Molecular Biology of the Cell Chapter 8 PDF

Molecular Biology of the Cell Sixth Edition Chapter 8 Analyzing Cells, Molecules, and Systems  CHAPTER CONTENTS ISOLATING CELLS AND GROWING THEM IN CULTURE PURIFYING PROTEINS ANALYZING PROTEINS ANALYZING AND MANIPULATING DNA STUDYING GENE EXPRESSION AND FUNCTIO...

Molecular Biology of the Cell Sixth Edition Chapter 8 Analyzing Cells, Molecules, and Systems  CHAPTER CONTENTS ISOLATING CELLS AND GROWING THEM IN CULTURE PURIFYING PROTEINS ANALYZING PROTEINS ANALYZING AND MANIPULATING DNA STUDYING GENE EXPRESSION AND FUNCTION MATHEMATICAL ANALYSIS OF CELL FUNCTIONS ANALYZING AND MANIPULATING DNA Restriction Nucleases Cut Large DNA Molecules into Specific Fragments C & C M sticky ends ANALYZING AND MANIPULATING DNA Gel Electrophoresis Separates DNA Molecules of Different Sizes SeparateshortT add GATC - terminating nu ANALYZING AND MANIPULATING DNA Genes Can Be Cloned Using Bacteria – DNA cloning: the act of making many identical copies of DNA / the isolation of a particular stretch of DNA from the rest of the cell’s genome Genes Can Be Cloned Using Bacteria Genes Can Be Cloned Using Bacteria ANALYZING AND MANIPULATING DNA An Entire Genome Can Be Represented in a DNA Library An Entire Genome Can Be Represented in a DNA Library synthesize 2 cDNA strands from 1 mRNA L ↳ An Entire Genome Can Be Represented in a DNA Library genomic cDNA citca cac doan (introns, exons, non chigoin exons -transcribed sLbang nhau SL m lai dia - Idok transcribe tren kIn transcribe ANALYZING AND MANIPULATING DNA Genomic and cDNA Libraries Have Different Advantages and Drawbacks – Genomic libraries are useful in determining the sequences of a whole genome – 13 years / $3 billion for the human genome project – cDNA libraries are used to produce the protein of interest in large quantities by expressing the cloned gene in a bacterial or yeast cell ANALYZING AND MANIPULATING DNA DNA Cloning Allows Any Protein to be Produced in Large Amounts highly active ↳ tao voi scion ANALYZING AND MANIPULATING DNA Hybridization Provides a Powerful, But Simple Way to Detect Specific Nucleotide Sequences 'renaturation - by heat/alkali ↳ reform inrand tie how can this be used on DNA libraries? ANALYZING AND MANIPULATING DNA Genes Can Be Cloned in vitro Using PCR Janio Genes Can Be Cloned in vitro Using PCR Genes Can Be Cloned in vitro Using PCR different from libraries Cell-free DNA (cfDNA), circulating tumor DNA (ctDNA) 41 type of CfDNA > produce CfDNA ANALYZING AND MANIPULATING DNA Both DNA and RNA Can Be Rapidly Sequenced o O Both DNA and RNA Can Be Rapidly Sequenced Both DNA and RNA Can Be Rapidly Sequenced C Both DNA and RNA Can Be Rapidly Sequenced Both DNA and RNA Can Be Rapidly Sequenced Both DNA and RNA Can Be Rapidly Sequenced (au library En PCR thay n bacteria ; PCR copies gan DNA gir e tao cluster & Both DNA and RNA Can Be Rapidly Sequenced u red ↳ can be removed chemically (unlike ddNTP) O Both DNA and RNA Can Be Rapidly Sequenced Both DNA and RNA Can Be Rapidly Sequenced ↳early diagnosis through liquid biopsy N Engl J Med 2018;379:1754-65  STUDYING GENE FUNCTION AND EXPRESSION Introduction Introduction Introduction O & N - M g Introduction (4) V V V  STUDYING GENE FUNCTION AND EXPRESSION Classical Genetics Begins by Disrupting a Cell Process by Random Mutagenesis – Before the advent of gene cloning technology, most genes were identified by the abnormalities produced when the gene was mutated – Mutagenesis (chemical, radiation, insertional) applied to thousands of individual organisms, which can then be screened for a particular defect of interest – such a search is called a genetic screen hoan vi – Transposable elements are used for insertional mutagenesis (molecular tag) – Because defects in many genes are lethal, the functions of these genes are often studied in individual with conditional mutations (figure 46)  STUDYING GENE FUNCTION AND EXPRESSION Mutations Responsible for a Phenotype Can Be Identified Through DNA Analysis – Insertional mutagenesis: DNA fragments that were inserted can be amplified by PCR and the flanking sequences can be determined Inverse PCR – Chemical or radiation: Genome sequencing > identification of causative mutation candidates > introduction of each mutation back into a normal organism for testing  STUDYING GENE FUNCTION AND EXPRESSION Rapid and Cheap DNA Sequencing Has Revolutionized Human Genetic Studies – Classical genetics with human subjects? – The human population is so large, there is a great deal of variation – With the recent advances in DNA sequencing, mutations can be identified easily Linked Blocks of Polymorphisms Have Been Passed Down from Our Ancestors ↳ single nu polymorphism  STUDYING GENE FUNCTION AND EXPRESSION Polymorphisms Can Aid the Search for Mutations Associated with Disease – Any two individuals differ in 1 nucleotide in 1000 – When two sequence variants (alleles) coexist in the population and both are common, the variants are called polymorphisms – The majority are SNPs (figure 50) – In population studies, investigators collect DNA from a large number of people who have the disease and compare them to a group of people who do not have the disease – They look for SNPs that are more common among the people who have the disease – Such genome-wide association studies (GWAS) have been used to search for genes that predispose individuals to common diseases (>1000 traits so far)  STUDYING GENE FUNCTION AND EXPRESSION Reverse Genetics Begins with a Known Gene and Determines Which Cell Processes Require Its Function – Classical genetics starts with a mutant phenotype