The beta subunit of human chorionic gonadotropin is encoded by multiple genes.

P Policastro,C E Ovitt,M Hoshina,H Fukuoka,M R Boothby,I Boime

doi:10.1016/s0021-9258(17)44254-2

Abstract

Two recombinant phage clones bearing sequences corresponding to the beta subunit of human chorionic gonadotropin (hCG beta) were isolated from a human genomic library. The beta sequences were mapped by blot hybridization of restriction digests of these phage DNAs and the nonoverlapping inserts were subcloned in pBR322 and sequenced. The nucleotide-sequencing data show that the hCG beta subunit is encoded by at least three nonallelic genes. Moreover, based on restriction analyses of human placental DNA, these genes may be linked in a single cluster with four other hCG beta-like genes. The sequenced genes all differ in their 5' flanking regions, and none of them is completely homologous in sequence to either of two hCG beta cDNA clones used here. In the translated region of one of these genes, three base substitutions result in two changes from the reported amino acid sequence. In the family of beta-containing glycoprotein hormones, the hCG beta subunit is unique in that it contains an extension of 29 amino acids at its COOH end. The DNA sequence corresponding to this region in the sequenced genes is part of a larger exon. These data show that the COOH-terminal extension does not result from splicing of the primary RNA transcript.

Highlights

Two recombinant phage clones bearing sequences corresponding tothe 8 subunit of human chorionic gonadotropin ere isolated from a human genomic library
In the family o&f containing glycoprotein hormones, the hCGp anLdH(3subunits is abou8t0% (3).The 12 cysteine residues that form intrachain disulfide bonds are highly conserved positions in thep chain family, presumably reflecting their importance to the secondary structure required for association with the a subunit (3).The hCGP subunit is unique in that it contains an extensoifo2n9 amino acids a t its COOH end (4)
The nucleotide sequence of a cDNA clone to hCGp mRNA suggests this extension derivesfrom loss of an upstream stop codon, since the triplet encoding glutamine, the terminalamino acid of theextension,formspart of the polyadenylation signal (5)

Summary

METHODS

Preparation and Isolation of cDNA Clones-The construction of PUNY, acDNA clone derived from hCGp mRNA, has been previously described (8). A human genomic DNAlibrary was provided by Dr Tom Maniatis, Harvard University It was prepared from 15-20-kb fragments of a nonlimit HaeIII-Ah1 digest of human fetal liverDNA ligated to bacteriophage Charon 4A "arms" via EcoRI linkers (16).E. coli strain LE392 (17) was grown in Luria-Bertani (LB) broth and used as host in plating phage at a density of 1-2 X lo plaques/gO-mm diameter dish. Transformation of HBlOl was performed as for cDNA clones; colonies growing on LB-ampicillin (20 Fg/ml) were screened for phage fragment inserts by hybridization of filter replicas (12) to nick-translated PUNY and phage clones. Positive colonies were further screened by small scale plasmid purifications (15) followed by EcoRI digestion, 5% polyacrylamide-TBE gel electrophoresis, and EtBr staining. Tetracycline-resistant, ampicillin-sensitive colonies were screened by restriction endonuclease digestion of small scale plasmid preparations followed by polyacrylamide-TBE gel electrophoresis and EtBr staining. Phage insert subclones were sequenced according to Maxam and Gilbert (24), using both 5' and 3' end labeling methods as described

RESULTS

G TCGLCATTTCC AG G h

DISCUSSION