Genomes

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Chapter 16

• Genomes

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Chapter 16
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Figure 16.1
ARCHAEA
EUKARYA
BACTERIA
Gram-positive bacteria
Green sulfur bacteria
Methanobacterium
Microsporidians
Methanococcus
Purple bacteria
Archeaoglobus
Kinetoplastids
Cyanobacteria
Diplomonads
Flavobacteria
Apicomplexa
Slime molds
Land plants
Thermotoga
Sulfolobus
Amoebae
Aquifex
Animals
Fungi
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Whole-Genome Sequencing Technology

• Large scale DNA sequencing uses the dideoxy
  method with some improvements for increased
  efficiency and speed.
• The use of fluorescently-tagged
  dideoxynucleotides enables all four reactions to
  occur in one tube and the sequence can be read
  by machines. (Fig. 16.2)

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Figure 16.2a
1. Do one sequencing reaction instead of four.
2. Fragments that result have distinctive labels.
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Figure 16.2b
3. Separate fragments via electrophoresis in
mass produced, gel-filled capillary tubes.
Automated sequencing machine reads output.
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Whole-Genome Sequencing Technology

• Large scale DNA sequencing uses the dideoxy
  method with some improvements for increased
  efficiency and speed.
• DNA fragments are separated on mass-produced
  capillary gels.

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Whole-Genome Sequencing Technology

• Two different sequencing strategies were employed
  to derive the human genome.
• Map-based Sequencing orders large fragments of
  DNA on a physical map of the chromosome, and
  then orders smaller fragments within the larger
  ones.

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Whole-Genome Sequencing Technology

• Two different sequencing strategies were employed
  to derive the human genome.
• Shotgun Sequencing skips the initial mapping
  step and sequences the ends of large fragments to
  find overlaps and order them on the chromosome
  map. (Fig. 16.3)

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Figure 16.3 upper
1. Cut DNA into fragments of 160 kb, using
different restrictions enzymes.
BAC library
2. Insert fragments into bacterial
artificial chromosomes, grow in E. coli cells.
3. Analyze fragments, locate each on map
of genome.
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Figure 16.3 middle
4. Cut each 160 kb fragment into 1 kb fragments.
Shotgun clones
5. Insert 1 kb fragments into plasmids, grow
in E.coli cells.
6. Sequence each fragment (note that ends of
fragments overlap).
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Figure 16.3 lower
7. Assemble 1 kb fragments from within each 160
kb fragment by matching overlapping ends.
...ATTTAGACTCGATAAGGATGC...
Draft sequence
8. Assemble fragments from different BACs
by matching overlapping ends.
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Whole-Genome Sequencing Technology

• Two different sequencing strategies were employed
  to derive the human genome.
• Sequencing of eukaryotic genomes is complicated
  by the presence of repeated sequences occurring
  in multiple locations in the genome.

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Whole-Genome Sequencing Technology

• Annotating Genomes
• Prokaryotic genomes the locations of genes are
  identified by searching for promoters and
  translation start/stop sites. (Fig. 16.4)

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Figure 16.4
Open reading frame (ORF)
Stop
Start
5 C T C A A T G G G T A C G T A G G AT C G G
G A A T C G T A C A G G A A C G T T T G A A A T
C G... 3
G A G T T A C C C A T G C A T C C T A G C
CC T T A G C A T G T C C T T G C A A A C T T T
A G C...
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Whole-Genome Sequencing Technology

• Annotating Genomes
• Eukaryotic genomes genes are identified by
  searching for sequences that match those in a
  cDNA library of expressed genes. (Fig. 16.5)

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Figure 16.5
CREATING A cDNA LIBRARY
1. Isolate mRNAs from cells.
mRNA
2. Use reverse transcriptase to make a DNA that
is complementary to each RNA. Use DNA polymerase
to make the single stranded cDNA double-stranded.
cDNA
mRNA
Reverse transcriptase
3. Add each DNA to a plasmid and insert into
E.coli cells.
cDNA library
Collection of cDNAs in library represents
DNA from each actively transcribed gene.
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Bacterial and Archaeal Genomes

• Features of prokaryotic genomes
• There is a strong correlation between genome size
  and metabolic capabilities.
• Bacteria with larger genomes colonize a wider
  variety of habitats.
• Tremendous genetic diversity exists between
  species.
• A significant portion of their genomes appears to
  have been acquired by lateral gene transfer via
  plasmids, transformation, or transfer by
  viruses. (Fig. 16.6)

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Figure 16.6
BACTERIA
ARCHAEA
Gram-positive bacteria
Green sulfur bacteria
Purple bacteria
Thermotoga
Methanobacterium
Archaeoglobus
Methanococcus
Thermoproteus
Methanopyrus
Thermus
Cyanobacteria
Thermococcus
Flavobacteria
Pyrodictium
Aquifex
Halococcus
Sulfolobus
pSL17
pJP78
When genes are transferred laterally, they move
between species that are not necessarily closely
related
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Bacterial and Archaeal Genomes

• Comparative genomics of pathogenic and benign
  bacteria are underway to understand the
  mechanisms of virulence.

