Molecular Cell Biology

1. Molecular Cell Biology Professor Dawei Li daweili@sjtu.edu.cn 3420-4744 Textbook: MOLECULAR CELL BIOLOGY 6th Ed Lodish • Berk • Kaiser • Krieger • Scott • Bretscher •Ploegh • Matsudaira Part 2. Genetics and Molecular Biology 1. Quiz Analyzing Data Chapter 5 2. Student Presentations 3. Chapter 5.2-5.5: Key Figures 4. Answer Questions

2. Cloned DNA Molecules Are Sequenced Rapidly by the Dideoxy Chain-Termination Method FIGURE 5-20 Structures of deoxyribonucleoside triphosphate (dNTP) and dideoxyribonucleoside triphosphate (ddNTP)

3. EXPERIMENTAL FIGURE 5-21(a) Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs)

4. EXPERIMENTAL FIGURE 5-21(b) Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs)

5. EXPERIMENTAL FIGURE 5-21(c) Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs)

6. Strategies for Assembling Whole Genome Sequences FIGURE 5-22 Two Strategies for Assembling Whole Genome Sequences

7. The Polymerase Chain Reaction Amplifies a Specific DNA Sequence from a Complex Mixture EXPERIMENTAL FIGURE 5-23 The polymerase chain reaction (PCR) is widely used to amplify DNA regions of known sequence

8. Direct Isolation of a Specific Segment of Genomic DNA EXPERIMENTAL FIGURE 5-24 A specific target region in total genomic DNA can be amplified by PCR for use in cloning

9. Tagging of Genes by Insertion Mutations EXPERIMENTAL FIGURE 5-25 The genomic sequence at the insertion site of a transposon is revealed by PCR amplification and sequencing

10. KEY CONCEPT OF SECTION 5.2 DNA Cloning and Characterizaiton(p190)

11. 5.3 Use Cloned DNA Fragments to Study Gene Expression EXPERIMENTAL FIGURE 5-26 Southern blot technique can detect a specific DNA fragment in a complex mixture of restriction fragments

12. Hybridization Techniques Permit Detection of Specific DNA Fragments and mRNAs EXPERIMENTAL FIGURE 5-27 Northern blot analysis reveals increased expression of β-globin mRNA in differentiated erthroleukemia cells

13. In Situ Hybridization EXPERIMENTAL FIGURE 5-28 In situ hybridization can detect activity of specific genes in whole and sectioned embryos

14. Using Microarrays to Compare Gene Expression under Different Conditions EXPERIMENTAL FIGURE 5-29(a) DNA microarray analysis can reveal differences in gene expression in fibroblasts under different experimental conditions

15. EXPERIMENTAL FIGURE 5-29(b) DNA microarray analysis can reveal differences in gene expression in fibroblasts under different experimental conditions

16. Cluster Analysis of Multiple Expression Experiments Identifies Co-regulated Genes EXPERIMENTAL FIGURE 5-30 Cluster analysis of data from multiple microarray expression experiments can identify co-regulated genes

17. E.Coli Expression Systems Can Produce Large Quantities of Proteins from Cloned Genes EXPERIMENTAL FIGURE 5-31 Some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac promoter

18. EXPERIMENTAL FIGURE 5-31(a) Some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac promoter

19. EXPERIMENTAL FIGURE 5-31(b) Some eukaryotic proteins can be produced in E.coli cells from plasmid vectors containing the lac promoter

20. Plasmid Expression Vectors Can Be Designed for Use in Animal Cells EXPERIMENTAL FIGURE 5-32(a) Transient and stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells

21. EXPERIMENTAL FIGURE 5-32(b) Transient and stable transfection with specially designed plasmid vectors permit expression of cloned genes in cultured animal cells

22. Retroviral Expression Systems EXPERIMENTAL FIGURE 5-33 Retroviral vectors can be used for efficient integration of cloned genes into the mammalian genome

