Chap. 5 Molecular Genetic Techniques (Part C)

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Chap. 5 Molecular Genetic Techniques (Part C)

• Topics
• Identifying and Locating Human Disease Genes
• Inactivating the Function of Specific Genes in
  Eukaryotes

Goals Learn genetic and recombinant DNA methods
for isolating genes and characterizing the
functions of the proteins they encode.
Use of RNA interference (RNAi) in analysis of
planarian regeneration
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Overview of Identifying Locating Human Disease
Genes
Human geneticists use multiple approaches to find
genes responsible for inherited diseases.
Typically, the family inheritance pattern of the
disease initially is determined (Fig. 5.35).
Subsequently, researchers try to find genetic
markers (e.g., SNPs, Fig. 5.36) that consistently
are linked to the disease. This information and
sometimes linkage disequilibrium analysis (Fig.
5.37) further helps investigators home in on the
gene's approximate chromosomal location. Finally,
DNA sequencing and other procedures are used to
pinpoint the location of the responsible gene.
The relationship between the cytogenetic and
physical maps of a human chromosome is
illustrated in Fig. 5.38.
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Common Hereditary Human Diseases
Most inherited diseases are caused by preexisting
mutant alleles that have been passed down from
one generation to the next. Examples of common
autosomal recessive, autosomal dominant, and
X-linked recessive human diseases of monogenic
origin are listed in Table 5.2. At least some
fraction of diseases such as cancers, diabetes,
obesity, and heart disease are hereditary and
polygenic in origin. The molecular bases of these
diseases are even harder to solve than those of
monogenic diseases.



