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Synthetic double-stranded DNAs (sDNAs) were prepared from sheep globin mRNA templates isolated from reticulocytes producing either hemoglobin B (HbB) (alpha 2 beta B2), HbC (alpha 2 beta C2), or HbF (alpha 2 gamma 2). These DNAs were inserted into the Eco RI site of plasmid pMB9 by the homopolymer tailing method and used to transform Escherichia coli X1776 to tetracycline resistance. Recombinant clones were identified by colony hybridization and further characterized by molecular hybridization and restriction endonuclease analysis. All plasmids analyzed thus far contained either beta- or gamma-globin DNA sequences. Moreover, sDNAs used for cloning yielded restriction endonuclease fragments consistent with the presence of predominantly beta- or gamma-sDNA, indicating that formation of double-stranded alpha-sDNA proceeds much less efficiently under our conditions than the formation of non-alpha-sDNAs. Three recombinant plasmids, pS beta B2, pS beta C69, and pS gamma 56, were selected for detailed study. These were shown to contain, respectively, beta B-, beta C-, and gamma-DNA sequences by molecular hybridization and by protection of the appropriate cDNAs from S1 nuclease digestion. Each contained all of the restriction endonuclease sites defined for the synthetic sDNAs and protected at least 90% of the sequence length of homologous cDNA. Restriction endonuclease maps of the beta B- and beta C-globin genes were identical at all 12 sites that were mapped, whereas four differences were identified in the gamma gene compared to the two others; three of these corresponded to differences in amino acid sequence of the globins. A method was developed to isolate the anti-mRNA strand of the insert for use as a specific molecular hybridization probe analogous to complementary DNA.

Specific complementary DNAs (cDNAs) for the messenger RNAs coding for sheep alpha-, betaA-, betaB-, betaC-, and gamma-globins were prepared by thermal denaturation of heterologous hybrids (e.g. alphabetaB-cDNA-alphagamma-mRNA) followed by hydroxylapatite chromatography. Each cDNA represented a nearly full-length copy of its globin mRNA complement as determined by electrophoretic analysis in polyacrylamide gels containing 98% formamide. The purity of each cDNA fraction was estimated by hybridization analysis and thermal denaturation. The beta- and gamma-cDNAs contained 5 to 20% contaminating alpha-cDNA while the alpha-cDNA was 25 to 30% contaminated with non-alpha-cDNA. The melting temperatures (Tm) of homologous duplexes between each non-alpha chain cDNA and its mRNA complement ranged from 69.5-71.5 degrees in 50% formamide while alpha-alpha duplexes melted with a Tm of 75-76 degrees. The Tm values of heterologous duplexes formed between each non-alpha-cDNA and the various globin mRNAs (e.g. betaB-cDNA-Hb C mRNA) ranged between 64.5 degrees and 68 degrees and thus were only 1.5-5.0degrees below that of homologous duplexes. These results suggest that the nucleotide sequence divergence among the various non-alpha-mRNAs (or cDNAs) is not greatly different from the minimum predicted from the amino acid sequence differences of the corresponding globins. When annealing reactions were performed above the Tm of the heterologous hybrids (68 degrees), each non-alpha-cDNA hybridized only to its own complementary mRNA. Thus the purified cDNAs provide molecular probes for the quantitation of alpha-, beta-, and gamma-globin-specific nucleotide sequences.

Genomic DNA from a fetal sheep homozygous for the beta A gene was used to construct a library of one million cloned DNA fragments using the bacteriophage vector, Charon 4A. Screening of 150,000 plaques from this library using radioactive beta-globin gene sequences resulted in the isolation of two recombinant bacteriophage containing globin genes. One of these, S beta AG-21, contains the complete adult beta A-globin gene as demonstrated by hybridization and restriction endonuclease analysis. In common with adult globin genes from other species, the beta A gene contains small (105 base pairs) and large (900 base pairs) intervening sequences. The second recombinant bacteriophage, SG-4, contains a complete embryonic beta-like globin gene which is expressed in the sheep embryo as demonstrated by hybridization analysis with cDNA made from sheep embryonic globin mRNA. Although differing in its restriction endonuclease map from the adult beta-globin genes, SG-4 appears to contain a large intervening sequence of at least 750 base pairs in length. Finally, preliminary evidence is discussed which indicates that a Pvu II site just 5' to the Cap site may be a common feature of sheep globin genes.