Use of the EST Database Resource to Identify and Clone Novel Mono(ADP-Ribosyl)Transferase Gene Family Members

Abstract

We searched the database of expressed sequence tags (dbEST) for relatives of the known human and murine mono(ADP-ribosyl)transferases (mADPRT), poly(ADP-ribosyl)polymerases (PARP), ADP-ribosyl cyclases, and ADP-ribosylarginine hydro lases (ARH). By May 31, 1996, all of the known enzymes except for RT6 were represented in dbEST by exact sequence matches from mouse and/or human tissues. Several ESTs show significant sequence similarity but not identity to known mADPRTs. We isolated, cloned, and sequenced the corresponding genes. Our results show that seven human ESTs stem from a novel gene, provisionally designated LART, which is specifically expressed in lymphatic tissues. Five human ESTs stem from a novel gene, here designated TARTJ, which is specifically expressed in testis. This gene is also represented by a single mouse EST. One other mouse EST stems from a distinct gene, here designated TART2, which is also expressed in testis. These genes have similar exon/intron structures. The predicted LART and TARTI gene products contain hydrophobic N-and C-terminal signal peptides characteristic for GPI-anchored surface proteins, TART2 lacks the GPI-anchor signal peptide. The predicted native proteins show 28-42% sequence identity to one another. They each contain four cysteine residues that probably form conserved disulfide bonds. They each also contain a conserved glutamic acid residue within the proposed active site motif. LART and TARTI show interesting deviations from the surrounding consensus sequence.

Transcriptional Control

“…Human and murine mono (ADP) ribosyl transferases are testis specific. In human one specific gene (TART 1) and the other in murine (TART2) are testes specific (Braren et al, 1997). The predicted TART1 product contains hydrophobic Nand C-terminal signal peptides characteristic for GPI anchored surface proteins, where as TART;!…”

“…The predicted TART1 product contains hydrophobic Nand C-terminal signal peptides characteristic for GPI anchored surface proteins, where as TART;! contain 4 cysteine residues and also a glutamic acid conserved residue at active site (Braren et al, 1997).…”

Digital cloning: Identification of human cDNAs homologous to novel kinases through expressed sequence tag database searching

“…The catalytic domain of a kinase is further divided into 12 smaller subdomains, defined as regions uninterrupted by large insertions and containing characteristic, highly conserved amino acid residues [23][24][25]. Subdomains I-IV, located at the NH2-terminus of the catalytic domain, are involved in anchoring and orienting the nucleotide ATP [7][8][9][10]. Subdomains VI-XI form a large lobe structure at the COOH-terminus of the catalytic domain and are involved in the binding of substrates and catalyzing the phospho-transfer reaction [8,12,46,47].…”

“…ESTs are short cDNA sequences (only a few hundred base pairs in length), which are derived by partial, single-pass sequencing of the inserts of randomly selected cDNA clones [3,7]. It has been estimated that 40-80% of the total number of human genes are represented in dbEST, and a significant percentage of EST clones are derived from yet uncharacterized genes [26]; thus, the dbEST represents a significant resource for identifying new genes [5,9,34,43]. Such an extraordinary database represents valuable, low-priced, and easily accessible resources.…”

Digital Cloning: Identification of Human cDNAs Homologous to Novel Kinases through Expressed Sequence Tag Database Searching

“…Such an extraordinary database represents valuable, low-priced, and easily accessible resources. It has been estimated that 40-80% of the total number of human genes are represented in dbEST, and a significant percentage of EST clones are derived from yet uncharacterized genes [26]; thus, the dbEST represents a significant resource for identifying new genes [5,9,34,43].…”