Chapter 10: Genomics

1
Chapter 10 Genomics

• Linnea Fletcher Ph.D.
• BIOL 2316

2
Principal Points

• Genomics is the development and application of
  new mapping, sequencing, and computational
  procedures for the analysis of the entire genome
  of organisms
• Three distinct subfields
• Structural genomics genetic mapping, physical
  mapping, and sequencing
• Functional genomics function of gene and of
  nongene sequences in entire genomes
• Comparative genomics better understand function
  including evolutionary relationships

3

• Approaches to Genome Sequencing
• Generate high-resolution genetic and physical map
  then sequence the segments in an orderly manner
• Whole genome shotgun approach is to break up the
  genome into random, overlapping fragments and
  then sequence the fragments and assemble the
  sequences by means of computer algorithms (the
  scientists who figured out this program in
  combination with the company that designed and
  produced the computers that were used determined
  the success of this approach)

4

• In Bacteria and Archaea, gene density is high. In
  Eukarya, there is a wide range of gene densities,
  showing a trend of decreasing gene density with
  increasing complexity
• Gene expression is analyzed at two levels, mRNA
  (transcriptome) and proteins (proteome).

5
Google the Human Genome Project and find out why
DOE was the Lead partner. (www.ornl.gov/TechResour
ces/Human_Genome/home.html)
6

• Who were the participants of the Human Genome
  Organization, and what were the six goals of the
  Human Genome Project?
• Why is this project considered to be descriptive
  science and not hypothesis-driven science? (a
  website that has a lot of info on this project is
  www.DNAi.org)
• What were the two general sequencing approaches
  used in the project?
• Have these goals been met?
• How much of the human genome has been sequenced,
  and at what accuracy?
• What sequences remain to be determined?
• What are the special concerns of the following
  fields in genomics?
• structural genomics
• functional genomics
• comparative genomics

7

• What are some types of markers used in the
  mapping approach to sequencing a large genome?
• What problem must be overcome to allow
  restriction mapping of a human chromosome, and
  how successful was this approach in the HGP?
• How is a clone contig map generated? Include in
  your answer the role the following play in the
  process
• mechanical shearing
• YACs BACs
• electroporation
• flow cytometry
• FISH
• STSs
• ESTs

8
YAC clone contig mapping using STSs. Each letter
represents An STS.
The final map is A F D M T V P W Z
YAC contig mapping was used for chromsome 21 and
Y and what else? What were some of the problems
with YAC clone contig mapping?
9
Figure 10.1 A representative YAC contig map
assembled by STS mapping. The YAC contig map
shown is of the telomere proximal region of the
small arm of chromosome 7 (7pter). The contig is
oriented such that the 7p telomere is leftward
and the chromosome 7 centromere is rightward.
Maps such as this are difficult to read because
of the amount of information they contain. On the
map, each STS is positioned along the top (with
its name given vertically, starting with y),
and each YAC is indicated as a horizontal line
below, with its name given to the left and its
measured YAC size (in kb) in parentheses. The
presence of an STS in a YAC is indicated by a
closed blue circle at the appropriate position.
STSs in red correspond to YAC insert ends, with a
square placed around the corresponding circle
both at the top (under the STS name) and at the
end of the YAC from which the STS was derived.
All other STSs are shown in blue.
10
Generating the Sequence of a Genome

• What are two advantages of PCR-based DNA
  sequencing (cycle sequencing)?
• What length of sequence can be gotten from a
  single sequencing reaction?
• How are DNA sequences assembled into the full
  length of a BAC insert?

11
Whole-Genome Shotgun Sequencing

• What was the first organism shot-gun sequenced?
• Be able to briefly explain this approach to
  sequencing.
• Who invented this approach?
• Why couldnt the entire human genome be assembled
  with the shotgun approach?

12
Figure 10.2 The whole-genome shotgun approach to
obtaining the genomic DNA sequence of an
organism.
13
Selected Examples of Genomes Sequenced

• What are the organisms profiled in the chapter
  and why were they picked to be sequenced (e.g.
  the fruit fly for development)?
• What is one insight per organism gained as a
  result of sequencing that organism? (review the
  questions and information on the next slides)
• When reading the summary and the keynote on page
  256 to 257, rewrite it in your own words so you
  can summarize what has been learned as a result
  of sequencing these organisms

14
The annotated genome of H. influenzae.

• What types of organisms have the highest gene
  density (ie the least amount of non-coding DNA)?

Figure 10.3 The annotated genome of H.
influenzae. The figure shows the location of each
predicted ORF containing a database match, as
well as selected global features of the genome.
Outer perimeter Key restriction sites. Outer
concentric circle Coding regions for which a
gene was identified. Each location of a coding
region is color coded with respect to its
function. Second concentric circle Regions of
high GC content are shown in red (gt42 percent)
and blue (gt40 percent) and regions of high AT
content are shown in black (gt66 percent) and
green (gt64 percent). Third concentric circle The
locations of the six ribosomal RNA gene clusters
(green), the tRNAs (black), and the cryptic
mu-like prophage (blue). Fourth concentric
circle Simple tandem repeats. The origin of
replication is illustrated by the
outward-pointing arrows (green) originating near
base 603,000. Two possible replication
termination sequences are shown near the opposite
midpoint of the circle (red).

