SNPs in the porcine <Emphasis Type="Italic">PPARGC1a</Emphasis> gene: Interbreed differences and their phenotypic effects

Due to its function, the peroxisome proliferative activated receptor-γ, coactivator-1α (PPARGC1A) gene is a candidate in the search for genes that may affect production traits in the pig. The purpose of this study was to screen for new SNPs in exon 8 of the porcine PPARGC1A gene and to test their possible association with production traits. Altogether 736 pigs representing five breeds Polish Landrace, n=242; Polish Large White, n=192; Hampshire, n=27; Duroc, 21; Pietrain, n=12) and synthetic line 990 (n=242) were scanned via SSCP assay. Four SNPs were found; two new ones: C/G (His338Gln) and G/A Thr359Thr), and two previously reported ones: C/A (Arg369Arg) and T/A Cys430Ser). The missense T/A and C/G SNPs demonstrated pronounced interbreed variability in terms of allele frequencies, including the exclusive presence of the C/G substitution in the Hampshire breed. The tested SNPs occurred in five putative haplotypes, and their frequency also differed substantially between breeds. The association of the SNPs with production traits was tested for G/A (Thr359Thr), C/A (Arg369Arg) and T/A (Cys430Ser) substitutions in Polish Large White, Polish Landrace and line 990. The analysis revealed only breed-specific associations. The T/A (Cys430Ser) SNP was related to the feed conversion ratio in the Polish Large White (P=0.02), and the silent G/A and C/A substitutions were respectively associated with abdominal fat in line 990 and backfat thickness in Polish Landrace (P=0.04). The combined effects of the substitutions were estimated as haplotype effects. Three significant contrasts between haplotypes were calculated, but the observed associations were again only breed-specific.     

Polymorphisms in OATP-C: identification of multiple allelic variants associated with altered transport activity among European- and African-Americans

The human organic anion transporting polypeptide-C (OATP-C) (gene SLC21A6) is a liver-specific transporter importantly involved in the hepatocellular uptake of a variety of endogenous and foreign chemicals. In this study, we demonstrate the presence of multiple functionally relevant single-nucleotide polymorphisms (SNPs) in OATP-C in a population of African- and European-Americans. Moreover, examination of 14 nonsynonymous polymorphisms indicated that genotypic frequencies were dependent on race. Functional assessment of 16 OATP-C alleles in vitro revealed that several variants exhibited markedly reduced uptake of the OATP-C substrates estrone sulfate and estradiol 17beta-d-glucuronide. Specifically, alterations in transport were associated with SNPs that introduce amino acid changes within the transmembrane-spanning domains (T217C (Phe-73 --> Leu), T245C (Val-82 --> Ala), T521C (Val-174 --> Ala), and T1058C (Ile-353 --> Thr)) and also with those that modify extracellular loop 5 (A1294G (Asn-432 --> Asp), A1385G (Asp-462 --> Gly), and A1463C (Gly-488 --> Ala)). Cell surface biotinylation experiments indicated that the altered transport activity of some OATP-C variants was due, in part, to decreased plasma membrane expression. Given the relatively high genotypic frequency of the T521C (14%) transition in European-Americans and the G1463C (9%) transversion in African-Americans, SNPs in OATP-C may represent a heretofore unrecognized factor influencing drug disposition.     

Mutations in lipopolysaccharide-binding protein (LBP) gene change the susceptibility to clinical mastitis in Chinese Holstein

