牦牛MC1R基因的多态性研究

旨在为探究牦牛MC1R基因多态性与毛色形成的相关性,利用PCR-SSCP和DNA测序技术,对64头牦牛(33头黑色九龙牦牛,31头白色天祝白牦牛)的MC1R基因多态性进行检测。结果表明:天祝白牦牛和九龙牦牛均有3种基因型(AA、BB、AB),但天祝白牦牛的多态性较低,而九龙牦牛表现为中度多态。经χ2适合性检验,2个牦牛品种在该基因多态位点上均偏离Hardy-Weinberg平衡。测序结果表明BB型与AA型在该片段的第179位碱基处存在C→A单碱基突变;第214位碱基处发生T→C突变。     

天祝白牦牛OXGR1基因多态性与体尺性状的关联性分析

为了研究牦牛α-酮戊二酸（盐）受体1（Oxoglutarate receptor1, OXGR1）基因多态性与其体尺性状的相关性,本文以4-8岁天祝雄性（阉）白牦牛的血液DNA（n=192）为实验材料并构建DNA混合池。通过DNA直接测序法检测OXGR1的核苷酸序列潜在的多态位点;利用高分辨率熔解曲线分析技术 (High Resolution Melting,HRM ) 进行分型。采用SHESIS软件对OXGR1基因多态位点进行连锁不平衡分析;运用 PIC-Calc 0.6软件分析多态信息含量;采用卡方检验检测 Hardy- Weinberg平衡;运用SPSS20.0软件对多态位点与体尺性状进行关联分析;运用RNAFOLD、ExPASy和Swiss-model软件对OXGR1基因突变前后的蛋白结构进行预测分析。结果表明：天祝白牦牛OXGR1基因发现有两个多态位点,分别为347(A/G)和678(G/A),每个位点均有3种基因型（AA、AG、和GG）,其优势基因型分别为GG和AA。两个SNPs均达到Hardy-Weinberg 平衡（P＞0.05）,存在强连锁不平衡（D’＞0.75, R²＞0.33）,且均表现为中度多态（0.25< PIC< 0.5）。上述位点不同基因型在体斜长、体高、胸围和管围存在显著差异（P＜0.05）。分子结构预测显示：347 位点处突变为错义突变,其编码的氨基酸由天冬酰胺变为丝氨酸。突变后OXGR1 mRNA二级结构、蛋白质二级结构及三级结构均发生改变。上述结果表明: OXGR1基因可作为牦牛分子育种的候选分子标记,为今后牦牛遗传资源的保护、开发以及新品种的选育提供依据。     

牦牛MC1R基因的克隆测序及其分析研究

以麦洼牦牛、斯布牦牛、天祝牦牛和九龙牦牛为研究对象,对黑色素皮质素受体1(Melanocortin receptor I,MCIR)基因编码区进行了克隆测序及分析.结果表明,牦牛的MC1R基因编码区全长954 bp,编码317个氨基酸:4个牦牛品种间及与普通牛间在MC1R基因的编码区内共有13个碱基差异,无碱基的插入和缺失现象,编码蛋白共有9个氨基酸差异.MC1R蛋白为亲水性蛋白,无信号肽,有糖基化位点和7个跨膜区.系统进化分析显示,麦洼牦牛与斯布牦牛的MC1R基因相似性最近.本研究结果时今后开展MC1R基因与牦牛毛色性状的相关性分析以及牦牛的毛色遗传机理、基因定位、基因表达调控等研究具有重要的意义.     

