宗地花猪和从江香猪ADRP基因多态性及生物信息学分析

为研究宗地花猪和从江香猪ADRP基因的单核苷酸多态性,为选种选育提供理论参考,试验以宗地花猪和从江香猪为对象,构建DNA池,采用PCR产物测序法对ADRP基因8个外显子进行单核苷酸多态性检测,并利用生物信息学方法对ADRP基因编码产物进行结构功能预测。结果显示,在两个猪种ADRP基因中筛查到5个SNPs,分别是T37C、T140C、G777A、A1061G、A1117G,其中T37C位于非编码区内,T140C和A1061G为同义突变,G777A(Val→Ile)和A1117G(Asn→Ser)为错义突变;突变前后ADRP基因m RNA二级结构、编码蛋白二级结构及三级结构均发生了变化。研究结果表明,宗地花猪和从江香猪ADRP基因具有较高的遗传多样性,可为猪种的选育和创新提供参考。     

海胆Runx基因保守序列的克隆与初步分析

Runx(Runt box)是Runt结构域转录因子家族的成员之一。马粪海胆Runx基因第663位和728位碱基发生突变,分别为A→T和C→T。第一个突变为错义突变,使密码子编码的氨基酸由天冬氨酸变为酪氨酸,而第二个突变为同义突变,密码子编码的氨基酸未发生变化。海胆Runx基因保守序列的突变可能会导致其表达产物的变化,进而会影响海胆的生理机能。     

Leptin基因多态性与绵羊季节性发情的相关分析

根据leptin基因在GenBank中的已知序列设计两对引物,采用PCR-SSCP技术在常年发情的湖羊和季节性发情的阿勒泰羊群体中进行单核苷酸多态性(SNPs)检测,对筛查到的SNP位点进行基因型与绵羊季节性发情的关联分析。结果表明,与湖羊相比,阿勒泰羊leptin基因第1内含子上有3个连续碱基TTG的插入和C/T碱基突变;第3外显子3上发生G/T碱基突变,编码氨基酸由缬氨酸变成亮氨酸。Leptin外显子2扩增片段上检测到AA、AB、BB三种基因型,BB基因型在阿勒泰羊群体中属于优势基因型;对两个群体进行基因型频率独立性χ2检验,差异极显著(P0.001),说明BB基因型是影响季节性发情的有利基因型。研究结果提示,绵羊品种中Leptin基因序列的差异性可能是造成绵羊季节性发情的原因之一,可作为常年发情绵羊品种选育的辅助标记。     

猪H-FABP基因多态片段的序列分析

利用PCR-RFLP方法在猪H-FABP基因内确定了3个变异酶切位点,分别为5′-上游区HinfI-RFLP、内含子2的HaeIII-RFLP和HinfI*-RFLP（HinfI和HinfI*代表不同区域的HinfI酶切反应）。对每个多态片段进行克隆测序分析,结果表明,5′-上游区HinfI-RFLP是由于1324位的碱基T→C的突变引起;在内含子2中,HaeIII-RFLP变异酶切位点在1811位,发生了C到G的突变;HinfI*-RFLP是由于1970位发生T→C的碱基替换。 Abstract:Three variant restriction sites of porcine H-FABP gene,including HinfI-RFLP in 5′-upsream,HaeIII-RFLP and HinfI*-RFLP in intron 2,were confirmed by PCR-RFLP method.The polymorphic fragments were cloned and sequenced.The results revealed a single nucleotide substitution of T→C at position 1324 for HinfI-RFLP,C→G at position 1811 for HaeIII-RFLP and T→C at position 1970 for HinfI*-RFLP,respectively.     

