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LA Lund and GB Sherman

Luteotropic glycoprotein hormones (LGH) include luteinizing hormone (LH) and chorionic gonadotropin (CG). The order Primates is the only phylogenetic clad known to exhibit more than one LGHbeta subunit gene per haploid genome. In the present study, we report the discovery of a second case of LGHbeta gene replication, in the white (w) rhinoceros (r or rhino). The presence of more than one gene was strongly suggested by a complex banding pattern observed on Southern blots of DNA prepared from two unrelated white rhinos. The existence of two LGHbeta genes per haploid genome was estimated by genomic equivalence assay. However, genomic restriction-site mapping studies, together with other findings, suggested that the replicates are probably not tandemly arranged as occurs in primates. A simple band pattern was observed in Southern blots of four other perissodactyl species, indicating that a single-copy LHbeta gene is the consensus condition. Two distinct white rhino LHbeta genomic clones (wrLHbeta1 and wrLHbeta2) were isolated. The nucleotide sequence of wrLHbeta1 was identical with that of wrLHbeta2, except that the latter lacked the consensus mammalian LGHbeta second intron. Sequences of the TATA-containing proximal 5'-flanking regions of the two genes were homologous to at least -57 relative to the site of pituitary transcriptional initiation. We conclude that wrLHbeta1 is the extant form of the ancestral perissodactyl LHbeta gene, whereas wrLHbeta2 is a randomly integrated cDNA element (processed gene) reverse transcribed from a partially spliced ancestral wrLHbeta1 mRNA. That wrLHbeta2 was heritable demonstrates that wrLHbeta1 was transcribed in gametes or early conceptus cells contributing to the germline at some point in time since the divergence of white rhinos from other members of the family Rhinocerotidae. Furthermore, because homologous proximal (pituitary) promoter sequence is present in wrLHbeta2, it can be concluded that the wrLHbeta1 mRNA template from which wrLHbeta2 is derived was transcribed from a secondary promoter located upstream of the consensus TATA-regulated pituitary promoter.

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GB Sherman, DF Heilman, AJ Hoss, D Bunick, and LA Lund

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.