Reaction-single Strand Conformation Polymorphism Analysis Research Articles

Expression and Variations in EPAS1 Associated with Oxygen Metabolism in Sheep.

Endothelial PAS domain protein 1 gene (EPAS1) is a member of the HIF gene family. This gene encodes a transcription factor subunit that is involved in the induction of oxygen-regulated genes. Several studies have demonstrated that a mutation in EPAS1 could affect oxygen sensing, polycythemia, and hemoglobin level. However, whether EPAS1 mutation affects sheep oxygen metabolism is still unknown. Therefore, we explored the relationship between the variation of EPAS1 and oxygen metabolism in sheep. In this study, variations in ovine EPAS1 exon 15 were investigated in 332 Tibetan sheep and 339 Hu sheep by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. In addition, we studied the effect of these variations on blood gas in 176 Tibetan sheep and 231 Hu sheep. Finally, the mRNA expression of EPAS1 in six tissues of Hu sheep and Tibetan sheep living at different altitudes (2500 m, 3500 m, and 4500 m) was analyzed by real-time quantitative PCR (RT-qPCR). Four alleles (A, B, C, and D) were detected, and their distributions highly differed between Tibetan sheep and Hu sheep. In Tibetan sheep, B was the dominant allele, and C and D alleles were rare, whereas all four alleles were common in Hu sheep. Six single nucleotide polymorphisms (SNPs) were identified between the four alleles and one of them was non-synonymous (p.F606L). While studying the blood gas levels in Tibetan sheep and Hu sheep, one variant region was found to be associated with an elevated pO2 and sO2, which suggested that variations in EPAS1 are associated with oxygen metabolism in sheep. RT-qPCR results showed that EPAS1 was expressed in the six tissues of Hu sheep and Tibetan sheep at different altitudes. In addition, the expression of EPAS1 in four tissues (heart, liver, spleen, and longissimus dorsi muscle) of Hu sheep was lower than that in Tibetan sheep from three different altitudes, and the expression of EPAS1 was positively correlated with the altitude. These results indicate that the variations and expression of EPAS1 is closely related to oxygen metabolism.

Identification of the KAP27-1 gene in sheep and its effect on wool traits

ABSTRACT The keratin-associated proteins (KAPs) are structural components of wool and hair fibres, with over 90 genes that encode these proteins having been identified in mammalian species. A KAP gene called KRTAP27-1 was recently identified in goats, but the gene has not been reported in the closely related ruminant species sheep. In this study, we used a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis to find ovine KRTAP27-1 and investigate nucleotide sequence variation in the gene. Our analysis revealed four sequence variants (named A to D) in 399 New Zealand Merino × Southdown-cross sheep. Within these sequence variants there were five single nucleotide polymorphisms (SNPs) in the coding region, including two non-synonymous SNPs. Six genotypes (AA, AB, AC, BB, BC and BD) of KRTAP27-1 were detected, and for the three genotypes with a frequency of over 5.0%, sheep of genotypes AB and BB produced wool of higher mean staple length (MSL) and higher greasy fleece weight (GFW) than sheep of genotype AA. These results suggest that variation in ovine KRTAP27-1 may be affecting wool growth and thus production.

Comparison of merits of DNA sequencing, PCR-SSCP and MFP assays in the detection of drug resistance in Mycobacterium leprae

