Study of conformational properties of solid phase synthesized ovine kisspeptin-14 using Circular Dichroism spectroscopy.

Kisspeptins are peptide products of Kiss-1 gene and have been substantially associated with the initiation of puberty by virtue of their ability to cause pulsatile release of GnRH. Kisspeptin consists of 54 amino acids domain called metastin and its biological activity may be localized to the C-terminal (C-10, C-13, and C-14) segments. Kisspeptin binds to its cognate receptor GPR54 in the hypothalamic neurons and it is a G-coupled membrane receptor. This study is an attempt to understand the tentative conformation of the peptides in its native membrane mimicking environment. A 14 amino-acid derivative of kisspeptin (Asp-58-Val59-Ser60-Ala61-Tyr62-Asn63-Trp64-Asn65-Ser66-Phe67-Gly68-Leu69-Arg70-Tyr71NH2) was synthesized by f-moc (9-fluorenyl methoxy carbonyl) solid phase synthesis strategy. The synthetic peptide was cleaved and purified by Reverse phase-HPLC. CD spectroscopic analysis of secondary structure of the peptide revealed random coil disordered conformation in the aqueous environment. However, the peptide adopted more ordered ß-sheet conformation in the solvents such as TFE and HFIP.

Characterization of MTNR1A gene in terms of genetic variability in a panel of subtemperate and subtropical Indian sheep breeds.

Seasonality of animals is an important adaptive trait for successful survival and production during limited food availability and extreme environmental conditions. Photoperiodic changes in day length are utilized by these seasonal animals as an important environmental cue for regulating their annual rhythms of reproduction cycles. Melatonin is an important hormone which is secreted by the pineal gland in proportion to darkness and its effect is mediated by melatonin receptor subtypes, principally MTNR1A. In the present study, polymorphism in the coding sequence at two important SNPs (C606T and G612A),known to be markers for out of season breeding in sheep were studied by PCR-RFLP in a panel of four breeds of sheep from subtemperate and subtropical arid conditions, respectively. The frequencies of 'G' and 'A' alleles with reference to G612A SNP did not differ considerably among all the breeds of sheep. Frequency of 'T' allele of the C606T SNP was found to be dom inantly higher in subtemperate sheep breeds in comparison to subtropical sheep breeds. Identified SNPs in the coding region were mostly synonymous and did not lead to any change in conformation of the MTNR1A receptor protein.

Sequence analysis and identification of new variations in the coding sequence of melatonin receptor gene (MTNR1A) of Indian Chokla sheep breed.

Melatonin receptor 1A gene is the prime receptor mediating the effect of melatonin at the neuroendocrine level for control of seasonal reproduction in sheep. The aims of this study were to examine the polymorphism pattern of coding sequence of MTNR1A gene in Chokla sheep, a breed of Indian arid tract and to identify new variations in relation to its aseasonal status. Genomic DNAs of 101 Chokla sheep were collected and an 824 bp coding sequence of Exon II was amplified. RFLP was performed with enzyme RsaI and MnlI to assess the presence of polymorphism at position C606T and G612A, respectively. Genotyping revealed significantly higher frequency of M and R alleles than m and r alleles. RR and MM were found to be dominantly present in the group of studied population. Cloning and sequencing of Exon II followed by mutation/polymorphism analysis revealed ten mutations of which three were non-synonymous mutations (G706A, C893A, G931C). G706A leads to substitution of valine by isoleucine Val125I (U14109) in the fifth transmembrane domain. C893A leads to substitution of alanine by aspartic acid in the third extracellular loop. G931C mutation brings about substitution of amino acid alanine by proline in the seventh transmembrane helix, can affect the conformational stability of the molecule. Polyphen-2 analysis revealed that the polymorphism at position 931 is potentially damaging while the mutations at positions 706 and 893 were benign. It is concluded that G931C mutation of MTNR 1A gene, may explain, in part, the importance of melatonin structure integrity in influencing seasonality in sheep.