intro to molbiol and tics

8/3/2019 Intro to Molbiol and tics

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The Hierarchical Structure of an organism

L

evelofOrganization

organism

organs

tissues

cellschromsomes
http://images.google.co.il/imgres?imgurl=www.chromosome5.com/chromosome.jpg&imgrefurl=http://www.chromosome5.com/&h=500&w=551&prev=/images%3Fq%3Dchromosome%2Bpicture%26svnum%3D10%26hl%3Den%26lr%3D%26ie%3DUTF-8%26oe%3DUTF-8%26sa%3DNhttp://images.google.com/imgres?imgurl=www.washington.edu/newsroom/news/images/heart.jpg&imgrefurl=http://www.washington.edu/newsroom/news/images/&h=1392&w=1040&prev=/images%3Fq%3Dheart%2Btissue%26svnum%3D10%26hl%3Den%26lr%3D%26ie%3DUTF-8


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http:/

/www.brooklyn.cuny.e

du/bc/ahp/LAD/C4/C4

_Chromosomes.html

Chromosomes are made up of protein and DNA


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Chromosomes

Figure 4-14. Two closely related species of deer with very different chromosome numbers. In the evolution of the Indianmuntjac, initially separate chromosomes fused, without having a major effect on the animal. These two species have roughlythe same number of genes. (Adapted from M.W. Strickberger, Evolution, 3rd edition, 2000, Sudbury, MA: Jones & Bartlett

Publishers


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dista

ncelearning.ksi.edu/

demo/bio378/lecture

.html

DNA is a macromolecule composed of four basic units


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A chromosome is a long sequence


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A simple view of the central dogma


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All organisms on earth use the same operating system
http://www.cbs.dtu.dk/staff/dave/roanoke/central_new_dogma.gif


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Central Dogma of GeneExpression


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Dinner discussion: Integrative Bioinformatics & Genomics VU

metabolome

proteome

genome

transcriptome

physiome

Genomics


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Genetic information is carried as three base genetic code

Four bases (A G C T/U) must encode for 20 a.a.

Therefore a combination is required: 43 = 64

Triplet code is called a CODON that must begin at a precise site

Of 64, 61 specify individual a.a.; and three are STOP codons

starting codon is AUG (methionine)

Code is universal, synonymous,degenerate

Reading frame

3rd base in codon wobble

frameshifts/deletions/insertions

(MUTATIONS)


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The genetic code


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All organisms use the same genetic code


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Three different reading frames


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Codon Bias

Gene Findersare oftenorganism

specific Coding

regions oftenmodelled by

5th orderMarkov chain(hexamers/di-codons)


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Important Features

DNA contains genetic template" forproteins.

DNA is found in the nucleus

Protein synthesis occurs in thecytoplasm - ribosome.

"Genetic information" must betransferred to the cytoplasm where

proteins are synthesized.


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DNA proteingenetic code

*

There are between 30,000 to 40,000 genes in the human genome

The human gene inventory corresponds to ~1.5% of the genome

(coding regions)
http://www.iacr.bbsrc.ac.uk/notebook/courses/guide/images/dna.gifhttp://www.missouri.edu/~jesse105/ar2001/Images/protein.gif


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The average human gene is 1.4 kb long,

but distributed in exons over an average of 30 kb.

There are about 11 genes/ Mb DNA

Chromosome 19 having the highest density (close to 30).

The average gene

Ashurst, J.L. and Collins, J.E. 2003.Annu Rev Genomics Hum Genet 4: 69-88.


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Component Regions of RNA

The leading and trailing regions, the 5 and 3 untranslated regions (UTR), areimportant for cellular positioning, message stability, and the efficiency with whichprotein can be made from the mRNA. The open reading frame codes for the protein.


