Download Chapter 13

Chapter 13
Genetic Engineering
Selective Breeding
- Humans use selective breeding, which takes advantage of naturally occurring genetic variation in plants,
animals, and other organisms, to pass desired traits on to the next generation of organisms.
- hybridization, crossing dissimilar individuals to bring together the best of both organisms.
- Inbreeding is the continued breeding of individuals with similar characteristics.
Increasing Variation
- Selective breeding would be nearly impossible without the wide variation that is found in natural
populations.
- This is one of the reasons biologists are interested in preserving the diversity of plants and animals in the
wild.
- Breeders can increase the genetic variation in a population by inducing mutations, which are the ultimate
source of genetic variability.
Producing New Kinds of Bacteria
- Their small size enables millions of organisms to be treated with radiation or chemicals at the same time.
This increases the chances of producing a useful mutant.
Producing New Kinds of Plants
-
Drugs that prevent chromosomal separation during meiosis have been particularly useful in plant
breeding.
-
- Sometimes these drugs produce cells that have double or triple the normal number of chromosomes.
-
Polyploidy may produce new species of plants that are often larger and stronger than their diploid
relatives. (bananas and citrus fruits).
Manipulating DNA
-
animal and plant breeders could not control the modifications the genetic code of living things.
-
They were limited by the variation that already exists in nature.
-
the changes they produced in the DNA were random and unpredictable.
-
How are changes made to DNA? Scientists use their knowledge of the structure of DNA and its
chemical properties to study and change DNA molecules. Different techniques are used to
extract DNA from cells, to cut DNA into smaller pieces, to identify the sequence of bases in a
DNA molecule, and to make unlimited copies of DNA.
1
The Tools of Molecular Biology
In 1973, biochemists Stanley Cohen and Herbert Boyer constructed a creature that was part bacterium and
part frog
- isolating the gene that codes for ribosomal RNA from the DNA of a frog; they inserted it into the DNA
of the bacterium Escherichia coli.
- During transcription, the bacterium produced frog rRNA
- Never before had such a genetically altered organism existed.
The Basics of Genetic Engineenng
-
The Cohen and Boyer experiments were revolutionary
genetic engineering is the process used to isolate a gene from the DNA of one organism and transfer it
into the gene of another DNA molecule
Genetic engineering involves building recombinant DNA, a molecule made from pieces of the DNA
from separate organisms.
Every genetic engineering experiment presents unique problems, but all share four distinct steps:
1. Cleaving DNA or Cutting DNA : The DNA containing the gene of interest (the gene to be
transferred) is cut into fragments using, special enzymes that cleave, or separate, sequences of
nucleotides.
2. Producing recombinant DNA: recombinant DNA is when a DNA fragment is put into the
DNA of a vector, an agent that Is used to carry the fragment into another cell. Commonly
used vectors include viruses and plasmids A plasmid is a circular DNA molecule, usually
found in bacteria, that can replicate independently from the main chromosome.
3. Cloning cells: A culture of bacteria is infected with the fragment-containing vectors. Some of
the bacteria will take in the vectors. These cells are isolated and allowed to reproduce.
Growing a large number of genetically identical cells from a single cell is called cloning.
4. Screening cells or Separating DNA : Bacterial cells that have received the particular gene of
interest are identified and isolated.
2
How did Cohen and Boyer transfer the frog rRNA gene into the DNA of a bacterium?
Cleaving DNA
- Cohen and Boyer wanted to transfer a gene that codes for ribosomal RNA in the African clawed frog
- using bacterial enzymes called restriction enzymes that cleave DNA at specific sequences, produces a
set of small fragments of DNA
- These sequences are unusual because they are made of two strands of DNA that have the same
nucleotides running in opposite directions.
- For example, Cohen and Boyer used a restriction enzyme called EcoR1, which recognizes the sequence
GAATTC which has the complementary strand CTTAAG
- CTTAAG is the same as the original sequence written backward
- Many restriction enzymes do not make their incision in the center of the sequence; rather, the cut is
staggered, made to one side of the sequence.
- For example, in the sequence GAATTC, EcoR1 makes its cut after the nucleotide G
- The cuts produce fragments of DNA with short single strands dangling from each end
- Because these dangling tails are complementary to each other, they are called cohesive ends, or "sticky
ends."
- These ends can pair with each other and sealed with the aid of an enzyme called DNA ligase
- Or, the sticky ends can pair with any other DNA fragment cut by the same restriction enzyme because
these would have the same complementary sticky ends
- Any two fragments of DNA cut by the same restriction enzyme can be joined together because they have
the same complementary sequences at their ends
3
4
Producing Recombinant DNA
- Cohen and Boyer used the restriction enzyme EcoR1 to cut apart a plasmid for the vector to carry the
ribosomal RNA frog gene
- From this plasmid DNA they isolated the genes for plasmid DNA replication and resistant to the
antibiotic tetracycline
- both ends of this plasmid were cut by the same restriction enzyme
- so they could be joined together to form a circular molecule of DNA
- This plasmid was called pSC101
- Cohen and Boyer produced recombinant DNA by mixing the frog's DNA fragments and pSC101
-
-
-
5
Cloning Cells
-
after the recombinant DNA was made they treated growing cultures of bacteria
some of the cells take up the vectors
then the gene for tetracycline resistance became important
the only cells that were not killed by the antibiotic were those that were resistant to tetracycline because
they had taken up the vector
All bacterial cells without vectors were eliminated
The surviving cell were allowed to reproduce, forming clones, all of which had the recombinant DNA
Screening Cells
-
Cohen and Boyer eliminated the bacteria that did not contain the recombinant then they needed to test
each clone to see if the frog rRNA gene was indeed present.
