Chapter 9 Biotechnology and Recombinant DNA

mumpsimuskneesΒιοτεχνολογία

12 Φεβ 2013 (πριν από 4 χρόνια και 5 μήνες)

163 εμφανίσεις

Chapter 9
Biotechnology and
Recombinant DNA
Introduction to Biotechnology
Recall from Chapter 8 that recombination, the reshuffling of genes between two DNAmolecules, form-
ing recombinant DNA,occurs naturally in microorganisms. It is also possible to manipulate DNAartifi-
cially to combine genes from two different sources, even from vertebrates to bacteria. Artificial gene
manipulation is known as genetic engineering,and the term biotechnology usually means the indus-
trial use of genetically engineered microorganisms.
Overview of Recombinant DNA Procedures
The gene of interest is first inserted into vector DNA (or cloning vector). This DNAmolecule used as a
carrier must be self-replicating, such as a plasmid or viral genome. This recombinant vector DNAmust
enter a cell where it can multiply, forming a clone of genetically identical cells. The gene itself may be
the desired product, or it may be a protein product expressed by the gene.
Tools of Biotechnology
Restriction Enzymes
Aspecial class of DNA-cutting enzymes, restriction enzymes,are the technical basis of genetic engineering.
Restriction enzymes have the natural function of protecting the bacteria from attack by phages by hydrolyz-
ing their DNA. The DNAof the bacteria is protected from the enzyme by addition of a methyl group to
some cytosines, methylates.The enzyme recognizes and cuts only one particular sequence of nucleotide
bases. Many restriction enzymes make staggered cuts in the two DNAstrands—cuts that are not directly
opposite each other (Figure 9.2 in the text). The stretches of single-stranded DNAat the ends of the DNA
fragments are called sticky ends.They stick to complementary stretches of single-stranded DNAby base
pairing. If two fragments of DNAfrom different sources have been produced by the same restriction
enzymes, the sets of sticky ends can be spliced (recombined) easily. DNAligase then links the DNApieces.
Vectors
Figure 9.1 diagrams the cloning of a DNAfragment by using a plasmid for a vector. The host cell can
be induced to take up the plasmid vector by chemical treatment. Plasmids that can exist in several
species are shuttle vectors.
Polymerase Chain Reaction
Arecent development in DNAanalysis is the polymerase chain reaction (PCR) (Figure 9.4 in the text).
Starting with just one gene-sized piece of DNA, PCR can make billions of copies in only a few hours.
The target piece of DNAis heated to separate the DNAstrands, which serve as templates for DNA
103
104
Chapter 9
F
IGURE
9.1 Typical genetic engineering procedures with examples of applications.
Amyase, cellulase, and other
enzymes prepare fabrics for
clothing manufacture
Bacterium
Bacterial
chromosome
Plasmid
1
Vector such as a
plasmid is isolated
Recombinant DNA
(plasmid)
2
DNA is cleaved
by an enzyme
into fragments
3
Gene is inserted
into plasmid
4
Plasmid is taken up by a
cell such as a bacterium
Recombinant
bacterium
Gene of
interest
DNA containing gene
of interest
5
Cells with gene
of interest are cloned
OR
Goal may be to make
copies of gene
Goal may be to make protein
product of gene
6A
Copies of gene are harvested
7
Copies of protein
are harvested
Plasmid
RNA
Protein product
Cells make a
protein product
Gene for pest resistance
is inserted into plants
Gene alters bacteria for
cleaning up toxic waste
Human growth hormone
treats stunted growth
6B
synthesis. DNA polymerase enzyme, which forms DNAby linking the nucleotides, is supplied with
DNA’s four nucleotides and short pieces of primer nucleic acid. Each newly synthesized piece of DNA
serves in turn as a template for more new DNA.
Techniques of Genetic Engineering
Inserting Foreign DNA into Cells
Plasmid vectors can be inserted into many cells by chemical treatments that make transformation possi-
ble. Such cells are then called competent,or able to take up external DNA.
