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
5. divergent genes: is the process in which two or more populations of an ancestral species accumulate independent genetic changes (mutations) through time, often after the populations have become reproductively isolated for some period of time. http://en.wikipedia.org/wiki/Genetic_divergence #Hybridization: (biology) The act or process of mating organisms of different varieties or species to create a hybrid. (molecular biology) The process of forming a double stranded nucleic acid from joining two complementary strands of DNA (or RNA). (chemistry) The mixing of atomic orbitals to form new orbitals suitable for bonding. http://www.biology-online.org/dictionary/Hybridization
6. Operon: A group of genes or a segment of DNA that functions as a single transcription unit. It is comprised of an operator, a promoter, and one or more structural genes that are transcribed into one polycistronic mRNA. http://www.biology-online.org/dictionary/Operon
7. upregulation/downregulation: (1) Downregulation is the process by which a cell decreases the quantity of a cellular component, such as RNA or protein, in response to an external variable. An increase of a cellular component is called (2) upregulation. http://en.wikipedia.org/wiki/Downregulation_and_upregulation

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
   • CGH of H. pylori strain CCUG17874
     • Performed genetic and microbiological analysis of H. pylori motility in strain CCUG17874
     • Because the genome has not been sequenced they used H. pylori’s DNA array to investigate its genome content
     • CGH was performed to analyze the level of genetic conservation between CCUG178874 and the sequenced strains.
     • Genes were classified into 3 groups
       • Absent (highly divergent)
       • Uncertain (have values that cannot be confidently assigned to a group [Transition Region]. Consists of 130 genes)
       • Absolutely present
     • Figure one shows a PCR analysis of CGH. They used Seven genes from all three groups and amplified them using the genomic DNA of CCUG17874 and NCTC26695.
       • You can see that omp27 and flgl were assigned as divergent, groES, flag and omp13 were classified as uncertain. cheA and omp22 were classified as present.
       • PCR provided empirical data to allow rational adjustment of the cut-off value for spot intensity in the array data…to narrow the breakpoint between genes classified as present and divergent.
     • Some divergent genes in CCUG17874 may be present but their sequences may be poorly conserved compared with the sequenced reference strain.
     • The uncertain gene list was reduced from 130 to 7 genes after the PCR screening. Five of these genes encoded hypothetical proteins. The other two encoded transketolase and geranyltranstransferase.
     • A very important structure component of flagellar super-structure is fllgI, encoding the flagellar basal-body P-ring protein.
     • according to CGH data, flgI was absent from the genome strain of CCUG128774. The CGH output value for this gene was not reliable because the standard deviation of hybridization values was very high.
       • PCR confirmed the presence of flgl, which is consistent with the motile phenotype of the CCUG178774 strain used.
     • Table one
       • lists genes present in NCTC26695 and J99 but missing in CCUG178774.
       • 14.25% of H. pylori NCTC26695 genes were absent in the CCUG178774 genome.
       • Genes that appear to be missing: encode hypothetical protein are strain and H. pylori-specific. Also missing; genes that encode for restriction enzymes, outer-membrane proteins, insertion elements, virulence-associated proteins and transposases.
       • Transposable elements may be associated with disruption of operons and large regions in the CCUG17874. They are present in multiple copies in the genome.
     • Figure two:
       • location of genes idenfified as missing by the CGH analysis revaled 5 large regions have been disrupted in the genome of CCUG relative to NCTC
       • Region consist of genes encoding hypothetical proteins and or mobile and extrachromosomal elements.
       • Genes designated as divergent in CCUG were filtered out in further type II array exp.
     • Table two:
       • Global transcription analysis of the HP0906 insertional mutant.
       • The microarray data showed upregulation of stress-related proteins.
       • Omp22 is highly immunoreactive and was shown to be downregulated.
       • Three Rpon-depended genes, flab, flgE and HP1076 were upregulated.
     • Table Three:
       • Examined fold changes of flagellar genes in the HP0906 mutant. This was done because fold-changes has a likely role in the flagellar regulation.
       • Class I genes:
         • only one genes from this sector was significantly down regulated, flip/HP0684).
       • Class II genes:
         • fliK mutation affected the transcription of almost all class II genes in the flagellar regulon with only three exceptions.
       • Class III genes:
       • Intermediate level
         • only one gene here was significantly upregulated, hp367
     • Figure 3:
       • shows the transcription analysis of the flab gene region. (A) Shows the probability profile of stress induced DNA duplex destabilization of the flab gene and other linked genes. This also shows the expression ratio of the relevant genes in the flik mutant. (B) Results from the PCR analysis of the flab operons structure. Here we can see that primer pairs were designed to amplify the intergenic region between the genes hp0114 and flab and the garment of the flab transcription.
     • Figure 4:
       • the flgE gene of the H. pylori is controlled by the RpoN sigma factor. Completion of the hook in important as a morphogenetic check point in pylori flagellum biogensis. A targeted analysis of RpoN dependent transcription was carried out by performing qRT PCR for the rpoN and flab genes. It is seen that Mutation on the flgE caused a significant increase in rpoN and flab transcription