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Eukaryotic Genomes

• Features of eukaryotic genomes
• The size is typically orders of magnitude larger
  than prokaryotic genomes.
• Exons comprise a small percentage of the genome,
  whereas repeated sequences make up the majority
  of the DNA.

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Eukaryotic Genomes

• Features of eukaryotic genomes
• Most repeated sequences are derived from
  transposable elements.
• Simple sequence repeats are derived from
  replication errors, and are the basis for DNA
  fingerprinting since they are hypervariable.

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Eukaryotic Genomes

• Features of eukaryotic genomes
• Gene families are thought to arise by gene
  duplication and may be the most important way of
  generating new genes in eukaryotes. (Fig. 16.9)

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Figure 16.7
HOW TRANSPOSABLE ELEMENTS SPREAD
1. A long interspersed element (LINE) exists in
DNA.
DNA
Original location of LINE
Cytoplasm
Nuclear envelope
Line mRNA
RNA polymerase
2. RNA polymerase transcribes LINE.
LINE protein
3. LINE mRNA exits nucleus and is ranslated.
Two LINE proteins
LINE mRNA
4. LINE proteins and mRNA enter nucleus.
Ribosome
cDNA
Reverse transcriptase
5. One protein nicks DNA. The other protein,
reverse transcriptase, makes LINE cDNA from RNA.
DNA polymerase makes cDNA double stranded.
mRNA
6. New copy of LINE is integrated into genome.
New copy
Original copy
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Figure 16.8
HOW UNIQUE NUMBERS OF SIMPLE SEQUENCE REPEATS
ARE GENERATED
1. Start with two chromosome selections
containing the same simple sequence repeats.
1
2
3
4
5
6
7
8
8 repeats
8 repeats
1
2
3
4
6
7
5
8
1
2
3
2. The repeats misalign during meiosis l.
Crossing over and recombination occurs.
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5
8
6
7
5
4
6
3
2
1
7
8
1
2
3
4
6
7
5
8
6
5
10 repeats
3. Meiotic products have unique number of repeats.
1
2
3
4
7
8
6 repeats
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Figure 16.9
Globin gene family
e
yb2
Gg
Ag
yb1
b
d
Coding genes
Pseudogene
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Eukaryotic Genomes

• Eukaryotic genome size does not show strong
  correlation to the complexity of the organism,
  perhaps because alternative splicing enables
  multiple proteins to be produced from one locus.

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Future Prospects

• Functional genomics is the study of how the
  products of all the genes interact in the
  organism.
• DNA microarrays show changes in gene
  expression.(Fig. 16.10a,b)
• Proteomics uses a protein microarray to identify
  protein-protein interactions.

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Figure 16.10a, upper
Determining which genes are being transcribed
Probe microarray with labeled single-stranded
cDNAs
Each spot contains a different section of
single-stranded coding sequence
If labeled DNA binds to sequence on microarray,
that sequence is being transcribed in the cell
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Figure 16.10a, lower
Output looks like this
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Figure 16.10b
Evidence for alternative splicing
Suppose 5 spots on microarray contain sequences
from 5 exons of same locus. If hybridization with
cDNA gives...
Exons
1
2
3
4
5
Time 1
(Exons 1, 3, 4 in mRNA)
Time 2
(Exons 2, 3, 4, 5 in mRNA)
Time 3
(Exons 15 in mRNA)
... then alternative splicing is occurring.
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Future Prospects

• Genome studies provide information that is useful
  in biomedical research
• DNA sequence data can be used to identify targets
  for rational drug design.
• Genomic studies of pathogens are being used for
  rational vaccine design. (Fig. 16.11)

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Figure 16.11
FINDING POSSIBLE VACCINE COMPONENTS
1. Isolate open reading frames (ORFs) from
pathogen genome sequence.
2. Introduce ORFs into E.coli cells.
3. Isolate proteins that result
from transcription and translation.
4. Inject proteins into mice.
If mice develop immune system response to
protein, it may be an effective vaccine component.
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Future Prospects

• Serious ethical/legal/privacy questions will need
  to be addressed as new technology expands the
  types of genetic information that can be obtained
  on individuals.