Fundamentals of Molecular Biology 4 4 Fundamentals of Molecular Biology Heredity, Genes, and DNA Expression of Genetic Information Recombinant DNA Detection of Nucleic Acids and Proteins Gene Function in Eukaryotes Introduction Molecular biology focuses on the mechanisms of transmission and expression of genetic information. Fundamental experiments on model organisms such as bacteria and viruses have provided information on molecular mechanisms that operate in all organisms. Introduction Recombinant DNA technology allowed fundamental principles and experimental approaches to be extended to eukaryotic cells. Recombinant DNA allows isolation and characterization of individual genes, and determination of complete genome sequences. Heredity, Genes, and DNA All organisms inherit genetic information specifying their structure and function from their parents. All cells arise from preexisting cells, so the genetic material must be replicated and passed from parent to progeny at each cell division. Heredity, Genes, and DNA The classical principles of genetics were deduced by Gregor Mendel in 1865, from experiments with pea plants. He studied well-defined traits such as seed color, and could predict patterns of inheritance by assuming that each trait is determined by a pair of inherited factors, now called genes. Heredity, Genes, and DNA One gene copy (allele) specifying each trait is inherited from each parent. Example: Plants with identical alleles specifying yellow (YY) or green (yy) seeds, are crossed. The progeny (F 1 generation) are hybrids (Yy) and have yellow seeds: yellow is dominant, green is recessive. Figure 4.1 Inheritance of dominant and recessive genes Heredity, Genes, and DNA Genotype is the genetic makeup of an individual. Phenotype is the resulting physical appearance. The genotype of the F 1 generation is Yy. The phenotype is yellow. Heredity, Genes, and DNA Mendels results were ignored until 1900 when their importance was recognized. Shortly afterward, chromosomes were identified as the carriers of genes. Heredity, Genes, and DNA Most cells of plants and animals are diploid: they have two copies of each chromosome. Formation of germ cells (sperm and egg) involves a type of cell division (meiosis) in which only one member of each chromosome pair is transmitted to each progeny cell. Figure 4.2 Chromosomes at meiosis and fertilization Heredity, Genes, and DNA The sperm and egg are haploid: they have only one copy of each chromosome. At fertilization, two haploid cells are combined to make a new diploid cell. Heredity, Genes, and DNA Fundamentals of mutation, genetic linkage, genes, and chromosomes were established by experiments with the fruit fly Drosophila melanogaster. In the early 1900s, mutations (genetic alterations) were observed in Drosophila that affected characters such as eye color and wing shape. Heredity, Genes, and DNA Breeding experiments showed that some genes are inherited independently of each other. This suggested that the genes are on different chromosomes that segregate independently during meiosis. Genes located on the same chromosome are inherited together and are said to be linked. Figure 4.3 Gene segregation and linkage (Part 1) Figure 4.3 Gene segregation and linkage (Part 2) Figure 4.3 Gene segregation and linkage (Part 3) Figure 4.3 Gene segregation and linkage (Part 4) Heredity, Genes, and DNA The relationship between genes and proteins began to emerge in 1909: It was realized that the inherited disease phenylketonuria results from a genetic defect in the enzyme needed to metabolize the amino acid phenylalanine. Heredity, Genes, and DNA In 1941, experiments with the fungus Neurospora crassa by Beadle and Tatum found more evidence linking genes with the synthesis of enzymes. Mutant strains of Neurospora required particular amino acids for growth. Heredity, Genes, and DNA Each mutation resulted in a deficiency in a specific metabolic pathway, which is governed by enzymes. This led to the conclusion that genes specify enzyme structure. It is now known that some genes specify RNAs rather than proteins. Heredity, Genes, and DNA Evidence that DNA is the genetic material first came from experiments with the bacterium that causes pneumonia (Pneumococcus). A transforming principle was responsible for inducing the genetic transformation of one strain of the bacteria to another. Figure 4.4 Transfer of genetic information by DNA Heredity, Genes, and