Polymerase Chain Reaction-single Strand Conformation Polymorphism Research Articles

Identification of the Caprine Keratin-Associated Protein 20-2 (KAP20-2) Gene and Its Effect on Cashmere Traits.

The gene encoding the high glycine/tyrosine keratin-associated protein 20-2 (KAP20-2) gene has been described in humans, but has not been identified in any livestock species. A search for similar sequences in the caprine genome using the human KAP20-2 gene (KRTAP20-2) revealed a homologous sequence on chromosome 1. Three different banding patterns representing distinct sequences (A–C) in Longdong cashmere goats were identified using polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis. These sequences shared high sequence similarity with the human and mouse KRTAP20-2 sequences, suggesting that A–C are caprine variants of the human and mouse genes. Four single nucleotide polymorphisms (SNPs) were identified, and three of them were non-synonymous. KRTAP20-2 was found to be expressed in secondary hair follicles, but not in heart, liver, lung, kidney, spleen, or longissimus dorsi muscle. The presence of A was associated with increased cashmere fibre weight, while the presence of B was associated with a decrease in cashmere fibre weight and curly fibre length. Goats with genotype AA had a higher cashmere fibre weight and a higher curly fibre length than those with genotypes AB or BB. These results indicate that caprine KRTAP20-2 variation may have value as a genetic marker for improving cashmere fibre weight.

New polymorphisms of PAPPA and PAPPA2 genes and their associations with egg production traits in Chinese Dagu chickens

Pregnancy-associated plasma protein A, pappalysin1 (PAPPA) and pappalysin2 (PAPPA2) genes were implicated in regulation of hen ovarian follicular development and growth. Four novel single nucleotide polymorphisms (SNPs) were identified using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. Among them, A/G transition at position 172864 and T/C mutation at position 172952 in 3‘-untranslated region (UTR) of PAPPA named SNP A172864G and T172952C, respectively. A/G transition at position 77421 and T/C at position 77455 in 3‘-UTR of PAPPA2 gene named as SNP A77421G and T77455C, respectively. For SNP A172864G and T172952C (PAPPA), 360 Dagu hens were classified as AA, AB and BB genotypes based on PCR-SSCP patterns, and BB genotype correlated significantly (P less than 0.05) with higher hen-housed egg production (HHEP) at 30 and 57 weeks (wks) of age and higher egg weight (EW) at 43 wks of age. Consequently, these SNPs identified will be potential genetic markers to improve egg productivity in chicken breeding.

Variation in the Toll-like Receptor 4 (TLR4) gene affects milk traits in dairy cows.

The objective of this Research Communication was to use polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis to investigate a region of the bovine TLR4 gene (TLR4) in pasture-fed New Zealand (NZ) Holstein-Friesian × Jersey (HF × J) cross dairy cows and to determine whether gene variation was associated with milk production traits. Genetic variation was observed, with two variants (A and B) containing a single nucleotide polymorphism (SNP) (c.2021C/T) that was non-synonymous and putatively results in a p.Thr674Ile substitution in the transmembrane/cytoplasmic domain of TLR4. Variant A was associated with higher milk yields, but lower milk fat percentages, whereas B was associated with lower milk yields, but higher fat and protein percentages. Cows of genotype AA produced more milk than AB or BB cows, but the milk produced by AA cows contained less fat than AB or BB cows.

SNPs of CD14 change the mastitis morbidity of Chinese Holstein.

