Download Presentation

Document related concepts

Primary transcript wikipedia , lookup

DNA vaccination wikipedia , lookup

Frameshift mutation wikipedia , lookup

Human genetic variation wikipedia , lookup

Minimal genome wikipedia , lookup

Gene therapy wikipedia , lookup

Cre-Lox recombination wikipedia , lookup

Genomic library wikipedia , lookup

Extrachromosomal DNA wikipedia , lookup

Gene wikipedia , lookup

Mutagen wikipedia , lookup

Mutation wikipedia , lookup

RNA-Seq wikipedia , lookup

No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup

Cell-free fetal DNA wikipedia , lookup

Human genome wikipedia , lookup

Non-coding DNA wikipedia , lookup

Cancer epigenetics wikipedia , lookup

Genomics wikipedia , lookup

Epigenetics of neurodegenerative diseases wikipedia , lookup

Genome evolution wikipedia , lookup

Therapeutic gene modulation wikipedia , lookup

Genetic engineering wikipedia , lookup

Nutriepigenomics wikipedia , lookup

Helitron (biology) wikipedia , lookup

Polycomb Group Proteins and Cancer wikipedia , lookup

Site-specific recombinase technology wikipedia , lookup

Vectors in gene therapy wikipedia , lookup

Point mutation wikipedia , lookup

Public health genomics wikipedia , lookup

Genome editing wikipedia , lookup

Artificial gene synthesis wikipedia , lookup

Designer baby wikipedia , lookup

NEDD9 wikipedia , lookup

Microevolution wikipedia , lookup

Oncogenomics wikipedia , lookup

History of genetic engineering wikipedia , lookup

Genome (book) wikipedia , lookup

Transcript
17
Genome Sequencing, Molecular
Biology, and Medicine
17 Genome Sequencing, Molecular Biology, and Medicine
• 17.1 How Do Defective Proteins Lead to
Diseases?
• 17.2 What Kinds of DNA Changes Lead to
Diseases?
• 17.3 How Does Genetic Screening Detect
Diseases?
• 17.4 What Is Cancer?
• 17.5 How Are Genetic Diseases Treated?
• 17.6 What Have We Learned from the Human
Genome Project?
17.1 How Do Defective Proteins Lead to Diseases?
Genetic mutations are often expressed
as proteins that differ from wild-type.
Genetic diseases can result from
abnormalities in enzymes, receptor
proteins, transport proteins, structural
proteins, etc.
17.1 How Do Defective Proteins Lead to Diseases?
Phenylketonuria (PKU) was traced to its
molecular phenotype in the 1950s.
Results from an abnormal enzyme
phenylalanine hydroxylase—normally
catalyzes conversion of dietary
phenylalanine to tyrosine.
The abnormal enzyme has tryptophan
instead of arginine in position 408.
Figure 17.1 One Gene, One Enzyme
17.1 How Do Defective Proteins Lead to Diseases?
People with PKU have light skin and hair
color.
Melanin—pigment in dark skin and hair,
is made from tyrosine, which people
with PKU can not synthesize.
17.1 How Do Defective Proteins Lead to Diseases?
Polymorphism in proteins does not
always mean disease.
There can be numerous normal alleles of
a gene which produce normally
functioning proteins.
17.1 How Do Defective Proteins Lead to Diseases?
The first human disease known to be
caused by an abnormal protein was
sickle-cell disease.
The abnormal allele produces abnormal
hemoglobin that results in sickle-shaped
blood cells.
The sickle-shaped cells block blood flow
in capillaries.
17.1 How Do Defective Proteins Lead to Diseases?
Hemoglobin—protein with quaternary
structure; 2 α and 2 β chains.
In sickle-cell disease, one of 146 amino
acids in the β-globin chain is different:
glutamic acid (negatively charged) is
replaced by valine (neutral).
Changes shape of the hemoglobin and
causes anemia.
17.1How Do Defective Proteins Lead to Diseases?
Variation in hemoglobin has been well
documented.
There are many amino acid substitutions;
many have no effect on the protein
function.
Figure 17.2 Hemoglobin Polymorphism
17.1 How Do Defective Proteins Lead to Diseases?
Some diseases result from altered
membrane receptors or transport
proteins.
Familial hypercholesterolemia (FH)—
excess cholesterol can accumulate on
artery walls and block them, causing
heart attacks and strokes.
17.1 How Do Defective Proteins Lead to Diseases?
People with FH are unable to transport
cholesterol to the liver and other cells
that use it.
Cholesterol travels as a lipoprotein (LDL).
LDL binds to a receptor on a liver cell,
