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Recombinant DNA Technology Recombinant DNA technology allows us to isolate specific genes from the genome so that we can study their function. Recombinant DNA molecules can be made from any organism by inserting DNA fragments into a gene cloning vector. (Figure 12-1) Vector (plasmid or virus) Contains an origin of replication for gene amplification. Usually contains an antibiotic resistance gene for selection. Restriction Enzymes Make sequence specific cuts in DNA by cleaving each strand of the duplex (Digestion). (e.g.) Eco RIcohesive "sticky" ends 5’...GAATTC...3’ ...G + AATTC... 3’...CTTAAG...5’ ...CTTAA G... (e.g.) Hin dIIblunt ends 5’...GTPyPuAC...3’ ...GTPy + PuAC... 3’...CAPuPyTG...5’ ...CAPu + PyTG... Characteristics of Restriction Sites A. 180° axis of symmetry. B. Usually a 4 (1/256) or 6 nt sequence (1/4096).
Gene Cloning (Figure 12-4)
- Digest chromosomal and vector DNA with an enzyme.
- Mix together. Sticky ends anneal due to base complementarity. Can also use blunt ends.
- Seal the "nicks" with DNA Ligase.
- Transform E. coli and select for drug resistance or by complementation (compensation) of a mutant defect. (Figure 12-5)
- Amplify and purify recombinant DNA. Vectors A. Plasmids small, circular, origin of replication, antibiotic resistance gene. (Can clone several kb) (Figure 12-6) B. Expression Vectors contain transcription and translation signals to allow overproduction of the protein encoded by the gene. Express eukaryotic genes in bacteria. C. Shuttle Vectors contains origins for two organisms (e.g.) E. coli and SV40 virus. Clone in E. coli , purify DNA, transfect mammalian cell line. D. Yeast Artificial Chromosomes (YACs) contain an ARS, telomeres, and a centromere. 1000 kb can be cloned. DNA Library Random chromosomal or cDNA fragments cloned into one of the above vectors. A random population of clones should contain every gene. Isolate a specific gene by selection or screening. Cloning by complementation
- Isolate a mutant strain giving the desired phenotype.
- Transform the mutant strain with a DNA library and directly select for the positive clone by its ability to complement the mutant defect.
Restriction Mapping Restriction sites in a DNA fragment can be used to subclone fragments within the fragment.
- Digest DNA with one of several enzymes.
- Run digested DNA on an agarose gel to separate fragments.
- Stain DNA with ethidium bromide (EtBr) which intercalates between bases.
- View under UV light (EtBr fluoresces).
- Single, double or partial digests. (Figure 12-27) A linear 13 kb fragment of DNA is digested with various restriction enzymes. The results of single and double digests are shown below. Here is a sample problem to work on your own Enzyme(s) Fragment Sizes (kb) Bam HI 3 and 10 Eco RI 6 and 7 Hin dIII 1 and 12 Bam HI and Eco RI 3, 4 and 6 Bam HI and Hin dIII 1, 3 and 9 What fragment sizes are expected if the 13 kb fragment is digested with Eco RI and Hin dIII? A. 1, 3 and 9 B. 3, 4 and 6 C. 1, 5 and 7 D. 1, 4 and 8 E. 4, 4 and 5 Site-Directed Mutagenesis Directing point mutations, insertions or deletions into cloned DNA fragments. (Figure 13-1) Eukaryotic Gene Expression in Bacteria Use specialized vectors to specifically overexpress biologically important human proteins in bacteria (usually E. coli ). (e.g.) Insulin
Eukaryotic Transgenic Technology E. coli4.2 million bp Human3 billion bp PlantsSome even larger Specialized techniques were developed to handle large genomes. Transgenic Technology Methods used to transfect eukaryotic cells. Transgenic Organism Organism that develops from the transfected cell. Gene Inactivation (Suicide vector) (Figure 13-12)
- Clone selectable marker in the middle of a gene.
- Linearize with restriction enzyme.
- Transform organism.
- Double X-over results in replacement of WT gene with disrupted gene.
- Study the effect of the mutation. Studying Gene Regulation (Figure 13-13)
- Clone regulatory (5') region adjacent to a reporter gene (gene whose protein is easy to assay). Expression of reporter gene depends on cloned regulatory elements.
- Study regulation.
