Lecture Presentation to accompany Principles of...

Biotechnology

13

Concept 13.1 Recombinant DNA Can Be Made in the Laboratory

It is possible to modify organisms with

genes from other, distantly related

organisms.

Recombinant DNA is a DNA molecule

made in the laboratory that is derived from

at least two genetic sources.


Three key tools:

• Restriction enzymes for cutting DNA into

fragments

• Gel electrophoresis for analysis and

purification of DNA fragments

• DNA ligase for joining DNA fragments

together in new combinations


Restriction enzymes recognize a specific

DNA sequence called a recognition

sequence or restriction site.

5′…….GAATTC……3′

3′…….CTTAAG……5′

Each sequence forms a palindrome: the

opposite strands have the same sequence

when read from the 5′ end.

Figure 13.1 Bacteria Fight Invading Viruses by Making Restriction Enzymes


Some restriction enzymes cut DNA leaving a short sequence of single-stranded DNA at each end.

Staggered cuts result in overhangs, or “sticky ends;” straight cuts result in “blunt ends.”

Sticky ends can bind complementary sequences on other DNA molecules.

Methylases add methyl groups to restriction sites and protect the bacterial cell from its own restriction enzymes.


Many restriction enzymes with unique

recognition sequences have been purified.

In the lab they can be used to cut DNA

samples from the same source.

A restriction digest combines different

enzymes to cut DNA at specific places.

Gel electrophoresis analysis can create a

map of the intact DNA molecule from the

formed fragments.


DNA fragments cut by enzymes can be

separated by gel electrophoresis.

A mixture of fragments is placed in a well in

a semisolid gel, and an electric field is

applied across the gel.

Negatively charged DNA fragments move

towards the positive end.

Smaller fragments move faster than larger

ones.


DNA fragments separate and give three

types of information:

• The number of fragments

• The sizes of the fragments

• The relative abundance of the fragments,

indicated by the intensity of the band

Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 1)

Figure 13.2 Separating Fragments of DNA by Gel Electrophoresis (Part 2)


After separation on a gel, a specific DNA

sequence can be found with a single-

stranded probe.

The gel region can be cut out and the DNA

fragment removed.

The purified DNA can be analyzed by

sequence or used to make recombinant

DNA.


DNA ligase is an enzyme that catalyzes the

joining of DNA fragments, such as Okazaki

fragments during replication.

With restriction enzymes to cut fragments

and DNA ligase to combine them, new

recombinant DNA can be made.

Figure 13.3 Cutting, Splicing, and Joining DNA


Recombinant DNA was shown to be a

functional carrier of genetic information.

Sequences from two E.coli plasmids, each

with different antibiotic resistance genes,

were recombined.

The resulting plasmid, when inserted into

new cells, gave resistance to both of the

antibiotics.

Figure 13.4 Recombinant DNA (Part 1)

Concept 13.2 DNA Can Genetically Transform Cells and

Organisms

Recombinant DNA technology can be used

to clone (make identical copies) genes.

Transformation: Recombinant DNA is

cloned by inserting it into host cells

(transfection if host cells are from an

animal).

The altered host cell is called transgenic.


Organisms

Usually only a few cells exposed to

recombinant DNA are actually

transformed.

To determine which of the host cells are

transgenic, the recombinant DNA includes

selectable marker genes, such as genes

that confer resistance to antibiotics.


Organisms

Most research has been done using model organisms:

• Bacteria, especially E. coli

• Yeasts (Saccharomyces), commonly used as eukaryotic hosts

• Plant cells, able to make stem cells—unspecialized, totipotent cells

• Cultured animal cells, used for expression of human or animal genes—whole transgenic animals can be created


Organisms

Methods for inserting the recombinant DNA

into a cell:

• Cells may be treated with chemicals to

make plasma membranes more

permeable—DNA diffuses in.

• Electroporation—a short electric shock

creates temporary pores in membranes,

and DNA can enter.


Organisms

• Viruses and bacteria can be altered to

carry recombinant DNA into cells.

• Transgenic animals can be produced by

injecting recombinant DNA into the nuclei

of fertilized eggs.

• “Gene guns” can “shoot” the host cells with

particles of DNA.


Organisms

The new DNA must also replicate as the

host cell divides.

DNA polymerase does not bind to just any

sequence.

The new DNA must become part of a

segment with an origin of replication—a

replicon or replication unit.


Organisms

New DNA can become part of a replicon in

two ways:

• Inserted near an origin of replication in

host chromosome

• It can be part of a carrier sequence, or

vector, that already has an origin of

replication


Organisms

Plasmids make good vectors:

• Small and easy to manipulate

• Have one or more restriction enzyme

recognition sequences that each occur

only once

• Many have genes for antibiotic resistance

which can be selectable markers


Organisms

• Have a bacterial origin of replication (ori)

and can replicate independently of the

host chromosome

Bacterial cells can contain hundreds of

copies of a recombinant plasmid. The

power of bacterial transformation to

amplify a gene is extraordinary.

