Transcript Chapter 9 Genome Analysis

Peter J. Russell
CHAPTER 9
Genome Analysis
edited by Yue-Wen Wang Ph. D.
Dept. of Agronomy,台大農藝系
NTU
遺傳學 601 20000
Chapter 9 slide 1
Structural Genomics
1. The advent of DNA sequencing techniques changed experimental
biology, and automation has enhanced the rate of change.
2. Genomics is the development and application of techniques for:
a. Mapping chromosomes.
b. Sequencing genomes.
c. Computational analysis of entire genomes.
3. Subfields of genomics are:
a. Structural genomics, the genetic and physical mapping and sequencing
of chromosomes.
b. Functional genomics, comprehensive analysis of gene functions and of
non-gene sequences in entire genomes.
c. Comparative genomics, comparison of entire genomes across species,
looking at functions and evolutionary relationships.
4. This section focuses on structural genetics, specifically genome
sequencing.
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Chapter 9 slide 2
Genomic Sequencing Using a Mapping
Approach
1. Genome projects use two general approaches:
a. The mapping approach divides the genome into
segments with genetic and physical mapping, refines
the map of each segment, and finally sequences the
DNA.
b. A “shotgun” approach breaks the genome into random,
overlapping fragments, and sequences each fragment.
Based on overlaps, the sequences are assembled by
computer. An advantage is that physical mapping is
not required.
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Chapter 9 slide 3
Genetic Mapping of a Genome
1. A genetic map derives from the frequency of recombination between
genes. Genetic mapping involves determining, the location of genes on
a chromosome relative to other genes, using genetic crosses and
pedigree analysis.
2. A genetic map of the human genome has 24 different maps, one for each
autosomal pair, plus X and Y.
3. Marker alleles in genetic crosses help determine the crossover rate
between linked genes:
a. Individuals with different alleles at two or more loci are crossed, and
their offspring examined.
b. Most of the offspring will have phenotypes corresponding to the linked
alleles. A few progeny will be recombinant.
c. The frequency of the recombinant phenotype is calculated as a
percentage of the total offspring, giving the recombination frequency or
genetic distance. Units are map units (mu) or centiMorgans (cM).
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Chapter 9 slide 4
4. Experimental crosses are not done in humans and so genetic mapping
relies on pedigree analysis, and is limited by rarity of large,
multigenerational pedigrees showing segregation of defined linked
traits.
5. Usually, the lod (logarithm of odds) score method is used for statistical
analysis of pedigree data.
a. A lod score compares the expected distributions of traits if they are
linked, and if they are not linked.
b. The lod score is the log10 of the ratio of the two probabilities. The
higher the lod score, the closer the two genes.
6. The map distance for linked markers is computed from the
recombination frequency given by the highest lod score, by solving lod
scores for a range of proposed map units. For the human genome, 1 mu
is approximately 1 megabase (Mb).
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Chapter 9 slide 5
Genetic Markers for Genetic Mapping
Experiments
1. Genes have historically been used as markers for genetic mapping experiments, but in
humans only a few allelic forms are easily studied, and the genes are spaced too widely
on the chromosome for high- density mapping.
2. DNA markers are used in association with gene markers for genetic and physical mapping
of chromosomes. DNA markers are distinguishable polymorphic alleles that do not
encode proteins, and therefore are neither dominant nor recessive. Four major types are
used for humans:
a. Restriction fragment length polymorphisms (RFLPs) result from mutations that create or abolish
restriction sites, or from insertions or deletions of DNA between sites under study (Figure 8.5).
The procedure to detect polymorphisms is:
i. Isolate genornic DNA and digest with a restriction enzyme.
ii. Electrophorese and transfer DNA to a membrane filter.
iii. Probe with labeled DNA from the polymorphism region.
iv. Monozygotes show one band, heterozygotes two.
v. PCR amplification is an alternative method.
vi. RFLP probes are discovered by chance when random DNA fragments are used in
Southern blots.