The walls of cells can be enzymatically removed (forming a protoplast) and exchange DNAby pro-
toplast fusion.Polyethylene glycol may be used to improve efficiency. Transfer of DNAin this manner
can also be enhanced by using an electric field to form minute pores in the protoplast membranes—
electroporation.Foreign DNAcan be introduced into plant cells by coating microscopic particles of
tungsten with DNAand firing it through the plant cell wall using a “gene gun.” DNAcan be
introduced into a cell through a minute glass micropipette by microinjection.
Obtaining DNA
Genes from a particular organism are isolated by cutting up the entire genome with restriction enzymes,
splicing as many as possible into vectors, and then introducing the genes into bacterial cells. Acollec-
tion of bacterial clones containing different DNAfragments is called a gene library.
Cloning genes from a eukaryotic organism generally requires removal of introns,stretches of DNA
that do not code for protein. This can be done by splicing;introns are removed when an RNAtranscript
of a gene is converted to mRNA. What remains are exons,stretches of DNAthat code for protein. An
artificial gene that contains only exons can be made with the enzyme reverse transcriptase.This will
synthesize complementary DNA (cDNA) from an mRNAtemplate. This is the most common method of
obtaining eukaryotic genes.
Genes of synthetic DNA can also be made with the help of DNA-synthesizing machines. The smaller
chains of about 40 nucleotides synthesized in this way can be linked together to make an entire gene.
Selecting a Clone
One of the most common methods of selecting a desired gene is blue/white screening—from the color
of the bacterial colonies formed at the end of the process. Briefly, only white colonies are of interest,
because they contain foreign DNAincorporated into a plasmid. Further work is then needed to identify
this foreign DNA.
For example, colony hybridization is a common method. Short segments of single-stranded DNA,
consisting of a sequence of nucleotides unique to the gene sought, can be synthesized. These molecules,
called DNA probes,are radioactively labeled so they can be located later. The clone-carrying bacteria
from the library are grown into colonies on a plate of nutrient medium treated to break open the cells
and separate the DNAinto single strands. The labeled probe, added to the plate, will react with DNAin
any bacterial colony that base-pairs with the probe (colony hybridization).The radioactive tag allows the
colonies containing the desired gene to be identified. Asimilar probe based on labeled antibodies
against protein products of the cells is also used.
Making a Gene Product
Escherichia coli is often used as the genetically engineered organism to produce a desired gene product.
It has disadvantages; it produces endotoxins that cause fever and shock in animals. Also, it does not
secrete the product; the cells must be harvested, ruptured, and the product recovered.
Biotechnology and Recombinant DNA
105
Organisms such as the gram-positive bacterium Bacillus subtilis and yeasts are more likely to secrete
their products. Animal viruses, such as the vaccinia virus, have been genetically engineered to produce
vaccines. Mammalian cells and plant cells are often engineered to produce useful products.
Applications of Genetic Engineering
Medically important products made by genetic engineering are insulin, somatotropin growth hormone,
tissue-plasminogen activator (t-PA), and subunit vaccines.
Therapeutic, Scientific, and Medical Applications
Recombinant DNAtechnology is also the basis of DNAanalysis of genetic abnormalities responsible for
various diseases, and it contributes to advances in gene therapy, in which abnormal genes might be
replaced with normal genes in a living individual.
To isolate a fragment of DNAcontaining a gene, the method of Southern blotting is commonly
used. DNAfragments from each candidate clone are treated with the same restriction enzyme, and the
resulting fragments separated by gel electrophoresis.The fragments are called restriction fragment
length polymorphisms (RFLPs).The bands representing the fragments are then blotted onto a special
filter, which is bathed with radioactively tagged probes of DNAcomplementary to the gene desired.
The desired bands can be cut out of the gel and recovered by soaking in solvent. The genes may be
studied by DNA sequencing to determine the sequence of nucleotides.
In random shotgun sequencing small pieces of the genome are sequenced and then assembled by
computer analysis. These techniques are vital to the Human Genome Project discussed in the next para-
graph. DNAsequencing, which is often highly automated, can be combined with PCR. This permits
recovery of detectable, identifiable DNAfrom extremely small samples. These procedures are used for
so-called DNA fingerprinting.This is used for analysis of crime-scene samples, tissue identification,
and paternity testing.
