Gene Expression [M.Tevfik DORAK]

1
Gene Expression M.Tevfik DORAK http//www.dorak.i
nfo
2
(No Transcript)
3
(No Transcript)
4
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
5
(No Transcript)
6
From Gene Quantification Page by MW Pfaffl
7
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
10
(No Transcript)
11
(No Transcript)
12
Medical Biochemistry Pages http//www.indstate.edu
/thcme/mwking/gene-regulation.html
13
(No Transcript)
14
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). Humana Press, 1998
16
University of Arizona Biology Project http//www.b
iology.arizona.edu/molecular_bio/molecular_bio.htm
l
17
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
18
Chromosome
DNA unpackingOther changes to DNA
GENE
GENE
TRANSCRIPTION
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
19
(No Transcript)
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
21
(No Transcript)
22
(No Transcript)
23
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
24
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"
25
Regulatory SNPs
26
Medical Biochemistry Pages http//www.indstate.edu
/thcme/mwking/rna.html
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
30
(No Transcript)
31
(No Transcript)
32
(No Transcript)
33
Gene Expression in Prokaryotes
Glick and Pasternak Fig. 3.10
34
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/t
rna.jpeg Tobin and Duschek, Asking About Life
http//www.tokyo-ed.ac.jp/genet/mutation/nort.gif
35
(No Transcript)
36
(No Transcript)
37
(No Transcript)
38
From Principles of Molecular Medicine. LL
Jameson (Ed). Humana Press, 1998
39

(No Transcript)
40
Transcription
41
(No Transcript)
42
Maston GA et al. 2006 (www)
43
Maston GA et al. 2006 (www)
44
Maston GA et al. 2006 (www)
45
Maston GA et al. 2006 (www)
46
Maston GA et al. 2006 (www)
47
(WWW)
48
(No Transcript)
49
(No Transcript)
50
(No Transcript)
51
(No Transcript)
52
(No Transcript)
53
(No Transcript)
54
(No Transcript)
55
(No Transcript)
56
(No Transcript)
57
(No Transcript)
58
(No Transcript)
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 http//www.wellcome.ac.uk/en/genome
/thegenome/hg02b002.html
62
(No Transcript)
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
64
(No Transcript)
65
(No Transcript)
66
Nigel Walker, NIEHS (www)
67
Traditional gene expression analysisNorthern
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.htm
l
71
(No Transcript)
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.
75
(No Transcript)
76
Ambion http//www.ambion.com/techlib/resources/mi
RNA/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.
79
(No Transcript)
80
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

81

• 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

82
Why are they so different?
Quantity or Quality?
83
(www)
84
(No Transcript)