Chap. 5 Molecular Genetic Techniques (Part A)

1. Chap. 5 Molecular Genetic Techniques (Part A) • Topics • Genetic Analysis of Mutations to Identify and Study Genes • DNA Cloning and Characterization Goals Learn about genetic and recombinant DNA methods for isolating genes and characterizing the functions of the proteins they encode. Use of RNA interference (RNAi) in analysis of planarian regeneration

2. Leptin Receptor Knockout Mice db/dbDB/DB

3. Importance of Mutations in Gene Analysis One of the most important ways in which the function of a gene can be learned is by the study of a mutant in which the gene has been inactivated. Currently, mutants can be generated by classical “forward” genetic methods, and by more modern “reverse” genetic approaches (Fig. 5.1). In forward genetic analyses one generates a mutant organism and then uses molecular biological techniques to isolate the mutant gene and characterize the protein responsible for the phenotype of the mutant. In reverse genetic approaches, a gene is inactivated and the function of the gene is learned by study of the properties of the mutant organism.

4. Genetics Terms Alleles-Different versions (sequences) of a gene. Mutant-Newly created allele made by mutagenesis. Genotype-The complete set of alleles for all genes carried by an individual. Wild type-Standard reference genotype. Most common allele for a certain trait. Phenotype-Observable trait specified by the genotype. Point mutation-A change in a single base pair (e.g., a G.C to A.T transition). Silent mutation-A point mutation in a codon that does not change the specified amino acid. Missense mutation-A point mutation that changes the encoded amino acid. Nonsense mutation-A point mutation that introduces a premature stop codon into the coding sequence of a gene. Recessive & dominant mutant alleles-(next slide)

5. Recessive and Dominant Mutant Alleles Diploid organisms have two copies of each gene; haploid organisms (e.g., some unicellular organisms) contain only one. A recessive mutant allele must be present in two copies (be homozygous) to cause a phenotype in a diploid organism (Fig. 5.2). Only one copy of a recessive allele must be present for the phenotype to be observable in a haploid organism. In contrast, a dominant mutant allele needs to be present in only one copy (heterozygous) in a diploid organism for the phenotype to be observable. Most recessive alleles cause gene inactivation and phenotypic loss of function. Some dominant alleles change or increase activity causing a gain of function. However, a dominant affect can be caused by gene inactivation if two copies of the gene are needed for proper function (haplo-insufficiency). Lastly, a dominant negative mutation refers to a situation where the product of the mutant gene inactivates the product of the wild-type gene. This can occur if a gene encodes one subunit of an oligomeric protein.

6. Review of Mitosis Mating experiments provide important information about gene function. These experiments demand a thorough knowledge of meiosis and production of gametes (sperm & egg cells in higher eukaryotes). In Fig. 5.3, mitosis is described to contrast it with meiosis. In mitosis, one round of DNA replication in a diploid somatic cell is followed by one cell division. The paternal and maternal homologous chromosomes (homologs) first are duplicated. The sister chromatids then are separated by a cell division. The daughter cells end up with one copy of each paternal and maternal chromosome and are diploid (2n).

7. Review of Meiosis In meiosis, one round of DNA replication in a diploid germ cell is followed by two cell divisions, resulting in four haploidgametes (Fig. 5.3). Paternal and maternal homologous chromosomes first are copied as in mitosis. However, after alignment (synapsis) and crossing over (recombination) of homologous chromosomes, paternal and maternal chromosomes are randomly segregated between the daughter cells formed in the first cell division. Subsequently, the sister chromatids of each chromosome are separated in a second cell division, which produces the gametes (1n). The two sets of gametes each contain a random assortment of the paternal and maternal chromosomes.

8. Identification of Dominant Mutations Dominant mutations can be identified by mating strains that each are homozygous for two alleles of a given gene. Because all gametes from each parent are of one type (Fig. 5.4a), all members of the first filial generation from the cross (F1) necessarily will be heterozygotes. If the mutation is dominant, all F1 offspring will display the mutant phenotype. On self crossing of F1 cells, 3/4 of the second filial generation (F2) will display the mutant phenotype, if it is dominant.

9. Identification of Recessive Mutations Recessive mutations also can be identified by mating strains that are homozygous for two alleles of a given gene. Again, because all gametes from each parent are of one type (Fig. 5.4b), all members of the F1 generation from the cross necessarily will be heterozygotes. None of the F1 offspring will display the mutant phenotype if it is recessive. On self crossing of F1 cells, only 1/4 of the F2 generation will display the phenotype, if it is recessive.

10. Analysis of Mutant Alleles in Yeast The yeast Saccharomyces cerevisiae is an ideal experimental organism for analysis of dominant and recessive alleles. First, cells can exist in either a haploid or diploid state. Second, haploid cells occur in two mating types (a and a) that are useful for performing crosses. The diploid cells resulting from matings can be examined to determine if a mutation is dominant or recessive (Fig. 5.5). Finally, haploid cells can be regenerated by meiotic sporulation of diploid cells grown under starvation conditions.