The Human Genome Project
The goal of the Human Genome Project is to map the 35,000–70,000 genes of the human genome,
approximately 3 billion nucleotide pairs. This is now nearly complete. The next goal is the Human
Proteome Project.This will map all the proteins expressed in human cells.
Agricultural Applications
The most elegant method of introducing recombinant DNAinto a plant cell is by the Ti plasmid.A
bacterium that infects plants normally carries this plasmid. The infection causes a tumorlike growth
called a crown gall (Ti means tumor inducing). The plasmid also serves as a vehicle for insertion of
genetically engineered DNAinto a plant. Other applications are to make crop plants resistant to herbi-
cides that then selectively kill weeds, and to improve the ability to fix nitrogen in certain symbiotic bac-
teria. Abacterium, Bacillus thuringiensis (Bt),has been engineered into plants to produce a toxin that kills
certain plant pathogens that feed on the plant. Agenetically engineered product, bovine growth hor-
mone, increases milk production in dairy herds.
Safety Issues and the Ethics of Genetic Engineering
Laboratories engaged in recombinant DNAresearch must meet rigorous safety standards to avoid acci-
dentally releasing genetically engineered microbes. The microbes may also be engineered to contain
suicide genes that prevent them from surviving outside the laboratory environment. Genetic screening
for hereditary diseases and birth defects in the fetus introduces ethical questions not yet resolved.
106
Chapter 9
Self-Tests
In the matching section, there is only one answer to each question; however, the lettered
options (a, b, c, etc.) may be used more than once or not at all.
I. Matching
II. Matching
III. Matching
a.Blue
b.White
c.Exon
d.Intron
1.In the blue/white screening procedure, the foreign DNAis
in these colonies.
2.These stretches of DNAof eukaryotes do not code for
proteins.
a.Recombinant
b.Complementary
c.Probe
d.Protoplast fusion
e.Plasmid
1.Probably the most common cloning vector used in genetic
engineering.
2.The reshuffling of genes between two DNAmolecules
forms DNAof this type.
3.The kind of DNAsynthesized by using mRNAas a
template.
4.DNAexchange between cells in this process uses
polyethylene glycol to improve efficiency.
5.Short segments of single-stranded DNAused to recognize
a DNAsequence in a gene.
a.Restriction enzymes
b.DNAligase
c.DNApolymerase
d.Vector
1.DNA-cutting enzymes that often form sticky ends.
2.Aself-replicating DNAmolecule used as a carrier to
transmit a gene from one organism to another.
3.An enzyme that links short pieces of DNAinto longer
pieces.
4.An enzyme that links nucleotides to form DNA.
Biotechnology and Recombinant DNA
107
108
Chapter 9
Fill in the Blanks
1.Sticky ends stick to each other by complementary stretches of single-stranded DNAby
pairing.
2.Acollection of bacterial clones each containing a different DNAfragment is called a
.
3.To isolate a fragment of DNAcontaining a gene, DNAfragments of clones are separated by gel elec-
trophoresis. This is an early step in the technique for DNAanalysis.
4.The procedure by which billions of copies of a sequence of DNAcan be made in a few hours is
called the reaction.
5.The most elegant way of introducing recombinant DNAinto a plant cell is by means of the
plasmid, carried naturally by Agrobacterium tumefaciens.
6.The Human Proteome Project has the goal of identifying all the
produced by human cells.
Label the Art
R
ecogn
iti
on s
it
es
Cut
G A A T T C
C T T A A G
Cut
Cut
G A A T T C
C T T A A G
Cut
AAT TC
G
C T T A A
G C T T A A
GA A T T C
G
CT T AA
G
A A T T C
G
G C T T A A
GA A T T C
G C T T A A
GA A T T C
Sticky end
G
A
A
T
T
C
G
C
T
T
A
A
A
A
T
T
C
G
C
T
T
A
A
G
G
A
A
T
T
G
C
T
T
A
A
A
A
T
T
C
C
T
T
A
A
G
G
3 When two such fragments
of DNA cut by the same
restriction enzyme come
together, they can join
by .
