Recombinant DNA and Biotechnology
Recombinant DNA and Biotechnology
Cleaving and Rejoining DNA
Getting New Genes into Cells
Sources of Genes for Cloning
Some Additional Tools for DNA Manipulation
Biotechnology: Applications of DNA Manipulation
Cleaving and Rejoining DNA
Recombinant DNA technology is the ________ and combination of DNA molecules
from different sources.
Recombinant DNA technology uses the techniques of sequencing, rejoining,
________ and locating DNA fragments, making use of complementary base pairing.
Cleaving and Rejoining DNA
Bacteria defend themselves against invasion by viruses by producing
restriction enzymes which catalyze the cleavage of DNA into small fragments.
The enzymes cut the bonds between the 3’ hydroxyl of one nucleotide, and the
5’ phosphate of the next.
There are many such enzymes, each of which recognizes and cuts a specific
sequence of bases, called a ________ sequence or restriction site (4 to 6 base
pairs long).
Cleaving and Rejoining DNA
Host DNA is not damaged due to methylation of certain bases at the restriction
sites; this is performed by enzymes called methylases.
The enzyme EcoRI cuts DNA with the following paired sequence:
§ 5’ ... GAATTC ... 3’
§ 3’ ... CTTAAG ... 5’
Notice that the sequence is ________ It reads the same in the 5’-to-3’
direction on both strands.
Cleaving and Rejoining DNA
Using EcoRI on a long stretch of DNA would create fragments with an average
length of 4,098 bases.
Using EcoRI to cut up small viral genomes may result in only a few fragments.
For a eukaryotic genome with tens of millions of base pairs, the number of
fragments will be very large.
Hundreds of restriction enzymes have been purified from various organisms, and
these enzymes serve as "knives" for genetic surgery.
Cleaving and Rejoining DNA
The fragments of DNA can be separated using gel electrophoresis. Because of
its phosphate groups, DNA is negatively charged at neutral pH.
When DNA is placed in a ________ gel and an electric field is applied, the DNA
molecules migrate toward the positive pole.
Smaller molecules can migrate more quickly through the porous gel than larger
ones.
After a fixed time, the separated molecules can then be stained with a
fluorescent dye and examined under ultraviolet light.
Cleaving and Rejoining DNA
Electrophoresis gives two types of information:
§ Size of the DNA fragments can be determined by ________ to DNA fragments of
known size added to the gel as a reference.
§ A specific DNA sequence can be determined by using a complementary labeled
single-stranded DNA probe.
The specific fragment can be cut out as a lump of gel and removed by diffusion
into a small volume of water.
Cleaving and Rejoining DNA
Some restriction enzymes cut DNA strands and leave ________ ends of
single-stranded DNA, or "sticky" ends, that attract complementary sequences.
If two different DNAs are cut so each has sticky ends, fragments with
complementary sticky ends can be recombined and sealed with the enzyme DNA
ligase.
These simple techniques, which give scientists the power to manipulate genetic
material, have revolutionized biological science in the past 30 years.
Getting New Genes into Cells
The goal of recombinant DNA work is to produce many copies (clones) of a
particular gene.
To make protein, the genes must be introduced, or transfected, into a host
cell.
The host cells or organisms, referred to as transgenic, are transfected with
DNA under special conditions.
The cells that get the DNA are distinguished from those that do not by means
of genetic markers, called reporter genes.
Getting New Genes into Cells
Bacteria have been useful as hosts for recombinant DNA.
§ Bacteria are easy to manipulate, and they grow and divide quickly.
§ They have genetic markers that make it easy to select or screen for insertion.
§ They have been intensely studied and much of their molecular biology is known.
Getting New Genes into Cells
Bacteria have some disadvantages as well.
§ Bacteria lack splicing machinery to excise introns.
§ Protein modifications, such as glycosylation and phosphorylation, fail to
occur as they would in a eukaryotic cell.
§ In some applications, the expression of the new gene in a eukaryote (the
creation of a transgenic organism) is the desired outcome.
Getting New Genes into Cells
Saccharomyces, baker's and brewer's yeast, are commonly used eukaryotic hosts
for recombinant DNA studies.
In comparison to many other eukaryotic cells, yeasts divide quickly, they are
easy to grow, and have relatively small genomes (about 20 million base pairs).
Getting New Genes into Cells
Plants are also used as hosts if the goal is to make a ________ plant.
It is relatively easy to regenerate an entire plant from differentiated plant
cells because of plant cell totipotency.