Purpose: To compare the merits of conventional Mouse Foot Pad (MFP) assay, Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) analysis, and Direct-DNA sequencing in identification of drug resistance in M. leprae.
 Methods: A total of 41 leprosy cases were investigated for drug resistance using MFP assay, whereby growth results were obtained in 23 cases. The DNAs extracted from these samples were amplified in polymerase chain reaction (PCR) using specific primers in order to determine the drug resistance-loci. Furthermore, PCRSSCP analysis was carried out using PCR amplicons, while gel electrophoresis was performed to identify any shift in mobility in any one of the DNA strands, as well as the pattern of mutation.
 Results: A total of 5 isolates exhibited the highest degree of resistance R100, whereas 4 isolates showed intermediate level of resistance R10. In contrast, the least resistance was seen in R1. There were no mutations in 4 out of 10 strains which were dapsone-resistant, i.e., 1 HR, 2 IR and 1 LR. Moreover, there was no mutation in 305 bp sequence of the rpoB gene. DNA sequencing sensitivity was 60 %, whereas the sensitive isolates tested in the experiments exhibited no mutation, resulting in 100 % specificity.
 Conclusion: These results indicate the advantages of molecular techniques over conventional MFP technique. The study has revealed that PCR-SSCP analysis, a rapid, specific and less expensive method, has greater potential for use in routine testing of the susceptibility of M. leprae to rifampicin and dapsone, than the other tests.

Variation in a Newly Identified Caprine KRTAP Gene Is Associated with Raw Cashmere Fiber Weight in Longdong Cashmere Goats.

Keratin-associated proteins (KAPs) and keratins determine the physical and chemical properties of cashmere fibers as they are the main components of the fibers. It has been reported that ovine KRTAP1-2 affects clean fleece weight, greasy fleece weight and yield in sheep, but the gene has not been described in goats and its effects on fiber traits are unknown. In this study, we identify the keratin-associated protein 1-2 gene (KRTAP1-2) in the goat genome and describe its effect on cashmere fiber traits in 359 Longdong cashmere goats. Six sequence variants (named CAPHI-KRTAP1-2*A to CAPHI-KRTAP1-2*F) were revealed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. These sequences have the highest homology with ovine KRTAP1-2 sequences. There were a 60-bp deletion, a 15-bp insertion and five single nucleotide polymorphisms (SNPs) including two non-synonymous SNPs in the coding sequence. The caprine KRTAP1-2 gene was expressed in the skin tissue, but a signal was not observed for the kidneys, liver, lungs, spleen, heart and longissimus dorsi muscle. Variation in caprine KRTAP1-2 was found to be associated with raw cashmere fiber weight, but not with fiber diameter and length.

Identification of the Ovine Keratin-Associated Protein 2-1 Gene and Its Sequence Variation in Four Chinese Sheep Breeds.

Keratin-associated proteins are important components of wool fibers. The gene encoding the high-sulfur keratin-associated protein 2-1 has been described in humans, but it has not been described in sheep. A basic local alignment search tool nucleotide search of the Ovine Genome Assembly version 4.0 using a human keratin-associated protein 2-1 gene sequence revealed a 399-base pair open reading frame, which was clustered among nine previously identified keratin-associated protein genes on chromosome 11. Polymerase chain reaction–single strand conformation polymorphism analysis revealed four different banding patterns, with these representing four different sequences (A–D) in Chinese sheep breeds. These sequences had the highest similarity to human keratin-associated protein 2-1 gene, suggesting that they represent variants of ovine keratin-associated protein 2-1 gene. Nine single nucleotide variations were detected in the gene, including one non-synonymous nucleotide substitution. Differences in variant frequencies between fine-wool sheep breeds and coarse-wool sheep breeds were detected. The gene was found to be expressed in various tissues, with the highest expression level in skin, and moderate expression levels in heart and lung tissue. These results reveal that the ovine keratin-associated protein 2-1 gene is variable and suggest the gene might affect variation in mean fiber diameter.

Variation in the Lipin 1 Gene Is Associated with Birth Weight and Selected Carcass Traits in New Zealand Romney Sheep.

