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26.1 Mapping the Human Genome
26.2 A Trip Along a Chromosome
26.3 Mutations and Polymorphisms
26.4 Recombinant DNA
26.5 Genomics: Using What We Know
© 2013 Pearson Education, Inc.
Goals
1. What is the working draft of the human genome and the
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circumstances of its creation?
Be able to describe the genome mapping projects and the major
accomplishments of their working drafts.
What are the various segments along the length of the DNA in a
chromosome?
Be able to describe the double helix and base pairing in DNA.
What are mutations?
Be able to define mutations, identify what can cause them, and also identify
their possible results.
What are polymorphisms and single nucleotide polymorphisms
(SNPs) and how can identifying them be useful?
Be able to define polymorphisms and SNPs, and explain the significance of
knowing the locations of SNPs.
What is recombinant DNA?
Be able to define recombinant DNA and explain how it is used for
production of proteins by bacteria.
What does the future hold for uses of genomic information?
Be able to provide an overview of the current and possible future
applications of the human genome map.
© 2013 Pearson Education, Inc.
26.1 Mapping the Human Genome
• A genomic map is a physical representation
of landmarks in a genome and where they
are with respect to one another.
• Mapping the genes on a eukaryotic
chromosome is no easy feat; the nucleotides
that code for proteins (the exons) are
interrupted by noncoding nucleotides (the
introns).
• There is neither spacing between “words” in
the genetic code, nor any “punctuation.”
© 2013 Pearson Education, Inc.
26.1 Mapping the Human Genome
• Two organizations led the effort to map the
human genome: the Human Genome Project (a
collection of 20 groups at not-for-profit institutes
and universities) and Celera Genomics (a
commercial biotechnology company).
• The Human Genome Project created a series of
maps of finer and finer resolution.
• Celera fragmented DNA and then relied on
instrumental and computer-driven techniques to
establish the sequence.
© 2013 Pearson Education, Inc.
26.1 Mapping the Human Genome
• In 2001, 90% of the human genome
sequence had been mapped in 15 months
instead of the originally anticipated four
years.
• By October 2004, 99% of the genome was
sequenced and declared to be 99.999%
accurate.
• The mapped sequence correctly identifies
almost all known genes, allowing researchers
to rely on highly accurate sequence
information.
© 2013 Pearson Education, Inc.
26.1 Mapping the Human Genome
Human Genome Project Strategy
• A genetic map was generated, showing the
physical location of markers, identifiable DNA
sequences known to be inherited.
• The physical map refined the distance between
markers to about 100,000 base pairs.
• To proceed to a map of finer resolution, a
chromosome was cut into large segments and
multiple copies of the segments were produced.
• The overlapping clones, which covered the
entire length of the chromosome, were arranged
in order to produce the next level of map.
© 2013 Pearson Education, Inc.
26.1 Mapping the Human Genome
Human Genome Project Strategy
• In the next step, each clone was cut into
500 base-pair fragments, and identity and
order of bases in each fragment was
determined.
• In the final step, all 500 base-pair
sequences were assembled into a
completed nucleotide map of the
chromosome.
© 2013 Pearson Education, Inc.
26.1 Mapping the Human Genome
Celera Genomics Project Strategy
• In what has come to be known as the shotgun
approach, Celera broke the human genome into
fragments without identifying the origin of any given
fragment.
• The fragments were copied many times to generate
many clones of each area of the genome; ultimately
they were cut into 500-base long pieces and modified
with fluorescently labeled bases that could be
sequenced by high-speed machines.
• The sequences were reassembled by identifying
overlapping ends. This monumental task was carried
out using the world’s largest nongovernmental
supercomputing center.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
• At both ends of every chromosome are
specialized regions of DNA called telomeres.
• Each telomere is a long, noncoding series of a
repeating sequence of nucleotides.
• Telomeres act as “endcaps,” or “covers,”
protecting the ends of the chromosome from
accidental changes that might alter the more
important DNA coding sequences.
• Telomeres also prevent the DNA ends from
fusing to the DNA in other chromosomes or to
DNA fragments.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
• As the DNA in each chromosome is
duplicated in preparation for cell division,
the two copies remain joined together at a
constricted point in the middle of the
chromosome.
• This is the centromere.
• The duplicated chromosomes bound
together at the centromere are known as
sister chromatids.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
• As the DNA in each
chromosome is duplicated in
preparation for cell division,
the two copies remain joined
together at a constricted point
in the middle of the
chromosome.
