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Biomedical subjects

H Aviv

Publications and source records attributed to H Aviv.

At least 37 records · Page 2Linked to original sources
PubMed1982

Expression of a cloned bovine growth hormone gene in Escherichia coli minicells.

The synthesis of polypeptides in Escherichia coli minicells, directed by a pBR322 plasmid and its derivative-carrying bovine growth hormone cDNA insert, was studied. Two polypeptides coded by the ampicillin-resistance (Apr) gene (32 000 and 28 000 daltons) and a tetracycline-resistance (Tcr) polypeptide (36 000 daltons) were identified by insertion inactivation. Two additional polypeptides of 37 000 and 34 000 daltons of as yet unknown function were detected in all extracts regardless of the presence of the Apr or Tcr genes in the plasmid. The pBR322-BGH recombinant plasmid coded for several novel polypeptides, among them one of 46 000 daltons, presumably a fused product of the BGH and beta-lactamase genes. This protein, however, was not secreted into the periplasmic space of the cells as was the beta-lactamase.

Animals↗
PubMed1981

Large globin RNA molecules and their processing.

RNA containing beta-globin message sequences larger than 2000 nucleotides could be detected in nuclei of murine erythroid cells using cloned beta-globin cDNA. Under steady-state conditions, when nuclear RNA was separated on denaturing agarose gels and covalently bound to diazobenzyloxymethyl-paper, a 4200-nucleotide and a approximately equal to 3500-nucleotide band could be seen. The presence of these large molecules could also be visualized under the electron microscope after hybridization to a beta-globin genomic DNA fragment. We suggest that these molecules are precursors to mature mRNAs. In addition to these large molecules, a series of molecules smaller than 2000 nucleotides were seen. These are postulated to be processing intermediates in the maturation of beta-globin mRNA.

Animals↗
PubMed1981

Cloning of bovine growth hormone gene and its expression in bacteria.

A hybrid plasmid was constructed containing beta-lactamase gene of plasmid pBR322 and cloned coding sequences of bovine growth hormone (BGH). The constructed plasmid contains all DNA sequences required to encode BGH, and when used as a hybridization probe it detects one growth hormone gene in the bovine genome. The cloned DNA sequences are inserted into the beta-lactamase gene in the correct reading frame for BGH synthesis. The hybrid gene is expressed in bacteria and the product, a fused beta-lactamase-bovine growth hormone protein, is specifically immunoprecipitated with anti-serum to BGH. Unlike beta-lactamase, very little growth hormone containing sequences can be detected in the periplasmic space.

Amino Acid Sequence↗
PubMed1981

Modulation of globin gene expression by a tumor promoter during induced differentiation of Friend erythroleukemic cells.

Friend erythroleukemic cells were induced to differentiate by dimethylsulfoxide (Me2SO) in the absence or presence of the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate. The effects of the latter on the molecular parameters related to globin mRNA metabolism were examined. When differentiation was scored by benzidine staining, it had an inhibitory effect on Me2SO-treated cells. On the other hand, when differentiation was followed by determination of globin mRNA accumulation, it had a pleiotropic effect on Me2SO-treated cells. At the early phase of differentiation (2--3 days) the rate of globin mRNA accumulation was higher in the promoter-treated cells than in the control. This unexpectedly high level of accumulation was followed by a sharp reduction and most of the globin RNA sequences disappeared at later stages of differentiation (days 4--5). The reduction can be related to the effect of the promoter on the stability of globin RNA in the cytoplasm which was reduced from a half-life of 16 h to that of 8 h only. Other parameters, such as the rate of globin mRNA synthesis and its capability to serve as a template for cell-free protein synthesis were not affected by treatment with the promoter throughout the differentiation process.

Animals↗
PubMed1980

Isolation of viable deletion mutants of Streptomyces actinophage (Pal 6) and their molecular characterization.

Deletion mutants of bacteriophage Pal 6 were isolated by successive treatments of either heat (60 degrees C) or pyrophosphate (10 mM). These mutants were characterized by restriction enzyme cleavage analysis. The pyrophosphate resistant clones lost the whole Eco R1 fragment in which the Sal I site is located, as well as an unrelated Hind III fragment. These results show that the region containing the Sal I site in the phage genome is not essential for phage viability. This single Sal I site is therefore suitable as a potential insertion site for DNA cloning. On the other hand, the heat resistant clones that were isolated and characterized do not appear to have detectable deletions as indicated by their Eco R1 DNA digestion pattern.