Simple SummaryLipin 1 plays an important role in lipid metabolism. It not only acts as a phosphatidate phosphatase and directly participates in the synthesis of glycerol and osteolipid, but also acts as a transcriptional co-activator to indirectly regulate the expression of genes related to lipid metabolism. In livestock species, variation in the lipin 1 gene (LPIN1) has been reported in pigs, chickens, and dairy cows, but has not been investigated in sheep, and little is known about whether it might affect production traits in this globally important meat-producing species. In this study, we used polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analyses to search for variation in ovine LPIN1, and its effects on production and carcass traits were investigated in New Zealand Romney sheep. The results suggest that ovine LPIN1 is variable and it may have value as a genetic marker for improving meat production and carcass traits.Lipin 1 plays an important role in lipid metabolism. In this study; we searched for variation in the ovine lipin 1 gene (LPIN1) in three gene regions (a 5′ non-coding region; a region containing an alternatively spliced exon in intron 4; and a region containing coding exon 6) using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The greatest amount of alleles was found in coding exon 6; with five sequences being detected. The effect of variation in this exon was investigated in 242 New Zealand Romney lambs derived from 12 sire-lines. The presence of variant E3 was associated with a decrease in birth weight (p = 0.005) and the proportion of leg yield (p = 0.045), but with an increase in hot carcass weight (p = 0.032) and the proportion of loin yield (p = 0.014). The presence of variant B3 was associated with an increased pre-weaning growth rate (p = 0.041), whereas the presence of variant C3 was associated with an increase in shoulder yield (p < 0.001). These results suggest that ovine LPIN1 variation may have value as a genetic marker for improving meat production and carcass traits.

Analysis of the VSX1 gene in sporadic keratoconus patients from China

BackgroundKeratoconus normally presents as a sporadic disease. Although different studies have found sequence variants of the visual system homeobox 1 (VSX1) gene associated with keratoconus in humans, no research has detected such variants in sporadic keratoconus patients from China. To investigate the possibility of VSX1 being a candidate susceptibility gene for Chinese patients with sporadic keratoconus, we performed sequence screening of this gene in such patients.MethodsWhole DNA was obtained from the leukocytes in the peripheral venous blood of 50 patients with sporadic keratoconus and 50 control subjects without this ocular disorder. Polymerase chain reaction single-strand conformation polymorphism analysis and direct DNA sequencing technology were used to detect sequence variation in the five exons and splicing regions of the introns of the VSX1 gene. The sequencing results were analyzed using DNAstar software.ResultsOne novel missense heterozygous sequence variant (p.Arg131Pro) was found in the first exon of the VSX1 gene in one keratoconus patient. Another heterozygous sequence variant (p.Gly160Val) in the second exon was found in two keratoconus patients. These variants were not detected in the control subjects. In the third intron of the VSX1 gene, c.8326G > A nucleotide substitution (including heterozygous and homozygous change) was also discovered. The frequency of this variation did not differ significantly between patients and controls, it should belong to single-nucleotide polymorphism of the VSX1 gene. Bioinformatic analysis also predicted that one missense sequence variation (p.Arg131Pro) may not cause a pathogenic change.ConclusionsIn this study, we added one novel missense sequence variation (p.Arg131Pro) in the coding region of the VSX1 gene to the range of VSX1 coding region variations observed in patients with sporadic keratoconus from China. Our work suggests that VSX1 sequence variants might be involved in the pathogenesis of sporadic keratoconus, but their precise role in disease causation requires further investigation.

A 2-base insertion in exon 5 is a common mutation of the TP53 gene in dogs with histiocytic sarcoma.

Canine histiocytic sarcoma (HS) is a malignancy originating from the histiocytic cell lineage and characterized by poor response to chemotherapy and short survival time. Mutation of the TP53 gene and its association with poor prognosis has been reported in several canine tumors. However, the mutation of this gene has not been investigated in canine HS. The aim of this study was to examine a TP53 gene mutation in dogs with HS. Aberrations of the TP53 gene were examined by polymerase chain reaction-single strand conformational polymorphism analysis and DNA sequence analysis, revealing mutations of the TP53 gene in 12 (46%) of 26 dogs affected by HS. The incidence of the TP53 gene mutation was relatively high in canine HS compared with other canine tumors. Among these mutations, 10 of 12 dogs (83%) with a TP53 gene mutation harbored the same mutation: a 2-base (AT) insertion in exon 5, resulting in the introduction of a stop codon (c.446_447insAT, p.Tyr150SerfsX8). Further studies are needed to examine the functional change due to the mutation and its association with the pathogenesis of canine HS.