In-Text Art, Ch. 13, p. 249


Organisms

A plasmid from the soil bacterium

Agrobacterium tumefaciens is used as a

vector for plant cells.

A. tumefaciens contains a plasmid called Ti

(for tumor-inducing).

The plasmid has a region called T DNA,

which inserts copies of itself into

chromosomes of infected plants.


Organisms

T DNA genes are removed and replaced

with foreign DNA.

Altered Ti plasmids transform

Agrobacterium cells, then the bacterium

cells infect plant cells.

Whole plants can be regenerated from

transgenic cells, or germ line cells can be

infected.

In-Text Art, Ch. 13, p. 250


Organisms

Most eukaryotic genes are too large to be

inserted into a plasmid.

Viruses can be used as vectors—e.g.,

bacteriophage. The genes that cause host

cells to lyse can be cut out and replaced

with other DNA.

Because viruses infect cells naturally they

offer an advantage over plasmids.


Organisms

Usually only a small proportion of host cells

take up the vector (1 cell in 10,000) and

they may not have the appropriate

sequence.

Host cells with the desired sequence must

be identifiable.

Selectable markers such as antibiotic

resistance genes can be used.


Organisms

If a vector carrying genes for resistance to

two different antibiotics is used, one

antibiotic can select cells carrying the

vector.

If the other antibiotic resistance gene is

inactivated by the insertion of foreign DNA,

then cells with the desired DNA can be

identified by their sensitivity to that

antibiotic.

Figure 13.5 Marking Recombinant DNA by Inactivating a Gene


Organisms

Selectable markers are a type of reporter

gene—a gene whose expression is easily

observed.

Green fluorescent protein, which normally

occurs in a jellyfish, emits visible light

when exposed to UV light.

The gene for this protein has been isolated

and incorporated into vectors as a reporter

gene.

Figure 13.6 Green Fluorescent Protein as a Reporter

Concept 13.3 Genes and Gene Expression Can Be Manipulated

DNA fragments used for cloning come from

three sources:

• Gene libraries

• Reverse transcription from mRNA

• Products of PCR

• Artificial synthesis or mutation of DNA


A genomic library is a collection of DNA

fragments that comprise the genome of an

organism.

The DNA is cut into fragments by restriction

enzymes, and each fragment is inserted

into a vector.

A vector is taken up by host cells which

produce a colony of recombinant cells.


Smaller DNA libraries can be made from complementary DNA (cDNA).

mRNA is extracted from cells, then cDNA is produced by complementary base pairing, catalyzed by reverse transcriptase.

A cDNA library is a “snapshot” of the transcription pattern of the cell.

cDNA libraries are used to compare gene expression in different tissues at different stages of development.

Figure 13.7 Constructing Libraries


DNA can be synthesized by PCR if

appropriate primers are available.

The amplified DNA can then be inserted into

plasmids to create recombinant DNA and

cloned in host cells.

Artificial synthesis of DNA is now fully

automated.


Synthetic oligonucleotides are used as

primers in PCR reactions.

Primers can create new sequences to

create mutations in a recombinant gene.

Longer synthetic sequences can be used to

construct an artificial gene.


Synthetic DNA can be manipulated to create

specific mutations in order to study the

consequences of the mutation.

Mutagenesis techniques have revealed

many cause-and-effect relationships (e.g.,

determining signal sequences).


A knockout experiment inactivates a gene so that it is not transcribed and translated into a functional protein.

In mice, homologous recombination targets a specific gene.

The normal allele of a gene is inserted into a plasmid—restriction enzymes are used to insert a reporter gene into the normal gene.

The extra DNA prevents functional mRNA from being made.


The recombinant plasmid is used to

transfect mouse embryonic stem cells.

Stem cells—unspecialized cells that divide

and differentiate into specialized cells

The original gene sequences line up with

their homologous sequences on the

mouse chromosome.


The transfected stem cell is then

transplanted into an early mouse embryo.

The knockout technique has been important

in determining gene functions and studying

human genetic diseases.

Many diseases have a knockout mouse

model.

Figure 13.8 Making a Knockout Mouse


Complementary RNA:

Translation of mRNA can be blocked by

complementary microRNAs—antisense

RNA.

Antisense RNA can be synthesized and

added to cells to prevent translation—the

effects of the missing protein can then be

determined.


RNA interference (RNAi) is a rare natural mechanism that blocks translation.

RNAi occurs via the action of small interfering RNAs (siRNAs).

An sRNA is a short, double stranded RNA that is unwound to single strands by a protein complex, which also catalyzes the breakdown of the mRNA.

Small interfering RNA (siRNA) can be synthesized in the laboratory.

Figure 13.9 Using Antisense RNA and siRNA to Block the Translation of mRNA


DNA microarray technology provides a large

array of sequences for hybridization

experiments.

A series of DNA sequences are attached to

a glass slide in a precise order.

The slide has microscopic wells, each

containing thousands of copies of

sequences up to 20 nucleotides long.


