ppt

1. Chap. 5 Molecular Genetic Techniques
(Part A)
Topics
• Genetic Analysis of Mutations to Identify and Study Genes
• DNA Cloning and Characterization
Goals
Learn about 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

2. Leptin Receptor Knockout Mice
db/db DB/DB

3. Importance of Mutations in Gene Analysis
One of the most important ways in which the function of a gene can be
learned is by the study of a mutant in which the gene has been
inactivated. Currently, mutants can be generated by classical “forward”
genetic methods, and by more modern “reverse” genetic approaches (Fig.
5.1). In forward genetic analyses one generates a mutant organism and
then uses molecular biological techniques to isolate the mutant gene and
characterize the protein responsible for the phenotype of the mutant. In
reverse genetic approaches, a gene is inactivated and the function of the
gene is learned by study of the properties of the mutant organism.

4. Alleles-Different versions (sequences) of a gene.
Mutant-Newly created allele made by mutagenesis.
Genotype-The complete set of alleles for all genes carried by
an individual.
Wild type-Standard reference genotype. Most common allele
for a certain trait.
Phenotype-Observable trait specified by the genotype.
Point mutation-A change in a single base pair (e.g., a G.C to
A.T transition).
Silent mutation-A point mutation in a codon that does not
change the specified amino acid.
Missense mutation-A point mutation that changes the encoded
amino acid.
Nonsense mutation-A point mutation that introduces a
premature stop codon into the coding sequence of a gene.
Recessive & dominant mutant alleles-(next slide)
Genetics Terms

5. Recessive and Dominant Mutant Alleles
Diploid organisms have two copies of each gene; haploid organisms
(e.g., some unicellular organisms) contain only one. A recessive
mutant allele must be present in two copies (be homozygous) to
cause a phenotype in a diploid organism (Fig. 5.2). Only one copy
of a recessive allele must be present for the phenotype to be
observable in a haploid organism. In contrast, a dominant mutant
allele needs to be present in only one copy (heterozygous) in a
diploid organism for the phenotype to be observable. Most
recessive alleles cause gene inactivation and phenotypic loss of
function. Some dominant alleles change or increase activity causing
a gain of function. However, a dominant affect can be caused by
gene inactivation if two copies of the gene are needed for proper
function (haplo-insufficiency). Lastly, a dominant negative mutation
refers to a situation where the product of the mutant gene
inactivates the product of the wild-type gene. This can occur if a
gene encodes one subunit of an oligomeric protein.

6. Review of Mitosis
Mating experiments provide
important information about gene
function. These experiments
demand a thorough knowledge of
meiosis and production of
gametes (sperm & egg cells in
higher eukaryotes). In Fig. 5.3,
mitosis is described to contrast
it with meiosis. In mitosis, one
round of DNA replication in a
diploid somatic cell is followed by
one cell division. The paternal
and maternal homologous
chromosomes (homologs) first are
duplicated. The sister
chromatids then are separated
by a cell division. The daughter
cells end up with one copy of
each paternal and maternal
chromosome and are diploid (2n).

7. Review of Meiosis
In meiosis, one round of DNA
replication in a diploid germ cell is
followed by two cell divisions,
resulting in four haploid gametes
(Fig. 5.3). Paternal and maternal
homologous chromosomes first are
copied as in mitosis. However, after
alignment (synapsis) and crossing
over (recombination) of homologous
chromosomes, paternal and maternal
chromosomes are randomly
segregated between the daughter
cells formed in the first cell
division. Subsequently, the sister
chromatids of each chromosome are
separated in a second cell division,
which produces the gametes (1n).
The two sets of gametes each
contain a random assortment of the
paternal and maternal chromosomes.

8. Identification of Dominant Mutations
Dominant mutations can be
identified by mating strains
that each are homozygous
for two alleles of a given
gene. Because all gametes
from each parent are of
one type (Fig. 5.4a), all
members of the first filial
generation from the cross
(F1) necessarily will be
heterozygotes. If the
mutation is dominant, all F1
offspring will display the
mutant phenotype. On self
crossing of F1 cells, 3/4 of
the second filial generation
(F2) will display the mutant
phenotype, if it is dominant.

9. Identification of Recessive Mutations
Recessive mutations also can
be identified by mating
strains that are homozygous
for two alleles of a given
gene. Again, because all
gametes from each parent are
of one type (Fig. 5.4b), all
members of the F1 generation
from the cross necessarily
will be heterozygotes. None
of the F1 offspring will
display the mutant phenotype
if it is recessive. On self
crossing of F1 cells, only 1/4
of the F2 generation will
display the phenotype, if it is
recessive.

10. Analysis of Mutant Alleles in Yeast
The yeast Saccharomyces
cerevisiae is an ideal
experimental organism for
analysis of dominant and
recessive alleles. First, cells
can exist in either a haploid
or diploid state. Second,
haploid cells occur in two
mating types (a and a) that
are useful for performing
crosses. The diploid cells
resulting from matings can
be examined to determine if
a mutation is dominant or
recessive (Fig. 5.5). Finally,
haploid cells can be
regenerated by meiotic
sporulation of diploid cells
grown under starvation
conditions.

