Abstract
We have used a myosin heavy chain gene fragment to probe two chicken genomic libraries. The fragment, derived from a gene expressed in the fast-white fibers of adult chickens, contains 1400 bases upstream from the translational start site and 1600 bases downstream from the initiation codon. Thirty-one unique nonoverlapping clones were isolated. All of the genes showed homologies in the nucleotide sequences which code for the globular head portion of the myosin protein while no extensive homologies were detected in the 5'-flanking sequences. The relationships between the genes were studied using oligomeric sequences as probes. The hybridization patterns showed that seven of the genes fall within a well defined subgroup. The exon containing a domain of the nucleotide (ATP) binding site was sequenced for all seven of these genes and shown to be, except for 2 nucleotides in one of the genes, completely conserved. The lack of sequence conservation in the 5'-nontranslated portions of the genes was exploited in the preparation of transcript-specific probes. We have used these probes to show that two of the isoforms are expressed in a tissue-specific and developmental stage-specific manner in the chicken.
Highlights
From the Department of Pharmacologyand Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575 and the Departmentof Biochemistw- ., University of Missouri-ColumbiaSchoolof Medicine, Columbia, Missouri 65212
5”nontranslated portions of the genes was exploited some of the different myosin heavy chain (MHC) protein isoforms
Screening the Chicken GenomicLibraries Using the 5'-End of a Myosin Gene-We reported previously the characteristics of a myosin heavy chain gene which codes for an adult isoform
Summary
Isolation of the Cloms-Two genomic libraries [17,41] both made in Charon 4A using chicken genomic DNA were screened. The 3000-bp fragment used to probe the libraries was labeled to a specific activity of 5 X 10' cpm/ pg by the method of nick translation [30] and added to the hybridization buffer a t a final concentrationof 1X lo cpm/ml. Duplicates and overlaps between the clones (a total of 92were isolated) were determined by restriction endonuclease digestion. A combination of single, double, and triple digestions using the restriction endonucleases EcoRI, AccI, BglI, HincII, Hinfl, PstI, and SphI proved to be sufficient in determining which clones shared common fragments. Partial maps of these clones are described elsewhere [31, 32]. The precipitate was collected by centrifugation, and the products were subsequently analyzed on 8%polyacrylamide gels [38]
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