小型猪与梅山猪Prop1基因的比较分析

Prop1是一种新发现的参与早期胚胎垂体发育的特异性转录因子。在人和动物均已发现由于该基因突变而引起的综合性垂体功能障碍,进而影响生长和繁殖。小型猪与梅山猪比较,其繁殖力和生长性能差异显著,为了了解这些差异的遗传基础,本试验对五指山猪、藏猪和版纳猪三种小型猪的Prop1基因进行克隆测序,并与已知梅山猪的序列进行对比,结果为:五指山猪与藏猪外显子1第69位发生G-A碱基替换,编码同义氨基酸;藏猪外显子2第115位发生A-G碱基替换,编码终止密码子;藏猪与版纳猪外显子3第466位和第631位均发生C-T碱基替换,第466位藏猪编码同义氨基酸;第631位版纳猪编码丝氨酸(TCC),梅山猪编码脯氨酸(CCC),即两种猪的Prop1基因产物不同。在Prop1基因终止密码下游250左右有一个腺苷酸丰富区,五指山猪与藏猪有15个腺苷酸串联,版纳猪有12个。上述结果为进一步研究大猪和小型猪之间生产性能差异的遗传基础提供了新线索。     

中国荷斯坦牛ABCG2基因编码区多态性检测及生物信息学分析

目的：研究中国荷斯坦牛ABCG2基因编码区（CDS）多态性,并进行生物信息学分析。方法：以中国荷斯坦牛为材料,利用PCR-SSCP技术对ABCG2基因CDS多态性进行检测,然后预测蛋白质序列的改变,并用生物信息学软件对蛋白质序列突变前后的结构及性质进行分析。结果：在外显子9中存在一个A→G碱基突变,导致氨基酸由酪氨酸突变为半胱氨酸,将此突变命名为Y367C;在外显子14中存在一个G→A突变,导致氨基酸由精氨酸突变为谷氨酰胺,将此突变命名为R578Q。2个突变一个位于功能区与跨膜区之间,一个位于跨膜区。生物信息学分析发现,蛋白质二级结构增加了1个卷曲（C）和2个转角（T）,同时减少了3个β折叠（E）,且ABCG2蛋白的组成和一些性质也发生了改变。结论：检测到的2个单核苷酸多态性引起了ABCG2蛋白性质和二级结构的改变;为进一步研究ABCG2蛋白对产乳性状的影响奠定了基础。     

可乐猪ApoA5基因多态性及其对肉质性状的遗传效应分析

根据GenBank中报道的猪apoA5基因序列设计2对引物,应用PCR技术从可乐猪肌肉组织基因组中扩增出了apoA5基因第三外显子的特异片段,采用直接测序法对apoA5基因进行单核甘酸多态性检测,并分析该基因与可乐猪12项肉质性状的关联性,结果表明:在可乐猪apoA5基因第三外显子上共检测到3个SNPs-突变位点:A~(226)G、G~(535)C、和C~(1514)T,其中A~(226)G和G~(535)C突变发生在编码区内,没有引起氨基酸的改变,为沉默突变。C~(1514)T突变发生在3'-UTR,χ~2检验表明,发现的3个多态位点均处于Hardy-Weinberg平衡状态(p0.05)。A~(226)G位点为低度多态,其余两个突变位点均为中度多态。最小二乘分析显示,apoA5基因第三外显子226位点AA基因型个体的肌内脂肪显著高于AG基因型个体(p0.05),535位点GG基因型个体的肉色显著高于CC和GC型个体(p0.05),1514多态位点与可乐猪肉质性状没有显著关系(p0.05)。     

西藏小型猪IGF-1基因的克隆测序分析

目的克隆西藏小型猪的肝脏组织中的IGF-1基因的c DNA序列,并与Pubmed中查询到的猪c DNA进行比对分析。方法提取了西藏小型猪肝脏组织的总RNA,应用RT-PCR技术扩增了IGF-1基因的c DNA序列,将扩增出的片段克隆到p MD18-T载体上,构建重组质粒p MD18-T-IGF-1,进行测序分析。结果克隆出西藏小型猪肝脏组织中的IGF-1的c DNA序列,获得了大小为567 bp长的片段,编码了186个氨基酸,与Pubmed中查询到的猪(NM_214256.1)的IGF-1基因高度同源,比对序列发现,在440、455 bp处发生了G→A、C→T的突变,该位点的突变引起相应编码氨基酸的变化,分别由组氨酸变成了精氨酸、亮氨酸转变成了丝氨酸。结论为西藏小型猪的生长发育机制研究提供了分子学依据。两个位点的突变引起的氨基酸的改变是否是导致西藏小型猪矮小的原因,需要进一步论证。     

