Determination of Gardnerella vaginalis Genome Size by Pulsed-Field Gel Electrophoresis

The chromosomal DNA of four strains of Gardnerella vaginaliswere digested with rare cutting restriction enzymes and analyzedby pulsed-field gel electrophoresis (PFGE). The four strainsstudied were two clinical isolates (GVP 004 & GVP 007) andtwo American Type Culture Collection strains (ATCC 14018 &ATCC 14019). The restriction enzyme SfiI generated two DNA fragmentsof about 0.6 Mb and 1.1 Mb in all four strains giving a G. vaginalisgenome size of about 1.7 Mb. A similar genome size was calculatedutilizing two more GC-rich sequence specific restriction endonucleases,NotI and AscI. When digested with AscI, the chromosomal DNAof all four strains gave rise to 11 to 12 DNA fragments rangingbetween 0.01 Mb to 0.43 Mb. DNA from the two clinical isolateswere digested by NotI (yielding 7 to 9 fragments), while theDNA from the two ATCC strains were resistant to NotI digestion.In contrast to the clinical isolates, DNA from the two ATCCstrains gave an identical profile for all restriction endonucleasestested. From double digestion experiments, the two SfiI sitescould be localized on two AscI fragments. From these PFGE studies,it is concluded that the G. vaginalis genome is a circular DNAthat ranges between 1.67 Mb and 1.72 Mb in size.     

Studies on the Flavor of Green Tea

With an assumption that the laver-like odor of green tea is due to dimethyl sulfide, an attempt to isolate dimethyl sulfide from commercial green tea was made, and the identification of dimethyl sulfide was successful by making the co-ordinated compound with mercuric chloride, 2 (CH₃) ₂S·3HgCl₂. In addition, the presence of methylmethionine sulfonium salt in tea extract as a precursor of dimethyl sulfide was examined.     

Direct Determination of NotI Cleavage Sites in the Genomic DNA of Adult Mouse Kidney and Human Trophoblast Using Whole-Range Restriction Landmark Genomic Scanning

Restriction landmark genomic scanning (RLGS) is a method forvisualizing restriction landmarks, employing direct labelingof restriction sites of genomic DNA and high-resolution two-dimensionalelectrophoresis. We determined the conditions for both the firstand second dimensions of RLGS that define all of the restrictionfragments which carry the NotI landmark. Using this system,we determined the number of cleavable NotI sites of genomicDNA from the mouse kidney (C57BL/6) and from the human placenta.The mouse and human genomes were cleaved at 2,380±80sites (4,760±160 spots) and 3,240±110 sites (6,480±220spots), respectively with NotI.     

LRRC3B gene is frequently epigenetically inactivated in several epithelial malignancies and inhibits cell growth and replication

Chromosome 3 specific NotI microarrays containing 180 NotI linking clones associated with 188 genes were hybridized to NotI representation probes prepared using matched tumor/normal samples from major epithelial cancers: breast (47 pairs), lung (40 pairs) cervical (43 pairs), kidney (34 pairs of clear cell renal cell carcinoma), colon (24 pairs), ovarian (25 pairs) and prostate (18 pairs). In all tested primary tumors (compared to normal controls) methylation and/or deletions was found. For the first time we showed that the gene LRRC3B was frequently methylated and/or deleted in breast carcinoma - 32% of samples, cervical - 35%, lung - 40%, renal - 35%, ovarian - 28%, colon - 33% and prostate cancer - 44%. To check these results bisulfite sequencing using cloned PCR products with representative two breast, one cervical, two renal, two ovarian and two colon cancer samples was performed. In all cases methylation was confirmed. Expression analysis using RT-qPCR showed that LRRC3B is strongly down-regulated at the latest stages of RCC and ovarian cancers. In addition we showed that LRRC3B exhibit strong cell growth inhibiting activity (more than 95%) in colony formation experiments in vitro in KRC/Y renal cell carcinoma line. All these data suggest that LRRC3B gene could be involved in the process of carcinogenesis as a tumor suppressor gene.