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BB30055: Genes and genomes
Genomes - Dr. MV Hejmadi (bssmvh)
BB30055: Genomes - MVH
Recommended texts:
1) Genetics from genes to genomes 2e - Hartwell et al
2) Human Molecular Genetics 3 – Strachan and Read
4) Genomes 2 - TA Brown
5) Genes VII – Benjamin Lewin
Special issue Journals:
Nature (2001) 15th Feb Vol 409
Science (2001) Vol 291 No 5507
Full text of both above journals available at
http://www.bath.ac.uk/library/subjects/bs/links.html#hgp
BB30055: Genomes - MVH
3 broad areas
(A)Genomes, transcriptomes, proteomes
(B) Applications of the human genome
project
(C) Genome evolution
A) Genomes, transcriptomes, proteomes
Genome projects
- Human Genome Project (HGP): a history
- Other genome projects: why do it
- Genome organisation
-
insights from HGP
Repeat elements
Transposable elements
Mitochondrial genomes
Y chromosome
Post-genomics
-transcriptomes
- proteomes
(A) Genomes, transcriptomes and proteomes
genome
Entire DNA complement of any organism which
include organelle DNA
transcriptome
proteome
All RNA transcribed from genome of a cell
or tissue
all proteins expressed by a genome, cell or
tissue
Why study the genome?
3 main reasons
• description of sequence of every gene valuable.
Includes regulatory regions which help in
understanding not only the molecular activities of the
cell but also ways in which they are controlled.
• identify & characterise important inheritable disease
genes or bacterial genes (for industrial use)
• Role of intergenic sequences e.g. satellites, intronic
regions etc
History of Human Genome Project (HGP)
1953 – DNA structure (Watson & Crick)
1972 – Recombinant DNA (Paul Berg)
1977 – DNA sequencing (Maxam, Gilbert and Sanger)
1985 – PCR technology (Kary Mullis)
1986 – automated sequencing (Leroy Hood & Lloyd Smith
1988 – IHGSC established (NIH, DOE) Watson leads
1990 – IHGSC scaled up, BLAST published (Lipman+Myers)
1992 – Watson quits, Venter sets up TIGR
1993 – F Collins heads IHGSC, Sanger Centre (Sulston)
1995 – cDNA microarray
1998 – Celera genomics (J Craig Venter)
2001 – Working draft of human genome sequence published
2003 – Finished sequence announced
HGP
Goal: Obtain the entire DNA sequence of human genome
Players:
(A) International Human Genome Sequence Consortium
(IHGSC)
- public funding, free access to all, started earlier
- used mapping overlapping clones method
(B) Celera Genomics
– private funding, pay to view
- started in 1998
- used whole genome shotgun strategy
Whose genome is it anyway?
(A) International Human Genome Sequence Consortium
(IHGSC)
- composite from several different people generated
from 10-20 primary samples taken from numerous
anonymous donors across racial and ethnic groups
(B) Celera Genomics
– 5 different donors (one of whom was J Craig
Venter himself !!!)
Genomicists looked at two basic features
of genomes: sequence and polymorphism
Major challenge - to determine sequence of each
chromosome in genome and identify polymorphisms
– How does one sequence a 500 Mb chromosome
600 bp at a time?
– How accurate should a genome sequence be?
• DNA sequencing error rate is about 1% per 600 bp
– How does one distinguish sequence errors from
polymorphisms?
• Rate of polymorphism in diploid human genome is about
1 in 500 bp
– Repeat sequences may be hard to place
– Unclonable DNA cannot be sequenced (30%)
Divide and conquer strategy
meets most challenges
• Chromosomes are broken into small
overlapping pieces and cloned
• Ends of clones sequenced and reassembled
into original chromosome strings
• Each piece is sequenced multiple times to
reduce error rate
– 10-fold sequence coverage achieves a rate of
error less than 1/10,000
Strategies for sequencing the human genome
Strategies for sequencing the human genome
Whole-genome shotgun sequencing
Private company Celera used to sequence whole human genome
•
•
•
•
•
• Whole genome randomly
sheared
three times
Whole genome randomly
sheared
three times
– Plasmid library
– Plasmid library constructed
with ~
constructed
with ~ 2kb
2kb inserts
inserts
– Plasmid library with ~10 kb
– Plasmid library with ~10
inserts
kb kbinserts
– BAC library with ~ 200
inserts
– BAC library with ~ 200 kb
Computer program assembles
sequences into chromosomes
inserts
No physical map
• construction
Computer program assembles
sequences into chromosomes
Only one BAC library
Overcomes problems
repeat
• Noofphysical
map construction
sequences
• Only one BAC library
• Overcomes problems of
repeat sequences
Fig. 10.13 Genetics by Hartwell
Fig. 10.13
sequencing larger genomes
Mapping phase
Sequencing phase
http://www.DNAi.org
Other genomes sequenced
1997
4,200 genes
2002
36,000 genes
1998
19,099 genes
2002
38,000 genes
Sept 2003
18,473
human orthologs
Science (26 Sep 2003)Vol301(5641)pp1854-1855
Human genome – size and structure
Nuclear genome (3.2 Gbp)
24 types of chromosomes
Y- 51Mb and chr1 -279Mbp
Base composition – 41% GC
Mitochondrial genome
Nuclear genome organisation (human)
Genomes 2 by TA Brown
pg 23
Nuclear genome organisation (human)
1) Gene and gene related sequences
Coding regions – Exons (5%)
Non-coding regions
RNA genes
Introns
Pseudogenes
Gene fragments
RNA genes -
Nuclear genome organisation
Major classes of RNA involved in gene expression
(human)
rRNA 16S, 23S, 28S, 18S etc
tRNA 22 types of mitochondrial
& 49 cytoplasmic
snRNA U1,U2.U4,U5,U6 etc
snoRNA > 100 types
Other RNA classes
• microRNA
• XIST RNA
• Imprinting associated RNA
• Nervous system specific
• Antisense RNA
• Others
Non-coding regions…..
introns
Non-coding regions…..
Pseudogenes ()
A non functional copy of most or all of a gene
Inactivated by mutations that may cause either

