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Biomedical subjects

G Zubay

Publications and source records attributed to G Zubay.

At least 37 records · Page 2Linked to original sources
PubMed1981

Negative regulation of beta and beta' synthesis by RNA polymerase.

The genes for the beta and beta' subunits of RNA polymerase, rpoB and rpoC, and the genes for the two ribosomal proteins, rplL and rplJ, are part of the beta operon. Although this operon and contains a single strong promoter, the genes of the operon are not always coordinately expressed in vivo. This has now been confirmed in vitro where the lack of coordinate expression has been shown to be correlated with the selective inhibition of rpoB and rpoC gene expression by RNa polymerase. Rifampicin, which stops the initiation of transcription, also relieves this autogenous inhibition of beta and beta' (beta beta') synthesis. The inhibitory action of RNA polymerase and its reversal by rifampicin most likely occurs at a posttranscriptional or translation level.

Bacteriophage lambda↗
PubMed1980

Cell-free coupled transcription-translation system for investigation of linear DNA segments.

Heretofore the DNA-directed coupled transcription-translation system, most useful in gene expression analysis, has been limited to the use of circular or long linear DNAs. Linear DNAs are degraded in this system by an exonucleolytic activity that can be eliminated by making the synthetic extracts from a suitable recB mutant of Escherichia coli. Using these extracts, we have examined the gene expression of a variety of linear DNAs. In particular, the complex pattern of expression of ribosomal protein genes and RNA polymerase genes in the rpoBC-rplLJ region has been analyzed by comparing the protein products obtained when using lambda rifd18 DNA with the product obtained when using the same DNA segmented with various restriction enzymes. The results obtained confirm the conclusions of others obtained by much more elaborate in vivo techniques. It seems highly likely that this cell-free system will have extensive applications in the area of analysis of gene expression.

Base Sequence↗
PubMed1980

The replication pattern of adenovirus DNA in vivo reproduced in vitro.

Temperature-sensitive mutants of adenovirus type 5 (H5ts125 and H5ts149), which are conditionally inhibited in the initiation of viral DNA synthesis, have been exploited to investigate the possibility of the initiation of replication in a cell-free system. Nuclei were isolated from human KB cells which had been infected with wild-type or mutant adenovirus. More than 90% of the DNA synthesis taking place in such nuclei was virus-specific and the pattern of drug inhibition suggested that the synthesis required DNA polymerase gamma. Nuclei prepared from cells infected with the H5ts125 temperature-sensitive mutant which have been shifted from 33 degrees C to 39.5 degrees C showed a pattern of synthesis in vitro which began at both ends of the viral genome and gradually spread through the rest of the molecule.

Adenoviruses, Human↗
PubMed1979

Differential sensitivity of gene expression in vitro to inhibitors of DNA gyrase.

We have used the antibiotics coumermycin A1, novobiocin, and oxolinic acid, which are specific inhibitors of DNA gyrase, to study the coupled transcription and translation of several bacterial and plasmid genes in a DNA-directed cell-free system. The expression of different genes is reduced to different extents by inhibition of DNA gyrase activity. Among the genes tested, the lac operon, an rRNA gene, and the colicin gene of colicin E1 plasmid were found to be most sensitive, while the trp operon and some other genes in colicin E1 plasmid were relatively unaffected by the inhibitors. These results, together with earlier work on the transcription of circular DNA templates, indicate that DNA supercoiling can significantly enhance transcription from certain promoters.

Anti-Bacterial Agents↗
PubMed1976

In vitro synthesis and repression of argininosuccinase in Escherichia coli K12; partial purification of the arginine repressor.

Phi80dargECBH DNA has been used to direct cell-free synthesis of argininosuccinase, the argH gene product in Escherichia coli K12. In vitro enzyme synthesis is sensitive to repression by partially purified preparations from an argR+ strain but not by corresponding preparations from an argR- strain. Using DNA-cellulose chromatography, approximately seventyfold purification of repressor has been obtained. The partially purified preparation represses argininosuccinase synthesis but has no effect on beta-galactosidase synthesis.

Arginine↗
PubMed1976

Synthesis of an R plasmid protein associated with tetracycline resistance is negatively regulated.

Synthesis of proteins encoded by the R222 plasmid was observed in a DNA-directed cell-free system and the products were compared to those plasmid proteins synthesized in Escherichia coli minicells. A greater number of plasmid-specified proteins was detected in the in vitro system than in the minicell, suggesting the presence of control factors for plasmid gene expression in the minicell. Synthesis of a newly detected plasmid protein (TET protein) is induced by tetracycline in minicells containing tetracycline-resistant plasmids, including R222, and this induced synthesis correlates with induced host resistance to the drug. This TET protein was synthesized in vitro from R222 DNA in the absence of tetracycline, indicating that no positive regulatory role for tetracycline is required for the protein's synthesis. TET proteon synthesis was inhibited in vitro when cell-free extracts prepared from cells containing the R222 plasmid were used.