• This is the centromere.
• The duplicated chromosomes
bound together at the
centromere are known as
sister chromatids.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
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One Genome To Represent Us All?
Using the DNA of a single individual to represent the entire
human genome is a bad idea.
To avoid this, the path chosen by both genome mapping
groups was to employ DNA from a group of anonymous
individuals.
In the Human Genome Project, researchers collected blood
(female) or sperm (male) samples from a large number of
donors of diverse backgrounds.
The Celera project relied on anonymous donors of European,
African, American (North, Central, South), and Asian ancestry.
As a result, one of the most frequently asked questions about
the human genome, “Whose DNA was sequenced?” can never
truly be answered.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
• Each new cell starts life with a long stretch of
telomeric DNA.
• Some of this repeating sequence is lost with each
cell division, so that as the cell ages, the
telomere gets shorter and shorter.
• A very short telomere is associated with
senescence.
• Continuation of shortening beyond this stage is
associated with DNA instability and cell death.
• Telomerase adds telomeres to DNA. It is active
during embryonic development. In adults,
telomerase is only active in germ cells.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
• There is widespread speculation that telomere shortening
plays a role in aging.
• Some support for this concept comes from experiments
with mice whose telomerase activity has been “knocked
out” (in genetic research vernacular).
• These mice age prematurely, and if they become
pregnant, their embryos do not survive.
• The majority of cancer cells are known to contain active
telomerase, which is thought to confer immortality on
these tumor cells.
• Current research suggests that it is the genes responsible
for regulating telomerase expression that are altered in
cancer cells. There are ongoing experiments on the
consequences of telomerase inactivation on cancer cells.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
Noncoding DNA
• There are noncoding promoter sequences, which are
regulatory regions of DNA that determine which of its
genes are turned on.
• Only the genes needed by any individual cell will be
activated in that cell.
• Current data suggests that only about 2% of all DNA in
the human genome actually codes for protein.
• The human genome has much more noncoding DNA than
do the genomes known for other organisms.
• The function of noncoding DNA remains to be discovered,
and the debate over its role continues.
© 2013 Pearson Education, Inc.
26.2 A Trip Along a Chromosome
Genes
• The nucleotides of a single gene are not consecutive along a stretch
of DNA, having coding segments (exons) that alternate with
noncoding segments (introns).
• Chromosome 22 was the first to have all of its nonrepetitive DNA
sequenced and mapped.
• The chromosome map identified 49 million bases containing 693
genes, with an average of 8 exons and 7 introns per gene.
• Chromosome 22 carries genes known to be associated with the
immune system as well as congenital heart disease, schizophrenia,
leukemia, cancers, and many other genetically-related conditions.
• The map also revealed several hundred previously unknown genes.
• With the signal (exon) to noise (intron) ratio being so low (meaning
more noise to hide the signal) in the human genome, it will be
challenging to completely identify all the coding sequences present.
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
• An error in base sequence that is carried along
during DNA replication is called a mutation.
• Mutation commonly refers to variations in DNA
sequence found in a very small number of
individuals of a species.
• Some mutations result from spontaneous and
random events.
• Others are induced by exposure to a mutagen—an
external agent that can cause a mutation.
• Viruses, chemicals, and ionizing radiation can all be
mutagenic.
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
• Polymorphisms are variations in the nucleotide
sequence of DNA that are common within a
given population.
• Most polymorphisms are simply differences in
the DNA sequence between individuals due to
geographical and ethnic differences and are part
of the biodiversity exhibited by life on earth.
• The vast majority of polymorphisms seen have
neither advantageous nor deleterious effects,
some have been shown to give rise to various
disease states.
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
FIGURE 26.2 A human chromosome map
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26.3 Mutations and Polymorphisms
• The replacement of one nucleotide by another in
the same location along the DNA sequence is a
single-nucleotide polymorphism.
• The biological effects of SNPs range from
negligible to normal variations such as those in
eye or hair color, to genetic diseases.
• In addition to producing a change in the identity
of an amino acid, a SNP might specify the same
amino acid (for example, changing GUU to
GUC, both of which code for valine), or it might
terminate protein synthesis by introducing a stop
codon.
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
• An international team of scientists is compiling a
catalog of SNPs.
