AP Ch 19

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WHAT PRACTICAL USES HAS OUR KNOWLEDGE OF GENETICS
PROVIDED?
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VIDEO(S) Genetics, longevity, and computer science
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AP Ch 20

• Biotechnology and genetic engineering

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• Biotechnology use of organisms genes and
  current technology to advance society
• Genetic Engineering manipulation of genes for
  practical purpose
• Genomics study of genomes and proteins
  (proteomics)
• Genomics is HOT
• Now lets explore some DNA manipulation and usage
  lab techniques

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General Applications of DNA technology
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General Applications of DNA technology

• Diagnosis of diseases
• Gene therapy
• Forensics matching crime scene DNA to suspects
  and victims
• Genetic engineering of food/animals
• adding traits of other organisms to hosts
• Removing, modifying, or enhancing pre-existing
  genes
• Inserting designer genes into organism (e.g.
  antibiotic production in corn)
• http//www.pbs.org/wgbh/nova/genome/program.html
  (long vid)

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• Key tools of the trade

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• Key tools of the trade
• Restriction enzymes protective enzymes from
  bacteria are used to cut other DNA segments at
  specific locations
• often used to make plasmids with genes of
  interest, p 398
• Vectors delivers chosen gene into a host cell
  where it will be replicated (e.g. bacterial
  plasmid, virus)
• Electroporation, microscopic needles, and bullets
  can also introduce foreign DNA into host

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Restriction enzymes in use
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Microtiter plates with 96, 384 and 1536 wells
(often called 96-well plate). How helpful?
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• Probes piece of single-stranded DNA or RNA of a
  known gene (p. 400)
• used to find a specific DNA sequence by
  hybridization
• Probe can be traced because it is labeled with a
  glowing isotope

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• Genomic libraries genetic library of an
  organisms DNA, over 1000 completed
• Can be genomic libraries or complementary DNA
  (cDNA made in reverse transcription from mRNA)
• What would an advantage of having an entire
  genome on file have over cDNA?
• Human Genome Project - (accomplished 2001)

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• Genomic libraries genetic library of an
  organisms DNA, over 1000 completed
• Can be genomic libraries or complementary DNA
  (cDNA made in reverse transcription from mRNA)
• What would an advantage of having an entire
  genome on file have over cDNA?
• Human Genome Project - (accomplished 2001)
• What would be next step to make this knowledge
  useful?

9/14/2015
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• Genomic libraries genetic library of an
  organisms DNA, over 1000 completed
• Can be genomic libraries or complementary DNA
  (cDNA made in reverse transcription from mRNA)
• What would an advantage of having an entire
  genome on file have over cDNA?
• Human Genome Project - (accomplished 2001)
• What would be next step to make this knowledge
  useful?
• How would they acquire such knowledge?

9/14/2015
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• DNA techniques
• PCR- see diagram to right, make copies of chosen
  of DNA segments

9/14/2015
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• DNA techniques
• PCR- see diagram to right, make copies of chosen
  of DNA segments
• How long until you have 100 DNA copies? 1
  billion?
• Gel electrophoresis see diagram next pg.,
  separates DNA based on size,
• moves by electric charge as DNA is -,
• Used for DNA fingerprinting

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• Gene Cloning producing copies of chosen gene
• Benefits 1. amplifying a chosen gene
• 2. produce a chosen protein product

Give an example of 2 for practical purpose
9/14/2015
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Practical uses of cloned genes, including
cellular transformation
9/14/2015
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Use of restriction sites on DNA segment in gel
electrophoresis
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A technique called Southern blotting combines gel
electrophoresis of DNA fragments with nucleic
acid hybridization?Specific DNA fragments can be
identified by Southern blotting, using labeled
probes that hybridize to the DNA immobilized on a
blot of gel
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Figure 20.13
TECHNIQUE
cDNA synthesis
mRNAs
cDNAs
Primers
PCR amplification
?-globingene
Gel electrophoresis
Embryonic stages
RESULTS
2
1
3
4
5
6
Reverse PCR compares gene expression between
samples (such as 6 stages of organismal
development)
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• Microarrays tests thousands of genes in tissue
  under different environmental conditions
• Can reveal profiles of genes over a lifetime of
  an organism
• How can this technique be used for medical
  discovery?

