[The study of inter-simple sequences repeat genotyping based on one pentanucleotide repeat sequences in mycobacteria].

OBJECTIVE: To establish inter-simple sequences repeat (ISSR) molecular makers based on (CAGCG)n repeat sequence in mycobacteria. METHODS: The distribution of pentanucleotide repeat sequence (CAGCG)n in mycobacterial genomes was analyzed by MICdb 2.0 software in the microsatellite database. ISSR primer MISP6 based on (CAGCG)n sequences was designed and tested in mycobacterial strains, which included 17 mycobacterial strains and 41 Mycobacterium tuberculosis clinical strains. RESULTS: The abundances of pentanucleotide repeat sequences (CAGCG)n were high in most of the mycobacterial genomes and they were mainly located in the coding regions. The results of ISSR analysis in mycobacteria showed that 15 reference strains from mycobacteria were clustered into 2 major clusters. The first cluster contained 2 subtypes and the second cluster contained 4 subtypes. Forty-one clinical strains from Mycobacterium tuberculosis were divided into 2 major clusters by the analysis of MISP6 primer, and each cluster had 2 subtypes. CONCLUSION: ISSR primer MISP6 based on (CAGCG)n sequences can be used as a genetic marker to genotype mycobacterial strains.

[Identification and evaluation of a new nucleic acid amplification test target for specific detection of Mycobacterium tuberculosis complex].

OBJECTIVE: To identify and evaluate a new nucleic acid amplification (NAA) test target for specific detection of Mycobacterium tuberculosis (MTB) complex (MTC). METHODS: MTC-specific fragment was obtained by ISSR genotyping technology. Primer pairs were designed based on the sequences of MTC-specific fragment and tested in 211 mycobacterial strains including 107 MTC strains and 104 nontuberculous mycobacteria (NTM) strains. IS6110 element (specific identification of MTC strains) and 16s rRNA gene (specific identification of Mycobacterium) amplification were used as a control to evaluate the efficacy of the NAA test target in the detection of MTC strains. RESULTS: One MTC-specific fragment with the length of 588 bp, located in 315947 - 316534 of the genome from MTB reference strain H(37) Rv, were obtained, cloned and sequenced. MTC-specific primer pairs MTCF/R were designed based on these sequences. All 211 mycobacterial strains accurately produced the genus-specific 16s rRNA amplicon. All MTC strains were positive in the MTCF/R PCR amplification while 99% MTC strains (106/107) were positive in the amplification of IS6110 sequences. All NTM strains were negative in both IS6110 and MTCF/R PCR amplification. CONCLUSIONS: The MTC-specific fragment developed in this study can be used as a new NAA test target to correctly distinguish MTC from NTM.

[Expression of estrogen receptor alpha in the testis of infertile men with spermatogenic arrest].

OBJECTIVE: To explore the association of spermatogenic arrest with the expression of estrogen receptor alpha (ERalpha) in human testes. METHODS: We examined the testicular biopsy specimens of 120 infertile men by HE staining, detected the expression of ERalpha in the specimens of those with spermatogenic arrest by the two-step immunohistochemical method, and compared the results with those of 10 healthy men. RESULTS: Of the 120 specimens from the infertile men, 31 (25.8%) met the diagnostic criteria of spermatogenic arrest. In the testis tissue of normal men, ERalpha expressed in Sertoli, myoid and Leydig cells, but not in spermatogenic cells, while in the testis tissues of those with spermatogenic arrest, ERalpha expressed lowly in Sertoli, myoid and Leydig cells, with statistically significant differences in immunostaining intensity between the two groups (P < 0.05). CONCLUSION: Androgen receptor (AR) and ERalpha may play a coordinating role in facilitating spermatogenesis. Spermatogenic arrest may be related to a complex series of disorders in cell signal transduction involving AR, ERalpha and HSP90.

[Comparison of the transcriptional levels of mannitol PTS operon between epidemic and non-epidemic strains of Vibrio cholerae].

OBJECTIVE: To compare the transcription difference of the mannitol PTS genes between epidemic and non-epidemic strains of Vibrio cholerae El Tor in mannitol ferment tests. METHODS: Growth curves of 10 epidemic strains (slow-ferment) and 10 non-epidemic strains (rapid-ferment) of Vibrio cholerae were detected in the process of fermentation test, and the transcriptional level of mannitol PTS operon of these strains were determined with quantitative reverse-transcriptional PCR. RESULTS: After 4 hours of test, the non-epidemic strains became positive and the average growth density of the non-epidemic strains was higher than that of the epidemic strains; however, some were still lower than the epidemic strains. In contrast, at the eighth hour of test, when epidemic strains got positive, they showed higher average growth density. Compared to the epidemic strains, the transcription of mannitol PTS genes of the non-epidemic strains were much more active at the 1st and 2nd hour and were lower at the 4th and 8th hour. CONCLUSIONS: The difference of mannitol PTS operon transcription level should be an important feature to identify the epidemic and non-epidemic strains of Vibrio cholerae, which directly influences the mannitol fermentation rate during the test. The growth rate is not a key factor that affect such difference.

[Transcriptional repressor gene--mtlR of mannitol PTS operon in Vibrio cholerae].

The fermentation rates of mannitol in toxigenic and non-toxigenic El Tor strains of Vibrio cholerae are obviously different, which is a valuable indicator in the rapid identification of toxigenic strain. To determine the regulating role of mtlR in transcription of mannitol PTS operon in V. cholerae, and whether it plays a role in the ferment difference of the toxigenic and non-toxigenic strains, the mtlR deletion mutants from the mannitol rapid-ferment strain (non-toxigenic strain) and slow-ferment strain (toxigenic strain) were constructed. Comparisons of the growth in M9 containing 0.2% mannitol as the sole carbon source and pH change in mannitol fermentation media of these wild strains and their mutants, indicated that mtlR is a repressor. Its repression in mtlCBA transcription was further verified with the analyses of quantitative reverse-transcriptional PCR. However, the regulation of mtlR is not the immediate cause of the ferment difference of the toxigenic and non-toxigenic strains. The study also provides the necessary data in the analyses of mannitol ferment difference between the toxigenic and non-toxigenic V. cholerae strains.

[Sequencing and analysis of Vibrio cholerae typing phage VP3 genome].

DNA sequence and the genome of phage VP3 (a typing phage of V. cholera) were analyzed. A random library of VP3 DNA was constructed by shot-gun library method. The VP3 genome sequence was assembled with contigs sequences, the gaps between different contigs were filled with sequencing data from primer walking. ORFs were predicted; Phylogeny of DNA polymerase sequences was analyzed to determine the class of VP3; The activity of putative promoter genes were analyzed using lacZ report system. VP3 genome is a 39504bp of circular double-stranded DNA. Twenty-seven out of forty-nine putative ORFs were annotated; twenty gene products were homologous with T7-like phages, including DNAP, DNA replicative protein, capsid, tail tubular, tail fiber protein, and DNA packaged protein. The activity of the putative promoter regions was confirmed through cloning those regions to LacZ-fuse plasmid pRS1274 and analysis of the expression of beta galactosidase. The complete genomic sequence of VP3 and phylogenetic tree analysis suggests VP3 is a member of T7 phage family.