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

Abstract 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&gt;T; c.226A&gt;G; c.242G&gt;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&gt;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.

Detection of Genetic Diversity in Four Candidate Genes by Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) Analysis of Goats in Sri Lanka

Genetic diversity of Sri Lankan goats (Capra hircus) was evaluated using Polymerase Chain Reaction-Single Strand Conformation Polymorphism (PCR-SSCP) method to detect polymophism in four candidate genes (LALBA, k-CSN3, GnRHR and BMP4) in a sample of 219 goats representing three phenotypic groups. All studied loci were polymorphic, having three morphs for BMP4, LALBA and two morphs for k-CSN3 and GnRHR in the study population. A significant difference between genotypic frequencies for BMP4 and GnRHR genes was found while PIC criterion revealed an intermediate polymorphism for all analyzed phenotypic groups except for k-CSN3 and GnRHR genes. Cross-bred animals for BMP4 gene and nondescript animals for LALBA gene were comparatively more polymorphic according to the effective allele number criterion. This study provides insight into the genetic diversity of Sri Lankan goats, which can be utilized to develop single nucleotide polymorphism markers to be used in association studies, and marker assisted selection.Int J Appl Sci Biotechnol, Vol 4(1): 133-138

Genetic variability of bovine GHR, IGF-1 and IGFBP-3 genes in Indian cattle and buffalo

The identification of genetic polymorphisms in the genes that play a crucial role in regulatiing growth and development of livestock enables us to evaluate the biological similarities and to acquire a better perspective of quantitative traits. The present study was undertaken to characterize genetic variability in the bovine growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) genes among Bos indicus (Malnad Gidda, Khillar), Bos taurus (Holstein Friesian, Jersey) cattle and Asian water buffalo Bubalus bubalis (Murrah, Surti) using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. These polymorphisms were confirmed by direct sequencing. The comparative gene sequence analysis in cattle and buffalo breeds revealed 18 single nucleotide polymorphisms (SNPs) across different loci. Eight SNPs were detected in the bovine growth hormone receptor (GHR) gene, of which four were found in the promoter region and four in the exon 4 region. In the IGF-1 gene, two SNPs were observed in the 5ˈUTR, three SNPs in the intron 3 region and two SNPs in the coding region of exon 4. Three SNPs were detected in the exon 2 region of the bovine IGFBP-3 gene. The frequency of rare alleles observed in the present study ranged from 0.04 to 0.16. The present results revealed high levels of genetic variability in the GHR, IGF-1 and IGFBP-3 genes in cattle and buffalo reared in India. Keywords: PCR-SSCP, genetic polymorphism, cattle, buffalo

Single Nucleotide Polymorphism in the 5′-Upstream Region of Activin A Receptor, Type IIB in Aseel Chicken

Activin A receptor, type IIB (ACVR2B) is a protein that is encoded by the ACVR2B gene, which plays a crucial role during muscle development in chicken. The present investigation was conducted to ascertain the nucleotide variability in the 5′-upstream regulatory region of ACVR2B and its association with body weight at different ages in Aseel chicken. Polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) analysis of the regulatory region revealed two novel mutations (T68A and C172T) in the 5′-untranslated region of ACVR2B. However, both the mutations observed were common to all the birds involved in the current study, indicating that 5′-upstream regulatory region of ACVR2B gene was monomorphic in Aseel. Being monomorphic, no relationship was found with growth traits in Aseel. Absence of polymorphism in the regulatory region suggests that the regulation pattern of ACVR2B gene might be consistent with all birds in this region.