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Chapter 9 slide 6
b. Variable Number of Tandem Repeats (VNTRs), also called minisatellites, are short
sequences (5-lOs) repeated 10s-to-l000s of times (Figure 9.1).
i. DNA is digested with a restriction enzyme that cuts flanking the VNTR.
ii. Fragments are electrophoresed, and blotted to a filter.
iii. The blot is probed with the VNTR repeating sequence.
iv. Some VNTR sequences are in only one genomic locus, corresponding to a
monolocus probe.
v. Other VNTR sequences map to a number of genomic loci, corresponding to a
multilocus probe.
vi. PCR can also find VNTR lengths, if the flanking sequences are known.
c. Short Tandem Repeats (STRs), or microsatellite sequences, contain very short (1-4)
tandem repeats, and are highly polymorphic.
i. STR alleles are more useful than VNTRs for human genetic mapping.
ii. STRs are typed by probing or by PCR, to which they are more suited than
VNTRs.
iii. STRs are distributed widely in the genome, providing about 105 loci.
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Chapter 9 slide 7
Fig. 9.1 Variable number of tandem repeats (VNTRs), also known as minisatellite
sequences
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 9 slide 8
d. Single Nucleotide Polymorphisms (SNPs, “snips”) are base-pair differences between
individuals. If at least 1% of the population has an altered base pair at a particular
site, it is an SNP. About 98% of human DNA polymorphisms are SNPs, with about
3 million in the human genome.
i. SNPs are typed by oligonucleotide hybridization analysis, where an oligo
complementary to the common sequence detects polymorphisms by failing to
bind at high stringency (Figure 9.2).,
ii. DNA microarrays or oligonucleotide arrays can type hundreds of SNPs in one
experiment.
(1) A DNA microarray has DNAs of known sequence fixed at known
locations to a solid substrate (usually a silicon chip or glass).
(2) There are two major types of DNA microarray technology:
(a) Mechanical spotting of DNAs onto the substrate using specially designed spotting pins or ink jets.
(b) Oligonucleotides synthesized at defined positions on the substrate using a light-directed process,
creating an even greater density of DNA sequences in the array (Figure 9.4).
(3) An experiment using a commercially available
GeneChips® probe array (the fixed DNAs are the probes, and the unknown
free DNA that binds is the target) is outlined (Figure 9.5):
(a) In this experiment the chip has an array of oligonucleotide probes, and the target is a population
of cDNAs.
(b) Target cDNAs are labeled with a fluorescent tag, and after hybridization the fluorescence pattern
is recorded by laser scanning and analyzed. Combinations of fluorescent dyes may be used
depending on the goals of the experiment.
(c) In just one experiment, fluorescence patterns
can show allele
identities
for thousands of
loci, and9 slide 9
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Chapter
indicate whether the individual is homozygous or heterozygous for each.
Fig. 9.2 Typing of an SNP by oligonucleotide hybridization analysis
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 10
Fig. 9.3 Preparing a DNA microarray by using robot-driven, mechanical microspotting
of premade DNA molecules onto glass
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 11
Fig. 9.4a Preparing a GeneChip®
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 12
Fig. 9.4b Preparing a GeneChip®
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 13
Fig. 9.5 Illustration of an experiment using a GeneChip®
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 9 slide 14
A High-density Genetic Map of the Human
Genome
1. Human genetic mapping was revolutionized by discovery of many
polymorphic DNA markers, and development of molecular tools to type
them. Hundreds may be typed in a given cross, and computer
algorithms then determine linkage relationships.
2. High-density genetic mapping has been important in the human genome
project. Some aspects of this procedure:
a. A sequence tagged site (STS) is a unique genomic DNA sequence used
as a genetic marker. STRs (short tandem repeats) are extensively used
for STS mapping, but nonpolymorphic markers are also used.
b. A consortium of laboratories works on the same set of DNA samples
(mapping panel), so their data may be combined.
c. A high-density human genetic map completed in 1994 localizes 5,264
STRs to 2,335 chromosomal loci, with an average density of one
marker per 599 kb.
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Chapter 9 slide 15
Fig. 9.6 A high-density genetic map with 5,264 microsatellites localized to 2,335
chromosomal loci
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 9 slide 16
Physical Mapping of a Genome
1. Genetic maps generated for some species (e.g.. E. coli )
are sufficient to begin sequencing, but in humans even the
detailed genetic map described above lacks the required
resolution. Therefore, a physical map derived directly
from genomic DNA rather than analysis of recombinants
has been generated.
