Genomes

1
Genomes
2
Figure 17.7 Synthetic Cells
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17 Genomes

• 17.1 How Are Genomes Sequenced?
• 17.2 What Have We Learned from Sequencing
  Prokaryotic Genomes?
• 17.3 What Have We Learned from Sequencing
  Eukaryotic Genomes?
• 17.4 What Are the Characteristics of the Human
  Genome?
• 17.5 What Do the New Disciplines of Proteomics
  and Metabolomics Reveal?

4
17 Genomes
No other mammal shows as much phenotypic
variation as dogs. The Dog Genome Project
sequences entire genomes of different breeds and
identifies genes that control specific traits,
such as size.
Opening Question What does dog genome
sequencing reveal about other animals?
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17.1 How Are Genomes Sequenced?

• Genome sequencing determine the nucleotide base
  sequence of an entire genome.
• The information is used to
• Compare genomes of different species to trace
  evolutionary relationships
• Compare individuals of the same species to
  identify mutations that affect phenotypes

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17.1 How Are Genomes Sequenced?

• Identify genes for particular traits, such as
  genes associated with diseases
• The Human Genome Project was proposed in 1986 to
  determine the normal sequence of all human DNA.
• Methods used were first developed to sequence
  prokaryotes and simple eukaryotes.

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17.1 How Are Genomes Sequenced?

• To sequence an entire genome, the DNA is first
  cut into millions of small, overlapping
  fragments.
• Then many sequencing reactions are performed
  simultaneously.

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17.1 How Are Genomes Sequenced?

• High-throughput sequencing uses miniaturization
  techniques, principles of DNA replication, and
  polymerase chain reaction (PCR).
• It is fully automated, rapid, and inexpensive.

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Figure 17.1 DNA Sequencing
10
17.1 How Are Genomes Sequenced?

1. DNA is cut into small fragments physically or
   using enzymes.
2. The fragments are denatured using heat,
   separating the strands.
3. Short, synthetic oligonucleotides are attached to
   each end of each fragment, and these are attached
   to a solid support.

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17.1 How Are Genomes Sequenced?

• Fragments are amplified by PCR.
• Sequencing
• Universal primers, DNA polymerase, and the 4
  nucleotides (dNTPs, tagged with fluorescent dyes)
  are added.
• One nucleotide is added to the new DNA strand in
  each cycle, and the unincorporated dNTPs are
  removed.

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17.1 How Are Genomes Sequenced?

1. Fluorescence color of the new nucleotide at each
   location is detected with a camera.
2. Fluorescent tag is removed and the cycle repeats.

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17.1 How Are Genomes Sequenced?

• Then the sequences must be put together.
• The DNA sequence fragments, called reads, are
  overlapping, so they can be aligned.

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17.1 How Are Genomes Sequenced?

• Example Using a 10 bp fragment, cut three
  different ways
• TG, ATG, and CCTAC
• AT, GCC, and TACTG
• CTG, CTA, and ATGC
• The correct order is ATGCCTACTG.

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Figure 17.2 Arranging DNA Fragments
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17.1 How Are Genomes Sequenced?

• The field of bioinformatics was developed to
  analyze DNA sequences using complex mathematics
  and computer programs.

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Figure 17.3 The Genomic Book of Life
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17.1 How Are Genomes Sequenced?

• Genome sequence information is used in two
  research fields
• Functional genomicssequence information is used
  to identify functions of various parts of
  genomes
• Open reading framesgene coding regions

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17.1 How Are Genomes Sequenced?

• Amino acid sequences, deduced from sequences of
  open reading frames
• Regulatory sequences, such as promoters and
  terminators.
• RNA genes
• Other noncoding sequences

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17.1 How Are Genomes Sequenced?