3个猪品种黑素皮质素受体1(MC1R)基因变异研究

利用测序、PCR-RFLP和PCR-SSCP等技术对杜洛克、长白、大白猪MC1R基因进行研究发现了5个多态位点。其中,668位点G→C突变发生在5′UTR,其余4个多态位点nt894insCC(894位点CC插入),1318C→T,1554G→A和1197G→A发生在编码区。nt894insCC导致编码蛋白过早终止。1318C→T,1554G→A和1197G→A突变分别导致a164Val,Ala243Thr和Asp124Asn氨基酸的改变。所有长白、大白猪个体在894位点均存在CC插入,其余多态位点基因型分别为668GG,1197AA,1318CC,1554GG。所有杜洛克个体在894位点均不存在CC插入,其余多态位点基因型分别为668CC,1197GG,1318TT,1554AA。所有突变位点无杂合子出现。由此可以推测,668G→C,1318C→T和1554G→A可能与杜洛克的红毛色存在相关,导致1197G→A突变无意义的894位点CC插入可能与长白、大白猪白毛色存在相关。     

催乳素受体基因与羊驼繁殖性能关系的初探

通过氯仿/异戊醇法制备羊驼血液基因组DNA,采用PCR方法首次扩增出羊驼催乳素受体基因(prolactin receptor gene,PRLR)exon8-exon9序列(GenBank登录号为DQ198164),该片段长度为622bp。通过NCBI blast(http://www.ncbi.nlm.nih.gov/BLAST/)比较,结果表明:该序列包括exon8的82bp、intron8全序列472bp和exon9的68bp。同源性比较发现,羊驼PRLR基因exon8和exon9核苷酸序列与其它哺乳动物的相应区域的同源性特高,均≥92%;同时还发现羊驼exon8引物后第19个碱基为G,而其它哺乳动物(猪除外)均为A,猪则是在羊驼exon8引物后的第34个碱基处由G变为A,通过推导氨基酸序列分析发现,这种单碱基的突变使得羊驼与其它哺乳动物相比,该处的氨基酸由亮氨酸取代了异亮氨酸;在羊驼exon9引物前第22个碱基处也发生了A-G碱基替换现象,但这个碱基的突变发生在密码子的第3个碱基上,编码的氨基酸均为脯氨酸。在这些动物中只有羊驼为单胎动物,羊驼exon8核苷酸序列中A-G的碱基替换并引起编码氨基酸序列发生改变是否与羊驼繁殖性能有关还有待进一步研究。     

Transition and transversion rate in the evolution of animal mitochondrial DNA

We present a further application of the stochastic model previously described (Lanave et al., 1984, 1985) for measuring the nucleotide substitution rate in the mammalian evolution of the mitochondrial DNA (mtDNA). The applicability of this method depends on the validity of "stationarity conditions" (equal nucleotide frequencies at first, second and third silent codon positions in homologous protein coding genes). In the comparison of homologous sequences satisfying the stationarity condition at the silent sites, only the four codon families (quartets) for which both transitions and transversions are silent at the third position are considered here. This has allowed us to estimate the transition and transversion rates for any pair of species. We have analyzed the third silent codon position of the triplet rat-mouse-cow, of a series of slightly divergent primates and of two Drosophila species. In terms of two external dating input we have then determined the phylogenetic trees for rat, mouse, and cow as well as for a number of primates including man. The phylogenetic tree that we have derived for the triplet rat, mouse and cow agrees with that we had previously determined by analyzing the first, second and third silent codon positions (in both duets and quartets) of mt genes (Lanave et al., 1985). For primates our method leads to the following branching order from the oldest to the most recent: Gibbon, Orangutan, Gorilla, Chimpanzee and Man. In absolute time, fixing the distance Chimpanzee-Man as 5 million years (Myr) we estimate the dating of the divergence nodes as: Gorilla 7 Myr; Orangutan 16 Myr; Gibbon 20 Myr. In all cases analyzed, the transition rate has been found to be substantially higher than the transversion rate. Moreover we have found that the transition/transversion ratio is different in the various lineages. We suggest that this fact is probably related to the nucleotide frequencies at the third silent codon position.     

Alpha-catulin maps to the familial dysautonomia region on 9q31.