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• Real-time PCR
• (or quantitative PCR, a.k.a. Q-PCR)
  simultaneously amplifies and quantifies segments
  of DNA

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Use of entire genome in biotechnology

• Cloning
• Stem cells

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Wholeorganism cloning Done by nuclear transfer
How is this useful?
9/14/2015
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Stem cells

• Unspecialized cells that can reproduce
  indefinitely and under can become other types of
  specialized cells?
• What would determine the type of cell a stem cell
  becomes?
• Can be multipotent, pluripotent, omnipotent stem
  cells

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Figure 20.21
Embryonicstem cells
Adultstem cells
Cells generatingall embryoniccell types
Cells generatingsome cell types
Culturedstem cells
Differentcultureconditions
Livercells
Bloodcells
Nervecells
Differenttypes ofdifferentiatedcells
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Figure 20.22
Remove skin cellsfrom patient.
Reprogram skin cellsso the cells becomeinduced
pluripotentstem (iPS) cells.
Patient withdamaged hearttissue or otherdisease
Treat iPS cells sothat they differentiateinto a
specificcell type.
Return cells topatient, wherethey can
repairdamaged tissue.
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Figure 20.23
Gene Therapy
Cloned gene
Insert RNA version of normal alleleinto
retrovirus.
Viral RNA
Let retrovirus infect bone marrow cellsthat have
been removed from thepatient and cultured.
Retroviruscapsid
Viral DNA carrying the normalallele inserts into
chromosome.
Bonemarrowcell frompatient
Bonemarrow
Inject engineeredcells into patient.
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Protein Production by Pharm Animals

• Transgenic animals are made by introducing genes
  from one species into the genome of another
  animal
• Transgenic animals can be pharmaceutical
  factories, producers of large amounts of
  otherwise rare substances for various uses
  (medical, nourishment)

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Figure 20.24
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Do you consume genetically modified foods?
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Food Properties of the genetically modified variety Modification Percent Modified in US Percent Modified in world
Soybeans Resistant to herbicides Herbicide resistant gene taken from bacteria inserted into soybean 93 77
Corn, Resistant to herbicides and insects. Vitamin-enriched corn New genes, some from the bacterium added/transferred into plant genome. 86 26
Cotton (cottonseed oil) Pest-resistant cotton Bt crystal protein gene added/transferred into plant genome 93 49
Alfalfa Resistant to herbicides New genes added/transferred into plant genome. Planted 2005-07, unbanned 1/2011
Tomatoes enzyme (PG) is suppressed, retarding fruit softening after harvesting. RNAi of PG enzyme added into plant genome Failed commercially in US Small quantities grown in China
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More examples
Food Properties of the genetically modified variety Modification Percent Modified in US Percent Modified in world
Sugar beet Resistance to herbicides New genes added/transferred into plant genome 95 9
Golden Rice contain beta-carotene (a source of vitamin A) contain gene from daffodils and from a bacterium on the market in 2013
Zucchini Resistance to yellow mosaic viruses Contains coat protein genes of viruses. 13
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Is genetically modified food (GM) safe? Why or
why not?
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AP Ch 21

• Genomes and Their Evolution

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• Genomics is the study of whole sets of genes and
  their interactions
• Bioinformatics is the application of
  computational methods to the storage and analysis
  of biological data

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What genomic information distinguishes a human
from a chimpanzee?
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Concept 21.1 New approaches have accelerated the
pace of genome sequencing

• The most ambitious mapping project to date has
  been the sequencing of the human genome
• Officially begun as the Human Genome Project in
  1990, the sequencing was largely completed by
  2003
• The project had three stages
• Genetic (or linkage) mapping
• Physical mapping
• DNA sequencing

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Three-Stage Approach to Genome Sequencing

• Step 1 A linkage map (genetic map) maps the
  location of several thousand genetic markers on
  each chromosome
• Recombination frequencies are used to determine
  the order and relative distances between genetic
  markers

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Three-stage approach to sequencing an entire
genome.
Chromosomebands
Cytogenetic map
Genes locatedby FISH
Linkage mapping
Geneticmarkers
Physical mapping
Overlappingfragments
DNA sequencing
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Whole-Genome Shotgun Approach to Genome Sequencing

• The whole-genome shotgun approach was developed
  by J. Craig Venter in 1992
• This approach skips genetic and physical mapping
  and sequences random DNA fragments directly
• Powerful computer programs are used to order
  fragments into a continuous sequence

