Molecular anatomy of the cytoplasmic domain of bovine growth hormone receptor, a quantitative trait locus

Quantitative trait loci (QTL) studies have indicated growth hormone receptor (GHR) as a candidate gene affecting cattle milk yield and composition. In order to characterize genetic variation at GHR in cattle, we studied European and East African breeds with different histories of selection, and Bos grunniens, Ovis aries, Sus scrofa, Bison bison and Rangifer tarandus as references. We sequenced most of the cytoplasmic domain (900 bp of exon 10), 89 bp of exon 8, including the putative causative mutation for the QTL effect, and 390 bp of intron 8 for comparison. In the cytoplasmic domain, seven synonymous and seven non-synonymous single nucleotide polymorphisms (SNP) were identified in cattle. Three non-synonymous SNPs were found in sheep and one synonymous SNP in yak, while other studied species were monomorphic. Three major haplotypes were observed, one unique to African breeds, one unique to European breeds and one shared. Bison and yak haplotypes are derivatives of the European haplotype lineage. Most of the exon 10 non-synonymous cattle SNPs appear at phylogenetically highly conserved sites. The polymorphisms in exon 10 cluster around a ruminant-specific tyrosine residue, suggesting that this site may act as an additional signalling domain of GHR in ruminants. Alternative explanations for the persistent polymorphism include balancing selection, hitch-hiking, pleiotropic or sexually antagonistic fitness effects or relaxed functional constraints.     

Comparison of cAMP receptor protein (CRP) and a cAMP-independent form of CRP by Raman spectroscopy and DNA binding.

The secondary structures of the cAMP receptor protein (CRP), a complex of CRP and cAMP, and a cAMP-independent receptor protein mutant (CRP*141 gln) were examined by using Raman spectroscopy. Spectra were obtained from CRP and CRP*141 gln dissolved in 0.3 M NaCl and 30 mM sodium phosphate at protein concentrations of 30-40 mg/mL. CRP and CRP.cAMP1 were compared at lower protein concentrations (10-12 mg/mL) in a solvent of 0.35 M NaCl and 20 mM sodium phosphate. Raman analysis indicates that CRP structural changes induced by one bound cAMP or by the Gly to Gln mutation at residue 141 are small. Spectra of the three CRP samples are essentially identical from 400 to 1900 cm-1. This result differs from the Raman spectroscopy study of CRP and CRP.cAMP2 cocrystals [DeGrazia et al. (1990) Biochemistry 29, 3557]. The latter work showed spectral differences between CRP and CRP.cAMP2 consistent with alterations in the protein conformation. These studies indicate that CRP and CRP.cAMP1 in solution are similar in structure and differ from CRP.cAMP2 cocrystals. Protease digestion and a DNA binding assay were also employed to characterize the wild-type and mutant proteins. CRP*141 gln exhibited the same conformational characteristics of previously reported cAMP-independent mutant proteins. It was sensitive to proteolytic attack in the absence of cAMP, or upon addition of cGMP. In the absence of cAMP, both wild-type and mutant CRPs bound noncooperatively to a 62 bp lac promoter DNA. The equilibrium constants were approximately 10(6) M-1 in 0.1 M Na+. CRP*141 gln had a 2-4-fold higher affinity for the 62 bp DNA than CRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

猪PRDX6基因编码区的多态性及遗传效应分析

PRDX6基因属于抗氧化蛋白超家族,主要在应答氧化压力、脂分解代谢、磷脂分解代谢中发挥作用。根据PRDX6基因的生理生化功能,文章选择其为影响猪肉质性状的候选基因,探索PRDX6基因的多态性与猪肉质性状的关系。首先分离了猪PRDX6的部分编码区序列,并在不同猪种间进行序列比对,发现编码区第4外显子有2个潜在的SNPs分别位于第417 bp处(C/T突变)和第423 bp处(A/G突变),但均没有引起氨基酸的改变。采用Pyrosequencing焦磷酸测序方法对这两个位点在6个猪种和247头F2"大白×梅山"资源家系中进行了遗传变异分析和性状关联分析。结果表明:417C/T位点国外品种均以C等位基因为主,而国内品种均以T等位基因为主,该位点与肌内脂肪、肌肉水分存在显著关联(P<0.05);423A/G位点国外品种均以A等位基因为主,国内品种以G等位基因为主,该位点与失水率、系水力、肌内脂肪及肌肉水分存在显著关联(P<0.05)。由此推断:这两个位点很可能是影响猪肉质性状(尤其是肌肉嫩度)的分子标记。     

