Sherman G B, Heilman D F, Hoss A J, Bunick D, Lund L A
Great Plains Veterinary Educational Center, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, PO Box 148, Clay Center, Nebraska 68933, USA.
J Mol Endocrinol. 2001 Jun;26(3):267-80. doi: 10.1677/jme.0.0260267.
Neither gene locus nor gene sequence characterizations have been reported for the beta subunits of guinea pig (gp) LH and putative gp chorionic gonadotropin (CG). Descriptions of this locus would allow comparison with functionally relevant molecular genetic features of other species' homologous loci including the single-copy equid LH/CGbeta gene and the primate LHbeta-CGbeta gene cluster locus. Contiguous cDNA and genomic DNA fragments spanning the entire mature coding sequence of gpLHbeta mRNA, gpCGbeta mRNA and a homologous gpLH/CGbeta gene were amplified using PCR methodologies. With the exception of one silent mutation, the two cDNA and the genomic sequences were identical where they overlapped. Comparison of guinea pig coding sequence with LHbeta, CGbeta and LH/CGbeta sequences of other vertebrate species revealed the following order of similarity expressed as per cent coding sequence identity: rhinoceros LHbeta (83.6%)>pig LHbeta (81.8%)>donkey LH/CGbeta=bovine LHbeta (81.5%)> horse LH/CGbeta (80.6%)>dog LHbeta (79.7%)>human LHbeta (78.2%)>rat LHbeta (77.9%)>human CGbeta (75.8%)>turkey LHbeta (52.7%); values that are generally consistent with recently postulated phylogenetic relationships. Like the consensus mammalian LHbeta gene, the 5'-flanking region of the gpLH/CGbeta gene contains a single TATA sequence 37 bp upstream of the translation start codon. The first in-frame stop codon occurred at codon position +122 which is consistent with the 121 amino acid residue length of the consensus mammalian mature LHbeta peptide. To estimate gene copy number, full-length gpLHbeta cDNA was radiolabeled and hybridized to Southern blots of guinea pig genomic DNA digested with a panel of six restriction endonucleases. The resulting simple hybridization pattern strongly suggested that there is a single-copy gpLH/CGbeta gene. Northern analysis of total pituitary RNA using the same probe indicated that gpLHbeta transcript size is indistinguishable from that of consensus mammalian pituitary LHbeta mRNAs ( approximately 750 nucleotides). Despite amplifying gpCGbeta from placental RNA, positive signal was not detected in Northern blot lanes containing guinea pig total RNA prepared from placentae collected at three gestational ages (17.3 days, 24.3 days and 68 days (term)). Other data suggest that inability to detect Northern blot signal could have been due to low relative tissue concentrations of gpCGbeta transcript and/or sampling at gestational time-points that missed peak periods of mRNA expression. We conclude that, with respect to gene copy number, coding sequence and pituitary mRNA size, the gpLH/CGbeta gene locus reflects the CTP-less consensus mammalian LHbeta condition. However, based on the capacity of this single-copy gene to express in both pituitary and placental tissues, gpLH/CGbeta also exhibits functional similarities with the single-copy equine LH/CGbeta locus.
豚鼠(gp)促黄体生成素(LH)和推测的gp绒毛膜促性腺激素(CG)的β亚基的基因座和基因序列特征均未见报道。对该基因座的描述将有助于与其他物种同源基因座的功能相关分子遗传特征进行比较,包括单拷贝马科动物LH/CGβ基因和灵长类动物LHβ - CGβ基因簇基因座。使用PCR方法扩增了跨越gpLHβ mRNA、gpCGβ mRNA和同源gpLH/CGβ基因整个成熟编码序列的连续cDNA和基因组DNA片段。除了一个沉默突变外,两个cDNA和基因组序列在重叠处是相同的。将豚鼠编码序列与其他脊椎动物物种的LHβ、CGβ和LH/CGβ序列进行比较,发现相似性顺序如下,以编码序列同一性百分比表示:犀牛LHβ(83.6%)>猪LHβ(81.8%)>驴LH/CGβ = 牛LHβ(81.5%)>马LH/CGβ(80.6%)>狗LHβ(79.7%)>人LHβ(78.2%)>大鼠LHβ(77.9%)>人CGβ(75.8%)>火鸡LHβ(52.7%);这些值与最近推测的系统发育关系总体一致。与哺乳动物LHβ基因共识序列一样,gpLH/CGβ基因的5'侧翼区域在翻译起始密码子上游37 bp处含有一个单一的TATA序列。第一个框内终止密码子出现在密码子位置+122,这与哺乳动物成熟LHβ肽的121个氨基酸残基长度一致。为了估计基因拷贝数,将全长gpLHβ cDNA进行放射性标记,并与用六种限制性内切酶消化的豚鼠基因组DNA的Southern印迹杂交。所得的简单杂交模式强烈表明存在单拷贝的gpLH/CGβ基因。使用相同探针对垂体总RNA进行Northern分析表明,gpLHβ转录本大小与哺乳动物垂体LHβ mRNA的共识序列(约750个核苷酸)无法区分。尽管从胎盘RNA中扩增出了gpCGβ,但在包含从三个胎龄(17.3天、24.3天和68天(足月))收集的胎盘制备的豚鼠总RNA的Northern印迹泳道中未检测到阳性信号。其他数据表明,无法检测到Northern印迹信号可能是由于gpCGβ转录本的相对组织浓度较低和/或在错过mRNA表达高峰期的胎龄时间点进行采样。我们得出结论,就基因拷贝数、编码序列和垂体mRNA大小而言,gpLH/CGβ基因座反映了无CTP的哺乳动物LHβ基因共识情况。然而,基于这个单拷贝基因在垂体和胎盘组织中均能表达的能力,gpLH/CGβ也与单拷贝的马科动物LH/CGβ基因座表现出功能相似性。