7Measuring protein might be better, but is currently harder. Idea: measure the amount of mRNA to see which genes are being expressed in (used by) the cell.Measuring protein might be better, but is currently harder.Gene expression does not always result in a protein product !
14Expression of the human b-globin gene Expression of the human b-globin gene. Exons 1 and 3 each contain noncoding sequences (shaded bars) at their extremities, which are transcribed and are present at the 5’ and 3’ ends of the b-globin mRNA, but are not translated to specify polypeptide synthesis. Such 5’ and 3’ untranslated regions (5’ UTR and 3’ UTR), however, are thought to be important in ensuring high efficiency of translation. The stop codon UAA represents the first three nucleotides of the 3’ untranslated region. Note that the initial translation product has 147 amino acids, but that the N-terminal methionine is removed by post-translational processing to generate the mature b-globin polypeptide.From: Human Molecular Genetics by Strachan & Read. NCBI Books Online
15From: Principles of Molecular Medicine. LL Jameson (Ed) From: Principles of Molecular Medicine. LL Jameson (Ed). Humana Press, 1998
17Complex assemblies of proteins control eukaryotic transcription A variety of regulatory proteins interact with DNA and each otherEnhancersDNAActivator proteinsOther proteinsTranscription factorsRNA polymeraseBending of DNATranscriptionPromoterGeneCampbell NE et al (Eds):Biology: Concepts & Connections4th Edition, 2003
18Campbell NE et al (Eds): Biology: Concepts & Connections ChromosomeDNA unpacking Other changes to DNAGENETRANSCRIPTIONGENEExonRNA transcriptAddition of cap and tailIntronSplicingTailCapmRNA in nucleusNUCLEUSFlow through nuclear envelopemRNA in cytoplasmCYTOPLASMBreakdown of mRNATranslationBroken-down mRNAPolypeptideCleavage/modification/ activationACTIVE PROTEINBreakdown of proteinBroken-down proteinCampbell NE et al (Eds):Biology: Concepts & Connections4th Edition, 2003
20A eukaryotic promoter: This promoter contains three promoter elements upstream of the TATA box that are required for efficient transcription: a CCAAT box and two GC boxes (consensus sequence GGGCGG). From: The Cell by GM Cooper. NCBI Online Books
27Generating Protein Diversity from the “Small” Human Genome Alternative Splicing Can Generate Very Large Numbers ofRelated Proteins From a Single GeneExon 412 alternativesExon 848 alternativesExon 933 alternativesExon 172 alternativesDSCAM geneand pre-mRNAsplicingmRNA38,01612 X 48 X 33 X 2 = alternative mRNAs49 of 50 cDNAs sequenced showed alternative splicing suggestingthousands of different proteins from the same gene.Black, Cell 103: 367, 2000
28Generating Protein Diversity from the “Small” Human Genome
29Generating Protein Diversity from the “Small” Human Genome
33Gene Expression in Prokaryotes Glick and Pasternak Fig. 3.10
34Three kinds of RNA mRNA tRNA rRNA mRNA: a copy of the gene; is translated to make protein.tRNA: smallest RNA, does actual decoding.mRNAtRNArRNA: 3 sizes that, along with proteins, make up a ribosomerRNATobin and Duschek, Asking About Life;
59The role of signal sequences in membrane translocation: Signal sequences target the translocation of polypeptide chains across the plasma membrane of bacteria or into the endoplasmic reticulum of eukaryotic cells. The signal sequence, a stretch of hydrophobic amino acids at the amino terminus of the polypeptide chain, inserts into a membrane channel as it emerges from the ribosome. The rest of the polypeptide is then translocated through the channel and the signal sequence is cleaved by the action of signal peptidase, releasing the mature translocated protein.From: The Cell by GM Cooper: NCBI Online Books
67Traditional gene expression analysis: Northern Blotting Northern blotting detects specific RNAsRNA is isolated from cells and separated using electrophoresisprobed with radioactive cDNA from a specific geneMethod can be used to determine steady-state level of a transcript in a specific RNA mixture
68Serial analysis of gene expression (SAGE) • 9 to 11 base “tags” correspond to genes• measure of gene expression in differentbiological samples• SAGE tags can be compared electronically
69Serial analysis of gene expression (SAGE) Page 169
74NUCLEASE PROTECTION ASSAY In this example, an mRNA containing a point mutation (indicated by the inverted triangle in the mRNA on the right) is distinguished from its normal, non-mutated counterpart (mRNA on the left). The mRNA is mixed with a single-stranded 32P-labeled DNA or RNA probe that (1) has sequences perfectly complementary to the nonmutated region of interest in the mRNA, and (2) extends for some length beyond the mRNA. The mixture is heated then cooled to allow the probe to anneal to its complementary sequences in the mRNA. The annealed mixture is then treated with single-strand specific nucleases (S1 nuclease for a DNA probe, or RNAses for an RNA probe). This results in digestion of the probe at all single-stranded areas: the extension beyond the mRNA sequences, and the single base-pair mismatch overlying the mutation (right). The radioactive digestion products are then separated by electrophoresis through a urea-containing polyacrylamide gel. The probe that annealed to normal, nonmutated mRNA is smaller than the undigested probe (by the length of the extended region not complementary to the mRNA) and will therefore migrate farther than undigested probe. The probe that annealed to the mutated mRNA will have been digested into two fragments whose summed length will equal that of the digested probe that annealed to nonmutated mRNA.
77DNA MICROARRAY ANALYSIS From: Gene Quantification Page by MW Pfaffl
78DNA MICROARRAY ANALYSIS RNA extracted from a tumour is end-labelled with a fluorescent marker, then allowed to hybridise to a chip consisting of cDNAs or oligonucleotides. The precise location of RNA hybridisation to the chip can be determined using a laser scanner. Since the position of each unique cDNA or oligonucleotide is known, the presence of a cognate RNA for any given unique sequence can be determined.
80MIAME Guidelines “Minimum Information About a Microarray Experiment” Provides a minimum standard that should be followed to objectively interpret findings from array experiments and ensure reproducibility of resultsGuidelines are provided for:Experimental designSamples used and preparationHybridization techniquesMicroarray protocolProcessing and Analysis of Data
81Davidson University (Microarray Animation) Imagecyte (Microarray Animation)Microarray Data Analysis (Microarray Bibliography)