Mastitis is an unsolved human challenge all dairy farms facing with, which leads to immeasurable economic loss to the farmers. LBP gene plays a vital role in the innate immune recognition of Gram-negative bacterium that is a major cause of bovine clinical mastitis, but little is known about LBP mutations and their effects on cows' susceptibility to clinical mastitis. In this study, PCR-SSCP method was adopted to analyze SNPs of LBP gene in Chinese Holstein for the first time. 17 SNPs were found in the promoter core region, exon1, exon2, exon3, exon4 and exon8. The mutation g.-81C?→?T in promoter leads to an AP-2 binding site lost. Two mutations, g.11T?→?C (4 Leu?→?Ser) and g.68G?→?C (23Gly?→?Ala) in signal peptide brought about molecular secondary structural change, meanwhile, g.11T?→?C made a Big-1 domain lost, and there was an N-myristoylation site at the g.68G/C locus. The three mutations above were in complete linkage disequilibrium in allele A. In mature LBP protein, five mutations were found: g.3034G?→?A(36Asp?→?Asn), g.3040A?→?G(38Asn?→?Asp), g.3056T?→?C(43Ile?→?Thr) in allele D; g.4619G?→?A(67Ala?→?Thr) in allele F; 19975G?→?A (282Val?→?Met) in allele J. And SNPs in allele D and F were in complete linkage disequilibrium, also in which 38Asn?→?Asp and 67Ala?→?Thr influenced the protein secondary structure. Prediction of the 3-D structure shows mutations 36Asp?→?Asn, 38Asn?→?Asp and 43 Ile?→?Thr were on the concave surface of LBP protein at barrel-N, 67Ala?→?Thr was in the apolar pocket at barrel-N. Motif analysis shows 36Asp?→?Asn causes loss of a CK2 phosphorylation site, 67 Ala?→?Thr forms a new PKC phosphorylation site. And 43Ile?→?Thr, 67Ala?→?Thr made hydrophobic amino acids to be hydrophilic amino acids. Interestingly, the morbidity of AB (mixed type g.-81C/T, g.11T/C, g.68G/C), CD (mixed type g.3034G/A, g.3040A/G, g.3056T/C) and EF (mixed type g.4619G/A) genotype cows are significant higher than others in this study (P?相似文献   

Identification of three SNPs in the porcine myostatin gene (MSTN)

Thirteen pairs of primers were designed for the entire porcine MSTN gene to enable PCR amplification for the detection of single nucleotide polymorphisms (SNPs) by a PCR-SSCP approach. Altogether 96.5% (1089/1128) of the encoding regions and 971 bp of the non-coding regions were screened. A total of three polymorphisms were identified with PCR-SSCP. They were located in the promoter, intron one and exon three regions of the gene. These polymorphisms were then confirmed to be point mutations (T --> A transversion, G --> A transition and C --> T transition respectively) by sequencing. Allele frequencies were determined for all three SNPs in several different pig breed populations. The polymorphisms were found to be rare in Western breeds, but much more common in Chinese breeds. Whether they have any relationship with the marked difference in lean meat mass between Western and Chinese breeds requires further study.     

Single nucleotide polymorphisms in the open reading frame of the stearoyl-CoA desaturase gene and resulting genetic variants in Canadian Holstein and Jersey cows.

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) from their saturated counterparts in the mammary gland and adipose tissue of ruminant animals. We hypothesize that single nucleotide polymorphisms (SNPs) in the SCD gene account for some of the differences in SCD activity, and consequently for some of the variations in CLA and MUFA content of milk fat between Holsteins and Jersey cows and within these two breeds. We analyzed the open reading frame of the SCD gene of 44 Holsteins and 48 Jerseys for SNPs by sequencing. Three SNPs: 702A --> G, 762T --> C and 878C --> T were identified in both breeds and a further SNP, 435G --> A, was unique to Holsteins. The SNPs characterized four different genetic variants in Holsteins: A (G(435)A(702)T(762)C(878)), A1 (A(435)A(702)T(762)C(878)), B (G(435)G(702)C(762)T(878)) and B1 (A(435)G(702)C(762)T(878)), with only variants A and B in Jerseys. SNP 878C --> T resulted in a non-synonymous codon change while the rest resulted in synonymous codon changes giving rise to two protein variants, A having alanine and B having valine. Allele A was the most prevalent in the two breeds. These differences may, therefore, contribute to existing variations in CLA and fat content between and within Canadian Holstein and Jersey cows.     

