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Abstract
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.
Highlights
From the Clinical Hematology Branch andSLaboratory of Molecular Hematology, National Heart,Lung, and Blood Institute, Bethesda, Maryland 20205
Features characteristic ofglobin genes in other species that were identified in both genes included a sequAeTnAceA, AA, 30 nucleotides from the presumed site of initiation of and rabbit, the sheep globin genes are not closely linked to one another, and, there is considerable homology in the flanking sequences of the y and PAgenes despite their highly selective expression during development
Polymerase (Klenow fragment) was purchased from Boehringer Mannheim, DNase I and RNase A from Worthington Biochemicals, and restriction endonucleases from Bethesda Research Laboratories, of sheep lie within segments of homologous DNA at New England Biolabs, and Boehringer Mannheim
Summary
A Pst I 1 Pvu II fragments from Barn HI and the 7.55 end fragment for Hind111 are those defined by sites for these enzymes within the inserted DNA andthe Eco RI site which markstheend of theinserted DNA segment. C, comparison of the relative positions of the Isco RI, Barn HI, PstI, and PCJUI1 sites in the globin-coding sequence derived from cDNA in the recombinant plasmid pSy56 [25] to those found in the y-globin gene in hSyC31. 8.5, and 1 mM EDTA and the resulting filmpacked onto 3.5% hybridization to the 32P-labeledy-Hha I fragment suggested collodion-coated copper grids. Grids were shadowed with platinum/ that this was the sheep y-globin gene. Carbon (95%:5%by weight) at a 5" angleusingaBalzers BAF 300 Sequence Comparison of the Sheep y- and pA-Globin high vacuum freeze-etch unit equipped with quartz and crystal thin fiim monitor QSG2Ol. The unit was modified for rotary and low angle shadowing (details available on request). Molecules majority of the coding regionsand the sequences immediately weremeasured with a Numonics digitizer.Under these conditions, surrounding the two geneswere determined. We expected the ratio of double-stranded to single-strandedDNA was 1.19:l
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