• As of 2010, over 5 million SNPs had been
recorded. Their frequency is roughly one SNP
for about every 2000–5000 bases, with many of
them in coding regions.
• The SNP catalog has been used to locate SNPs
responsible for total color blindness, one type of
epilepsy, and susceptibility to breast cancer.
• It is hoped that this information will inspire the
development of new treatments for diseases.
© 2013 Pearson Education, Inc.
26.3 Mutations and Polymorphisms
• The cataloging of SNPs has ushered in
the era of genetic medicine.
• The SNP catalog may allow physicians to
predict for an individual the potential age
at which inherited diseases will become
active, their severity, and their reactions to
various types of treatment.
• The therapeutic course will be designed to
meet the distinctive genomic profile of the
person.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
• Using recombinant DNA technology, it is
possible to cut a gene out of one organism
and splice it into (recombine it with) the
DNA of a second organism.
• Bacteria provide excellent hosts for
recombinant DNA.
• Bacterial cells contain part of their DNA in
small circular pieces called plasmids, each
of which carries just a few genes.
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26.4 Recombinant DNA
• The ease of isolating and manipulating plasmids
plus the rapid replication of bacteria create ideal
conditions for production of recombinant DNA
and the proteins whose synthesis it directs.
• The plasmid is cut open with a restriction
endonuclease or restriction enzyme, that
recognizes a specific sequence.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
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Serendipity and the Polymerase Chain Reaction
Kary Mullis figured out that combining DNA polymerase, target DNA,
nucleoside triphosphates, and short synthetic nucleotide chains
(oligonucleotides) in just the right way would massively amplify the
target.
The polymerase chain reaction (PCR) is now carried out automatically
by instruments in every molecular biology lab. In 1993, Mullis shared
the Nobel Prize in chemistry for this work.
The reaction is carried out in three steps: heating the sample to cause
the helix to unravel into single strands, adding primers complementary
to the target, and extending the primers with DNA polymerase.
The reactants are combined in a closed container and the temperature
cycled from about 90 °C for Step 1, to about 50 °C for Step 2, and to
about 70 °C for Step 3. The temperature cycle requires only a few
minutes and can be repeated over and over again for the same mixture.
Automation of the PCR was made possible by the discovery of a heatstable polymerase (Taq polymerase) isolated from a bacterium that
lives in hot springs.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
• Recombinant DNA is produced by cutting the
two DNA segments to be combined with the
same restriction endonuclease. The result is
DNA fragments with complementary sticky ends.
• The two are mixed in the presence of a DNA
ligase enzyme that joins them together by reforming their phosphodiester bonds,
reconstituting the now-altered plasmid.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
• The altered plasmid is inserted back into a
bacterial cell where the normal processes of
transcription and translation synthesize the
protein encoded by the inserted gene.
• Bacteria multiply rapidly; there are soon a
large number of them, all containing the
recombinant DNA and all manufacturing the
protein encoded by the recombinant DNA.
• Huge numbers of the bacteria can be put to
work as a protein factory.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
• One hurdle is getting the recombinant
plasmid back into a bacterium.
• Host organisms may modify the protein:
yeast cells attach carbohydrates to various
amino acids.
• The protein of interest must be isolated
from endotoxins—potentially toxic natural
compounds found inside the host
organism.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
• Even small amounts of endotoxins can lead to
serious inflammatory responses, so rigorous
purification and screening protocols are
necessary.
• Proteins manufactured in this manner have
already reached the marketplace, including
human insulin, human growth hormone, and
blood clotting factors for hemophiliacs.
• A major advantage of this technology is that
large amounts of these proteins can be made,
thus allowing their practical therapeutic use.
© 2013 Pearson Education, Inc.
26.4 Recombinant DNA
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DNA Fingerprinting
DNA fingerprinting relies on variations between two or more DNA samples.
The repetitive patterns used in DNA fingerprinting are variable number
tandem repeats (VNTRs), short DNA sequences that are repeated multiple
times. For any given VNTR, the number of copies of the repeated sequence
varies between individuals.
The probability of a match with someone other than the correct individual is
estimated at 1 in 1.5 billion.
There are two common techniques used for DNA fingerprinting today: the
restriction fragment length polymorphism (RFLP) approach and the
polymerase chain reaction (PCR) method.
RFLP relies on use of a restriction endonuclease (an enzyme used to cut
DNA) that recognizes and cuts sequences on either side of a given VNTR.