CD44 knock-down in bovine and human chondrocytes results in release of bound HYAL2

CD44 shedding occurs in osteoarthritic chondrocytes. Previous work of others has suggested that the hyaluronidase isoform HYAL2 has the capacity to bind to CD44, a binding that may itself induce CD44 cleavage. Experiments were developed to elucidate whether chondrocyte HYAL2: (1) was exposed on the extracellular plasma membrane of chondrocytes, (2) bound to CD44, (3) underwent shedding together with CD44 and lastly, (4) exhibited hyaluronidase activity within a near-neutral pH range. Enhancing CD44 shedding by IL-1β resulted in a proportional increase in HYAL2 released from human and bovine chondrocytes into the medium. CD44 knockdown by siRNA also resulted in increased accumulation of HYAL2 in the media of chondrocytes. By hyaluronan zymography only activity at pH 3.7 was observed and this activity was reduced by pre-treatment of chondrocytes with trypsin. CD44 and HYAL2 were found to co-immunoprecipitate, and to co-localize within intracellular vesicles and at the plasma membrane. Degradation of hyaluronan was visualized by agarose gel electrophoresis. With this approach, hyaluronidase activity could be observed at pH 4.8 under assay conditions in which CD44 and HYAL2 binding remained intact; additionally, weak hyaluronidase activity could be observed at pH 6.8 under these conditions. This study suggests that CD44 and HYAL2 are bound at the surface of chondrocytes. The release of HYAL2 when CD44 is shed could provide a mechanism for weak hyaluronidase activity to occur within the more distant extracellular matrix of cartilage.     

Genotyping of Novel SNPs in BMPR1B,BMP15, and GDF9 Genes for Association with Prolificacy in Seven Indian Goat Breeds

Goats form the backbone of rural livelihood and financial security systems in India but their population is showing decreasing trend. Improvement of reproductive traits such as prolificacy offers a solution to stabilize the decreasing goat population and to meet the nutritional needs of growing human population. In the present study, six novel SNPs in three candidate genes for prolificacy (BMPR1B, BMP15, and GDF9) were genotyped in seven breeds of Indian goats to evaluate their association with litter size. Tetra primer ARMS-PCR and PCR-RFLP based protocols were developed for genotyping six novel SNPs, namely, T(-242)C in BMPR1B; G735A and C808G in BMP15; and C818T, A959C, and G1189A in GDF9 gene. The effect of breed was highly significant (p ≤ 0.01) on litter size but the effect of genotype was nonsignificant. The effect of parity on litter size was also significant in the prolific Black Bengal breed. The litter size differences observed between breeds are attributed to breed differences. Novel mutations observed at different loci in GDF9, BMP15, and BMPR1B genes do not contribute to the reproductive capability of the investigated breeds. Further studies with more number of breeds and animals exploring association of these novel SNPs with reproductive traits may be fruitful.     

Expression Analysis of Six Paralogous Human Hyaluronidase Genes Clustered on Chromosomes 3p21 and 7q31

Two new members of a family of putative hyaluronidase genes involved in glycosaminoglycan catabolism have been identified and mapped by FISH and YAC library screening to chromosome 7q31.3. One of these (HYALP1) is an expressed pseudogene with mutations in the genomic DNA and cDNA. The six members of the hyaluronidase family are grouped into two tightly linked triplets on human chromosomes 3p21.3 (HYAL1, HYAL2, and HYAL3) and 7q31.3 (HYAL4, SPAM1 (PH-20), and HYALP1). This arrangement could arise by an ancient cluster formation, followed by a more recent cluster block-duplication. All of the hyaluronidase genes have unique tissue-specific expression patterns as determined by Northern blot analysis of 23 human tissues. HYAL1, HYAL2, and HYALP1 are widely expressed, but HYAL3 is differentially expressed in bone marrow and testis, while HYAL4 is differentially expressed in placenta and skeletal muscle. SPAM1 (PH-20) was detectable only in testis by Northern blot as previously reported, but was detectable in fetal and placental cDNA libraries by PCR, suggesting a possible role for this gene during embryonic development.     

Polymorphisms in genes involved in the inflammatory response and interaction with NSAID use or smoking in relation to lung cancer risk in a prospective study