2. As in human genetic mapping, there are 24 physical maps,
for the autosomes plus X and Y. Types of physical maps
are presented in order of increasing resolution:
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Chapter 9 slide 17
Cytogenetic Maps: Chromosomal Banding
Patterns
1. Microscopic examination of stained
chromosomes reveals a pattern of bands that
average about 6 Mb. Regions are designated
based on their chromosomal position relative to
the centromere:
a. Regions designated “q” are on the chromosome’s long
arm.
b. Regions designated “p” are on the short arm.
c. Regions are numbered from the centromere outward,
with “q1” and “p1” nearest.
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Chapter 9 slide 18
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Chapter 9 slide 19
FISH (Fluorescent in situ Hybridization) Maps
• 1. Individual metaphase chromosomes are probed in situ
with specific fluorescently labeled DNA sequences,
identifying homologous sequences in the chromosome.
• 2. Different probes labeled with different fluorescent dyes
may be used in the same experiment. Fluorescence
microscopy provides data for computer imaging analysis
to determine binding site(s) for each probe.
• 3. With a resolution of 2-5 Mb in metaphase
chromosomes, FISH can localize markers to subregions
of chromosomal bands. Less condensed chromosomes
may be. resolved in the 5-700 kb range.
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Chapter 9 slide 20
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Chapter 9 slide 21
Restriction Maps
1. Restriction enzymes are used that cut rarely, due
either to a large (7-8 bp) recognition sequence or
to scarcity of the recognition sequence in the
DNA under study.
2. The map for even a rarely cutting restriction
enzyme is very complex, and so far has been
obtained for only the smallest human
chromosome.
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Chapter 9 slide 22
Radiation Hybrid Maps
1. A radiation hybrid (RH) is a rodent cell line carrying a
small genomic DNA molecule from another organism
(e.g., a human). In this technique (Figure 9.8):
a. Exposure to X rays breaks the DNA in human cells. The
fragments become smaller with more X ray exposure, and
fragment length determines the, map resolution.
b. Irradiation kills the human cells, which are then fused with
rodent cells, rescuing chromosomal fragments that are typically a
few Mb in length.
c. Human DNA in the RH is analyzed for gene and/or DNA
markers. The closer two markers are to each other on the
chromosome, the more likely they are to be found together in an
RH.
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Chapter 9 slide 23
Fig. 9.8 Making a radiation hybrid
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 24
Clone Contig Maps
1. A partial restriction digest produces a set of large, overlapping DNAs,
which are cloned into YAC vector cut with a compatible restriction
enzyme. Shearing may also be used to make high-molecular-weight
DNA that is blunt-end cloned into a YAC.
2. An entire genome or a single chromosome may be represented in a YAC
clone library.
3. YAC clones are then assembled into a map either by matching with a
FISH-generated chromosome map or by DNA fingerprinting and
assembly based on overlaps. Nonpolymorphic STSs are especially
useful for YAC contig mapping (Figure 9.9).
4. A complete library should yield a complete contig map that indicates the
order in which the cloned fragments occur in the chromosome.
5. Problems arise when some of the YAC inserts contain DNA from more
than one chromosomal location. This has complicated efforts at
generating a YAC contig map of human chromosomes.
6. Many labs have switched to BAC (bacterial artificial chromosome)
vectors with a capacity of 300 kb and the ability to replicate in E. coli
as a resource for their sequencing projects.
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Chapter 9 slide 25
Fig. 9.9 A representative YAC contig map assembled by STS mapping
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 26
Generating the Sequence of a Genome
1. When a high-resolution map is available, sequencing is possible.
(Automated DNA sequencing is discussed in Chapter 7.) Briefly:
a. Dideoxy sequencing is used. DNA is synthesized from a template, and
terminates with incorporation of a fluorescently labeled ddNTP.
b. All four reactions (ddA, ddG, ddC and ddT) occur in the same tube.
Each ddNTP carries a different fluorescent label.
c. Products are separated electrophoretically, colored bands are detected
with lasers and the data are converted to a computer sequence file.
d. PCR-based sequencing uses one oligonucleotide primer and
thermostable DNA polymerase. The advantages of this approach are:
i. Double-stranded DNA is sequenced directly.
ii. Only a small amount of template DNA is required.
