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Chapter 11
Chapter 12
Genomics,
Proteomics,
and
Transgenics
Jones and Bartlett Publishers © 2005
Programmed DNA rearrangements
• Genes amplification: rRNA genes in oocytes
(insects, amphibians, and fish) increase in
number.
– (600 copies tandemly duplicated in normal
toad genome, but more are needed: 4000-fold
increase in gene copy number via rolling circle
replicating extrachromosomal rRNA genes,
over 3 weeks during oogenesis).
Programmed DNA rearrangements
• Antibody and T-cell receptor variability:
– Normal mammals can produce 108 different
antibodies. How?
Structure of an immunoglobulin G (IgG) molecule
The distribution of variable, joining and
constant sequences which are spliced to
create many different light chain proteins
Antibody
diversity
in
humans
Mating type switching during the life cycle
of some strains of Saccharomyces
Both mating type genes are located on chromosome III
of Saccharomyces. The mating type of the cell
is determined by the sequence present at the MAT site
Regulation of a-specific, a-specific and
haploid-specific genes in Saccharomyces
Three proteins (a1,
a1 and a2) are
involved in
regulating the
expression of these
3 classes of genes.
• Genomics, Proteomics, and
Transgenics
Restriction nuclease cutting followed by
ligation of sticky ends creates closed
circles from linear DNA fragments
Restriction nuclease cutting may generate
sticky (with overhangs)- or blunt-ends
DNA fragments may be amplified (cloned) by
joining with plasmid DNA and replication
of the recombinant DNA in bacteria
Foreign DNA and vector DNA both
must have matching sticky ends
Size limits of foreign DNA that can be
inserted into different cloning vectors
Different DNA fragments created by a restriction
nuclease may be joined in many different
arrangements since they all have the same sticky ends
RNA templates may be copied into double
stranded DNA and then cloned
[complementary DNA (cDNA) cloning]
After being
copied into
DNA, the
RNA template
is usually
destroyed
(rather than
displaced)
before the
synthesis of
the second
DNA strand.
Useful features of a plasmid cloning vector
Use of lacZ a-peptide coding sequence for
color-dependent selection of recombinant clones
Use of a radioactive probe and
hybridization to immobilized DNA on a
filter for selection of desired clones
Use of overlapping clones to create “contigs”
and physical mapping of genes
The contig from
these 24
overlapping
clones is ~500 kb
long.
The sizes of the 16 Saccharomyces chromosomes
A F-Factor (sex-plasmid)-derived vector
(BAC, Bacterial Artificial Chromosome)
A BAC vector can
accept very large
inserts (several
hundred Kb)
YAC vectors can
take even bigger
inserts
Genetic and physical maps of a chromosome
at various levels of resolution
Functional classification of expressed genes
A listing of number of sequenced cDNA clones
(and the unique expressed genes they represent)
in various human organs and tissues
Genes in the Mycoplasma genitalium
classified by function
Use of DNA microarrays (chips)
Fluorescently tagged
cDNA probes are
hybridized to DNA
spots in the microarray
for studying
differential expression
of thousands of genes at
a time in two mRNA
samples
Patterns of transcriptional regulation
of about 2500 genes
Use of a transposon (P-element) for cloning
of foreign genes in Drosophila
chromosome
Steps in the creation of a transgenic mouse
Methodology for gene knockout or gene
replacement using a “targeting” vector
Tumor-inducing (Ti)-plasmid introduces
part of the plasmid DNA (T DNA) into the
infected cell’s chromosome
Site-specific mutagenesis of a cloned DNA
sequence using a synthetic mutagenic primer
Steps in the construction of transgenic rice plant
capable of producing b-carotene
Medical applications of recombinant
DNA technology