Variation in bovine leptin gene affects milk fatty acid composition in New Zealand Holstein Friesian × Jersey dairy cows

Leptin is a protein hormone secreted from white adipose tissue. It regulates food/feed intake, body weight, immune function and reproduction. In our investigation, the polymerase chain reaction (PCR) amplification coupled with single-strand conformational polymorphism (SSCP) analysis was used to reveal variation in bovine leptin gene (LEP) in New Zealand (NZ) Holstein Friesian × Jersey (HF × J) dairy cows. Subsequent sequence analysis of a 430 bp amplicon spanning the entirety of exon 3 and part of the intron 2 region revealed three variant sequences (A3, B3 and C3) containing a total of five nucleotide substitutions, all of which have been reported previously. Using general linear mixed-effect model analyses, the presence of variant A3 (the most common variant) was associated with a decreased level of C15:1, C18:1 trans-11, C18:1 all trans, C18:2 trans-9, cis-12, C22:0 and C24:0 levels but increased levels of C12:1 and C13:0 iso (p<0.05). Variant B3 was associated with reduced levels of C6:0, C8:0, C11:0, C13:0 and C20:0 but increased C17:0 iso and C24:0 levels (p<0.05). Variant C3 was associated with decreased C17:0 iso levels but increased C20:0 (p<0.05) levels. In a genotype model, the A3B3 genotype was associated with increased levels of C22:0 and C24:0 but decreased C8:0, C10:0, C11:0, C13:0, C15:0 and grouped medium-chain fatty acid (MCFA) levels (p<0.05). Genotype A3C3 was found to be associated with decreased levels of C10:0, C11:0, C13:0 and grouped MCFA (p<0.05). This is the first report of findings of this kind in NZ HF × J cows, and they suggest that variation in exon 3 of bovine leptin gene could be explored as a means of decreasing the concentration of saturated fatty acids in milk.

CAG repeats and one polymorphism in androgen receptor gene are associated with renal calcium stone disease

Purpose: Evidence suggests that androgens can be involved in the pathogenesis of renal stones. This study aimed at investigating coding region polymorphisms and CAG repeats in androgen receptor (AR) and their association with active renal calcium stone disease. Materials and Methods: Male patients with calcium kidney stones ( N = 106) with at least two episodes of stone recurrence or size increase during the past 5 years (ASF) were enrolled from December 2008 to April 2009. Control individuals were recruited after matching for age and gender from healthy individuals without current stone or history of stone disease. Genetic sequencing and single strand conformational polymorphism (SSCP) were used to determine AR polymorphisms in the patients and controls. Results: Two polymorphisms were identified in the AR gene: Silent G to A polymorphism in the first exon of the AR gene and C to G polymorphism in intron 4. CAG repeats ranged from 12 to 37. The C/G polymorphism in intron 4 and CAG repeats were associated with the status of active renal calcium stone disease (all p < 0.05). The CC variant of C/G polymorphism was not observed in patients with stone disease. CAG repeats less than 20 and more than 28 were mostly observed in ASF patients ( p < 0.05). Conclusions: CAG repeats and intron 4 C/G polymorphism in the AR gene have an association with renal calcium stone disease.

Genetic Differentiation of the Blue Swimming Crab Portunus pelagicus Along the Coastal Thai Waters Revealed by SSCP Analysis of Cytochrome c Oxidase Subunit I

The basic information on genetic diversity and population structure is essential for the construction of appropriate management schemes leading to sustainable fisheries of the blue swimming crab (Portunus pelagicus). Here, genetic heterogeneity of P. pelagicus (N=174) was examined by single-strand conformational polymorphism (SSCP) analysis of mitochondrial cytochrome c oxidase subunit I (PpCOI270). Seven SSCP genotypes were found across all investigated samples. The average genetic distance between pairs of geographic samples was 0.0014-0.7247. Significant geographic heterogeneity (P<0.05) and restricted levels of female gene flow between paired samples (0.03-1.60 individuals per generation) were observed except between Chanthaburi - Prachuap Kriri Khan and Ranong - Krabi (P>0.05; 6.54 and 16.17 individuals per generation) located in the same coastal regions. Therefore, the gene pool of P. pelagicus in Thai waters was genetically differentiated to different stocks even though it is biologically regarded as a potential dispersal species. Five geographic samples of P. pelagicus in Thai waters could be differentiated to three genetic stocks; Chanthaburi and Suratthani (stock A), Prachuap Khiri Khan (stock B) and Ranong and Krabi (stock C).