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Chapter 9 slide 27
2. One sequencing reaction is limited to about 500 nucleotides, and for
accurate sequences both strands must be sequenced several times.
3. Progress on the human genome and other projects has been accelerated
by improved technologies for sequencing and analysis.
4. Human genome sequencing by the mapping approach used BACs, but a
BAC insert is far too large to sequence in one reaction. Instead, the
inserts were each sequenced using a shotgun approach:
a. Each insert is cut from the vector, sheared into fragments that will be
partially overlapping and cloned into a plasmid vector.
b. Each subclone is sequenced, and overlaps are used by a computer to
assemble the data into one contiguous sequence representing the BAC
insert.
c. Using the chromosomal map for BAC clones, the BAC insert sequences
are put in order to yield the complete chromosome sequence.
5. In theory, sequencing contigs for a total length of 6.5-8 times the
genome will span more than 99.8% of the genomic sequence.
6. In practice, the HGP (human genome project) did its sequencing 7times over, and has obtained 97% of the genome, although assembly of
the sequences is still incomplete.
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Chapter 9 slide 28
Genome Sequencing Using a Direct Shotgun
Approach
Animation: Direct Shotgun Sequencing of Genomes
1. The shotgun approach obtains a genomic sequence by
breaking the genome into overlapping fragments for
cloning and sequencing. A computer is then used to
assemble the genomic sequence.
2. Advances that have made this approach practical for large
genomes include:
a. Better computer algorithms for assembling sequences.
b. Automation in the actual sequencing.
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Chapter 9 slide 29
3. A pioneer of this approach is J. Craig Venter, whose Celera Genomics
has also sequenced (5-fold) the human genome to 97%, with complete
assembly of the fragments except for gaps caused by the missing 3%.
4. Direct shotgun sequencing involves (Figure 9.10):
a. Mechanical shearing and cloning of small (about 2 kb) genomic DNA
fragments.
b. Sequencing about 500 bp on each end of the insert DNA. Sequences in
the center of the cloned DNA are obtained from an overlapping clone
rather than directly.
c. Computer analysis gives the sequence of most of the genome, with
gaps caused by sequences missing from the library.
d. A second library is made with larger (about 10 kb) random fragments,
allowing resolution of repeated sequences.
5. The shotgun approach is a successful option for genomic sequencing
that does not require genetic mapping. With both approaches, however,
finishing is required to correct errors and fill in gaps.
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Chapter 9 slide 30
Fig. 9.10 The direct shotgun approach to obtaining the genomic DNA sequence of an
organism
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 31
Overview of Genomes Sequenced
1. The human mitochondrial genome was the first
sequenced, in 1981. Many viral genomes have
been sequenced, as have a number of genomes
from living organisms, the focus of this section.
2. Some features of genomic sequences are noted
when the sequence is published. Published
genomic sequences are usually only mostly
complete, and work continues to fill in gaps and
resolve ambiguities.
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Chapter 9 slide 32
Bacterial Genomes
1. Haemophilus influenzae, the first cellular organism to have its genome
sequenced, was selected for its typical bacterial genome size and its GC
content close to humans.
a. No genetic or physical map existed, so a shotgun approach was used.
b. The H. influenzae genome is 1.83 Mb, with 38% GC content.
c. Annotation of the sequence involved computer analysis to find
significant sequences, including:
i. Open reading frames, regions with no stop codon in a particular
reading frame. Arbitrarily, ORFs over 100 codons are considered
likely to encode proteins (Figure 15.2).
ii. Repeated sequences.
iii. Operons.
iv. Transposable elements.
v. rRNA and tRNA genes.
d. Nearly half of the predicted genes have no “role assignment” meaning
that no function is yet verified for them.
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Chapter 9 slide 33
Fig. 9.11 The annotated genome of H. influenzae
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 34
2. Mycoplasma genitalium was selected because
mycoplasmas have the smallest known genomes
of any living cells, and they often are significant
pathogens.
a. A shotgun approach was used.
b. The genome is 0.58 Mb with a GC content of 32%.
c. Only 470 genes occur in this organism, comprising
88% of the genome.