A missense mutation in the bovine leptin receptor gene is associated with leptin concentrations during late pregnancy

The leptin receptor (LEPR) gene consists of 20 exons divided over 1.75 Mb. Parts of bovine LEPR exon 4 (79 bp), exon 11 (95 bp) and exon 20 (513 bp) of 20 cows (Holstein-Friesian) were sequenced (AJ580799; AJ580800; AJ580801) in an attempt to find polymorphisms. In exons 4 and 11 no SNPs were found. In exon 20, a T to C missense mutation was found at nucleotide 115, which causes an amino acid substitution at residue 945 (T945M). Frequencies for alleles C and T were 0.93 and 0.07 respectively, in a population of 323 Holstein-Friesian cows and TT animals were not detected. Using genotypes of these cows an association study was performed for leptin concentrations during late pregnancy and lactation. Leptin concentrations were determined by radioimmunoassay (RIA). The T945M mutation showed an association with circulating leptin concentrations only during late pregnancy (P < 0.05) but not during lactation (P > 0.05). The CC genotype had higher concentrations than the CT genotype during this period. A combined effect with previously described leptin polymorphisms on prepartum leptin concentrations was observed, with one genotype combination having significantly lower levels of leptin up to 50 days, but interaction effects were not significant. The T945M polymorphism may have induced a structural change in the intracellular domain of the LEPR, which may have influenced the signal transduction pathway. However, the effect was found only for the heterozygous genotype because the TT genotype was not detected in this population of 323 Holstein-Friesian cows.     

Molecular characterization of the Rocky Mountain elk (Cervus elaphus nelsoni) PRNP putative promoter

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting deer (Odocoileus spp.), moose (Alces alces), and Rocky Mountain elk (Cervus elaphus nelsoni). Leucine homozygosity at elk PRNP codon 132 has been associated with reduced CWD susceptibility. However, naturally acquired CWD has been detected in elk possessing the 132 Leu/Leu genotype. Recent human and bovine studies indicate that PRNP regulatory polymorphisms may also influence TSE occurrence. Therefore, we generated sequences for the elk PRNP putative promoter (2.2 kb), exon 1 (predicted; 54 bp), intron 1 (predicted; 193 bp), and exon 3 (771 bp). Promoter prediction analysis using CpGProD yielded a single elk PRNP promoter that was homologous to regions of known promoter activity in cow and sheep. Molecular interrogation of the elk PRNP putative promoter revealed 32 diallelic single-nucleotide polymorphisms (SNPs). No variation was detected within the predicted exon 1 or intron 1 sequences. Evaluation of elk PRNP exon 3 revealed 3 SNPs (63Y, 312R, 394W-->Met/Leu). Bayesian haplotype reconstruction resulted in 3 elk PRNP haplotypes, with complete linkage disequilibrium observed between all PRNP putative promoter SNPs and codon 132. The results of this study provide the initial genomic foundation for future comparative and haplotype-based elk PRNP studies.     

Basal cell carcinoma and variants in genes coding for immune response, DNA repair, folate and iron metabolism

Basal cell carcinoma (BCC) is one of the most common neoplasms in the world and its incidence has been increasing worldwide in recent years. BCCs are caused by an interplay between genetic and environment factors. We conducted a case-control association study in BCC patients and controls from Sweden and Finland. Fifteen single nucleotide polymorphisms (SNPs), IL-6-174G/C, -634G/C, and -597G/A; IL-10-1082G/A and -592C/A; IL-1beta-511C/T; NBS1 exon 5 Glu185Gln; XPC exon 15 Lys939Gln; XPD exon 23 Lys751Gln; XRCC1 exon 10 Arg399Gln; XRCC3 exon 7 Thr241Met; cyclin D1 exon 4 G870A; MTHFR exon 4 Ala222Val and exon 7 Glu429Ala; HFE exon 4 C282Y were performed by Pyrosequencing and RFLP techniques. Most of the genotype distributions were in accordance with the Hardy-Weinberg equilibrium (HWE), except for IL-10-1082G/A, where cases with BCC showed a significant deviation from HWE (P = 0.04). Linkage disequilibrium was observed between the -174 and -597 alleles in the IL-6 gene in the present populations. No difference between BCC and controls appeared in any of the SNPs analyzed. Only the combined distributions of TT/AA genotypes in MTHFR exon 4 (C/T) and exon 7 (A/C) showed slight increase in BCC compared to controls (P < 0.07, OR: 1.94; 95% CI: 0.96-3.89).     

