Sequencing Entire Genomes (p. 256)

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13
THE NEW BIOLOGY

CHAPTER OUTLINE

Sequencing Entire Genomes
(p. 256)

13.1

Genomics

(p. 256; Fig. 13.1; Table 13.1)

A.

The new field of genomics is concerned with sequencing the genomes of organisms.

1.

The first genome to be sequenced was that of a small bacter
ial virus; this was
accomplished by Frederick Sanger in 1977.

2.

The advent of automated sequencing machines has made the sequencing of larger
genomes practical.


B.

Sequencing DNA

1.

To sequence DNA, the DNA is first cut with restriction enzymes, and then a fra
gment of
unknown sequence is amplified to get thousands of copies of the fragment.

2.

The fragment copies are then mixed with primers, DNA polymerase, the four
nucleotides, and four different chain
-
terminating chemical tags.

3.

Heating denatures the double
-
stran
ded DNA fragments, and then cooling allows the
primers to bind and DNA polymerase to synthesize complementary strands of DNA.

4.

Synthesis stops whenever a chain
-
terminating chemical tag is added instead of a
nucleotide base; this results in a series of doubl
e
-
stranded DNA fragments of different
lengths.

5.

Gel electrophoresis is used to separate the fragments by size.

6.

Computer analysis helps to read the gel and determine the DNA sequence.

13.2

The Human Genome
(p. 258; Figs. 13.2, 13.3)

A.

The Number of Genes Is Qu
ite Low

1.

There are 20,000

25,000 genes in the human genome.

2.

Coding regions are called exons while noncoding regions are introns.

3.

Alternative splicing of genes allows there to be four times as many proteins as there are
genes.

B.

Some Chromosomes Have Few Ge
nes

Genes Exist in Many Copy Numbers



DNA That Codes for Proteins

1.

Four different classes of protein
-
encoding genes are found in the human genome: single
-
copy genes, segmental duplications, multigene families, and tandem clusters.

D.

Most Genome DNA Is Non
coding

1.

The human genome possesses a startling amount of noncoding DNA, up to 99% of the
total.

2.

There are four major types of noncoding human DNA: noncoding DNA within genes
(introns), structural DNA, repeated sequences, and transposable elements.

3.

There are

five types of transposable elements, some of which lead to harmful mutations.


Genetic Engineering
(p. 260)

13.3


A Scientific Revolution

(p. 260; Figs. 13.4
-
13.7)

A.

Transferring genes from one organism to another is often called genetic engineering.

B.

Geneti
c engineering is having a major impact on medicine and agriculture.


C. Restriction Enzymes

1.

The first step in a genetic engineering experiment is to cleave the DNA that the geneticist
wishes to transfer.

2.

This process involves the use of restrict
ion enzymes that bind to specific sequences of
nucleotides and cut the DNA at a certain position in the sequence.

3.

Since DNA is made up of complementary bases, both strands do not split at the same
position, and “sticky” ends result.

4.

These “sticky”
ends can then be joined with any other complementary sequence using
ligase, a sealing enzyme.

5.

Since only the ends are involved, the combining of DNA from different sources (i.e.,
human and mouse, or human and bacteria) is possible.


D.
For
mation of cDNA

1.

Prokaryotes do not have exons interspersed with introns the way eukaryotes do, but
instead have continuous information encoded in the DNA.

2.

Thus, bacteria do not have the means to remove introns from eukaryotic DNA or newly
transcribed mRNA.


3.

When researchers want to reproduce eukaryotic genes inside bacteria, they isolate the
processed mRNA from the cytoplasm of eukaryotic cells.

4.

Reverse transcriptase is then used to make DNA from the processed mRNA, which
produces a version of the gene call
ed copy DNA (cDNA) that is made up only of exons.

E. DNA Fingerprinting and Forensic Science

1.

DNA can be analyzed for its unique pattern of fragments produced by restriction
endonucleases.

2.

Different probes can yield information that can be used to identi
fy suspects from crimes.


F. PCR Amplification

1.

Geneticists can use the polymerase chain reaction (PCR) to produce many copies of a
target gene.

2.

This procedure first involves identifying short sequences of nucleotides, called primers,
on eith
er side of the target gene.

3.

Next, a solution of target DNA, the primers, nucleotides, and DNA polymerase is heated,
which disrupts the hydrogen bonds of DNA and produces single strands.

4.

As the solution cools, the primers bind to their complementary seque
nces near the target
gene.

5.

DNA polymerase then begins at a primer and replicates the single
-
stranded DNA.

6.

Many copies of the desired gene can be made in this manner.

13.4 Genetic Engineering and Medicine

(p. 264; Figs. 13.8, 13.9; Table 13.2 )

A.


With
the advent of genetic engineering, major medical advances have been made.

B.

Making “Magic Bullets”

1.

Bacteria now can produce human insulin, the hormone that is under produced in
diabetics.

2.

Other products, such as anticoagulants to dissolve blood clots and fa
ctor VIII to promote
clotting, are now safely produced by bacteria, which eliminates the possibility of
transferring diseases from a human donor.

C.

Piggyback Vaccines

1.

Vaccines have been used for years to trigger immunity to a wide variety of diseases.

2.

Vacc
ines can now be made more safely by inserting the gene for a pathogen's surface
protein into the DNA of a harmless virus.

3.

Such piggyback vaccines are being developed for a variety of diseases.


