rho protein.

When bacteriophage lambdapga18 DNA is transcribed in a purified in vitro system by E. coli RNA polymerase (nucleoside triphosphate: RNA nucleotidyl-transferase, EC 2.7.7.6), several major transcripts are synthesized. We have investigated transcriptional termination of one of these transcripts, the 4S, or "oop" RNA. Analysis by two-dimensional "fingerprinting" of T1 oligonucleotides reveals that transcription of the 4S RNA terminates at a specific site on the lambdapga18 DNA template, t-L with an efficiency of approximately 80%, i.e. 20% of transcripts are extended into larger RNAs. Addition of the E. coli protein rho to our transcription reactions has two effects: a) the efficiency of termination at the t-L site is increased to 100%; b) the number of 4S transcripts synthesized is increased by greater than 5-fold. Rho appears to stimulate 4S RNA synthesis by facilitating more rapid release of RNA polymerase from the t-L' termination site.

Binding Sites↗
PubMed1976

ATPase activity required for termination of transcription by the Escherichia coli protein factor rho.

The relationship between the RNA-dependent beta-gamma ATPase in purified rho preparations and rho-mediated termination of transcription has been investigated. In a purified in vitro system, transcription from lambdagal DNA has been carried out using either ribonucleoside triphosphates (NTPs) or four ribonucleoside 5'-(beta-gamma-imino)triphosphates (NMP-P(NH)Ps) as RNA precursors. In the presence of NTPs, rho termination activity results in (a) the synthesis of rho-dependent transcripts which are of discrete size by polyacrylamide gel analysis and (b) a marked reduction by hybridization assay in RNA transcribed distal to the rho-sensitive termination site tR. In the presence of four NMP-P(NH)Ps, which are not substrates for the beta-gamma ATPase, termination by rho is completely abolished, whereas rho-independent termination occurs normally. Addition of ATP to transcription reactions containing four NMP-P(NH)Ps restores termination, ruling out the possibility that the termination activity of rho is nonspecifically inhibited by the analog preparations. We interpret our data as strongly suggesting that the RNA-dependent beta-gamma ATPase activity of rho is required for rho-mediated termination of transcription.

Adenosine Triphosphatases↗
PubMed1976

Desulfovibrio of the sheep rumen.

A sulfate-reducing bacterium has been isolated in pure culture from sheep rumen contents. Its properties agree in all respects tested with those ascribed to Desulfovibrio desulfuricans. The populations observed (about 10(8)/ml) are sufficient to account for published rates of ruminal sulfide production.

Animals↗
PubMed1975

The amino acid sequence of the alpha chain of the major haemoglobin of the rat (Rattus norvegicus).

1. A partial amino acid sequence of the alpha chain from the rat (Wistar, Rattus norvegicus) major haemoglobin is reported. The soluble tryptic peptides prepared from aminoethylated alpha-globin were separated by peptide 'mapping'. Sequencing of the tryptic peptides was carried out by the dansyl-Edman method and by the overlapping of smaller peptide fragments derived from secondary enzymic digestion. The insoluble 'core' peptides were further digested with chymotrypsin, thermolysin and pepsin to give smaller soluble peptides for sequencing. The tryptic peptides were ordered on the basis of their homology with the corresponding peptides of human alpha chain. 2. The proposed sequence is compared with that obtained by using an automated sequencer [Garrick et al. (1975) Biochem. J. 149, 245-258]. The differences in sequence resulting from the two methods are discussed. 3. It is suggested that the externally situated cysteine (residue 13) is responsible for the observed inhibition of crystallization of rat haemoglobin at alkaline pH. 4. Detailed evidence for the sequence has been deposited as Supplementary Publication SUP 50047 (9 pages) at the British Library (Linding Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from which copies can be obtained on the terms given in Biochem. J. (1975) 145, 5.

Amino Acid Sequence↗