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Insulin gene

promoter

+1

poly-A

signal

100 bp

+1411

1 exon 2intron 1 intron 2 exon 3

primary transcript (1431 nt)

transcription

splicing

1 exon 2 exon 3

capping, polyadenylation

mRNA (465 nt + poly-A tail)

AAAAAAAAAAAAAAAAAAAAAAAAAAG

1 exon 2 exon 3

Most eukaryotic genes contain introns

5 end 3 end

5 end 3 end


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AGCCCUCCAGGACAGGCUGCAUCAGAAGAGGCCAUCAAGCAGAUCACUGUCCUUCUGCCAUGGCCCUGUGGAUGCG

CCUCCUGCCCCUGCUGGCGCUGCUGGCCCUCUGGGGACCUGACCCAGCCGCAGCCUUUGUGAACCAACACCUGUGC

GGCUCACACCUGGUGGAAGCUCUCUACCUAGUGUGCGGGGAACGAGGCUUCUUCUACACACCCAAGACCCGCCGGG

AGGCAGAGGACCUGCAGGUGGGGCAGGUGGAGCUGGGCGGGGGCCCUGGUGCAGGCAGCCUGCAGCCCUUGGCCCU

GGAGGGGUCCCUGCAGAAGCGUGGCAUUGUGGAACAAUGCUGUACCAGCAUCUGCUCCCUCUACCAGCUGGAGAAC

UACUGCAACUAGACGCAGCCUGCAGGCAGCCCCACACCCGCCGCCUCCUGCACCGAGAGAGAUGGAAUAAAGCCCU

UGAACCAGC

Insulin Gene mature mRNA sequence

1

465

3

5

AAAAAAAAAAAAAAAAAAAAAAAAAAAAA

59

392

159 = 5 UTR

60-388 = protein-coding region

392-465 = 3 UTR


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RNA Processing, including capping and splicing,is co-transcriptional


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TYPES OF INTRONS

GUAG INTRONS;

AU AC INTRONS;


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The GT-AG (or GU-AG) Rule

Intron boundaries are defined by the nucleotides GU

(GT in DNA) and AG. Called the GT-AG rule.

Splicing enhancers (and silencers) are found in the exons.

The majority of animal and plant introns are removed by thespliceosome that recognizes GT-AG introns.

However, plants and animals (but not fungi) have a second alternativespliceosome that is responsible for splicing non-canonical introns.

After removal from the primary transcript, virtually all introns aredegraded.

Alternative splicing is thought to explain our complexity despite ourlimited number of protein coding genes (~30,000).


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HOW THE SPLICING STARTS?

Andrew P. ReadHuman Molecular Genetics


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The Splicing Reaction

An unusual 5-2linkage is madebetween thebranch pointnucleotide and the5 splice site.

The the free 3 endof the 5 exon willdisplace the 3splice site.

This liberates theso-called lariatstructure, which isdegraded.


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The Branch Point Attack in More Detail


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A mature mRNA transcript looks like this


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Alternative Splicing

The pre-mRNA contains the introns and the exons encoded in the DNA. For the mRNAto produce a functional protein, the introns must be removed. In removing the introns,a variety of potential exon combinations are possible, ie, different combinations ofexons may be joined together to generate different forms of the same protein.


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female

male

Alternative splicing can generate different polypeptides

487 amino acidpolypeptide

549 amino acidpolypeptide


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a-tropomyosin splicing in different cell types


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Summary

A Drosophilahomolog of human Down syndrome cell adhesion molecule (DSCAM), an immunoglobulinsuperfamily member, is required for the formation of axon pathways in the embryonic central nervoussystem. cDNA and genomic analyses reveal the existence of multiple forms of Dscam with a conservedarchitecture containing variable Ig and transmembrane domains. Alternative splicing can potentiallygenerate more than 38,000 Dscam isoforms. This molecular diversity may contribute to the specificityof neuronal connectivity.

(12) (2)(48) (33)

A huge amount of diversity can be derivedfrom a single gene


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Forms of alternative splicing

Exon skipping / inclusion

Alternative 3 splice site

Alternative 5 splice site

Mutually exclusive exons

Intron retention

Constitutive exon Alternatively spliced exons


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Alt splicing as a mechanism of gene regulation

Functional domains can be added/subtracted protein diversity

Can introduce early stop codons, resulting in truncated proteins or

unstable mRNAs

It can modify the activity of the transcription factors, affecting theexpression of genes

It is observed nearly in all metazoans

Estimated to occur in 30%-60% of human


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How to study alternative splicing?