Lung cancer risk was investigated in relation to single nucleotide polymorphisms in genes involved in the inflammatory response. The aim was to see if polymorphisms modifying the inflammatory response are associated with risk of lung cancer and if there were interactions between the same polymorphism and factors, which modify an inflammatory response, such as smoking status, duration, and intensity, and use of NSAID. The functional SNPs IL-1B T-31C, IL6 G-174C, IL8 T-251A, IL10 C-592T, COX2 C8473T, COX2 A-1195G and PPARgamma2 Pro(12)Ala were included. A case-cohort study including 428 lung cancer cases and a sub-cohort of 800 persons was nested within a population-based prospective study of 57,053 individuals. Variant allele carriers of IL-1B T-31C were at increased risk of lung cancer (IRR=1.51, 95% CI=1.08-2.12). There was interaction between the polymorphism COX-2 T8473C and smoking status. Thus, non-smoking variant allele carriers were at 5.75-fold (95% CI=1.25-26.43) higher risk of lung cancer than for homozygous wild type allele carriers. Lung cancer risk was similar for all genotype carriers among past and current smokers. There were, however, very few non-smoking lung cancer cases. There was interaction between IL-1B T-31C, COX-2 A-1195G and PPARgamma2 Pro(12)Ala and NSAID use in relation to lung cancer risk. For the two latter, NSAID use was only associated with a lower cancer risk among homozygous wild type allele carriers. p for interaction was 3 x 10(-6) for COX-2 A-1195G and 9 x 10(-5) for PPARgamma2 Pro(12)Ala. The results suggest that NSAID use may modify risk of lung cancer differently depending on the genotype.     

Porcine skeletal muscle differentially expressed gene CMYA1: isolation, characterization, mapping, expression and association analysis with carcass traits

To investigate the differences in gene expression between some obese and lean pig breeds, differential display of mRNA was employed in our previous research. One differentially expressed EST ( BI596262 ) was further identified as the porcine cardiomyopathy associated 1 ( CMYA1 ) gene because of its homology to the human CMYA1 gene. The full-length DNA of the porcine CMYA1 gene encompasses 9379 bp, including a complete open reading frame encoding 1839 amino acid residues, a 158-bp 5'-untranslated region and a 630-bp 3'-untranslated region. The porcine CMYA1 gene was assigned to chromosome 13 by the radiation hybrid panel (IMpRH). The porcine CMYA1 gene was expressed only in the striated muscle. Single nucleotide polymorphism (SNP) scanning in the coding region identified one synonymous mutation (c.1053C>T) and three missense mutations, c.1394A>G (p.His465Arg), c.1751A>G (p.Asp582Gly) and c.3290C>A (p.Thr1097Asp). The allele frequencies were tested among about 200 unrelated pigs from several pig breeds. Linkage mapping was further conducted with the SNP c.1751A>G (p.Asp582Gly) in a Berkshire × Yorkshire resource family and this confirmed that porcine CMYA1 is closely linked with Sw344 (distance  =  2 cM, LOD score is 129.47), an interesting region harbouring a QTL for back fat thickness. Association analysis in our experimental pig population showed that different genotypes of CMYA1 gene were associated with different back fat thicknesses ( P  <   0.05). Our results suggest that the porcine CMYA1 gene has effects on porcine back fat deposition and further investigation will be necessary to illustrate the underlying mechanisms.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

ACDC/Adiponectin and PPAR‐γ Gene Polymorphisms: Implications for Features of Obesity

Objective: The main purpose of this study was to investigate associations of single‐nucleotide polymorphisms (SNPs) in the adipocyte C1q and collagen domain‐containing (ACDC) gene and its regulator, the nuclear peroxisome proliferator‐activated receptor (PPAR)‐γ gene, with body fat mass and its topographical distribution in postmenopausal women. Research Methods and Procedures: Participants were 1501 healthy women, 60 to 85 years old, who were genotyped for four SNPs in the ACDC gene (−11391G/A, −11377C/G, +45T/G, +276G/T) and the Pro12Ala SNP in the PPAR‐γ gene. Total body fat mass and the central to peripheral fat mass ratio (CFM/PFM ratio) were measured using DXA. Adiponectin and homeostasis model assessment of insulin resistance were measured in 287 subjects. Results: The −11377C/G SNP was associated with adiponectin (p < 0.001) and the CFM/PFM ratio (p = 0.005); the G allele being associated with low adiponectin and high CFM/PFM ratio. Similar associations of adiponectin (p = 0.0001) and the CFM/PFM ratio (p = 0.002) characterized the 1_2 (G_G) promoter haplotype (11391G/A_−11377C/G). Genotype variation of SNP Pro12Ala was associated with total body fat mass (p = 0.04); women with GG being the most obese (p = 0.01). The Ala/Ala (GG) genotype of Pro12Ala SNP interacted with the CC genotype of SNP‐11377C/G in the determination of BMI (p = 0.001), when analyzed using a codominant model. Discussion: Polymorphisms in the ACDC gene are associated with body fat distribution, whereas the Pro12Ala polymorphism in PPAR‐γ is associated with overall adiposity, apparently in interaction with an ACDC promoter SNP.     