• How did genome sequences of archaea confirm that
  they are an evolutionary branch distinct from
  bacteria and from eukaryotes?

15
Scanning electron micrograph of the yeast
Saccharomyces cerevisiae.

• What types of organisms have introns in their
  genes?

Functionally it resembles mammals in many ways-
and it is easy to culture.
16
nematode worm Caenorhabditis elegans.
First multicellular organism to be sequenced. An
important model organism for studying the genetic
and molecular aspects of embryogenesis,
morphogenesis, development, nerve development and
function, aging, and behavior.
17
Three mammals Human, mouse, rat

• What is a gene desert and how many are there in
  the human genome?

All three genomes have the approximately the same
number of protein-coding genes Sequence analysis
reveals that approximately 90 of the genes in
the mouse And rat have direct counterparts in the
human
18
The pufferfish, Fugu rubripes.

• How does the pufferfish assist in identifying
  ORFs in other vertebrate genomes? What is an ORF?

19
Differences in gene density when comparing
organisms
20
Functional Genomics

• Define bioinformatics.
• What is bioinformatics used for?
• Identifying Genes in DNA Sequences..
• What is the next step after obtaining the
  complete sequence of a genome?
• How are ORFs of putative genes identified in DNA
  sequence data?
• Approximately how many of the ORFs found in
  prokaryotes are of unknown function?
• What proportion of yeast ORFs have been
  identified as genes of known function?

21

• Whose DNA did the HGP and the Celera sequencing
  project actually sequence?
• How many ORFs were found in the finished
  sequence, and how did this compare with
  predictions?
• How does this number compare with other mammals,
  and other non-mammalian species?
• What percent of rat and mouse genes have homologs
  in the human genome?

22

• Explain why especially large and especially small
  genes are more likely to be missed by computer
  algorithms searching for ORFs.
• How does the BLAST programs at NCBI assist in
  identifying ORFs?
• Why is a gene search for amino acid sequence
  similarity often more effective than the same
  search done at the nucleotide sequence level?
• What is assigned to an orphan family and a
  single orphan when using bioinformatics
  approaches to identifying genes?

23
Distribution of predicted ORFs in the genome of
yeast
24

• Assigning Gene Function Experimentally
• How can model organisms be used to identify the
  function of a newly-discovered gene?
• How is a kanamycin resistance gene used to make a
  deletion mutation and how is G418 used to select
  for knock-out recombinants?
• How is PCR used to verify a knockout?
• How successful has the YKO project been in
  identifying function of yeast genes?

25
Figure 10.10 Creating and verifying a gene
knockout in yeast. (a) Schematic of a PCR-based
gene deletion strategy involving a DNA fragment
constructed by PCR from gene sequences flanking
the kanR selectable marker that is transformed
into yeast and replaces the chromosomal ORF by
homologous recombination. (b) Verification of
gene deletion. PCR-based screening method to
confirm (1) unsuccessful deletion (ORF still
present) and (2) successful deletion (ORF
replaced with kanR DNA segment).
26
Figure 10.10 Creating and verifying a gene
knockout in yeast. (a) Schematic of a PCR-based
gene deletion strategy involving a DNA fragment
constructed by PCR from gene sequences flanking
the kanR selectable marker that is transformed
into yeast and replaces the chromosomal ORF by
homologous recombination. (b) Verification of
gene deletion. PCR-based screening method to
confirm (1) unsuccessful deletion (ORF still
present) and (2) successful deletion (ORF
replaced with kanR DNA segment).
27
Describing Patterns of Gene Expression

• How can probe arrays be used to study gene
  function?
• In transcriptome analysis by DNA microarray,
  explain why genes giving a yellow signal are
  not as interesting as the green or red
  signaling genes.
• Explain how transcriptome analysis using DNA
  microarrays may be used for accurate screening
  and diagnosis of cancerous tumor types in the
  future.
• Explain how genetic analysis using DNA
  microarrays can be used to screen for exact types
  of mutations of cancer genes and other complex
  diseases, and how this can lead to
  pharmacogenomics.
• How successful has the field of pharmacogenomics
  been in developing custom drug treatments to
  match the individual genetic and biochemical
  makeup of the patient?

28
Read and then click to the next slide to see the
picture

• Figure 10.11 Global gene expression analysis of
  yeast sporulation via a DNA microarray. (a) The
  stages of sporulation in yeast, correlated with
  the sequential transcription of at least four
  classes of genes. (Adapted from Chu et al. 1998.
  Science 282699705.) (b) Outline of the DNA
  microarray experiment. (c) Example of results of
  a global gene expression analysis in yeast,
  obtained a DNA microarray. The entire yeast
  genome is represented on the DNA chip, and the
  colored dots represent levels of gene expression,
  as described in the text.

29
(No Transcript)
30

• What is bioinformatics and what is its role in
  structural, functional, and comparative genomics?
• What is pharmacogenetics and what gene family
  does it target and why?
• Why is the study of proteomics so much more
  complex than that of genomics or transcriptomics?
• What are the limitations of 2-dimensional
  electrophoresis coupled to mass spectroscopy
  techniques in the study of proteomics?
• What are two types of protein microarrays?
• What are some ethical, legal, and social issues
  (ELSI) associated with genomics projects, and
  what is being done to address them?
• Dont forget to review end of chapter questions
  for practice