• Comparative genomics comparison of a newly
  sequenced genome with sequences from other
  organisms.
• This provides more information about functions of
  sequences and can be used to trace evolutionary
  relationships.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• The first life forms to be sequenced were the
  simplest viruses with small genomes.
• The first complete genome sequence of a
  free-living cellular organism was for the
  bacterium Haemophilus influenzae in 1995.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Bacterial and archaeal genomes are
• Small, and usually organized into a single
  chromosome
• Compact85 is coding sequences
• Usually do not have introns
• Have plasmids, which may be transferred between
  cells

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Table 17.1
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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Functional genomics
• H. influenzae chromosome has 1,727 open reading
  frames.
• When it was first sequenced, only 58 coded for
  proteins with known functions.
• Since then, the roles of many other proteins have
  been identified.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Highly infective strains of H. influenzae have
  genes for surface proteins that attach the
  bacterium to the human respiratory tract.
• These surface proteins are now a focus of
  research on treatments for H. influenzae
  infections.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Comparative genomics
• M. genitalium lacks enzymes to synthesize amino
  acids, so it must obtain them from the
  environment.
• E. coli has 55 genes that encode transcriptional
  activators, whereas M. genitalium has only 7a
  relative lack of control over gene expression.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Genome sequencing provides insights into
  microorganisms that are important in agriculture
  and medicine.
• Surprising relationships between organisms
  suggests that genes may be transferred between
  different species.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Rhizobium bacteria form symbiotic relationships
  with plants. The bacteria fix N into forms
  useable by plants.
• Sequencing has identified genes involved in
  successful symbiosis, and may broaden the range
  of plants that can form these relationships.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• E. coli strain O157H7 causes illness in humans.
• 1,387 genes are different from those in the
  harmless strains of this bacterium, but are also
  present in other pathogenic bacteria, such as
  Salmonella.
• This suggests genetic exchange among species.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Severe acute respiratory syndrome (SARS) was
  first detected in southern China in 2002 and
  rapidly spread in 2003.
• Isolation and sequencing of the virus revealed
  novel proteins that are possible targets for
  antiviral drugs or vaccines.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Genome sequencing of organisms involved in global
  ecological cycles
• Some bacteria produce methane, a greenhouse gas,
  in cow stomachs.
• Others remove methane from the air.
• Understanding the genes involved in methane
  production and consumption may help us slow the
  progress of global warming.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Traditionally, microorganisms have been
  identified by culturing them in the laboratory.
• Now, PCR and DNA analysis allow microbes to be
  studied without culturing.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• DNA can also be analyzed directly from
  environmental samples.
• Metagenomicsgenetic diversity is explored
  without isolating intact microorganisms.
• Sequencing is used to detect presence of known
  microbes and previously unidentified organisms.

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Figure 17.4 Metagenomics
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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• It is estimated that 90 of the microbial world
  has been invisible to biologists and is only
  now being revealed by metagenomics.
• The increased knowledge of the microbial world
  will improve our understanding of ecological
  processes and better ways to manage environmental
  problems.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Transposable elements (transposons) are DNA
  segments that can move from place to place in the
  genome or to a plasmid.
• If a transposable element is inserted into the
  middle of a gene, it will be transcribed, and
  result in abnormal proteins.

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Figure 17.5 DNA Sequences That Move (A)
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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Composite transposons transposable elements
  located near one another will transpose together
  and carry the intervening DNA sequence with them.
  
• Genes for antibiotic resistance can be multiplied
  and transferred between bacteria in this way, via
  plasmids.

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Figure 17.5 DNA Sequences That Move (B)
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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Certain genes are present in all organisms
  (universal genes) and some universal gene
  segments are present in many organisms.
• This suggests that a minimal set of DNA sequences
  is common to all cells.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• Efforts to define a minimal genome involve
  computer analysis of genomes, the study of the
  smallest known genome (M. genitalium), and using
  transposons as mutagens.
• Transposons can insert into genes at random the
  mutated bacteria are tested for growth and
  survival, and DNA is sequenced.

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Figure 17.6 Using Transposon Mutagenesis to
Determine the Minimal Genome
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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• M. genitalium can survive in the laboratory with
  only 382 functional genes.
• One goal of the research is to design new life
  forms for specific purposes, such as cleaning up
  oil spills.