With PCR, primers are directed towards regions of the DNA that are known
to contain variations. These are amplied about 30 times (at 4 minutes per
cycle) so that in two hours more than a billion copies are produced. These
fragments can then be separated according to size, stained, and compared
against other samples.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
• Genomics is the study of whole sets of genes and their
functions.
• The study of bacterial genomics has been instrumental
in linking the three domains of life—Archaea (formerly
archeabacteria), Bacteria, and Eukarya—to one another
from an evolutionary standpoint.
• The study of bacterial genomics is giving us a better
understanding of how bacteria cause disease, it is also
helping in the development of new therapies.
• Plant genomics is enhancing the value and utility of
agricultural crops.
• The genomic study of farm animals is improving their
health and availability.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
Genetically Modified Plants and Animals
• The mapping and study of plant and animal genomes
can greatly accelerate our ability to generate crop plants
and farm animals with desirable characteristics and
lacking undesirable ones.
• Some genetically modified crops are planted in large
quantities in the United States.
– Each year millions of tons of corn are destroyed by the European
corn borer. To solve this problem, a bacterial gene (from Bacillus
thuringiensis, Bt) has been transplanted into corn. The gene
causes the corn to produce a toxin that kills the caterpillars.
– Soybeans genetically modified to withstand herbicides are also
widely grown. The soybean crop remains unharmed when the
surrounding weeds are killed by the herbicide.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
Genetically Modified Plants and Animals
• Tests are under way with genetically modified coffee
beans that are caffeine-free, potatoes that absorb less
fat when they are fried, and “Golden Rice,” a yellow rice
that provides the vitamin A desperately needed in poor
populations where insufficient vitamin A causes death
and blindness.
• Fish farming is an expanding industry as natural
populations of fish diminish. There are genetically
engineered salmon that can grow to marketable size in
one-half the time of their unmodified cousins.
• Similar genetic modifications are anticipated for other
varieties of fish, and there is the prospect of cloning
leaner pigs.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
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Genetically Modified Plants and Animals
Will genetically modified plants and animals
intermingle with natural varieties and cause
harm to them?
Should food labels state whether the food
contains genetically modified ingredients?
Might unrecognized harmful substances enter
the food supply?
These questions and have led to the
establishment of the Non-GMO Project, the goal
of which is to offer consumers a non-GMO
choice for organic and natural products.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
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Gene Therapy
Gene therapy is based on the premise that a
disease-causing gene can be corrected or
replaced by inserting a functional, healthy gene.
The most clear-cut expectations for gene
therapy lie in treating monogenic diseases.
The focus has been on using viruses as vectors,
the agents that deliver therapeutic quantities of
DNA directly into cell nuclei.
The Food and Drug Administration (FDA) has,
as of July 2011, not yet approved any human
gene therapy product for sale.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
A Personal Genomic Survey
• If a patient lacks an enzyme needed for a drug’s
metabolism or has a monogenic defect, therapies
could be individually tailored.
• In cancer therapy, understanding the genetic
differences between normal cells and tumor cells
could assist in chemotherapy.
• Genetic screening of infants might permit the use of
gene therapy to eliminate the threat of a
monogenically-based disease, or a lifestyle adjustment
for an individual with SNPs that predict a susceptibility
to a disease that results from combinations of genetic
and environmental influences.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
Snips and Chips
• Our understanding of SNPs is already at work in
screening implemented by DNA chips. Different
individuals may have no effect from a drug, the expected
effect, or a greater-than-normal response to the drug.
• Genomic screening can determine whether particular
polymorphisms are linked to a patient’s ability to respond
to the medication.
• Once such connections have been established,
screening could be a diagnostic test carried out by a
DNA chip in a doctor’s office.
• DNA chip screening has already revealed genetic
variations responsible for two types of pediatric leukemia
which require quite different therapies.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
Snips and Chips
• A DNA chip is a solid support bearing large numbers of
short, single-stranded bits of DNA of known composition.
• The DNA is organized for a particular type of screening.
• A sample is labeled with a fluorescent tag and applied to
the chip.
• During an incubation period, sample DNA and chip DNA
with complementary nucleic acid sequences will bond to
each other.
• After excess sample DNA is washed away, the
fluorescence remaining on the chip is read to discover
where the bonding has occurred and thus, what DNA
variations are present in the sample DNA.
© 2013 Pearson Education, Inc.
