Sequence analysis of Orientia tsutsugamushi DNA from mites collected in the Xisa archipelago, China.

The genotype of Orientia tsutsugamushi DNA from mites in the Xisa archipelago of China were identified. A natural focus of tsutsugamushi disease in the archipelago was found. The DNA sequence that codes for the 56 kDa protein of O. tsutsugamushi was amplified by nested polymerase chain reaction (N-PCR). The purified positive products were cloned into a pGEM-T vector and sequenced. The DNA sequence was compared with various sequences on the internet for sequence homology. A 507 bp DNA fragment encoding the 56 kDa protein was amplified from the samples. The sequence homology was 85% (Karp strain), 68% (Gilliam strain), 65% (Kato strain), and 67% (Yonchon strain). Orientia tsutsugamushi is carried by the mites of the Xisa archipelago; the main genotype is the Karp strain.

[The analysis of S gene phylogenetic tree of HBV in transmission from father to infant].

OBJECTIVE: To study HBV transmission from father to infants, the study was carried out. METHODS: The study contained 16 pairs of fathers who was HBV carriers, and infants whose mothers wasn't HBV carriers. The infants infected HBV in womb. The homogenous of HBV S gene were compared between fathers and infants, meanwhile HBV gene phylogenetic tree were analyzed. RESULTS: The genotype of 16 pairs fathers and infants were HBV adw. The homogenous of HBV S gene were 98% approximately 100%. The mutation of 488, 491, 494, 530, 531, 546, 581, 621 nucleotide of HBV S gene caused in 112, 113, 114, 126, 131, 143, 156 amino acid substitution. The mutation of 126 amino acid from Threonine to Alanine didn't existed in the gene bank. The sequence was difference from gene classification. CONCLUSION: The homogenous of HBV S gene of fathers and infants was very high. The HBV gene characteristic in China was difference from gene bank.

[Application of microarrays in screening the tumor-specific genes in the genome of K562 cells].

OBJECTIVE: To screen tumor-specific genes of K562 cells using DNA microarray technique. METHODS: The genomic DNA of normal white blood cells and cultured K562 cells were respectively purified and digested with Sau3A I, and the digested DNA fragments of K562 cells were cloned into TA cloning vector to construct the corresponding genomic DNA library. The insert genomic DNA fragments were amplified from the library to prepare the microarray using Cartesian 5500 Microarrayer. The digested genomic DNA fragments of normal white blood cells were labeled with fluorescent Cy3 by restriction display PCR (RD-PCR), followed by hybridization with the microarray, after which the slide was washed and scanned with ScanArray. RESULTS: Among the 426 target genes, 42 differential genes were identified in the genomic DNA of K562 cells in comparison with the normal white blood cells. One of the genes was identified as the breakpoint cluster gene (BCR) after sequence analysis. CONCLUSIONS: The DNA microarrays we constructed may effectively identify the tumor-specific genes in K562 cells, and DNA microarray technique can be helpful in elucidating the molecular mechanisms of tumorigenesis at the genomic level.

[HBV C gene mutation in the transmission from father to infant].

OBJECTIVE: Hepatitis B virus (HBV) DNA was detected from infants whose mothers were negative for all HBV markers and the fathers were HBV carrier, the homology of HBV sequence of fathers and fetus was high, and HBV mutations concentrated on some points, and the transmission of HBV from father to fetus was also identified in some reports. The present study aimed to study HBV transmission from father to infant. METHODS: The study enrolled 16 pairs of fathers who were HBV carriers and infants whose mothers were negative for HBV markers. The infants had evidences for intrauterine HBV infection. The five HBV serum markers HBsAg, HBeAg, anti-HBe, anti-HBs, and anti-HBc were detected with ELISA. The positive results for HBsAg and/or HBeAg were regarded as markers of HBV infection. Amplification of HBV DNA was done using a nested PCR method. The first amplification was carried out using primer C1 (nt 2394-2370), and primer C3 (nt 1730-1754). The second amplification was carried out using primer C2 (nt 1955-1974) and primer C6 (nt 2348-2330). Both primers were designed to amplify the part of sequence coding for the hepatitis B C antigen. The size of the amplified fragment obtained by the nested PCR was expected to be 394 bp. The PCR products were electrophoresed on 1.5% agarose gels, which were then stained with ethidium bromide and observed with ultraviolet transillumination. When 394 bp specific band was detectable, the sample was designated positive. Then the positive samples were identified by dot blot. The second PCR products were extracted by phenol-chloroform and 70% ethanol precipitation, then resuspended in TE buffer (pH8.0), and used as the template for cloning. The template was connected into pGEM-T vector by ligase. The ligated products were cloned into fresh competent JM109 cells, and incubated for 90 minutes at 37 degrees C on roller drum. Finally several dilutions were plated on plates containing ampicillin, X-Gal and IPTG, and incubated at 37 degrees C overnight. The white colony on plates was used for identification by the nested PCR with the above primers. When the 394 bp band was detectable by electrophoresis of PCR products in 1.5% agarose gels, the colony was designated positive; a positive colony was incubated in LB medium for 8 to 12 hrs, then plasmid was extracted using the Wizard Plus SV Minipreps DNA Purification System Kit (Promega). The purified plasmid was sent to Beijing Saibaisheng Company for sequencing. The homology of HBV C nt 2022-2301 sequence was compared between fathers and infants. RESULTS: The homology of HBV C nt 2022-2301 sequence were 99% - 100% in 16 pairs of fathers and infants. The results were referred to the published sequence of HBV adw/adr clones, and the nucleic acid databases were searched for homology by using BLAST tool on Internet. HBV of the sixteen pairs of father/infant was closely related to the Japan strain (Genebank accession number AF121249), but there were still 17 more mutations at nucleotide positions 2029, 2034, 2044, 2059, 2078, 2095, 2104, 2154, 2161, 2169, 2189, 2201, 2233, 2251, 2284, 2288, 2293. Moreover the mutations at positions 2189, 2288 resulted in the substitution of the encoded amino acid (corresponding to amino acid positions 97 and 130, respectively), the other mutations at the position were nonphenotypic. The mutation of 2189, 2288 nucleotide of HBV C gene caused 97, 130 amino acid substitution for isoleucine to leucine and proline to threonine. The mutation of 2189, 2288 nucleotide of HBV C gene were detected in 6 (37.5%) of 16 pairs of fathers and infants. CONCLUSION: The HBV transmission from father to infants did exist. The main HBV C gene mutation strains also existed in the transmission.

Sequence analysis of Orientia tsutsugamushi DNA from mites collected in the Xisa archipelago, China.

The genotype of Orientia tsutsugamushi DNA from mites in the Xisa archipelago of China were identified. A natural focus of tsutsugamushi disease in the archipelago was found. The DNA sequence that codes for the 56 kDa protein of O. tsutsugamushi was amplified by nested polymerase chain reaction (N-PCR). The purified positive products were cloned into a pGEM-T vector and sequenced. The DNA sequence was compared with various sequences on the internet for sequence homology. A 507 bp DNA fragment encoding the 56 kDa protein was amplified from the samples. The sequence homology was 85% (Karp strain), 68% (Gilliam strain), 65% (Kato strain), and 67% (Yonchon strain). Orientia tsutsugamushi is carried by the mites of the Xisa archipelago; the main genotype is the Karp strain.