大菱鲆MC1R基因克隆与序列分析

黑素皮质素受体1(melanocortin-1-receptor,MC1R)是黑色素形成调控中的一个关键因子.MC1R通过调节色素产生的数量和类型,决定皮肤表型.本研究根据鱼类的MC1R基因保守区的核苷酸序列设计引物,利用PCR技术扩增出大菱鲆MC1R基因部分片段,纯化后进行克隆测序.经生物软件拼接后,得到大菱鲆MC1R基因编码区951 bp片段.序列分析表明:此片段与GenBank上公布的部分鱼类的MC1R基因序列同源性较高(97%～86%).本试验结果为进一步研究该基因的结构与生物学功能奠定了基础.     

野猪MC4R基因的克隆及变异初步研究

黑素皮质素受体4是在人类肥胖研究中发现的重要调节因子,参与调节动物的体重、采食量和能量稳态,缺失MC4R基因的突变纯合体小鼠出现遗传性肥胖。为了进一步揭示其群体遗传变异,寻找新的遗传标记,本研究对野猪(Sus scrofa ussuricus)MC4R基因进行了克隆(GenBank accession NoDQ388767)和序列分析,并对所发现的错义突变进行了基于限制性内切酶HindⅢ的PCR-RFLP分析。序列分析表明野猪与民猪MC4R基因的编码区序列完全相同,与大白猪相比存在4个SNPs;对14头野猪的酶切多态性分析表明该突变位点是多态位点,并且3种基因型的分布符合Hardy-Weinberg定律。结果表明,野猪具有独特的遗传信息。     

用放射杂交板定位鸡的MC4R基因以及其在鸡和人染色体上同源区的比较分析

黑素皮质素受体(melanocortin-4 receptor,MC4R)基因的突变与猪、鼠和人等的食欲、肥胖和生长有关联性,然而对鸡的MC4R基因的功能却知之甚少。为了确定鸡的MC4R基因在染色体上的位置,使用鸡-仓鼠杂交板(ChickRH6)做了该基因的定位工作。通过扩增ChickRH6杂交板上的93个样品,然后经整合分析将mC4R基因定位在2号染色体上的标记MCW0062、BCL2和OVY附近,即2q12。这个连锁图上的5个标记基于两点分析与MC4R的LOD值都大于5。同时,以MC4R基因为标记做了鸡和人的染色体比较分析。结果显示鸡的2号染色体(GGA2)和人的18号染色体(HSA18)存在同源区,且基因BCL2和肥胖基因(obesity)位于MC4R基因附近。推测鸡的MC4R基因与人的MC4R基因可能具有相似的功能。该研究揭示了鸡和人MC4R基因的染色体分布,并用杂交放射板将鸡的MC4R基因定位在2号染色体的12区带。     

藏獒MC1R基因多态性与毛色表型的关联研究

目的:通过检测藏獒黑素皮质激素受体1(MC1R)基因的单链构象多态性(SSCP)在不同毛色群体中的分布,探讨MC1R基因多态性与毛色表型的相关性。方法:采用DNA测序技术,选择不同毛色藏獒的DNA为样本,根据GenBank发布的荷斯坦牛MC1R基因序列设计一对引物,采用PCR-SSCP技术分析MC1R基因在藏獒中的SSCP。结果:MC1R基因在藏獒中具有PCR-SSCP多态性,分别检测到3种基因型(AA、AB和BB);对MC1R基因多态性片段DNA克隆测序后发现,MC1R基因在编码区第313位存在单碱基突变(G→A),该突变导致第105位氨基酸发生由丙氨酸向苏氨酸的改变(T105A)。结论:MC1R基因的多态性与毛色性状不存在显著的相关性。     

比格犬MC4R基因多态性与体重相关性的研究

为了分析比格犬黑素皮质素受体-4基因多态性与犬体重的关系, 根据犬MC4R基因DNA外显子序列, 设计MC4R基因特异PCR引物1对, 犬DNA经PCR扩增, 克隆和测序, 寻找和确定犬MC4R基因的多态性位点, 分析多态性与犬体重的关系。结果在比格犬MC4R基因中发现2处单碱基缺失突变, 1个单碱基颠换变异, 存在Psh AⅠ酶切位点, 并基于PshAⅠ酶切位点建立了PCR-RFLP技术。统计分析显示犬MC4R基因型与体重显著相关, 可以考虑将MC4R基因作为犬体重的候选基因。     

