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Biomedical subjects

A Das

Publications and source records attributed to A Das.

At least 379 records · Page 21Linked to original sources
PubMed1985

Autogenous regulation of transcription termination factor Rho.

We present evidence that the transcription termination factor Rho is autogenously regulated in Escherichia coli. The steady-state level of Rho is increased approximately tenfold in rho mutant cells. In the rho+ revertants, the content of Rho is similar to the wild-type level. A rho-/rho+ merodiploid produces equimolar amounts of the mutant and the wild-type Rho polypeptides, both at a reduced level compared to the mutant. The steady-state level of rho messenger RNA is also increased in a rho mutant. A rho-galK transcriptional fusion produces at least tenfold more galactokinase in a rho- strain than in a rho+ strain. In vitro, in a coupled transcription-translation system, the synthesis of Rho protein is specifically inhibited by wild-type Rho but not by Rho15 mutant protein. Anti-Rho antibody specifically stimulates Rho synthesis in the rho+ extract but not in a rho- extract. We suggest that the autogenous regulation of Rho involves premature transcription termination within the rho gene. Regulation of Rho level may provide the cell a mechanism to modulate the expression of genes which are separated from their promoters by Rho-dependent termination signals.

Adenosine Triphosphatases↗
PubMed1985

Analysis of the requirements for transcription pausing in the tryptophan operon.

RNA polymerase pausing during transcription of the tryptophan (trp) operon leader region is postulated to be the key event that synchronizes transcription of this region with translation of the coding region for the trp leader peptide. Coupling of transcription to translation enables the cell to monitor the intracellular concentration of charged tRNATrp and determine whether polymerase should terminate transcription at the attenuator or proceed into the structural genes of the operon. We used mutant templates containing deletions of DNA segments corresponding to sequences that are predicted to form alternative RNA secondary structures to show that formation of an RNA hairpin in the leader transcript, and the concentration of the next nucleoside triphosphate to be added to the paused transcript, both markedly affect the kinetics of pausing in vitro. A model is presented that accounts for many of the findings obtained in this and other pausing studies.

Base Sequence↗
PubMed1985

Evidence that ribosomal protein S10 itself is a cellular component necessary for transcription antitermination by phage lambda N protein.

Bacteriophage lambda N gene product acts to modify host RNA polymerase allowing the formation of a termination-resistant transcription apparatus. Previous studies have demonstrated that the nusE71 mutation that has altered the ribosomal protein S10 prevents N action in vivo. Using a coupled transcription-translation system, we demonstrate here that purified S10 protein as well as the 30S ribosomal subunit is sufficient to restore N activity in the nusE mutant extract, allowing antitermination of Rho-dependent and Rho-independent terminators. This provides direct biochemical evidence that the S10 protein itself is one of the cellular components necessary for the formation of an antitermination apparatus.

Bacterial Proteins↗
PubMed1985

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate.

A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.

Aspergillus niger↗
PubMed1984

A ribosome binding site sequence is necessary for efficient expression of the distal gene of a translationally-coupled gene pair.

Expression of trpB and trpA of the Escherichia coli tryptophan operon is shown to be "translationally coupled", i.e., efficient translation of the trpA coding region is dependent on prior translation of the trpB coding region and termination of translation at the trpB stop codon. To examine the dependence of trpA expression on the ribosome binding site sequence in the distal segment of trpB, deletions were produced that replaced this trpB sequence. Analysis of trpA expression in these deletion mutants established that the ribosome binding site sequence is required for efficient translation of the trpA segment of trp mRNA. A modest effect of translation over the trpA ribosome binding site on independent initiation at that site was also observed.

Amino Acid Sequence↗
PubMed1984

Diabetic retinopathy and insulin therapy in a rural diabetic population.

The prevalence of diabetic retinopathy was lower in a rural diabetic population when compared to the prevalence of diabetic retinopathy in medical center clinic populations. Adult-onset (Type II) diabetics were at greater risk for developing diabetic retinopathy shortly after diagnosis than newly diagnosed cases of juvenile diabetes (Type I). The use of insulin positively correlated with the prevalence of diabetic retinopathy in this rural population.

Adult↗
PubMed1984

Transcription antitermination in vitro by lambda N gene product: requirement for a phage nut site and the products of host nusA, nusB, and nusE genes.

Employing specifically engineered plasmids in which the expression of E. coli galK cistron is regulated by transcription termination, we have analyzed the antitermination function of phage lambda N gene product in S30 extracts. Antitermination by N, dependent on its site of action, nutL, is defective in the extracts prepared from nusA, nusB, and nusE mutants. By complementation analysis, we demonstrate that none of the these nus mutations affects the synthesis of N or the other nus gene products to cause a defect in antitermination. Rather, these mutations have inactivated a set of specific host components, the Nus factors, which are essential for N activity. Curiously, an appreciable portion of N and Nus complementation activities of an S30 extract is ribosome-associated. The significance of this finding remains to be uncovered.