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Mapping the human genome

1953 DNA double helix (Watson and Crick)1972 Recombinant DNA (Berg, et al.)1977 DNA sequencing (Maxam and Gilbert, and Sanger)1980 Physical mapping by RFLPs (Bostein, Davis, and White)1985 PCR (Mullis)1986 Automated DNA sequencing machine (Hood and Smith)1987 YACs (Burke, Olson, and Carle) Fluorescent chain-

terminating dideoxynucleotides (DuPont)Commercial DNA sequencing machine (AppliedBiosystems)

1991 Expressed Sequence Tag (EST, Venter et al.)1992 Bacterial Artificial Chromosomes (BACs, Shizuya et al.)1997 Capillary sequencing machine (Molecular Dynamics)

Cloning and Sequencing


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What is bioinformatics?

Interface of biology and computers

Analysis of proteins, genes and genomes

using computer algorithms andcomputer databases

Genomics is the analysis of genomes.

The tools of bioinformatics are used to makesense of the billions of base pairs of DNAthat are sequenced by genomics projects.


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Mapping the human genome

Bioinformatics1970 Global alignment, dynamic programming (Needleman and Wunsch)1981 Local alignment (Smith and Waterman)1990 Basic Local Alignment Search Tool (BLAST, Altschul et. al.)

1994 Hidden Markov Model (HMM, Krogh et. al.)protein domaingene structure

1995 Phred/phrap (Phil Green and Brent Ewing)Phred: assign confidence score to sequenced nucleotidePhrap: assemble sequences

Align genome and cDNA/EST: sim4, spidey, BLATGene prediction: GeneScan, FgeneSH, Genie, GeneWise


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

20

30

4050

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

Chromosome number

Length of the human genome

Estimated base-pairs in the genome: 3,231,365,992

Basic characteristics of the human genome


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g

T841,181,383

A839,853,043

C

581,077,516

G

581,490,131

It is estimated that 88% is in finished form.

Currently in finished form: 2,843,602,073 base-pairs

10 20 30 40 50 60 70

A

C

G

T

Mbp (mega base-pairs)

Estimated base-pairs in the genome: 3,231,365,992

Colors correspondto chromosomes

GC content of the human genome


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IHGSC. Nature (2001) 409 860-921

GC content of the human genome


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The average chromosome 6

167,000,000 bp

6p21Major Histocompatibility Complex

2,190 genes 61% of the genes have CpG

islands 2kb upstream and

1kb downstream of 5 and 3

ends of genes. Mean gene length 32,530 bp

Mean exon length 318 bp

Mean transcript per gene

1.79

Mean exons per gene 5.28Mungall, A.J. et al. 2003. The DNA sequence and analysis of human chromosome 6.Nature 425: 805-811.

Human genome: individuals 0 1%


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Human genome: individuals 0.1%different

Lots of variation!3.2 x 109 bp/genome x 0.001 changes/bp =

3.2 x 106 changes/genome

Two major types of variationSNPs, RFLPsRepeated DNA - short to long repeats

For every person . . .

Di t ib ti f DNA A di


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Distribution of DNA Accordingto Function


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Genome structure: GC content

GC vs. genes GC vs. introns/exons


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Protein-coding genes


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Pawson, T. et al., Trends in Cell Biology Vol.11 No.12 December 2001

The SH2 domain is found embedded in a wide variety of metazoan proteins that

regulate functionally diverse processes.

Sequence definition

Distinct regions of proteinsequence that are highlyconserved in evolution.

Recurrence of domains


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Recurrence of chromosomal segments

Science v. 291, pp 1304-1351


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Recurrence of segments

Science v. 291, pp 1304-1351


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There are dead genes in the genome

www.people.virginia.edu/ ~rjh9u/hbmut.html


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Finding genes -- computer searches

Computer searches locate most genes in prokaryotes,Archeae, and yeast, but only ~1/3 of human genes areidentified correctly.

CriteriaProtein start, stop signals, splicing signals . . .Codon biasComparisons to other genomes (mouse, rat, fish, fly,

mosquito, worm, yeast . . .)

Some hard problems: small genes, post-translationalmodifications,unique genes, spliced genes, alternative splicing, generearrangements (e.g. IgGs) . . .


