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Material Type: Notes; Class: Genetics; Subject: Biology; University: Penn State - Main Campus; Term: Fall 2004;
Typology: Study notes
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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)
Restriction Mapping Restriction sites in a DNA fragment can be used to subclone fragments within the fragment.
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)
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
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.
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.
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.
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.
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).