Complementary DNA

About: Complementary DNA is a research topic. Over the lifetime, 55301 publications have been published within this topic receiving 2752650 citations. The topic is also known as: cDNA & DNA, Complementary.

Isolation and expression of an altered mouse dihydrofolate reductase cDNA

Abstract: We have constructed a cDNA library from a murine cell line expressing high levels of a dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) that displays an abnormally low affinity for methotrexate. From this library we have isolated a cDNA clone similar to, but distinguishable from, a cDNA clone previously demonstrated to encode the wild-type enzyme. Analysis of the nucleotide sequence of this cDNA clone allows us to predict that the altered dihydrofolate reductase differs from the wild-type enzyme at a single amino acid, reflecting the substitution of an arginine for a leucine residue in a region of the polypeptide thought to form a hydrophobic pocket essential for inhibitor binding. To confirm that this substitution was responsible for the altered properties of the enzyme, we genetically localized the region of the cDNA that specified resistance to methotrexate by in vitro recombination. These results reveal that a single nucleotide change in the codon specifying amino acid 22 of the enzyme was sufficient to alter the methotrexate sensitivity of the enzyme. We demonstrate that this altered gene can be employed as a dominant selectable marker in cultured cells expressing normal levels of wild-type dihydrofolate reductase.

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.

Sequence similarity of phospholipase C with the non-catalytic region of src

Abstract: The production of the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate1 is mediated by activated phos-phatidylinositol-specific phospholipase C (PLC) enzymes2,3. Here we report the cloning of a bovine brain complementary DNA encoding an enzyme PLC-148 that is characterized by calcium-dependent and phosphatidylinositol-specific phospholipase C activity when expressed in mammalian cells. Bovine brain messenger RNA contains a 7.5-kilobase transcript corresponding to the isolated cDNA; a related transcript of the same size is present in mRNA from some but not all human cell lines tested. Southern blot analysis of the bovine genome indicated that one or possibly two genes hybridize to the cloned PLC-148 cDNA. There is a striking sequence similarity between specific regions of PLC-148 and the non-catalytic domain of the non-receptor tyrosine kinases. The newly characterized crk transforming gene of the avian sarcoma virus CT10 also contains extensive sequence similarities with PLC-148.

Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin, and Kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2.

Abstract: Using a 796-basepair cDNA fragment obtained from a mouse pituitary library we have screened two mouse insulinoma libraries and isolated a full-length cDNA clone (2516 basepairs; 753 amino acids), designated mPC1. The cDNA sequence of mPC1 codes for a protein containing 753 amino acids and three potential N-glycosylation sites. This cDNA encodes a putative novel subtilisin-like proteinase, exhibiting within its presumed catalytic domain 64%, 55%, and 47% amino acid sequence identity to the recently characterized candidate prohormone convertases human Furin, mouse PC2, and yeast Kex2 gene products, respectively. An identical sequence to mPC1 was derived from a cDNA library of mouse corticotroph AtT-20 tumor cells. An ArgGlyAsp tripeptide identical to the recognition sequence of integrins was observed in the structures of the mammalian PC1, PC2, and Furin. In situ hybridization results demonstrated a distinct localization of the mPC1 and mPC2 transcripts in pituitary and brain. Thus, whereas both mPC1 and mP...