Polymorphism identification within 50 equine gene-specific sequence tagged sites

The continued discovery of polymorphisms in the equine genome will be important for future studies using genomic screens and fine mapping for the identification of disease genes. Segments of 50 equine genes were examined for variability in 10 different horse breeds using a pool-and-sequence method. We identified 11 single nucleotide polymorphisms (SNPs) in 9380 bp of sequenced exon, and 25 SNPs, six microsatellites, and one insertion/deletion in 16961 bp of sequenced intron. Of all genes studied 52% contained at least one polymorphism, and polymorphisms were found at an overall rate of 1/613 bp. Several of the putative SNPs were tested and verified by restriction enzyme analysis using natural restriction sites or ones created by primer mutagenesis. The lowest allele frequency for a SNP detected in pooled samples was 10%. Three of the SNPs verified in the diverse horse pool were further tested in six breed-specific horse pools and were found to be reasonably variable within breeds. The pool-and-sequence method allows identification of polymorphisms in horse populations and will be a valuable tool for future disease gene and comparative mapping in horses.     

First polymorphisms in JY-1 gene in cattle (Bos taurus indicus) and their association with sexual precocity and growth traits

Protein JY-1 is a bovine oocyte-specific protein that regulates granulosa cell function and is involved in early embryonic development, influencing the chance of pregnancy. This study investigated molecular markers for the JY-1 gene. Seven SNPs were identified in exon 3 of the gene. The positions of the SNPs in the exon and the respective substitutions are: 163 (T/C), 281 (T/C), 321 (T/C), 532 (T/C), 652 (A/G), 679 (T/C), and 722 (G/C) (GenBank: JN592587 and JF262042.2). SNP 163 is located in a coding region and causes a proline-to-leucine substitution. The other SNPs are located in the 3′UTR region. SNPs 163, 281, 321, and 679 were genotyped in 297 Nellore heifers and the haplotypes were constructed. The haplotypes of JY-1 were not correlated with the traits studied at 5?%.     

Novel SNPs of the Bovine PRLR Gene Associated with Milk Production Traits

The single nucleotide polymorphisms (SNPs) within exon 10 of the prolactin receptor gene (PRLR) were detected in Chinese Holstein cows using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing methods, and their genetic effects on milk production traits were evaluated in this study. Two newly detected SNPs (g.9206G→A and g.9681C→T) caused amino acid variations E378K and A536V, respectively, which were then preliminarily predicted at the topological level. Statistical results indicated that the two SNPs were significantly associated with milk yields, and cows with the combined genotype GGCC showed superior milk performance. A putative phosphorylation site was identified at residue 378K ([ST]-×-[RK]), which offers a partial explanation for the associations. These results suggest that the two novel SNPs within exon 10 of the PRLR gene associated with milk production traits are useful genetic markers in a selection program for Holstein dairy cattle.     

秦川牛脂联素基因SNPs检测及其与胴体、肉质性状的相关性

利用PCR-SSCP结合测序技术对405头24月龄秦川牛脂联素基因SNPs位点进行检测, 运用SPSS统计程序中的GLM模型将检测到的SNPs位点与部分胴体及肉质性状的相关性进行了分析。结果检测到AA、AB、BB、CC、CD 5种基因型, 其中AB、BB型个体在脂联素基因第2外显子 64 bp处发现G→C突变, CD型个体第3外显子50 bp处发现C→T的突变, G→C导致谷氨酸(GGA)转化为谷氨酰胺(GCA), C→T导致丝氨酸(TCA)转化为亮氨酸(TTA)。方差分析结果表明: AA型个体的宰前活重、胴体重、眼肌面积显著高于BB型(P<0.05), 而在胴体腿臀围方面, AA型个体极显著高于AB型、BB型个体(P<0.01)。CD型个体的宰前活重、胴体腿臀围、皮下脂肪厚、背膘厚、嫩度都显著优于CC型个体(P<0.05)。脂联素基因该位点可能是影响秦川牛胴体及肉质性状的主效QTL或与之紧密连锁, 可作为秦川牛高档牛肉生产的候选分子标记。     