猪资源家系MC4R基因扫描及其与脂肪性状的相关分析

黑素皮质激素受体－4（Melanocortin-4 Receptor,MC4R)是在人类肥胖研究中发现的重要调节因子,它可以与瘦蛋白（Leptin),神经肽Y（Neuropeptide Y,NPY),α－黑素细胞刺激激素（Alpha-melanocyte-stimulating hormone,α-MSH)第一起调节动物体重和采食量。采用PCR－RFLP技术,分析了MC4R基因部分片段在猪资源家系群体中的TaqⅠ酶切片段多态性分析。MC4R基因多态性与生长肥育性状,肉质性状,胴体性状的相关性分析的结果表明,MC4R基因型频率在不同品种群体中的分布不同;MC4R基因与猪胸腰椎间膘厚,臀部膘厚,平均背膘,眼肌宽度,眼肌面积,皮率呈显著性相关。MC4R基因主要以显性作用方式发挥作用,加性作用不显著。     

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.     

黑皮质素受体-4调节通路的研究进展

黑皮质素系统来自阿片-促黑素细胞皮质素原,在中枢摄食行为和能量平衡代谢中起到重要作用,此系统生理功能的发挥主要通过与下丘脑神经元细胞上特定膜受体（黑皮质素受体）结合完成。黑皮质素受体（MCR）有五种亚型（MC1R-MC5R）,其中参与体重调节的受体主要是黑皮质素受体3（MC3R）和黑皮质素受体4（MC4R）。MC4R属于G蛋白耦联受体,具有七次跨膜结构。作为一种膜受体,MC4R发挥体重调节作用,一方面受外界激动剂或拮抗剂的调节;另一方面,此受体活化后会影响到细胞内的信号调节通路。研究MC4R的功能首先要了解受体的结构,本文对G蛋白耦联受体的结构进行了较详细的叙述,MC4R经信号调节通路,激活腺苷酸环化酶,增加cAMP的浓度,最终通过影响细胞内基因的转录和翻译,来调节体重和能量的消耗。     

MC1R基因多态性与豚鼠隐性黄毛色的相关性分析

豚鼠Cavia porcellus的隐性黄毛色表型是由编码黑素皮质激素受体1(MC1R)的extension基因座位的等位基因e控制。本研究对野生型和黄毛色豚鼠MC1R基因位点所在区域进行PCR扩增与测序发现,在黄毛色豚鼠中存在1个2 760 bp的基因组缺失,该缺失涵盖了MC1R基因的整个编码区。采用三引物扩增体系对豚鼠MC1R基因缺失突变进行群体基因分型,在随机选择的58只野生型个体中,36只为EE纯合子,22只为Ee杂合子,而31只黄毛色个体均为ee纯合子;在15只测交后代中,8只黄毛色个体均为ee纯合子,而7只野生型个体均为Ee杂合子。基因分型结果表明,MC1R基因2 760 bp的缺失与隐性黄毛色完全相关。本研究为进一步探究MC1R基因在哺乳动物毛色遗传机制中的作用以及豚鼠的分子标记辅助育种提供了理论依据。     

鸡MC4R基因的SNPs及其与屠体性状的相关研究

黑素皮质素受体(MC4R, melanocortin-4 receptor)基因的突变与猪、鼠和人等的食欲、肥胖、生长等性状有关, 而鸡MC4R基因的功能却知之甚少. 利用PCR-SSCP(single strand conformation polymorphism)和DNA测序的方法, 对资源家系F₂代鸡群MC4R基因多态性进行了分析, 发现存在4个单核苷酸多态(SNPs, single nucleotide polymorphisms)位点. 其中, 在MC4R基因5′调控区-524 nt发生了碱基的转换突变(C→T), 导致突变型基因比野生型基因多了一个NF-E2和一个cap转录因子结合位点; 在MC4R编码区(61 nt)发生了碱基的错义突变(G→A), 导致此处蛋白质的氨基酸由甘氨酸变为精氨酸; 在MC4R编码区315和336 nt发生了碱基的颠换突变(G→T)和转换突变(C→T), 这两个突变为同义突变. 通过最小二乘分析SNPs与屠体性状的关系, 结果是突变的BB, DD和FF等基因型与鸡的体重、全净膛重(或半净膛重)、腿肉重等存在显著(P<0.05)或极显著(P<0.01)的关系, 但与腹脂重不显著. 结果表明, MC4R基因可以作为影响和控制鸡体重、生长等屠体性状的主要候选基因.     