Bacterial Proteins↗
PubMed1975

Effects of bacteriophage T4-induced modification of Escherichia coli RNA polymerase on gene expression in vitro.

After T4 bacteriophage infection of E. coli a complex series of events take place in the bacterium, including gross inhibition of host transcription and discrete changes in the classes of the genes of T4 that are transcribed. Accompanying these changes in the pattern of transcription one finds T4-induced changes in the RNA polymerase (EC 2.7.7.6; nucleosidetriphosphate:RNA nucleotidyltransferase). The effects of modified polymerase on transcription can be advantageously analyzed in a DNA-directed cell-free system for protein synthesis. In this system gene activity is measured indirectly by the amounts and types of proteins sythesized. In the DNA-directed cell-free system this modified polymerase, like normal polymerase, transcribes T4 DNA with a high efficiency but transcribes bacteriophage lambda and host DNA very poorly. Polymerase reconstruction experiments show that modification of the alpha subunit of the RNA polymerase is sufficient for inhibition of host transcription. Host transcription is also inhibited in vitro by T4 DNA. This latter type of inhibition is presumed to involve competition between host DNA and T4 DNA for some factor essential for transcription. The T4-modified polymerase transcribes from T4 DNA many of the same genes as normal unmodified polymerase; it also shows a capability for transcribing certain "non-early" T4 genes which is enhanced in the presence of protein-containing extracts from T4-infected cells.

Coliphages↗
PubMed1975

Effects of guanosine tetraphosphate on cell-free synthesis of Escherichia coli ribosomal RNA and other gene products.

A cell-free system derived from E. coli is described in which mature-sized 16S and 23S ribosomal RNAs (rRNA) are synthesized at a high relative rate, comprising 17-25% of the total transcription. The addition of guanosine tetraphosphate (ppGpp) to this system results in up to a 5-fold selective inhibition of rRNA accumulation. This effect is exerted at the level of synthesis rather than degradation. It is concluded that ppGpp, which is produced in large amounts by E. coli during amino-acid deprivation, could mediate the decrease in rRNA synthesis that accompanies such deprivation. The expression of other genes has also been investigated. No selective reduction of transfer RNA synthesis by ppGpp is observed. The trp and lac operons are found to be stimulated at the transcriptional level by the presence of this nucleotide. It is hypothesized that ppGpp interacts with the RNA polymerase in such a manner as to alter the affinity of the enzyme for promoters in an operon-specific fashion.

Cell-Free System↗
PubMed1974

Effects of guanosine tetraphosphate, guanosine pentaphosphate, and beta-gamma methylenyl-guanosine pentaphosphate on gene expression of Escherichia coli in vitro.

The effects of guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), both produced by E. coli, were measured on the activities of several genes in a cell-free system. Gene activity is measured as gene-directed synthesis of biochemically competent protein or transfer RNA. Both ppGpp and pppGpp stimulated the activities of the ara, lac, and trp operons and inhibited the arg operon. Production of transfer-RNA(Tyr) was unaffected by moderate levels of either ppGpp or pppGpp and only slightly inhibited at higher levels of ppGpp. Since the cell-free reaction mixtures hydrolyze pppGpp to ppGpp, we performed similar studies with a hydrolysis-resistant analog of pppGpp, the beta-gamma methylenyl derivative (pcppGpp). In general, pcppGpp shows the same inhibitory potency as pppGpp for the arg operon, but lacks the stimulatory effects on the ara, lac, and trp operons. This result suggests that the stimulation of these gene activities is specific for ppGpp.Under similar conditions, pppGpp and ppGpp show a slight inhibitory effect on the messenger-directed synthesis of beta-galactosidase and no effect on the messenger-directed synthesis of MS2 viral-coat protein. These observations, together with the fact that in the same system these nucleotides affect coupled transcription and translation, lead us to surmise that the activities of pppGpp and ppGpp are exerted at the level of RNA polymerase activity.

Acetamides↗
PubMed1974

Prolonged transcription in a cell-free system involving nuclei and cytoplasm.

A cell-free system for nuclear-directed transcription has been developed that gives prolonged synthesis in the presence of cytoplasm. The nuclear and cytoplasmic components have been prepared from Krebs II ascites tumor cells for most experiments but further observations indicate that components prepared from other cell types may be used. After an initial 5- to 10-min period of relatively rapid RNA synthesis a linear rate ensues for 2-3 hr. In the absence of cytoplasm no net RNA synthesis occurs after the initial 10-min period. Experiments with alpha-amanitin suggest that about half of the cell-free synthesized RNA is made by RNA polymerase II, the enzyme believed to be responsible for messenger synthesis in vivo.The conditions used for RNA synthesis were derived from conditions found to be optimal for protein synthesis that proceeds linearly for 2-3 hr. It has not yet been possible to demonstrate the synthesis of protein from cell-free synthesized RNA in this system. A major problem here is that isolated nuclei, even when carefully washed, contain a great deal of translatable RNA.

Animals↗