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Chapter 9 slide 35
3. Escherichia coli was selected because it is an important
model system for molecular biology, genetics and
biotechnology, as well as a common bacterium in animal
intestines and the environment.
a. A shotgun approach was used.
b. The genome is 4.64 Mb with a GC content of 50.8%.
c. Analysis of the genome sequence shows that:
i. 88% of the genome is ORFs.
ii. 0.8% encodes rRNAs (7 operons) and tRNAs (86 genes).
iii. 0.7% is repeated sequences.
iv. About 11% is regulatory and other sequences.
d. Of 4,288 ORFs, 38% are of unknown function.
e. The sequence correlates with the extensive genetic mapping
already in existence for this well-studied organism.
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Chapter 9 slide 36
4. Examples of other bacterial genomes sequenced,
and associated disease or traits:
a. Treponema pallidum (syphilis).
b. Rickettsia prowazekii (typhus).
c. Deinococcus radiodurans (survives heat, cold, poison
and radiation).
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Chapter 9 slide 37
Archaeon Genomes
1. Methanococcus jannaschii is an anaerobic, hyperthermophilic
methanogen that reduces CO2 to methane.
a. A shotgun approach was used.
b. The genome has 31% GC, and three parts:
i. A large circular chromosome of about 1.66 Mb, with 1,682 ORFs.
ii. A circular extrachromosomal element (ECE) of about 58 kb, with
44 ORFs.
iii. A smaller circular ECE of about 17 kb, with 12 ORFs.
c. Only 38% of the 1,738 ORFs have assigned functions.
2. Analysis of the sequence confirms Archaea’s unique taxonomic
position, showing that:
a. Most M. jannaschii genes involved in energy production, metabolism
and cell division are similar to those of eubacteria.
b. Most of the genes involved in DNA replication, transcription and
translation are similar to those of eukaryotes.
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Chapter 9 slide 38
Eukaryotic Genomes
1. The yeast, Saccharomyces cerevisiae, is a model eukaryote for many
types of research. It was the first eukaryotic genome to be completely
sequenced.
a. The mapping approach was used.
b. The 16-chromosome genome is 12 Mb, with individual chromosomes
ranging from 230 kb to 1.5 Mb. An estimated 969 kb of repeated
sequences are missing from the published sequence.
c. Analysis reveals:
i. 6,183 ORFs, 233 with introns.
ii. 120–150 rRNA genes.
iii. 37 snRNA genes.
iv. 262 tRNA genes, 80 with introns.
d. ORFs comprise about 70% of the total genome, and about 1⁄3 have no
known function.
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Chapter 9 slide 39
2. Caenorhabditis elegans, a nematode, has been important
in both genetic and molecular study of embryogenesis,
morphogenesis, development, nerve development and
function, aging and behavior.
a. The nearly-complete genome sequence spans 97 Mb distributed
between six chromosomes (five autosomes and an X
chromosome).
b. Analysis shows:
i.19,099 ORFs, with an average of five introns; 27% of the
sequenced genome consists of exons.
ii. 659 tRNA genes.
iii. One tandem array of rRNA genes.
iv. One tandem array of 5S rRNA genes.
v. A number of non-coding RNA genes in introns of proteincoding genes.
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Chapter 9 slide 40
3. Drosophila melanogaster, the fruit fly, has been important
in both classical genetics and the molecular genetics of
development.
a. Sequencing used the direct shotgun approach, supported by
clone-based sequencing and a BAC-derived physical map.
b. The genome is estimated at 180 Mb. About 1⁄3 (60 Mb) is
heterochromatin located near centromeres. This heterochromatin
is so far unclonable, blocking completion of genomic
sequencing.
c. Remaining 2⁄3 (120 Mb) contains more than 99% of
Drosophila’s 13,600 genes. Comparison with genomic sequences
from other species indicates:
i. Drosophila (fruit fly) has about twice the number of genes
found in Saccharomyces cerevisiae (yeast).
ii. Of 289 genes known to be involved in human disease,
Drosophila has homologs for 177.