Familial dysautonomia is a severe autosomal-recessive neurodegenerative disease that primarily affects the Ashkenazi Jewish population. We present the mapping of alpha-catulin and show that it maps precisely to the familial dysautonomia candidate region on 9q31. Patient sequence analysis identified two new sequence variants, which show linkage disequilibrium with this disease. A G to A transition at nucleotide 423 in exon 3 is a silent base change that does not alter the Val residue at position 141. A G to C transversion at nucleotide 1579 changes the Glu at postion 527 to Gln. These base changes were analyzed in several patients, unaffected Ashkenazi Jewish controls, and non-Jewish controls. Because of the presence of these sequence variants in several unaffected individuals, alpha-catulin is unlikely to be the causative gene in this disease.     

Nucleotide sequence changes in polyoma virus A gene mutants.

The mutational alterations in polyoma virus mutants ts-a and ts-25E which cause their large T-antigens to be thermolabile have been identified. In ts-a, a G leads to A transition at nucleotide 2193 causes the replacement of Ala (GCT) by Thr (ACT). In ts-25E, a G leads to T transversion at nucleotide 2883 causes the replacement of Gly (GGC) by Cys (TGC). Revertants of both mutants have been isolated and shown to have the original nucleotides restored at these positions.     

Repair of a Mismatch Is Influenced by the Base Composition of the Surrounding Nucleotide Sequence

Heteroduplexes with single base pair mismatches of known sequence were prepared by annealing separated strands of bacteriophage lambda DNA and used to transfect Escherichia coli. A series of transition (G:T and A:C) and transversion (G:A and C:T) mismatches located throughout most of the bacteriophage lambda cI gene has been examined. The results suggest that the transition mismatches are generally better repaired than the transversion mismatches and that, at least for the transversion mismatches studied, repair efficiency increases with increasing G:C content in the neighboring nucleotide sequence. This specificity of the E. coli mismatch repair system can account, in part, for the similar frequencies of base substitution mutations throughout the E. coli genome.     

Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome). An intermediate clinical phenotype caused by substitution of valine for glycine at position 137 of arylsulfatase B.

The Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI) is a lysosomal storage disease with autosomal recessive inheritance caused by deficiency of the enzyme arylsulfatase B. Severe, intermediate, and mild forms of the disease have been described. The molecular correlate of the clinical heterogeneity is not known at present. To identify the molecular defect in a patient with the intermediate form of the disease, arylsulfatase B mRNA from his fibroblasts was reverse-transcribed, amplified by the polymerase chain reaction, and subcloned. Three point mutations were detected by DNA sequence analysis, two of which, a silent A to G transition at nucleotide 1191 and a G to A transition at nucleotide 1126 resulting in a methionine for valine 376 substitution, were polymorphisms. A G to T transversion at nucleotide 410 causing a valine for glycine 137 substitution (G137V) was identified as the mutation underlying the Maroteaux-Lamy phenotype of the patient, who was homozygous for the allele. The kinetic parameters of the mutant arylsulfatase B enzyme toward a radiolabeled trisaccharide substrate were normal excluding an alteration of the active site. The G137V mutation did not affect the synthesis but severely reduced the stability of the arylsulfatase B precursor. While the wild type precursor is converted by limited proteolysis in late endosomes or lysosomes to a mature form, the majority of the mutant precursor was degraded presumably in a compartment proximal to the trans Golgi network and only a small amount escaped to the lysosomes accounting for the low residual enzyme activity in fibroblasts of a patient with the juvenile form of the disease.     

Nucleotide correspondence between protein-coding sequences of Helicobacter pylori 26695 and J99 strains

Comparison of open-reading frames (ORFs) H. pylori 26695 and J99 strains has been revealed prevalence of nucleotide replacements as transitions (more than 3%) above transversions (less than 1%). Prevalence of nucleotide transitions is caused by high speed of C : G to T : A transitions in a coding strand of DNA (3.5-5.3%) and not coding strand (2.9-3.9%). The correspondence rate of transversion (A --> C, A --> T, C --> A, C --> G, G --> C, G --> T, T --> A and T --> G) did not exceed 0.84%. The highest correspondence frequency between C and T was detected in ACGT-ATGT (28.3%) - the site of methylation by active methyltransferase M.Hpy99XI in H. pylori 26695 and J99. Thus one can speculate that predominant transition taking place in H. pylori is mutation of C into T, which is realized through cytosine methylation-deamination mechanism.     