Bacteriophages↗
PubMed1980

Radioimmunoassay of gentamicin in Micromonospora medium extracts.

Medium extracts of Micromonospora adversely affected a radioimmunoassay which was used for the measurement of gentamicin in the medium. An overestmation or an underestimation resulted, as judged by the addition of extracts to a gentamicin standard sample.

Chromatography↗
PubMed1980

Gentamicin bioautography assay vs. the microbiological disk test.

The use of bioautography for quantitative measurement of gentamicin concentrations was compared with the disk test. Following chromatographic separation and bioautography, gentamicin produced inhibition zones, 2 approximately 7 times larger than the inhibition zones formed by the same amounts of gentamicin in the disk test. Bioautography, therefore, is a more sensitive assay method.

Bacillus subtilis↗
PubMed1979

Evaluation of several enrichment procedures for the isolation of recombinant plasmid DNA.

A number of methods for the selective enrichment of recombinant plasmids were examined; these include alkaline phosphatase treatment of the restricted pBR322 vector, as well as a combination of this and S1 nuclease treatment of the ligated mixture of pBR322 and pCR1 plasmids or S. griseus DNA followed by D-cycloserine treatment to enrich for cells carrying recombinant molecules. The relative efficiencies of these methods were compared.

Alkaline Phosphatase↗
PubMed1978

Methylation and capping of RNA polymerase II primary transcripts by HeLa nuclear homogenates.

HeLa nuclear homogenates incubated in vitro incorporate [beta-32P]ATP and S-[methyl-3H]-adenosylmeth-ionine ([3H]SAM) into blocked methylated 5' termini of newly synthesized RNA. Approximately 10% of the RNA chains initiated in vitro with [beta-32P]ATP are subsequently blocked by condensation of GMP to di- or triphosphate terminated RNA. The blocked termini can then be methylated by transfer of methyl groups from [3H]SAM to the 7 position of the guanosine and 2'-O position of the adenosine to form m7Gpp*pAm- capped terminus. In addition to conventional triphosphate caps, HeLa nuclear homogenates produce capping structures containing two phosphate residues in the pyrophosphate bridge. The two distinct cap forms were separated by DEAE-cellulose chromatography and analyzed. In contrast to triphosphate caps (m7GpppXm) in which X can be any one of the four nucleosides (G, A, C, or U), in diphosphate caps (m7GppXm), more than 95% of the penultimate nucleoside Xm is G. Incorporation of both [beta-32P]ATP and [3H]SAM into caps was markedly reduced by low concentrations of alpha-amanitin. However, an ammonium sulfate fraction of the nuclear homogenate can cap beta-32P-labeled RNA (pp*pA-RNA) to form m7Gpp*pA-RNA, in the presence of 0.5 microgram/mL of alpha-amanitin. Therefore, the nuclear capping enzyme is resistant to this drug. Our results indicate that RNA polymerase II primary transcripts are the substrate for the cellular capping enzyme and that the beta phosphate in the pyrophosphate bridge (m7GgammapbetapalphapXm) is derived from the 5' ends of the RNA chains.

Amanitins↗
PubMed1977

Initiation of RNA synthesis in isolated nuclei.

Ribonucleotide triphosphates, labeled at the beta position, were synthesized and used directly to quantify RNA chain initiation in nuclei isolated from Friend cells grown in tissue culture. At the optimal salt concentration, low-molecular-weight RNAs (4-5 S) synthesized by RNA polymerase III were the predominant species initiated. Less than 5% of the molecules were initiated by polymerase II. We calculate that 50-80% of the small RNA molecules synthesized in vitro were also initiated in vitro. Assuming that a substantial fraction of the nuclei were active in vitro, the number of 4-5 S RNA molecules initiated per nucleus was about 100 molecules/min.

Acetates↗
PubMed1977

Globin RNA precursor molecules: biosynthesis and process in erythroid cells.