牦牛生态类型的分类

为进一步弄清中国牦牛的遗传资源及其类型划分,利用微卫星DNA、随机扩增多态性（RAPD）、扩增片断长度多态性（AFLP）等3种分子遗传标记技术研究了麦洼牦牛、九龙牦牛、大通牦牛和天祝白牦牛的分类;并结合作者对牦牛染色体和血液蛋白多态性的研究结果探讨了中国牦牛类群的分类.结果：①根据微卫星位点的等位基因频率进行聚类分析,表明麦洼牦牛和九龙牦牛的遗传距离最大（1.506）,麦洼牦牛2个群体之间的遗传距离最小（1.062）.5个牦牛群体被聚为两大类,四川九龙牦牛单独成一大类,其他牦牛群体聚为一类.②根据RAPD和AFLP两种分子遗传标记的分析,表明天祝牦牛和大通牦牛的遗传距离最小（0.0336）,九龙牦牛和天祝牦牛的遗传距离最大（0.0414）,4个牦牛品种被聚为两大类,九龙牦牛品种聚为一类,其它3个牦牛品种聚为一类.大通牦牛和天祝白牦牛在较近的水平上首先聚为一类,然后在较远处与麦洼牦牛聚为一大类.③根据染色体特征和血液蛋白位点的基因频率进行聚类的结果与微卫星DNA、RAPD、AFLP的聚类结果相似.中国牦牛可分为以九龙牦牛和麦洼牦牛为代表的两个类群（型）.这与蔡立等将中国牦牛分为“青藏高原型”和“横断高山型”的结果是一致的.而与其他学者的分类结果有较大的差异.结合中国牦牛品种（群体）的地理分布、生态条件、育成史及其分化的实际情况,作者认为中国牦牛分为两个大的生态类型是合理的.     

我国黄牛属(Bos)五个种的染色体比较研究

本文应用外周血淋巴细胞短期培养方法,研究了我国黄牛属中五种牛的染色体组型,结果:黄牛(Bos taurus)2n=60;牦牛(Bos grunniens)2n=60;高峰牛(Bos indicus)2n=60;大额牛(Bos frontalis)2n=58;云南野牛(Bos gaurus readei)2n=56。并从染色体数目和形态上比较了它们之间的差异和类同。最后,从细胞遗传学角度讨论了这几种牛彼此之间杂交育种的可能性,为畜牧业上培养新的优良牛种提供了细胞遗传学的依据。     

血清白蛋白与抗肿瘤药物莪术醇的相互作用关系

莪术醇是近年来发现的重要的新的抗肿瘤中药单体之一。分析莪术醇与转运蛋白—血清白蛋白之间的相互作用能够帮助研究者更好的理解药物的作用机制。本文通过用多序列分析、进化关系和分子对接技术等分析莪术醇与人血清白蛋白相互作用位点及其在其他亲缘关系较近的物种中的特点。结果表明,莪术醇与人血清白蛋白相互作用的结合位点II和III处周围几乎都是疏水性的氨基酸,分子间的疏水作用起着很重要的作用,其最低结合能分别是-7.22 Kcal/mol和-8.34 Kcal/mol。莪术醇与人血清白蛋白之间的作用位点在其他亲缘关系较近的狼(Canis lupus)、绵羊(Ovine)、牦牛(Bos mutus)、家牛(Bos Taurus)物种中都较为保守,少数有变化的氨基酸基本是在极性相同的氨基酸之间发生的。与分子相互作用前的结构相比,莪术醇中的羟基的结构在活性位点处发生了最明显的变化。     

牦牛HYOU1基因克隆及组织表达分析

为探究HYOU1基因在牦牛中的组织表达谱,通过对西藏牦牛HYOU1基因的CDS区进行克隆测序,分析该基因的结构和功能,采用RT-PCR技术检测黄牛和牦牛肺脏、心脏、肝脏、乳腺、大脑及肌肉中HYOU1基因的相对表达量。结果表明:(1)HYOU1基因CDS区长度为3 006 bp,其中A、G、C和T含量分别为25.0%、31.1%、26.7%和17.1%,存在一定碱基偏好性;发现1个(ACG→ACA)SNP位点,为同义突变。(2)生物信息学分析表明该蛋白呈中性,为较不稳定、亲水性分泌信号蛋白;存在一个跨膜螺旋(TMhelix)区位于13-35氨基酸位置;二、三级蛋白结构分析发现其主要的空间构象为:无规则卷曲及α-螺旋。(3)聚类分析显示,西藏牦牛与野牦牛的遗传距离最近,与瘤牛及普通牛的遗传距离次之,与水牛最远。(4)RT-PCR结果显示HYOU1基因在黄牛和牦牛肝脏、乳腺、肺脏、大脑、心脏、肌肉6个组织中均有表达,且相对表达量依次递减;在相同组织中,黄牛组织的表达量均显著高于牦牛组织中的表达量(P0.05),其中黄牛肝脏组织的表达量为牦牛表达量的4.4倍。     