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Figure 21.3-1
Cut the DNA intooverlapping frag-ments short
enoughfor sequencing.
Clone the fragmentsin plasmid or phagevectors.
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Figure 21.3-2
Cut the DNA intooverlapping frag-ments short
enoughfor sequencing.
Clone the fragmentsin plasmid or phagevectors.
Sequence eachfragment.
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Figure 21.3-3
Cut the DNA intooverlapping frag-ments short
enoughfor sequencing.
Clone the fragmentsin plasmid or phagevectors.
Sequence eachfragment.
Order thesequences intoone overallsequencewith
computersoftware.
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• Both the three-stage process and the whole-genome
  shotgun approach were used for the Human Genome
  Project and for genome sequencing of other
  organisms
• A complete haploid set of human chromosomes
  consists of 3.2 billion base pairs

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• Technological advances have also facilitated
  metagenomics, in which DNA from a group of
  species (a metagenome) is collected from an
  environmental sample and sequenced

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(No Transcript)
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Concept 21.2 Scientists use bioinformatics to
analyze genomes and their functions

• The Human Genome Project established databases to
  make data available on the Internet
• Bioinformatics resources are provided by
• National Library of Medicine and the National
  Institutes of Health (NIH) created the National
  Center for Biotechnology Information (NCBI)
• European Molecular Biology Laboratory
• DNA Data Bank of Japan
• BGI in Shenzhen, China

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NCBI database can compare DNA, RNA, or even
proteins
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Understanding Genes and Gene Expression at the
Systems Level

• Proteomics is the systematic study of all
  proteins encoded by a genome

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The systems biology approach to protein
interactions
Glutamatebiosynthesis
Serine-relatedbiosynthesis
Mitochondrialfunctions
Translation andribosomal functions
Vesiclefusion
Amino acidpermease pathway
RNA processing
Peroxisomalfunctions
Transcriptionand chromatin-related functions
Metabolismand amino acidbiosynthesis
Nuclear-cytoplasmictransport
Secretionand vesicletransport
Nuclear migrationand proteindegradation
Protein folding,glycosylation, andcell wall
biosynthesis
Mitosis
DNA replicationand repair
Cell polarity andmorphogenesis
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Application of Systems Biology to Medicine

• A systems biology approach has several medical
  applications
• The Cancer Genome Atlas project is currently
  seeking all the common mutations in 13 types of
  cancer by comparing gene sequences and expression
  in cancer versus normal cells

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Silicon and glass chips have been produced that
hold a microarray of most known human genesThe
expression of all/most genes at the same
time.What are the pros/cons of evaluating
someones entire genome?
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Table 21.1
What stands out to you from these data?
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Types of DNA sequences in the human genome
Exons (1.5)
Introns (5)
Regulatorysequences(?20)
RepetitiveDNA thatincludestransposableelements
and relatedsequences(44)
UniquenoncodingDNA (15)
L1sequences(17)
RepetitiveDNA unrelated totransposableelements
(14)
Alu elements(10)
Large-segmentduplications (5?6)
Simple sequenceDNA (3)
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• Intergenic DNA is noncoding and found between
  genes
• Pseudogenes are former genes that have
  accumulated mutations and are nonfunctional
• Repetitive DNA is present in multiple copies in
  the genome
• About three-fourths of repetitive DNA is made up
  of transposable elements (a.k.a. jumping genes)
  and sequences related to them

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Figure 21.8
Barbara McClintock and transposable DNA
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Types of transposable elements

• Eukaryotic transposable elements are of two types
• Transposons, which move by means of a DNA
  intermediate
• Retrotransposons, which move by means of an RNA
  intermediate

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Figure 21.9
New copy oftransposon
Transposon
DNA ofgenome
Transposonis copied
Insertion
Mobile transposon
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Figure 21.10
New copy ofretrotransposon
Retrotransposon
Formation of asingle-strandedRNA intermediate
RNA
Insertion
Reversetranscriptase
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Gene Families
DNA
RNA transcripts
?-Globin
Nontranscribedspacer
?-Globin
Transcription unit
Heme
DNA
?-Globin gene family
?-Globin gene family
18S
5.8S
28S
Chromosome 16
Chromosome 11
rRNA
5.8S
G?
A?
??
?
??
??
?
?
?
?1
?2
28S
Fetusand adult
18S
Embryo
Fetus
Adult
Embryo
(a) Part of the ribosomal RNA gene family
(b) The human ?-globin and ?-globin gene families
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Alterations of Chromosome Structure Humans have
23 pairs of chromosomes, while chimpanzees have
24 pairsHow?
Humanchromosome 2
Chimpanzeechromosomes
Telomeresequences
Centromeresequences