- Repeat with deletions or point mutations in regulatory region. Transgenic Plants Ti Plasmid from Agrobacterium tumefaciens Causes crown gall (plant tumors). (Figure 13-14) Bacteria infects plant and injects part of plasmid called T DNA (T=tumor). Clone gene in middle of T DNA so that the gene is inserted into plants with T DNA. (e.g.) Firefly luciferase geneglow in the dark plants. (Chapter 13 cover)
Wild type (WT) compared to a mutant or variant. Mutant An individual or strain carrying a mutation. Mutation Change from one hereditary state to another. Used to genetically dissect biological functions and to study the process of mutation. Gene Mutation A mutation in a specific gene resulting in a new allele. Forward Mutation Any change from the WT allele. Reverse Mutation Change to the WT allele (true reversion). Second Site Suppressor A change in the same gene or a second gene resulting in a complete or partial phenotypic reversion to WT (second site reversion). Loss of Function Mutation A. Null mutation No activity. B. Leaky mutation Some residual activity. Gain of Function Mutation Results in a new activity. Silent Substitution The mutation changes one codon for an AA into another codon for the same AA. Missense Mutation The codon for one AA is replaced by a codon for another AA. Nonsense Mutation The codon for an AA is replaced by a stop codon. Somatic Mutation A mutation in any tissue other than the germinal tissue. Clone Population of identical cells derived from one mutant progenitor (asexual). (i.e.) Mitosisnot transmitted to progeny Often visualized as a sector (Figures 15-3 to 15-5).
Germinal Mutation A mutation in tissue that forms gametes. An individual with the “new” germinal mutation will not show the phenotype but the mutation can be transmitted to progeny. (Figure 15-8) Conditional Mutation The allele only expresses the mutant phenotype under certain environmental conditions. (e.g.ts t emperature s ensitive) Auxotrophic Mutation The individual must be supplied with certain nutrients (amino acids, nucleotides, vitamins). Commonly used when studying microorganisms. WT is prototrophic (nutritionally self-sufficient). Resistance Mutation Confers the ability to grow in the presence of an inhibitor.(e.g.) antibiotic or pathogen Point Mutation Single base pair change in DNA. Deletion Removal of one or more bases of DNA. Insertion Addition of one or more bases of DNA. Mutation Rate
of mutations OR # of mutations
cell division gamete Human Genetics Germinal mutations are detected by the sudden appearance of the abnormal phenotype in a pedigree with no previous record of abnormality. Dominant mutations are easy to detect. Recessive mutations can go unnoticed for several generations. X-linked recessive mutations are easier to detect than autosomal. (Queen Victoria-Hemophilia) Different genes have different mutation rates. (See Tables 15-2 & 15-3)
Penicillin Enrichment Auxotrophic selection in bacteria. Penicillin kills actively growing cells.
- Grow cells in rich medium.
- Transfer to minimal medium.
- Add penicillin (prototrophs die, auxotrophs survive).
- Plate cells on minimal medium + supplement. Resistance Mutations
- Grow cells in liquid culture.
- Plate cells on selective medium (drug or virus) Fluctuation Test Used to determine if resistant mutations were due to random mutations or changes in bacterial physiology.
- Liquid culture of E. coli.
- Mix individual cultures with bacteriophage T1.
- Plate cell-phage mixture.
- WT cells killed by phage T1.
- Resistant mutants form colonies. Fluctuation Test Predictions A) Random mutationRare mutations could occur early or late in the culture. Predict a large variation in the number of resistant colonies. B) Physiological changeTime for physiological adaptation would be relatively constant. Predict small variation in number of colonies. Answer: Random Mutation (Figure 15-21; Table 15-4)
Somatic Cell Genetics Applying mutagenic and selective techniques to animal and plant cell cultures. Often only identify dominant mutations because the organism is diploid. Mutation and Cancer Cancer is a genetic disease caused by mutations in proto-oncogenes (dominant) or tumor suppressor genes (recessive). Proto-oncogenes and Tumor Suppressor Genes Normally carry out functions related to regulation of cell division. A mutation in a proto-oncogene can lead to uncontrolled cell division (mutant clone) resulting in a tumor (cancer). Cancer can spread by metastasis. Genetic Predisposition A mutant gene causes an increase in mutation frequency of other genes leading to cancer.