DNA The transforming principle was later identified as DNA when it was shown that the activity of the transforming principle is abolished by enzymatic digestion of DNA, but not by digestion of proteins. Heredity, Genes, and DNA Other studies with viruses confirmed that DNA was the genetic material. It was shown that when a bacterial virus infects a cell, the viral DNA must enter the cell in order for the virus to replicate (not the viral protein). Heredity, Genes, and DNA The structure of DNA was determined in 1953 by Watson and Crick, using information from X-ray crystallography and ideas on hydrogen bonding in the helix of proteins. Heredity, Genes, and DNA DNA is a double helix with the sugar- phosphate backbones on the outside of the molecule. The bases are on the inside; hydrogen bonds are formed between purines and pyrimidines on opposite chains. The base pairing is very specific: A always pairs with T and G with C. Figure 4.5 The structure of DNA (Part 1) Figure 4.5 The structure of DNA (Part 2) Heredity, Genes, and DNA Complementary base pairing suggested the mechanism for DNA replication. The DNA molecule separates, and each strand becomes a template for synthesis of a new strand: Semiconservative replication one strand of parental DNA is conserved in each progeny DNA molecule. Figure 4.6 Semiconservative replication of DNA Heredity, Genes, and DNA Experimental support for semiconservative DNA replication: Meselson and Stahl grew E. coli in medium labeled with the heavy isotope 15 N. The heavier DNA could be separated from light DNA (with 14 N) by equilibrium ultracentrifugation in a CsCl solution. Heredity, Genes, and DNA E. coli grown with 15 N were transferred to 14 N medium and allowed to replicate once more. The DNA from these bacteria was intermediate in weight. Figure 4.7 Experimental demonstration of semiconservative replication Heredity, Genes, and DNA Further evidence: an enzyme purified from E. coli (DNA polymerase) could catalyze DNA replication in vitro. In the presence of DNA to act as a template, DNA polymerase directed the incorporation of nucleotides into a complementary DNA molecule. Expression of Genetic Information Genes determine the structure of proteins. The information in DNA is specified by the order of the four bases. Proteins are made up of 20 different amino acids, the sequence of which determines the protein function. Expression of Genetic Information The first direct link between a genetic mutation and alteration in a protein was made in Patients with inherited sickle-cell anemia had hemoglobin that differed from normal hemoglobin by a single amino acid substitution. Expression of Genetic Information The next step was determining the relationship between DNA and proteins. The simplest explanation: the order of nucleotides in DNA specifies the order of amino acids in proteins (colinearity). Mutations (changes in the nucleotide sequence) should lead to corresponding changes in the protein. Expression of Genetic Information Using E. coli, Yanofsky and colleagues mapped a series of mutations in the gene that encodes an enzyme for tryptophan synthesis. The relative positions of the amino acid alterations were the same as those of the corresponding mutations. Expression of Genetic Information Although DNA appeared to specify the order of amino acids in proteins, it did not necessarily follow that DNA itself directs protein synthesis. DNA is located in the nucleus of eukaryotic cells, but protein synthesis occurs in the cytoplasm. Expression of Genetic Information RNA was the likely intermediate, because of its similarity to DNA. RNA is single-stranded, its sugar is ribose, and it contains uracil (U) instead of thymine (T), but this does not affect base-pairing. Figure 4.9 Synthesis of RNA from DNA Expression of Genetic Information The pathway for the flow of genetic information: DNA RNA Protein is now known as the central dogma of molecular biology. RNA is synthesized from DNA templates (transcription); proteins are synthesized from RNA templates (translation). Expression of Genetic Information RNA polymerase catalyzes synthesis of messenger RNA (mRNA) from a DNA template. Ribosomal RNA (rRNA) is a component of ribosomes, sites of protein synthesis. Transfer RNAs (tRNAs) serve as adaptor molecules that align amino acids along the mRNA template. Expression of Genetic Information Each amino acid is attached by a specific enzyme to