11. Use of Conditional Mutations to Study
Essential Genes
The study of essential genes
(needed for life) requires special
genetic screening techniques. In
diploid organisms, such as the
fruit fly Drosophila, lethal
mutations in essential genes can
be maintained in the diploid state
and identified by inbreeding
experiments. In haploid
organisms, such as haploid yeast
(Fig. 5.6), defects in essential
genes can be isolated and
maintained through the use of
conditional mutations. Very often, conditional
mutations that display temperature-sensitive
(ts) phenotypes are used. ts mutations often
result from substitution mutations that cause
an essential protein to be unstable and
inactive at high (nonpermissive), but not low
(permissive) temperatures. A number of yeast
cell-division cycle (cdc) mutants have been
isolated via this technique (Fig. 5.6).

12. Complementation Analysis of Recessive
Mutations
Many processes, including
cell division involve the
combined actions of
multiple genes. Thus, a
genetic screen for
mutations affecting such
processes will turn up a
collection of genes.
Through mating haploid
yeast containing the
defective genes, one can
establish in the diploid
cells whether the
mutations fall in the same
or separate genes. As
shown in Fig. 5.7, diploid
cells will grow under
nonpermissive conditions if
the mutations reside in
different genes (the wild
type genes complement
the defective ones).
However, diploids with two
defective copies of the
same gene will not survive.

13. Double Mutant Analysis of Biosynthetic
Pathways
Genetic experiments can be used to determine the order in
which gene products act in carrying out a process. Double
mutant analysis can be applied to order the enzyme-catalyzed
steps in a metabolic pathway. As shown in Fig. 5.8a, the
accumulation of intermediate 1 in the double mutant strain
indicates enzyme A operates prior to enzyme B.

14. Suppressor Mutations
Suppressor mutation analysis is a powerful tool for identifying
proteins that interact with one another in the performance of a
certain cellular process. In genetic suppression, a loss of function
mutation in Protein A is corrected by a compensating mutation in
Protein B. The resulting gain of function phenotype of the double
mutant results from the recreation of interaction sites between
two proteins that are disrupted by each individual amino acid
substitution mutation (Fig. 5.9a).

15. Synthetic Lethal Mutations
The analysis of synthetic lethal mutations also is an important
tool for identifying proteins that must interact to carry out a
cellular process (Fig. 5.9b). It also is a powerful method to
identify proteins that function in redundant pathways needed for
the production of an essential cell component (Fig. 5.9c). Unlike
suppressor mutations, synthetic lethal double mutants display a
loss of function phenotype.

16. Intro to DNA Cloning by Recombinant
DNA Methods
To study a gene, one must first prepare and purify its DNA in
relatively large amounts. This is accomplished via the
recombinant DNA (rDNA) technology method known as DNA
cloning. In cloning, a DNA molecule of interest is spliced into a
vector such as a bacterial plasmid or virus forming a rDNA
molecule which can be propagated in bacterial cells such as E.
coli. After replication and amplification of the rDNA in the
bacterium, it is purified for sequencing and other manipulations
used in gene characterization.

17. DNA Cleavage by Restriction Enzymes
Restriction enzymes are nucleases that are very important in
rDNA technology. These enzymes make double-stranded cuts in
DNA molecules at specific 4-8 bp palindromic (two-fold
symmetrical) sequences called restriction sites. Many restriction
enzymes make staggered cuts in DNA molecules resulting in
single-stranded complementary sticky ends (Fig. 5.11). Sticky-
ended fragments can be readily joined together to synthesize
rDNA molecules (Fig. 5.12). In many cases, cleavage at the
restriction site is blocked by methylation of bases in the site.

19. Joining of DNA Molecules by Ligation
Plasmid vectors containing a
DNA of interest (e.g.,
genomic DNA) can be
readily constructed by
ligating restriction
fragments to vector DNA
that has been digested with
the same restriction enzyme
(Fig. 5.12). Base-pairing
between the complementary
sequences of the sticky
ends aligns the fragments
for covalent linkage by a
DNA ligase, typically T4
DNA ligase. This enzyme
uses 2 ATP to provide
energy for joining the 3'-
hydroxyl and 5'-phosphate
groups of the base-paired
fragments together in 2 new
3'-5' phosphodiester bonds.
Note, all restriction
enzymes produce a 5'-
phosphate and 3'-hydroxyl
group at the cut site.

20. E. coli Plasmid Cloning Vectors
Plasmids are autonomously replicating circular DNAs found in
bacterial cells. Naturally occurring plasmids contain an origin of
replication (ori) for propagation in the host cell and one or more
genes that specify a trait that may be useful to the host. Cloning
vectors are plasmids that have been genetically engineered to
reduce unneeded DNA and to introduce selectable markers such
as antibiotic resistance genes (e.g., ampr) that are used to force
cells to maintain the plasmid. Polylinker sequences that encode
several unique restriction sites for cloning purposes also are
engineered into these vectors (Fig. 5.13).