天祝白牦牛IGF-2基因的cDNA克隆

旨在克隆天祝白牦牛胰岛素样生长因子2(IGF-2)基因编码区全长cDNA序列,为研究该基因的生理功能奠定基础。运用cDNA末端快速扩增(RACE)技术获得天祝白牦牛IGF-2基因全长cDNA序列。扩增获得天祝白牦牛IGF-2基因全长cDNA序列为1 060 bp(GenBank登录号:KF682139),ORF长540 bp,编码179个氨基酸。其编码的氨基酸与已报道哺乳动物IGF-2氨基酸序列同源性在80%-92%之间。天祝白牦牛IGF-2基因的成功克隆为进一步研究该基因的功能奠定了基础。     

大通牦牛ApoA1基因克隆及生物信息学分析与组织表达谱分析

本试验旨在分析大通牦牛(Bos grunniens)ApoA1基因的分子特征,并检测其在不同组织的表达规律,为进一步研究ApoA1基因的功能提供理论依据。试验以牦牛肝脏cDNA为模板,通过PCR扩增和基因克隆获得牦牛ApoA1基因编码区序列(coding region sequence, CDS),并进行生物信息学分析;利用RT-qPCR技术检测ApoA1基因mRNA在心脏、肌肉、肝脏、脾脏、肺脏、肾脏、子宫角、脂肪和输卵管等组织中的mRNA表达水平。结果表明,大通牦牛ApoA1基因编码区序列长798 bp,编码265个氨基酸;ApoA1蛋白质的分子质量为30 324.414 Da,理论等电点为5.71;蛋白质二级结构以α-螺旋(93.58%)为主;牦牛ApoA1蛋白为不稳定亲水蛋白,存在信号肽,为分泌蛋白,不存在跨膜结构域;牦牛ApoA1基因的核苷酸序列与黄牛相应序列相似度最高,同源性为99.64%;组织表达谱分析表明,ApoA1基因在大通牦牛不同组织中都表达,但在肝脏中表达量最高,显著高于其他组织(P<0.05)。这些结果为进一步研究大通牦牛ApoA1基因在大通牦牛结构和功能...     

牦牛AQP4基因CDS序列的克隆及其生物信息学特征分析

为了研究高原动物对青藏高原高寒、低氧等极端生境的适应机理,进一步探讨高原动物对高原反应——高原脑水肿抗性的分子机理,运用基因克隆与生物信息学相关技术和方法,对牦牛脑AQP4(水通道蛋白4,AQP4)基因CDS全长序列进行克隆、基因序列比对及其生物信息学特征分析。结果表明,牦牛AQP4的CDS含有一个966 bp的开放阅读框,编码322个氨基酸;牦牛AQP4基因编码蛋白分子量34.69 k D,理论等电点(p I)7.59,其编码蛋白含有6次跨膜结构,属于疏水性蛋白;二级结构主要由α-螺旋、延伸及无规则卷曲构成;AQP4基因编码产物氨基酸同源性及系统进化分析发现,牦牛AQP4基因编码氨基酸序列与黄牛、绵羊等物种间同源性较高,系统进化情况与其亲缘关系远近一致。     

Comprehensive investigation of nucleotide diverdity in yaks

To understand the maternal genetic diversity of Tianzhu white yak better, we analyzed mtDNA D‐loop sequences of 209 Tianzhu white yaks, which are from the central region of Tianzhu white yak habitat. Accordingly, a total of 45 haplotypes were identified in Tianzhu white yaks in this study, and 18 of them were unique. The nucleotide diversity and haplotype diversity of population studied were 0.024 ± 0.003 and 0.946 ± 0.007 respectively, revealing that Tianzhu white yak possess a relatively high genetic diversity. The phylogenetic analysis, combining D‐loop sequences in this study with 533 previous published D‐loop sequences of 13 yak breeds, indicated that Tianzhu white yaks fell mainly into haplogroup A and that a small portion belonged to haplogroups B, C, D and E. Moreover, six haplotypes of 20 individuals identified in Tianzhu white yak were in the taurine haplogroup, indicating hybridization between Bos taurus and Tianzhu white yaks. In summary, this study supplies a comprehensive maternal genetic pattern for Tianzhu white yak and provides a basic reference for future breeding programs to conserve the purebred Tianzhu white yak.     

Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp:Asn170Gln with benzylpenicillin and cephaloridine

The serine-beta-lactamases hydrolyze beta-lactam antibiotics in a reaction that proceeds via an acyl-enzyme intermediate. The double mutation, E166D:N170Q, of the class A enzyme from Staphylococcus aureus results in a protein incapable of deacylation. The crystal structure of this beta-lactamase, determined at 2.3 A resolution, shows that except for the mutation sites, the structure is very similar to that of the native protein. The crystal structures of two acyl-enzyme adducts, one with benzylpenicillin and the other with cephaloridine, have been determined at 1.76 and 1.86 A resolution, respectively. Both acyl-enzymes show similar key features, with the carbonyl carbon atom of the cleaved beta-lactam bond covalently bound to the side chain of the active site Ser70, and the carbonyl oxygen atom in an oxyanion hole. The thiadolizine ring of the cleaved penicillin is located in a slightly different position than the dihydrothiazine ring of cephaloridine. Consequently, the carboxylate moieties attached to the rings form different sets of interactions. The carboxylate group of benzylpenicillin interacts with the side chain of Gln237. The carboxylate group of cephaloridine is located between Arg244 and Lys234 side chains and also interacts with Ser235 hydroxyl group. The interactions of the cephaloridine resemble those seen in the structure of the acyl-enzyme of beta-lactamase from Escherichia coli with benzylpenicillin. The side chains attached to the cleaved beta-lactam rings of benzylpenicillin and cephaloridine are located in a similar position, which is different than the position observed in the E. coli benzylpenicillin acyl-enzyme complex. The three modes of binding do not show a trend that explains the preference for benzylpenicillin over cephaloridine in the class A beta-lactamases. Rather, the conformational variation arises because cleavage of the beta-lactam bond provides additional flexibility not available when the fused rings are intact. The structural information suggests that specificity is determined prior to the cleavage of the beta-lactam ring, when the rigid fused rings of benzylpenicillin and cephaloridine each form different interactions with the active site.     

Identification of a novel bond between a histidine and the essential tyrosine in catalase HPII of Escherichia coli.

A bond between the N delta of the imidazole ring of His 392 and the C beta of the essential Tyr 415 has been found in the refined crystal structure at 1.9 A resolution of catalase HPII of Escherichia coli. This novel type of covalent linkage is clearly defined in the electron density map of HPII and is confirmed by matrix-assisted laser desorption/ionization mass spectrometry analysis of tryptic digest mixtures. The geometry of the bond is compatible with both the sp3 hybridization of the C beta atom and the planarity of the imidazole ring. Two mutated variants of HPII active site residues, H128N and N201H, do not contain the His 392-Tyr 415 bond, and their crystal structures show that the imidazole ring of His 392 was rotated, in both cases, by 80 degrees relative to its position in HPII. These mutant forms of HPII are catalytically inactive and do not convert heme b to heme d, suggesting a relationship between the self-catalyzed heme conversion reaction and the formation of the His-Tyr linkage. A model coupling the two processes and involving the reaction of one molecule of H2O2 on the proximal side of the heme with compound 1 is proposed.     

Probing protein structure and dynamics with resonance Raman spectroscopy: cytochrome c peroxidase and hemoglobin

Because vibrational frequencies are sensitive to structure, RR spectroscopy can provide structural information about kinetic steps in protein transformations when carried out in a time-resolved mode. UVRR spectroscopy has shown that the aromatic groups of the HbCO photoproduct respond with a delay of 20 microseconds and has provided direct structural evidence that the 20-microseconds kinetic step is the R-T quaternary re-arrangement of the subunits. RR bands of the porphyrin ring show that the core relaxes via a 0.1-microsecond protein motion, which probably allows the Fe atom to attain its full out-of plane displacement. The Fe-His stretching frequency has an elevated value immediately after CO photolysis, in part, perhaps, because of the protein constraint on the Fe displacement. It relaxes on both the 0.1- and 1-microsecond time scales to its value in R-state Hb and then decreases further to its T-state value. These changes may be connected with reorientation of the proximal His side chain. At very early times after a photolysis pulse, heating effects may be an important aspect of the protein dynamics, but further experiments are needed to understand the RR response.     

