Biomedical subjects

A V Sivaprasad

Publications and source records attributed to A V Sivaprasad.

6 recordsLinked to original sources

PubMed1995

Expression of bacterial cysteine biosynthesis genes in transgenic mice and sheep: toward a new in vivo amino acid biosynthesis pathway and improved wool growth.

It is possible to improve wool growth through increasing the supply of cysteine available for protein synthesis and cell division in the wool follicle. As mammals can only synthesise cysteine indirectly from methionine via trans-sulphuration, expression of transgenes encoding microbial cysteine biosynthesis enzymes could provide a more efficient pathway to cysteine synthesis in the sheep. If expressed in the rumen epithelium, the abundant sulphide, produced by ruminal microorganisms and normally excreted, could be captured for conversion to cysteine. This paper describes the characterisation of expression of the cysteine biosynthesis genes of Salmonella typhimurium, cysE, cysM and cysK, and linked cysEM, cysME and cysKE genes as transgenes in mice and sheep. The linked transgenes were constructed with each gene driven by a separate promoter, either with the Rous sarcoma virus long terminal repeat (RSVLTR) promoter or the mouse phosphoglycerate kinase-1 (mPgk-1) promoter, and with human growth hormone (hGH) polyadenylation sequences. Transgenesis of mice with the RSVLTR-cysE gene afforded tissue-specific, heritable expression of the gene. Despite high levels of expression in a number of tissues, extremely low levels of expression occurred in the stomach and small intestine. Results of a concurrent sheep transgenesis experiment using the RSVLTR-cysEM and -cysME linked transgenes revealed that the RSVLTR promoter was inadequate for expression in the rumen. Moreover, instability of transgenes containing the RSVLTR sequence was observed. Expression of mPgk-cysME and -cysKE linked transgenes in most tissues of the mice examined, including the stomach and small intestine, suggested this promoter to be a better candidate for expression of these transgenes in the analogous tissues of sheep. However, a subsequent sheep transgenesis experiment indicated that use of the mPgk-1 promoter, active ubiquitously and early in development, may be inappropriate for expression of the cysteine biosynthesis transgenes. In summary, these results indicate that enzymically active bacterial cysteine biosynthesis gene products can be coexpressed in mammalian cells in vivo but that expression of the genes should be spatio-temporally restricted to the adult sheep rumen epithelium.

Acetyltransferases↗

PubMed1994

Transgenic sheep and wool growth: possibilities and current status.

Merino wool is the result of generations of selection, yet improvements in wool quality and performance are still being sought. Through gene manipulation, sheep transgenesis offers possibilities of understanding the relationship between wool keratin protein composition and fibre structure and properties and of introducing novel changes to fibre properties and growth rates. We have established an efficient sheep transgenesis programme with an overall transgenic rate of 2.1% of zygotes injected. However, by incorporating in vitro culture and assessment of injected zygotes, this equates to a transgenic rate of 13% from 516 lambs born. With the first keratin gene construct, a wool keratin type II intermediate filament gene, four live F0 transgenic sheep have been produced and all express the transgene. In one of them, the highest expressor, phenotypic and ultrastructural changes were evident in the fleece. To improve wool growth rate by increasing the supply of cysteine to the follicle, transgenic sheep are being produced carrying the two genes necessary for endogenous cysteine synthesis. Three promoters have been tested driving the cysteine synthesis genes: two general promoters, the Rous sarcoma virus long terminal repeat and mouse phosphoglycerate kinase promoter, and a rumen-specific promoter from the sheep small proline-rich protein gene. To date, one transgenic sheep (bearing the small proline-rich protein promoter constructs) has produced cysteine in the rumen, although the amount was low at 3 months of age and not detectable at 6 months.

Animals↗

PubMed1992

Coexpression of the cys E and cys M genes of Salmonella typhimurium in mammalian cells: a step towards establishing cysteine biosynthesis in sheep by transgenesis.

The Salmonella typhimurium genes for serine acetyltransferase (cys E) and O-acetylserine sulphydrylase B (cys M) were isolated and characterized in order to express these as transgenes in sheep to establish a cysteine biosynthesis pathway and, thereby, to achieve an increased rate of wool growth. Comparison of the S. typhimurium and Escherichia coli genes showed considerable homology, both at the nucleotide and amino acid sequence levels. The in vitro and in vivo expression studies showed that both genes could be transcribed and translated in eukaryotic cells and that their products could function as active enzymes. The cys M gene of S. typhimurium possessed a GUG initiation codon, like its E. coli counterpart, but translation could be initiated using this codon in eukaryotic cells to give an active enzyme product. Chinese hamster ovary cells, stably transfected with a tandem arrangement of the two genes, showed a capacity to synthesize cysteine in vivo, indicating the establishment of a cysteine biosynthesis pathway in these cells. The measured levels of activity of the gene products suggest that improved wool growth is possible by transgenesis of sheep with these genes.