CORRELATION ANALYSIS BETWEEN MYOSTATIN GENE POLYMORPHISMS AND CARCASS TRAITS IN NEW ZEALAND ROMNEY SHEEP

Using genetic markers can aid identifying those animals with the highest values for economically important traits in sheep. The current study was designed to detect the allelic and genotypic polymorphisms of the intron 1 of myostatin gene and to test their associations with carcass traits (slaughtering weight, dressing%, shoulder yield, loin yield, leg yield, total yield, shoulder yield%, loin yield% and leg yield%) in 529 male lambs of New Zealand Romney sheep. The polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to identify the allelic and genotypic polymorphisms in intron 1 of myostatin gene for 529 males from New Zealand Romney lambs. Associations of the variation in the intron 1 of myostatin gene with carcass traits were determined using the general linear model (GLM) procedure of SAS (2000). The SSCP analysis revealed six SSCP genotypes: AA, AB, AC, BB, BC and CC with frequencies of 0.107, 0.368, 0.100, 0.289, 0.129 and 0.005, respectively, that derived from three identified alleles: A, B and C with frequency 0.34, 0.54 and 0.12, respectively. Myostatin genotype significantly affected (P˂0.05) slaughtering weight and total yield, and highly significant affected (P˂0.001) dressing%, leg yield, shoulder yield%, loin yield% and leg yield%. The presence of A allele in animal genotype was associated with higher leg yield and leg yield%, however, the presence of B allele was associated with higher loin yield and loin yield%. The LSM showed that, lambs with two copies of A allele had the highest dressing%, leg yield, leg yield% and total yield, however, lambs with two copies of B allele had the highest shoulder yield, shoulder yield% and loin yield%. The results presented here give valuable information to select for A and B alleles and against C allele to improve the most important primal cuts of lambs across most production systems.

Variation in the bovine FABP4 gene affects milk yield and milk protein content in dairy cows.

Fatty acid binding proteins (FABPs) bind long-chain fatty acids and are involved in their intracellular transport. Of the known bovine FABP genes, FABP4 has been mapped to a region on chromosome 14 that contains quantitative trait loci for milk traits. This study investigated the association of FABP4 haplotypes with milk production traits in 719 Holstein-Friesian × Jersey cows. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis of a variable region of the gene revealed three haplotypes (A, B and C). Five single nucleotide polymorphisms (SNPs) were identified: two in exon 3 and three in intron 3. A was associated (P = 0.032) with increased milk protein percentage (present: 4.00 ± 0.02%; absent: 3.95 ± 0.02%) and B was associated (P = 0.009) with increased milk yield (present: 23.81 ± 0.23 kg/d; absent: 23.06 ± 0.21 kg/d), but tended to be associated with a decrease in protein percentage and an increase in protein yield. Cows with genotypes AA, AB and AC produced less milk, but with a higher protein percentage than BC cows. This suggest that FABP4 affects milk yield and milk protein content, both economically important traits, and that further study of this gene is warranted.

Molecular characterization of the HSPA1A gene by single-strandconformation polymorphism and sequence analysis in Holstein-Friesiancrossbred and Deoni cattle raised in India

A group of proteins called heat shock proteins (HSPs) are synthesized during heat stress. They are expressed at high levels when exposed to stress. Polymorphism in HSPs has been reported to be associated with heat tolerance and reproductive performance in cattle. The present study aimed to investigate the polymorphisms in the coding region of the HSPA1A gene in Holstein-Friesian (HF) crossbreds and the Deoni breed of cattle. Genomic DNA was extracted from 94 animal blood samples and was subjected to polymerase chain reaction-single strand conformation polymorphism analysis, which revealed 14 band patterns in Deoni cattle and 8 band patterns in HF crossbreds. Sequence data were analyzed using BioEdit software for detecting single nucleotide polymorphisms. Sequence analysis showed 12 single nucleotide polymorphisms in the coding region of the HSPA1A gene, which included 5 transitions (G456A, A972G, A1098G, C1766T, and G1788A) and 2 transversions (C312G and G2033C) in Deoni cattle and 2 indels (C at positions 574?575 and 624?625), 2 transitions (A480G and A1098G), and 1 transversion (C312G) in HF crossbred cattle. The study indicated a high degree of genetic variability in the HSPA1A gene in the cattle populations under study.

