Over-expression of Bim alpha3, a novel isoform of human Bim, result in cell apoptosis.

Bim proteins are essential factors of apoptosis. Nine isoforms of Bim have been submitted to GenBank database. In order to improve the understanding of the regulation of Bims' proapoptotic activity, we screened a multiple tissue cDNA panels for Bim isoforms and tested their proapoptotic activity by over-expression. Two novel cDNA isoforms of Bim family are generated by alternative splicing. One isoform encodes a 79 residue putative protein with a BH3 domain, named Bim alpha3. There is not any significant ORF found in another isoform, named Bim beta5. Subcellular localized analysis of EGFP-Bim fusion protein suggests Bim alpha3 distributed to both plasma and nucleus of HEK 293 cell. HEK 293 cells transfected with pcDNA-Bim alpha3 presented a similar proapoptotic activity (32.05 +/- 6.42%) with Bim alpha2 (30.14 +/- 2.66%). The proapoptotic activity of Bim alpha3 was obviously weaker than that of Bim S (46.52 +/- 5.07%) and Bim L (55.53 +/- 1.99%). Anti-sense over-expression of Bim in HEK 293 cells results in a weak down-regulated proapoptotic level. Expression pattern analysis reveals that both the novel cDNAs are expressed widely in normal tissue just like the other reported isoforms. The expression pattern of Bim isoforms shows tissue specific obviously. The results suggest that BH3 domain is sufficiency for proapoptotic activity of Bim proteins. The functional state of Bims might be regulated both in the transcript and post transcript process.

Cloning and identification of a novel cDNA which encodes a putative protein with a DnaJ domain and a thioredoxin active motif, human macrothioredoxin.

A 3345 bp cDNA was isolated from the fetal brain cDNA library by high throughput cDNA sequencing. The cDNA with an open reading fragment (ORF) of 2241 bp encodes a 747 amino acids putative protein with a DnaJ N-terminus domain and four thioredoxin active sets. So it is named human macrothioredoxin (hMTHr). We used Northern blot to detect a band with a length of about 5 kb, which was ubiquitously expressed in human adult tissues with different intensities. The expression pattern was verified by RT-PCR, revealing that the transcripts were ubiquitously expressed in fetal tissues and human tumor tissues also. The transcripts in fetal tissues were more numerous than in adult tissues. The transcripts were high in adult testis, adult pancreas, fetal thymus, and fetal kidney. We have found three splice isoforms with lengths of 3483, 3345, and 2982 bp in the sequencing analysis of the RT-PCR products. They encode three putative proteins with 793, 747, and 275 amino acids, respectively. The first two putative proteins contain a DnaJ domain and four thioredoxin domains. The third putative protein only contains an N-terminus DnaJ domain and one thioredoxin active set. Blast analysis against the NCBI database revealed that the gene spans a genome sequence of about 75 kb that contains at least 25 exons and is located in human chromosome 2q32.1. The organization of the functional motifs of hMTHr suggests that the protein might be a member of a molecular chaperone family.