Sheep Genomic DNA Research Articles

The Booroola F gene mutation in sheep is not located close to the FSH-β gene

A cDNA probe for the beta subunit of bovine FSH (FSH-beta) detects multiple restriction fragment length polymorphisms (RFLPs) in sheep genomic DNA consistent with an insertion/deletion polymorphism around the FSH-beta locus. The presence of the insertion/deletion was confirmed by screening over 100 individuals with two restriction enzymes detecting RFLPs. All individuals showed the same patterns of fragments with both enzymes. A partial restriction map of the FSH-beta gene in sheep suggests that the insertion/deletion is approximately 2 kb in size and located downstream from the third exon. Individual DNA samples were analysed from two flocks where the Booroola F gene is known to be segregating. Individuals that were heterozygous for the F gene were shown to be homozygous for one or other of the two alleles. Genetic recombination between the FSH-beta locus and the F gene was observed in four pedigrees and there was no evidence that the insertion/deletion is closely linked genetically to the Booroola F gene. A major gene transcript of 2.2-2.3 kb was detected on Northern blots of sheep RNA. Neither the insertion/deletion polymorphism nor the presence of the F gene appeared to influence the size of the FSH-beta gene transcript.

Two alleles of a neural protein gene linked to scrapie in sheep.

Sheep are the natural hosts of the pathogens that cause scrapie, an infectious degenerative disease of the central nervous system. Scrapie-associated fibrils [and their major protein, prion protein (PrP)] accumulate in the brains of all species affected by scrapie and related diseases. PrP is encoded by a single gene that is linked to (and may be) the major gene controlling the incubation period of the various strains of scrapie pathogens. To investigate the role of PrP in natural scrapie, we have determined its gene structure and expression in the natural host. We have isolated two sheep genomic DNA clones that encode proteins of 256 amino acids with high homology to the PrPs of other species. Sheep PrPs have an arginine/glutamine polymorphism at position 171 that may be related to the alleles of the scrapie incubation-control gene in this species.

The Gene Coding for Tropoelastin is Represented as a Single Copy Sequence in the Haploid Sheep Genome

The identity of the primary in vitro translation products of fetal sheep nuchal ligament elastin mRNA was confirmed as two distinct polypeptides of 63 Kdal and 65Kdal in both rabbit reticulocyte and wheat germ extract cell-free translation systems. Both polypeptides were co-translationally processed by a microsomal membrane signal peptidase, with the removal of 20–25 amino acid residues. A single (3.5 kb) RNA species encodes both tropoelastin polypeptides. Restriction endonuclease mapping of sheep genomic DNA by hybridisation with two radiolabelled genomic DNA fragments containing sequences coding for sheep tropoelastin (pSE1-1.3 and pSE1-0.7,) indicated the presence of a single elastin gene. The elastin gene copy number was further quantitated by comparison of hybridisation of pSE1-1.3 and pSE1-0.7 to slot-blots and Southern transfers of sheep genomic DNA and to standard curves constructed with each clone. These results clearly demonstrate that each of these sequences is represented only once per haploid genome, suggesting that the two tropoelastin polypeptides are products of a single elastin gene.

Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone.

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabelled mRNAE by hydridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.

Hemoglobin switching in sheep. Isolation of the fetal gamma-globin gene and demonstration that the fetal gamma- and adult beta A-globin genes lie within eight kilobase segments of homologous DNA.

A recombinant DNA library of sheep genomic DNA fragments inserted into the bacteriophage vector, Charon 4A, was screened by plaque hybridization with a probe for sheep gamma-globin gene sequences. Three clones containing overlapping segments of DNA, the total length of which was 25 kb, were identified; each included all or a part of the same globin gene. Nucleotide sequencing of substantial portions of the globin gene in one clone, lambda S gamma G31, and of the gene in a previously isolated recombinant, lambda S beta AG21, established that the genes in these recombinants encoded for the gamma- and beta A-globin genes of sheep, respectively. Features characteristic of globin genes in other species that were identified in both genes included a sequence, ATAAAA, 30 nucleotides from the presumed site of initiation of transcription, a region of "capping homology," two introns at positions corresponding to amino acids 29-30 and 103-104 and the polyadenylation sequence, AATAAA, in the 3' untranslated region. Electron microscopic analysis of heteroduplexes formed between lambda S gamma G31 and lambda S beta AG21 revealed that the gamma and beta A genes of sheep lie within segments of homologous DNA at least 8 kb in length within which were identified small regions of nonhomology both 5' and 3' to the genes.