and is taken up by endocytosis.
In FH, the receptor protein is
nonfunctional.
Figure 17.3 Genetic Diseases of Membrane Proteins (A)
17.1How Do Defective Proteins Lead to Diseases?
In cystic fibrosis, thick dry mucous lines
surfaces such as the respiratory tract
and prevents passage of air, and
prevents cilia from functioning.
This is caused by a nonfunctional
chloride transporter protein. Normally,
this ion channel releases Cl– outside the
cell. Water leaves cell by osmosis,
providing moist surfaces.
Figure 17.3 Genetic Diseases of Membrane Proteins (B)
17.1 How Do Defective Proteins Lead to Diseases?
Duchenne muscular dystrophy and
hemophilia are diseases caused by
altered structural proteins.
In Duchenne muscular dystrophy, there is
no functional dystrophin, which normally
connects actin fibers of muscle cells to
the extracellular matrix. Without it,
muscle cells are structurally
disorganized, and stop working.
17.1 How Do Defective Proteins Lead to Diseases?
Hemophilia is caused by the absence of
a blood clotting protein.
Normally, clotting proteins are always
present in the blood.
17.1 How Do Defective Proteins Lead to Diseases?
Transmissible spongiform
encephalopathies (TSEs) are
degenerative diseases in which holes
develop in the brain—includes mad cow
disease.
Results from errors in conformation of
proteins.
Figure 17.4 Mad Cow Disease in Britain
17.1How Do Defective Proteins Lead to Diseases?
TSEs can be transferred by eating
animals that had the disease.
Kuru—a TSE is found in a tribe in New
Guinea that practiced ritual cannibalism.
The infectious agent is a prion—
proteinaceous infective particle.
17.1 How Do Defective Proteins Lead to Diseases?
Normal brain cell membranes have a
protein called PrPc.
In TSE infected tissue, the protein has a
different shape, called PrPsc. This
protein piles up as fibers and causes
cell death.
The abnormal PrPsc causes the normal
protein to change conformation.
Figure 17.5 Prion Diseases are Disorders of Protein Conformation
17.1 How Do Defective Proteins Lead to Diseases?
Most human diseases are multifactorial—
caused by interactions of many genes and
proteins and the environment.
Alleles that cause genetic diseases may be
inherited in a dominant or recessive pattern,
and may be carried on autosomes or sex
chromosomes.
Some diseases result from extensive
chromosomal abnormalities.
17.1 How Do Defective Proteins Lead to Diseases?
PKU, sickle-cell disease, and cystic
fibrosis are autosomal recessive.
If both parents are carriers (heterozygotes
with normal phenotypes), every time a
child is conceived there is a one in four
chance that it will have the disease.
17.1 How Do Defective Proteins Lead to Diseases?
Familial hypercholesterolemia is caused
by an autosomal dominant allele.
Presence of only one mutant allele is
enough to cause the disease.
17.1 How Do Defective Proteins Lead to Diseases?
Hemophilia is X-linked recessive.
A son that inherits the allele from the
mother will have the disease, because
there is no allele on the Y chromosome.
All rare X-linked diseases are much more
common in men than women.
17.1 How Do Defective Proteins Lead to Diseases?
Chromosomal abnormalities include the
gain or loss of chromosomes
(aneuploidy), deletions, and
translocations.
Some are inherited, some result from
meiotic events.
Fragile-X syndrome is a constriction at
the tip of the X chromosome. Causes
mental retardation in some people.
Figure 17.6 A Fragile-X Chromosome at Metaphase
17.2 What Kinds of DNA Changes Lead to Diseases?
Some disease-causing mutations are
determined when the abnormal protein
phenotype is known, the gene can be
cloned.
In other cases, the defective protein is
unknown until the gene is isolated.
17.2 What Kinds of DNA Changes Lead to Diseases?
For sickle-cell disease, mRNA was
isolated from immature red blood cells,
a cDNA copy was made and used to
probe a human gene library.
Then gene sequencing was used to
compare normal and sickle-cell genes.
17.2 What Kinds of DNA Changes Lead to Diseases?
Duchenne muscular dystrophy was
thought to be X-linked, but the abnormal
protein nor the gene could be identified.
A small chromosome deletion was
discovered in the X chromosome.