Evaluation of RDS/Peripherin and ROM1 as candidate genes in generalised progressive retinal atrophy and exclusion of digenic inheritance

Generalised progressive retinal atrophy (gPRA) is a heterogeneous group of hereditary diseases causing degeneration of the retina in dogs and cats. As a combination of mutations in the RDS/Peripherin and the ROM1 genes leads to the phenotype of retinitis pigmentosa in man we first performed mutation analysis to screen these genes for disease causing mutations followed by the investigation of a digenic inheritance in dogs. We cloned the RDS/Peripherin gene and investigated the RDS/Peripherin and ROM1 genes for disease causing mutations in 13 gPRA-affected dog breeds including healthy animals, obligate gPRA carriers and gPRA-affected dogs. We screened for mutations using single strand conformation polymorphism (SSCP) analysis. Sequence analysis revealed several sequence variations. In the coding region of the RDS/Peripherin gene three nucleotide exchanges were identified (A277C; C316T; G1255A), one of which leads to an amino acid substitution (Ala339Thr). Various silent sequence variations were found in the coding region of the ROM1 gene (A536G, G1006A, T1018C, T1111C, C1150T, C1195T), as well as an amino acid substitution (G252T; Ala54Ser). By excluding the respective gene as a cause for gPRA several sequence variations in the intronic regions were investigated. None of these sequence variations cosegregated with autosomal recessively (ar) transmitted gPRA in 11 breeds. The candidate gene RDS/Peripherin obviously does not harbour the critical mutation causing the autosomal recessive form of gPRA because diseased individuals show heterozygous genotypes for sequence variations in the Miniature Poodle, Dachshund, Australian Cattle Dog, Cocker Spaniel, Chesapeake Bay Retriever, Entlebucher Sennenhund, Sloughi, Yorkshire Terrier, Tibet Mastiff, Tibet Terrier and Labrador Retriever breeds. In the following breeds the ROM1 gene was also excluded indirectly for gPRA: Miniature Poodle, Dachshund, Australian Cattle Dog, Sloughi, Collie, Tibet Terrier, Labrador Retriever and Saarloos/Wolfhound. Digenic inheritance for gPRA is practically excluded for both these genes in four breeds: Miniature Poodle, Dachshund, Labrador Retriever and Saarloos/Wolfhound.     

The primary structure of the COOH-terminal half of cholera toxin subunit A1 containing the ADP-ribosylation site

The sequence of 96 amino acid residues from the COOH-terminus of the active subunit of cholera toxin, A₁, has been determined as PheAsnValAsnAspVal LeuGlyAlaTyrAlaProHisProAsxGluGlu GluValSerAlaLeuGlyGly IleProTyrSerGluIleTyrGlyTrpTyrArg ValHisPheGlyValLeuAsp GluGluLeuHisArgGlyTyrArgAspArgTyr TyrSerAsnLeuAspIleAla ProAlaAlaAspGlyTyrGlyLeuAlaGlyPhe ProProGluHisArgAlaTrp ArgGluGluProTrpIleHisHisAlaPro ProGlyCysGlyAsnAlaProArg(OH). This is the largest fragment obtained by BrCN cleavage of the subunit A₁ (M_r 23,000), and has previously been indicated to contain the active site for the adenylate cyclase-stimulating activity. Unequivocal identification of the COOH-terminal structure was achieved by separation and analysis of the terminal peptide after the specific chemical cleavage at the only cysteine residue in A₁ polypeptide. The site of self ADP-ribosylation in the A₁ subunit [C. Y. Lai, Q.-C. Xia, and P. T. Salotra (1983) Biochem. Biophys. Res. Commun.116, 341–348] has now been identified as Arg-50 of this peptide, 46 residues removed from the COOH-terminus. The cysteine that forms disulfide bridge to A₂ subunit in the holotoxin is at position 91.     

