Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy.

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96-98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis).

A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3' region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

Cloning of partial putative gonadotropin hormone receptor sequence from fish.

A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.

Construction, electroporatic transfer and expression of ZpfJypGH and ZpfJrtGH in zebrafish.

Recombinant transformation vectors (ZPPypGH and ZpprtGH) consisting of fish growth hormone cDNA, and a reporter gene p-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 J.1F capacitance; 00 ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 J.1g circular DNNml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen Fo individuals was screened (by Southern blot hybridization); the transgenes were retained in the host-. genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F] progenies was screened for germ-line transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZPPypGH or ZpPrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpPrtGH and 53% of F1 ZPPypGH) of transmission revealed the degree of mosaicism i!1 F 0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic Fo and F1 progenies.

Interspecific hybridization in poeciliids.

Seventy per cent attempts to ensure interspecific hybridization between Poecilia velifera and P. sphenops were successful and led to the production of true hybrids, but not gynogens or triploids, as evidenced by the mottled or striped colour, chromosome number (2n = 46) and response from scale transplantation. Most hyrbids were infertile as they failed to cross among themselves or with their respective parents; however they were more closely related to P. sphenops as indicated by mating responses and scale transplantation studies. Heterospecific impregnation resulted in 40% reduction in fecundity but retention of interparturition period characteristic of the female species. A skewed ratio of 3 Female Female:2 Male Male, observed in the laboratory populations of both P. velifera and P. sphenops, was also sustained among the progenies sired from heterospecifically inseminated females. The colour patterns of hybrids were of two types: mottled and striped, the latter one being reported for the first time.

Sublethal effects of textile dye stuff effluent on selected oxidative enzymes and tissue respiration of Cyprinus carpio (Linn.).

Toxic effects of sublethal concentration of dye stuff effluent on succinic dehydrogenase (SDH) and lactic dehydrogenase (LDH) activities and tissue respiration were studied in C. carpio. While the sublethal exposure significantly reduced SDH activity and tissue respiration, LDH activity increased in gill, brain, liver, muscle and kidney. The maximum inhibition of SDH activity (74%) was recorded in gill and the minimum (38%) in liver. The percentage reduction of oxygen consumption in the tested tissues was in the order of gill greater than brain greater than liver greater than muscle greater than kidney. The muscle showed the highest level (96%) of increase in LDH activity whereas the kidney cells showed the minimum increase. Exposure to sublethal concentration suppressed the aerobic respiration and triggered the anaerobic respiration.