Bacteriophage lambda↗
PubMed1984

Mutagenesis and mutation transfer induced by ultraviolet light in plasmid-cloned DNA.

We describe here simple techniques for increasing the frequency of UV-induced mutations in a DNA fragment cloned in plasmid pBR322. Irradiation of both the host and the plasmid DNA before transformation is necessary to produce new mutations in the plasmid DNA, presumably because the UV-damaged pBR322 replicon cannot efficiently induce the error-prone repair pathway of Escherichia coli. In contrast, UV irradiation of the plasmid DNA alone before transformation primarily causes the transfer of preexisting mutations from the host chromosome to homologous DNA present in the plasmid. The only other kind of mutants obtained were large deletions of the plasmid DNA. Two chromosomal mutations from the host galK gene and one from the lacZ gene have been transferred to the plasmid by UV irradiation of the plasmid DNA alone. The technique can thus be of general use.

Bacterial Proteins↗
PubMed1984

Formation of termination-resistant transcription complex at phage lambda nut locus: effects of altered translation and a ribosomal mutation.

Transcription antitermination by lambda N gene product is affected in a mutant Escherichia coli with altered ribosomal protein S10, caused by the nusE71 mutation. To study the role of translation in antitermination, we have fused the phage nutR locus, the site of action of N, with the lac regulatory region. We have monitored N action by measuring galactokinase, whose synthesis depends on suppression of terminators located between nutR and the galK cistron. We show that a deletion removing potential ribosome binding signals and AUG codons from the upstream region of nut site does not affect N action. Moreover, the lack of translation upstream of nutR does not overcome the antitermination defect caused by nusE mutation. When the upstream region is translated, however, N action is impaired if translation terminates 19 base pairs upstream of nutR . Termination of translation at further upstream sites, such as 23 or 97 base pairs upstream, does not interfere with N action. Our results suggest that the S10 ribosomal protein is required for N action without involving translation. These results also suggest that the nut site RNA itself plays an important role in the formation of a termination-resistant transcription complex.

Bacteriophage lambda↗
PubMed1984

nusB: a protein factor necessary for transcription antitermination in vitro by phage lambda N gene product.

We demonstrate that the protein product of the Escherichia coli nusB gene is essential for transcription antitermination in vitro by phage lambda N gene product. We recently have described a convenient biochemical assay for N protein activity in the S30-coupled transcription translation system and demonstrated that N action requires the 69-kDa L factor (nusA), the product of E. coli nusA gene. Using a complementation assay for the restoration of N activity specifically in the nusB mutant extract, we have purified the nusB complementing activity. This activity is due to a 15-kDa polypeptide that is overproduced in E. coli containing multiple copies of the nusB gene. We find that nusA and nusB are required for N activity to suppress a rho-dependent as well as a rho-independent terminator. The requirement for nusB protein in antitermination could not be overcome by an excess of nusA or N protein, nor could an excess of nusB overcome the requirements for nusA in antitermination. Our results suggest that the formation of an antitermination apparatus by N requires nusA and nusB proteins in equimolar amounts.

Bacterial Proteins↗
PubMed1984

Overproduction of tryptophanyl-tRNA synthetase relieves transcription termination at the Escherichia coli tryptophan operon attenuator.

Overproduction of tryptophanyl-tRNA synthetase increased trp operon expression by reducing transcription termination at the trp attenuator. The total cellular level of charged tRNATrp was not affected by increased levels of the synthetase. We propose that excess synthetase binds charged tRNATrp and reduces the concentration available for translation.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Significance of carcinoembryonic antigen in retinoblastoma.

The role of estimating the serum carcinoembryonic antigen (CEA) in the diagnosis and prognosis of retinoblastoma cases has been evaluated. Although the mean serum CEA titre in children with retinoblastoma was higher than that in control children (p = 0.01), the serum CEA level itself was found not to be a useful marker in the diagnosis of retinoblastoma. A significant correlation of serum CEA titre with progression or regression of the disease observed during therapy in most cases indicated that serial assays of serum CEA may be important in the follow-up of cases with retinoblastoma. The lower CEA values in aqueous humour and subretinal fluid from eyes with retinoblastoma than in the serum suggests that the tumour does not secrete the CEA.

Carcinoembryonic Antigen↗
PubMed1984

An application of the health belief model toward educational diagnosis for VD education.