Spontaneous Lesions Mutations can occur due to DNA damage. Depurination When the N-glycosidic bond between the base and the sugar is broken. The resulting apurinic site (AP site) can’t specify a complementary base during replication. 10,000/cell/generation in mammals. (DNA repair required) Deamination Loss of an amino group from the base. Deamination of dC yields dU. (Figure 16-8a) dU pairs with dA (GC--->AT transition). (DNA repair) Oxidative Damage Byproducts of aerobic metabolism produces compounds that cause oxidative DNA damage. Induced Mutations Produced when a cell or organism is exposed to a mutagenic agent (mutagen). Replace a base in DNA Molecules which are similar in structure to bases (base analogs) but have different pairing properties can replace the normal base in the DNA during replication. Specific Mispairing Some chemicals alter the structure of a base resulting in mispairing during replication. Intercalating Agents Chemicals that are planar can mimic bases and slip in (intercalate) between bases in the double helix. Results in frameshifts.
Loss of Pairing due to Chemical Alteration Chemical structure is altered so it can't pair with any base. Results in a replication block. Lethal unless the block is bypassed. UV light can cause several types of DNA damage. (e.g.) UV light can generate cyclobutane pyrmidine dimers which distorts the structure of DNA such that base pairing is not possible. Most carcinogens result in chemical alteration of DNA. Cancer can be caused by mutations in genes whose protein products regulate cell division. If this check point is abolished it will lead to uncontrolled cell division (i.e.) cancer. p53 About 50% of all cancerous tissues contain mutations in the p53 gene. p53 arrests cell division when DNA mismatches are recognized. (i.e.) involved in cell cycle control.
III. Excision-Repair Pathways General Excision Repair Removal of altered bases, along with several neighboring bases, and then repairing the gap by DNA synthesis. (Figure 16-27) E. coli An endonuclease cuts on both sides of the damaged base removing ssDNA containing the damaged base(s). Gap filled in by DNA pol I. DNA ligase seals the nick. Animation 1601 Specific Excision Repair AP Endonuclease Repair Pathway AP endonuclease removes AP site by breaking a phosphodiester bonds at the AP site. (Figure 16-30) Then the general excision repair pathway takes over. DNA Glycosylase Repair Pathway DNA glycosylases recognize certain damaged bases and cleave the N-glycosidic bond between the base and the sugar leaving an AP site. (Figure 16-29) (e.g.) Uracil DNA Glycosylase The resulting AP site is cleaved by AP endonuclease. Then the general excision repair pathway takes over.
IV. Postreplication Repair (Figure 16-32) Mismatch Repair DNA editing by DNA pol III did not occur.
- Recognition of the mismatch.
- Determine which mismatched base is incorrect.
- Excise the incorrect base.
- General excision repair takes over. Adenine methylase methylates A residues following replication in the sequence: 5' GATC 3' But it takes a few minutes for the newly synthesized strand to be methylated. The old methylated strand is distinguished from the newly synthesized unmethylated strand. An enzyme introduces a cleavage in the backbone of the unmethylated strand near the mismatch and adjacent to the nearest GATC sequence. ssDNA gap is filled in by DNA pol I. Ligase seals the nick.
Chromosomal Rearrangements Deletions Loss of a region caused by a chromosomal break. Duplication Reciprocal change of a deletion. Inversion Chromosomal region rotated 180°. Translocation Exchange of parts of non-homologous chromosomes. Deletions Usually fatal if homozygous. Often fatal if heterozygous. Some small deletions are viable as a heterozygote. Deletions can never revert to WT. Visualized as a deletion loop during meiosis.
Homologous
Chromosomes
Pseudodominance Deletion will “uncover” recessive alleles on the other chromosome, thus the recessive phenotype is expressed. Small deletions can be mapped due to pseudodominance. Useful in correlating linkage and cytological maps. (Figure 17-4) Humans Usually caused by a new germinal mutation in one parent. (e.g.) cri du chat syndrome. Tip of chromosome 5 deleted.
Duplications Can be adjacent to each other or the second copy may be in a novel location on the same or a different chromosome. 3 copies/cell in a diploid. Duplication heterozygotes can result in unusual pairing structures during meiosis. (Figure 17-9) Duplications can arise from breaking, adding new DNA, then rejoining OR by unequal crossing over (Figure 17-11). Usually difficult to detect phenotypically. A loop structure may be detected during meiosis.
Homologous
Chromosomes
Tandem Duplication ABCBCD Reverse Duplication ABCCBD The hemoglobin (Hb) gene family provides evidence for duplications generated by unequal crossing over. (Figure 17-13) Human homozygous duplications have never been detected (probably lethal).
