Ph.D. Tezi Görüntüleme

Student:	ELİF SEVİM
Supervisor:	Prof. Dr. Ali Osman BELDÜZ
Department:	Biyoloji
Institution:	Graduate School of Natural and Applied Sciences
University:	Karadeniz Technical University, Turkey

Title of the Thesis:	Cloning and Promoter Architecture of Flagellin Gene (hag) Expressed at High-level in Aeribacillus pallidus AC6 and Isolation and Purification of Aeribacillus pallidus AC6 Flagellar-specific Transcription Factor (σD).
Level:	Ph.D.
Acceptance Date:	24/9/2010
Number of Pages:	142
Registration Number:	Di803

Summary:

Aeribacillus pallidus AC6 , a thermophilic bacteria, isolated from hot spring water was used in this study. An over-expressed protein was determined and results of MALTI-TOF analyses showed that the over-expressed protein is similar flagellin protein. The AC6 flagellin gene was amplifioed based on this protein sequences. Rest of the flagellin gene was amplified by inverse PCR.

5’-RACE expreriment showed that transcriptional start bases of hag gene are G and T residue located 79 and 80 bp upstream from translational start codon. When Upstream sequences were examined, we have seen that upstream sequence revealed potential sequence recognized σD-dependent RNA polymerase. After β-galactosidase experiment, We determined that AC6 hag gene has TAAA sequences at -35 region and CCGATAT sequences at -10 region and these region recognized by σD of bacterial RNA polymerase.

For describing interactions between AC6 hag promoter and σD factor as in-vitro, σD protein of AC6 and B. subtilis and kor RNAP of B. subtilis were purified. In vitro transcription assay showed that σD proteins and kor RNAP enzyme reconstructed as holoenzyme form and this holoenzyme was activated transcription at AC6 hag promoter region. We determined that CCG motif of a long -10 region (CCGATAT) of promoter is important for the recognition and binding by σD.

When secondary structure of mRNA of AC6 hag gene was examined, we noted the prensence of a potetial steem-loop structure at 5’ and 3’ ends of hag gene transcripts. As a result of investigation, while AC6 hag gene includes same high frequencies codons of B. subtilis, it does not include same high frequencies codons of G. kaustophilus and G. stearothermophilus.

Key Words: Flagellin gene, σD, Aeribacillus pallidus AC6, promoter elements of hag gene, in vitro transcription