The transgenic plant can then reproduce naturally in the field and will carry
and express the gene on the recombinant DNA.
Getting New Genes into Cells
New DNA can be introduced into the cell's genome by integration into a
chromosome of the host cell.
If the new DNA is to be ________ it must become part of a segment of DNA that
contains an origin of replication called a replicon, or replication unit.
Getting New Genes into Cells
New DNA can be incorporated into the host cell by a vector, which should have
four characteristics:
§ The ability to replicate independently in the host cell
§ A recognition sequence for a restriction enzyme, permitting it to form
recombinant DNA
§ A reporter gene that will announce its presence in the host cell
§ A small size in comparison to host chromosomes
Getting New Genes into Cells
Plasmids are ideal ________ for the introduction of recombinant DNA into
bacteria.
A plasmid is small and can divide separately from the host's chromosome.
They often have just one restriction site, if any, for a given restriction
enzyme.
Cutting the plasmid at one site makes it a linear molecule with sticky ends.
If another DNA is cut with the same enzyme, it is possible to insert the DNA
into the plasmid.
Plasmids often contain antibiotic resistance genes, which serve as genetic
markers.
Getting New Genes into Cells
Only about 10,000 base pairs can be inserted into plasmid DNA, so for most
eukaryotic genes a vector that accommodates larger DNA inserts is needed.
For inserting larger DNA sequences, viruses are often used as vectors.
If the genes that cause death and lysis in E. coli are eliminated, the
bacteriophage l can still infect the host and inject its DNA.
The deleted 20,000 base pairs can be replaced by DNA from another organism,
creating recombinant viral DNA.
Getting New Genes into Cells
Bacterial plasmids are not good vectors for yeast hosts because prokaryotic
and eukaryotic DNA sequences use different origins of replication.
A yeast artificial chromosome, or YAC, has been made that has a yeast origin
of replication, a centromere sequence, and telomeres, making it a true
eukaryotic chromosome.
YACs have been engineered to include specialized single restriction sites and
selectable markers.
YACs can accommodate up to 1.5 million base pairs of inserted DNA.
Getting New Genes into Cells
Plasmid vectors for plants include a plasmid found in the Agrobacterium
tumefaciens bacterium, which causes the tumor-producing disease, crown gall, in
plants.
Part of the tumor-inducing (Ti) plasmid of A. tumefaciens is T DNA, a
transposon, which inserts copies of itself into the host chromosomes.
If T DNA is replaced with the new DNA, the plasmid no longer produces tumors,
but the transposon still can be inserted into the host cell's chromosomes.
The plant cells containing the new DNA can be used to generate transgenic
plants.
Getting New Genes into Cells
When a population of host cells is treated to introduce DNA, just a fraction
actually incorporate and express it.
In addition, only a few vectors that move into cells actually contain the new
DNA sequence.
Therefore, a method for selecting for ________ cells and screening for inserts
is needed.
A commonly used approach to this problem is illustrated using E. coli as
hosts, and a plasmid vector with genes for resistance to two antibiotics.
Getting New Genes into Cells
Other methods have since been developed for screening.
The gene for luciferase, the enzyme that makes fireflies glow in the dark, has
been used as a reporter gene.
Green fluorescent protein, which is the product of a ________ gene, glows
without any required substrate.
Cells with this gene in the plasmid grow on ampicillin and ________ when
exposed to ultraviolet light.
Sources of Genes for Cloning
Gene libraries contain fragments of DNA from an organism's genome.
Restriction enzymes are used to break chromosomes into fragments, which are
inserted into vectors and taken up by host cells.
Sources of Genes for Cloning
Using plasmids for insertion of DNA, about one million separate fragments are
required for the human genome library.
Phage l, which carries four times as much DNA as a plasmid, is used to hold
these random fragments.
It takes about 250,000 different phage to ensure a copy of every sequence.
This number seems large, but just one growth plate can hold as many as 80,000
phage colonies.
Sources of Genes for Cloning
A smaller DNA library can be made from complementary DNA (cDNA).
Oligo dT primer is added to mRNA tissue where it hybridizes with the poly A
tail of the mRNA molecule.
Reverse transcriptase, an enzyme that uses an RNA template to synthesize a
DNARNA hybrid, is then added.
The resulting DNA is ________ to the RNA and is called cDNA. DNA polymerase
can be used to synthesize a DNA strand that is complementary to the cDNA.
Sources of Genes for Cloning
If the amino acid sequence of a protein is known, it is possible to synthesize
a DNA that can code for the protein.