DNA from another source,
perhaps a plasmid.
4 The joined fragments will
usually form either a linear
molecule or a circular one,
as shown here for a plasmid.
Other combinations of
fragments can also
occur.
5 The enzyme is
used to unite the backbones
of the two DNA fragments,
producing a molecule of
.
DNA
C
1
cuts (arrows)
double-stranded DNA at its particular
, which are shown
in dark.
a.
b.
2
These cuts produce a DNA
fragment with two .
c.
d.
e.
f.
Biotechnology and Recombinant DNA
109
Critical Thinking
1.During the investigation of a robbery, police discover a small quantity of blood and skin on a piece
of broken glass from the window through which the perpetrator gained entry. There was insuffi-
cient blood on the glass to type conventionally. No usable fingerprints were obtained. How might
the police tie their prime suspect to the crime?
2.What are some of the advantages of using genetic engineering to produce human hormones such as
insulin and somatotropin?
3.What method would be most appropriate for inserting foreign DNAin each of the following examples?
a.Inserting DNAinto an animal cell.
b.Inserting DNAinto a plant cell.
c.Inserting DNAinto a yeast.
4.You are asked to develop a protocol for the industrial production of a protein from a genetically
engineered bacterium. Abacterium of what Gram designation would most likely be the best choice?
Why?
5.What are subunit vaccines? Are they safer than avirulent vaccines? Why?
110
Chapter 9
Answers
Matching
I.1. a 2. d 3. b 4. c
II.1. e 2. a 3. b 4. d 5. c
III.1. b 2. d
Fill in the Blanks
1.base 2.gene library 3.Southern blot 4.polymerase chain 5.Ti 6.proteins
Label the Art
a.Restriction enzyme b.Recognition sites c.Sticky ends d.Base pairing e.DNAligase
f.Recombinant DNA
Critical Thinking
1.Police could use recombinant DNAtechniques to amplify the DNAfrom the skin sample or from
the white cells in the blood sample. This material would be compared to the suspect’s DNAusing
the Southern blot method to do a DNAfingerprint. If the samples match, then the police have posi-
tive identification tying the suspect to the crime—that is, unless the suspect has an identical twin!
2.Genetic engineering produces human hormones that are less expensive, less allergenic, available in
large quantities, and pose no risk of transmitting disease.
3.a.Animal cells may be made “competent” to take up external DNAby soaking them in a solution
of calcium chloride. After this treatment many of the animal cells will take up the DNA; recombi-
nation will occur in some of the cells. Direct introduction of foreign DNAinto animal cells can be
achieved by microinjection using a glass micropipette.
b.Plant cells may have foreign DNAintroduced by first enzymatically removing the cell wall to
create protoplasts. Protoplasts are then fused by adding polyethylene glycol to form a hybrid cell.
The DNAin the hybrid cell may then undergo recombination naturally. Another method utilizes
a “gene gun” that shoots DNA-coated tungsten or gold particles through the cell wall. Some cells
will incorporate and express the new DNA.
c.Yeast cells may also be made competent by soaking them in a calcium chloride solution or
through the process of electroporation. This involves using an electrical current to form micro-
scopic pores in the cell membrane. DNAis able to enter through these pores.
4.Agram-positive bacterium would be the best choice for two reasons. First, gram-positive bacteria
lack a cell-wall component called endotoxin that is found in gram-negative bacteria. Endotoxin
causes fever and shock in animals and would pose a serious problem if present in gene products.
Second, gram-negative cells such as E. coli don’t usually secrete protein products, and the process
used to harvest them is too expensive for industrial applications.
5.Subunit vaccines consist of a protein portion of a pathogen produced by a genetically engineered
organism—for example, a yeast. Because the protein is made by a yeast rather than a treated
pathogen or a portion of the actual pathogen, there is no chance that the disease will be transmitted.
Biotechnology and Recombinant DNA
111