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17.2 What Have We Learned from Sequencing
Prokaryotic Genomes?

• An artificial genome has been created and
  inserted into bacterial cells.
• The entire genome of Mycoplasma mycoides was
  synthesized, then transplanted into empty cells
  of Mycoplasma capricolum.
• The new cells genome had extra sequences, so it
  was a new organism Mycoplasma mycoides
  JCV1-syn.1.0.

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Figure 17.7 Synthetic Cells
46
Working with Data 17.1 Using Transposon
Mutagenesis to Determine the Minimal Genome

• In the experiment to create a synthetic genome
  and determine the minimum set of genes necessary
  for survival, transposon mutagenesis was used
  with Mycoplasma genitalium, which had the
  smallest known genome.

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Working with Data 17.1 Using Transposon
Mutagenesis to Determine the Minimal Genome

• Growth of M. genitalium strains with gene
  insertions (intragenic) was compared with strains
  with insertions in noncoding regions (intergenic).

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Working with Data 17.1 Using Transposon
Mutagenesis to Determine the Minimal Genome

• Question 1
• Explain these data in terms of genes essential
  for growth and survival.
• Are all of the genes in M. genitalium essential
  for growth?
• If not, how many are essential?
• Why did some of the insertions in intergenic
  regions prevent growth?

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Working with Data 17.1 Using Transposon
Mutagenesis to Determine the Minimal Genome

• Question 2
• If a transposon inserts into the following
  regions of a gene, there might be no effect on
  the phenotype.
• Explain in each case
• a. near the 3' end of a coding region
• b. within a gene coding for rRNA
• How does this affect your answer to Question 1?

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• There are major differences between eukaryotic
  and prokaryotic genomes
• Eukaryotic genomes are larger and have more
  protein-coding genes.
• Eukaryotic genomes have more regulatory
  sequences. Greater complexity requires more
  regulation.

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Much of eukaryotic DNA is noncoding, including
  introns, gene control sequences, and repeated
  sequences.
• Eukaryotes have multiple chromosomes each must
  have an origin of replication, a centromere, and
  a telomeric sequence at each end.

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Table 17.2
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Several model organisms have been studied
  extensively.
• Model organisms are easy to grow and study in a
  laboratory, their genetics are well studied, and
  they have characteristics that represent a larger
  group of organisms.

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• The yeast, Saccharomyces cerevisiae
• Yeasts are single-celled eukaryotes.
• Yeasts and E. coli appear to use about the same
  number of genes to perform basic functions.
• Compartmentalization of the eukaryotic yeast cell
  requires it to have many more genes.

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Table 17.3
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• The nematode, Caenorhabditis elegans
• A millimeter-long soil roundworm.
• The transparent body is made up of about 1,000
  cells, yet has complex organ systems.
• It has about 3.3 times as many protein-coding
  genes as do yeasts.

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Table 17.4
58
17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• The fruit fly, Drosophila melanogaster
• Studies of fruit flies led to formulation of many
  basic principles of genetics. More than 2,500
  mutations have been described.
• It has 10 times more cells and a larger genome
  than C. elegans, but fewer coding genes.

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Figure 17.8 Functions of the Eukaryotic Genome
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• The thale cress, Arabidopsis thaliana
• A small plant with a small genome.
• Many of the genes found in animals have homologs
  in plants, suggesting a common ancestor.
• But many genes are also unique to plants.

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Table 17.5
62
17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Rice (Oryza sativa) and a poplar tree (Populus
  trichocarpa) have also been sequenced.
• Comparison of the genomes shows many genes in
  common, comprising the basic minimal plant genome.

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Figure 17.9 Plant Genomes
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Eukaryotes have closely related genes called gene
  families.
• These arose over evolutionary time when different
  copies of genes underwent separate mutations.
• Example Genes encoding the globin proteins all
  arose from a single common ancestral gene.