VARIATION IN THE FORKHEAD BOX CLASS O3 (FOXO3) GENE AND ITS ASSOCIATION WITH LIFESPAN TRAITS IN BARKI EWES

In this study the polymorphisms in exon 2 of ovine Forkhead Box Class O3 (FOXO3) gene were detected in 96 Barki ewes by the polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis. The association of FOXO3 gene polymorphisms with lifespan traits was tested using general linear mixed effect models. Three alleles (A, B and C) were detected with frequencies of 0.57, 0.28 and 0.15, respectively. Also five genotypes (AA, AB, AC, BB and CC) were identified with frequencies of 0.26, 0.44, 0.18, 0.06 and 0.06, respectively. Genotypes of the FOXO3 gene were shown to be associated with age, twining rate, total number of lambs born per ewe (TNLB) and total weight of lambs born per ewe (TWLB). The presence of allele C was associated with longer age and increased TNLB, whereas the presence of allele B was associated with shorter age, increased twining rate and decreased rearing ability and total number of lams weaned per ewe (TNLW). The effect of the number of allele copies present was tested and it was found that the presence of two copies of allele C was associated with longer age and increased TNLB and the presence of two copies of allele B was associated with shorter age, increased twining rate and decreased rearing ability and TNLW. These results suggest that variation in ovine FOXO3 gene is associated with lifespan traits of Barki ewes.

Effects of genetic variants and mapping assignments of the ovine calpain regulatory subunit gene on chromosome 14

The aim of this study was to search genetic variants, to map the ovine calpain regulatory subunit gene (CAPN4S) on chromosomes, and to find associations between single nucleotide (nt) polymorphisms (SNPs) and weight traits. Genotyping was performed with 226 purebred, 133 crossbred and 122 animals from eight sheep reference families. The polymerase chain reaction–single strand conformational polymorphism analysis detected genetic variants in the genomic regions between exons 4 and 8. Nt polymorphisms were confirmed at positions 115 (G/C) with CAPN445, 71 (A/G) with CAPN456, and 25 (A/G) and 44 (A/T) with the CAPN478 segments. The Polypay and Targhee purebreds showed significantly different allele frequencies while the mixed population was placed between pure breeds. The two-point linkage analysis using the CRI-MAP version 2.4 identified locations of the CAPN4S segments, showing that CAPN4S is closely linked with the LSCV30 microsatellite marker on chromosome 14. The genotypes of g.45923178G>C had significant mean differences for BWT and WWT, meaning that the identified SNP may be used in marker assisted selection programs. A significant haplo-block was constructed between g.45924950T>C and g.45924969A>T, and major frequencies were observed as AA (0.727) and GT (0.267).

Genetic variations in the myostatin gene (MSTN) in New Zealand sheep breeds

Myostatin, which is also known as growth and differentiation factor 8 (GDF8), acts as a negative regulator of skeletal muscle growth. Variation in the myostatin gene (MSTN) has been associated with variation in muscularity in many animals including sheep. Polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) analysis was used to investigate MSTN in a diverse range of sheep breeds including the New Zealand (NZ) Romney, Coopworth, Corriedale, Dorper, Perendale, Suffolk, Merino, Dorset Down, Poll Dorset, Texel and other NZ cross-bred sheep. A total of 28 nucleotide substitutions were identified from nucleotide c.-1199 in the promoter region to c.*1813 (based on NCBI GenBank accession number DQ530260) and including the well-described substitution c.*1232G>A (MSTN g+6223G>A). Of these 28 substitutions, 3 were located in the promoter region, 3 in the 5'UTR, 11 in intron 1, 5 in intron 2 and 5 in the 3'UTR. One substitution in exon 1 (c.101G>A) potentially results in an amino acid substitution of glutamic acid (Glu) with glycine (Gly) at codon 34. Ten of these substitutions have not been reported previously. The genetic variation revealed in this study suggests this gene is more variable than hitherto reported and provides a foundation for future research into how this variation affects muscle and growth traits.