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Chapter 9 slide 41
4. Homo sapiens. DNA from a variety of anonymous donors has been sequenced.
The “human genome sequence” does not exactly match the genome of any
human being.
a. A “working draft” of the human genome was announced in June 2000 jointly by:
i. Francis Collins for the HGP (Human Genome Sequencing Project Consortium),
an effort involving 16 institutions located in 5 countries.
ii. J. Craig Venter of Celera Genomics.
b. By June 2000, the sequencing effort had generated 7-fold coverage of the genome,
with about 50% of the genome sequence considered to be near-finished, and 24%
completely finished.
c. The sequencing approaches:
i. The HGP consortium focused on sequencing the gene-rich euchromatin regions,
ignoring the generally unclonable heterochromatin, using existing genetic and
physical maps.
ii. Celera Genomics used shotgun sequencing followed by a very large computer
calculation looking for overlaps in the random DNA fragments (enough to
represent 4.6-fold coverage of the human genome). Shotgun assembly results
were verified by comparison with BAC clone sequences available in public
databases.
d. The next step in the human genome project is annotating the sequence, analyzing its
genes and other features.
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Chapter 9 slide 42
Functional Genomics
1. Functional genomics analyzes all genes in genomes to determine their
functions and their gene control and expression.
2. Classically, genetic analysis has started with a phenotype and gone in
search of a gene. New approaches are needed to work in the opposite
direction, from gene to phenotype.
3. Current functional genomics relies on molecular biology lab research
and sophisticated computer analysis by bioinformatics researchers.
4. This fusion of biology with math and computer science is used for
many things. Examples:
a. Finding genes within a genomic sequence.
b. Aligning sequences in databases to determine matching.
c. Predicting structure and function of gene products.
d. Describing interactions between genes and gene products in the cell,
between cells and between organisms.
e. Considering phylogenetic relationships.
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Chapter 9 slide 43
Identifying Genes in DNA Sequences
1. Annotation begins the process of assigning
functions to genes, especially protein-coding
genes, using computer algorithms to search both
strands for ORFs. Introns complicate analysis of
eukaryotic genes.
2. ORFs exist in all sizes, and not all encode
proteins. To focus on sequences most likely to
encode proteins, a minimum ORF size is
arbitrarily set and shorter sequences are not
analyzed.
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Chapter 9 slide 44
Homology Searches to Assign Gene Function
1. Computers are used to find homology between sequences in a database (e.g., a BLAST
search). Similarity reflects evolutionary relationships and often shared functions.
2. Either DNA or amino acid sequences can be searched, but amino acids yield more
specific information, since there are 20 possible matches, rather than just four. Often no
convincing match is found, due in part to the limitations of current databases.
3. Sometimes matches are found only at the domain level, when a region in the new protein
matches protein domains in the database. This provides clues to the new protein’s
function and the evolution of its gene.
4. As databases grow, so does our knowledge of gene functions. The current distribution of
knowledge about the genes of yeast is (Figure 9.14):
a. About 30% of the genes have known functions.
b. Of the remaining 70% of ORFs:
i. 30% encode a protein that either has homology to protein(s) of known function, or has
domains related to functionally characterized domains.
ii. 10% are FUN (function unknown) genes. They have homologs in databases, but
function(s) of the homologs are unknown. Groups of homologous genes of unknown
function are orphan families.
iii. 30% of ORFs have no homologs in the databases. These include 6–7% that may not
actually encode proteins. The remainder may represent genes known only in yeast, the
single orphans.
5. Every genome sequenced contains “function unknown” genes, but as databases are
expanded the problem should decrease.
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Chapter 9 slide 45
Fig. 9.14 The distribution of predicted ORFs in the genome of yeast
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 46
Assigning Gene Function Experimentally
1. One approach to determining gene function is to delete the gene, and observe the
phenotype when that gene’s function is knocked out. PCR may be used to produce a
gene knockout (Figure 9.15):
a. Using known genome sequences, PCR primers are designed to construct an artificial linear
DNA deletion module. It consists of:
i. The gene sequence upstream and through the start codon.
ii. A kanR (kanamycin) marker gene conferring resistance to a chemical, G418.
iii. The gene sequence downstream of and including the stop codon.
b. The amplified linear DNA is transformed into yeast, and G418-resistant colonies selected.
These are generated when the new DNA replaces the gene of interest in the genome by
homologous recombination.
c. They now express kanR instead of the gene under study, producing a loss-of-function mutation.