Telomere-likesequences
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Humanchromosome 16
Mousechromosomes
Centromere-likesequences
13
7
8
16
17
(a) Human and chimpanzee chromosomes
(b) Human and mouse chromosomes
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Alterations of Chromosome Structure Humans have
23 pairs of chromosomes, while chimpanzees have
24 pairsHow?
Humanchromosome 2
Chimpanzeechromosomes
Telomeresequences
Centromeresequences
Telomere-likesequences
12
Humanchromosome 16
Mousechromosomes
Centromere-likesequences
13
7
8
16
17
(a) Human and chimpanzee chromosomes
(b) Human and mouse chromosomes
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• The rate of duplications and inversions seems to
  have accelerated about 100 million years ago
• WHY?

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• The rate of duplications and inversions seems to
  have accelerated about 100 million years ago
• This coincides with when large dinosaurs went
  extinct and mammals diversified
• Chromosomal rearrangements are thought to
  contribute to the generation of new species
• We still have recombination hot spots now.
  What types of traits are being selected for now
  intensively?

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Transposable elements can provide sites for
crossover between nonsister chromatids
Nonsisterchromatids
Transposableelement
Gene
Crossoverpoint
Incorrect pairingof two homologsduring meiosis
and
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How do Transposable Elements Contribute to Genome
Evolution?
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How Transposable Elements Contribute to Genome
Evolution

• may facilitate crossing over between different
  chromosomes
• Insertion of transposable elements within a
  protein-coding sequence may block protein
  production
• Insertion of transposable elements within a
  regulatory sequence may increase or decrease
  protein production
• changes are usually detrimental but may on
  occasion prove advantageous to an organism

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Concept 21.6 Comparing genome sequences provides
clues to evolution and development
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Figure 21.16
Bacteria
Most recentcommonancestorof all livingthings
Eukarya
Archaea
4
3
2
0
1
Billions of years ago
Chimpanzee
Human
Mouse
40
0
10
20
30
50
60
70
Millions of years ago
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Comparing Distantly Related Species

• Highly conserved genes have changed very little
  over time
• These help clarify relationships among species
  that diverged from each other long ago

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• Human and chimpanzee genomes differ by 1.2 at
  single base-pairs
• Several genes are evolving faster in humans than
  chimpanzees
• Which do you think these would control?

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• Human and chimpanzee genomes differ by 1.2 at
  single base-pairs
• Several genes are evolving faster in humans than
  chimpanzees
• These include genes involved in defense against
  malaria and tuberculosis and in regulation of
  brain size, and genes that code for transcription
  factors

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Comparing Genomes Within a Species

• As a species, humans have only been around about
  200,000 years and have low within-species genetic
  variation
• Variation within humans is due to single
  nucleotide polymorphisms, inversions, deletions,
  and duplications

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Comparing Developmental Processes

• Evolutionary developmental biology, or evo-devo,
  is the study of the evolution of developmental
  processes in multicellular organisms
• Genomic information shows that minor differences
  in gene sequence or regulation can result in
  striking differences in form

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Widespread Conservation of Developmental Genes
Among Animals

• Molecular analysis of the homeotic genes in
  Drosophila has shown that they all include a
  sequence called a homeobox
• An identical or very similar nucleotide sequence
  has been discovered in the homeotic genes of both
  vertebrates and invertebrates
• Homeotic genes in animals are called Hox genes

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review
Adultfruit fly
Fruit fly embryo(10 hours)
Fly chromosome
Mousechromosomes
Mouse embryo(12 days)
Adult mouse
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Differences in Hox genes influences body
plan.Artemia has coexpression in the thorax
region.
Genital segments
Thorax
Abdomen
Thorax
Abdomen
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Summary Genomes vary in size, number of genes,
and density
Archaea
Eukarya
Bacteria
Most are 10?4,000 Mb, but a few are much larger
Genome size
Most are 1?6 Mb
Number ofgenes
5,000?40,000
1,500?7,500
Genedensity
Lower than in prokaryotes(Within eukaryotes,
lowerdensity is correlated with largergenomes.)
Higher than in eukaryotes
None inprotein-codinggenes
Present insome genes
Introns
Unicellular eukaryotespresent, but prevalent
only insome speciesMulticellular
eukaryotespresent in most genes
OthernoncodingDNA
Can be large amountsgenerally more
repetitivenoncoding DNA inmulticellular
eukaryotes
Very little