its appropriate tRNA. Base pairing between the tRNA and a complementary sequence on the mRNA directs the attached amino acid to its correct position on the mRNA template. Figure 4.10 Function of transfer RNA Expression of Genetic Information How can four nucleotide bases specify the sequence of 20 amino acids? The nucleotides are used as triplets to encode the different amino acids: the genetic code. Expression of Genetic Information Evidence for the triplet code came from bacteriophage T4 with mutations in a gene called rII. Mutants with the addition of one or two bases always exhibited the mutant phenotype. Changes in three bases frequently led to the wild-type phenotype. Figure 4.11 Genetic evidence for a triplet code Expression of Genetic Information Assigning nucleotide triplets to their corresponding amino acids was done using in vitro systems that could carry out protein synthesis (in vitro translation). The systems contain ribosomes, amino acids, tRNAs, enzymes, and synthetic mRNA with known base sequences. Figure 4.12 The triplet UUU encodes phenylalanine Expression of Genetic Information All 64 possible triplets (called codons) were assigned in this way. 61 specify an amino acid; three are stop codons that signal the termination of protein synthesis. The code is degenerate: many amino acids are specified by more than one codon. Table 4.1 The Genetic Code Expression of Genetic Information With few exceptions, all organisms utilize the same genetic codestrong support for the conclusion that all present-day cells evolved from a common ancestor. Expression of Genetic Information Some viruses contain RNA instead of DNA. How viral RNA replicates was determined using RNA bacteriophages of E. coli. These viruses encode an enzyme that catalyzes synthesis of RNA from an RNA template (RNA-directed RNA synthesis). Expression of Genetic Information Most animal viruses replicate in this way, but one group (RNA tumor viruses) requires DNA synthesis in infected cells. These viruses (now called retroviruses) replicate via a DNA intermediate, called a DNA provirus. Figure 4.13 Reverse transcription and retrovirus replication Expression of Genetic Information This hypothesis was initially met with disbelief because it reverses the central dogma. Later, an enzyme that catalyzes synthesis of DNA from an RNA template (reverse transcription) was discovered. Key Experiment, Ch. 4, p. 125 (2) Expression of Genetic Information Reverse transcription has other broad implications. It occurs in cells and is critical for replicating the ends of eukaryotic chromosomes, and is frequently responsible for transposition of DNA from one chromosomal location to another. Expression of Genetic Information Reverse transcriptases are used experimentally to generate DNA copies of any RNA molecule. This has allowed study and manipulation of eukaryotic mRNAs. Recombinant DNA Recombinant DNA technology allows scientists to isolate, sequence, and manipulate individual genes from any type of cell. It has enabled detailed molecular studies of the structure and function of eukaryotic genes and genomes, and revolutionized our understanding of cell biology. Recombinant DNA Restriction endonucleases: enzymes that cleave DNA at specific sequences. First identified in bacteria, where they provide defense against the entry of foreign DNA. Bacteria have a variety of restriction endonucleases that cleave DNA at more than 100 distinct recognition sites. Table 4.2 Recognition Sites of Representative Restriction Endonucleases Recombinant DNA EcoRI recognizes the sequence GAATTC. This sequence is present at five sites in DNA of bacteriophage , so the DNA is digested into 6 fragments ranging from 3.6 to 21.2 kb long. 1 kilobase (kb) = 1000 base pairs Recombinant DNA The fragments can be separated by gel electrophoresis: A gel of agarose or polyacrylamide is placed between two electrodes and the sample is added to the gel. Nucleic acids are negatively charged so they migrate toward the positive electrode. Smaller molecules move more rapidly, allowing the fragments to be separated by size. Figure 4.14 EcoRI digestion and gel electrophoresis of DNA Recombinant DNA The order of restriction fragments can also be determined, and maps of restriction sites generated. Detailed restriction maps of viral DNA molecules have been produced. DNA fragments can also be isolated for sequencing. Figure 4.15 Restriction maps of and adenovirus