Isaiah M. Castaneda Week 11
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Template: Isaiah M. Castaneda]
Journal Club 3
Definitions
- Adenocarcinoma: Carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
- Ablation: Damage and removal to part of an organism either in part or in whole.
- http://www.biology-online.org/dictionary/Ablation. Nov 09, 2011
- CGH: A cytogenetic technique in which reference DNA and the DNA to be studied, as from a tumor or an embryo, are labeled with green- and red-fluorescing fluorochromes, respectively. Genetic abnormalities are detected by changes in the green-to-red ratio.
- SSD: (Saline Sodium-Citrate) Na3C6H5O7 -2H2O; trisodium citrate;used as diuretic, antilithic, systemic and urinary alkaliser, expectorant, and anticoagulant.
- http://www.biology-online.org/dictionary/Sodium_citrate. Nov 09, 2011.
- SDS: (Sodium dodecyl sulfate) An anionic surfactant, usually a mixture of sodium alkyl sulfates, mainly the lauryl; lowers surface tension of aqueous solutions; used as fat emulsifier, wetting agent, detergent in cosmetics, pharmaceuticals and toothpastes; also as research tool in protein biochemistry.
- Triplicate:Having three copies; triple; threefold. Triplicates in scientific experiments are important to validate empirical data or the observed results. In general, a research plan entails three replicates so that the results obtained from them can be verified. Thus, the relative differences of data from the three replicates can be measured and compared. A low deviation is ideal to represent a veritable data from where a conclusive inference can be derived upon.
- http://www.biology-online.org/dictionary/Triplicate. Nov 15, 2011.
- Transposase: an enzyme involved in the movement of a DNA fragment from one site in the genome to another.
- http://medical-dictionary.thefreedictionary.com/Transposases. Nov 15, 2011.
- Motility:Motility is spontaneous movement. One example is the automatic stomach contractions that move the food content along from the stomach into the intestines. A motility disease is one that involves changes in the way the stomach contracts.
- http://medical-dictionary.thefreedictionary.com/motility. Nov 15, 2011.
- Upregulation: An increase in the number of receptors on the surface of target cells, making the cells more sensitive to a hormone or another agent.
- http://www.medterms.com/script/main/art.asp?articlekey=24470. Nov 16, 2011.
- Basal-body: A short cylindrical array of microtubules and other proteins, found at the base of a eukaryotic cilium or flagellum, that organises the assembly of the axoneme (the bundle of microtubules and other proteins forming the core of each cilium or flagellum).
Outline
Effect of FliK mutation on the transcriptional activity of the s54 sigma factor RpoN in Helicobacter pylori
I. Helicobacter pylori: An infection that causes gastrointestinal disorders such as peptic and duodenal ulcers.
- Mostly affects those residing in developing nations
- -They cannot afford treatment
- New antibiotic resistant strains are manifesting
- -There is a need to develop new treatments
- Known to be quite motile
- -Use flagella
- fliK gene
- -In salmonella, compares length of hook
- -In H. pylori, motility is adversely affected by disruption of this gene
- This is accompanied by reduced flagellin production
- And increased flgE production
- Significantly increased flgE & flaB transcription levels
- -The above genes are controlled by RpoN
- -fliK may be needed to turn off RpoN
II. Methods
- Cultured H. pylori
- -Mutants defective in fliK
- Extract & quantify DNA
- Isolate RNA
- -Used a Qiagen RNeasy Mini kit
- Designed/constructed microarray
- -Used robotic spotting of PCR products
- CGH was used to study CCUG17874 macrodiversity
- -Genome of strain hadn’t been sequenced
- Performed hybridizations in duplicate
- Followed quintile normalization
- -Divergent genes, uncertain genes, present genes
- Confirm CGH results with PCR
- Type II Microarray analysis
- Cy5-labeled cDNA co-hybridized to Cy3-labeled genomic DNA
- CCUG17874 genomic DNA labeled with dCTP Cy3
- -random primers
- Cy5-labelled cDNA made from total RNA
- -random primers
- Co-purified the labeled nucleic acids
- -Mixed them in SSC-SDS solution
- -Wash microarray slides in SSC-SDS solution once
- -Wash microarray slides in SSC twice for 2 mins
- -Dry, centrifuge, laser scan
- -Analyza images & data with a special software
- -Filter out genes that were missing or uncertain
- -Hybridizations performed in triplicate
- -Wt DNA vs mutant RNA data entered into Excel
- -Within-slide normalization
- Discard genes with empty values
- Quantile normalization
- One-way ANOVA was used to calculate statistics
- -P-value<0.05 + P-value>2.00 = differentially expressed
III. Results
- Seven genes were analyzed by PCR
- Table 1 compares shows gene absence in CCUG17874 compared to NCTC26695
- HP1192 was present in PCR data, but not CGH data
- -5/7 genes encode hypothetical proteins
- -Other two genes encode transketolase & geranyltransferase
- -Although CGH data indicated that flgI was not present in test strain, PCR confirmed that it was
- 14.2% of H. pylori genes were missing in the CCUG17874
- -Mot of these were strain and H. pylori specific
- -It was revealed that 5 regions were disrupted in the test strain compared to the reference strain
- -Fig. 2 shows present genes in green and missing genes in red
- Global transcript analysis of HP0906 mutant was performed
- -54 genes were differentially transcribed
- 34 of these were possibly proteins
- -Table 2 shows fold-changes and thus differentially expressed gene
- -7 genes which encoded ribosomal proteins were upregulated in FliK mutant
- Stress related proteins were also upregulated
- RpoN-dependent genes were upregulated (also shown in Table 2)
- -Table 3 shows the data calculated for differential expression in HP0906 compared to wild type
- Only one class I gene was down regulated (HP0684)
- Class III genes were transcribed at wt level
- Just 1 gene in intermediate class was upregulated (hp0367)
- -There are regions where DNA duplex is destabilized
- Promoter
- Terminator
- Fig 3
- -Duplex destabilization is brought on by stress
- -Flab & linked genes
- Indicates that HP0114 & flaB are not co-transcribed
- -Fig. 4
- Mutation of flgE causes an increase in rpoN and flaB transcription
IV. Discussion
- FliK protein HP0906 controls hook length
- Instead of performing analyses on multiple strains (previous studies) with the same mutation, the genes components were identified
- -Showed that all known flagella genes were present in CCUG17874
- -Mot divergent genes were in regions of low G+C content
- Insertional inactivation of fliK mutant increased expression in most RpoN-dependent genes
- -HP0870 & HP0115 were most affected
- Class I genes were transcribed at WT levels
- Upregulation of RpoN-dependent genes suggests that activation is triggered by FlgS/FlgR
- Result of completion MS ring or export apparatus
- FliA-dependent mechanisms ensue
- -Termination of RpoN-dependent mechanisms does not exist in fliK mutants
- It is hypothesized that signal which induces FlgR/FlgS activation stop until rod & hook are complete
- -FliK is secreted during hook polymerization
- -After hook is completed, fliK probably interacts with FlgR/FlgS system or RpoN sigma factor
- RpoN regulon should then be turne doff
- No protein-protein interactions between FlgR/FlgS with FliK
- -Signals controlling class II genes does not directly involve fliK
- Future experiments will work to discover the unknown mechanisms in flagella control (fig 5)
