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Biomedical subjects

G Zubay

Publications and source records attributed to G Zubay.

At least 55 records · Page 3Linked to original sources
PubMed1973

Cell-free synthesis of rat growth hormone.

Growth hormone has been synthesized in a cell-free system derived from Krebs II ascites cells, under the direction of RNA prepared from rat pituitary tumor (GC) cells. Growth hormone synthesized in the cell-free system was identified by precipitation with antiserum against growth hormone developed in baboon, followed by electrophoretic analysis of the dissolved precipitate on sodium dodecyl sulfate-polyacrylamide gels. RNA from both the membrane and the post-membrane fractions of the cytoplasm of GC cells stimulated protein synthesis in the cell-free system, but only RNA from the membrane fraction was found to direct the synthesis of growth hormone.

Animals↗
PubMed1972

Detection and isolation of the repressor protein for the tryptophan operon of Escherichia coli.

DNA from a transducing bacteriophage carrying a fusion of the tryptophan and lactose operons of E. coli (lambdadtrp-lac) has been used to direct cell-free synthesis of beta-galactosidase (EC 3.2.1.23). Whereas normal lac operon (lambdadlac) DNA requires adenosine-3':5'-cyclic monophosphate (cAMP) for beta-galactosidase synthesis, trp-lac DNA is unaffected by cAMP. This difference in cAMP dependence verifies the presence of a cAMP-requiring promoter in the lac operon that has been removed from the trp-lac DNA. Synthesis with trp-lac DNA is controlled by the protein product of the tryptophan repressor gene (trpR). Synthesis in extracts of trpR(-) (repressor-negative) cells is progressively reduced by increased additions of extract from trpR(+) cells. No trpR(-) product repression is seen when beta-galactosidase synthesis is programmed by normal lac DNA. This highly sensitive and specific assay has facilitated quantitation and partial purification of the trp repressor.

Bacterial Proteins↗
PubMed1971

Purification and DNA-binding properties of the catabolite gene activator protein.

A protein required for the activation of the lac operon has been extensively purified and partly characterized. This protein, called CGA protein (catabolite gene activator protein, sometimes named CAP), is a dimer with subunits of 22,000 daltons. Purified CGA protein has a substantial affinity for DNA; this affinity is greatly strengthened by cAMP and strongly inhibited by cGMP. Other studies have shown that these cyclic nucleotides compete for a binding site on CGA protein. The opposing effects of the two cyclic compounds in DNA-CGA protein binding show a parallel behavior to their effects on the expression of the lac operon. Thus cAMP, in addition to CGA protein, is required for expression of the lac operon, whereas cGMP inhibits the expression. The obvious inference is that CGA protein activates the lac operon by binding to the DNA under the influence of cAMP. Thus, CGA protein seems to be a new type of regulatory protein: a DNA-binding activator.

Bacterial Proteins↗
PubMed1971

DNA-directed cell-free synthesis of biologically active transfer RNA: su + 3 tyrosyl-tRNA.

Biologically active su(+) (III) tyrosyl-tRNA has been synthesized in a DNA-directed cell-free system from Escerichia coli. Such a system should be most useful for studying the mechanism of tRNA synthesis. This tRNA is capable of suppressing amber mutations in the gene coding for beta-galactosidase (EC 3.2.1.23) and, therefore, must be capable of being charged and transferring amino acids. A 4-fold stimulation in the activity of the tRNA formed de novo is obtained with isopentenyl pyrophosphate, a compound involved in the post-transcriptional acylation of an adenine base adjacent to the anticodon. It has been suggested elsewhere that formation of RNA subject to stringent control may be inhibited by guanosine tetraphosphate (ppGpp). However, guanosine tetraphosphate did not affect the synthesis of su(+)III tyrosyl-tRNA, even though the synthesis of this tRNA is subject to stringent control.

Cell-Free System↗
PubMed1970

Mechanism of activation of catabolite-sensitive genes: a positive control system.

