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Gene Expression M.Tevfik DORAK http://www.dorak.info.

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1 Gene Expression M.Tevfik DORAK

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4 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

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6 From: Gene Quantification Page by MW Pfaffl

7 Measuring 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 !

8 Transcribed and Nontranscribed Strands

9 From: Vlad Bajic at BioDiscovery Group, Singapore

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12 Medical Biochemistry Pages

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14 Expression 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

15 From: Principles of Molecular Medicine. LL Jameson (Ed)
From: Principles of Molecular Medicine. LL Jameson (Ed). Humana Press, 1998

16 University of Arizona Biology Project

17 Complex assemblies of proteins control eukaryotic transcription
A variety of regulatory proteins interact with DNA and each other Enhancers DNA Activator proteins Other proteins Transcription factors RNA polymerase Bending of DNA Transcription Promoter Gene Campbell NE et al (Eds): Biology: Concepts & Connections 4th Edition, 2003

18 Campbell NE et al (Eds): Biology: Concepts & Connections
Chromosome DNA unpacking Other changes to DNA GENE TRANSCRIPTION GENE Exon RNA transcript Addition of cap and tail Intron Splicing Tail Cap mRNA in nucleus NUCLEUS Flow through nuclear envelope mRNA in cytoplasm CYTOPLASM Breakdown of mRNA Translation Broken-down mRNA Polypeptide Cleavage/modification/ activation ACTIVE PROTEIN Breakdown of protein Broken-down protein Campbell NE et al (Eds): Biology: Concepts & Connections 4th Edition, 2003

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20 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

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23 Chromosomal Location: 6p21.4 EDN1 Locus: ID 1906
Morey AK et al. JBC 1998 (http://www.biochemj.org/bj/330/1097/ 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

24 EDN1 (GeneID 1906) GI: 340555 repeat_region 98...383 /rpt_family="Alu"
protein_bind /bound_moiety="acute phase reactant regulatory element" misc_feature /note="Z-DNA region; putative" protein_bind /bound_moiety="acute phase reactant regulatory element" protein_bind /bound_moiety="TPA/JUN" protein_bind /bound_moiety="TPA/JUN" protein_bind /bound_moiety="NF-1" protein_bind /bound_moiety="TPA/JUN" CAAT_signal /gene="EDN1" TATA_signal /gene="EDN1" Exon /gene="EDN1"

25 Regulatory SNPs

26 Medical Biochemistry Pages

27 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 33 alternatives Exon 17 2 alternatives DSCAM gene and pre-mRNA splicing mRNA 38,016 12 X 48 X 33 X 2 = alternative mRNAs 49 of 50 cDNAs sequenced showed alternative splicing suggesting thousands of different proteins from the same gene. Black, Cell 103: 367, 2000

28 Generating Protein Diversity from the “Small” Human Genome

29 Generating Protein Diversity from the “Small” Human Genome

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33 Gene Expression in Prokaryotes
Glick and Pasternak Fig. 3.10

34 Three kinds of RNA mRNA tRNA rRNA
mRNA: a copy of the gene; is translated to make protein. tRNA: smallest RNA, does actual decoding. mRNA tRNA rRNA: 3 sizes that, along with proteins, make up a ribosome rRNA Tobin and Duschek, Asking About Life;

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38 From: Principles of Molecular Medicine. LL Jameson (Ed)
From: Principles of Molecular Medicine. LL Jameson (Ed). Humana Press, 1998

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40 Transcription

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42 Maston GA et al (www)

43 Maston GA et al (www)

44 Maston GA et al (www)

45 Maston GA et al (www)

46 Maston GA et al (www)

47 (WWW)

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59 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

60 EPIGENETIC CROSSTALK From: Weissmann & Lyko. BioTechniques 2003

61 Wellcome Trust

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63 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

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66 Nigel Walker, NIEHS (www)

67 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

68 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

69 Serial analysis of gene expression (SAGE)
Page 169

70 SAGENet: http://www.sagenet.org/findings/index.html

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72 DNA FOOTPRINTING ANALYSIS

73 RIBONUCLEASE PROTECTION ASSAY

74 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 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.

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76 Ambion: http://www.ambion.com/techlib/resources/miRNA/mirna_gen.html

77 DNA MICROARRAY ANALYSIS From: Gene Quantification Page by MW Pfaffl

78 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.

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80 MIAME 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 results Guidelines are provided for: Experimental design Samples used and preparation Hybridization techniques Microarray protocol Processing and Analysis of Data

81 Davidson University (Microarray Animation)
Imagecyte (Microarray Animation) Microarray Data Analysis (Microarray Bibliography)

82 Why are they so different?
Quantity or Quality?

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