DNAs Recombinant DNA For larger DNA molecules, restriction endonuclease digestion alone does not provide enough resolution. For example, the human genome would yield more than 500,000 EcoRI fragments, which cant be separated by gel electrophoresis. Recombinant DNA Purified DNA fragments can be obtained by molecular cloning. In molecular cloning, a DNA fragment is inserted into a DNA molecule (a vector) that can replicate independently in a host cell. The result is a recombinant molecule or molecular clone. Recombinant DNA Fragments of human DNA can be cloned in plasmid vectors: small circular DNA molecules that can replicate independently in bacteria. Recombinant plasmids with human DNA inserts can be introduced into E. coli, where they replicate along with the bacteria to yield millions of copies of plasmid DNA. Figure 4.16 Generation of a recombinant DNA molecule Recombinant DNA The fragment can be isolated from the plasmid DNA by restriction endonuclease digestion and gel electrophoresis. This allows a pure fragment of human DNA to be analyzed and further manipulated. Recombinant DNA Restriction endonucleases cleave recognition sequences at staggered sites, leaving single-stranded tails that can associate with each other by complementary base pairing. DNA ligase can then seal the ends permanently. Figure 4.17 Joining of DNA molecules Recombinant DNA Synthetic DNA linkers containing desired restriction endonuclease sites can be added to the ends of any DNA fragment. This allows virtually any fragment of DNA to be ligated to a vector and isolated as a molecular clone. Recombinant DNA RNA can also be cloned. RNA is copied using reverse transcriptase. The resulting DNA (cDNA) is ligated to a vector DNA. This allows mRNA to be isolated as a molecular clone, and the noncoding sequences in eukaryotic genes (introns) can be explored. Figure 4.18 cDNA cloning Recombinant DNA Plasmids are often used for cloning DNA inserts up to a few thousand base pairs long. Plasmids have an origin of replication (ori)the DNA sequence that signals the host DNA polymerase to start replication. Figure 4.19 Cloning in plasmid vectors (Part 1) Figure 4.19 Cloning in plasmid vectors (Part 2) Figure 4.19 Cloning in plasmid vectors (Part 3) Recombinant DNA Plasmid vectors also carry genes that confer resistance to antibiotics, so bacteria carrying the plasmids can be selected for. Recombinant DNA Bacteriophage vectors can accommodate larger DNA fragments. Sequences not needed for virus replication are removed and replaced with unique restriction sites for insertion of cloned DNA. Recombinant DNA The recombinant molecules are then put into E. coli, where they replicate to yield millions of progeny phages containing a single DNA insert. Recombinant DNA For even larger fragments of DNA, five major types of vectors are used: 1. Cosmid vectors contain bacteriophage sequences, origins of replication, and genes for antibiotic resistance, so they are able to replicate as plasmids in bacterial cells. Recombinant DNA 2. Bacteriophage P1 vectors allow recombinant molecules to be packaged in vitro into P1 phage particles and replicated as plasmids in E. coli. 3. P1 artificial chromosome (PAC) vectors also have bacteriophage P1 sequences, but are introduced directly as plasmids into E. coli. Recombinant DNA 4. Bacterial artificial chromosome (BAC) vectors are derived from a naturally occurring plasmid of E. coli (the F factor). 5. Yeast artificial chromosome (YAC) vectors have yeast origins of replication and other sequences that allow them to replicate as linear chromosome-like molecules in yeast cells. Table 4.3 Vectors for Cloning Large Fragments of DNA Recombinant DNA Nucleotide sequencing aids the study of protein structure, gene sequences that regulate expression, and gene function. DNA sequencing is usually done with automated systems. Recombinant DNA The basic method is based on premature termination of DNA synthesis. DNA synthesis is initiated with a synthetic primer. Dideoxynucleotides are included along with the normal nucleotides. They are labeled with different fluorescent dyes. Recombinant DNA The dideoxynucleotides stop DNA synthesis because no 3 OH group is available for addition of the next nucleotide. The fragments are separated by gel electrophoresis, and a laser beam excites the fluorescent dyes. The data is analyzed by computer. Figure 4.20 DNA sequencing Recombinant DNA Next-generation