The growth hormone (GH) gene belongs to a gene family that also includes the chorionic somatomammotropin (placental lactogen) gene, the prolactin gene, and several prolactin-like genes, all evolved through series of gene duplications (Ohta 1993; Wallis 1993, 1994, 1996). The ovine GH gene is about 1.8 kb long and contains five exons and four introns (Byrne et al. 1987; Orian et al. 1988). Previous studies (Valinsky et al. 1990; Gootwine et al. 1993) have shown that gene duplication occurred at the ovine GH locus, and two alleles are found: the GH1 allele with a single GH copy, and the GH2 allele with two gene copies designated GH2-N and GH2-Z. The GH2 allele is found in wild sheep (Valinsky et al. 1990) and is the more frequent allele in most of the domesticated sheep breeds studied (E. Gootwine, unpublished results), suggesting that carrying the duplicated GH2 allele may have a selective advantage. It is not yet clear whether the GH2-N and the GH2-Z gene copies are similar in their structure or whether they code for different peptides. In sheep carrying the duplicated GH2 allele, only the GH2-N copy is expressed in the pituitary (Gootwine et al., 1996). Recently, it was found that GH is expressed also in the ovine placenta (Lacroix et al. 1996) where three GH mRNA variants were found. The relationship between these GH variants and the GH1, GH2-N, and GH2-Z is not yet clear. In the present study we looked at the nucleotide sequences of each of the GH1, GH2-N, and GH2-Z copies of the ovine GH and compared them with DNA sequences of pituitary and placental oGH expressed genes. Genomic DNA samples were obtained from an Awassi ewe and a Romney ewe homozygous for the GH1 allele, and from an Awassi ewe and a Romanov ewe homozygous for the GH2 allele. The sheep were selected from breeds that are unrelated and which were separated early in sheep domestication. The Awassi is a fat-tailed Asiatic breed, the Romanov belongs to the north European short-tailed breeds, and the Romney originated from the southeast England Romney Marsh breed. Regions of the GH1 gene copy were amplified from DNA extracted from GH1/GH1 sheep by means of the PCR reaction as described previously (Gootwine et al. 1993), with sets of primers (Table 1, Fig. 1A) that were designed on the basis of the published sequence of the oGH gene (Byrne et al. 1987; Orian et al. 1988). Regions of the GH2-N and GH2-Z copies were amplified from DNA extracted from GH2/GH2 sheep, after the DNA was fractionated. Genomic DNA fractions containing either the GH2-N or the GH2-Z copies were obtained by HindIII digestion: 150 mg of DNA from a homozygous GH2/GH2 sheep was digested with HindIII (Boehringer Mannheim, Germany) and separated on an 0.8% (wt/vol) agarose gel. Gel slices containing 8.3-kb DNA fragments that included the GH2-N copy only, or 5.8-kb DNA fragments that included the GH2-Z copy only, were excised, and the DNA was electroeluted and ethanol precipitated. DNA for sequencing was prepared from PCR products after purification with DS Primer Remover (Adv. Gen. Tech. Corp., Gathersburg, MD, USA). Direct PCR cycle sequencing was performed with the original amplification primers and dye termination labeling in an Applied Biosystems Inc. Model 373A automated DNA sequencer. Templates were sequenced from both ends. The full length of the GH genes of the GH1/GH1 Awassi and Romney ewes, 80% of the lengths of the GH2-N and GH2-Z gene copies of the GH2/GH2 Awassi ewe, and 53% and 76% lengths respectively, of the GH2-N and GH2-Z gene copies of the GH2/ GH2 Romanov ewe were obtained (GeneBank Accession numbers AF002110-AF002129; Fig. 1B). The structure of the two GH1 gene copies was identical and exactly matched (100%) the previously published oGH sequence (Orian et al. 1988), which will be considered here as a reference sequence. The structure of the GH2-N copy of the Awassi ewe was also identical in its coding region to the reference sequence, while the sequence of the coding region of the GH2-N copy of the Romanov sheep differed at one position, predicting histidine instead of asparagine at amino acid No. 148 of the hormone molecule. Two to eight nucleotide differences were found at the noncoding regions between the GH2-N sequences and the reference sequence. Five substitutions were found in the coding region of the Awassi GH2-Z gene copy compared with the reference sequence. Three were nonsynonymous substitutions leading to the presence of leucine, arginine, and serine instead of proline, glycine, and glycine in amino acid positions Nos. −7, 9, and 63, respectively. Two sequence differences leading to the same amino acid substiCorrespondence to: E. Gootwine
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Dec 15, 2002
James De Jersey + 4
Open Access