23. Gene and Protein Tagging EXPERIMENTAL FIGURE 5-34 Gene and protein tagging facilitate cellular localization of proteins expressed from cloned genes

24. KEY CONCEPTS OF SECTION 5.3 Using Cloned DNA Fragments to Study Gene Expression(p198)

25. Applications of Molecular Technology Examples

26. 5.4 Identifying and Locating Human Disease Genes

27. Many Inherited Diseases Show One of Three Major Patterns of Inheritance FIGURE 5-35 Three common inheritance patterns of human genetic diseases

28. DNA Polymorphisms Are Used in Linkage-Mapping Human Mutations Restriction fragment length polymorphisms EXPERIMENTAL FIGURE 5-36(a) Restriction fragment length polymorphisms (RFLPs) can be followed like genetic markers

29. EXPERIMENTAL FIGURE 5-36(b) Restriction fragment length polymorphisms (RFLPs) can be followed like genetic markers

30. Linkage Studies Can Map Disease Genes with a Resolution of About 1 Centimorgan FIGURE 5-37 Linkage disequilibrium studies of human populations can be used to map genes at high resolution

31. Further Analysis Is Needed to Locate a Disease Gene in Cloned DNA FIGURE 5-38 The relationship between the genetic and physical maps of a human chromosome

32. KEY CONCEPTS OF SECTION 5.4 Identifying and Locating Human Disease Genes(p204)

33. 5.5 Inactivating the Function of Specific Genes in Eukaryotes Gene Knockout Normal Yeast Genes Can Be Replaced with Mutant Alleles by Homologous Recombination EXPERIMENTAL FIGURE 5-39(a) Homologous recomnination with fransfected disruption constructs can inactivate specific target genes in yeast

34. Study essential genes by conditional knockout Gal1 Promoter-Essential Gene Grow in Galactose medium Gal1 Promoter-Essential Gene Grow in Glucose medium Mutant phenotype EXPERIMENTAL FIGURE 5-39(b) Homologous recomnination with fransfected disruption constructs can inactivate specific target genes in yeast

35. Specific Genes Can Be Permanently Inactivated in the Germ Line of Mice EXPERIMENTAL FIGURE 5-40(a) Isolation of mouse ES cells with a gene-targeted disruption is the first stage in production of knockout mice

36. EXPERIMENTAL FIGURE 5-40(b) Isolation of mouse ES cells with a gene-targeted disruption is the first stage in production of knockout mice

37. EXPERIMENTAL FIGURE 5-41 ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

38. EXPERIMENTAL FIGURE 5-41(a) ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

39. EXPERIMENTAL FIGURE 5-41(b) ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

40. EXPERIMENTAL FIGURE 5-41(c) ES cells heterozygous for a disrupted gene are used to produce gene-targeted knockout mice

41. Conditional Knockout: To Study Embryonic Lethal Essential Gene KO

42. Somatic Cell Recombination Can Inactivate Genes in Specific Tissues EXPERIMENTAL FIGURE 5-42 The loxP-Cre recombination system can knock out genes in specific cell types

43. Dominant-Negative Alleles Can Functionally Inhibit Some Genes EXPERIMENTAL FIGURE 5-43 Transgenic mice are produced by random integration of a foreign gene into the mouse germ

44. FIGURE 5-44 Inactivation of the function of a wild-type GTPase by the action of a dominant-negative mutant allele

45. RNA Interference Causes Gene Inactivation by Destroying the Corresponding mRNA EXPERIMENTAL FIGURE 5-45 RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

46. KEY CONCEPTS OF SECTION 5.5 Inactivating the Function of Specific Genes in Eukaryotes(p211)

47. Discussion: • Answer Chapter 5 Questions • Homework: Review Chapter 5 • Key Terms (p212) • Concepts p212 (will be tested in Final) • Analyzing the data p213-214 • (These will be tested in Final)

48. KEYWORDS