4.

Finally, a nucleic acid probe with a sequence complementary t
o that of the gene of
interest is washed over a filter containing replica colonies, and the clone containing the
gene can be identified.

13.5

Genetic Engineering and Agriculture

(p. 266; Fig. 13.10, 13.11, 13.12; Table 13.3)

A.

Bacteria can be genetically

engineered to produce bovine somatotropin, which when fed to
dairy cows, greatly enhances milk production.

B.

Also, livestock can be genetically engineered to produce pharmaceuticals.



C. Pest Resistance

1.

Certain crops, like cotton, have been engine
ered to be resistant to insect pests, which
means these crops will not require pesticides.

2.

Bacterial genes that produce proteins toxic to certain plant pests have been inserted into
tomatoes and other crops; the proteins are not toxic to humans.





D. Herbicide Resistance

1.

Agriculture has benefited from the genetic engineering of herbicide
-
resistant crops.

2.

The active ingredient in Roundup, called glyphosate, is easily broken down in the
environment and is thus a comparatively safe herbicid
e.

3.

Making crops resistant to glyphosate means less tilling is needed, thus soils are saved
from erosion and less fuel and expense are needed to raise the crop.


E. More Nutritious Crops

1.

The cultivation of genetically modified (GM) crops has become com
monplace.

2.

Rice can be modified to contain more minerals, such as iron, and vitamins.


F. How Do We Measure the Potential Risks of Genetically Modified Crops?

1.

Consumers worry that eating GM food might be dangerous or that GM crops are
harmful to the enviro
nment.

2.

Other than allergic reactions to modified proteins, dangers to the consumer appear to be
slight.

3.

Whether GM products are potentially harmful to the environment is not yet clear.

4.

The first concern is the possibility of harming other organisms, and so
me studies have
shown that in certain cases, GM modified crops may adversely affect insect populations
by removing sources of food and cover.

5.

The potential of resistance developing to GM crops is a possibility.

6.

Because of gene flow, it may be difficult to
control the spread of modified genes from
the cultivated populations to the wild populations.

The Revolution in Cell Technology (p. 270)

13.6

Reproductive Cloning

(p. 270; Figs. 13.13, 13.14, 13.15)

A.


Wilmut’s Lamb

1.

In the 1990s, Campbell proposed th
at the egg cell and donated nucleus need to be at the
same stage in the cell cycle.

2.

Wilmut applied Campbell’s idea and was able to successfully clone an adult mammal.

3.

Wilmut removed mammary cells from the udder of a six
-
year
-
old sheep and egg cells
from a
different sheep and starved the cells; he then combined them surgically and
applied an electrical shock to kick
-
start the cell cycle.



B. Progress with Reproductive Cloning

1.

Many animals have now been successfully cloned.

2.


Most of thes
e animals have been cloned using the technique of nuclear transfer.



C. The Importance of Gene Reprogramming

13.7

Stem Cells

Therapy

(p. 272; Figs. 13.16, 13.17, 13.18)

A.

Embryonic Stem Cells are Totipotent

1.

Embryonic stem cells are totipotent,

capable of developing into any type of tissue or an
adult animal.

2.

As development progresses, some stem cells commit to form specific types of tissue; in
this way, each major tissue is formed from its own kind of tissue
-
specific adult stem cell.

B.

Using Embr
yonic Stem Cells to Repair Damaged Tissues

1.

Because embryonic stem cells are totipotent, they offer the possibility of restoring
damaged tissue.

13.8

Therapeutic Use of Cloning

(p. 274; Figs.13.19, 13.20)

A.

Therapeutic use of embryonic stem cells involves sur
gically transferring embryonic stem cells
to a damaged area, where the stem cells can then form healthy replacement tissue.

B.


Cloning to Achieve Immune Acceptance

1.

In therapeutic cloning, the nucleus from a patient is inserted into an enucleated egg cell.

2.

Th
e egg develops in vitro.

3.

The embryonic stem cells are removed and used to replace the patient’s damaged or lost
tissue.

4.

Cloned stem cells are readily accepted by the body as they pass the immune system’s
“self” identity check.


C. Gene Re
programming to Achieve Immune Acceptance

13.
9

Gene Therapy

(p. 276; Figs. 13.21, 13.22)

A.

Gene therapy involves introducing “healthy” genes into cells that lack them.

B.

Early Success

1.

In 1990, two girls were cured of a blood disorder by gene therapy, prov
ing that genetic
disorders can be cured by gene therapy.

2.

The girls were treated with injections of gene
-
modified proliferated bone marrow.

C.

The Rush to Cure Cystic Fibrosis

1.

Adenovirus vectors have been used to transfer healthy
cf

genes into lung cells of mi
ce
with some success.

2.

Human research has not been as promising, as the transformed cells are attacked by the
patient’s immune system.

D.

A More Promising Vector

1.

The adeno
-
associated virus, a small parvovirus, is less likely to evoke a strong immune
response o
r cause cancerous changes.

2.

In 1999, AAV successfully cured anemia in rhesus monkeys

5.

AAV cured dogs of a hereditary disorder leading to retinal degeneration and blindness.

6.

In 2000, the first gene therapy for muscular dystrophy began.

7.

Trials are also underwa
y for cystic fibrosis, rheumatoid arthritis, hemophilia, and types of
cancer.

E.

Success with New Vectors