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8 How Cellular Information is Altered 8.1. Introduction DNA Replication in Ch. 4. Exchange of genetic information from one generation to another Additional genetic information through natural of artificial means Some mechanisms causing alterations in cells’ content of genetic information 8.2. Evolving Desirable Biochemical Activities through Mutation and Selection Mutation: genotype change, irreversible Genotype: the sum of the genetic consortium of an organism Phenotype: the characteristics expressed by a cell 8.3. How Mutations Occur Point mutation: the change of a single base (CAA:glutamine – UAA:stop) Deletion mutation: alter the whole composition of a protein Insertion mutation: addition of about 700 to 1400 base pairs in length IS: insertion elements Reversion: original wild-type phenotype restored 8.2.2. Selecting for Desirable Mutants Natural (spontaneous) rates of mutation vary greatly Selectable mutation: mutants can survive under a set of specific set of environmental conditions Direct selection: an example of direct selection to find a mutant resistant to an antibiotic or toxic compound Indirect selection: isolate mutants that are deficient in their capacity to produce a necessary growth factor Auxotrophic mutants would not grow on such a simple medium unless it were supplemented with the growth factor (ex: lysine auxotroph, replicate plating method) 8.3. Natural Mechanisms for Gene Transfer and Rearrangement Gene transfer from one organism to another Large rearrangements in chromosomal DNA 8.3.1. Genetic Recombination A process that brings genetic elements from two different genomes into one unit. Three main mechanisms for gene transfer Transformation: free DNA taken by cells Transduction: bacteriophage transfer DNA Conjugation: DNA transfer between two intact cells 8.3.2. Transformation (Figure 8.4) Competent cells are transformable bacteria strain E.coli are not normally competent, but treating them with high concentrations of Ca++ coupled with temperature manipulation induces competency of E.coli cells Plasmid is autonomous, self-replicating, double-strand piece of DNA that is normally extrachromosal. 8.3.3. Transduction (Figure 8.5) Generalized transduction Specialied transduction Lysogenic cell: one carrying a prophage or phage DNA incorporated into chromosomal DNA Phage lamda: temperature phage Prophage → lytic cell 8.3.4. Episomes and conjugation (Figure 8.6) Episome: DNA molecule that may exist either integrated into the chromosome or separate from it (ex: F factor) E.coli F(+) F(-) Sex pilus conduit for transfer copy of the F plasmid to the F- cell Hfr (high frequency recombination) 8.3.5. Transposons: Internal gene transfer Gene or genes that have the ability to jump from one piece of DNA to another piece of DNA, or to another position on the original piece of DNA Many of the transposons encode antibiotic resistance Can induce mutations Bring once-separate genes together Movement of genes between unrelated bacteria Transposon mutagenesis 8.4. Genetically Engineering Cells The purposeful, predetermined manipulation of cells at the genetic level – idea before 1970, now grasp of beginning college students Genetic engineering A set of tools Not a scientific discipline Manipulation of DNA outside the cell to create artificial genes or novel combinations of genes 8.4.1. Basic elements of genetic engineering Obtain the gene of insert (Figure 8.7) Shotgun cloning Hybridization: Probe using chemical synthesis Total chemical synthesis of a gene Insertion (Figure 8.8) cDNA synthesized from mRNA by reverse transcriptase Vector: plasmid is typical vector Restriction enzyme: cut DNA at prespecific site Ligase Construction of desired vector-donor DNA Screen: selectable markers such as antibiotic resistance/ expression Cloning and Amplificaton of genes Electrophoresis: agarose, SDS-PAGE Immnunoblot (Western blot): Ag-Ab, radioactive marker Shotgun cloning (gene library/gene bank) – radiolabeled DNA/RNA probes complementary to the cloned gene Phage displays & bacterial displays PCR (polymerase chain reaction) – two short primer sequences (<20mer nucleotides) and Taq polymerase 8.4.2. Genetic Engineering of Higher Organisms Transfection: introduction of foreign gene into higher organisms Agrobacterium tumefaciens: T-DNA Biolistic process: plant, very successful Electroporation Protoplasts: without outer cell envelope Protoplast fusion A baculovirus – insect cell system 8.5. Genomics: the set of experimental & computational tools which allow the genetic blueprints of life to be read. Human genome projects Functional genomics Protemics (up-, down- regulation) DNA sequencing, mRNA & protein expression Micorarrays: mRNA level detection 2D gel electrophoresis (pI & MW) Bioinformatics Metabolomics Highly nonlinear, dynamic cellular system: mathematical modeling