ABSTRACT
Zinc finger protein, one of the most important transcription factors, plays an essential role in regulating gene expression. C(2)H(2) type zinc protein with KRAB domain contains two parts, one is C(2)H(2) zinc finger motif which is use to binding to the DNA, while the other is KRAB associated box which mostly performs as a transcription repressor. In this study, we report the cloning and characterization of a novel splice variant of the human ZNF300 gene (ZNF300-B). The ZNF300-B gene cDNA is 2293 bp in length, encoding a putative protein with 619 amino acid residues. ZNF300-B gene is mapped to chromosome 5q32-5q33 with seven exons. Reverse transcription polymerase chain reaction (RT-PCR) analysis showed that ZNF300-B and ZNF-300 were expressed highly in human testis.
Subject(s)
Alternative Splicing/genetics , Cloning, Molecular , Repressor Proteins/chemistry , Repressor Proteins/genetics , Testis/metabolism , Zinc Fingers/genetics , Amino Acid Sequence , Base Sequence , Humans , Male , Molecular Sequence Data , Protein Isoforms/biosynthesis , Protein Isoforms/genetics , Repressor Proteins/biosynthesisABSTRACT
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.
Subject(s)
Apoptosis , Carrier Proteins/genetics , Cloning, Molecular/methods , Membrane Proteins/genetics , Proto-Oncogene Proteins/genetics , Apoptosis Regulatory Proteins , Base Sequence , Bcl-2-Like Protein 11 , Brain , Carrier Proteins/physiology , Cell Compartmentation , DNA, Complementary , Fetus , Gene Components , Humans , Membrane Proteins/physiology , Molecular Sequence Data , Protein Isoforms/genetics , Protein Isoforms/physiology , Proto-Oncogene Proteins/physiology , Tissue Distribution , TransfectionABSTRACT
Thermostable p-nitrophenylphosphatase from Bacillus stearothermophilus has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C(2), with unit-cell parameters a = 67.17 A, b = 57.84 A, c = 62.49 A and alpha = 90.0 degrees, beta = 95.4 degrees, gamma = 90.0 degrees. Diffraction data were collected to 1.40 A resolution with a completeness of 94.7% (96.6% for the last shell), an R(fac) value of 0.074 (0.341) and an I/sigma (I) value of 30.1 (2.67).