Gram‑negative (GN) bacterial infection is a main cause of bovine mastitis. The cluster of differentiation (CD) 14 gene serves an essential role in GN bacterium‑induced innate immune response. CD14 works as a bacterial lipopolysaccharide (LPS) receptor, combines with LPS‑liposaccharide binding protein complex, and causes cellular activation. However, the effects of CD14 single nucleotide polymorphisms (SNPs) on morbidity of clinical mastitis remain unclear. In the present study, To investigate the polymorphisms of CD14 gene and its effects on cows' susceptibility to mastitis, polymerase chain reaction‑single‑strand conformation polymorphism (PCR‑SSCP) assay was used to detect SNPs of CD14 gene in 134 Chinese Holsteins. SNPs were identified in PCR products amplified with 3 sets of primers in CD14 exon 2. A total of three SNPs were located in that exon: g.528 A→C (147Ser→Arg) in allele B; g.612 A→G (175Asn→Asp) in allele D; and g.1022 A→G in allele F (synonymous mutation). The SNPs in alleles B and D affected the secondary structure of CD14. A 3‑dimensional (3D) structural analysis predicted three potential protein forms with a similar structure and indicated that the changes of the above‑mentioned alleles were on the concave surface of the protein. In more detail, 147 Ser→Arg induced a protein kinase C phosphorylation site to move forward, as assessed by the motif analysis. The morbidity rate of AB (mixed type g.528 A/C) and CD (mixed type g.612 A/G) was the highest among all genotypes presented in the current study, and via of tumor necrosis factor‑α and interleukin‑6 mRNA levels were upregulated in animals of this genotype compared with others. Taken together, the CD14 SNPs identified in the present study, may be closely associated with the morbidity of mastitis.

Gene Effects on Body Weight, Carcass Yield, and Meat Quality of Thai Indigenous Chicken.

The selection of rapidly growing animals in breeding programs has had inadvertent detrimental effects on meat quality. Thus, the aim of the present study was to investigate the relationship between body weight (BW) and meat quality traits, and the effects of genes encoding insulin-like growth factor I (IGF-I), insulin-like growth factor II (IGF-II), melanocortin-4 receptor (MC4R), and calpain 1 (CAPN1) on BW, carcass yield, and meat quality of the Thai indigenous chicken, Leung Hang Khao. Five hundred and ten chickens were used for genotyping. PCR-restriction fragment length polymorphism and PCR-single strand conformation polymorphism were used to determine the genotypes of IGF-I, IGF-II, MC4R, and CAPN1. BWs were collected from 0–16 weeks of age. The chickens were sacrificed at 16 weeks and individual carcass yields and meat qualities (drip loss, cooking loss, and shear force) were recorded. The correlations between BW and meat qualities were determined. Significant correlation between BW and cooking loss and shear force of breast meat and between BW and drip loss of thigh meat were detected (P<0.05); however, the magnitude of the association was low (−0.1–0.1). IGF-I was eliminated from the association analysis because genotype AA was lost and the frequency of occurrence of the AC genotype was low (0.04). Significant associations between IGF-II, CAPN1, and BW, and CAPN1 and meat quality were detected, while non-significant association between MC4R and BW was observed. The results indicated a low, negative relationship between BW and meat quality, and that the IGF-II and CAPN1 could be used as genetic markers in Leung Hang Khao chickens to improve growth and meat quality through breeding.

Polymorphism of KRT83 and its association with selected wool traits in Merino-cross lambs

Keratins are structural proteins in the cortex of wool fibres. It is thought that variation in the keratin genes (KRTs) affects wool structure and characteristics. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis was used to investigate three regions of ovine KRT83. These regions were a portion of the promoter, the exon 2 region (including part of intron 1) and a region encompassing exon 3-4 (including all of exon 3, intron 3, exon 4 and part of intron 4). Initially, in 300 New Zealand Romney, Merino and White Dorper sheep obtained from 26 farms, one, two and four PCR-SSCP banding patterns were observed for these regions respectively. The exon 2 region contained two single nucleotide polymorphisms (SNPs) and the exon 3-4 region contained five SNPs. Investigation of the effect of the variation in the exon 3-4 region on variation in some wool traits were subsequently undertaken in 489 Merino×Southdown-cross sheep from seven sire-lines. The four variants identified in the original 300 sheep (designated A-D) and a new variant (E containing a new SNP) were observed with a frequency of 64.6%, 15.4%, 6.6%, 10.1% and 3.3%, respectively. General linear mixed-effects models (GLMMs) were used to investigate associations between the presence or absence of the variants and the wool traits, with a second set of models testing associations between common genotypes and those traits. The presence of A was associated with a decrease in fibre diameter standard deviation (FDSD) and coefficient of variation of fibre diameter (CVFD). The presence of C was associated with an increase in mean fibre diameter (MFD), mean fibre curvature (MFC) and prickle factor (PF), and a decrease in wool yield (Yield) [clean fleece weight (CFW)/greasy fleece weight (GFW)×100%]. A trend for association between the presence of C and increased FDSD was also detected. The presence of D was associated with an increase in MFD and PF, and a decrease in Yield. The presence of E was associated with a decrease in CVFD. Genotype AD had a higher GFW and a lower Yield than AA, and AC and AD tended to have increased MFD compared to AA and AB. These results suggest that ovine KRT83 might be a useful candidate gene for improving wool traits.

