Isaiah M. Castaneda Week 11

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BIOL368/F11:Week_11

Contents

Journal Club 3

Media:H.pylori_ICSH.ppt

Definitions

  1. Adenocarcinoma: Carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.
    http://medical-dictionary.thefreedictionary.com/adenocarcinoma. Nov 09, 2011.
  2. 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
  3. 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.
    http://medical-dictionary.thefreedictionary.com/Comparative+genomic+hybridization+%28CGH%29. Nov 09, 2011.
  4. 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.
  5. 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.
    http://www.biology-online.org/dictionary/Sodium_dodecyl_sulfate. Nov 09, 2011.
  6. 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.
  7. 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.
  8. 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.
  9. 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.
  10. 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).
    http://groups.molbiosci.northwestern.edu/holmgren/Glossary/Definitions/Def-B/basal_body.html. Nov 16, 2011.

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)
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