1. rest of Chapter 11 Chapter 12 Genomics, Proteomics, and Transgenics Jones and Bartlett Publishers © 2005

2. 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).

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

4. Structure of an immunoglobulin G (IgG) molecule

5. The distribution of variable, joining and constant sequences which are spliced to create many different light chain proteins

6. Antibody diversity in humans

7. Mating type switching during the life cycle of some strains of Saccharomyces

8. 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

9. 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.

10. Genomics, Proteomics, and Transgenics

11. Restriction nuclease cutting followed by ligation of sticky ends creates closed circles from linear DNA fragments

12. Restriction nuclease cutting may generate sticky (with overhangs)- or blunt-ends

13. DNA fragments may be amplified (cloned) by joining with plasmid DNA and replication of the recombinant DNA in bacteria

14. Foreign DNA and vector DNA both must have matching sticky ends

15. Size limits of foreign DNA that can be inserted into different cloning vectors

16. Different DNA fragments created by a restriction nuclease may be joined in many different arrangements since they all have the same sticky ends

17. 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.

18. Useful features of a plasmid cloning vector

19. Use of lacZ  -peptide coding sequence for color-dependent selection of recombinant clones

20. Use of a radioactive probe and hybridization to immobilized DNA on a filter for selection of desired clones

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

22. The sizes of the 16 Saccharomyces chromosomes

23. 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

24. Genetic and physical maps of a chromosome at various levels of resolution

25. Functional classification of expressed genes

26. A listing of number of sequenced cDNA clones (and the unique expressed genes they represent) in various human organs and tissues

27. Genes in the Mycoplasma genitalium classified by function

28. 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

29. Patterns of transcriptional regulation of about 2500 genes

30. Use of a transposon (P-element) for cloning of foreign genes in Drosophila chromosome

31. Steps in the creation of a transgenic mouse

32. Methodology for gene knockout or gene replacement using a “targeting” vector

33. Tumor-inducing (Ti)-plasmid introduces part of the plasmid DNA (T DNA) into the infected cell’s chromosome

34. Site-specific mutagenesis of a cloned DNA sequence using a synthetic mutagenic primer

35. Steps in the construction of transgenic rice plant capable of producing  -carotene

36. Medical applications of recombinant DNA technology