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Virus: 1,377 viral genomes and 36 viroids Organelle: mitochondria (~ 600); chloroplasts (~40) Microbial: archaea (~20); bacteria (~700) Eukaryota:

YeastSaccharomyces cerevisiae(bakers); Schizosaccharomyces pombe(fission)

MetazoaHomo sapiens(human); Pan troglodytes (chimp); Musmusculus(mouse); Rattus norvegicus(rat); Gallus gallus(chicken); Drosophilamelanogaster(fly);Anopheles gambiae(mosquito); Caenorhabditis elegans(worm);Fugu rubripes (Puffer fish); Danio rerio(zebrafish);

PlantsArabidopsis thaliana(thale-cress); Oryza sativa(rice);Avenasativa(oat); Glycine max(soybean); Hordeum vulgare(barley);Lycopersicon esculentum(tomato); Triticum aestivum(breadwheat); Zea mays(corn)

OthersEncephalitozoon cuniculi; Guillardia theta nucleomorph;

Plasmodium falciparum;Leishmania major

Completed Genomes (as of 2004)

Si il f d i


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Similar genes are found across organisms

Protein kinase, cAMP-dependent, catalytic, alpha

Mus TCTTAGACAAGCAGAAGGTGGTGAAGCTAAAGCAGATCGAGCACACTCTGAATGAGAAGC

Rat TCTTGGACAAGCAGAAGGTGGTGAAGCTGAAGCAGATCGAGCACACTCTGAATGAGAAGC

Chinese TCTTGGACAAACAGAAGGTGGTGAAGCTGAAGCAGATTGAGCACACTCTAAATGAGAAGC

Oryctolagus TCCTCGACAAACAGAAGGTGGTGAAGCTGAAACAGATCGAGCACACCCTGAACGTTAAAC

Canis TCCTCGACAAACAGAAGGTCGTGAAGCTGAAACAGATTGAGCATACCCTGAACGAAAAGC

Ovis TCCTCGACAAACAGAAGGTGGTGAAGCTGAAACAGATTGAGCACACCCTGAACGAGAAGC

B.taurus TCCTCGACAAACAGAAGGTGGTGAAGCTGAAACAGATTGAGCACACCCTGAATGAGAAGC

Homo TCCTCGACAAACAGAAGGTGGTGAAACTGAAACAGATCGAACACACCCTGAATGAAAAGC

** * ***** ******** ***** ** ** ***** ** ** ** ** ** * ** *


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The Minimal Genome

E. coli

H.influenzae M.genitalium

1,146

1,129

88918

10

239

1

O i # Ch # G E I t


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Organism # Chromosomes # Genes Exons Introns

Mycoplasmagenitalium

1 500 500

1/gene

0

Deinococcusradiodurans

2 3200 3500

1.02/gene

61

Saccharomyces

cerevisiae

16 6200 6500

1.04/gene

220

C. elegans 6 18,000 91,000

5/gene

73,000

4/gene

Drosophila

melanogaster

5 14,000 54,000

4/gene

44,000

3/gene

60 bp/intron

Arabodopsis

thaliana

5 25,000 133,000

5/gene

247/exon

107,000

4/gene

169 bp/intron

Homo sapiens 23 30,000 310,000

8+/gene

455 bp/exon

250,000

7/gene

3400 bp/intron


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Needles in Haystacks...

Only 2% of human genome is

coding regions

Intron-exon structure of genes Large introns (average 3365 bp )

Small exons (average 145 bp)

Long genes (average 27 kb)


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ESTs (Expressed Sequence Tags)

Single-pass sequencing of a small (end) piece of cDNA

Typically 200-500 nucleotides long

It may contain coding and/or non-coding region


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ESTs

Cells from a specific

organ, tissue ordevelopmental stage

AAAAAA 35

AAAAAA 35

TTTTTT53

AAAAAA 35

TTTTTT53

TTTTTT

53

AAAAAA 35

TTTTTT53

mRNA extraction

RNA

DNA

Double stranded cDNA

Add oligo-dT primer

Reverse transcriptase

Ribonuclease H

DNA polimeraseRibonuclease H


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ESTs

AAAAAA 35

TTTTTT53Clone cDNA into a vector

Multiple cDNA clones5 EST

3 EST

Single-pass sequence reads


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Splice variants

Genomic

Primary transcript

Splicing

cDNA clones

(double stranded)