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Figure 17.10 The Globin Gene Family
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• During development, different members of the
  globin gene family are expressed at different
  times in different tissues.
• Example Hemoglobin of the human fetus contains
  ?-globin, which binds O2 more tightly than adult
  hemoglobin.

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Many gene families include nonfunctional
  pseudogenes (?), resulting from mutations that
  cause a loss of function.
• A pseudogene may simply lack a promoter, and thus
  fail to be transcribed, or a recognition site
  needed for the removal of an intron.

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Eukaryotic genomes have repetitive DNA sequences
• Highly repetitive sequencesshort sequences (lt
  100 bp) repeated thousands of times in tandem
  not transcribed.
• Short tandem repeats (STRs) of 15 bp can be used
  in DNA fingerprinting.

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Moderately repetitive sequences are repeated
  101,000 times.
• Includes genes for tRNAs and rRNAs
• Single copies of the tRNA and rRNA genes would
  be inadequate to supply the large amounts of
  these molecules needed by cells.

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Figure 17.11 A Moderately Repetitive Sequence
Codes for rRNA (Part 1)
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Figure 17.11 A Moderately Repetitive Sequence
Codes for rRNA (Part 2)
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Transposons (transposable elements) are
  moderately repetitive sequences.
• Three types are retrotransposons
• SINEs (short interspersed elements)
• LINEs (long interspersed elements)
• LTRs (long terminal repeats)

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Table 17.6
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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Retrotransposons are transcribed into RNA, which
  is a template for new DNA. The new DNA becomes
  inserted at a new location, resulting in two
  copies of the transposon.
• DNA transposons are excised from the original
  location and become inserted at a new location
  without being replicated.

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17.3 What Have We Learned from Sequencing
Eukaryotic Genomes?

• Insertion of a transposon at a new location can
  have important consequences, such as mutations
  and gene duplications.
• They can result in shuffling the genetic material
  and creating new genes.
• Transposons may have played a role in
  endosymbiosis.

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17.4 What Are the Characteristics of the Human
Genome?

• Sequencing of the human genome revealed many
  interesting facts
• Protein-coding regions make up about 1.2, or
  21,000 genes.
• The average gene must code for several different
  proteins, and posttranscriptional mechanisms
  result in different proteins.

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17.4 What Are the Characteristics of the Human
Genome?

• An average gene has 27,000 base pairs.
• All human genes have many introns.
• About half of the genome is transposons and other
  repetitive sequences.

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17.4 What Are the Characteristics of the Human
Genome?

• 99.5 of the genome is the same in all people.
• Variation among individuals is due to single
  nucleotide polymorphisms (SNPs), and differences
  in sequence copy number from chromosomal
  deletions, duplications, or translocations.

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17.4 What Are the Characteristics of the Human
Genome?

• Genes are not evenly distributed over the genome.
  
• The Y chromosome has the fewest genes (231)
  chromosome 1 has the most (2,968).

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17.4 What Are the Characteristics of the Human
Genome?

• Comparisons of prokaryote and eukaryote genomes
  have revealed evolutionary relationships between
  genes.

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Figure 17.12 Evolution of the Genome
82
17.4 What Are the Characteristics of the Human
Genome?

• The genomes of many primates have been sequenced,
  and biologists are interested in which genes make
  humans unique.
• Chimpanzees are our closest living relative they
  share almost 99 of our DNA sequences.

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17.4 What Are the Characteristics of the Human
Genome?

• DNA from the bones of Neanderthals, who lived in
  Europe up to 50,000 years ago, has also been
  sequenced.
• It is 99 identical to human DNA, justifying
  classification of Neanderthals as part of the
  same genus, Homo.

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17.4 What Are the Characteristics of the Human
Genome?

• Comparisons of human and Neanderthal genes
• A mutation in MC1R in Neanderthals causes lower
  activity of MC1R, known to result in fair skin
  and red hair.
• FOXP2, involved in vocalization, is identical in
  humans and Neanderthals, suggesting that
  Neanderthals were capable of speech.