Variation in the Ovine KAP6-3 Gene (KRTAP6-3) Is Associated with Variation in Mean Fibre Diameter-Associated Wool Traits.

Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) analysis was used to investigate variation in the ovine KAP6-3 gene (KRTAP6-3) in 383 Merino × Southdown-cross lambs from four sire-lines, and to determine whether this variation affects wool traits. Five PCR-SSCP banding patterns, representing five different nucleotide sequences, were detected, including four previously identified (named A, B, C, and F) variants and one newly identified (named G) variant. A new non-synonymous single nucleotide polymorphism (SNP) and a 45-bp deletion were detected in variant G. Of the three common genotypes (AA, AB, and AG) identified in these sheep, wool from sheep that were AG, on average, had a lower mean fibre diameter (MFD), fibre diameter standard deviation (FDSD), and prickle factor (PF) than wool from AA sheep, whereas wool from AB sheep, on average, had a higher MFD, FDSD, and PF than wool from AA sheep. This suggests that variation in ovine KRTAP6-3 affect MFD, FDSD, and PF, and that this gene may have potential for use as a gene-maker for improving fibre diameter-associated wool traits.

Identification of the ovine keratin-associated protein 15-1 gene (KRTAP15-1) and genetic variation in its coding sequence

Keratin-associated proteins (KAPs) are the major component of wool fibres. The gene encoding the high sulphur-protein KAP15-1 has been described in humans and mice, but it has not been identified in sheep. A BLAST search of the Ovine Genome Assembly v4.0 using the coding sequence of caprine KRTAP15-1, identified a putative ovine KAP15-1 gene clustered within eleven other KAP genes on chromosome 1. PCR-Single Stranded Conformational Polymorphism (PCR-SSCP) analysis was used to screen amplicons derived from the gene in 732 sheep from six Chinese sheep breeds. Four different PCR-SSCP patterns, representing four unique DNA sequences (designated A-D) were detected. These sequences shared lower identity with other ovine KRTAP sequences, but the highest similarity was found with goat and human KRTAP15-1 sequences. This suggests that the sequences are variants of ovine KRTAP15-1. Among these four sequences, six nucleotide substitutions were identified in the coding region and four of the substitutions were non-synonymous. Breed differences in variant frequencies were observed. A was the most common variant in all the breeds. The next common variant differed in frequency between breeds. These results indicate that ovine KRTAP15-1 is variable and the differences between the Chinese breeds investigated suggest the variation might underpin variation in wool traits.

CDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India.

Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the full-length coding sequence of the caprine IGF1 gene from Attappady Black and Malabari breeds, two indigenous goat breeds of south India, to analyse its structure, and to ascertain the relative abundance of IGF1 mRNA in different tissues. The caprine IGF1 cDNA (GenBank accession nos: KJ549851 and KJ549852) contained a 465-bp open reading frame encoding IGF1 protein with 154 amino acid residues. A novel SNP was detected in the 3'UTR region, g.931A>G. Genotyping was performed in 277 goats from the two genetic groups using the PCR-single strand conformational polymorphism (SSCP) and two genotypes, AA and AG were observed at this locus. IGF1 is a secretary pathway protein with 49 amino acid-long signal peptide with 19 phosphorylation sites. Caprine IGF1 amino acid sequence was 83-99% identical to other species with highest identity with the ruminants. Relative expression of IGF1 was highest in uterus and liver (P < 0.05), followed by oviduct and muscle. This work provided an important experimental basis for further research on the functions of IGF1 in goats.