EST sequences

(Single-pass sequence reads)5 3 5 3

Sampling the Transcriptome with ESTs

oligo-dT primer

Reverse transcriptase

Large scale EST-sequencing coupled to Genome


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sequencing


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EST sequencing

Is fast and cheap

Gives direct information about the gene sequence

Partial information

Resulting ESTs Known gene

(DB searches) Similar to known geneContaminant

Novel gene


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Number of public entries: 20,039,613

Summary by organism

Homo sapiens (human) 5,472,005 Mus musculus + domesticus (mouse) 4,056,481

Rattus sp. (rat) 583,841

Triticum aestivum (wheat) 549,926

Ciona intestinalis 492,511

Gallus gallus (chicken)460,385

Danio rerio (zebrafish) 450,652

Zea mays (maize)391,417

Xenopus laevis (African clawed frog)

359,901

dbEST release 20 February 2004


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EST lengths

Human EST length distribution(dbEST Sep. 2003 )

~ 450 bp


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ESTs provide expression data

eVOC Ontologies http://www.sanbi.ac.za/evoc/

AnatomicalSystem

Cell Type

The tissue, organ or anatomical system from which the sample was prepared.Examples are digestive, lungand retina.

Pathology

The precise cell type from which a sample was prepared. Examples are: B-

lymphocyte, fibroblastand oocyte.

DevelopmentalStage

The pathological state of the sample from which the sample was prepared.Examples are: normal, lymphoma, and congenital.

Pooling

The stage during the organism's development at which the sample was prepared.Examples are: embryo, fetus, and adult.

Indicates whether the tissue used to prepare the library was derived from single ormultiple samples. Examples are pooled, pooled donorand pooled tissue.

J Kelso et al. Genome Research 2002


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Exon Size

0

5

10

15

20

25

30

35

1-

100

100-

200

200-

300

300-

500

>500

Fungi

Verterbrate


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Intron Size

0

10

20

30

40

50

60

70


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Intron Prevalence

0

10

20

30

40

50

60

70

80

90

100

0 1 >1

Yeast

Fungi

Mammal


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Gene Finding Challenges

Need the correct reading frame Introns can interrupt an exon in mid-

codon

There is no hard and fast rule foridentifying donor and acceptorsplice sites

Signals are very weak


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Codon Bias

Gene Findersare oftenorganism

specific Coding

regions oftenmodelled by

5th orderMarkov chain(hexamers/di-codons)


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Overpredicting Genes

Easy to predict all exons

Report all sequences

flanked by ..AG and GT.. asexons

Sensitivity = 100%

Specificity ~ 0%


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Locating ORFs

Simplest method of predicting coding regions isto search for open reading frames (ORFs)

open reading frames begin with a start (AUG)codon, and ends with one of three stop codons

Six total reading frames


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Locating ORFs

Example from HW#1:

AUUGCAAUGGAAUUAGUAAUCUCUAUUUCCGCCCUUAUUAUAGUUGAAUAGAUAGCCGUA

E L V I S I S A L I I V E

O


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Locating ORFs

Prokaryotes: DNA sequences coding for proteinsgenerally transcribed into mRNA which is translated intoprotein with very little modification

Locating an open reading frame from a start codon to astop codon can give a strong suggestion into proteincoding regions

Longer ORFs are more likely to predict protein-coding

regions than shorter ORFs.

L i ORF


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Locating ORFs

Eukaryotes: mRNA undergoes processing toremove introns before the protein is translated

ORF corresponding to a gene may containregions with stop codons found within intronicregions

Posttranscriptional modification makes geneprediction more difficult

L i Si il S


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Locating Similar Sequences

Take the new DNA sequence, translatinginto six reading frames

Compare each to protein sequence

databases

Locates known open reading frames

L i Si il S


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Locating Similar Sequences


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Top ten challenges for bioinformatics

[1] Precise models of where and when transcriptionwill occur in a genome (initiation and termination)

[2] Precise, predictive models of alternative RNA splicing

[3] Precise models of signal transduction pathways;ability to predict cellular responses to external stimuli

[4] Determining protein:DNA, protein:RNA, protein:proteinrecognition codes

[5] Accurate ab initio protein structure prediction

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