Samantha M. Hurndon Week 11: Week 11: DNA Microarray Journal Club

Definitions

1. Adenocarcinoma is a cancer of an epithelium that originates in the glandular tissue.
2. MALT lymphoma: development of cancer in the B Cells. Rare amoung people under the age of 50 and affects men much more than women. Signs and symptoms: Swelling in the neck , groin and armpit due to enlargement of lymph nodes. Loss of unger and body fatigue, high fever and weight loss. http://maltlymphoma.net/
3. B cells: Help the immune system fight against the invasion of harmful microorganisms into the body. http://maltlymphoma.net/
4. Ablation: Removal or excision. Ablation is usually carried out surgically. For example, surgical removal of the thyroid gland (a total thyroidectomy) is ablation of the thyroid. http://www.medterms.com/script/main/art.asp?articlekey=2089

Outline

1. Abstract
   • Helicobacter pylori is a motile Gram-negative bacterium. This bacterium persists in the human gastric mucosa
   • FliK is the gene that controls hook length during flagellar assembly.
   • Microarry analysis was done on fliK-null mutant, this revealed that under the control of the sigma 54 factor RpoN there is an incase in transcription of genes.
   • Sigma 45 fact is responsible for transcription on the class II flagellar genes (flgE and flgB).
   • Flik is involved in three processes:
     • hook-length control
     • export substrate specificity
     • control of RpoN transcriptional activity
2. Introduction
   • Helicobacter pylori is an infection that causes gastrointestinal disorders (peptic and duodenal ulcers). It is also a predisposing factor of gastric adenocarcinoma and B-cell MALT lymphoma.
   • This is seen often in developing countries due to their living conditions and lack of health care.
   • A key feature of H. pylori is motility, which is required for colonization and persistence. The motility in this bacteria is due to flagella.
   • three RNA polymerase sigma factors (80, 54, 28) control the transcription of H. pulori flagellar genes.
   • There are three classes of regulation
     • Class one: Sigma 80 modulates the transcription of the early flagullar genes.
     • Class two: RpoN deals with the regulation of the middle flagellar structural genes. RpoN is regulated FlgR/FlgS activation system and HP0958
       • Class two regulation encodes hook-length-control proteins.
     • Class three: Transcription of these genes is controlled by FliA (sigma 28) and an anti sigma factor (FlgM).
   • It was observed that the initial annotation of H.pylori genome sequences did not identify all flagellar genes expected by comparison with the Salmonella/Escherichia coli paradigm.
   • Flik in Salmonella controls hook length. In h pylori fliK gene impairs motility. The cells, in both H. pylori and Salmonella, harbor polyhook structures
   • it is suggested that the FliK protein is required to turn off the RpoN regulation during flagullar assembly.
   • In their study they used pan-H.pylori array. The array was based on the genomes of strains NCTC26695 and J99.
3. Methods
   • Used a pan-H. pylori array based on the genomes of strains NCTC26695 and J99
   • They preformed array comparative genomic hybridization to identify specific chromosomal regions that were missing in CCUG17874
   • Global transcipt analysis of a CCUG17847 mutant lacking the fliK gene was performed inorder to find out more infomation on the role of FliK in flagellar biogenesis in H. pylori.
   • Bacterial strains, media and growth conditions
     • The mutants used in this study were cultured on Columbia agar base plates containing the appropriate antibiotic
   • Extraction of genomic DNA frp, H. pylori
     • Genomic DNA was isolated using the DNeasy tissue kid. Then was quantified using Nanodrop ND 1000 spectrophotometer.
   • RNA isolation from H. pylori
     • RNA was isolated from 20 liquid cultures, the cells were harvested and centrifuged for 15 secs.
     • Cell plates were then resuspended in bacteria RNA protected reagent.
     • total RNA samples DNase-treated
   • H. pylori microarray design and construction
     • PCR products were designed to represent all ORFs in H pylori NCTC26695 aand J99.
     • Micro arrays were constructed by robotic spotting of PCR products on UltraGaps amino-silane-coated glass slides and post-print-processed
   • CGH
     • Strain CCUG17874 was used to study global transcription patterns of different flagellar mutants (the genome of this strain as not been sequenced).
     • The macro diversity of this strain was investigated using CGH.
     • Array hybridiztions for the wt. and the mutant were performed in duplicate.
     • Quantile normalization was performed
     • The output files give three lists: divergent genes, uncertain genes and present genes.
   • Confirmatory analysis of selected CGH data by PCR
     • under standard conditions, confirmed selected results obtained from CGH.
     • Seven genes were selected from CGH data
     • Primer pair sequences were designed using the Primer3 online software.
   • Type II Microarray analysis
   • Quantitative analysis of transcription by real-time PCR
   • Reverse transcription and amplification of intergenic regions by PCR
   • Bioinformatics analysis
4. Results