Comparing with normal chromosomes
allowed the gene to be isolated.
Figure 17.7 Two Strategies for Isolating Human Genes
17.2 What Kinds of DNA Changes Lead to Diseases?
When no abnormal protein or
chromosome deletion can be identified,
positional cloning is used.
Genetic markers can be positioned
anywhere on the DNA. They must be
polymorphic (more than one allele).
17.2 What Kinds of DNA Changes Lead to Diseases?
RFLPs (Restriction Fragment Length
Polymorphisms)
If there is a mutation in a restriction site,
it will not be cut by a restriction enzyme,
resulting in a larger fragment. These
can be seen in gel electrophoresis.
An RFLP band pattern is inherited in
Mendelian fashion.
Figure 17.8 RFLP Mapping (Part 1)
Figure 17.8 RFLP Mapping (Part 2)
17.2 What Kinds of DNA Changes Lead to Diseases?
SNPs (single nucleotide polymorphisms)
are widespread in eukaryotic genomes.
SNPs can be detected by direct
sequence comparisons or chemical
methods such as mass spectrometry.
17.2 What Kinds of DNA Changes Lead to Diseases?
Genetic markers such as RFLPs and
SNPs can be used to find genes if the
genes are polymorphic too.
The gene and the marker must always be
inherited together—pedigrees are
constructed to determine this.
17.2 What Kinds of DNA Changes Lead to Diseases?
To isolate a gene, the neighborhood
around an RFLP might be screened
with other restriction enzymes.
When a relatively short sequence of DNA
is identified as a candidate for the gene,
it can be cut in fragments and tested
with probes made from mRNA from
affected cells.
17.2 What Kinds of DNA Changes Lead to Diseases?
DNA sequencing has shown that
mutations occur most often in certain
base pairs—“hot spots” for mutation.
Often where cytosine has been
methylated to 5-methylcytosine
Unmethylated cytosine can lose its amino
group to form uracil—this error is
detected and repaired.
17.2 What Kinds of DNA Changes Lead to Diseases?
When 5-methylcytosine loses its amino
group, it forms thymine, which is
ignored by DNA repair mechanism.
Mismatch repair recognizes the mistake,
GT instead of GC, but cannot tell which
member of the pair was incorrect.
Figure 17.9 5-Methylcytosine in DNA Is a “Hot Spot” for Mutations (Part 1)
Figure 17.9 5-Methylcytosine in DNA Is a “Hot Spot” for Mutations (Part 2)
17.2 What Kinds of DNA Changes Lead to Diseases?
Larger mutations can involve many base
pairs.
In Duchenne muscular dystrophy, the
deletion may be small, covering only
part of the gene for dystrophin, or the
entire gene may be deleted.
Other mutations involve millions of base
pairs.
17.2 What Kinds of DNA Changes Lead to Diseases?
About 1/5 of males and their daughters
with fragile X chromosome are
phenotypically normal, but their sons
are mentally retarded.
Later generations tend to show earlier
onset and more severe symptoms.
17.2 What Kinds of DNA Changes Lead to Diseases?
The gene for fragile-X, FMR1, contains a
repeated triplet (CGG) in the promoter
region.
In normal people it is repeated six to 54
times.
In mentally retarded people with fragileX, it is repeated 200 to 2,000 times.
17.2 What Kinds of DNA Changes Lead to Diseases?
Males with a moderate number of
repeats (55–200) have no symptoms
and are said to be premutated.
The repeats become more numerous in
successive generations.
With more than 200 repeats, increased
methylation of cytosine results in
transcriptional inactivation of FMR1.
17.2 What Kinds of DNA Changes Lead to Diseases?
Normal function of protein made by
FMR1 is to bind to mRNAs involved in
neuron function and regulate
translation.
If the mRNAs are not translated in
sufficient amounts, the nerve cells die.
Figure 17.10 The CGG Repeats in the FMR1 Gene Expand with Each Generation
17.2 What Kinds of DNA Changes Lead to Diseases?
Expanding triplet repeats has been
found in other diseases—myotonic
dystrophy, Huntington’s disease.
How the repeats expand is unknown;
possibly DNA polymerase slips after
copying the triplet, and copies it again.
17.2 What Kinds of DNA Changes Lead to Diseases?
Groups of genes differ in their phenotypic
effect depending on which parent they
came from—genomic imprinting.
Raises the possibility that male and
female genomes are not functionally
equivalent.