26.5 Genomics: Using What We Know
Bioethics
• The ELSI program of the National Human Genome
Research Institute deals with the Ethical, Legal, and
Social Implications of human genetic research such as:
– Who should have access to personal genetic information and
how will it be used?
– Who should own and control genetic information?
– Should genetic testing be performed when no treatment is
available?
– Are disabilities diseases? Do they need to be cured or
prevented?
– Preliminary attempts at gene therapy are exorbitantly
expensive. Who will have access to these therapies? Who will
pay for their use?
– Should we re-engineer the genes we pass on to our children?
© 2013 Pearson Education, Inc.
Chapter Summary
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What is the working draft of the human genome and the
circumstances of its creation?
The Human Genome Project, an international consortium of not-for-profit
institutions, and Celera Genomics, a for-profit company, have both
announced completion of working drafts of the human genome. With the
exception of large areas of repetitive DNA, the DNA base sequences of
all chromosomes have been examined.
The Human Genome Project utilized a series of progressively more
detailed maps to create a collection of DNA fragments with known
location. Celera began by randomly fragmenting all of the DNA without
first placing it within the framework of a map. In both groups the
fragments were cloned, labeled, ordered, and the individual sequences
assembled by computers.
The results of the two projects are generally supportive of each other.
There are about three billion base pairs and 20,000–25,000 genes in the
human genome, each able to direct the synthesis of more than one
protein. The bulk of the genome consists of noncoding, repetitive
sequences. About 200 of the human genes are identical to those in
bacteria.
© 2013 Pearson Education, Inc.
Chapter Summary, Continued
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What are the various segments along the length of the
DNA in a chromosome?
Telomeres, which fall at the ends of chromosomes, are regions
of noncoding, repetitive DNA that protect the ends from
accidental changes. At each cell division, the telomeres are
shortened, with significant shortening associated with
senescence and death of the cell.
Telomerase, the enzyme that lengthens telomeres, is typically
inactivated in adult cells, but becomes reactivated in cancer
cells.
Centromeres are the constricted regions of chromosomes that
form during cell division and also carry noncoding DNA.
Exons are the protein coding regions of DNA and together
make up the genes that direct protein synthesis. The repetitive
noncoding segments of DNA are of either no function or
unknown function.
© 2013 Pearson Education, Inc.
Chapter Summary, Continued
3. What are mutations?
• A mutation is an error in the base
sequence of DNA that is passed along
during replication.
• Mutations arise by random error during
replication but may also be caused by
ionizing radiation, viruses, or chemical
agents (mutagens).
• Mutations can cause inherited diseases
and the tendency to acquire others.
© 2013 Pearson Education, Inc.
Chapter Summary, Continued
4. What are polymorphisms and single nucleotide
polymorphisms (SNPs) and how can identifying
them be useful?
• A polymorphism is a variation in DNA that is found
within a population. An SNP is the replacement of one
nucleotide by another.
• The result might be the replacement of one amino acid
by another in a protein, no change because the new
codon specifies the same amino acid, or the
introduction of a stop codon.
• Many inherited diseases are known to be caused by
SNPs, but they can also be beneficial or “neutral”.
• Understanding the location and effect of SNPs is
expected to lead to new therapies.
© 2013 Pearson Education, Inc.
Chapter Summary, Continued
5. What is recombinant DNA?
• Recombinant DNA is produced by joining DNA
segments that do not normally occur together.
• A gene from one organism is inserted into the
DNA of another organism. Recombinant DNA
techniques can be used to create large
quantities of a particular protein. The gene of
interest is inserted into bacterial plasmids
(small, extrachromosomal circular DNA).
• Bacteria carrying these plasmids then serve as
factories for the synthesis of large quantities of
the encoded protein.
© 2013 Pearson Education, Inc.
Chapter Summary, Continued
6. What does the future hold for uses of genomic
information?
• Mapping the human genome holds major promise for
applications in health and medicine.
• Drugs can be precisely chosen based on a patient’s
own DNA, thereby avoiding drugs that are ineffective
or toxic for that individual. Perhaps one day inherited
diseases will be prevented or cured by gene therapy.
• By genetic modification of crop plants and farm
animals, the productivity, marketability, and health
benefits of these products can be enhanced.
• Progress in each of these areas is bound to be
accompanied by controversy and ethical dilemmas.
© 2013 Pearson Education, Inc.