Variation in the ovine MYF5 gene and its effect on carcass lean meat yield in New Zealand Romney sheep

Myogenic factor 5 (MYF5) plays an important role in regulating skeletal muscle, but to date there have been no reports on whether the gene is variable and whether this variation is associated with meat yield in sheep. In this study, four variants (A to D) of ovine MYF5 containing two Single Nucleotide Polymorphisms (SNPs) and one basepair (bp) insertion/deletion were detected by Polymerase Chain Reaction – Single Stranded Conformational Polymorphism (PCR-SSCP) analysis. Breed differences in variant frequencies were observed. The effect of variation in ovine MYF5 on lean meat yield, predicted using VIAScan® technology, was investigated in 388 male NZ Romney lambs. Only genotypes AA and AB were found in these lambs. Lambs with genotype AA had a higher leg yield (P=0.044), loin yield (P=0.002) and total yield (P=0.012) than those with genotype AB. No association with shoulder yield was detected. These results suggest that ovine MYF5 may be a valuable genetic marker for improved lean meat yield.

Mannose-Binding Lectin (MBL) gene polymorphisms in susceptibility to pulmonary tuberculosis among the Lur population of Lorestan Province of Iran

ObjectiveTuberculosis (TB) is caused by infection of Mycobacterium tuberculosis. Host genetic variability is an important determinant of the risk of developing TB in humans. Although the association between MBL polymorphisms and TB has been studied in various populations, the results are controversial. The aim of this study was to investigate mannose-binding lectin (MBL) gene polymorphisms with susceptibility to pulmonary tuberculosis (PTB) in a Lur population of Iran. MethodsIn this case-control study, four functional MBL gene polymorphisms (HL, XY, PQ and AB) were genotyped by using PCR Single Strand Conformation Polymorphism (SSCP) technique in a Lur population living in Lorestan Province, consisting of 100 patients with pulmonary tuberculosis (PTB) age and sex matched 100 healthy controls (HCs). Association analyses were performed with the SPSS 21 statistical software. ResultsWe found that MBL (HH) genotype polymorphism significantly was associated with increased susceptibility to TB (35% in patients vs. 22% in controls, P=0.0417, OR=1.909, %95 CI=1.020–3.573). Additionally, H allele showed a significant association with increased risk of TB (56.5% in patients vs. 46% in controls, P=0.0357, OR=1.525, %95 CI=1.028–2.262). Also, the distribution of L allele in patients was significantly lower frequency in TB patients compared to controls (43.5% vs. 54%, P=0.0357, OR=0.656, %95 CI=0.442–0.973). However, the allelic and genotypic frequencies of AB, XY and PQ polymorphisms were not significantly different between the patients and the controls. We couldn't detect any significant differences between haplotypes among TB patients and healthy controls. ConclusionsOur findings demonstrated that HH genotype and H allele may increase the susceptibility to pulmonary TB in the Lur population of Iran, although L allele may decrease the susceptibility to pulmonary TB in this population. We suggest that it is necessary to further more studies with larger sample size and other ethnic population.

The clinicopathologic relevance of RECK gene polymorphisms in ameloblastoma

ObjectiveTo investigate the relationship between RECK gene polymorphisms and the clinicopathologic features of ameloblastoma. DesignNormal gingival mucosa specimens were obtained from 10 healthy volunteers. Ameloblastomas were surgically removed from 30 patients and part of the tumor specimens were used to detect RECK gene polymorphisms by using PCR-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing analysis. Expression of RECK and MMP-9 protein was measured using western blot. ResultsThe overall SNP rate was 46.7% (14/30). Four polymorphisms were detected in exon 9, 11, 13, 15 of the RECK gene: two synonymous (P520P and R625R) and two missense SNPs (V275I and I395V). RECK protein expression in specimens with minor RECK SNPs was lower than that in specimens without RECK SNPs (P<0.05), and, RECK protein expression in specimens with and without RECK SNPs was lower than that in the normal gingiva specimens (P<0.05). MMP-9 protein expression in specimens with minor RECK SNPs was higher than that in specimens without RECK SNPs (P<0.05), and MMP-9 protein expression in specimens with and without RECK SNPs was higher than that in normal gingiva specimens (P<0.05). RECK gene polymorphisms were closely associated with active proliferation, capsular invasion, and clinical recurrence of ameloblastoma. ConclusionThe rs16932912(G/A) SNP in the RECK gene was closely associated with active proliferation, capsular invasion, and clinical recurrence of ameloblastoma. RECK protein expression was closely associated with the presence of the rs16932912(G/A) SNP.