17.2 What Kinds of DNA Changes Lead to Diseases?
A small deletion on mother’s chromosome
15 results in Angleman syndrome—thin
child with prominent jaw and wide mouth.
Same deletion on father’s chromosome 15
results in a short, obese child with small
hands and feet—Prader-Willi syndrome.
Both are heterozygotes—one deleted and
one “normal” allele.
17.3 How Does Genetic Screening Detect Diseases?
Genetic screening: using tests to
determine if an individual has a genetic
disease, or is predisposed to one, or is
a carrier.
• Prenatal screening
• Screening of newborns
• Screening asymptomatic people with
relatives who have genetic diseases.
17.3 How Does Genetic Screening Detect Diseases?
Enzymes can be checked for low
activity—suggests mutation.
Phenylketonuria can be detected in
newborns—treatment can begin
immediately.
Before birth, excess phenylalanine
diffuses across placenta to mother’s
blood; she has adequate phenylalanine
hydrolase.
17.3 How Does Genetic Screening Detect Diseases?
Screening method uses auxotrophic
bacteria that require phenylalanine to
grow. If bacteria grow in presence of
baby’s blood—there is too much
phenylalanine in it.
Now being replaced by direct chemical
tests.
Mandatory screening of newborns is now
done for 25 diseases.
Figure 17.11 Genetic Screening of Newborns for Phenylketonuria
17.3 How Does Genetic Screening Detect Diseases?
DNA testing is the most direct and
accurate way to detect abnormal alleles.
Any cell can be scanned at any time for
mutations.
Works best for diseases caused by only
one or a few mutations.
17.3 How Does Genetic Screening Detect Diseases?
Fetal cells can be screened
preimplantation (rare), or after
implantation.
Fetal cells can be analyzed at 10 weeks
by chorionic villus sampling, or by
amniocentesis during the 13th to 17th
weeks.
17.3 How Does Genetic Screening Detect Diseases?
DNA testing of adults is also used to
screen for heterozygotes.
For example, a sister of a boy with
Duchenne muscular dystrophy can be
screened to determine whether she is a
carrier.
17.3 How Does Genetic Screening Detect Diseases?
Two main methods for DNA testing:
Allele specific cleavage method: normal
and mutant alleles have different
restriction recognition sequences.
A restriction enzyme may cut a normal
allele, but not the mutant allele,
resulting in a larger DNA fragment.
Figure 17.12 DNA Testing by Allele-Specific Cleavage
17.3 How Does Genetic Screening Detect Diseases?
Allele-specific oligonucleotide hybridization
uses short artificial DNA strands, or
oligonucleotides, that will hybridize with
either the normal or mutant allele.
The oligonucleotide probe can be labeled
with radioisotopes or florescent dyes.
Easier and faster than allele-specific
cleavage.
Figure 17.13 DNA Testing by Allele-Specific Oligonucleotide Hybridization (Part 1)
Figure 17.13 DNA Testing by Allele-Specific Oligonucleotide Hybridization (Part 2)
17.4 What Is Cancer?
Cancer is caused primarily by genetic
changes—mostly by mutations of DNA
in the somatic cells.
Cancer cells differ from normal cells in
two main ways:
• Cancer cells lose control over cell
division.
• Cancer cells can invade other tissues.
17.4 What Is Cancer?
Most cells divide only when exposed to
external factors such as hormones or
growth factors.
Cancer cells divide continuously, forming
tumors (large masses of cells).
Benign tumors resemble the tissue they
start from, grow slowly, and remain
localized (e.g., a lipoma is a tumor of fat
cells, but not a cancer).
17.4 What Is Cancer?
Malignant tumors do not resemble
parent tissue and often have irregular
structures.
Many malignant cells express the gene
for telomerase, and do not shorten the
ends of chromosomes after each DNA
replication.
Figure 17.14 A Cancer Cell with Its Normal Neighbors
17.4 What Is Cancer?
Cancer cells can invade other tissues—
called metastasis.
Occurs in stages:
• First extends into surrounding tissue by
secreting digestive enzymes.
• Then some cells enter the blood stream
or lymphatic system. Only a few cells
survive this.
17.4 What Is Cancer?
• If a cancer cell finds new suitable tissue,
it expresses cell surface proteins to bind
to and invade the new tissue.
• Cancer cells at a new site secrete
chemical signals that cause blood