High polymorphism at the human melanocortin 1 receptor locus

Variation in human skin/hair pigmentation is due to varied amounts of eumelanin (brown/black melanins) and phaeomelanin (red/yellow melanins) produced by the melanocytes. The melanocortin 1 receptor (MC1R) is a regulator of eu- and phaeomelanin production in the melanocytes, and MC1R mutations causing coat color changes are known in many mammals. We have sequenced the MC1R gene in 121 individuals sampled from world populations with an emphasis on Asian populations. We found variation at five nonsynonymous sites (resulting in the variants Arg67Gln, Asp84Glu, Val92Met, Arg151Cys, and Arg163Gln), but at only one synonymous site (A942G). Interestingly, the human consensus protein sequence is observed in all 25 African individuals studied, but at lower frequencies in the other populations examined, especially in East and Southeast Asians. The Arg163Gln variant is absent in the Africans studied, almost absent in Europeans, and at a low frequency (7%) in Indians, but is at an exceptionally high frequency (70%) in East and Southeast Asians. The MC1R gene in common and pygmy chimpanzees, gorilla, orangutan, and baboon was sequenced to study the evolution of MC1R. The ancestral human MC1R sequence is identical to the human consensus protein sequence, while MC1R varies considerably among higher primates. A comparison of the rates of substitution in genes in the melanocortin receptor family indicates that MC1R has evolved the fastest. In addition, the nucleotide diversity at the MC1R locus is shown to be several times higher than the average nucleotide diversity in human populations, possibly due to diversifying selection.     

黑素皮质素受体-4的研究进展

黑素皮质素受体 4 (MC4R)是人类中枢神经系统中参与调节肥胖症发生的重要因素 ,可调节动物的体重和采食量。自MC4R基因克隆以来 ,学者们对MC4R的结构 ,生理功能 ,调控 ,作用机制及其基因突变与体重的关系等方面进行了大量的研究。     

Synthesis and biological evaluation on hMC3, hMC4 and hMC5 receptors of gamma-MSH analogs substituted with L-alanine.

To elucidate the molecular basis of the interaction of the native dodecapeptide gamma-MSH with the melanocortin receptors, we performed a structure-activity study in which we systematically replaced l-Ala in each position of this peptide. Here we report the binding affinity and agonist potency on human MC3R, MC4R and MC5R. Intracellular cAMP concentration was measured on CHO cells, and binding assays were carried out using membranes prepared from these cell lines which stably express hMC3R, hMC4R and hMC5R. Our results indicate that the last four amino acids in the C-terminal region of gamma-MSH are not important determinants of biological activity and selectivity at human melanocortin receptors. Interesting results were obtained when l-Ala was substituted for His6, Phe7, Arg8 and Trp9. For these peptides, the affinity and activity at all three human receptors (MC3R, MC4R and MC5R) decreased significantly, demonstrating that the His-Phe-Arg-Trp sequence in gamma-MSH is important for activity at these three melanocortin receptors. Similar results were obtained when Met3 was replaced with l-Ala, suggesting the importance of this position in the interaction with all three receptors. This study highlights the role played by the His-Phe-Arg-Trp sequence in receptor binding and in agonist activity of gamma-MSH.     

Functional characterization of mutations in melanocortin-4 receptor associated with human obesity

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor implicated in the regulation of body weight. Genetic studies in humans have identified two frameshift mutations of MC4R associated with a dominantly inherited form of obesity. We have generated and expressed the corresponding MC4R mutants in 293T cells and found that cells transfected with the truncation mutants failed to exhibit agonist binding or responsiveness despite retention of structural motifs potentially sufficient for binding and signaling. Immunofluorescence studies showed that the mutant proteins were expressed and localized in the intracellular compartment but absent from the plasma membrane, suggesting that these mutations disrupted the proper cellular transport of MC4R. Further studies identified a sequence in the cytoplasmic tail of MC4R necessary for the cell surface targeting. We further investigated a possible dominant-negative activity of the mutants on wild-type receptor function. Co-transfection studies showed that the mutants affected neither signaling nor cell surface expression of wild-type MC4R. We also characterized three human sequence variants of MC4R, but these exhibited identical affinities for peptide ligands and identical agonist responsiveness. Thus, unlike the obesity-associated MC4R truncation mutants, the polymorphisms of MC4R are unlikely to be contributors to human obesity.