Download Gene Expression - M.Tevfik DORAK`s

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)