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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. Campbell NE et al (Eds): Biology: Concepts & Connections

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slide1

Gene Expression

M.Tevfik DORAK

http://www.dorak.info

slide4

DNAdoublehelix(2-nmdiameter)

Histones

“Beads ona string”

Nucleosome(10-nm diameter)

Tight helical fiber(30-nm diameter)

Supercoil(200-nm diameter)

700nm

Campbell NE et al (Eds):

Biology: Concepts & Connections

4th Edition, 2003

Metaphase chromosome

slide7

Idea: measure the amount of mRNA to see which genes are being expressedin (used by) the cell.

Measuring protein might be better, but is currently harder.

Gene expression does not always result in a protein product !

slide12

Medical Biochemistry Pages

http://www.indstate.edu/thcme/mwking/gene-regulation.html

slide14

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

slide16

University of Arizona Biology Project

http://www.biology.arizona.edu/molecular_bio/molecular_bio.html

complex assemblies of proteins control eukaryotic transcription

Enhancers

Promoter

Gene

DNA

Activatorproteins

Transcriptionfactors

Otherproteins

RNA polymerase

Bendingof DNA

Transcription

Complex assemblies of proteins control eukaryotic transcription

A variety of regulatory proteins interact with DNA and each other

Campbell NE et al (Eds):

Biology: Concepts & Connections

4th Edition, 2003

slide18

Chromosome

DNA unpackingOther changes to DNA

GENE

TRANSCRIPTION

GENE

Exon

RNA transcript

Intron

Addition of cap and tail

Splicing

Tail

Cap

mRNA in nucleus

NUCLEUS

Flowthroughnuclear envelope

mRNA in cytoplasm

CYTOPLASM

Breakdown of mRNA

Translation

Broken-down mRNA

Polypeptide

Cleavage/modification/activation

ACTIVE PROTEIN

Breakdownof protein

Broken-down protein

Campbell NE et al (Eds):

Biology: Concepts & Connections

4th Edition, 2003

slide20

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

slide23

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

slide24

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"

slide26

Medical Biochemistry Pages

http://www.indstate.edu/thcme/mwking/rna.html

slide27

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

gene expression in prokaryotes
Gene Expression in Prokaryotes

Glick and Pasternak Fig. 3.10

three kinds of rna
Three kinds of RNA

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

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

slide59

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

slide60

EPIGENETIC CROSSTALK

From: Weissmann & Lyko. BioTechniques 2003

slide61

Wellcome Trust

http://www.wellcome.ac.uk/en/genome/thegenome/hg02b002.html

slide63

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

slide67

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
slide68

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

slide74

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.

slide76

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

slide77

DNA MICROARRAY ANALYSIS

From: Gene Quantification Page by MW Pfaffl

slide78

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
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
slide81
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

slide82

Why are they so different?

Quantity or Quality?