Cloning and sequencing of a deoxyribonucleic acid copy of glyceraldehyde 3-phosphate dehydrogenase messenger ribonucleic acid isolated from chicken muscle

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified from the breast muscles of 3-week-old chickens and used to raise a specific antiserum in rabbits. This antiserum was coupled to an in vitro translation assay to monitor the purification of GAPDH mRNA. RNA was isolated from identical breast muscles and consecutively fractionated with several techniques to yield a preparation of GAPDH mRNA which was at least 50% pure. Double-stranded cDNA was made against this purified RNA, inserted into pBR322, and used to transform Escherichia coli. Recombinants were screened by colony filter hybridization with a cDNA probe made against the purified RNA. The hybridization-positive clone with the largest insert, pGAD-28, was then characterized by using pGAD-28-cellulose to select complementary RNA from total poly(A) RNA and then translating the hybridization-selected RNA in vitro. The single translation product was shown to be GAPDH by (1) comigration with pure GAPDH on sodium dodecyl sulfate-polyacrylamide gels, (2) precipitation with specific anti-GAPDH antiserum, (3) cyanylation fingerprinting, and (4) AMP-agarose affinity chromatography. pGAD-28 was mapped with several restriction enzymes and then sequenced by the method of Maxam and Gilbert [Maxam, A. M., & Gilbert, W. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 560]. The 1261-nucleotide insert was found to contain 29 nucleotides of noncoding sequence at the 5' end, the entire coding region, and 230 nucleotides of the 3'-noncoding region including a poly(A) addition signal (AATAAA) and the first five residues of the poly(A) tail.

Identification and Characterization of a Novel Squamous Cell-associated Gene Related to PMP22

“…The plasmid pGas3 94EH8 (23) containing an EcoRI-HindIII fragment encoding gas3 was kindly provided by Dr. C. Schneider (International Center for Genetic Engineering and Biotechnology, Trieste, Italy). Glyceraldehyde-3-phosphate dehydrogenase (pGAD-28) served as a control probe (40).…”

A Myb dependent pathway maintains Friend murine erythroleukemia cells in an immature and proliferating state

“…Ten micrograms of total cellular RNA from each sample was fractionated on 0.8% agarose-formaldehyde gels, transferred to nitrocellulose ®lters and hybridized to [a-32 P]dCTP nick-translated probes as we have previously described . The probes used were a 600 bp BamHI/EcoRI murine c-myb cDNA fragment encoding the DNA binding domain ), a 1.5 kb XhoI murine c-myc second and third exon cDNA fragment (Stanton et al, 1983), a 9.6 kb EcoRI fragment of murine a-globin cDNA (McClinton et al, 1990), a 1.2 kb PstI fragment of chicken glyceraldehydes 3-phosphate dehydrogenase (GAPHD) cDNA (Dugaiczyk et al, 1983), and a 900 bp Nco1/Sst murine mad1 cDNA fragment (the kind gift of Dr Shoshana Segal, National Cancer Institute). The ®lters were washed and processed for autoradiography as previously described .…”

Bcl-2 interferes with the execution phase, but not upstream events, in glucocorticoid-induced leukemia apoptosis

“…The ®lters were prehybridized to block unspeci®c binding and hybridized to heat-denatured 32 P-labeled cDNA probes for human bcl-2 (donated by Dr SJ Korsmeyer) (Seto et al, 1988), human c-myc (gift from Dr M Eilers) (Eilers et al, 1989), chicken GAPDH (Dugaiczyk et al, 1983), or human a-tubulin (donated by Dr A Helmberg) (Cowan et al, 1983), respectively. The washed blots were exposed to an Agfa Curix X-ray ®lm with an amplifying screen at 7908C for 3 h to several days.…”