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Human Disease Inheritance Patterns
One of the first procedures performed in
identifying a disease gene is to establish its
inheritance pattern in affected families (Fig.
5.35). Analysis of inheritance patterns can
quickly establish if the disease gene is carried
on an autosome or a sex chromosome. Examples of
inheritance patterns (family segregation) for
autosomal dominant (Huntington's disease),
autosomal recessive (cystic fibrosis), and
X-linked recessive (Duchenne's muscular
dystrophy) genetic diseases are
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25
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illustrated in the diagram. In autosomal dominant
diseases, male and female children each have a
50 chance of developing the disease. In
autosomal recessive diseases, both sexes have a
25 chance of developing the disease. In X-linked
recessive diseases, males have a 50 chance of
developing the disease. In this case, the
defective allele is inherited from the mother.
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Human Karyotype Metaphase Chromosomes
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Linkage Mapping Using Molecular Markers
The next step in gene ID is to genetically map
its position with respect to known genetic
markers in the genome. This method can be
performed by breeding studies in simple
experimental organisms in which genetic markers
confer readily detectable phenotypes. However
such phenotypic markers are uncommon in humans,
and instead DNA-based molecular markers are used.
Molecular markers caused by DNA polymorphisms
(sequence differences) occur at a frequency of
about 1/1,000 nucleotides. Polymorphisms are used
as landmarks in locating the position of a
disease gene. In some cases, polymorphisms change
the locations of restriction sites. This results
in restriction fragment length polymorphisms
(RLFPs) which can be used in linkage studies.
Other DNA polymorphisms do not affect restriction
sites. These molecular markers--called single
nucleotide polymorphisms (SNPs) and simple
sequence repeats (SSRs)--can be identified and
studied by PCR amplification and sequencing of
genomic DNA.
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SNP Analysis of Pedigrees
The use of SNP analysis in following disease gene
inheritance in a family is illustrated in Fig.
5.36. In the family shown, the region of the
chromosome being studied occurs in 3 forms based
on the 3 different SNPs observed via sequencing
of this region (A, T, or C). The analysis
indicates that the disease trait segregates with
a C at the SNP site.
Currently, about 104 DNA polymorphisms have been
mapped in the human genome. Researchers screen
DNA from individuals with genetic diseases to try
to find polymorphisms that are linked to a
disease gene. Pooled data from multiple families
helps in identifying polymorphisms linked to the
gene. SNP analysis also is used in diagnostics
and genetic counseling.
Key Circles indicate females squares indicate
males. Blue indicates unaffected individuals
orange indicates individuals with the disease.
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Linkage Mapping of Disease Gene Location by
Recombination Analysis
Although it is not commonly used in analysis of
human diseases, it is instructive to consider the
procedure known as recombination analysis (Fig.
5.10), which is often applied in linkage analysis
in simple model organisms. This method relies on
the facts that, phenotypic traits that segregate
together during meiosis more frequently than
expected based on random segregation typically
are specified by genes residing on the same
chromosome. In addition, the less frequently
recombination occurs between two markers on a
chromosome, the more tightly they are linked and
the closer together they are. One genetic map
unit is defined as the distance between two genes
along a chromosome that results in a 1 (1/100
gametes) recombination frequency (1 centimorgan,
cM). In humans, 1 cM corresponds to a physical
distance of 750,000 bp.
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Linkage Disequilibrium Analysis of Disease Gene
Location
In linkage disequilibrim analysis, investigators
map the location of a disease gene on a
chromosome by looking for regions that maintain
the same grouping of linked genetic markers as in
the ancestral chromosome in which the disease
gene arose (Fig. 5.37). Markers that are unlinked
to the mutant gene recombine extensively over
generations, whereas markers closely linked to
the disease allele do not. Linkage disequilibrium
analysis can sometimes improve the resolution of
genetic mapping studies to 0.1 cM.
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Final Steps of Mutant Gene Isolation
Often mutations can be mapped only to 1 cM
regions of human DNA using the methods discussed
above (Fig. 5.38). Regions of this length can
contain dozens of genes. The final identification
of the disease gene typically involves sequencing
and mapping of all SNPs, etc. in a long region of
DNA. The responsible gene is likely to be located
in regions where SNPs associated with the disease
consistently are found in a number of affected
individuals. The mutation itself eventually is
identified by DNA sequencing. The analysis of
gene expression by Northern blotting and in situ
hybridization in affected tissues also may help
in identifying a disease gene in cases where
grossly defective mRNA transcripts are produced
from a gene.
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Leptin Receptor Knockout Mice
db/db DB/DB
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Gene Replacement by Homologous Recombination
In bacteria and yeast, specific genes can be
replaced or disrupted by homologous recombination
methods (Fig. 5.39). To disrupt yeast genes, a
disruption construct consisting of the kanMX
antibiotic resistance marker fused to 20-nt
sequences that flank the targeted gene is made by
PCR and transformed into diploid yeast.
Recombinants in which the disruption construct
has replaced one wild type allele are selected by
plating cells on G-418. On sporulation, half of
the haploid spores receive the disrupted gene. If
the gene is essential, these two spores will be
nonviable. This method has been used to show that
only 1,500 of the total 6,000 yeast genes are
essential at least under laboratory conditions.
Synthetic lethality screens are now being
conducted to group genes with redundant functions.
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Knockout Mice Stem Cell Manipulations I
In knockout mice, both copies of a gene of
interest are disrupted by targeted homologous
recombination. First, a DNA construct containing
a disrupted allele of a particular gene (X) is
introduced into embryonic stem (ES) cells (Fig.
5.40a). In a small percentage of cells,
homologous recombination occurs and the target
gene is replaced with a copy of the neo r gene
(G-148 resistance). In most cells, the DNA
construct recombines nonhomologously
and inserts at another site in the genome. The
entire DNA construct typically inserts into the
genome via nonhomologous recombination. This
results in cointegration of neo r and a copy of
the herpes simplex virus thymidine kinase gene
(tkHSV).
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Knockout Mice Stem Cell Manipulations II
ES cells carrying the neo r gene then are
selected by growing the mixed population of cells
on G-148 (positive selection) (Fig. 5.40b).
Subsequently, cells obtained by nonhomologous
recombination are eliminated (negative selection)
by growing cells in the presence of the
nucleoside analog, ganciclovir. Ganciclovir is
converted into a toxic nucleotide by the tkHSV
gene product which blocks DNA replication in
these cells. The resulting culture contains only
ES cells with a targeted disruption in one copy
of gene X (genotype X-/X).
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Knockout Mice In Vivo Manipulations I
In the next step, the ES cells are injected into
the blastocoel cavity of a 4.5-day old mouse
embryo (Fig. 5.41). The embryo then is introduced
into a pseudopregnant female (foster mother) who
gives birth to some chimeric progeny containing
the knockout gene. In chimeras, different tissues
have different genotypes.To facilitate isolation
of genetically pure lines of knockout mice, the
original ES cells are obtained from dominant
brown (A) mice of the A/A, X-/X genotype. A
recessive black (a) mice of the a/a, X/X
genotype is the source of the blastocyst.
Homozygous knockout mice are isolated after
performing the crosses shown on the next two
slides.
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Knockout Mice In Vivo Manipulations II
In the first cross, a chimeric mouse is mated
with a black mouse that is homozygous for the
wild type X allele. Possible germ cells from the
chimeric mouse are shown in the figure. The brown
progeny of this cross will be derived from the ES
cells. Repeated matings and genotype screenings
are performed until X-/X- homozygous knockout
mice are obtained.
In many cases, germ-line knockout mice are
nonviable. Thus, alternative strategies for
knocking out a gene of interest in a later stage
of life have been developed (Fig. 5.42, not
covered).
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Transgenic Mice
In knockout mice, both copies of a wild type
allele are inactivated to observe a recessive
trait. In transgenic mice, an allele specifying a
dominant negative mutation is introduced into the
germ line DNA. Animals need express only one copy
of such an allele to exhibit a phenotype. The
method for introduction of transgenes into mice
is shown in Fig. 5.43. A copy of the dominant
negative allele typically inserts into the genome
via nonhomologous recombination. Often the
transgene is controlled by a regulated promoter,
allowing it to be expressed in certain tissues at
certain times.
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Example of a Dominant Negative Allele
Only one copy of a dominant negative allele is
required to cause a loss-of-function phenotype in
a diploid organism. A classic example of this
type of mutation is shown in Fig. 5.44. Certain
mutant forms of "small GTPases" form extremely
long-lived complexes with GEF proteins (guanine
nucleotide exchange factors). GEF proteins
catalyze exchange of GTP for GDP and activation
of GTPases. In cells expressing both the wild
type and dominant-negative GTPase alleles, all
copies of GEF become complexed with the mutant
GTPase, blocking the switching function of the
wild type GTPase.
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RNA Interference (RNAi)
In RNA interference, short interfering RNAs
(siRNAs, 21 nts) produced from longer dsRNAs
specifically block gene expression by binding to
a target mRNA and triggering its degradation.
dsRNAs can be transcribed in vitro and injected
into an embryo, for example, where processing by
the enzyme known as dicer produces the siRNA
(Fig. 5.45 a b). Alternatively, dsRNA can be
expressed in vivo in response to some signal.
Subsequent processing to siRNA by dicer then
triggers mRNA degradation (Fig. 5.45c).
RNAi-mediated gene inactivation is commonly
applied to silence gene expression in C. elegans,
Drosophila, plants, and even mice. The mechanism
by which siRNAs cause mRNA degradation is covered
in Chap. 8.