Screening for signatures of selection of Tianzhu white yak using genome‐wide re‐sequencing

The Tianzhu white yak, a domestic yak indigenous to the Qilian Mountains, migrated inland from the Qinghai‐Tibet Plateau. Specific ecological and long‐term artificial selection influenced the evolution of its pure white coat and physiological characteristics. Therefore, it is not only a natural population that represents a genomic selective region of environmental adaptability but is also an animal model for studying the pigmentation of the yak coat. A total of 24 261 829 variants, including 22 445 252 SNPs, were obtained from 29 yaks by genome‐wide re‐sequencing. According to the results of a selective sweep analysis of Tianzhu white yak in comparison to Tibetan yaks, nine candidate genes under selection in Tianzhu white yak were identified by combining π, Tajima's D, πA/πB and F_ST statistics, with threshold standards of 5%. These genes include PDCD1, NUP210, ABCG8, NEU4, LOC102287650, D2HGDH, COL4A1, RTP5 and HDAC11. Five of the nine genes were classified into 12 molecular signaling pathways, and most of these signaling pathways are involved in environmental information processing, organismal systems and metabolism. A majority of these genes has not been implicated in previous studies of yak coat color and high‐altitude animals. Our findings are helpful not only for explaining the molecular mechanism of yak coat pigmentation but also for exploring the genetic changes in Tianzhu white yak due to environmental adaptation.     

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.     

X-ray and functional studies of hemoglobins Nancy and Cochin-Port-Royal.

The mutations in hemoglobin Nancy beta145(HC2) Tyr leads to Asp and hemoglobin Cochin-Portal-Royal beta146(HC3) His leads to Arg involve residues which are thought to be essential for the full expression of allosteric action in hemoglobin. Relative to the structure of deoxyhemoglobin A, our x-ray study of deoxyhemoglobin Nancy shows severe disordering of the beta chain COOH-terminal tetrapeptide and a possible movement of the beta heme iron atom toward the plane of the porphyrin ring. These structural perturbations result in a high oxygen affinity, reduced Bohr effect, and lack of cooperatively in hemoglobin Nancy. In the presence of inositol hexaphosphate (IHP), the Hill constant for hemoglobin Nancy increases from 1.1 to 2.0. But relative to its action on hemoglobin A, IHP is much less effective in reducing the oxygen affinity and in increasing the Bohr effect of hemoglobin Nancy. This indicates that IHP does not influence the R in equilibrium T equilibrium as much in hemoglobin Nancy as in hemoglobin A, and this probably is due to the disordering of His 143beta which is known to be part of the IHP binding site. IHP is also known to produce large changes in the absorption spectrum of methemoglobin A, but we find that it has no effect on the spectrum of methemoglobin Nancy. In contrast to the large structural changes in deoxyhemoglobin Nancy, the structure of deoxyhemoglobin Cochin-Port-Royal differs from deoxyhemoglobin A only in the position of the side chain of residue 146beta. The intrasubunit salt bridge between His 146beta and Asp 94beta in deoxyhemoglobin A is lost in deoxyhemoglobin Cochin-Portal-Royal with the guanidinium ion of Arg 146beta floating freely in solution. This small difference in structure results in a reduced Bohr effect, but does not cause a change in the Hill coefficient, the response to 2,3-diphosphoglycerate, or the oxygen affinity at physiological pH.     

X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11?Å resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.     

Comparison of straight chain and cyclic unnatural amino acids embedded in the core of staphylococcal nuclease.

We have determined by X-ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side-chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM. Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026-1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over-packing of the protein core results in a 1.0 A shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.