Molecular Effects of Atmospheric Pressure Plasma Jet on the Double-Stranded DNA

Introduction The aim of this study was toinvestigate the sterilization potential of atmospheric pressure plasma jet (APPJ) and interactions of this technology with double-stranded DNA using the polymerase chain reaction (PCR) and single-strand conformation polymorphism (SSCP) techniques. Materials and Methods The plasma jet was produced through a high voltage sinusoidal power supplyusing a mixture of argon and oxygen gases with theflow rate of 1 L/min. Escherichia coli cells and double-stranded DNA (dsDNA) fragments were amplified by T7 universal primer through the PCR technique and treated with argon/oxygen APPJ at different exposure times. The data were analyzed by the agarose and polyacrylamide gel electrophoresis, SSCP and renewed PCR techniques. Results According to the results of the study, the APPJ could serve as an effective instrument for sterilization at > 30 sec discharge. The destruction of DNA was detectable by different techniques after 120 sec from APPJ discharge. Conclusion Our findings revealed that the active species of plasma can lead to cell death. These species may break or nick the dsDNA, exchange DNA nucleotides, and lead to transition and transversion mutations. These mutagenesis effects of APPJ might be the reason of microorganism cell death after the treatment in addition to other destructive effects of APPJ on macromolecules.

Identification of the Ovine Keratin-Associated Protein 22-1 (KAP22-1) Gene and Its Effect on Wool Traits.

Keratin-associated proteins (KAPs) are structural components of wool and hair fibers. To date, eight high glycine/tyrosine KAP (HGT-KAP) families have been identified in humans, but only three have been identified in sheep. In this study, the putative ovine homolog of the human KAP22-1 gene (KRTAP22-1) was amplified using primers designed based on a human KRTAP22-1 sequence. Polymerase chain reaction-single stranded conformational polymorphism (PCR-SSCP) was used to screen for variation in KRTAP22-1 in 390 Merino × Southdown-cross lambs and 75 New Zealand (NZ) Romney sheep. Three PCR-SSCP banding patterns were detected and DNA sequencing revealed that the banding patterns represented three different nucleotide sequences (A–C). Two single nucleotide polymorphisms (SNPs) were identified in these sequences. Variant B was most common with a frequency of 81.3% in NZ Romney sheep, while in the Merino × Southdown-cross lambs, A was more common with a frequency of 51.8%. The presence of B was found to be associated with increased wool yield and decreased mean fiber curvature (MFC). Sheep of genotype BB or AB had a higher wool yield than those of genotype AA. These results suggest that ovine KRTAP22-1 variation may be useful when developing breeding programs based on increasing wool yield, or decreasing wool curvature.

Associations of polymorphisms in the prolactin receptor gene with growth trait in Japanese quail (Coturnix coturnix japonica)

Prolactin receptor (PRLR), as an important regulatory gene about growth and differentiation, might be a condition gene for reproductive traits. The prolactin receptor is a specific receptor for prolactin (PRL), which is an anterior pituitary peptide hormone involved in various physiological activities and is essential for reproductive improvement. In chickens, the PRLR gene is on the Z chromosome. In the present study, the polymorphism of exon 2 of PRLR gene in Japanese Quail (Coturnix coturnix japonica) was investigated. Blood samples of 180 Japanese Quail were collected randomly. DNA was extracted from blood samples and amplified. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis of the exon 2 of PRLR gene. PCR-SSCP analysis of the exon 2 region revealed two banding patterns. Two different SSCP patterns, representing two different genotypes, were identified. The frequencies of the observed genotypes were 0.939 and 0.061 for AA and BB. Allele frequencies were 0.939 and 0.061 for A and B. Observed heterozygosity (Hobs) value was 0.0. The chi-square test showed significant (P&lt;0.05) deviation from Hardy-Weinberg equilibrium for this locus in studied population. The effect of these genotypes on weight gain was investigated, and the BB genotype was found to be associated with body weight at 46 days. This was the first study on polymorphism of PRLR gene in Japanese quail.