sequencing allows DNA to be sequenced faster and less expensively. These methods simultaneously determine sequences of millions of templates by parallel sequencing reactions in flow cells. Recombinant DNA Molecular cloning is also used to make large amounts of protein for study. Many proteins in cells are present in small amounts and cant be purified. Cloned genes can be used to engineer vectors that lead to high levels of gene expression in bacteria or eukaryotic cells. Recombinant DNA In bacteria, cDNA is cloned into a plasmid or phage vector (an expression vector). Inserted genes can be expressed at levels as high as 10% of the total bacterial protein. Figure 4.21 Expression of cloned genes in bacteria (Part 1) Figure 4.21 Expression of cloned genes in bacteria (Part 2) Recombinant DNA Expression in eukaryotic cells instead of bacteria may be needed, (e.g., if posttranslational modification of the protein is required). Cloned genes are inserted into virus vectors. Detection of Nucleic Acids and Proteins Detection of specific nucleic acids and proteins is important for a variety of studies: Analysis of gene expression Localization of proteins to subcellular organelles Detection of Nucleic Acids and Proteins To isolate large amounts of a single DNA molecule, an alternative to molecular cloning is the polymerase chain reaction (PCR), developed in DNA polymerase is used in vitro for repeated replication of a defined segment of DNA. Detection of Nucleic Acids and Proteins A specific region of DNA can be amplified if the nucleotide sequence surrounding the region is known. Two primers are designed to initiate DNA synthesis in opposite directions at the desired point. The synthetic primers are 1520 bases long. Detection of Nucleic Acids and Proteins The reaction starts by heating the template DNA to 95C to separate the strands. The DNA polymerase used (Taq polymerase) is a heat-stable enzyme from Thermus aquaticus, a bacterium that lives in hot springs. Detection of Nucleic Acids and Proteins The temperature is then lowered to allow primers to pair and DNA synthesis proceeds. Two DNA molecules are synthesized from one template. The cycle can be repeated multiple times, with a two-fold increase in DNA for each cycle. Figure 4.22 Amplification of DNA by PCR Detection of Nucleic Acids and Proteins PCR amplification can be performed rapidly and automatically. RNA can also be amplified by PCR if reverse transcriptase is used to synthesize a cDNA copy first. Detection of Nucleic Acids and Proteins If enough of a gene sequence is known so that primers can be made, PCR can detect small amounts of specific DNA or RNA in complex mixtures. The only DNA molecules that will be amplified are those containing sequences complementary to the primers used. Detection of Nucleic Acids and Proteins Real-time PCR can determine amounts of specific DNA in a sample. A fluorescent dye that binds to double- stranded DNA, and only fluoresces when bound, is incorporated. Fluorescence is monitored after each cycle of PCR, and the intensity of fluorescence is indicative of the amount of DNA that has been amplified. Detection of Nucleic Acids and Proteins The amount of DNA amplified after a given number of cycles is determined by the amount of template that was initially present. The sensitivity of real-time PCR has made it important for a variety of applications, including analysis of gene expression in cells and tissues. Detection of Nucleic Acids and Proteins The key to detection of specific nucleic acid sequences is base pairing. In nucleic acid hybridization, DNA strands are separated by high temperatures; when cooled, they re- form double-stranded molecules by complementary base pairing. Detection of Nucleic Acids and Proteins Cloned DNA can be labeled with radioactive nucleotides or nucleotides modified to fluoresce. This labeled DNA is then used as a probe that hybridizes with complementary DNA or RNA in complex mixtures. Figure 4.23 Detection of DNA by nucleic acid hybridization Detection of Nucleic Acids and Proteins Southern blotting: DNA is digested with a restriction endonuclease, and the fragments separated by gel electrophoresis. The gel is then overlaid with a nitrocellulose or nylon membrane to which the DNA fragments are transferred (blotted). The filter is then incubated with a labeled probe. Figure 4.24 Southern blotting Detection of Nucleic Acids and Proteins Northern blotting is used