Biomedical subjects

G Somasekhar

Publications and source records attributed to G Somasekhar.

10 recordsLinked to original sources

PubMed1989

Expression and characterization of the tau subunit of phosphorylase kinase.

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.

Animals↗

PubMed1988

Antisense RNA does not significantly affect expression of the galK gene of Escherichia coli or the N gene of coliphage lambda.

The effect of antisense RNA on the expression of genes galK and N was studied in vivo. These two genes were either present in the Escherichia coli chromosome, as single copies, or were cloned on plasmid vectors. Antisense RNA was supplied from multicopy vectors where the entire galK or N gene, or only their N-proximal portions, were cloned in the antisense orientation downstream from the strong PL, PR or lacZp promoters. In all of the experiments there was no significant inhibition of the galK or N expression by up to a 50-fold excess of the specific antisense RNAs, for both the in cis and in trans experimental designs. The excess of the antisense RNA was calculated as based on respective copy numbers, but was not experimentally measured. The apparent five-fold regulatory effect observed in one of the experiments was found to be artifactually caused by unexpected creation of a terminator in one of our constructs. To avoid such artifacts, all our constructs were equipped with the nut-N antitermination system. We conclude that the reported antimessenger-mediated inhibition of gene expression is not a general phenomenon, but must require some special features which are not present in the galK and N systems.

Bacteriophage lambda↗

PubMed1987

The functional boundaries of the Q-utilization site required for antitermination of late transcription in bacteriophage lambda.

Expression of the late genes of bacteriophage lambda requires, in addition to the host functions, the lambda p'R promoter, the antiterminator sequence qut, and the product of gene Q which interacts with the Q utilization (qut) site. In the absence of the Q function or qut site, the p'R-initiated transcription is blocked by the t'R terminator at the 194th nucleotide downstream of the start point, s'R, producing a short 6 S mRNA. In this study the position and boundaries of the qut site were deduced by constructing plasmids containing various portions of the p'R-qut region, the t'R1 terminator, and the reporter gene galK. We measured galK gene expression in response to the gamma Q gene product supplied in trans by a prophage or Q-expression plasmid. We show that among the lambda proteins, the Q gene product alone is necessary and sufficient for complete qut-mediated transcription antitermination in vivo. These antitermination experiments, employing plasmids that contain different lengths of lambda p'R-qut sequence, identified the right boundary of the qut site, which is located between +4 and +18 (for s'R = +1). The functional left boundary of qut does not extend upstream from the -26th nucleotide of the p'R promoter, as based on the following experiments. The promoter function of the truncated (-26)p'R-s'R-(+18) sequence can be restored by fusion to the complete but qut-less p'R, pp, or PLac promoter; however, no antitermination was observed for such a p-(-26)p'R-s'R-(+18)-t'R-galK plasmid. Thus we conclude that the qut site partially overlaps with the p'R promoter sequence. However, promoters that contain the -10 region of p'R, s'R, and the +1 to +18 qut sequence did mediate Q-dependent antitermination when properly fused to the homologous or heterologous -35 promoter regions. Only those transcripts that start at s'R (+1 or very near to it) and also contain at least the first 18 nucleotides (actually greater than 4 and less than or equal to 18) of 6 S RNA appear to be a target for the Q-qut-mediated transcription antitermination, which acts not only at t'R but also at other Rho-independent or Rho-dependent terminators.

Bacteriophage lambda↗

PubMed1983

Mapping of the Q-utilization site (qut) required for antitermination of late transcription in bacteriophage lambda.

To locate the site required for transcription antitermination by the gene Q product, we constructed a plasmid containing the p'R promoter, the t'R1 terminator, and gene galK. We measured the galK expression in response to the lambda Q product supplied in trans, while deleting various portions of lambda DNA adjacent to p'R. The presence of the lambda p'R promoter together with the downstream DNA coding for only a 34-bp segment of 5'-proximal 6S RNA permits antitermination to occur, whereas deletions removing this segment abolish antitermination, as measured by galK expression, but do not affect the p'R promoter. Thus the 34-bp segment must contain the p'R-distal (right) boundary of the Q-specific recognition site qut (Fig. 1). The Q-mediated antitermination appears to be p'R-qut specific but not t'R1 specific, since it does not operate with the pp-t'R1 assembly, but is also effective with terminators other than t'R1, e.g., with the combination of the p'R-qut-tL3 modules.

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↗

PubMed1981