vessels to grow to the tumor to supply it
with nutrients—angiogenesis.
17.4 What Is Cancer?
Different forms of cancer affect different
parts of the body:
• Carcinomas arise in surface tissues, skin
and linings of organs. Lung cancer, colon
cancer, breast cancer, liver cancer.
• Sarcomas occur in blood, bone, and
muscle.
• Leukemias and lymphomas affect cells
that give rise to blood cells.
17.4 What Is Cancer?
About 15 percent of human cancers are
virally induced.
Hepatitis B virus is associated with liver
cancer, but some gene mutations may
also be necessary for tumor formation.
Table 17. 1
17.4 What Is Cancer?
Papillomaviruses seem to act on their
own, not needing any gene mutations.
Occasionally the circular chromosome is
broken and the virus genome inserts
itself into a cell in the uterine cervix.
It disrupts a gene that normally blocks
cell division, and a tumor results.
17.4 What Is Cancer?
85 percent of cancers are not caused by
viruses.
Most cancers develop in older people—
one must live long enough for genetic
mutations to occur.
17.4 What Is Cancer?
DNA can be damaged in many ways.
Some mutations are spontaneous.
Mutagens called carcinogens can cause
mutations that lead to cancer.
Carcinogens include chemicals in
tobacco smoke, UV radiation, and
radiation from radioisotopes.
17.4 What Is Cancer?
Thousands of chemicals that occur
naturally in food are also carcinogens.
Cells that divide often, such as epithelial
cells and bone marrow stem cells, are
especially susceptible to cancer
because there is not as much time for
DNA repair in between cell cycles.
17.4 What Is Cancer?
Changes in the control of cell division lie
at the heart of cancer.
In the human genome, some genes act
to stimulate cell division—oncogenes;
others act to suppress cell division—
tumor suppressor genes.
17.4 What Is Cancer?
Oncogenes are normally turned off.
Products of oncogenes are involved in
pathways by which growth factors
stimulate division.
Some control apoptosis. Activation of
these genes by mutation prevents
apoptosis.
Figure 17.15 Oncogene Products Stimulate Cell Division
17.4 What Is Cancer?
About 10 percent of cancers are
inherited.
Noninherited cancers are usually a form
that occur later in life—sporadic form.
Inherited cancers show up earlier in life,
and as multiple tumors.
A tumor suppressor gene that normally
acts as a brake must be inactivated.
17.4 What Is Cancer?
Full inactivation requires two mutations—
both alleles must be turned off.
People with inherited cancer are born
with one mutant allele, and need only
one more mutational event for
inactivation of the tumor suppressor
gene.
Figure 17.16 The “Two-Hit” Hypothesis for Cancer
17.4 What Is Cancer?
Example: Women who inherit one mutant
allele of the gene BRCA1 have a 60
percent chance of developing breast
cancer by age 50; and an 82 percent
chance by age 70.
Chances for women who have two
normal alleles are 2 percent and 7
percent.
17.4 What Is Cancer?
Tumor suppressor genes regulate the cell
cycle.
Rb gene and gene for p53 keep the cell
in G1 phase.
These genes are mutated in many types
of cancer.
Figure 17.17 Tumor Suppressor Gene Products Inhibit Cell Division
17.4 What Is Cancer?
For a normal cell to become malignant, a
complex series of events must occur.
More than two mutations are usually
needed for cancer to develop.
Figure 17.18 Cancer Is the Result of Multiple Genetic Alterations
17.4 What Is Cancer?
Oncogene and tumor suppressor gene
mutations involved in colon cancer have
been described in detail.
At least four suppressor genes and one
oncogene must be mutated in succession.
Figure 17.19 Multiple Mutations Transform a Normal Colon Epithelial Cell into a Cancer Cell (1)
Figure 17.19 Multiple Mutations Transform a Normal Colon Epithelial Cell into a Cancer Cell (2)
17.4 What Is Cancer?
Many cancers are diagnosed using
specific oligonucleotide probes.
Mutations can be detected early in life.
New treatments for genetic diseases are
also being developed.
17.5 How Are Genetic Diseases Treated?
Two main approaches to treating genetic
diseases:
• Modify the disease phenotype
• Replace defective genes
17.5 How Are Genetic Diseases Treated?
Modifying the disease phenotype is done
in three ways:
• Restrict substrate of a defective enzyme
• Inhibit a harmful metabolic reaction
• Supply a missing protein product
17.5 How Are Genetic Diseases Treated?
In PKU, the substrate for the enzyme
phenylalanine hydroxylase
(phenylalanine) is restricted in the diet.
The low phenylalanine diet is crucial
during infancy and childhood when the
brain is still developing.
17.5 How Are Genetic Diseases Treated?
Statin drugs used to treat familial
hypercholesterolemia are examples of
metabolic inhibitors.
Statin blocks cholesterol synthesis.
17.5 How Are Genetic Diseases Treated?
Metabolic inhibitors are also used in
chemotherapy.
The strategy is to kill rapidly dividing cells
in tumors.
Many other cells in the body are also
affected—causing the side effects of
chemotherapy.
Figure 17.20 Strategies for Killing Cancer Cells
17.5 How Are Genetic Diseases Treated?
To treat hemophilia, the missing blood
clotting protein is supplied.
The pure clotting proteins are now made
using recombinant DNA technology.
17.5 How Are Genetic Diseases Treated?
In gene therapy, the aim is to supply the
missing allele(s) by inserting a new
gene that will be expressed in the host.
The challenges: must find appropriate
vector, ensure correct insertion into host
DNA, ensure appropriate expression,
and selection of cells to target.
17.5 How Are Genetic Diseases Treated?
The nonfunctional alleles cannot be
replaced in every cell of the body.
Ex vivo techniques—cells are removed
from the body, new genes inserted, cells
returned to the body.
17.5 How Are Genetic Diseases Treated?
Genes for adenosine deaminase have
been inserted (ex vivo) into white blood
cells via a viral vector.
The enzyme is required for maturation of
white blood cells.
Mature white blood cells were first used;
now use of bone marrow stem cells is
being investigated.
Figure 17.21 Gene Therapy: The Ex Vivo Approach (Part 1)
Figure 17.21 Gene Therapy: The Ex Vivo Approach (Part 2)
17.5 How Are Genetic Diseases Treated?
Skin cells have been taken from people
with hemophilia, and the gene for blood
clotting protein inserted by a plasmid.
Cells were reintroduced into body fat,
where they produced enough protein for
normal clotting.
17.5 How Are Genetic Diseases Treated?
The second approach to gene therapy is
in vivo—insert genes directly into body
cells.
Example: DNA or vectors can be
introduced to lungs as an aerosol to
treat lung cancer.
Vectors carry functional alleles and
antisense RNA targeting oncogene
mRNAs.
17.6 What Have We Learned from the Human Genome Project?
The Human Genome Project was
proposed in 1986—to determine the
normal sequence of all human DNA.
Private industries also launched a
sequencing effort in the 1990s.
In order to detect mutations, treat
cancers, and other applications, the
normal sequences must be known.
17.6 What Have We Learned from the Human Genome Project?
The 46 human chromosomes are
different sizes—easily separated and
identified.
The DNA is first cut into fragments about
500 bp long.
Haploid human genome has about 3.2
billion bp—results in 6 million
fragments.
17.6 What Have We Learned from the Human Genome Project?
Smaller fragments of DNA have
overlapping sequences and must be
aligned.
Two methods are used for this alignment:
• Hierarchical sequencing
• Shotgun sequencing
17.6 What Have We Learned from the Human Genome Project?
In hierarchical sequencing, short
marker sequences are identified—
ensuring that every DNA fragment
would have a marker.
Simplest markers are restriction sites.
Some restriction enzymes recognize a
sequence of 8–12 bp—results in larger
fragments.
17.6 What Have We Learned from the Human Genome Project?
The large fragments are added to a
vector—bacterial artificial
chromosome (BAC), and inserted into
bacteria to create a gene library.
The fragments are arranged in order along
the chromosome map by using marker
sequences.
Libraries made with different restriction
enzymes are compared and overlaps
determined.
17.6 What Have We Learned from the Human Genome Project?
The shotgun sequencing method cuts
DNA in random fragments.
Computers are used to search for
overlapping markers.
This approach is much faster.
Sophisticated computers and software
have refined the alignment process so
