1. Molecular Cell Biology
Professor Dawei Li
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 4
2. Student Presentations
3. Chapter 5-2: Key Figures
4. Answer Questions

2. © 2008 W. H. Freeman and Company
CHAPTER 5-2
Molecular Genetic Techniques
© 2008 W. H. Freeman and Company

3. Genetic Analysis of Mutations to Identify and Study Genes (Page175)
KEY CONCEPT OF SECTION 5.1
Genetic Analysis of Mutations to Identify and Study Genes (Page175)

4. 5.2 DNA Cloning and Characterization
Replication Vector +DNA Fragment
Recombinant DNA
Replication in Host Cells
Characterization and Manipulation of Purified DNA

5. Cutting DNA Molecules into Small Fragments
FIGURE 5-11
Cleavage of DNA by the restriction enzyme EcoRI

7. Inserting DNA Fragments into Vectors
FIGURE 5-12
Ligation of restriction fragments with complementary sticky ends

8. E.Coli Plasmid Vectors Are Suitable for Cloning Isolated DNA Fragment
FIGURE 5-13
Basic components of a plasmid cloning vector that can replicate within an E.coli cell
E.Coli Plasmid Vectors Are Suitable for Cloning Isolated DNA Fragment

9. FIGURE 5-14
DNA cloning in a plasmid vector permits amplification of a DNA fragment

10. FIGURE 5-14(a)
DNA cloning in a plasmid vector permits amplification of a DNA fragment

11. FIGURE 5-14(b)
DNA cloning in a plasmid vector permits amplification of a DNA fragment

12. cDNAs Prepared by Reverse Transcription of Cellular mRNAs Can Be Cloned to Generate cDNA Libraries
FIGURE 5-15
A cDNA library contains representative copies of cellular mRNA sequences

13. FIGURE 5-15(a)
A cDNA library contains representative copies of cellular mRNA sequences

14. FIGURE 5-15(b)
A cDNA library contains representative copies of cellular mRNA sequences

15. FIGURE 5-15(c)
A cDNA library contains representative copies of cellular mRNA sequences

16. FIGURE 5-15(d)
A cDNA library contains representative copies of cellular mRNA sequences

17. FIGURE 5-15(e)
A cDNA library contains representative copies of cellular mRNA sequences

18. DNA Libraries Can Be Screened by Hybridization to an Oligonucleotide Probe
FIGURE 5-16
cDNA libraries can be screened with a radiolabeled probe to identify a clone of interest

19. Yeast Genomic Libraries Can Be Constructed with Shuttle Vectors and Screened by Functional Complementation
FIGURE 5-17
A yeast genomic library can be constructed in a plasmid shuttle vector that can replication in yeast and E.coli

20. FIGURE 5-17(a)
A yeast genomic library can be constructed in a plasmid shuttle vector that can replication in yeast and E.coli

21. FIGURE 5-17(b)
A yeast genomic library can be constructed in a plasmid shuttle vector that can replication in yeast and E.coli

22. FIGURE 5-18
Screening of a yeast genomic library by functional complementation can identify clones carrying the normal form of a mutant yeast gene

23. Gel Electrophoresis Allows Separation of Vector DNA from Cloned Fragments
FIGURE 5-19
Gel electrophoresis separates DNA molecules of different lengths

24. FIGURE 5-19(d)
Gel electrophoresis separates DNA molecules of different lengths

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

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

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

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

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

30. 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

31. 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

32. 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

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

34. 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

35. 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

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

37. 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

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

39. 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

40. 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

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

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

43. 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

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

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

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

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

48. Applications of Molecular Technology
Examples

49. 5.4 Identifying and Locating Human Disease Genes

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

51. 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

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

53. 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

54. 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

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

56. 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

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

58. 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

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

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

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

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

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

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

65. 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

66. 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

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

68. 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

69. EXPERIMENTAL FIGURE 5-45(a)
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

70. EXPERIMENTAL FIGURE 5-45(b)
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

71. EXPERIMENTAL FIGURE 5-45(c)
RNA interference (RNAi) can functionally inactivate genes in C.elegans and other organisms

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

73. Discussion:
Answer Chapter 5 Questions
Homework: Review Chapter 5
Key Terms (p212)
Concepts p212 (will be tested in Final)
Analyzing the data p
(These will be tested in Final)
Next Monday in Class Quiz Chapter 5 questions

74. KEY WORDS Allete 等位基因 Mutation 突变 Mutagen 诱变剂 Genotype 基因型 Wild type
野生型
Phenotype 表型
Haploid
单倍体
Diploid
二倍体
Heterozygous
杂合子
Homozygous
纯合子
Recessive 隐性
Dominant 显性
Point mutation
点突变
Gamete 配子
Mitosis
有丝分裂

75. Germ cells
Meiosis
减数分裂
Homolog
纯合子
Homologous chromosome
同源染色体
Segregation
Type a
Temperature-sensitive mutation
温度敏感型突变株
Cell cycle
细胞周期
Genetic complementation
Suppressor mutation
Synthetic lethal mutation
Genetic mapping
Recombination 重组
Crossing over
Locus
Genetic marker
遗传标记
linkage

76. DNA cloning
基因克隆
Recombinant DNA
重组DNA
Restriction enzyme
限制性内切酶
DNA ligase
DNA连接酶
Restriction fragment
限制片段
Okazaki fragment
冈崎片段
Transformation 转化
DNA library
DNA文库
Complementary DNAs (cDNAs)
Reverse transcriptase
反转录酶
Probe 探针
Hybridization 杂交
Functional complementation
Shuttle vector
穿梭质粒
Electrophoresis 电泳
Polymerase chain reaction (PCR)
聚合酶链式反应
Mobile DNA element

77. Southern Blotting
Northern blotting
Hybridization 杂交
DNA microarray
DNA微阵列
Expression vector
表达载体
Promoter
启动子
Transfection 转染
Epitope
抗原决定簇
Autosome
DNA-based molecular marker
Restriction fragment length polymorphisms(RFLP)
限制片段长度多态性
Germ line
细胞系
Gene knockout
基因敲除
Embryonic stem cells
胚胎干细胞
Chimeras
Dominant-negative mutation
显隐性突变
transgene
转基因

78. Review the Concepts in p212 Analyze the Data in p213
Transgenic
转基因
RNA interference (RNAi)
Micro RNAs (miRNAs)
小RNA
Other key words in p212
Review the Concepts in p212
Analyze the Data in p213