Detection and Correlation of Single and Concomitant TP53, PTEN, and CDKN2A Alterations in Gliomas

Gliomas are the most frequent primary tumors of central nervous system and represent a heterogeneous group of tumors that originates from the glial cells. TP53, PTEN, and CDKN2A are important tumor suppressor genes that encode proteins involved in sustaining cellular homeostasis by different signaling pathways. Though genetic alterations in these genes play a significant role in tumorigenesis, few studies are available regarding the incidence and relation of concomitant TP53, PTEN, and CDKN2A alterations in gliomas. The purpose of this study was to evaluate the occurrence of mutation and deletion in these genes, through single-strand conformational polymorphism, array-comparative genomic hybridization, and fluorescence in situ hybridization techniques, in 69 gliomas samples. Molecular results demonstrated a significant higher prevalence of TP53, PTEN, and CDKN2A alterations in astrocytoma than other tumor subtypes, and heterozygous deletion was the most frequent event. In addition, a significant association was observed between TP53 and CDKN2A alterations (p = 0.0424), which tend to coexist in low grade astrocytomas (5/46 cases (10.9%)), suggesting that they are early events in development of these tumors, and PTEN and CDKN2A deletions (p = 0.0022), which occurred concomitantly in 9/50 (18%) patients, with CDKN2A changes preceding PTEN deletions, present preferably in high-grade gliomas.

Detection of Staphylococcal Species Diversity within Human Skin Microbiome Using a Simple PCR-SSCP Technique

Objective. The aim of this study was to detect the diversity of Staphylococcus species of a healthy human skin using a simple techniquevPCR-SSCP. Methods: Blood samples, saliva, and skin swaps samples were collected from 50 persons from Hilla City - Iraq. The genomic DNA was extracted from these samples using the Bacteria Genomic DNA Kit. The concentrations and purity of DNA extract estimated by NanoDrop spectrophotometer. Polymerase chain reaction – single-strand conformational polymorphism (PCR–SSCP) technique was performed to detect the diversity between Staphylococcal species in the human skin microbiome using a specific primer of the 16SrRNA gene. Results: The PCR results, indicated that the Staphylococcal species were found within the ski community, but it's not infected blood and mouth of test healthy individuals. SSCP-heteroduplex patterns of PCR products appeared the presence Staphylococcal species diversity within skin microbiome of test healthy individuals. Conclusion: In spite of the PCR-SSCP, heteroduplex method was simple and cheap and appeared the diversity betweenvStaphylococcalspeciesvin the human skin microbiome, but it's not diagnosed the bacterial strains. So these results required to confirm by DNAvsequencingvtechnique.

“Mirror” Method to Estimate Mutagenic Activity of DNA Lesions

We propose an improved method for detecting mutations that arise in DNA due to misincorporations of deoxyadenosine-5′-monophosphate (dAMP) opposite 7,8-dihydro-8-oxoguanine (8-oxoG). The method is based on the synthesis of complementary chains (“mirror” products) using a template containing 8-oxoG. The misincorporation of dAMP in the “mirror” product forms EcoRI sites. The restriction analysis of double-stranded DNAs obtained by PCR of “mirror” product allows quantification of the mutagenesis frequency. In addition, single-strand conformational polymorphism (SSCP) analysis of the single-stranded “mirror” products shows that different DNA polymerases only incorporate dA or dC opposite 8-oxoG. The proposed approach used in developing this technique can be applied in the study of other lesions as well, both single and clustered.

Molecular Characterization and Phylogeny Based Analysis of Intron I Sequence of Myostatin (MSTN) Gene in Iranian Makuei Sheep Breed

Myostatin (MSTN), a negative regulator of skeletal muscle development, acts as a potential candidate gene used to increase muscle mass. Likewise, sheep MSTN gene has an important role in meat production. MSTN is made up of 376 amino acids, and is synthesized as a precursor protein. To investigate the MSTN in Iranian native Makuei sheep, a polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used. Genomic DNA was isolated from the blood samples. A 417-bp of MSTN intron I segment was amplified using locus-specific primers. Four SSCP patterns were identified and nucleotide sequencing of the Makuei sheep MSTN gene was done and registered in the NCBI GenBank with “KJ526625” number. Three novel single nucleotide polymorphisms (SNPs) were found in the samples. These SNPs are found in 224bp, 226bp and 242bp locations. Accordingly three substitutions (c.224C>T; c.226A>G; c.242G>T) were observed in the intron 1 region of MSTN gene. The effects of the observed SNPs on breeding values of some biometric traits were investigated and the substitution of c.226A>G was found to be associated with heart girth (HG) and leg circumference (LC). Phylogenetic analysis, based on the nucleotide sequences indicated similar evaluation with the GenBank reference sequences. It seems that the observed polymorphisms of the ovine MSTN gene are associated with HG and LC traits.