[Genomic DNA sequences and functional expression, purification of BmalphaTX14 neurotoxin from scorpion Buthus martensii Karsch].

Based on the full-length cDNA of BmalphaTX14 from Chinese scorpion Buthus martensii Karsch (BmK), gene of the mature peptide of BmalphaTX14 was cloned into the yeast expression vector pPIC9K. After transforming, screening and inducing, tricine-SDS-PAGE and Western blot proved that rBmalphaTX14 protein was expressed in the medium for up to 84 hours, getting nearly 120 mg/L. Recombinant BmalphaTX14 was purified rapidly and efficiently through Ni-NTA-agarose, polyethylene glycol precipitation and gel filtration chromatography. The purified rBmalphaTX14 proved to have the anti-insect activity by toxicity assay. Meanwhile, genomic gene of BmalphaTX14 was cloned and sequenced by PCR method, sequence analysis of this gene showed that BmalphaTX14 had an intron of 408 base pairs located at the signal peptide encoding region, which was similar with the characteristic of other alpha-type sodium ion-channel toxin. Considering both the genomic organization and the peptide function, BmaTX14 proved to be a membership belonging to alpha-type sodium ion-channel toxin.

[Analysis of the genomic organization of a novel scorpion toxin BmTXK beta].

Scorpion venoms contain different types of low molecular mass toxic peptides acting on ion channels. Many cDNAs and genomic genes encoding these toxins have been isolated and sequenced while the mechanisms of expression and regulation of scorpion toxins is not well studied yet. BmTXK beta, one of the four putative long-chain potassium channel toxins, is isolated from the cDNA library of the venom gland of Chinese scorpion BmK (Buthus martensii Karsch). It has an 886 bp intron located in the mature peptide while other scorpion toxins' introns are located in the signal peptide. The special genomic organization of BmTXK beta makes it a good object to study the mechanism of expression and regulation of scorpion toxins. With primers designed according to the already known sequence of BmTXK beta, its 5' and 3' flanking regions are cloned by the Vecttorette II Staorette Pack method and sequenced. Analysis of the sequence shows that another intron longer than 997 bp is located in the signal peptide region of BmTXK beta, which makes BmTXK beta different from all the other scorpion toxins that have no intron or only one intron in the signal peptide. The special genomic organization of BmTXK beta indicates that BmTXK beta is a new membership of long-chain potassium channel toxin.

Differential expression of the Quox-1 gene in normal human cells, early human embryo, and tumor cells.

Quox-1 is the only gene in the hox family whose expression occurs throughout the developing central nervous system. The differential expression of the Quox-1 gene was studied in normal human tissues and tumor tissues. Marked expression of Quox-1 was detected in early human embryos, LCE cells, and HeLa cells, with weak to zero expression being detected in various normal human tissues. Immunocytochemistry analysis further confirmed that the Quox-1 protein was absent in normal human leukocytes. However, high levels of Quox-1 product were found in leukocytes of acute lymphocyte leukemia patients and in patients with a subtype of acute nonlymphocyte leukemia. In addition, Southern blot analysis showed that the genomic DNA of LCE, HeLa, and normal human leukocyte cells had a DNA rearrangement of the Quox-1 gene, suggesting that the rearrangement of genomic DNA might be the cause of differential expression in normal human tissues and tumor tissues. The data implied that the overexpression of Quox-1 was associated with tumors, and that there may be links between the processes of embryogenesis and carcinogenesis.

Sequence Analysis of 47 Low-abundance ESTs Expressed in Early Human Embryo.

The availability of high quality cDNA libraries has proven essential to positional gene cloning efforts differential gene expression studies and EST sequencing/mapping projects.In order to isolate and identify new genes expressed during early human development a 3-week-old human embryo cDNA library was constructed and a pre-screening procedure was used to select cDNAs corresponding to low abundance mRNAs. 6 508 clones were hybridized with a mixture of cDNA probes. Approximately 1 677 clones (26%) did not hybridize with the cDNA probes and represent low abundance mRNAs as well as empty vectors.Partial sequences were generated from one or both end of 47 low abundance cDNA clones and the sequences comparisons with genetic databases revealed that 38.3% of them was annotated human genes 10.6% was highly similar to those from either human or other species 40.4% was partial sequence matched with ESTs that had already been detected and 8.5% of the cDNAs appeared to be unknown in the genetic databases.

Construction of Early Human Embryo cDNA Libraries and Screening Objective Genes.

The construction, evaluation, and application of cDNA libraries from 3-, 4-, and 5-week-old human embryos are described. Total RNAs were extracted from whole embryos using a modified single-step method. mRNA purified by two passes through oligo (dT) columns was reverse-transcripted into single-stranded cDNA. Alkaline agarose electrophoresis showed that the double-strand cDNA fragments ranged from 0.4 9.0 kb and most of them were in the range of 1.0 2.0 kb. After separation on SizeSep 400 Spun columns to eliminate excess adaptors and small cDNA fragments(less than 400 bp), the cDNAs were ligated into pSPORT1 plasmid and lambdaZipLox phage. The plasmid libraries have complexities of 2.6x10(5), 1.7x10(5) and 2.1x10(5) clones and the phage cDNA libraries have complexities of 3.4x10(6), 3.7x10(6) and 2.3x10(6) clones, respectively. Three whole length cDNAs encoding human CD59, MCP and DAF were amplified by PCR using 3-week-old phage library as templates, and human tPA gene with whole length cDNA was screened from 4-week-old plasmid library by hybridization. It was shown that these libraries are of high quality and are suitable to screen rarely expressed genes. The libraries are a valuable source for the study of novel gene expression during human development.