Acetyltransferases↗

PubMed1990

DNA replication studies with coliphage 186. III. A single phage gene is required for phage 186 replication.

We have shown that the BglII to BamHI (79.6% to 95.8%) region of the coliphage 186 chromosome can direct 186-specific replication. DNA sequencing of the region revealed five presumptive genes, CP80, CP81, CP83, CP84 and CP87. Surprisingly, alleles of the previously defined replication gene, A, were localized in both CP84 and CP87. We have successfully constructed a 186 minichromosome using the single gene CP87, and determined that CP84 was not concerned with replication, neither of a minichromosome nor of the phage. Rather, the replication defect seen with amber mutants of CP84 reflects a polarity effect on the downstream expression of CP87. We have concluded that CP87 is the only phage gene necessary for 186 replication, and have called it gene A.

Amino Acid Sequence↗

PubMed1990

In vitro assessment of the viability of sheep zygotes after pronuclear microinjection.

Microinjected sheep zygotes were cultured in synthetic oviduct fluid medium (SOFM) for either 1 or 3 days and their subsequent developmental capacity was compared with that of microinjected zygotes cultured in vivo. Two experiments were carried out, using zygotes microinjected with one of three gene constructs containing the CysE and CysM genes from Salmonella typhimurium. In Experiment 1, microinjected zygotes were allocated to one of three treatments: (1) immediate transfer to recipient ewes (in vivo culture) followed by recollection 1 or 3 days later and subsequent transfer of viable embryos to other recipient ewes, (2) culture in SOFM (in vitro culture) for either 1 or 3 days before transfer to recipient ewes, and (3) immediate transfer to recipient ewes without subsequent interference. Recipient ewes were slaughtered on Day 14 of pregnancy and the number of elongated conceptuses determined. Although fewer zygotes failed to divide during in vitro culture than during in vivo culture, there were, overall, no significant differences between treatments in the percentage of zygotes that developed into elongated conceptuses (32.6-50.0%). In Experiment 2, microinjected zygotes were transferred immediately to recipient ewes or cultured in vitro for either 1 or 3 days before transfer. The number of fetuses per ewe on Day 50 of pregnancy and the number of lambs delivered per ewe were recorded. Neither the percentage of recipient ewes that became pregnant (overall 114/166, 68.7%) nor the percentage of zygotes that developed into lambs (overall 186/803, 23.2%) was significantly influenced by the culture treatment or by the gene construct microinjected.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Evaluating tests for acute pancreatitis.

The relative merits of various serum pancreatic enzymes, ultrasonography (US), and computerized tomography (CT) have been evaluated. In practice, the diagnosis of acute pancreatitis (AP) remains hinged on the clinical picture and elevated serum amylase. The advantages of total serum amylase are its technical simplicity, ready availability, and sensitivity. Within 24 h of onset of symptoms, elevation of amylase is as sensitive as that of lipase, pancreatic isoamylase, immunoreactive trypsin, or elastase. However, after the first hospital day, it is the least sensitive of the enzymatic tests. Its greatest disadvantage is its overall low specificity. Lipase assays are now fast, reliable, practical, more specific, almost as sensitive, and not more expensive than amylase assays. The current feeling is that lipase assays should be used more often or even should replace amylase assays. However, comparative studies using objective criteria for AP are required to evaluate the utility of lipase estimations over that of amylase. Other enzymes such as P-isoamylase, immunoreactive trypsin, chymotrypsin, or elastase are more cumbersome, expensive, and not better than lipase. They should be reserved for cases of doubtful diagnoses. The levels of these pancreatic enzymes neither correlate with the severity of the disease nor can they accurately predict the subsequent clinical course of the patients. The main role of ultrasonography remains in the evaluation of the biliary tract in AP. The contrast-enhanced computed tomography (CECT) is useful for estimating the presence and extent of pancreatic necrosis. Thereby, it enables prompt recognition of patients at high risk for systemic and local complications. Routine use of CECT may aid in the identification of pancreatitis when enzyme elevations are modest, but the utility of the procedure in all clinically mild cases is questionable. Patients who are seriously ill or who present a diagnostic problem should have a CECT. A normal CT under such circumstances excludes clinically severe AP. Serial CT should be done in patients demonstrating phlegmonous extrapancreatic spread.

Acute Disease↗