Isolation of 24 novel cDNA fragments from microdissected human chromosome band.

The strategy of isolating the band-specific expression fragments from a probe pool generated by human chromosome microdissection was reported. A chromosome 14q24.3 band-specific single copy DNA pool was constructed based on this probe pool. Using total DNA of the pool as probe to hybridize the human marrow cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Among these primary clones, 32 secondary clones were obtained after second-round screening and designed as cFD14-1-32. Finally, 24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization. Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but can't hybridize to 17q11-12 DNA. Partial sequences of 13 fragments of them were sequenced and identified as novel cDNA sequences, and these sequences were proved to have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

Effect of the esters of gallic acid on model and human blood platelet membranes studied by Fourier transform infrared spectroscopy.

Gallic acid is one of the components of Chinese herbal drug Radix paeoniae used for promoting blood circulation to remove blood stasis. This paper studied the effects of gallic acid and its esters (e.g. ethyl, propyl, isobutyl and butyl gallate) on model and human blood platelet membranes by FTIR which was used for monitoring the physical state of the acyl chain, interfacial and head group region of the membrane lipid bilayer. From the experimental results it can be seen that the gallic acid and its esters have the modifying function on the pure and cholesterol-containing DPPC model membranes, and have the quantity-effective and structural-effective relationships. In addition, it is discovered that these esters have the modifying effect on the structure of human blood platelet membrane and can reverse the effect of ADP. That the effect of the esters of gallic acid counteracts the effect of cholesterol and ADP on human blood platelet perhaps provides a new explanation of the mechanism of Chinese herbal drugs used for promoting blood circulation to remove blood stasis.

[Isolation and cloning of human beta 1, 4-galactosyltransferase III gene].

Using the conservative nucleotide sequences encoding the catalytic domain of the beta-1, 4-GalT genes in human, bovine, mouse, chick and snail as probes to search the NCBI GenBank EST database, several ESTs with high homology were obtained. Primers were designed in the flanking sequence of EST contig. Using the PCR product amplified in human placenta cDNA library as probe to perform "walking" hybridization with human placenta cDNA library, a cDNA fragment with the length of 1,907 bp was cloned. It contained an open reading frame (ORF) with the length of 1,179 bp, which encodes 393 amino acid residues. The deduced amino acid sequence of this gene shares 43.8% identity to the human beta-1, 4-GalTI and 60.9% in the catalytic domain especially. The expression mapping showed that it was expressed in human most tissues with a single 2.4 kb transcript, but the relative expression level of the transcript are vary. While this gene was mapped on chromosome 1 using the cDNA hybridization with human/rodent hybrid cell line DNA Southern blot panel.

[Isolation of the expression fragments with the probe pool of human chromosome 14 q 24.3 generated by microdissection].

The strategy of isolating the band-specific expression fragments from the probe pool of human chromosome generated by microdissection was reported in present paper. A chromosome 14 q 24.3 band-specific single copy DNA library was constructed based on this probe pool. Using this pool DNA as probe to hybridize the human bone marrow cell cDNA library, 68 primary positive clones were selected from 5 x 10(5) cDNA clones. Of them 32 clones were got in second-round screening and designed as cFD 14-1-32. Finally, 24 bandspecific expression fragments were identified from these 32 positive clones by analysing the results of DNA hybridization. Those band-specific clones can hybridize to both 14 q 24.3 DNA and human genomic DNA, but have no hybridization signal with 17 q 11-12 DNA. Partial sequences of 13 fragments of them were sequenced and were identified as novel cDNA sequences as well as have some homology with known genes in NCBI database. Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained can be used to isolate the genes not yet be cloned in 14 q 24.3 region.

[Isolation of novel expression sequences of C2H2 type zinc finger protein gene from human brain tissue according to the conservation of zinc finger motif].

By low stringency PCR amplification of genomic DNA using the primers designed based on the conservation of zinc finger motif, we got 8 gradient eletrophoretic bands. After recovery of the second and third bands, the DNA fragments in them were cloned and sequenced. Compared to the GenBank database, among these 60 segments containing zinc finger motif, 23 segments were novel zinc finger genes' genomic segments. Then the human brain tissue cDNA library was screened, using these segments as probes, and 44 positive clones were obtained. Rescreening 28 of them, we got 20 rescreened clones. All of them were sequenced and sent to the GenBank DNA database for sequence analysis, the results showed that 16 were novel C2H2 type zinc finger protein cDNA segments. The cDNA segments encoding the novel C2H2 type zinc finger proteins provide the basic materials for cloning of full length cDNA of valuable novel zinc finger protein genes.