Novel mutations of endothelin-B receptor gene in Pakistani patients with Waardenburg syndrome

Mutations in EDNRB gene have been reported to cause Waardenburg-Shah syndrome (WS4) in humans. We investigated 17 patients with WS4 for identification of mutations in EDNRB gene using PCR and direct sequencing technique. Four genomic mutations were detected in four patients; a G to C transversion in codon 335 (S335C) in exon 5 and a transition of T to C in codon (S361L) in exon 5, a transition of A to G in codon 277 (L277L) in exon 4, a non coding transversion of T to A at −30 nucleotide position of exon 5. None of these mutations were found in controls. One of the patients harbored two novel mutations (S335C, S361L) in exon 5 and one in Intronic region (−30exon5 A>G). All of the mutations were homozygous and novel except the mutation observed in exon 4. In this study, we have identified 3 novel mutations in EDNRB gene associated with WS4 in Pakistani patients.     

Negative correlation of G+C content at silent substitution sites between orthologous human and mouse protein-coding sequences.

We conducted a genome-wide analysis of variations in guanine plus cytosine (G+C) content at the third codon position at silent substitution sites of orthologous human and mouse protein-coding nucleotide sequences. Alignments of 3776 human protein-coding DNA sequences with mouse orthologs having >50 synonymous codons were analyzed, and nucleotide substitutions were counted by comparing sequences in the alignments extracted from gap-free regions. The G+C content at silent sites in these pairs of genes showed a strong negative correlation (r = -0.93). Some gene pairs showed significant differences in G+C content at the third codon position at silent substitution sites. For example, human thymine-DNA glycosylase was A+T-rich at the silent substitution sites, while the orthologous mouse sequence was G+C-rich at the corresponding sites. In contrast, human matrix metalloproteinase 23B was G+C-rich at silent substitution sites, while the mouse ortholog was A+T-rich. We discuss possible implications of this significant negative correlation of G+C content at silent sites.     

In vitro DNA replication implicates O2-ethyldeoxythymidine in transversion mutagenesis by ethylating agents.

A 36-nucleotide oligomer containing a single O2-ethyldeoxythymidine (O2-Et-dT) adduct at a specific site was synthesized. The oligomer, which corresponds to a specific DNA sequence in gene G of bacteriophage phi X174, was used as a template by T7 DNA polymerase to investigate the in vitro mutagenic specificity of O2-Et-dT. At 10 microM dNTP and 5 mM Mg++, the progress of T7 DNA polymerase was interrupted by O2-Et-dT: 80% 3' to O2-Et-dT and 14% after incorporating a nucleotide opposite O2-Et-dT (incorporation-dependent blocked product). DNA synthesis past the lesion was low (6%). Incorporation of a nucleotide opposite O2-Et-dT and subsequent postlesion synthesis were enhanced by increasing the dNTP concentration, with postlesion synthesis reaching 30% at 200 microM. Postlesion synthesis was further increased to 45% by addition of 10 mM dAMP to the polymerization reactions. DNA sequencing revealed that both dA and dT were incorporated opposite O2-Et-dT with dA incorporation impeding the progress of DNA synthesis. dT incorporation was efficiently extended implicating O2-Et-dT in transversion mutagenesis in vivo. These studies provide a basis for understanding the molecular mechanisms by which ethylating agents contribute to cytotoxicity, A.T transversion mutagenesis and activation of the oncogene neu by an A.T----T.A transversion event in rat neuroblastomas.     

Four Novel Cystic Fibrosis Mutations in Splice Junction Sequences Affecting the CFTR Nucleotide Binding Folds

Single cases of the four novel splice site mutations 1525-1G & rarr; A (intron 9), 3601 2A → G (intron 18), 3850 3T → G (intron 19), and 4374 → 1G → T (intron 23) were detected in the CFTR gene of cystic fibrosis patients of Indo-Iranian, Turkish, Polish, and German descent. The nucleotide substitutions at the +1, -1, and -2 positions all destroy splice sites and lead to severe disease alleles associated with features typical of gastrointestinal and pulmonary cystic fibrosis disease. The 3850 -- 3 T-to-G change was discovered in a very mildly affected 33-year-old ΔF508 compound heterozygote, suggesting that the T-to-G transversion at the less conserved -3 position of the acceptor splice site may retain some wildtype function.