Biomedical subjects

D Drahos

Publications and source records attributed to D Drahos.

4 recordsLinked to original sources

PubMed1982

Synthesis of the nutL DNA segments and analysis of antitermination and termination functions in coliphage lambda.

The nut antiterminator sequence, when present between a promoter and a terminator, permits the N-mediated antitermination of transcription in phage lambda. The efficiency of nutL was determined by assaying the activity of gene galK placed on a plasmid downstream from the promoter, nutL and terminator modules. As a reference and an estimate of the plasmid copy number, we have used an improved and very reproducible assay for bla activity. Sequences consisting of the 17-bp nutL core flanked by two HindIII cohesive sites were synthesized by the phosphite coupling method, and cloned in proper orientation between the Pp promoter of pBR322 and lambda gene N followed by the tL1 terminator on a galK-expression plasmid. The antitermination efficiencies for two synthetic 17-bp nutL sequences, one wild type and one point mutant at the base of the nutL stem, are similar but substantially reduced in comparison with the native 25-bp nutL sequence cloned at the same site in the otherwise identical galK-expression plasmid. Multiple tandem insertions of the synthetic 17-bp nutL segment successively increase antitermination efficiency, but also to levels below those of comparable plasmids carrying multiple copies of the native 25-bp nutL sequence. Thus, several specific base pairs in the flanking sequences appear to be important for the efficient nut function. In an inverted orientation the 17-bp nutL sequence has lost its antitermination function. It also lost the termination activity exhibited by inversion of the longer 25-bp and 74-bp native nutL sequences.

Bacteriophage lambda↗

PubMed1982

Sequence changes in coliphage lambda mutants affecting the nutL antitermination site and termination by tL1 and tL2.

The 17-bp sequence designated nutL is required for the N-mediated antitermination of transcription in the major leftward operon of coliphage lambda. The single-stranded sequence can be folded into a hairpin structure. Ten independently isolated spontaneous lambda nutL- mutants have changes that affect the same nucleotide, located in the loop of the hairpin structure, changing the guanine to adenine, thymine or cytosine. Another mutant (lambda nutL3), selected by a different means, has a deletion of one GC base pair and thus eliminates one C in the stem of the hairpin structure, destabilizing it -11.2 to -2.2 kcal/mol. True reversions of the nutL point mutations restore the guanine. The second-site revertant lambda ninL99 was found to have a deletion of 417 bp between the tL1 terminator and the N gene, removing bases +523 to +939 (counted from SL = +1). This deletion include codons for the six carboxy-terminal amino acids of gene N product, but the fusion allows continuation of translation for 53 additional amino acid residues beyond the truncated N gene before reaching a nonsense codon. The fused N product is active.

Bacteriophage lambda↗

PubMed1982

The B66.0 protein of Escherichia coli is the product of the dnaK+ gene.

B66.0 is one of the most abundant proteins of Escherichia coli. Its relative rate of synthesis is highly regulated depending on temperature and the growth rate of the culture. We identified the B66.0 protein to be the dnaK+ structural gene product since dnaK756 mutant bacteria synthesized a B66.0 protein with a more acidic isoelectric point.

Bacterial Proteins↗

PubMed1981

Antitermination and termination functions of the cloned nutL, N, and tL1 modules of coliphage lambda.

A plasmid containing a pp-galK operon was constructed to assay galK expression as a measure of transcriptional regulation by the cloned nutL, N and tL1 modules in a Rho+, Nus+ Escherichia coli host. Insertion of tL1 and rut, both carried on a lambda fragment located between the bamHI site and gene N, between the promoter and galK reduced expression of galK by 80--90%. Gene N alone, when controlled by pp, stimulated galK expression by about two-fold. Cloning of both gene N and nutL in the proper orientation resulted in about a 60% decrease in the tL1 termination. Additional tandem nutL sites increased efficiency of antitermination. A shortened nutL segment, containing only 25 bp of the original genomic nutL sequence, was found to have nearly equal ability to bring about antitermination. If the orientation of the nutL fragment is reversed, antitermination is abolished and the insert now displays a termination function. Termination efficiency is 60 to 73% for one to four tandem nutL modules in reverse orientation. Similarly, the inverted N module acts as a terminator, with 67% efficiency for one and 90% for two tandem inserts. Termination by inverted nutL or N fragments is probably unrelated to their normal functions, but indicates a fortuitous presence of a terminator sequence in the inverted orientation.

Bacteriophage lambda↗