The procedure for identifying a specific gene is known as the Southern blot
The Southern blot is a technique that uses radioactively labeled RNA or single-stranded DNA as a
"probe" to identify a specific gene.
In a Southern blot, the cloned DNA is first cleaved into fragments by restriction enzymes
The fragments are separated by gel electrophoresis a technique that uses an electrical field within a gel
to separate molecules in a mixture. Because DNA is negatively charged, the various fragments move
through the gel according to their size, forming a pattern of bands
The DNA fragments are then split into single-stranded DNA, which is then blotted onto filter paper
Afterward, the filter paper is moistened with a solution containing the radioactively labeled probes
Among the thousands of DNA fragments, only the fragments that contain the gene of interest will bind
with the probes, because of the complementary nucleotide sequence
all genetic engineering experiments employ the same basic strategy Cohen and Boyer used in this first
successful gene transfer.
6
7
Using the DNA Sequence
-
Once DNA is in a manageable form, its sequence can be read, studied, and even changed.
Knowing the sequence of an organism's DNA allows researchers to study specific genes, to compare
them with the genes of other organisms, and to try to discover the functions of different genes and gene
combinations.
Reading the Sequence
- Researchers “read” DNA by determining the order of its bases
- A strand of DNA whose sequence of bases is not known is placed in a test tube
- DNA polymerase uses the unknown strand as a template to make more new DNA strands
- The tricky part is that researchers also add a small number of bases that have a chemical dye attached
- Each time a dye-labeled base is added to a new DNA strand, the synthesis of that strand is terminated
- When DNA synthesis is completed, the new DNA strands are different lengths, depending on how far
synthesis had progressed when the dye-tagged base was added
- These fragments are then separated by length, often by gel electrophoresis
- The order of colored bands on the gel tells the exact sequence of bases in the DNA.
Cutting and Pasting
-
DNA sequences can be changed in a number of ways.
Short sequences can be assembled using laboratory machines known as DNA synthesizers.
“Synthetic” DNA sequences can then be joined to “natural” ones using enzymes that splice DNA
DNA molecules are sometimes called recombinant DNA because they are produced by combining DNA
from different sources.
Making Copies
- In order to study genes, biologists often need to make many copies of a particular gene.
- like a photocopy machine stuck on “print,” a technique known as polymerase chain reaction (PCR)
allows biologists to do exactly that.
8
Transforming Plant Cells
-
In nature, a bacterium inserts a small DNA plasmid that produces tumors into a plant's cells.
Researchers can inactivate the tumor-producing gene and insert a piece of foreign DNA into the plasmid.
The recombinant plasmid can then be used to infect plant cells
Transforming Animal Cells
-
Animal cells can be transformed in some of the same ways as plant cells
Many egg cells are large enough that DNA can be directly injected into the nucleus
Once inside the nucleus, enzymes normally responsible for DNA repair and recombination may help to
insert the foreign DNA into the chromosomes of the injected cell.
Like bacterial plasmids, the DNA molecules used for transformation of animal and plant cells contain
marker genes that enable biologists to identify which cells have been transformed.
it has become possible to eliminate particular genes
DNA molecules can be constructed with two ends that will sometimes recombine with specific sequences
in the host chromosome.
Once they do, the host gene normally found between those two sequences may be lost or specifically
replaced with a new gene.
9
-
This kind of gene replacement has made it possible to pinpoint the specific functions of genes in many
organisms.
Applications of Genetic Engineering
-
Genetic engineering makes it possible to transfer DNA sequences, including whole genes, from one
organism to another.
Steven Howell isolated the gene for luciferase, an enzyme that allows fireflies to glow, and inserted it
into tobacco cells.
When whole plants were grown from the recombinant cells and the gene was activated, the plants glowed
in the dark
The gene for luciferase, which comes from an animal, can specify a trait in a plant demonstrating that the
basic mechanisms of gene expression are shared by plants and animals.
Transgenic Organisms
-
transgenic, meaning that they contain genes from different species.
Genetic engineering has spurred the growth of biotechnology, which is a new industry that is
changing the way we interact with the living world.
Cloning
-
A clone is a member of a population of genetically identical cells produced from a single cell.
Cloned colonies of bacteria and other microorganisms are easy to grow, but this is not always true of
multicellular organisms, especially animals.
10
-
For many years, biologists wondered if it might be possible to clone a mammal—many scientists had
concluded that this was impossible.
-
In 1997, Scottish scientist Ian Wilmut stunned biologists by announcing that he had cloned a sheep.
In Wilmut's technique, the nucleus of an egg cell is removed. The cell is fused with a cell taken from
another adult. The fused cell begins to divide and the embryo is then placed in the reproductive system of
a foster mother, where it develops.
Cloned cows, pigs, mice, and other mammals have been produced by similar techniques.
Researchers hope that cloning will enable them to make copies of transgenic animals and even help save
endangered species.
On the other hand, the technology is controversial for many reasons, including studies suggesting that
cloned animals may suffer from a number of genetic defects and health problems.
-
11