Reverse polarization in amino acid and nucleotide substitution patterns between human-mouse orthologs of two compositional extrema.

Genome-wide analysis of sequence divergence patterns in 12,024 human-mouse orthologous pairs reveals, for the first time, that the trends in nucleotide and amino acid substitutions in orthologs of high and low GC composition are highly asymmetric and polarized to opposite directions. The entire dataset has been divided into three groups on the basis of the GC content at third codon sites of human genes: high, medium, and low. High-GC orthologs exhibit significant bias in favor of the replacements, Thr --> Ala, Ser --> Ala, Val --> Ala, Lys --> Arg, Asn --> Ser, Ile --> Val etc., from mouse to human, whereas in low-GC orthologs, the reverse trends prevail. In general, in the high-GC group, residues encoded by A/U-rich codons of mouse proteins tend to be replaced by the residues encoded by relatively G/C-rich codons in their human orthologs, whereas the opposite trend is observed among the low-GC orthologous pairs. The medium-GC group shares some trends with high-GC group and some with low-GC group. The only significant trend common in all groups of orthologs, irrespective of their GC bias, is (Asp)(Mouse) --> (Glu)(Human) replacement. At the nucleotide level, high-GC orthologs have undergone a large excess of (A/T)(Mouse) --> (G/C)(Human) substitutions over (G/C)(Mouse) --> (A/T)(Human) at each codon position, whereas for low-GC orthologs, the reverse is true.     

Detecting novel SNPs and breed-specific haplotypes at calpastatin gene in Iranian fat- and thin-tailed sheep breeds and their effects on protein structure

Calpastatin has been introduced as a potential candidate gene for growth and meat quality traits. In this study, genetic variability was investigated in the exon 6 and its intron boundaries of ovine CAST gene by PCR-SSCP analysis and DNA sequencing. Also a protein sequence and structural analysis were performed to predict the possible impact of amino acid substitutions on physicochemical properties and structure of the CAST protein. A total of 487 animals belonging to four ancient Iranian sheep breeds with different fat metabolisms, Lori-Bakhtiari and Chall (fat-tailed), Zel-Atabay cross-bred (medium fat-tailed) and Zel (thin-tailed), were analyzed. Eight unique SSCP patterns, representing eight different sequences or haplotypes, CAST-1, CAST-2 and CAST-6 to CAST-11, were identified. Haplotypes CAST-1 and CAST-2 were most common with frequency of 0.365 and 0.295. The novel haplotype CAST-8 had considerable frequency in Iranian sheep breeds (0.129). All the consensus sequences showed 98–99%, 94–98%, 92–93% and 82–83% similarity to the published ovine, caprine, bovine and porcine CAST locus sequences, respectively. Sequence analysis revealed four SNPs in intron 5 (C24T, G62A, G65T and T69-) and three SNPs in exon 6 (c.197A > T, c.282G > T and c.296C > G). All three SNPs in exon 6 were missense mutations which would result in p.Gln 66 Leu, p.Glu 94 Asp and p.Pro 99 Arg substitutions, respectively, in CAST protein. All three amino acid substitutions affected the physicochemical properties of ovine CAST protein including hydrophobicity, amphiphilicity and net charge and subsequently might influence its structure and effect on the activity of Ca2 + channels; hence, they might regulate calpain activity and afterwards meat tenderness and growth rate. The Lori-Bakhtiari population showed the highest heterozygosity in the ovine CAST locus (0.802). Frequency difference of haplotypes CAST-10 and CAST-8 between Lori-Bakhtiari (fat-tailed) and Zel (thin-tailed) breeds was highly significant (P < 0.001), indicating that these two haplotypes might be breed-specific haplotypes that distinguish between fat-tailed and thin-tailed sheep breeds.     

