Two New Human Genes Trpt1 And Pgl29 The Structure And Function Analysis

With the completion ot genome sequencing from human and a number of model organisms, the study of genomics enters post-genomics era. The identification and characterization of novel genes becomes the central mission to be accomplished before us. Here, by means of the analyses of bioinformatics, functional complementation and yeast two-hybrid screening, results of the two genes TRPTl and PGL29 isolated from human are reported as follows.TRPTl cDNA is 963bp in length containing an ORF encoding 253aa with the molecular mass of 27.8kDa and the pI of 10.13. TRPTl is mapped to I1q12.3 spanning approximately 2.5kb on the chromosome. It consists of 8 exons and 7 introns, with the exception of the boundary between the 3'-end of first exon and the 5'-end of first intron, all are consistent with the GT-AG rule.The amino acids sequence of TRPTl shows 40% identity with that of the Tptlp from Saccharomyces cerevisiae. Also, TRPTl is homologous to proteins from other organisms at different degrees over their entire protein sequence. Blast search revealed that a DUF60 /KptA domain was found in the middle part of TRPTl protein. A search by Profscan program showed that some predicted modification sites including four Protein kinase C phosphorylation sites, three Casein kinase II phosphorylation sites and so on distributed over the protein sequence of TRPTl.TPT1 is essential for the vegetative growth of S. cerevisiae and codes for the tRNA 2'-phosphotransferase By using the plasmid shuffling technique, we haveconstructed a TPT1 -deleted strain and transformed it with TRPT1 gene and found that the TRPT1 could complement the yeast TPT1. We believed that TRPT1 is a homolog of yeast TPT1 involved in the yeast tRNA splicing. This result indicated that there existed the same pathway of tRNA splicing in human as in S. cerevisiae, showing its extensively conversation and the presence of the two pathways implicated in tRNA maturation in human.PIT13, an interactor binding to TRPT1 protein, was screened by yeast two-hybrid, and confirmed by pull down and Co-IP assays. The association of TRPT1 with PIT 13 demonstrated that TRPT1 should probably participate in other activities besides in pre-tRNA splicing. More experiments will be required to determine the role of PIT 13 protein.To map the regions of TRPT1 and PIT13 responsible for their interaction with4each other, a series of deletions of TRPT1 or PIT 13 were constructed and examined by yeast two-hybrid. The results showed that the region comprising amino acids 80-220aa nearly within DUF60 from TRPT1 and the one from the amino acids 55-109 of PIT 13 protein were required for their binding to each other.The expression profile of TRPT1 was also examined using MTN blot from Clontech. The TRPT1 was highly expressed in skeletal muscle and in heart, but lower or undetectable in other tissues assayed. We thus suggested that the tRNA splicing may exhibit tissue specificity.Studies on the subcellular localization of GFP-TRPT1/PIT13 showed the TRPT1 and PIT 13 proteins localized predominantly to nuclear bodies. This is consistent with where the TRPT1 functions.Phylogenetic analysis using TRPT1 and some of its homologous proteins showed the TRPT1 gene may be used as a marker to describe the relationships of species.The PGL29 cDNA is 1698bp containing an ORF of 783bp encoding a protein with 260aa. The molecular mass of the predicated protein is 28.6kDa and the pI is5.07. PGL29 has eight exons and seven introns and is mapped to 16p13.3 with ca. 4.0kb in length along the chromosome. The amino acids sequence of PGL29 is 44% identical to Lst8p from S. cerevisiae and four WD40 domains found in its entire amino acids sequence.The resistance assays to all kinds of amino acids analogs of the strain CKY432 transformed with the PGL29 borne on an expression plasmid didn't showed the complementation of LST8 mutation by PGL29 gene. Nor protein partner interacting with PGL29 was detected by yeast two-hybrid screening. Thus further work needs to be made to understand the fu...