Ovine growth hormone gene duplication—structural and
Rachel Ofir, Elisha Gootwine
Institute of Animal Science, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel
Received: 20 January 1997 / Accepted: 2 June 1997
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sheep breeds studied (E. Gootwine, unpublished results), suggest-
ing 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
In sheep carrying the duplicated GH2 allele, only the GH2-N
copy is expressed in the pituitary (Gootwine et al., 1996). Re-
cently, 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 Euro-
pean 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 frac-
tionated. Genomic DNA fractions containing either the GH2-N or
the GH2-Z copies were obtained by HindIII digestion: 150 gof
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 frag-
ments that included the GH2-N copy only, or 5.8-kb DNA frag-
ments that included the GH2-Z copy only, were excised, and the
DNA was electroeluted and ethanol precipitated. DNA for se-
quencing 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 origi-
nal amplification primers and dye termination labeling in an Ap-
plied 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 in-
stead of asparagine at amino acid No. 148 of the hormone mol-
ecule. Two to eight nucleotide differences were found at the non-
coding regions between the GH2-N sequences and the reference
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 substi-
Correspondence to: E. Gootwine
Contribution from the Agricultural Research Organization, The Volcani
Center, Bet Dagan, Israel. No. 2035-E, 1996 series.
The nucleotide sequence data reported in this paper have been submitted to
Genbank and have been assigned the accession numbers AF002110–
Table 1. Primers used to amplify different regions of the ovine GH gene
Orientation Sequence (5Ј to 3Ј)
1 145 5Ј 3Ј TTATCCATTAGCACAGGCTGCCAGTG
2 144 5Ј 3Ј ATTATCCATTAGCACAGGCTGCCA
3 453 3Ј 5Ј TCAAACTTGGCCAAATGTCGGGTG
4 466 5Ј 3Ј GGCCAAGTTTGAAATGTTCTCAG
5 587 5Ј 3Ј ACCTCCCTGCTCCTGGCTTTCA
6 706 5Ј 3Ј GCATCAACTGGCTACTGACACC
7 752 3Ј 5Ј CTGGGGAGCTTACAAACTCTTT
8 987 5Ј 3Ј GGGACAGAGATACTCCATCCAG
9 993 3Ј 5Ј CTGTCCCTCCGGGATGTAGG
10 1416 5Ј 3Ј CGGACCTGGTCTATGAGAAGC
11 1448 3Ј 5Ј CCAGGTCCTTCAGCTTCTCATAG
12 1747 3Ј 5Ј GGGTAACATCTTCCAGCTCCTG
13 1749 5Ј 3Ј GGAGCTGGAAGATGTTACCCCC
14 1941 3Ј 5Ј GGAGTGGCACCTTCCAGGGTC
Position follows the designation of Orian and associates (1988).
Mammalian Genome 8, 770–772 (1997).
© Springer-Verlag New York Inc. 1997