Hyaluronidases and CD44 undergo differential modulation during chondrogenesis

Hyaluronan, a high-molecular-weight glycosaminoglycan of cartilage, is deposited directly into the extracellular space by hyaluronan synthases, while hyaluronan catabolism is mediated by the hyaluronidases. An in vitro cell culture system has been established in which human dermal fibroblasts are induced to undergo chondrogenesis. Here, we describe the differential modulation of the hyaluronidases and the up-regulation of the hyaluronan receptor, CD44, during such chondrogenesis. Dermal fibroblasts, plated in micromass cultures in the presence of lactic acid and staurosporine for 24 h, were then placed in serum-free, chemically defined medium. At 3 days, RNA was extracted and RT-PCR performed using primers for the hyaluronidase genes. Marked increase in HYAL1 expression was observed, with only moderate increases occurring in HYAL2 and HYAL3. No expression of HYAL4 and PH-20, the sperm-associated hyaluronidase, was detected. RNA levels correlated well with changes in hyaluronidase enzyme activity. Finally, greater expression and staining for the hyaluronan receptor, CD44s, the standard form, were detected. Differential expression of the somatic hyaluronidases and CD44-mediated hyaluronan turnover play a critical role in cartilage development from mesenchymal precursors.     

SNP detection and haplotype analysis in partial sequence of MSTN gene in sheep

Polymerase chain reaction (PCR) products of the MSTN gene amplified from sixty sheep of nine Chinese indigenous sheep breeds and one imported sheep breed were sequenced to identify the single-nucleotide polymorphisms (SNPs) in a 378-bp fragment including intron 2 and exon 3 of the MSTN gene. A total of fifteen SNPs (A1937C, T1942G, C1956T, A1972C, A1990G, A2008C, A2011G, C2019T, A2025C, A2027C, T2085G, T2173C, C2198T, C2210T and C2213T) were detected among the sixty sequenced individuals and they were all located in intron 2. Twelve haplotypes were identified from these fifteen SNPs, of which haplotype I (CGTCGCGTCCGCTTT) and VIII (ATCAAAACAATTCCC) were the two major and basic ones with frequencies of 12.25% and 77.80%, respectively. Haplotype VIII was distributed in all sheep breeds and all individuals of the meat or meat-wool type sheep breeds were homozygous with respect to this haplotype. This suggests that haplotype VIII might be related to meat production traits in sheep. Haplotype I was only distributed in the fur, lambskin type and fur-meat type sheep breeds. This suggests that haplotype I may have some relationship with fur traits in sheep.     

Comparison of the three primary structures of deoxyribonuclease isolated from bovine, ovine, and porcine pancreas. Derivation of the amino acid sequence of ovine DNase and revision of the previously published amino acid sequence of bovine DNase

Based on the published bovine DNase sequence (Liao, T.-H., Salnikow, J., Moore, S., and Stein, W. H. (1973) J. Biol. Chem. 248, 1489-1495), the ovine DNase sequence is derived from the amino acid compositions of isolated short peptides covering all regions of the intact polypeptide. The sequence is substantiated by results of automated Edman degradation of the intact polypeptide and of the two middle CNBr fragments, and by elucidation of the complete sequence of the COOH-terminal CNBr peptide. The 12 changes from bovine to ovine DNase are at residues 22 (Ala to Ser), 29 (Val to Leu), 35 (Val to Ala), 54 (Tyr to Asp), 62 (Thr to Ser), 83 (Leu to Val), 121 (His to Pro), 127 (Glu to Ala), 132 (Ala to Pro), 159 (His to Asp), 163 (Val to Ile), and 231 (Ala to Val). A minor genetic variant form of ovine DNase has Val at residue 163. The data from automated Edman degradation of the largest CNBr peptide of bovine DNase show that the published bovine DNase sequence is in error and that an Ile-Val-Arg tripeptide must be inserted between Arg-27 and Arg-28. The corrected sequence is substantiated by two peptides covering this region each with three amino acids more than the published sequence. Comparison of the bovine, ovine, and porcine DNase sequences reveals the following: with the revised bovine sequence, all three DNase sequences can be aligned without a gap; all three DNases have a carbohydrate side chain at Asn-18, but only porcine DNase has carbohydrate at Asn-106; there are 12 changes between bovine and ovine DNases, 56 between bovine and porcine, and 50 between ovine and porcine; there are six highly variable regions and four invariable ones; bovine and ovine DNases have the same length while porcine DNase is longer by 2 amino acid residues at the COOH terminus; the residues around the nucleotide-binding site, the four pairs of salt bridges, and the essential His-134 groups are not changed.