Isolation and sequencing

In 1985, a peptide was described in the supernatants of endothelial cells that mediated vasoconstriction [1]. This peptide was isolated and sequenced, and the cDNA was cloned. According to its origin from endothelial cells it was named endothelin. 

Note These are examples of important transporters involved in substrate and ADP uptake into the matrix compartment as indicated, and most are reversible. These transporters are proteins and several have been isolated and sequenced. Other specific carriers occur in mitochondria from other tissues. The inner membrane does not allow rapid exchange of NAD or CoA but there are mechanisms for the slow uniport of cofactors synthesized extramitochondrially. 

The expression of many of these molecules has been studied during various stages of differentiation of normal neutrophils and also of corresponding leukemic cells employing molecular biology techniques (eg, measurements of their specific mRNAs). For the majority, cDNAs have been isolated and sequenced, amino acid sequences deduced, genes have been localized to specific chromosomal locations, and exons and intron sequences have been defined. Some important proteinases of neutrophils are listed in Table 52-12. 

Homology screening. Using oligonucleotide probes based on known receptor sequences, search cDNA libraries for homologous sequences which may code for related receptors. The clones are then isolated and sequenced and used in expression studies to confirm the identity of the receptor. 

Carbonic anhydrase (CA) exists in three known soluble forms in humans. All three isozymes (CA I, CA II, and CA III) are monomeric, zinc metalloenzymes with a molecular weight of approximately 29,000. The enzymes catalyze the reaction for the reversible hydration of C02. The CA I deficiency is known to cause renal tubular acidosis and nerve deafness. Deficiency of CA II produces osteopetrosis, renal tubular acidosis, and cerebral calcification. More than 40 CA II-defi-cient patients with a wide variety of ethnic origins have been reported. Both syndromes are autosomal recessive disorders. Enzymatic confirmation can be made by quantitating the CA I and CA II levels in red blood cells. Normally, CA I and CAII each contribute about 50% of the total activity, and the CAI activity is completely abolished by the addition of sodium iodide in the assay system (S22). The cDNA and genomic DNA for human CA I and II have been isolated and sequenced (B34, M33, V9). Structural gene mutations, such as missense mutation, nonsense... 

Kwon, B. S., Haq, A. K Pomerantz, S. H., and Halaban, R. (1987b). Isolation and sequence of a cDNA clone for human tyrosinase that maps at the mouse c-albino locus. Proc. Natl. Acad. Sci. USA 84 7473-7477. 

Hu B, Trinh K, Figueira WF, Price PA 1996 Isolation and sequence of a novel human chondrocyte protein related to mammalian members of the chitinase protein family. J Biol Chem 271 19415-19420... 

Peptides that have been isolated and sequenced from C. elegans. 

The currently attainable level of serrsitivity allows to isolate and sequence peptides with ca. 0.1 nmol/g or higher concentratiorrs in the biological sample. The number of peptides present at these concentratiorrs is typically of the order of lOT Mass-spectral data indicate that the rrumber dramatically increases at lower concentrations as... 

The knowledge of the primary structure was the basis for the construction of models of the secondary structure of the RNA molecules. Different approaches have been used in several laboratories to get experimental support for developing secondary structure models for example, chemic modification of the RNA, treatment with single- or double-strand-specihc nucleases, intramolecular RNA cross-linking, isolation and sequence analysis of double-stranded RNA, and, last but not least, comparison of ribosomal RNA sequences from different organisms (reviewed by Brimacombe et al., 1983). 

Teutsch HG, Hasenfratz MP, Lesot A, Stoltz C, Gamier JM, Jeltsch JM, Durst F, Werck-Reichhart D (1993) Isolation and sequence of a cDNA encoding the Jemsalem artichoke cinnamate 4-hydroxylase, a major plant cytochrome P450 involved in the general phenyl-propanoid pathway. Proc Natl Acad Sci USA 90(9) 4102-4106... 

ACC 71 synthase, i. e. (S)-adenosylmethionine methylthioadenosine lyase (EC 4.4.1.14), has been purified from several plant tissues [116]. Recently, ACC synthase cDNA clones have been isolated and sequenced from wounded fruit tissues of tomato, winter squash, zucchini, ripening apple and tomato fruit. Using the polymerase chain reaction (PCR), four different ACC synthase gene fragments were obtained by amplification of cDNA derived from mRNA of tomato... 

Isolation and sequencing of the cellulose synthase gene(s) has not been accomplished yet however, DNA from Acetobacter xylinum containing this gene(s) was cloned into broad host-range plasmid vectors (82). These vectors were mobilized into Pel- mutants to test for complementation. To date, this approach has not produced a pellicle-forming transconjugant from a Pel- mutant of Acetobacter (82). The direct correlation between cellulose production and presence of plasmid DNA in Acetobacter has been reported... 

Human biotinidase is a monomeric glycoprotein with a molecular mass of 76 kDa when analysed by sodium dodecylsulphate-polyacrylamide gel electrophoresis [7]. The cDNA for human biotinidase was isolated and sequenced in 1994 by Cole and coworkers [6]. The deduced protein contains 502 amino acids, has a molecular mass of 57 kDa and contains 6 potential N-linked glycosylation sites. The biotinidase gene... 

Strong, S.J. Ellington, W.R. Isolation and sequence analysis of the gene for arginine kinase from the chelicerate arthropod, Limulus polyphemus insights into catalytically important residues. Biochim. Biophys. Acta, 1246, 197-200 (1995)... 

---