21. Cloning of DNA in Plasmid Vectors
An overview of the steps required
for DNA cloning in a plasmid
vector is presented in Fig. 5.14.
In Step 1, the DNA of interest
is ligated into a plasmid cloning
vector. In Step 2, the
recombinant plasmid is introduced
into E. coli host cells by
transformation. In Step 3, cells
that have taken up the plasmid
are selected on antibiotic
(ampicillin) agar. In Step 4, the
transformed cells replicate their
chromosomal and plasmid DNA
and multiply to form a colony.
Cells in the colony contain the
cloned DNA and are themselves
clones. The rDNA plasmid then is
harvested by growing a larger
culture of the cells.
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2
3
4
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22. Construction of cDNA Libraries (Part 1)
A genomic DNA library is a collection of cloned DNA fragments
representing all of the DNA of an organism. A cDNA library
(complementary DNA), is a collection of cloned DNA fragments
corresponding to all mRNAs transcribed in a certain tissue or
organism. Libraries can be constructed using plasmid cloning
vectors. To construct a cDNA library, one begins by isolating
mRNA from the cell or tissue of interest (Fig. 5.15). Because many
genes are transcribed at a low frequency, it is best to start with a
cell/tissue that expresses the
gene of interest at a relatively
high level. cDNAs are
transcribed from a mRNA
template by a retroviral enzyme
known as reverse transcriptase
(RT). In Step 1, mRNA isolated
by oligo-dT affinity
chromatography is hybridized via
its 3' poly(A) tail to an oligo-dT
primer. In Step 2, RT
synthesizes the first cDNA
strand. In Step 3, RNA is
destroyed and a poly(dG) tail is
added by terminal transferase.
In Step 4, the cDNA is
hybridized to an oligo-dC
primer. (Go to next slide).

23. Construction of cDNA Libraries (Part 2)
In Step 5, a DNA
polymerase is used to
synthesize the second
strand of the cDNA. In
Step 6, EcoRI sites that
might be present within
the mRNA coding region
are protected by
methylation using EcoRI
methylase. In Step 7,
unmethylated EcoRI
linkers, that encode
EcoRI restriction sites,
are ligated to the ends
of the fragment. In Step
8a, the cDNA is cleaved
with EcoRI restriction
enzyme, generating
sticky-ended cDNA
fragments. (See next
slide).

24. Construction of cDNA Libraries (Part 3)
In the last steps of cDNA library construction, the plasmid vector
is cut with EcoRI restriction enzyme (Step 8b), and then the
EcoRI-cut cDNA and plasmid are ligated together (Step 9).
Finally, the E. coli host strain is transformed and cells are plated
(Step 10) on selective medium. To be complete, both genomic and
cDNA libraries for higher eukaryotes must contain on the order of
a million individual clones.

25. Screening cDNA Libraries
To screen a plasmid library (Fig.
5.16), colonies representing each
cloned DNA first are plated on a
number of petri plates. Library
DNA then is lifted onto
nitrocellulose membranes which
serve as replicas of the plates.
Bound DNA is denatured and
hybridized with a radioactively-
labeled single-strand DNA probe
(next slide). After washing, spots
corresponding to colonies containing
the DNA of interest are detected
by autoradiography. Because not all
DNA gets lifted onto the
membranes, DNA for the clone can
be purified from the residual colony
on the original plate. Note, that
oligonucleotide probes must only be
~ 20 nucleotides long to recognize
unique sequences even in genomic
DNA. The probe sequence can be
derived from genome sequencing
databases, or designed based on
the known sequence of a protein.

26. DNA Detection by
Membrane Hybridization
The general method for
screening a membrane-bound
DNA sample for a gene of
interest is illustrated in Fig.
5.16. This involves fixation of
single-stranded DNA to the
membrane, hybridizing the
fixed DNA to a labeled DNA
probe complementary to the
gene of interest, removal of
un-hybridized probe by
washing, and detection of the
specifically hybridized probe
by autoradiography, etc.

27. Construction of a Yeast
Genomic Library in a
Shuttle Vector
Plasmids known as E. coli-yeast
shuttle vectors (Fig. 5.17a) can
replicate in both organisms.
Shuttle vectors contain 1) origins
of replication for both species
(ori, E. coli; ARS, yeast), 2)
markers for selection in E. coli
(ampr) and yeast (URA3), and 3) a
CEN sequence that ensures stable
replication and segregation in
yeast. The method for
construction of a yeast genomic
library in a E. coli-yeast shuttle
vector is illustrated in Fig. 5.17b.
A total of ~105 clones is needed
to include all genes, if the genomic
DNA is cut into fragments of
about 10 kb in length.

28. Screening by Functional Complementation
A yeast genomic library can be screened by the technique of
functional complementation to isolate the cloned version of a gene
of interest (Fig. 5.18). First, all recombinant plasmids from the
library are isolated from E. coli, pooled, and used to transform
haploid ura3- yeast that carry a conditional lethal ts copy of the
gene of interest. Transformants are selected by plating on
uracil-deficient agar at the permissive temperature. Second,
transformants are replica plated onto agar and incubated at the
nonpermissive temperature to identify colonies carrying a wild
type version of the gene of interest. Only cells containing the
library copy of the wild type gene can survive at high
temperature.