Sequence variations in estrogen receptor 1 and 2 genes and their association with egg production traits in Chinese Dagu chickens.

Estrogen receptors α (ESR1) and β (ESR2) play central roles in folliculogenesis and therefore in reproductive biology. In the present study, two single nucleotide polymorphisms (SNPs) were identified in the ESR1 and ESR2 genes using PCR-single strand conformation polymorphism (PCR-SSCP) and DNA sequencing. One of the identified SNPs, a T1101C transition located within exon 4 of the ESR1 gene, was significantly associated with hen-housed egg production (HHEP) at 30, 43, 57 and 66 weeks of age (P<0.05), and egg weight (EW) at 30 weeks (P<0.05). Another SNP, a G1755A transition leading to a non-synonymous substitution (valine 459-to-isoleucine) located within exon 8 of the ESR2 gene, was also markedly correlated with the HHEP at 30, 43, 57 and 66 weeks of age (P<0.05), and EW at 30 weeks (P<0.05). A greater proportion of the additive variance was explained by the SNPs for most of the associated egg production traits (>1%). Furthermore, the results of the combined genotype-based association analysis supported the finding that the two SNPs were associated with the traits under a study. Taken together, our findings suggest that the two sequence variations in the ESR1 and ESR2 genes may provide promising genetic markers for the early selection and prediction of advantageous phenotypes in chicken breeding.

Association of interleukin-17 single nucleotide polymorphisms with susceptibility to osteoarthritis

Objective To test single nucleotide polymorphisms (SNP) of IL-17A G-197A and IL-17F T7488C in the Chinese Han nationality population and analyze the association of the SNP with susceptibility to osteoarthritis. Methods Two hundred patients with primary knee OA underwent total knee arthroplasty and 200 healthy individuals were recruited into this study from January 2008 to December 2015. We obtained DNA from 2 ml of whole blood for 200 OA patients (OA group) and 200 healthy (healthy control group) to employed a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) assay to identify the genotypes IL-17A G-197A (rs2275913) and IL-17F T7488C (rs2275913). The distribution of IL-17A G-197A (rs2275913) and IL-17F T7488C (rs2275913) genotypes and allele frequencies were detected between the two groups. Chi-square test was used to compare genotype and allele frequency of IL-17A G-197A and IL-17F T7488C between the two groups. If the difference between the two groups was statistically significant, then odds ratio (OR) and 95% confidence interval (95%CI) would be calculated. Results The frequency of AA, AG and GG genotype of IL-17A G-197A(rs2275913) was 4.0%, 54.0% and 42.0% in OA group, which differed from control group (18.0%, 53.5% and 28.5%). The frequency of A and G allele were 31.0% and 69.0% in OA group, which differed from control group (44.8% and 55.3%). The risk of OA in carriers with an G allele (G/A or G/G genotypes) was statistically higher compared to that of carriers with the A/A genotype (AG: OR=0.862, 95%CI=0.729-1.019; GG: OR= 1.259, 95% CI=0.967-1.639; A:OR=0.693, 95% CI=0.577-0.831; G:OR=1.249, 95% CI=1.119-1.394). The distributions of TT, TC and CC genotypes for the IL-17F T7488C polymorphism (rs763780) in OA patients and controls shown 76.5%, 18.0%, 5.5% and 81.5%, 15.5%, 3.0%,respectively. The frequency of T and C allele were 85.5%, 14.5% and 89.3%, 10.8% in 2 group. There were no significant differences in the alleles frequency and genotypes distribution for IL-17F T7488C between OA patients and volunteers. Conclusion Polymorphism of IL-17A G-197A may be closely associated with susceptibility to the development of OA in the Chinese Han nationality population. Genotype G/A and G/G of rs2275913 is a high factor for OA while AA is a low risk factor. However, there is no relationship between rs763780 and OA susceptibility. IL-17F T7488C gene does not increase the risk of suffering from OA. Key words: Osteoarthritis, knee; Interleukins; Genetic testing; Disease susceptibility

