Sequence-tagged clones

The final step in TAG synthesis occurs by the action of acyl-GoA diacylglycerol acyltransferase (DGAT) witli DAG and fatty acyl-GoA as substrates (Fig. 11.2). DGAT is assumed to be a microsomal enzyme. The membrane-bound enzyme has been only partially purified from rat liver microsomes [124]. Gases and coworkers [125] have identified a mouse cDNA encoding DGAT and, in addition, an expressed sequence tag clone that shared regions of similarity with acyl-GoA cholesterol acyltransferase, an enzyme that also uses fatty acyl-CoA as a sub-... 

Expressed Sequence Tag. A short DNA sequence usually representing the most terminal regions of a cDNA clone. 

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). 

Empirically, ORFs have been identified through the high throughput single pass sequencing of individual clones within a cDNA library to rapidly and inexpensively generate Expressed Sequence Tags (ESTs). The ESTs can... 

The exonic-intronic structure of the dysbindin-1 gene is complex and not well established since the cDNA clones available for determining that structure (165 in humans and 207 in the mouse see Ace View) include many that derive only from partially expressed sequence tags or ESTs. Using conservative criteria,... 

EST Expressed sequence tag, a short (100s bp) partial nucleotide sequence from a full-length or nearly full-length cDNA. These sequences are usually from the 5 or 3 ends of directionally cloned inserts as part of high-throughput sequencing projects. The partial cDNA sequence provides annotation by similarity searches and the putative identification of function. 

To determine which protein has been tagged, RT-PCR (reverse transcription-polymerase chain reaction) can then be performed using a PCR primer from the GFP-coding sequence. A initial collection of over 100 tagged clones has been created (Jarvik et al. 2002) (http //cdtag.bio.cmu.edu). 

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