11. Use of Conditional Mutations to Study Essential Genes The study of essential genes (needed for life) requires special genetic screening techniques. In diploid organisms, such as the fruit fly Drosophila, lethal mutations in essential genes can be maintained in the diploid state and identified by inbreeding experiments. In haploid organisms, such as haploid yeast (Fig. 5.6), defects in essential genes can be isolated and maintained through the use of conditional mutations. Very often, conditional mutations that display temperature-sensitive (ts) phenotypes are used. ts mutations often result from substitution mutations that cause an essential protein to be unstable and inactive at high (nonpermissive), but not low (permissive) temperatures. A number of yeast cell-division cycle (cdc) mutants have been isolated via this technique (Fig. 5.6).

12. Complementation Analysis of Recessive Mutations Many processes, including cell division involve the combined actions of multiple genes. Thus, a genetic screen for mutations affecting such processes will turn up a collection of genes. Through mating haploid yeast containing the defective genes, one can establish in the diploid cells whether the mutations fall in the same or separate genes. As shown in Fig. 5.7, diploid cells will grow under nonpermissive conditions if the mutations reside in different genes (the wild type genes complement the defective ones). However, diploids with two defective copies of the same gene will not survive.

13. Double Mutant Analysis of Biosynthetic Pathways Genetic experiments can be used to determine the order in which gene products act in carrying out a process. Double mutant analysis can be applied to order the enzyme-catalyzed steps in a metabolic pathway. As shown in Fig. 5.8a, the accumulation of intermediate 1 in the double mutant strain indicates enzyme A operates prior to enzyme B.

14. Suppressor Mutations Suppressor mutation analysis is a powerful tool for identifying proteins that interact with one another in the performance of a certain cellular process. In genetic suppression, a loss of function mutation in Protein A is corrected by a compensating mutation in Protein B. The resulting gain of function phenotype of the double mutant results from the recreation of interaction sites between two proteins that are disrupted by each individual amino acid substitution mutation (Fig. 5.9a).

15. Synthetic Lethal Mutations The analysis of synthetic lethal mutations also is an important tool for identifying proteins that must interact to carry out a cellular process (Fig. 5.9b). It also is a powerful method to identify proteins that function in redundant pathways needed for the production of an essential cell component (Fig. 5.9c). Unlike suppressor mutations, synthetic lethal double mutants display a loss of function phenotype.

16. Intro to DNA Cloning by Recombinant DNA Methods To study a gene, one must first prepare and purify its DNA in relatively large amounts. This is accomplished via the recombinant DNA (rDNA) technology method known as DNA cloning. In cloning, a DNA molecule of interest is spliced into a vector such as a bacterial plasmid or virus forming a rDNA molecule which can be propagated in bacterial cells such as E. coli. After replication and amplification of the rDNA in the bacterium, it is purified for sequencing and other manipulations used in gene characterization.

17. DNA Cleavage by Restriction Enzymes Restriction enzymes are nucleases that are very important in rDNA technology. These enzymes make double-stranded cuts in DNA molecules at specific 4-8 bp palindromic (two-fold symmetrical) sequences called restriction sites. Many restriction enzymes make staggered cuts in DNA molecules resulting in single-stranded complementary sticky ends (Fig. 5.11). Sticky-ended fragments can be readily joined together to synthesize rDNA molecules (Fig. 5.12). In many cases, cleavage at the restriction site is blocked by methylation of bases in the site.

19. Joining of DNA Molecules by Ligation Plasmid vectors containing a DNA of interest (e.g., genomic DNA) can be readily constructed by ligating restriction fragments to vector DNA that has been digested with the same restriction enzyme (Fig. 5.12). Base-pairing between the complementary sequences of the sticky ends aligns the fragments for covalent linkage by a DNA ligase, typically T4 DNA ligase. This enzyme uses 2 ATP to provide energy for joining the 3'-hydroxyl and 5'-phosphate groups of the base-paired fragments together in 2 new 3'-5' phosphodiester bonds. Note, all restriction enzymes produce a 5'-phosphate and 3'-hydroxyl group at the cut site.

20. E. coli Plasmid Cloning Vectors Plasmids are autonomously replicating circular DNAs found in bacterial cells. Naturally occurring plasmids contain an origin of replication (ori) for propagation in the host cell and one or more genes that specify a trait that may be useful to the host. Cloning vectors are plasmids that have been geneticallyengineered to reduce unneeded DNA and to introduce selectable markers such as antibiotic resistance genes (e.g., ampr) that are used to force cells to maintain the plasmid. Polylinker sequences that encode several unique restriction sites for cloning purposes also are engineered into these vectors (Fig. 5.13).