Using the knowledge of the genetic code and known amino acid sequences, the
most likely base sequence for the gene may be found.
Often sequences are added to this sequence to promote expression of the
protein.
Human ________ has been manufactured using this approach.
Sources of Genes for Cloning
With synthetic DNA, mutations can be created and studied.
Additions, deletions, and base-pair substitutions can be manipulated and
tracked.
The functional importance of certain amino acid sequences can be studied.
The signals that mark proteins for passage through the ER membrane were
discovered by site-directed mutagenesis.
Some Additional Tools for DNA Manipulation
Homologous recombination is used to study the role of a gene at the level of
the organism.
In a knockout experiment, a gene inside a cell is replaced with an inactivated
gene to determine the inactivated gene's effect.
This technique is important in determining the roles of genes during
development.
Some Additional Tools for DNA Manipulation
The emerging science of genomics has to contend with two difficulties:
§ The large number of genes in eukaryotic genomes
§ The distinctive ________ of gene expression in different tissues at different
times
To find these patterns, DNA sequences have to be arranged in an array on some
solid support.
DNA chip technology provides these large arrays of sequences for
hybridization.
Some Additional Tools for DNA Manipulation
Analysis of cellular mRNA using DNA chips:
§ In a process called RT-PCR, cellular mRNA is isolated and incubated with
reverse transcriptase (RT) to make complementary DNA (cDNA). The cDNA is
amplified by PCR prior to hybridization.
§ The amplified cDNA is coupled to a fluorescent dye and then hybridized to the
chip.
§ A scanner detects glowing spots on the array. The combinations of these spots
differ with different types of cells or different physiological states.
Some Additional Tools for DNA Manipulation
DNA ________ technology can be used to detect genetic variants and to diagnose
human genetic diseases.
Instead of sequencing the entire gene, it is possible to make a chip with
20-nucleotide fragments including every possible mutant sequence.
Hybridizing that sequence with a person's DNA may reveal whether any of the
DNA hybridized to a mutant sequence on the chip.
Some Additional Tools for DNA Manipulation
Base-pairing rules can also be used to stop mRNA translation.
Antisense RNA is complementary to a sequence of mRNA.
The antisense RNA forms a double-stranded hybrid with an mRNA, which inhibits
translation.
These hybrids are broken down rapidly in the cytoplasm, so translation does
not occur.
In the laboratory, antisense RNA can be made and added to cells to block
translation.
Some Additional Tools for DNA Manipulation
A related technique uses interference RNA (RNAi) which inhibits mRNA
translation in the inactive X chromosome of mammals.
Scientists can synthesize a small interfering RNA (siRNA) to inhibit
translation of any known gene.
Some Additional Tools for DNA Manipulation
The two-hybrid system allows scientists to test for protein interactions
within a living cell.
A two-hybrid system uses a transcription factor that activates the
transcription of an easily detectable reporter gene.
This transcription factor has two domains: one that binds to DNA at the
promoter, and another that binds to the transcription complex to activate
transcription.
An example is the yeast two-hybrid system.
Biotechnology: Applications of DNA Manipulation
Biotechnology is the use of microbial, plant, and animal cells to produce
materialssuch as foods, medicines, and chemicalsthat are useful to people.
The use of yeast to create beer and ________ and bacterial cultures to make
yogurt and cheese are examples of centuries-old biotechnology.
Gene cloning techniques of modern molecular biology have vastly increased the
number of these products beyond those that are naturally made by microbes.
Biotechnology: Applications of DNA Manipulation
Expression vectors are typical vectors, but they also have extra sequences
needed for the foreign gene to be expressed in the host cell.
An expression vector might have an inducible promoter, which can be stimulated
into expression by responding to a specific signal such as a hormone.
A tissue-specific promoter is expressed only in a certain tissue at a certain
time.
Targeting sequences are sometimes added to direct the protein product to an
appropriate destination.
Biotechnology: Applications of DNA Manipulation
Many medical products have been made using recombinant DNA technology.
For example, tissue plasminogen activator (TPA), is currently being produced
in E. coli by recombinant DNA techniques.
TPA is an ________ that converts blood plasminogen into plasmin, a protein
that dissolves clots.
Recombinant DNA technology has made it possible to produce the naturally
occurring protein in quantities large enough to be medically useful.
Biotechnology: Applications of DNA Manipulation
Selective breeding has been used for centuries to improve plant and animal
species to meet human needs.
Molecular biology is accelerating progress in these applications.
There are three major advantages over traditional techniques:
§ Specific genes can be affected.
