Exon 2 Ovar-DRB1 gene polymorphism in the Iranian Sangsari sheep

The major histocompatibility complex [MHC] plays a central role in the control of disease resistance and immune response. Extensive genetic diversity in MHC genes provides a valuable source for genetic improvement, via selection, in many domestic animals. Exon 2 of the class II MHC, termed Ovar-DRBl in domestic sheep [Ovis aries] has been suggested as important disease resistance and immune response gene. We characterized Ovar-DRBl in DNA samples from 138 individuals of a population of the Iranian Sangsari sheep breed using PCR-RFLP. Eight DRB1 alleles were identified among Iranian Sangsari sheep, including one previously unrecognized allele. Eight homozygous genotypes were observed: a, b, c, d, f, g, h andN. Genotype bb was the most common pattern [46 of 138]. Heterozygous genotypes [ag, cb, cd, bf, and bN] were also observed. The observed homozygosity and heterozygosity values were 0.6377 and 0.3623, respectively, vs expected values of 0.220 and 0.779. Iranian Sangsari population deviate significantly from the theoretical proportions [FIS = 0.5283; p = 0.0005]. In conclusion, PCR-RFLP analysis allows rapid identification of Ovar-DRBl types and discrimination of homozygous and heterozygous genotypes. This study indicates that the exon 2 region of the Ovar-DRBl gene is highly polymorphic in the Iranian Sangsari sheep breed

Cloning and high level expression of bovine interferon gamma gene in eukaryotic cells [COS-7]

Interferon gamma [IFN-gamma] is one of the key cytokines in defining T helper 1 lymphocyte immune responses. In this study, the bovine IFN-gamma gene was cloned from spleen tissue RNA using the reverse transcription-polymerase chain reaction [RT-PCR]. IFN-gamma cDNA was sub-cloned and expressed in mammalian expression plasmid [pcDNA3.1[+]] under the control of the human cytomegalovirus [CMV] promoter. The predicted amino acid [aa] sequence of bovine IFN-gamma compared with corresponding known sequence from bovine [Bos taurus] was 100% identity and with ovine, caprine, camel, lama, equine, canine, feline, human, mice and chicken cytokine was 95, 95, 86, 83, 77, 75, 75, 61, 44 and 35%, respectively. Invitro expression of recombinant bovine IFN-gamma [rBoIFN-gamma] and secretion to culture medium was confirmed by ELISA test. Maximum expression of rBoIFN-gamma occurred at 96 and 144 h after transfection in COS-7 cells. These results showed that pcDNA3.1 expression vector and COS-7 cells transfected by diethylaminoethyl [DEAE]-dextran allowed the high level expression of bovine IFN-gamma gene and the release of protein in supernatant of cell culture

Typing of Ovar-DRB1 second exon with PCR-RFLP technique in Iranian Shaul Sheep

Several studies have focused on polymorphisms of major histocompatibility complex [MHC] in sheep, Ovar-MHC. This molecule plays a pivotal role in antigen presentation for eliciting immune responses against invading pathogens. The best-characterized genetic control of disease resistance and immune response in animals is associated with MHC. Numerous molecular genetic investigations have been undertaken to detect polymorphisms of MHC genes and their association with resistance to infectious diseases. We have examined Ovar-DRB1 in DNA samples of 82 Shaul Sheep using polymerase chain reaction [PCR] and restriction fragment length polymorphism [RFLP] method. Identities of 8 different patterns and 5 distinct DRB1 alleles among Iranian Shaul Sheep have been determined. PCR-RFLP analysis allows rapid identification of Ovar-DRB1 types and enables rapid discrimination between homozygotes and heterozygotes. Data obtained from the present study have revealed that the exon 2 region of Ovar-DRB1 was highly polymorphic in sheep. So PCR-RFLP can be applied to the analysis of this locus

RNA extraction and leptin receptor mRNA detection in bull ejaculated spermatozoa

Leptin, known as a potential satiety factor, plays an important role in both metabolism and reproduction. The presence of leptin in human seminal plasma and human spermatozoa has been shown; recently, leptin receptors [Ob-R] have been localized in human spermatozoa, thus suggesting a possible action of this hormone even on these cells. Our aim was to detect leptin receptor mRNA in bull ejaculated spermatozoa by reverse transcriptase-polymerase chain reaction [RT-PCR]. Total RNA was isolated from bull ejaculated spermatozoa and purified by different methods. Our results have revealed that sodium dodecylsulphate [SDS] and SDS/citric acid extraction methods are superior to guanidinium isothiocyanate in terms of yield and reproducibility of RNA recovery. The mRNA for Ob-Rb was detected in all samples examined. We conclude that Ob-R mRNA is present in bull spermatozoa where seminal plasma leptin can exert its effects

[Semiquantitative RT-PCR analysis to assess the expression levels of iNOS in chicken macrophages]

iNOS is inducible by a variety of factors related to inflammation and referred to as inducible NOS[iNOS]. It is regulated at the level of gene expression; once expressed, it produces NO at a high rate. iNOS gene-expression profiling is an important tool in understanding molecular markers of the responses of cells and tissues to external factors. In this article a semiquantitative reverse transcription-polymerase chain reaction [RT-PCR] protocol was optimized to extract RNA [ribonucleic acid] from chicken spleen and to measure the expression levels of iNOS mRNAs from each sample. Detailed procedure was described for the analysis of iNOS levels. beta-actin was used as an internal control to normalize for sample to sample variations in total RNA amounts and for reaction efficiency. Co-amplification of the iNOS gene with housekeeping gene [beta-actin] provides a quantitative result. Changes in gene expression level may be monitored, while avoiding sample-to-sample loading variation

Molecular epidemiology of Salmonella Abortusovis in Iran

Study of different genotypes amongS.abortusovis strains. Observation study. 58 Salmonella abortusovis strains belonged todifferent countries were studied. IS200 fingerprinting by Southern blothybridisation have been applied for genotyping. 8 different genotypes identified within strainsunder study. All genotypes contained 3 to 5 copies ofIS200 and revealed high relatedness to their origins. Using IS200 fingerprinting for IranianS.abortusovis strains showed high polymorphism becauseeach province at least had one distinct pattern. Also thismethod remains as a sensitive method for typing theS.abortusovis