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 4 2. Student Presentations 3. Chapter 5-2: Key Figures 4. Answer Questions

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Applications of Molecular Technology Examples

5.4 Identifying and Locating Human Disease Genes

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Conditional Knockout: To Study Embryonic Lethal Essential Gene KO

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

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

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

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

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

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

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

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

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) Next Monday in Class Quiz Chapter 5 questions

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

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

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

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

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