§ Genes can be introduced from other organisms.
§ Plants can be regenerated much more quickly by cloning than by traditional
breeding.
Biotechnology: Applications of DNA Manipulation
Insecticides tend to be nonspecific, killing both pest and beneficial insects.
They can also be blown or washed away to contaminate and pollute non-target
sites.
Bacillus thuringiensis bacteria produce a protein toxin that kills insect
larvae pests and is 80,000 times more toxic than the typical chemical
insecticide.
Transgenic tomato, corn, potato, and cotton plants have been made that produce
a toxin from B. thuringiensis.
Biotechnology: Applications of DNA Manipulation
The process of producing pharmaceuticals using agriculture is nicknamed
"pharming."
Transgenic sheep are being used to produce human a-1-antitrypsin (a-1-AT) in
their milk; this protein inhibits the enzyme elastase, which breaks down
connective tissue in the lungs. Treatment with a-1-AT alleviates symptoms in
people suffering from emphysema.
Other products of "pharming" include blood clotting factors and antibodies for
treating colon cancer.
Biotechnology: Applications of DNA Manipulation
Crops that are resistant to herbicides:
§ Glyphosate ("Roundup") is a broad-spectrum herbicide that inhibits an enzyme
system in ________ that is involved in the synthesis of amino acids.
§ A bacterial gene, which confers resistance to glyphosate, is inserted into
useful food crops (corn, cotton, soybeans) to protect them from the herbicide,
which otherwise would kill them along with the weeds.
Biotechnology: Applications of DNA Manipulation
Grains with improved nutritional characteristics:
§ Genes from bacteria and daffodil plants are transferred to rice using the
vector Agrobacterium tumefaciens.
§ Now a genetically modified strain of ________ produces b-carotene, a molecule
that is converted to vitamin A in animals.
Biotechnology: Applications of DNA Manipulation
Crops that adapt to the environment:
§ A gene was recently discovered in the thale cress (Arabidopsis thaliana) that
allows it to thrive in salty soils.
§ When this gene is added to tomato plants, they can grow in soils four times as
________ as the normal lethal level.
§ This finding raises the prospect of growing useful crops on previously
unproductive soils with high salt concentration.
§ Biotechnology may allow us to adapt plants to different environments.
Biotechnology: Applications of DNA Manipulation
There is public concern about biotechnology:
§ Genetically modified E. coli might share their genes with the E. coli bacteria
that live normally in the human intestines.
§ Researchers now take precautions against this. For example, the strains of E.
coli used in the lab have a number of mutations that make their survival in the
human intestine impossible.
Biotechnology: Applications of DNA Manipulation
There are concerns that genetic manipulation interferes with nature, that
genetically altered foods are unsafe, and that genetically altered plants might
allow transgenes to escape to other species and thus threaten the environment.
Regarding safety for human consumption, advocates of genetic engineering note
that typically only single genes specific for plant function are added.
As plant biotechnology moves from adding genes to improve plant growth to
adding genes that affect human nutrition, such concerns will become more
pressing.
Biotechnology: Applications of DNA Manipulation
The risks to the environment are more difficult to assess.
Transgenic plants undergo extensive field testing before they are approved for
use, but the complexity of the biological world makes it impossible to ________
all potential environmental effects of transgenic organisms.
Because of the potential benefits of agricultural biotechnology, most
scientists believe we should proceed, but with caution.
Biotechnology: Applications of DNA Manipulation
With the exception of identical twins, each human being is genetically
distinct from all other human beings.
Characterization of an individual by DNA base sequences is called DNA
fingerprinting.
Biotechnology: Applications of DNA Manipulation
Scientists look for DNA sequences that are highly polymorphic.
Sequences called VNTRs (variable number of tandem repeats) are easily
detectable if they are between two restriction enzyme recognition sites.
Different individuals have different numbers of repeats. Each gets ________
sequences of repeats, one from the mother and one from the father.
Using PCR and gel electrophoresis, patterns for each individual can be
determined.
Biotechnology: Applications of DNA Manipulation
The many applications of DNA ________ include forensics and cases of contested
paternity.
DNA from a single cell is sufficient to determine the DNA fingerprint because
PCR can amplify a tiny amount of DNA in a few hours.
PCR is used in diagnosing infections in which the infectious agent is present
in small amounts.
Genetic diseases such as sickle-cell anemia are now diagnosable before they
manifest themselves.
Animation 16.1 Separating Fragments of DNA by Gel Electrophoresis
Animation 16.2 DNA Chip Technology
Video 16-01