Study on the Relationship Between TSHR Gene and Thyroid Diseases

Thyroid stimulating hormone receptor (TSHR) is thought to play a critical role in the pathogenesis of certain thyroid diseases, including Graves' disease (GD), multinodular thyroid goiter (MTG), and Hashimoto's thyroiditis (HT). In order to understand whether single nucleotide polymorphisms in the TSHR gene contribute to thyroid diseases, we have conducted a case-control study in which, we examined 8 TSHR gene single-nucleotide polymorphisms in introns 1, 4, 5, 6 and exons 7 and 8, respectively, among patients with thyroid diseases. These included one family with GD (3 patients and 9 healthy members); 60 patients with familiar thyroid diseases (30 with GD, 20 with MTG, and 10 with HT patients), 48 sporadic patients with GD and 96 healthy control individuals. Direct sequencing of all 10 exons and part of introns of TSHR gene, in these patients as well as healthy controls revealed eight polymorphisms. A novel polymorphism in exon 8 AGA(Arg) → CGA(Arg). However, there were no significant differences between patients and controls in the incidence of these polymorphisms. These results suggest that the polymorphisms (polymorphism in intron 1 at 81 bp upstream of exon 2; polymorphism in intron 4 at 135 bp upstream of exon 5; polymorphism in intron 4 at 365 bp upstream of exon 5; polymorphism in intron 5 at 69 bp upstream of exon 6; means polymorphism in intron 6 at 13 bp downstream of exon 6; polymorphism in intron 6 at 187 bp upstream of exon 7; E7+16: polymorphism in 16 bp of exon 7; polymorphism in 40 bp of exon 8) of the TSHR gene may not contribute to the pathogenesis of thyroid diseases.     

Polymorphisms in the NPY2R gene show significant associations with BMI that are additive to FTO, MC4R, and NPFFR2 gene effects

Neuropeptide Y (NPY) is an appetite hormone that acts centrally to control feeding behavior. The 5' and exon 2 regions of NPY2R, one of five NPY receptor genes, have been weakly and inconsistently implicated with obesity. With the ATG start site of the gene at the beginning of exon 2, single-nucleotide polymorphisms (SNPs) across intron 1 may show stronger associations with obesity than expected. Two 5' SNPs, three intron 1 SNPs, and one synonymous exon 2 SNP were genotyped on 2,985 white Utah subjects. Previously associated FTO, NPY, NPY1R, MC4R, PPARGC1A, OR7D4, and four NPFFR2 SNPs were also genotyped and related to BMI. One NPY2R 5' SNP (rs12649641, P = 0.008), an exon 2 SNP (rs2880415, P = 0.009), and an intron 1 SNP (rs17376826, P = 7 × 10(-6)) were each significantly associated with BMI. All three SNPs, plus FTO (rs9939609, P = 1.5 × 10(-6)) and two NPFFR2 SNPs (rs4129733, P = 3.7 × 10(-13) and rs11940196, 4.2 × 10(-10)) remained significant in a multiple regression additive model. Diplotypes using the estimated haplotypes of NPY2R, NPFFR2, and MC4R were significantly associated with BMI (P = 1.0 × 10(-10), 3.2 × 10(-8), and 1.1 × 10(-4), respectively). Haplotypes of NPY2R, NPFFR2, and MC4R, plus the FTO SNP, explained 9.6% of the BMI variance. SNP effect sizes per allele for the four genes ranged from 0.8 to 3.5 kg/m(2). We conclude that haplotypes containing the rs17376826 SNP in intron 1 of NPY2R have strong associations with BMI, some NPFFR2 haplotypes are strongly protective against or increase risk of obesity, and both NPY2R and NPFFR2 play important roles in obesity predisposition independent of FTO and MC4R.     

