rest of 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).

Antibody and T-cell receptor variability: –Normal mammals can produce 10 8 different antibodies. How? Programmed DNA rearrangements

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,  -specific and haploid-specific genes in Saccharomyces Three proteins (a1,  1 and  2) 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  -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

The contig from these 24 overlapping clones is ~500 kb long. Use of overlapping clones to create “contigs” and physical mapping of genes

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  -carotene

Medical applications of recombinant DNA technology