that it is very accurate.
Figure 17.22 Two Approaches to Sequencing DNA
17.6 What Have We Learned from the Human Genome Project?
The complete human genome sequence
was finished in 2005.
Many surprises were revealed:
• Only about 2 percent of bp make up
coding sequences—24,000 genes.
Each gene must code for several
proteins.
• An average gene has 27,000 base
pairs.
17.6 What Have We Learned from the Human Genome Project?
• All human genes have many introns.
• Over 50 percent of the genome is
repetitive sequences.
• 99.9 percent of the genome is the same
in all people.
• Genes are not evenly distributed over
the genome.
• There are many genes with unknown
functions.
17.6 What Have We Learned from the Human Genome Project?
ENCODE project (Encyclopedia of DNA
Elements) will identify all the functional
sequences, not just protein coding
sequences.
This project will make use of sequences
from closely related species such as the
chimpanzee.
17.6 What Have We Learned from the Human Genome Project?
There are many applications of the
human genome project:
• Isolation of genes by positional cloning
is easier because of genetic markers.
Identification of disease-related genes.
• Pharmacogenomics studies variation in
drug metabolism.
17.6 What Have We Learned from the Human Genome Project?
• DNA chips are used to analyze gene
expression at different times, e.g.,
during the development of a tumor.
• “Genome prospecting” looks for genes
that predispose people to certain
conditions.
17.6 What Have We Learned from the Human Genome Project?
Ethical questions also arise:
Using genetic testing to deny health
insurance; laws prohibit discrimination
based on genetic information.
Questions of property rights; if a valuable
gene is discovered, is it the property of
the individual, the ethnic group, the
pharmaceutical company, or all
humanity?
17.6 What Have We Learned from the Human Genome Project?
Several human populations who have
descended from relatively few ancestors
are being studied to search for genetic
markers, genes that cause or predispose
to diseases, and other knowledge.
Examples: French Canadians in Quebec,
Costa Ricans, Sardinians, Ashkenazic
Jews, Icelanders
17.6 What Have We Learned from the Human Genome Project?
The proteome is the sum total of proteins
produced by an organism—more
complex than the genome.
Two techniques to analyze the proteome:
• Two-dimensional gel electrophoresis—
proteins are separated based on size and
electric charges.
• Mass spectrometry identifies proteins by
their atomic masses.
Figure 17.23 Proteomics
17.6 What Have We Learned from the Human Genome Project?
Recently, proteomics and DNA chip technology
was used to compare brain proteins in
humans and chimpanzees.
In 12,000 DNA sequences tested, only 1.4
percent were different in the two species.
But specific proteins expressed differed by
7.4%—probably due to alternative splicing.
Amounts of the proteins differed by 34 percent.
17.6 What Have We Learned from the Human Genome Project?
DNA sequencing and other molecular
approaches are reductionist—dissecting
biology into ever smaller parts.
Huge quantities of data are produced.
Systems biology aims to integrate
molecular biology data.
17.6 What Have We Learned from the Human Genome Project?
A system is a group of parts that interact,
forming a whole that is greater than the
sum of the parts.
Systems have emergent properties not
present in the parts by themselves.
Systems biologists try to discover
emergent properties; in order to predict
outcomes when physiological conditions
change.
17.6 What Have We Learned from the Human Genome Project?
Example: analysis of metabolic pathways
of fat metabolism in two strains of mice
Interactions between mRNA transcripts,
proteins, and metabolites show that one
protein is up-regulated in mutant mice,
while another is down-regulated in wildtype.
Figure 17.24 Applying Systems Biology
17.6 What Have We Learned from the Human Genome Project?
Systems biology uses information about
all the interactions of a protein to predict
consequences of a change in that
protein.
Requires sophisticated computational
techniques.
It will be useful in the study and treatment
of complex genetic diseases.