Catabolite repression is defined as the inhibition of enzyme induction by glucose or related substances. In the bacterium E. coli, the effect of glucose appears to be due to a lowering of the cyclic AMP level. A DNA-directed cell-free system for beta-galactosidase synthesis has served as a model system for studying the mechanism of action of cyclic AMP. Previously, it was reported that in this system cyclic AMP is required for normal initiation of mRNA synthesis. A protein factor which acts in conjunction with the cyclic AMP has been partially purified. This protein factor has a high affinity for cyclic AMP. These and other results presented herein lead us to the conclusion that cyclic AMP and a protein factor called the catabolite gene activator protein are part of a positive control system for activating catabolite-sensitive genes.

Adenine Nucleotides↗
PubMed1969

DNA-directed peptide synthesis. VI. Regulating the expression of the lac operon in a cell-free system.

The operon model was proposed by Jacob and Monod in 1961 to explain the regulation of enzyme synthesis in bacteria. The model requires two genetic elements: a locus i which directs the synthesis of a repressor, and a locus called the operon which contains an operator gene and associated structural genes. The repressor is believed to stop RNA and enzyme synthesis by combining with the operator site. The inhibiting effect of the repressor is reversed by an inducer which interacts directly with the repressor. A DNA-dependent cell-free system has been developed for studying the regulation of the operon containing the gene for beta-galactosidase enzyme. In this cell-free system, gene activity is indirectly measured by the synthesis of part of the enzyme. Qualitatively, the regulation of the operon in the cell-free system is similar to its regulation in intact cells. The cell-free system is used to advantage in experiments where it is desirable to use predetermined levels of repressor, operon, and inducer. Such quantitative studies suggest that two inducer molecules are most effective in removing the repressor from the gene.

Cell-Free System↗
PubMed1969

The stimulatory effect of cyclic adenosine 3'5'-monophosphate on DNA-directed synthesis of beta-galactosidase in a cell-free system.

A cell-free system allowing for synthesis of beta-galactosidase enzymatic activity has been developed. This system requires DNA containing the beta-galactosidase gene, a cell-free extract of Escherichia coli bacteria, and the low-molecular-weight components necessary for transcription of the DNA and translation of the resulting messenger RNA. Such a system is useful for studying enzyme synthesis, as well as its regulation. The gene for beta-galactosidase is part of the lac operon whose expression is under the control of the lac repressor. In whole cells the lac repressor inhibits almost all of the gene expression for beta-galactosidase. In the cell-free system, we had previously been able to repress about half the gene expression. Adding cyclic adenosine 3'5'-monophosphate to the cell-free system improved the yield of beta-galactosidase enzymatic activity by 8 to 30 times and the efficiency of repression from 50 to 95 per cent.

Adenine Nucleotides↗
PubMed1969

Characterization of a beta-galactosidase formed between a complementary protein and a peptide synthesized de novo.

In a cell-free system, phi80dlac can be transcribed, and the resulting ribonucleic acid can be translated to yield a product which interacts with an enzymatically inactive z protein to produce active enzyme. The inactive z protein is produced by Escherichia coli strain 21, which contains a deletion in the first part of the gene for beta-galactosidase and appears to exist as a dimer. The enzyme formed in the cell-free system appears to be composed of one strain 21 z protein dimer and one newly synthesized polypeptide chain with a molecular weight of about 3 x 10(4). The estimated size of this complementing segment is in good agreement with Ullmann, Jacob, and Monod's estimate of the size of the alpha region of beta-galactosidase. Using alpha fragments produced by autoclaving or guanidine treatments, we found that the active portion of alpha seems to be smaller than the full alpha region. We also found, using alpha produced by the autoclaving technique, that active dimer undergoes conversion to tetramer as the amount of alpha is increased. Evidently, the binding of alpha favors this conversion, but it is unlikely that the conversion of dimer to tetramer per se results in increased enzyme activity.

Bacterial Proteins↗