Nucleotide variability in Beta 2 Microglobulin (b2M) gene and its association with colostral IgG levels in buffaloes (Bubalus bubalis)

The present study was taken up to find out Beta 2 Microglobulin (b2M) gene variants and their association with IgG concentrations in colostrum of Murrah buffaloes. 40 newly parturated Murrah buffaloes dams were included in the present study. Polymerase chain reaction- single stranded conformational polymorphism (PCR-SSCP) technique was used to explore the polymorphism in b2M gene of buffaloes. The PCR-SSCP analysis and sequencing of different patterns obtained by silver staining of fragments pertaining to exon 1 through 4 and partial introns 1 through 3, revealed 9 SNPs which were further used to prepare the haplotypes. 7 different haplotypes were observed. Indirect ELISA was done to estimate IgG concentrations in colostrum (collected 1 hour before first milking) obtained from buffaloes. IgG levels in colostrum ranged from 11.22 to 185.1 mg/ml with a mean IgG concentration of 51.71 ± 5.99 mg/ml. The effect of different haplotypes was evaluated on IgG concentrations. The least square analysis of variance could not exhibit a significant effect of buffalo dam haplotype on colostral IgG concentration.

Sequence characterization and polymorphism detectionin AQP7 gene of Murrah buffalo

Aquaporin 7 (AQP7) gene is a member of aqua-glyceroporins which transports glycerol and water to spermatids. The present study aimed to investigate the polymorphisms within exons 2, 3, 4, 5 and their flanking intronic regions in AQP7 gene of Murrah bulls. Genomic DNA was extracted from 69 Murrah bulls blood samples and was subjected to polymerase chain reaction- single strand conformation polymorphism (PCR-SSCP) analysis. PCR-SSCP analysis revealed a total of eight different variants for amplicons of exons 4 and 5. Amplicon of exon 4 revealed three different patterns viz. E4P1, E4P2 and E4P3 with the frequency of 0.30, 0.22 and 0.48, respectively. Analysis of exon 5 revealed five unique SSCP patterns viz. E5P1, E5P2, E5P3, E5P4 and E5P5, with the frequency of 0.10, 0.37, 0.20, 0.20 and 0.13, respectively. Sequence analysis showed 16 single nucleotide polymorphisms, 7 of which were observed in coding region. Amplicons of exons 2 and 3 showed monomorphic patterns. However, compared to the reference sequence of taurine cattle one transition (C6878T) in exon 3 and 4 transitions (G2099A, C2116T, A2117G, G6848C) in intron 2 were observed for all the bulls under study. The genetic variability identified in the AQP7 gene may serve as potential genetic marker(s) for semen quality traits in buffalo.

Identification of fungal candidates for asthma protection in a large population-based study.

Exposure to molds has been related to asthma risk both positively and negatively, depending on the environmental setting. The pertinent results are based on generic markers or culturing methods although the majority of present fungi cannot be cultured under laboratory conditions. The aim of the present analysis was to assess environmental dust samples for asthma-protective fungal candidates with a comprehensive molecular technique covering also non-cultivable and non-viable fungi. Mattress dust samples of 844 children from the GABRIELA study were analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) of the fungus-specific internal transcribed spacer (ITS) region. Known asthma candidate species were tested for their associations with asthma, and further gel positions were sought to explain the above. As a second, data-driven, analysis, we tested the association of each individual gel position with asthma. In the hypothesis-driven approach, Penicillium chrysogenum emerged with an odds ratio of 0.80 (95% confidence interval 0.66-0.96; p=0.020). The effect size was changed by 39% toward the null when adjusting for the two bands 683 (DNA of Metschnikowia sp., Aureobasidium spp.) and 978 (DNA of Epicoccum spp., Galactomyces spp., uncultured Penicillium). The data-driven approach yielded an additional band (containing DNA of Pseudotaeniolina globosa) with reduced risk of asthma (OR=0.80 [0.66-0.96], p=0.012). A large population-based study revealed several fungal taxa with inverse associations with childhood asthma. Molds produce a variety of bioactive compounds with detrimental but also beneficial immunoregulatory capacities, which renders them promising targets for further asthma research.