Characterization and phylogenetic significance of rhinoceros luteinizing hormone beta (LHβ) subunit messenger RNA structure, complementary DNA sequence and gene copy number

The luteinizing hormone (LH) β subunit gene is expressed in the pituitary glands of all mammals, whereas the closely related chorionic gonadotropin (CG) β subunit genes have been identified only in primates and equids, and are expressed in placenta. In the case of horses, there is a single-copy equine (e) luteinizing hormone/chorionic gonadotropin hormone β subunit gene (eLH/CGβ) that (1) is expressed in both pituitary gland and placenta, (2) encodes a characteristic carboxyl terminal peptide (CTP) extension, and (3) transcribes an atypically elongated 5'-untranslated region (UTR) in both pituitary and placenta. However, it is not known whether similar expression patterns and gene locus characteristics may be exhibited by other members of the order Perissodactyla (equid, rhinoceros and tapir species). To begin to investigate these possibilities, we undertook analysis of the rhinoceros (rn or rhino) LH/CG?)β gene locus and the mLHβ cDNA. Total RNA isolated from the pituitary gland of a female white rhino was used as template for amplifying mLHβ cDNA by reverse transcription-polymerase chain reaction. Following cloning of the amplified cDNA, nucleotide (nt) and deduced amino acid sequences were determined. The first in-frame stop codon occurred at codon position + 122, suggesting that the rnLHβ subunit does not contain a CTP. To assess gene copy number, Southern blot analysis of Indian rhino genomic DNA was performed. The resulting simple hybridization pattern indicated that, as in the horse and donkey, there is a single-copy gene at the rnLH/(CG?) β gene locus. Primer extension mapping of the pituitary transcriptional start site of the rnLHβ subunit gene revealed an 8 nt 5'-UTR which is similar to that reported for the majority of mammalian LHβ transcripts. Northern analysis was consistent with the transcriptional start site findings. We postulate from these data that rhinos diverged from equids prior to the occurrence of the mutations causing CTP expression and adoption of a non-consensus 5'-UTR/proximal promoter region. However, these findings do not rule out the possibility of expression of a placental CGβ subunit lacking a CTP in rhinos.