Chapter 7: Trascriptomics Flashcards
The transcriptome•The proteome•The interactome
The transcriptome: The expression pattern of all gene transcripts;
The proteome: The expression pattern of all proteins;
The interactome: The complete set of molecular interactions (protein-protein, protein-DNA, protein-RNA)
DNA microarray or DNA-chip
A DNA microarray is a glass surface to which thousands of spots, each containing a different DNA sequence, have been fixed. Red and green cDNA Place on a DNA chip
data reorganized to define clusters of genes with similar expression 4
- Different clustering depending on treatment/condition;
- Uncover entire gene pathways affected (eg. yeast);
- Functional assignments to preciously unknown (eg. flower scent);
- Help genome assembly (are all exons expressed?)
Microarrays and cancer: DLBCL
(aggressive lymphona)
DLBCL = overexposes cell proliferation genes.
2 diffrent expression clusters for germinal center cells
[Red = Induced or activated germinal center (GC) genes Green = Repressed GC genes] (signature genes).
Patterns with activated expression—>poor prognosis.
Patients with low expression respond well to standard treatment
Microarrays and cancer: Child Leukaemia
classify into subtypes —>prognosis—>treatment—>current : Tumors lack asparagine synthetase gene expression. L-asparaginaseis an available drug that cleaves (destroys) asparagine thus starving cancer cells; overexpression of gene FLT3. Genes expression patterns correlate to length of remission (a predictor of survival)
Microarrays and cancer: Breast Cancer
normal breast tissue samples cluster with cancer tissue: indicating that tumors are very much like normal breast tissue.
signature genes
genes with a common pattern of expression
RNA sequencing
direct sequencing of RNA. Isolating RNA, fragmenting it, reverse transcribe it to cDNA and sequencing.
Advantages of RNA seq over microarrays
To make a microarray you must choose what probe sequences will go in it. So you only detect in your sample what matches the microarray. RNAseq reports whatever RNA sequence was in your sample. Sequencing methods give a more precise measurement of RNA concentration by recording how frequently each sequence appears. Microarrays have more limited sensitivity as it relies in hybridizations.
Lessons from RNAseq studies
1) New gene annotation (“gene discovery”)
2) Differential expression profiles between conditions. (miRNA cancer [only a small portion of tumor miRNAsare released into serum. They can be used as biomarkers], over expressed proteins in sterile males)
3) What is the nature of the regulatory difference?cis vs. trans regulation
Proteomics
The study of the expression of proteins, their interactions and their structure. ORF + motifs (PROSITE). Methodologically: 2D electrophoresis; Mass spec [Break down into peptide fragments (digestion),Separation by mass to charge ratio. Does the fingerprint of proteins in your gel matches the predicted fingerprints?]
PROSITE
A standard database of KNOWN protein domains. PROSITE uses a set of residues commonly found in a group of related proteins and uses it for comparisons to your query
The interactome
yeast two hybrid cell systems —>prot-prot interactions. Only if protein X and Y interact, then the binding and activation domains come together to turn on a reporter gene. (Alzheimer’s disease: βactin [ACTB] 10x higher expression in AD cerebral cortex than controls. Abnormalities in actin cytoskeleton may promote synaptic failure.)
Nomenclature: Nodes and hubs
Nodes are the protein interaction partners.
Hubs are proteins with a lot of interacting partners.
Hubs subdivided into:
-Party-hubs: Can interact with many other proteins at once.
-Date-hubs: Proteins that have many partners but interact with them at different times or locations
protein DNA interactions
CHIP (Chromatin Immunoprecipitation assays). Does a protein bind DNA under certain conditions? Treat cells grown under certain condition (e.g. high temperature) with a chemical that cross. Break chromatin and use antibodies as bait for your suspected proteins