鸡催乳素基因序列多态及生物信息学分析

选择繁殖性能具有明显差异的4个鸡品种（莱航鸡、阳山鸡、丝羽乌骨鸡和隐性白洛克鸡）构建品种DNA池,采用测序的方法快速筛查鸡催乳素基因（chicken prolactin,cPRL）5′侧翼调控区、外显子区和部分内含子区约4500 bp范围内可能与产蛋性能相关的序列多态,共检测到13个SNPs和两个短片段（24 bp和15 bp）插入/缺失多态,其中在5′侧翼序列筛查到9个SNPs及两个短片段插入/缺失多态,在第2外显子筛查到1个SNP,在第5外显子筛查到两个SNPs,在第2内含子筛查到1个SNP;进一步利用生物信息学分析cPRL基因的5′侧翼调控序列,发现24 bp短片段的插入使莱航鸡比阳山鸡多出了1个Evi-1可能的结合位点（93分）,C-2402T的变异则使阳山鸡比莱航鸡多出了1个C/EBPbeta可能的结合位点（94分）,这两个结合位点是否影响cPRL基因的表达,影响鸡的就巢性和产蛋性能,还需要进一步研究。Abstract：Four chicken breeds (White Leghorn, Yangshan, Taihe Silkies, White Recessive Rocks) with different reproduction were applied to screen potential SNPs related to laying performance in the 5′flanking region, exon region and partial intron region of chicken prolactin (cPRL) gene. Totally almost 4500 bp were screened rapidly based on DNA pooling and sequencing, and thirteen single nucleotide polymorphisms (SNPs) and two indels (24 bp and 15 bp) were found, including nine SNPs and two indels in the 5′flanking region, one SNP in Exon 2, two SNPs in Exon 5 and one SNP in Intron 2 respectively. Furthermore, 5′flanking region of cPRL gene was analyzed by the website of http://motif.genome.ad.jp/. A possible Evi-1 binding site (score 93) was found in White Leghorn cPRL gene because of the 24 bp insertion, another possible C/EBPbeta binding site (score 94) was found in Yangshan cPRL gene because of the variation of C-2402T. Further studies need to be carried out to verify their effects on the expression of cPRL gene, the broodiness and laying performance of chickens.     

Population genetic diversity of marble goby (<Emphasis Type="BoldItalic">Oxyeleotris marmoratus</Emphasis>) inferred from mitochondrial DNA and microsatellite analysis

Mastitis is an infectious disease of the mammary gland that leads to reduced milk production and change in milk composition. Complement component C3 plays a major role as a central molecule of the complement cascade involving in killing of microorganisms, either directly or in cooperation with phagocytic cells. C3 cDNA were isolated, from Egyptian buffalo and cattle, sequenced and characterized. The C3 cDNA sequences of buffalo and cattle consist of 5025 and 5019 bp, respectively. Buffalo and cattle C3 cDNAs share 99% of sequence identity with each other. The 4986 bp open reading frame in buffalo encodes a putative protein of 1661 amino acids—as in cattle—and includes all the functional domains. Further, analysis of the C3 cDNA sequences detected six novel single-nucleotide polymorphisms (SNPs) in buffalo and three novel SNPs in cattle. The association analysis of the detected SNPs with milk somatic cell score as an indicator of mastitis revealed that the most significant association in buffalo was found in the C >A substitution (ss: 1752816097) in exon 27, whereas in cattle it was in the C >T substitution (ss: 1752816085) in exon 12. Our findings provide preliminary information about the contribution of C3 polymorphisms to mastitis resistance in buffalo and cattle.     

Cloning, partial sequence, and single-nucleotide polymorphism of the ryanodine receptor gene of the Pacific white shrimp Litopenaeus vannamei (Penaeidae)

Ryanodine receptor/calcium release channel is a large protein that plays an essential role in muscle contraction; mutations in the ryanodine receptor gene affect sensitivity to stress. As a first step towards investigating the relationship between the ryanodine receptor and shrimp cramped muscle syndrome, we cloned, partially sequenced, and examined single-nucleotide polymorphisms (SNPs) of the ryanodine receptor gene of the Pacific white shrimp (Litopenaeus vannamei). The nucleotide sequence of a 15.06-kb L. vannamei genomic DNA segment containing a partial ryanodine receptor gene sequence was determined (deposited in GenBank nucleotide database: HM367069). Direct sequencing of PCR-amplified ryanodine receptor exons with their intron-flanking regions in 10 cramped muscle syndrome shrimp and 10 healthy shrimp, revealed seven SNPs. Five of them (1713A/G, 1749T/C, 1755T/C, 3965G/A, and 8737C/T) are located in exons; however, they appear to be neutral (synonymous), since they do not alter the encoded amino acid. The other SNPs (1553C/T and 13337A/G) are in introns. The SNPs identified in the ryanodine receptor gene could be useful for association studies aimed at determining the physiological role of the ryanodine receptor in cramped muscle syndrome of shrimp.     