for detection of RNA instead of DNA. It is often used in studies of gene expression, for example, to determine whether specific mRNAs are present. Detection of Nucleic Acids and Proteins Recombinant DNA libraries are collections of clones that contain all the genomic or mRNA sequences of a particular cell type. A genomic library of human DNA can be made by cloning random DNA fragments of about 15 kb in a bacteriophage vector. Figure 4.25 Screening a recombinant library by hybridization Detection of Nucleic Acids and Proteins Any gene for which a probe is available can be isolated from a recombinant library. cDNA clones can be used as probes to isolate corresponding genomic clones. Or a gene cloned from one species (e.g., mouse) can be used to isolate a related gene from a different species (e.g., human). Detection of Nucleic Acids and Proteins In situ hybridization can be used to detect homologous DNA or RNA sequences in cell extracts, chromosomes, or intact cells. Hybridization of fluorescent probes to specific cells or subcellular structures is analyzed by microscopic examination. Figure 4.26 Fluorescence in situ hybridization Detection of Nucleic Acids and Proteins Antibodies can be used as protein probes. Antibodies are proteins produced by immune system cells (B lymphocytes) that react against foreign molecules (antigens). Different antibodies recognize unique antigens. Detection of Nucleic Acids and Proteins Antibodies can be generated by inoculation of an animal with any foreign protein. Monoclonal antibodies can be produced by culturing clonal lines of B lymphocytes from immunized animals (usually mice). Detection of Nucleic Acids and Proteins Two common methods using antibodies: 1. Immunoblotting (Western blotting) Proteins are separated by size by SDS- polyacrylamide gel electrophoresis (SDS-PAGE). Detection of Nucleic Acids and Proteins The negatively charged detergent sodium dodecyl sulfate (SDS) denatures the protein and gives it an overall negative charge. The proteins will migrate towards the positive electrode and are then reacted with labeled antibodies. Figure 4.27 Immunoblotting (Part 1) Figure 4.27 Immunoblotting (Part 2) Figure 4.27 Immunoblotting (Part 3) Detection of Nucleic Acids and Proteins Antibodies can be used to visualize proteins in intact cells. Cells can be stained with antibodies labeled with fluorescent dyes or tags visible by electron microscopy. Figure 4.28 Immunofluorescence Gene Function in Eukaryotes In classical genetics, gene function has been revealed by the altered phenotypes of mutant organisms. It is now possible to study the function of a cloned gene directly by reintroducing it into eukaryotic cells. Gene Function in Eukaryotes Gene function can be studied by introducing cloned DNA into plant and animal cells (gene transfer). Methods were initially developed using infectious viral DNA; thus it is called transfection. Figure 4.29 Introduction of DNA into animal cells Gene Function in Eukaryotes Other methods include: Direct microinjection into the nucleus Coprecipitation of DNA with calcium phosphate to form small particles that are taken up by the cells Incorporation of DNA into liposomes that fuse with the plasma membrane Expose cells to brief electric pulses to open pores in the plasma membrane (electroporation) Gene Function in Eukaryotes In most of the cells, the DNA is transported to the nucleus, and is transcribed for several days: transient expression. In about 1% of cells, the foreign DNA is integrated into the genome and transferred to progeny cells at cell division. Gene Function in Eukaryotes If the transfected DNA contains a selectable marker, the stably transformed cells can be isolated and studied. Animal viruses, especially retroviruses, can be used as vectors to introduce cloned DNAs into a wide variety of cell types. Figure 4.30 Retroviral vectors (Part 1) Figure 4.30 Retroviral vectors (Part 2) Gene Function in Eukaryotes Cloned genes can also be introduced into the germ line of multicellular organisms. Mice that carry foreign genes (transgenic mice) are produced by microinjection of cloned DNA into the pronucleus of a fertilized egg. Figure 4.31 Production of transgenic mice Gene Function in Eukaryotes Embryonic stem (ES) cells are also used to get cloned genes into mice. Cloned DNA is introduced into ES cells in culture, then transformed cells are introduced back into mouse embryos. The offspring are