CDNA libraries sequencing

Nabeshima et al. (1987) have isolated two cDNA clones for LC17 mRNA from chicken gizzard and fibroblast cDNA libraries. Sequence analysis of the two... 

Now that we have briefly introduced some of the most important tools and techniques of molecularbiology, we can return to the isolation, cloningand identification of genes ofinterest from within prokaryotic genomic DNA or eukaryotic cDNA library sequences. There are two main approaches for this. 

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. 

The PE2 isoform has been purified and fully sequenced (Markovic and Joumval 1986). Using this sequence data Ray et al (1988) succeeded in isolating a clone fi om a tomato fruit cDNA library. The predicted amino acid sequence fi om this clone had high homology to the actual amino acid sequence determined for PE2 but was not identical. Subsequent screening of the tomato... 

The first Ca -ATPase clones were isolated by probing cDNA libraries with radiolabeled synthetic oligonucleotides [42] that represented an established amino acid sequence ((Trp-) Phe Met Tyr Ala) in the fast-twitch skeletal muscle... 

The amino acid sequence of the human erythrocyte glucose transporter was deduced from the nucleotide sequence of a cDNA clone in 1985 [106]. Polyclonal antibodies raised against the protein were used to screen a Xgtl I cDNA library prepared from the human hepatocellular carcinoma cell line HepG2. (Like many other transformed... 

The murine stromal-derived factor-la (SDF-1)/CXCL12 was originally cloned from a cDNA library derived from the bone marrow stromal cell tine ST2 by a method to clone cDNAs that carry specific amino terminal signal sequences, such as those encoding intercellular signaling-transducing molecules... 

To obtain the sequence for CBP, a cDNA library from the silk gland was prepared and screened using a anti-CBP antibody. One positive clone was identified from 200,000 plaques, and rapid amplification of 5 complementary DNA ends (5 -RACE) was performed to obtain the full length of the CBP cDNA (Figure 24.2a). The predicted sequence encodes a 297-residue polypeptide of... 

TES-32 is the most abundant single protein product secreted by the parasite. It is also heavily labelled by surface iodination of live larvae (Maizels et al., 1984, 1987), and is known by monoclonal antibody reactivity to be expressed in the cuticular matrix of the larval parasite (Page et al, 1992a). TES-32 was cloned by matching peptide sequence derived from gel-purified protein to an expressed sequence tag (EST) dataset of randomly selected clones from a larval cDNA library (Loukas et al., 1999). Because of the high level of expression of TES-32 mRNA, clones encoding this protein were repeatedly sequenced and deposited in the dataset (Tetteh et al., 1999). Full sequence determination showed a major domain with similarity to mammalian C-type (calcium-dependent) lectins (C-TLs), together with shorter N-terminal tracts rich in cysteine and threonine residues. Native TES-32 was then shown to bind to immobilized monosaccharides in a calcium-dependent manner (Loukas et al., 1999). 

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