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Gene Expression M.Tevfik DORAK http://www.dorak.info DNA double helix (2-nm diameter) Histones “Beads on a string” Nucleosome (10-nm diameter) Tight helical fiber (30-nm diameter) Supercoil (200-nm diameter) 700 nm Metaphase chromosome Campbell NE et al (Eds): Biology: Concepts & Connections 4th Edition, 2003 From: Gene Quantification Page by MW Pfaffl 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 ! Transcribed and Nontranscribed Strands From: Vlad Bajic at BioDiscovery Group, Singapore Medical Biochemistry Pages http://www.indstate.edu/thcme/mwking/gene-regulation.html 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 Nterminal methionine is removed by post-translational processing to generate the mature b-globin polypeptide. From: Human Molecular Genetics by Strachan & Read. NCBI Books Online From: Principles of Molecular Medicine. LL Jameson (Ed). Humana Press, 1998 University of Arizona Biology Project http://www.biology.arizona.edu/molecular_bio/molecular_bio.html Complex assemblies of proteins control eukaryotic transcription A variety of regulatory proteins interact with DNA and each other Enhancers Promoter Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Bending of DNA Transcription Campbell NE et al (Eds): Biology: Concepts & Connections 4th Edition, 2003 Chromosome DNA unpacking Other changes to DNA GENE GENE TRANSCRIPTION Exon RNA transcript Intron Addition of cap and tail Splicing Tail Cap mRNA in nucleus NUCLEUS Flow through nuclear envelope mRNA in cytoplasm CYTOPLASM Breakdown of mRNA Translation Brokendown mRNA Polypeptide Cleavage/modification/ activation ACTIVE PROTEIN Breakdown of protein Brokendown protein Campbell NE et al (Eds): Biology: Concepts & Connections 4th Edition, 2003 A 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 Morey AK et al. JBC 1998 (http://www.biochemj.org/bj/330/1097/3301097.pdf) Chromosomal Location: 6p21.4 EDN1 Locus: ID 1906 EDN1 Genome Annotation (chromosome 6 reference genomic contig) : NT_007592 EDN1 Genomic Sequence (including the promoter region): J05005 EDN1 (GeneID 1906) GI: 340555 repeat_region 98...383 /rpt_family="Alu" protein_bind 739...745 /bound_moiety="acute phase reactant regulatory element" misc_feature 979..1039 /note="Z-DNA region; putative" protein_bind 2183..2188 /bound_moiety="acute phase reactant regulatory element" protein_bind 2951..2958 /bound_moiety="TPA/JUN" protein_bind 3241..3248 /bound_moiety="TPA/JUN" protein_bind 3316..3328 /bound_moiety="NF-1" protein_bind 3499..3505 /bound_moiety="TPA/JUN" CAAT_signal 3510..3515 /gene="EDN1" TATA_signal 3577..3582 /gene="EDN1" Exon 1 3608..3939 /gene="EDN1" Regulatory SNPs Medical Biochemistry Pages http://www.indstate.edu/thcme/mwking/rna.html Generating Protein Diversity from the “Small” Human Genome Alternative Splicing Can Generate Very Large Numbers of Related Proteins From a Single Gene Exon 4 12 alternatives Exon 8 48 alternatives Exon 9 Exon 17 33 alternatives 2 alternatives DSCAM gene and pre-mRNA splicing mRNA 12 X 48 X 33 X 2 = 38,016 alternative mRNAs 49 of 50 cDNAs sequenced showed alternative splicing suggesting thousands of different proteins from the same gene. Black, Cell 103: 367, 2000 Generating Protein Diversity from the “Small” Human Genome Generating Protein Diversity from the “Small” Human Genome Gene Expression in Prokaryotes Glick and Pasternak Fig. 3.10 Three kinds of RNA mRNA: a copy of the gene; is translated to make protein. mRNA tRNA tRNA: smallest RNA, does actual decoding. rRNA: 3 sizes that, along with proteins, make up a ribosome http://www.cu.lu/labext/rcms/cppe/traducti/tjpeg/trna.jpeg; Tobin and Duschek, Asking About Life; http://www.tokyo-ed.ac.jp/genet/mutation/nort.gif rRNA From: Principles of Molecular Medicine. LL Jameson (Ed). Humana Press, 1998 Transcription Maston GA et al. 2006 (www) Maston GA et al. 2006 (www) Maston GA et al. 2006 (www) Maston GA et al. 2006 (www) Maston GA et al. 2006 (www) (WWW) The 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 EPIGENETIC CROSSTALK From: Weissmann & Lyko. BioTechniques 2003 Wellcome Trust http://www.wellcome.ac.uk/en/genome/thegenome/hg02b002.html Six steps at which eukaryote gene expression can be controlled From: Molecular Biology of the Cell by Alberts B, Bray D, Lewis J, Raff M, Roberts K, and Watson JD. NCBI Books Online Methods for mRNA Analysis Technique Northern Gel RNase Protect. RT-PCR Real Ti me PCR Micro-array Information on mRNA size/# yes no no no no Info. on splicing/ sequence no yes yes yes yes Quantitative Scalable yes yes no yes some no no some no yes a. Northern Ge l i) Size and number of transcripts ii) Little information about RNA s equence/splicing iii) Quantitative b. RNase protection ana lysis i). No information on size of full l ength mRNA ii) Good information on sequence/splicing iii) Quant itative c. Traditional R T-PCR i). No information on size of full-length mRNA ii) Good information on sequence/splicing iii) Very n on-quant itative d. Real Time RT-PCR i). No information on size of full-length mRNA ii) Good information on sequence/splicing iii) Very q uant itative e. New a rray techniques to detect alternatively spliced transcripts i). No information on size of full-length mRNA ii) Some information splicing iii) Semi-quant itative iv) Ammenable to scale-up Nigel Walker, NIEHS (www) Traditional gene expression analysis: Northern Blotting • Northern blotting detects specific RNAs • RNA is isolated from cells and separated using electrophoresis • probed with radioactive cDNA from a specific gene • Method can be used to determine steady-state level of a transcript in a specific RNA mixture Serial analysis of gene expression (SAGE) • 9 to 11 base “tags” correspond to genes • measure of gene expression in different biological samples • SAGE tags can be compared electronically Serial analysis of gene expression (SAGE) Page 169 SAGENet: http://www.sagenet.org/findings/index.html DNA FOOTPRINTING ANALYSIS RIBONUCLEASE PROTECTION ASSAY NUCLEASE 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 32Plabeled 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 singlestranded areas: the extension beyond the mRNA sequences, and the single basepair 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. Ambion: http://www.ambion.com/techlib/resources/miRNA/mirna_gen.html DNA MICROARRAY ANALYSIS From: Gene Quantification Page by MW Pfaffl DNA 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. MIAME Guidelines • “Minimum Information About a Microarray Experiment” • http://www.mged.org/Workgroups/MIAME/miame.html • Provides a minimum standard that should be followed to objectively interpret findings from array experiments and ensure reproducibility of results • Guidelines are provided for: • • • • • Experimental design Samples used and preparation Hybridization techniques Microarray protocol Processing and Analysis of Data Davidson University (Microarray Animation) http://www.bio.davidson.edu/courses/genomics/chip/chip.html Imagecyte (Microarray Animation) http://www.imagecyte.com/array2.html Microarray Data Analysis (Microarray Bibliography) http://www.nslij-genetics.org/microarray Why are they so different? Quantity or Quality? (www)