chimeric: a mixture of cells that arise from normal and transfected embryonic cells. Figure 4.32 Introduction of genes into mice via embryonic stem cells (Part 1) Figure 4.32 Introduction of genes into mice via embryonic stem cells (Part 2) Gene Function in Eukaryotes Cloned DNAs can also be introduced into plant cells. Since many plants can be regenerated from a single cell, transgenic plants are easily established. Many economically important plants, including corn, tomatoes, soybeans and potatoes, are transgenic varieties. Gene Function in Eukaryotes Classically, gene function has been studied by observing altered phenotypes of mutant organisms. Now, specific mutations can be introduced into cloned DNA (in vitro mutagenesis) to study gene function in eukaryotes. Gene Function in Eukaryotes In vitro mutagenesis allows detailed characterization of the functional roles of both regulatory and protein-coding sequences of cloned genes. The basic method uses synthetic oligonucleotides to generate changes in a DNA sequence. Figure 4.33 Mutagenesis with synthetic oligonucleotides Gene Function in Eukaryotes To determine the role of a cloned gene, activity of the normal gene copy must be eliminated. Homologous recombination: the mutated copy of the cloned gene replaces the normal gene copy. Mutations that inactivate (knockout) the cloned gene are introduced in place of the normal gene copy. Figure 4.34 Gene inactivation by homologous recombination Gene Function in Eukaryotes Genes can be inactivated in mouse embryonic stem cells, which can grow into transgenic mice. The mice yield progeny with mutated copies of the gene on both homologous chromosomes. Effects of gene inactivation can then be studied in the context of the intact animal. Figure 4.35 Production of mutant mice by homologous recombination in ES cells Gene Function in Eukaryotes Cells can be cultured from mouse embryos containing the mutated gene copies, so gene function can also be studied in cell culture. The function of more than 7000 mouse genes have been investigated in this way. Gene Function in Eukaryotes Methods have also been developed to conditionally knockout genes in specific mouse tissues, allowing the function of a gene to be studied in a defined cell type. Gene Function in Eukaryotes The CRISPR/Cas system is a new approach for introducing targeted gene mutations into mammalian cells. Specific target sequences are recognized by a synthetic RNA molecule, and Cas (CRISPR- associated system) proteins cleave the targeted DNA. Gene Function in Eukaryotes Plasmids expressing a guide RNA (gRNA) with sequences homologous to the target gene and the Cas9 nuclease are introduced into cells, along with a mutated copy of the gene. Figure 4.36 Gene targeting by CRISPR/Cas Gene Function in Eukaryotes Other approaches interfere with gene expression or function. Antisense nucleic acids are RNA or single-stranded DNA complementary to the mRNA of the gene of interest (antisense). They hybridize with the mRNA and block its translation into protein. Figure 4.37 Inhibition of gene expression by antisense RNA or DNA Gene Function in Eukaryotes RNA interference (RNAi) was first discovered in C. elegans. Fire and Mello found that injection of double-stranded RNA inhibited expression of a gene with a complementary mRNA sequence. Double-stranded RNA resulted in extensive degradation of the target mRNA, whereas single-stranded antisense RNA had only a minimal effect. Gene Function in Eukaryotes When double-stranded RNAs are introduced into cells, they are cleaved into short interfering RNAs (siRNAs) by an enzyme called Dicer. The siRNAs associate with a complex of proteins known as the RNA-induced silencing complex (RISC), where mRNA is cleaved. Figure 4.38 RNA interference Key Experiment, Ch. 4, p. 151 (3) Gene Function in Eukaryotes The discovery of RNA interference demonstrated a role for double- stranded RNAs in gene regulation. This has been developed into a powerful experimental tool for inhibiting expression of target genes. Gene Function in Eukaryotes It is sometimes possible to interfere directly with protein function: Microinject antibodies that block the activity of the protein. Gene Function in Eukaryotes Introduce cloned DNA that encodes mutant proteins (dominant inhibitory or dominant negative mutants) that interfere with the function of their normal counterparts. Figure 4.39 Direct inhibition of protein function