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Figure 17.13 A Neanderthal Child
86
17.4 What Are the Characteristics of the Human
Genome?

• There are some distinctive human DNA sequences
  and also distinctive Neanderthal sequences.
• There is some mixture of the two, indicating
  that humans and Neanderthals interbred.

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17.4 What Are the Characteristics of the Human
Genome?

• Rapid genotyping technologies are being used to
  understand the genetic basis of diseases such as
  diabetes, heart disease, and Alzheimers disease.
• Haplotype maps are used to identify SNPs that
  are linked to genes involved in disease.

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17.4 What Are the Characteristics of the Human
Genome?

• A haplotype is a piece of chromosome with a set
  of SNPs that are usually inherited as a unit.
• By comparing the haplotypes of individuals with
  and without a particular genetic disease, the
  loci associated with the disease can be
  identified.

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Figure 17.14 SNP Genotyping and Disease
90
17.4 What Are the Characteristics of the Human
Genome?

• New technologies analyze thousands or millions of
  SNPs to determine which ones are associated with
  specific diseases.
• As the cost of sequencing entire genomes
  decreases, SNP testing may be superseded.

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Table 17.7
92
17.4 What Are the Characteristics of the Human
Genome?

• Pharmacogenomics is the study of how an
  individuals genome affects response to drugs or
  other outside agents.
• SNPs that are associated with specific drug
  responses can be identified to personalize drug
  treatments and determine if a patient will
  respond to a drug.

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Figure 17.15 Pharmacogenomics
94
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Many genes encode more than one protein.
• Alternative splicing and posttranslational
  modifications increase the number of proteins
  that can be derived from one gene.
• But many proteins are produced only by certain
  cells under specific conditions.

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17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Proteome sum total of proteins produced by an
  organism it is more complex than the genome.
• Proteomics seeks to identify and characterize all
  the expressed proteins in an organism.

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17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Two techniques are used to analyze the proteome
• Two-dimensional gel electrophoresis separates
  proteins based on size and electric charges.
• Mass spectrometry identifies proteins by their
  atomic masses.

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Figure 17.16 Proteomics
98
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Comparisons of eukaryotic proteomes has revealed
  a common set of about 1,300 proteins that provide
  the basic metabolic functions.

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Figure 17.17 Proteins of the Eukaryotic Proteome
100
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Proteins have different functional regions or
  domains.
• Proteins that are unique to a particular organism
  are often just unique combinations of domains
  that exist in other organisms.
• This reshuffling of the genetic deck is a key to
  evolution.

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17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Gene and protein function are both affected by
  the internal and external environments of the
  cell.
• Enzyme activities affect concentrations of their
  substrates and products, called metabolites.
• As the proteome changes, so will the abundances
  of metabolites.

102
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Metabolome quantitative description of all of
  the small molecules in a cell or organism.
• Primary metabolitesinvolved in normal processes
  such as pathways like glycolysis. Also includes
  hormones and other signaling molecules.

103
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Secondary metabolitesoften unique to particular
  organisms or groups.
• Examples include antibiotics made by microbes and
  chemicals made by plants for defense.

104
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Measuring metabolites involves gas chromatography
  and high-performance liquid chromatography, which
  separate molecules.
• Mass spectrometry and nuclear magnetic resonance
  spectroscopy are used to identify them.

105
17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• A human metabolome database has been established
  and contains 6,500 metabolites.
• The challenge now is to relate levels of these
  substances to physiology.

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17.5 What Do the New Disciplines Proteomics and
Metabolomics Reveal?

• Plant metabolomics has been studied for many
  years.
• Tens of thousands of secondary metabolites have
  been identified.
• The metabolome of the model organism Arabidopsis
  thaliana is now being described.

107
17 Answer to Opening Question

• Myostatin is a protein that inhibits muscle
  growth.
• In dog breeds with highly developed leg muscles,
  the gene for myostatin has a mutation that makes
  the protein inactive.
• In humans it may be possible to manipulate
  myostatin to treat muscle-wasting diseases such
  as muscular dystrophy.