21. Cloning of DNA in Plasmid Vectors An overview of the steps required for DNA cloning in a plasmid vector is presented in Fig. 5.14. In Step 1, the DNA of interest is ligated into a plasmid cloning vector. In Step 2, the recombinant plasmid is introduced into E. coli host cells by transformation. In Step 3, cells that have taken up the plasmid are selected on antibiotic (ampicillin) agar. In Step 4, the transformed cells replicate their chromosomal and plasmid DNA and multiply to form a colony. Cells in the colony contain the cloned DNA and are themselves clones. The rDNA plasmid then is harvested by growing a larger culture of the cells. 1 2 3 4 4

22. Construction of cDNA Libraries (Part 1) A genomic DNA library is a collection of cloned DNA fragments representing all of the DNA of an organism. A cDNA library (complementary DNA), is a collection of cloned DNA fragments corresponding to all mRNAs transcribed in a certain tissue or organism. Libraries can be constructed using plasmid cloning vectors. To construct a cDNA library, one begins by isolating mRNA from the cell or tissue of interest (Fig. 5.15). Because many genes are transcribed at a low frequency, it is best to start with a cell/tissue that expresses the gene of interest at a relatively high level. cDNAs are transcribed from a mRNA template by a retroviral enzyme known as reverse transcriptase (RT). In Step 1, mRNA isolated by oligo-dT affinity chromatography is hybridized via its 3' poly(A) tail to an oligo-dT primer. In Step 2, RT synthesizes the first cDNA strand. In Step 3, RNA is destroyed and a poly(dG) tail is added by terminal transferase. In Step 4, the cDNA is hybridized to an oligo-dC primer. (Go to next slide).

23. Construction of cDNA Libraries (Part 2) In Step 5, a DNA polymerase is used to synthesize the second strand of the cDNA. In Step 6, EcoRI sites that might be present within the mRNA coding region are protected by methylation using EcoRI methylase. In Step 7, unmethylated EcoRI linkers, that encode EcoRI restriction sites, are ligated to the ends of the fragment. In Step 8a, the cDNA is cleaved with EcoRI restriction enzyme, generating sticky-ended cDNA fragments. (See next slide).

24. Construction of cDNA Libraries (Part 3) In the last steps of cDNA library construction, the plasmid vector is cut with EcoRI restriction enzyme (Step 8b), and then the EcoRI-cut cDNA and plasmid are ligated together (Step 9). Finally, the E. coli host strain is transformed and cells are plated (Step 10) on selective medium. To be complete, both genomic and cDNA libraries for higher eukaryotes must contain on the order of a million individual clones.

25. Screening cDNA Libraries To screen a plasmid library (Fig. 5.16), colonies representing each cloned DNA first are plated on a number of petri plates. Library DNA then is lifted onto nitrocellulose membranes which serve as replicas of the plates. Bound DNA is denatured and hybridized with a radioactively-labeled single-strand DNA probe (next slide). After washing, spots corresponding to colonies containing the DNA of interest are detected by autoradiography. Because not all DNA gets lifted onto the membranes, DNA for the clone can be purified from the residual colony on the original plate. Note, that oligonucleotide probes must only be ~ 20 nucleotides long to recognize unique sequences even in genomic DNA. The probe sequence can be derived from genome sequencing databases, or designed based on the known sequence of a protein.

26. DNA Detection by Membrane Hybridization The general method for screening a membrane-bound DNA sample for a gene of interest is illustrated in Fig. 5.16. This involves fixation of single-stranded DNA to the membrane, hybridizing the fixed DNA to a labeled DNA probe complementary to the gene of interest, removal of un-hybridized probe by washing, and detection of the specifically hybridized probe by autoradiography, etc.

27. Construction of a Yeast Genomic Library in a Shuttle Vector Plasmids known as E. coli-yeast shuttle vectors (Fig. 5.17a) can replicate in both organisms. Shuttle vectors contain 1) origins of replication for both species (ori, E. coli; ARS, yeast), 2) markers for selection in E. coli (ampr) and yeast (URA3), and 3) a CEN sequence that ensures stable replication and segregation in yeast. The method for construction of a yeast genomic library in a E. coli-yeast shuttle vector is illustrated in Fig. 5.17b. A total of ~105 clones is needed to include all genes, if the genomic DNA is cut into fragments of about 10 kb in length.

28. Screening by Functional Complementation A yeast genomic library can be screened by the technique of functional complementation to isolate the cloned version of a gene of interest (Fig. 5.18). First, all recombinant plasmids from the library are isolated from E. coli, pooled, and used to transform haploid ura3- yeast that carry a conditional lethal ts copy of the gene of interest. Transformants are selected by plating on uracil-deficient agar at the permissive temperature. Second, transformants are replica plated onto agar and incubated at the nonpermissive temperature to identify colonies carrying a wild type version of the gene of interest. Only cells containing the library copy of the wild type gene can survive at high temperature.