A protocol is developed which allows the translation of the Health Belief Model (HBM) to practice in supplying empirical needs assessment data on which to build responsive and effective VD education. The HBM is recognized as among the most important theoretical formulations to explain health behavior available in the health education literature, although there has to date been no information as to how to bridge the gap between theory and practice for actual, specific, and detailed programmatic input. The present study describes a procedure for using the HBM as a tool enabling practitioners to isolate those beliefs that need to be addressed for a given population. Prior attempts at belief identification have been limited to the four major dimensions of the HBM (perceptions of susceptibility, seriousness, barriers, and benefits), often with a single item to address each gross dimension. Such efforts are incapable of determining discrete areas of beliefs subject to change through direct educational intervention. The conceptualization and development of an extended view of these dimensions to include numerous individual entities enables the practitioner to create a data base which overcomes this limitation. The development of the VD Health Belief Scale is detailed. Subscales were operationalized by developing standardized attitude scales comprised of Likert-type items requiring a response on a five-point scale of agreement; instrument refinement occurred at several stages; and selected items were screened through internal consistency item analysis. Resultant scales had acceptable reliability with alphas of .89, .66, .91, .86, and .84. A straightforward procedure is described to explain how the measure is used to develop appropriate needs assessment data using the HBM framework. Studies performed by the authors utilizing the procedure are presented to illustrate the application of the research protocol.

Adult↗
PubMed1984

Immunocytochemistry of mouse and human retina with antisera to insulin and S-100 protein.

Immunocytochemistry using peroxidase antiperoxidase (PAP) techniques showed insulin-like immunoreactivity in the human retina, and in the mouse retina and optic nerve. The immunoreaction product was seen in the inner nuclear, ganglion cell, outer and inner plexiform layers of the retinas, and in glial cell bodies of the optic nerve. A similar staining pattern using antiserum to S-100 protein, a marker for glial elements, was also seen in these tissues. This demonstrates that insulin or insulin-like immunoreactivity appears to be limited to glial cells of the retina and optic nerve. Our study suggests that the presence of insulin or a similar peptide in retina and optic nerve may be important for their normal function and metabolism.

Animals↗
PubMed1983

In vitro synthesis of the tryptophan operon leader peptides of Escherichia coli, Serratia marcescens, and Salmonella typhimurium.

We used an in vitro DNA-dependent protein-synthesizing system to demonstrate de novo synthesis of the leader peptide specified by the tryptophan (trp) operons of several bacterial species. Peptide synthesis was directed by self-ligated short restriction fragments containing the trp promoter and leader regions. Synthesis of leader peptides was established by demonstrating that they were labeled in vitro only by those amino acids predicted to be present in the peptides. Leader peptide synthesis was abolished by the addition of the Escherichia coli trp repressor. The E. coli trp leader peptide was found to be extremely labile in vitro; it had a half-life of 3-4 min. In a highly purified DNA-dependent peptide-synthesizing system, synthesis of the di- and tripeptides predicted from the Salmonella typhimurium trp operon leader sequence, fMet-Ala and fMet-Ala-Ala, also was observed. Using this dipeptide synthesis system, we demonstrated that translation initiation at the ribosome binding site used for trp leader peptide synthesis was reduced 10-fold when the transcript contained a segment complementary to the ribosome binding site.

Bacterial Proteins↗
PubMed1983

lambda mutation in the Escherichia coli rho gene that inhibits the N protein activity of phage lambda.

Certain Escherichia coli rho mutations, exemplified by rho026, block the growth of phage lambda by interfering with phage gene expression. The phage gene N, whose product suppresses transcription termination, appears to be expressed normally in the mutants, and the functional stability of the N protein is not affected. Our data suggest that these rho mutations allow transcription to terminate despite the presence of N. Other E. coli mutants displaying a similar phenotype (Nus(-)) fail to propagate wild-type lambda but permit the growth of the lambda variant lambdanin5, which has undergone a deletion of the lambda terminator t(R2). The phenotype of the rho026 mutant differs: the growth of lambda is only marginally improved by the nin5 deletion. Interestingly, N activity at rho-independent terminators is not inhibited by the mutations, whereas its ability to suppress rho-dependent terminators is markedly reduced. The relevance of this specificity in terms of models of N action is discussed.

Bacteriophage lambda↗
PubMed1983

The pleiotropic ts15 mutation of E. coli is an IS1 insertion in the rho structural gene.

Rho protein regulates transcription termination in E. coli. Some of the temperature-sensitive mutants defective in Rho protein, e.g., ts15, show remarkable pleiotropic phenotypes. The ts mutations map between the ilv and cya loci on the E. coli chromosome. We have cloned the gene that restores the wild-type phenotypes of these mutants. Genetic and biochemical characterizations have shown that the cloned DNA segment carries the structural gene for the Rho polypeptide. Analysis of the rhots15 mutation has revealed the presence of an IS1 insertion in the carboxy terminal segment of the rho cistron, thereby truncating the 52-kilodalton (kd) Rho polypeptide to a 50-kd size and also making it thermolabile. This provides an example of how an IS1 insertion mutation can cause a TS phenotype. We have also shown that the multiple phenotypes of the mutant cell, including the temperature sensitivity, are caused by a single mutation (rhots15::IS1) in the rho structural gene. How a rho structural gene mutation may cause such pleiotropy is discussed.

Bacterial Proteins↗