Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.     

Seven novel single nucleotide polymorphisms identified within river buffalo (Bubalus bubalis) lactoferrin gene

The present study aimed at identifying single-nucleotide polymorphic (SNP) sites in different coding and non-coding regions of lactoferrin gene in Indian riverine buffaloes. A total of 102 animals from six different river buffalo breeds were screened at six bubaline lactoferrin gene loci. Single-strand conformation polymorphism (SSCP) analysis revealed monomorphic patterns at three loci LtfE2, LtfE11, and LtfE14 while a total of eight distinct patterns were observed in the other three loci viz. LtfE5, LtfE10, and LtfE16 which correspond to respective exons and their flanking regions. Sequence analysis of different SSCP variants revealed the presence of two SNP sites within the coding (exon 16) region and five SNP sites in flanking non-coding regions (intron 4 and intron 9). Both SNPs within exon 16 were found to be synonymous. The SNPs and haplotypes identified in the present study could serve as potential markers for association with susceptibility/resistance to mastitis in buffaloes.     

Purification of porcine beta-casein,N-terminal sequence,quantification in mastitic milk

The objectives of the study were to purify porcine beta-casein from sow's milk, to determine N-terminal amino acid sequence, to develop specific antisera against porcine beta-casein, and to use that antisera to evaluate milk samples from a mastitis study. Milk was collected by hand milking a Yorkshire by Duroc crossbred sow following oxytocin administration on d 27 of lactation. A casein-enriched fraction was then prepared by iso-electric precipitation. Porcine beta-casein was then purified by liquid chromatography on a Mono Q anion-exchange column, and checked for purity with SDS-PAGE. An apparent molecular weight of 29,000 Da was estimated from SDS-PAGE. N-Terminal amino acid sequence was determined by Edman degradation to be RAKEELNASGETVE. Rabbits (n = 2) were immunized with beta-casein mixed with Freund's complete (primary) or incomplete (boosters) adjuvant at 4-wk intervals. Antiserum collected from one rabbit 112 d after primary immunization detected 30 to 100 ng beta-casein by Western blot procedure when used at a dilution of 1:2 x 10(6). The antiserum was specific for porcine beta-casein, but showed some cross-reactivity with equine casein. It was determined by Western blot procedure that mammary inflammation induced by lipopolysaccharide infusion resulted in a 41% decrease in the beta-casein concentration of sow milk.