DNA microarrays can be used to identify

specific single nucleotide polymorphisms

or other mutations.

Microarrays can be used to examine gene

expression patterns in different tissues in

different conditions.

Example: Women with a propensity for

breast cancer tumors to recur have a gene

expression signature.

Figure 13.10 Using DNA Microarrays for Clinical Decision-Making

Concept 13.4 Biotechnology Has Wide Applications

Almost any gene can be inserted into

bacteria or yeasts and the resulting cells

induced to make large quantities of a

product.

Requires specialized expression vectors

with extra sequences needed for the

transgene to be expressed in the host cell.

Figure 13.11 A Transgenic Cell Can Produce Large Amounts of the Transgene’s Protein Product


Expression vectors may also have:

• Inducible promoters that respond to a

specific signal

• Tissue-specific promoters, expressed only

in certain tissues at certain times

• Signal sequences—e.g., a signal to

secrete the product to the extracellular

medium


Many medically useful products are being

made using biotechnology.

The two insulin polypeptides are

synthesized separately along with the β-

galactosidase gene.

After synthesis the polypeptides are

cleaved, and the two insulin peptides

combined to make a functional human

insulin molecule.

Figure 13.12 Human Insulin: From Gene to Drug (Part 1)

Figure 13.12 Human Insulin: From Gene to Drug (Part 2)


Before giving it to humans, scientists had to

be sure of its effectiveness:

• Same size as human insulin

• Same amino acid sequence

• Same shape

• Binds to the insulin receptor on cells and

stimulates glucose uptake


Pharming: Production of pharmaceuticals in

farm animals or plants.

Example: Transgenes are inserted next to

the promoter for lactoglobulin—a protein in

milk. The transgenic animal then produces

large quantities of the protein in its milk.

Figure 13.13 Pharming


Human growth hormone (for children

suffering deficiencies) can now be

produced by transgenic cows.

Only 15 such cows are needed to supply all

the children in the world suffering from this

type of dwarfism.


Through cultivation and selective breeding,

humans have been altering the traits of

plants and animals for thousands of years.

Recombinant DNA technology has several

advantages:

• Specific genes can be targeted

• Any gene can be introduced into any other

organism

• New organisms can be generated quickly

Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding (Part 1)

Figure 13.14 Genetic Modification of Plants versus Conventional Plant Breeding (Part 2)

Table 13.2 Potential Agricultural Applications of Biotechnology


Crop plants have been modified to produce

their own insecticides:

• The bacterium Bacillus thuringiensis

produces a protein that kills insect larvae

• Dried preparations of B. thuringiensis are

sold as a safe alternative to synthetic

insecticides. The toxin is easily

biodegradable.


• Genes for the toxin have been isolated,

cloned, and modified, and inserted into

plant cells using the Ti plasmid vector

• Transgenic corn, cotton, soybeans,

tomatoes, and other crops are being

grown. Pesticide use is reduced.


Crops with improved nutritional

characteristics:

• Rice does not have β-carotene, but does

have a precursor molecule

• Genes for enzymes that synthesize β-

carotene from the precursor are taken

from daffodils and inserted into rice by the

Ti plasmid


• The transgenic rice is yellow and can

supply β-carotene to improve the diets of

many people

• β-carotene is converted to vitamin A in the

body

Figure 13.15 Transgenic Rice Rich in -Carotene


Recombinant DNA is also used to adapt a

crop plant to an environment.

Example: Plants that are salt-tolerant.

Genes from a protein that moves sodium

ions into the central vacuole were isolated

from Arabidopsis thaliana and inserted into

tomato plants.

Figure 13.16 Salt-tolerant Tomato Plants (Part 1)

Figure 13.16 Salt-tolerant Tomato Plants (Part 2)


Instead of manipulating the environment to

suit the plant, biotechnology may allow us

to adapt the plant to the environment.

Some of the negative effects of agriculture,

such as water pollution, could be reduced.


Concerns over biotechnology:

• Genetic manipulation is an unnatural

interference in nature

• Genetically altered foods are unsafe to eat

• Genetically altered crop plants are

dangerous to the environment


Advocates of biotechnology point out that all

crop plants have been manipulated by

humans.

Advocates say that since only single genes

for plant function are inserted into crop

plants, they are still safe for human

consumption.

Genes that affect human nutrition may raise

more concerns.


Concern over environmental effects centers

on escape of transgenes into wild

populations:

• For example, if the gene for herbicide

resistance made its way into the weed

plants

• Beneficial insects can also be killed from

eating plants with B. thuringiensis genes

Answer to Opening Question

Bioremediation is the use, by humans, of

organisms to remove contaminants from

the environment.

Composting and wastewater treatment use

bacteria to break down large molecules,

human wastes, paper, and household

chemicals.

Recombinant DNA technology has

transformed bacteria to help clean up oil

spills.

Figure 13.17 The Spoils of War

Date post:	27-Jul-2020
Category:	Documents
Upload:	others
View:	0 times
Download:	0 times

Lecture Presentation to accompany Principles of...

Documents