甘肃现代肉羊新品种群羊GH基因外显子1多态性与体重关联分析

根据GenBank发表的绵羊生长激素(GH)基因外显子1的序列设计一对引物,采用PCR-SSCP技术分析GH基因外显子1在甘肃现代肉羊新品种选育群羊中的单核苷酸多态性,并与3月龄前的体重进行关联分析。结果表明,GH基因外显子1在新品种群羊中存在多态性,检测到两种基因型(AA、AB),其301bp处有一个T/A突变和305bp处有一个G/A突变,初生重、1月、2月、3月龄体重AA、AB型都无显著性差异(P>0.05),但3月龄AB型个体的体重相对于AA型偏高,由此初步推断GH基因可能是影响绵羊体重性状的主基因或与主基因相连锁,可用以对绵羊体重性状进行标记辅助选择。     

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   

Flanking nucleotide specificity for DNA mismatch repair-deficient frameshifts within activin receptor 2 (ACVR2)

We previously demonstrated that exonic selectivity for frameshift mutation (exon 10 over exon 3) of ACVR2 in mismatch repair (MMR)-deficient cells is partially determined by 6 nucleotides flanking 5' and 3' of each microsatellite. Substitution of flanking nucleotides surrounding the exon 10 microsatellite with those surrounding the exon 3 microsatellite greatly diminished heteroduplex (A(7)/T(8)) and full (A(7)/T(7)) mutation, while substitution of flanking nucleotides from exon 3 with those from exon 10 enhanced frameshift mutation. We hypothesized that specific individual nucleotide(s) within these flanking sequences control ACVR2 frameshift mutation rates. Only the 3rd nucleotide 5' of the microsatellite, and 3rd, 4th, and 5th nucleotides 3' of the microsatellite were altered from the native flanking sequences and these locations were individually altered (sites A, B, C, and D, respectively). Constructs were cloned +1bp out-of-frame of EGFP, allowing a -1bp frameshift to express EGFP. Plasmids were stably transfected into MMR-deficient cells. Non-fluorescent cells were sorted, cultured for 35 days, and harvested for flow cytometry and DNA-sequencing. Site A (C to T) and B (G to C) in ACVR2 exon 10 decreased both heteroduplex and full mutant as much as the construct containing all 4 alterations. For ACVR2 exon 3, site A (T to C), C (A to G), and D (G to C) are responsible for increased heteroduplex formation, whereas site D is responsible for full mutant formation by ACVR2 exon 10 flanking sequences. Exonic selectivity for frameshift mutation within ACVR2's sequence context is specifically controlled by individual nucleotides flanking each microsatellite.     

Single-strand conformation polymorphisms (SSCPs) detected in five bovine genes

Summary
Polymerase chain reaction (PCR) primers designed to amplify bovine specific sequences of the arginine-vasopressin ( ARVP ), glycoprotein hormone alpha ( CGA ), cytochrome oxidase c subunit IV pseudogene ( COXP ), prochymosin ( CYM ), coagulation factor X ( F10 ), inhibin βA ( INHBA ), low density lipoprotein receptor ( LDLR ) and oxytocin ( OXT ) genes in hybrid cells were used in a search for single strand conformation polymorphisms. DNA from 75 animals comprising crossbred and 7 purebred breeds were analysed. ARVP, COXP, CYM, LDLR and OXT were found to be polymorphic while CGA, F10 and INHBA were not. Polymorphic regions were identified within 206 bp of exon 1 of ARVP , 582 bp of the pseudogene COXP , 253 bp of exon 9 of CYM , 519 bp of LDLR cDNA and 160 bp of the upstream regulatory region of OXT. This is the first report of bovine polymorphisms for these genes and an important step in our goal to incorporate type I comparative anchor loci into the bovine linkage map. Polymorhic loci were subsequently analysed in pedigreed full-sib families and shown to be inherited in a Men-delian fashion.