Hybridization of labeled RNA with excess amounts of DNA complementary to globin mRNA, in conjunction with a pulse-chase technique, were used to investigate the biosynthetic pathway of globin mRNA in erythroid cells. Three species of molecules sharing common sequences with globin mRNA were detected in the nuclei of these cells, two of which are larger than the cytoplasmic globin mRNA. One species was approximately 7 times larger than globin mRNA ("27S"), and the other ("15S") was only about twice the size of cytoplasmic globin mRNA. The largest species lacked poly(A) sequences, while the others contained poly(A), After chase, the large RNA species gradually disappeared ( 1/2 = 5 min), while the cytoplasmic 10S species accumulated. From these results a model is proposed describing the biosynthetic pathway of globin RNA transcription: an early transcription product is the large molecule "27S" (approximately 5000 nucleotides long) which is then cleaved into a smaller species "15S" (approximately 1500 nucleotides). This intermediate precursor is then clipped, presumably at the 5' end, and finally converted to the exported "10S" molecule (approximately 750 nucleotides) which accumulates in the cytoplasm.

Cell Line↗
PubMed1976

Quantitation of labeled globin messenger RNA by hybridization with excess complementary DNA covalently bound to cellulose.

A method is described to quantitate labeled globin mRNA by hybridization with excess cDNA which was enzymatically polymerized on oligo(dT)-cellulose. In a large excess of cDNA-cellulose the rate of RNA hybridization was dependent on DNA concentration and not on RNA concentration. Nonhybridized RNA can be digested by RNase and washed from the cDNA which is covalently bound to cellulose. This enables the detection of labeled globin mRNA even when present in a porportion as low as 0.02-0.03% of the total RNA.

Animals↗
PubMed1976

Preferential synthesis of viral late RNA by nuclei isolated from SV40 lytically infected cells.

Nuclei from SV40-infected monkey cells were isolated late in lytic infection and their cell-free transcriptional activity was characterized. 3H-RNA synthesized in vitro was hybridized to excess quantities of separated SV40 DNA strands which were each covalently bound to Sepharose. It was found that 3-5% of the newly synthesized RNA is virus-specific and that the plus-strand DNA, coding for late RNA sequences, is transcribed at a rate about 15 times higher than that of the minus-strand DNA, which codes for early RNA sequences. This indicates that transcriptional control has a major role in determining the relative abundancy of early and late RNA classes in lytically infected cells.

Amanitins↗
PubMed1976

Biosynthesis and stability of globin mRNA in cultured erythroleukemic Friend cells.

Biosynthesis and stability of the mRNA population in DMSO-induced Friend erythroleukemic cells were studied after labeling the RNA with 3H-uridine and then chasing it with nonlabeled uridine. Globin RNA metabolism was studied by hybridization to excess complementary DNA convalently coupled to oligo(dT)-cellulose. After a labeling period of 120 min, 2-4% of the poly(A)-containing labeled RNA was in globin RNA; it decayed with a half-life of 16-17 hr. The rest of the poly(A)-containing RNA was composed to two kinetic populations: 85-90% decayed with a half-life of about 3 hr, while 10% decayed with a half-life of about 37 hr. The portion of globin RNA in labeled poly(A)-containing RNA behaved in an unexpected fashion during the chase period. During the initial chase period, the percentage of globin RNA increased rapidly, reaching a maximum of about 15% at 20 hr, but it subsequently declined gradually. Based on these findings, a model was built that describes the changes in the proportion of globin mRNA in poly(A)-containing RNA during continuous synthesis and after chase of the labeled RNA. It appears that if the parameters described remain constant during the maturation of erythroblasts, then this model would not account for the almost exclusive presence of globin RNA in the reticulocyte. By far the most effective way to achieve this high level of globin RNA is the destabilization of the mRNA population which is more stable than globin RNA, and not the stabilization of globin RNA itself.

Cell Line↗
PubMed1975

Purification of SV-40 messenger RNA by hybridization to SV-40 DNA covalently bound to Sepharose.

SV-40 DNA sheared form was coupled in a stable covalent bond to cyanogen bromide activated Sepharose. Under the conditions used at least 80% of the DNA was bound to Sepharose. The T 1/2 of hybridization of 0.5 mug/ml of SV-40 cRNA to SV-40 DNA-Sepharose was 1 hr. This rate of hybridization is sufficiently rapid to purify SV-40 sequences from solutions containing as little as 0.05-0.1 mug/ml. Nonspecific hybridization of RNA is in the range of 0.1-0.2% of the total input RNA. The DNA-Sepharose is fairly stable and can be reused several times to purify RNA. The SV-40 DNA-Sepharose was used to select large quantities of virus specific RNA from SV-40 infected BS-C-1 cells. The virus specific RNA when added to cell-free extracts from wheat germ was shown to direct the synthesis of the major viral structural protein VP-1.

Cells, Cultured↗