Biomedical subjects

D Ang

Publications and source records attributed to D Ang.

23 records · Page 2Linked to original sources

PubMed1988

Initiation of lambda DNA replication reconstituted with purified lambda and Escherichia coli replication proteins.

Using highly purified bacteriophage lambda and E. coli replication proteins, we were able to reconstitute an in vitro system capable of replication ori lambda-containing plasmid DNA. The addition of a new E. coli factor, the grpE gene product, to this replication system reduced the level of dnaK protein required for efficient DNA synthesis by at least 10-fold, and also allowed the isolation of a stable DNA replication intermediate. Based on all available information, we propose a molecular mechanism for the action of the dnaK and grpE proteins during the prepriming reaction leading to lambda DNA synthesis.

Bacterial Proteins↗

PubMed1988

Sequence analysis and transcriptional regulation of the Escherichia coli grpE gene, encoding a heat shock protein.

We have sequenced the Escherichia coli grpE gene and shown that it encodes a 197-amino acid residue protein of 21,668-Mr. The predicted N-terminal amino acid sequence, as well as the overall amino acid composition agree well with that of the purified protein. From Northern analysis, we have shown that transcription of the grpE gene is under heat shock regulation, i.e., there is a rapid and transient increase in the rate of synthesis of grpE mRNA upon a shift-up in temperature. Forty-six bases upstream of the structural gene is a sequence closely related to the consensus heat shock promoter identified by Cowing et al. [Proc. Natl. Acad. Sci. U.S.A, 82, 2679-2683]. We have shown by S1 mapping and RNA sequencing that this is indeed the promoter for the grpE mRNA. It appears that all discernable transcription initiates only from this promoter, even under non-heat shock conditions.

Amino Acid Sequence↗

PubMed1987

The grpE protein of Escherichia coli. Purification and properties.

The grpE gene of Escherichia coli was first identified because a mutation in it, grpE280, prevented bacteriophage lambda DNA replication in vivo. Subsequent work resulted in the identification of the grpE protein in two-dimensional gels and its classification as a heat shock protein. Here we report the purification of the grpE protein. We show that overproduction of grpE occurs in dnaK 103 bacteria which do not produce a functional Mr 72,000 dnaK protein. The grpE protein was purified from this strain primarily by its specific retention on a dnaK affinity column. The interaction between these two proteins, which is stable in the presence of 2 M KCl, allowed other proteins to be washed from this column. grpE was then eluted by ATP, which disrupts the interaction. During purification, grpE activity was monitored by its ability to complement an in vitro lambda dv DNA replication system dependent on the lambda O and lambda P proteins. The effect of ATP on the dnaK-grpE complex was also observed during sedimentation of the two proteins in glycerol gradients. Purified grpE protein has a Mr of approximately 23,000 under both denaturing and native conditions, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation, respectively. However, in the presence of dnaK under native conditions, grpE cosediments with dnaK. When ATP is added to the gradient, the complex is disrupted, and the two proteins sediment independently as monomers.

Adenosine Triphosphate↗

PubMed1986

Escherichia coli grpE gene codes for heat shock protein B25.3, essential for both lambda DNA replication at all temperatures and host growth at high temperature.

We have identified the grpE gene product as the B25.3 heat shock protein of Escherichia coli on the following evidence: (i) a protein similar in size and isoelectric point to B25.3 was induced after infection of UV-irradiated bacteria by lambda grpE+ transducing phage, (ii) mutant phage lambda grpE40, isolated by its inability to propagate on grpE280 bacteria, failed to induce the synthesis of the B25.3 protein, and (iii) lambda grpE+ revertants, derived from phage grpE40 as able to propagate on grpE280 bacteria, simultaneously recovered the ability to induce synthesis of the B25.3 protein. In addition, we show that E. coli bacteria carrying the grpE280 mutation are temperature sensitive for bacterial growth at 43.5 degrees C. Through transductional analysis and temperature reversion experiments, it was demonstrated that the grpE280 mutation is responsible for both the inability of lambda to replicate at any temperature tested and the lack of colony formation at high temperature. At the nonpermissive temperature the rates of synthesis of DNA and RNA were reduced in grpE280 bacteria.

Bacterial Proteins↗

PubMed1985

Mobile elements bounded by C4A4 telomeric repeats in Oxytricha fallax.

A novel family of micronuclear elements termed telomere-bearing elements (TBEs) is described. All 1900 family members are eliminated during macronuclear development. We conclude that they are transposons, first because the members are moderately conserved in sequence and probably dispersed in the genome. Second, in two cases, sequence comparison of the termini and flanks of the element with the corresponding empty site indicate that elements cause 3 bp target duplications (AAT) upon insertion; the 3 bp are part of the 5 bp target sequence, AATGA. Lastly, both elements carry 77 or 78 bp inverted terminal repeats. The tip of each inverted terminal repeat is the 17 bp telomere-like sequence 5' C1A4C4A4C4. At least half of the elements have these 17 bp or an extremely similar sequence. One possible pathway for transposition into new micronuclear sites starts in the developing macronucleus with excision to create a free linear form to which telomeres are added, followed by a low frequency of movement to the micronucleus, and insertion into the germ-line micronuclear DNA.

Animals↗