and identifies the mutations (GWAS for humans) – Instead, an investigator can start with a particular gene and proceed to make mutations in it - reverse genetics (more technologically oriented) Bacteria Use Small Noncoding RNAs to Protect Themselves from Viruses CRISPR Clustered regularly interspersed short palindromic repeat The Bacterial CRISPR System Has Been Adapted to Edit Genomes in a Wide Variety of Species n cas gues wi - can cleave giupphiet min ( DrAgenome ↳ Xmutated , casg wa ↳1 it se 2 genome varines F ↳ perform din ng F (VD turn gene : m/off) The Bacterial CRISPR System Has Been Adapted to Edit Genomes in a Wide Variety of Species O O ↳ edit genome  ↑ STUDYING GENE FUNCTION AND EXPRESSION Animals and Plants Can Be Genetically Altered – Animals and plants that have been genetically engineered by gene deletion or replacement are called transgenic organisms – The bacterial CRISPR system has been adapted to edit genomes in a wide variety of species (figure 7- 78; figure 55) – Advantages: relatively easy to design the guide RNA (following standard base pairing rules) / numerous v genes can be controlled simultaneously (Cas9 expressed only once with many guide RNAs) – The export of CRISPR from bacteria to virtually all other organisms has revolutionized the study of gene function UC Berkeley Max Planck MIT Feng Zhang  ~ ~ (not EcoRI) g ~ Small Noncoding RNA Transcripts Regulate Many Animal and Plant Genes Through RNA Interference common RNAi pathway transcriptio -X - gas gene expression  STUDYING GENE FUNCTION AND EXPRESSION RNA Interference Is a Simple and Rapid Way to Test Gene Function – RNA interference (RNAi) introduces into a cell or organism a double-strand RNA whole nucleotide sequence matches a part of the gene to be inactivated – How to introduce dsRNA? A short hairpin RNA or small hairpin RNA (shRNA) is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi) Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression  STUDYING GENE FUNCTION AND EXPRESSION Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression – The first tool that allowed investigators to analyze simultaneously thousands of different RNAs was the DNA microarray (figure 64) – Although microarrays are relatively inexpensive and easy to use, the sequences of the mRNA samples to be analyzed must be known in advance and represented by a corresponding probe on the array – With the development of improved sequencing technologies, investigators increasingly use RNA-seq as a more direct approach for cataloging the RNAs (alternative splicing, RNA editing, and noncoding RNAs can be detected)  STUDYING GENE FUNCTION AND EXPRESSION Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression – Comprehensive studies of gene expression provide information that is useful for predicting gene function – Cluster analysis can identify sets of genes that are coordinately regulated (genes that are turned on or off together under different circumstances are likely to work in concert in the cell) – Characterizing a gene whose function is unknown by grouping it with known genes that share its transcriptional behavior is called “guilt by association” (figure 65) Analysis of mRNAs by Microarray or RNA-seq Provides a Snapshot of Gene Expression  STUDYING GENE FUNCTION AND EXPRESSION Genome-wide Chromatin Immunoprecipitation Identifies Sites on the Genome Occupied by Transcription Regulators – Chromatin immunoprecipitation (ChIP) provides a way to experimentally determine all cis-regulatory sequences in a genome that are occupied by a given transcription regulator (figure 66) Genome-wide Chromatin Immunoprecipitation Identifies Sites on the Genome Occupied by Transcription Regulators to > - glue trans regn ,. DNA to freeze interactions * Fing antibodying - riting reg. # nhau D Genome-wide Chromatin Immunoprecipitation Identifies Sites on the Genome Occupied by Transcription Regulators Ribosome Profiling Reveals Which mRNAs Are Being Translated in the Cell (220nu/RNA) ribosome abundance ∝ protein level ribosome abundance > - ht translate / mRNA level = translation rate  STUDYING GENE FUNCTION AND EXPRESSION Ribosome Profiling Reveals Which mRNAs Are Being Translated in the Cell – mRNAs represent only one step in gene expression, and we are often more interested in the final level of the protein produced by the gene – An approach called ribosome profiling provides an instantaneous map of the position of ribosomes on each mRNA and thereby identifies those mRNAs that are being actively translated (figure 68) – Why do we study protein expression using nucleic acids instead of proteins themselves?  STUDYING GENE FUNCTION AND EXPRESSION Recombinant DNA Methods Have Revolutionized Human Health – Nucleic acid methodologies developed in the past 40 years have completely changed the way that mol & cell biology is studied – As DNA sequencing continues to drop in cost, more and more individuals will elect to have their genome sequenced; this information can be used to predict susceptibility to diseases or to predict the way an individual will respond to a given drug (classical genetics) – The genomes of cancer cells can be sequenced to determine the best type of anticancer treatment, and mutations that cause disease continue to be identified at an unprecedented pace – Using recombinant DNA technologies, these mutations can be introduced into animals that can be studied in the laboratory. The resulting transgenic animals can be used to explore molecular basis of the disease and to screen for drugs that could potentially be used therapeutically in humans (reverse genetics)