Transcript Brooker Chapter 19

PowerPoint Presentation Materials
to accompany
Genetics: Analysis and Principles
Robert J. Brooker
CHAPTER 19
BIOTECHNOLOGY
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
INTRODUCTION

Biotechnology is broadly defined as technologies that involve
the use of living organisms, or their products, to benefit
humans

It is not a new topic


It began about 12,000 years ago when humans began to domesticate
animal and plants for the production of food
Since the 1970s, molecular genetics has provided new,
improved ways to make use of organisms to benefit humans

An organism that has integrated recombinant DNA into its genome is
called transgenic
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-2
19.1 USES OF
MICROORGANISMS
IN BIOTECHNOLGY

Microorganisms are used to benefit humans in various ways

Refer to Table 19.1

Molecular genetic tools are very important in influencing and
improving our use of microorganisms

Overall, the use of recombinant microorganisms is an area of
great research interest and potential

However, there are problems such as safety concerns and negative
public perception
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-3
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-4
Many Important Medicines Are Produced
by Recombinant Microorganisms
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-16
Insulin

Insulin regulates several physiological processes,
particularly the uptake of glucose into fat and muscle cells


Persons with insulin-dependent diabetes have a defect in
their b cells




Therefore, they cannot synthesize enough insulin
Sources of insulin included


It is produced by the b cells of the pancreas
Cows
Human cadavers!
But now, patients can use insulin made by recombinant
bacteria
Refer to Figure 19.2
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-17

Insulin is a hormone composed of two polypeptide chains,
called the A and B chains
Figure 19.2 The use of bacteria to make human insulin
19-18
Transgenic animals
Gene
replacemen
t
Gene
addition
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Fig. 19.4
Gene Addition versus
Gene Replacement

Cloned genes can be introduced into plant and
animal cells

However, the gene will not be inherited stably if it does
not become integrated into the host cell’s genome


This integration occurs by recombination
The introduction of a cloned gene into a cell can
lead to one of two outcomes

Gene replacement
Gene addition

Refer to Figure 19.5

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-26
If rendered inactive
by mutation =>
gene knockout
Figure 19.5
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-27
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
Production of Mice That Contain
Gene Replacements

In bacteria and yeast, gene replacement is the
common outcome


These have relatively small genomes, so homologous
recombination occurs at a relatively high rate
In complex eukaryotes, gene addition is the norm

These have very large genomes, so homologous
recombination is rare

Only 0.1% of the time
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-28

To produce mice with gene replacements, molecular
biologists have resorted to trickery


Cells in which homologous recombination has occurred
are preferentially selected
This approach is shown in Figure 19.6

The cloned gene is altered using two selectable markers


A neomycin-resistant gene (NeoR) is inserted into the center of the
coding sequence of the target gene
A thymidine-kinase gene (TK) is inserted adjacent (not within) the
target gene


TK renders cells sensitive to killing by a drug called gancyclovir
The modified target gene is then introduced into mouse
embryonic cells which can be grown in the lab
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-29
Figure 19.6
Sensitive to
gancyclovir
Resistant to
both drugs
19-30
Figure 19.6
Blastocyst
A chimera is an organism
that contains cells from
two different individuals
Chimeric offspring
19-30
Use of Gene Replacements in Mice

Gene replacements were discussed in Figure 19.6

When a mouse is homozygous for an inactivated
gene, this is called a gene knockout


The inactive mutant gene has replaced both copies of the
normal gene
Gene replacements and gene knockouts have
become powerful tools for understanding gene
function
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-53

In some cases, gene knockouts produce phenotypic effect


This shows that a gene is critical in a certain tissue or during a specific
stage of development
In other cases, gene knockouts produce no detectable
phenotypic effect at all

This led to the conclusion that mammalian genomes have a fair
amount of gene redundancy

A particularly exciting avenue of gene replacement research
is its application in the study of human disease

Cystic fibrosis (CF)



Researchers have produced mice that are homozygous for the same type
of mutation that is found in humans with CF
These can be used as models organisms to study this human disease
Furthermore, these mice models have been used to test various
treatments for the disease
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-54
Transgenic Livestock
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-56

The production of proteins from mammals is
more advantageous than the from bacteria

1. Certain proteins are more likely to function
properly when expressed in mammals



Post-translational modifications occur in eukaryotes
Degradation and misfolding occur in bacteria
The strategy for expressing human genes in
animals is shown in Figure 19.13
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-57
And use it to treat humans
Figure 19.13
19-58
19.4 DNA FINGERPRINTING

DNA fingerprinting is a technology that identifies
particular individuals using properties of their DNA

It is also termed DNA profiling

The application of DNA fingerprinting to forensics
has captured the most public attention

In addition, DNA fingerprinting can also be used to
determine if two individuals are genetically related

For example, it is used routinely in paternity testing
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-62

When subjected to DNA fingerprinting, chromosomal
DNA gives rise to a series of bands on a gel


Refer to Figure 19.16
The order of bands is an individual’s DNA fingerprint


It is the unique pattern of these bands that makes it possible to
distinguish individuals
In the 1980s, Alec Jeffries found that certain loci in
human chromosomes are variable in length

These loci contain tandemly repeated sequences called
minisatellites

In humans, the number of tandem repeats varies substantially


Variable Number of Tandem Repeats (VNTRs)
Refer to Figure 19.17
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-63
Restriction enzyme sites
VNTRs
DNA probes are used
to hybridize
specifically to the
repeat sequence
located within VNTRs
Figure 19.17
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-64
Figure 19.18 Protocol
for DNA fingerprinting
19-65
The probe is called a
multilocus probe (MLS)
It binds to ~ 20 to 40
fragments of DNA that
contain the sequence
Figure 19.18 Protocol
for DNA fingerprinting
19-66
RFLPs as markers for diseasecausing alleles
RFLP marker
DNA
Disease-causing
allele
Normal allele

In the past decade, the technique of DNA
fingerprinting has become automated



It is now done using PCR, which amplifies short tandem
repeat sequences (STRs)
Like VNTRs, STRs are found in multiple sites in human
genomes and are variable among different individuals
The main difference between a VNTR and STR is size






STRs are much shorter, usually 100–450 bp
STRs are called microsatellites, and VNTRs minisatellites
The amplified STRs are fluorescently labeled
They are separated by gel electrophoresis
A laser excites the fluorescent molecule within the STR
A detector records the amount of emission for each STR
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-67
Each peak has a
characteristic mass
The pattern of peaks is an
individual’s DNA fingerprint
Figure 19.18 Protocol
for DNA fingerprinting
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-68
Uses of DNA Fingerprinting

Within the past decade, the uses of DNA
fingerprinting have expanded in many ways

In medicine, it is used identify different species of bacteria
and fungi and also different strains of the same species


This is useful for appropriate antibiotic treatment
DNA fingerprinting is also used in forensics and
relationship testing
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-69
Male 2 does not have
many of the child’s
paternal bands
Therefore he cannot
be the biological father
Male 1 has all the child’s
paternal bands
The probability of this
occurring by chance alone
is very small
Therefore he is the
biological father
Figure 19.19 The use of DNA fingerprinting to establish paternity
Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
19-71
DNA fingerprints from a murder case
Defendant’s
blood (D)
Blood from
defendant’s
clothes
4 g
D
Jeans
Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings
shirt
Victim’s
blood (V)
8 g
V