inhibition of signal for initiation or
transcription

prevent splicing at exon-intron boundary

premature termination of translation
Human Mol Gen 3 by Strachan & Read pgs 262-264
Non-coding regions…..
Pseudogenes ()
Different classes include
Non-processed:
contain non functional copies of genomic DNA
sequence incl exons and introns
arise from gene duplication events
E.g. rabbit pseudogene b2
Non-coding regions…..
rabbit pseudogene b2
Related to b1
Usual exon and intron organisation
b1
b2
Non-coding regions…
Pseudogenes - processed
Non-coding regions…
Pseudogenes - processed
non functional copies of exonic sequences of an active
gene. Thought to arise by genomic insertion of a cDNA
as a result of retroposition
Expressed processed: processed pseudogene
integrated adjacent to a promoter site
Contribute to overall repetitive elements
Non-coding regions…..
Gene fragments or truncated genes
Gene fragments: small
segments of a gene
(e.g. single exon from
a multiexon gene)
Truncated genes:
Short components
of functional genes
(e.g. 5’ or 3’ end)
Thought to arise due to unequal crossover or exchange
Nuclear genome organisation (human)
Nuclear genome organisation (human)
2) Extragenic (intergenic) DNA
(~62% of genome)
A) Unique or low copy number sequences
B) Repetitive sequences (~ 53%)
A) Unique or low copy number
sequences
Non –coding, non repetitive and single copy
sequences of no known function or
significance
B) Repetitive sequences
Significance
Evolutionary ‘signposts’
 Passive markers for mutation assays
 Actively reorganise gene organisation by
creating, shuffling or modifying existing
genes
Chromosome structure and dynamics
Provide tools for medical, forensic,
genetic analysis