| Budget Amount *help |
¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2006: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 2005: ¥3,400,000 (Direct Cost: ¥3,400,000)
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| Research Abstract |
Nid A and Nid B, which are subunits of dioxygenase participating in pyrene degradation, were obtained when cultivating Mycobacterium sp. H2-5 on pyrene as the sole source of carbon. The nid A and nid B genes were considered usable as the probe for pyrene-degrading bacteria, because Nid A and Nid B were specifically produced in pyrene degradation. DNA sequences of nid A and nid B were analyzed after PCR of the genes. In the first plan, in situ hybridization (FISH) analyses were to be performed using specific fluorescent probes for these genes. But, FISH targeting for plentiful 16S rRNA was conducted, because it was considered that FISH targeting for nid A and nid B would be more difficult for the small amount of mRNA of these genes.
Before FISH analysis of strain H2-5, FISH of E. coli was performed using universal probes of EUB 338 and NONEUB labeled by Alexa Fluore 488 on 5'-ends. Namely, cells were fixed by paraformaldehyde and were bound on the slide coated by gelatin. After hybridization, clear signals were observed by fluorescent microscopy when staining with EUB 338 and DAPI. In the case of strain H2-5 cultured in TSB medium, the signals were somewhat unclear compared with the results in E. coli. To improve permeability of the probe, lysozyme and achromopeptidase treatment of the cells fixed on a siliconized slide are being attempted.
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