2. Work is underway to systematically analyze by knockout mutation each gene in the
genome of yeast and other organisms.
a. Each knockout must be screened for possible phenotype change in every area of cell function,
making these studies a substantial undertaking.
b. Knockout mutations analyzed in yeast to date indicate about 1⁄3 of the genes are essential, 1⁄3
are nonessential but affect phenotype, and 1⁄3 show no significant change in phenotype
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Fig. 9.15 Creating a gene knockout in yeast
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
台大農藝系 遺傳學 601 20000
Chapter 9 slide 48
Describing Patterns of Gene Expression
1. Genomic sequencing makes it possible to
determine all genes that are expressed in a cell by
analyzing the total RNA transcripts of the cell, its
transcriptome. The transcriptome is an indicator
of cell phenotype and function. Similarly, the
complete set of proteins in a cell is its proteome.
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The Transcriptome
1. The transcriptome changes as the cell responds to stimulus and moves through its cell
cycle, and so is a tool for understanding cellular function.
2. Probe arrays are used to study gene expression. Yeast sporulation is one example:
a. Yeast sporulation produces four haploid spores, and involves four stages, each associated with its
own transcripts (Figure 9.16).
i. DNA replication and recombination.
ii. Meiosis.
iii. Meiosis II.
iv. Spore maturation.
b. Samples of mRNA taken at intervals during sporulation were converted to cDNAs and analyzed
on microarrays of PCR-amplifled ORF sequences. The. results were correlated with cellular
events.
c. Control cDNA was made from pre-induction mRNAs, and labeled green. The cDNAs from postinduction mRNAs were labeled red. Microarrays were probed with a mix of both, and results
were interpreted as follows:
i. Red spots indicate a gene induced during sporulation.
ii. Green spots indicate a gene repressed during sporulation.
iii. Yellow spots mark genes whose expression is unchanged during sporulation.
d. Results show more than 1,000 genes with altered expression during sporulation, about 1/2
repressed and the other 1/2 not repressed. Patterns of expression over time become apparent in
this type of experiment.
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Chapter 9 slide 50
Fig. 9.16a, b Global gene expression analysis of yeast sporulation using a DNA
microarray
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
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Chapter 9 slide 51
3. DNA microarrays are now widely used, although still
expensive. Examples of studies . that currently use this
technology:
a. Changes in Drosophila gene expression during morphogenesis.
b. Human cancers and their characteristic patterns of gene
expression (transcriptional fingerprints) that reveal distinctions
between different types of cancer.
c. Screening for genetic diseases, especially those resulting from
one of many alleles. A patient’s blood, for example, can be
screened for hundreds of possiblc mutations in the BRCA1 and
BRCA2 genes associated with breast cancer.
d. Optimizing drug therapies for patients using pharmacogenomics,
analyzing changes in transcription when the drug is present as a
means of developing drugs that target specific mutations.
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The Proteome
1. Proteomics is cataloging and analysis of the proteome, or complete set
of expressed proteins in a cell at a given time. Proteomics focuses on
which proteins are made and in what quantities, and their interactions
with other proteins.
2. Goals of proteomics are to:
a. Identify every protein in the proteome, using 2-D PAGE mapping,
isolating each protein and analyzing it by mass spectrometry.
b. Develop a database with the sequence of each protein.
c. Analyze protein levels in different cell types and stages of
development.
3. Protein identification and sequencing is very complex. Celera
Genomics is involved in identification, sequencing and computer
analysis of the data.
4. Proteomics stands to make a major contribution to understanding of
human diseases and development of biopharmaceutically based
diagnosis and treatment.
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Comparative Genomics
iActivity: Personalized Prescriptions for Cancer
Patients
1. Comparative genomics provides a way to study
functions of human genes by working with nonhuman homologs. Genes and their arrangement
also provide valuable clues to evolutionary
relationships between organisms.
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Ethics and the Human Genome Project
1. The ability to identify human genes raises complex ethical issues
involving the right to information about one’s own genome, access to
genomic information by employers, insurance companies and
government agencies, and concerns about the ability to diagnose but
not treat genetic disorders.
2. Federal agencies funding the HGP devote 3–5% of their budgets to
study of ethical, legal and social issues (ELSI), producing the world’s
largest bioethics program. Areas currently emphasized by the ELSI
program:
a. Privacy of genetic information.
b. Appropriate use of genetic information in the clinical setting.
c. Fair use of genetic information.
d. Professional and public education.
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