PTEN mutation, methylation and expression in breast cancer patients

The tumor suppressor gene, PTEN, has previously been demonstrated to be involved in breast tumorigenesis and tumor progression. The aim of the present study was to investigate the expression and significance of PTEN in breast carcinomas, to detect the mutation frequency of PTEN in sporadic breast carcinoma tissues and to determine the association between PTEN promoter methylation and gene expression. Immunohistochemical methods were used to analyze the expression of the PTEN gene in 146 cases of breast carcinoma and 10 cases of normal breast tissue closely adjacent to the carcinoma. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis was used to analyze conformation polymorphisms in 45 breast carcinoma and 10 normal breast tissues. Point mutations of abnormal single stranded conformation were detected by DNA sequencing. The methylation of the PTEN promoter was analyzed by methylation-specific PCR. Expression of PTEN was detected in 57.5% (84/146) of patients with breast carcinoma. By contrast, PTEN expression was detected in 100% of normal samples. Expression of PTEN was found to negatively correlate with the tumor size, the pathological stage and the expression of the estrogen receptor (ER) and the progesterone receptor (PR) in breast cancer. The 2-year disease-free survival of patients with a high expression of PTEN was higher compared with those with low PTEN expression (P<0.05). Missense mutations in exon 2 of PTEN were identified in 1/45 breast cancer cases. PTEN promoter methylation was detected in 31.1% (14/45) of breast carcinomas, of which 64.3% (9/14) were associated with a loss of PTEN expression. The tumor suppressor gene, PTEN, was abnormally expressed in the breast carcinomas. The number of PTEN mutations were low (1/45) in the sporadic breast cancer cases analyzed in the present study and PTEN promoter methylation may have been the main mechanism leading to the decreased expression of PTEN. These results indicate that PTEN is important for the tumorigenesis, development and prognosis of breast cancer.

Polymorphisms in MHC class Ia genes and resistance to IHNV in rainbow trout (Oncorhynchus mykiss)

Infectious haematopoietic necrosis virus (IHNV) is detrimental to the farming of rainbow trout (Oncorhynchus mykiss) and other salmonids in the Northern hemisphere. The major histocompatibility complex (MHC) plays a key role in immune response in invertebrates, as evidenced by the close correlation of MHC polymorphisms with disease resistance/susceptibility. To analyse the correlation between rainbow trout resistance and susceptibility to IHNV and genetic variation in exon 2 of MHC class Ia gene, UBA, we employed two approaches, namely, polymerase chain reaction-single strand conformation polymorphism analysis and cloning/sequencing. From 102 resistant and 82 susceptible individuals, a total of 12 alleles in UBA exon 2 (GenBank: JX136662–JX136673) were identified, including 11 novel alleles. The maximum number of these alleles in a single individual was four, suggesting that UBA exon 2 most likely resides on at least two loci in the genome. Most of the variations in UBA exon 2 were located in the peptide-binding region and were determined to have been subject to positive selection during evolution. Correlation analysis revealed that Onmy-UBA*0111 and Onmy-UBA*0107 are highly associated with IHNV susceptibility (P = 0.001), whereas Onmy-UBA*0101, Onmy-UBA*0102, and Onmy-UBA*0103 are highly related to IHNV resistance (P = 0.000). In addition, the three resistant alleles were predominant in the IHNV disease-resistant population; thus, these molecular markers can be used for anti-IHNV breeding of rainbow trout.