DNAbased information technologies Understand use of polymorphisms DNA

DNA-based information technologies Understand use of polymorphisms DNA fingerprinting Understand use of proteome to determine protein function Understand creation and use of DNA microarrays Describe use of recombinant technology in plant and animal cells Understand challenges in introducing DNA into animal cell

DNA technology - forensics DNA fingerprinting taking place of fingerprints - “DNA typing”, “DNA profiling” DNA fingerprinting based on sequence polymorphisms, single bp differences between 1 individual and another (between individuals 1 bp/1000 bp) - short tandem repeats (STRs) Sequence differences affect restriction endonuclease recognition sequences - and therefore DNA fragment sizes differ - called restriction fragment length polymorphisms - RFLPs Use of Southern blotting Genomic DNA sequences used in test are repetitive and are distinct (in sequence and # of repeats) from one person to another (can get rid of nonrepetitive by S 1 nuclease) Combine use of several probes and test can become so selective it can ID a single person in the entire human population If 3 STRs match a suspect likelihood is 2000: 1 that the police have the right person (nine matches makes the odds 1 billion: 1) FBI requires 13 matches (database exists) Drawback - Southern analysis needs large amount of DNA and must be relatively fresh To deal with this use PCR so can obtain DNA fingerprint from single hair follicle, drop of blood, and it can be years old Once we know what every DNA sequence in the genome does we can use sample found at a crime scene to determine suspects’ build, race, eye and hair color, inherited physical defects, personality traits, etc.

From genomes to proteomes Proteome = complement of proteins expressed by a genome Protein function described by: 1. Phenotypic function - effect of protein on entire organism 2. Cellular function - description of network of of interactions engaged in by the protein at the cellular level 3. Molecular function - precise biochemical activity of protein

Comparative genomics - assign gene function by genome comparisons Conserved gene order - synteny - between human and mouse

Cellular expression patterns can reveal cellular function of a gene DNA Microarrays - “DNA chips” - allow rapid and simultaneous screening of thousands of genes DNA segments from known genes (up to hundreds of bp long) are amplified by PCR and placed on a solid surface using robotic devices that accurately deposit nanoliter amounts of DNA solution Thousands of such spots are deposited in a pre-designed array on a surface area of just a few square centimeters Also can synthesize DNA directly on solid surface - photolithography Once chip constructed can be probed with m. RNAs or c. DNAs from a particular cell type or cell culture to ID genes being expressed

Photolithography

Use DNA microarray to answer: “which genes are expressed at a given stage in development? ”

DNA microarray Each spot contains DNA from one of the 6200 genes in S. cerevisiae Green dye - cells growing normally in culture Red dye - cells 5 hrs after begin to form spores Yellow - genes that do not change their level of expression during sporulation Actual chip - 1. 8 cm x 1. 8 cm

Genome alterations and New Products of Biotechnology PLANTS: Uses of recombinant technology in agriculture alter nutritional profile or yield of crops alter resistance of plants to insects, diseases, cold, salinity, drought Bacterial plant parasite aids in cloning of plants since no naturally occurring plant cell plasmids Use soil bacterium - Agrobacterium tumefaciens invades plants at site of wound, transforms nearby cells, induces tumor growth (called crown gall), bacterium contain large Ti plasmid where T DNA moves from bacterium and integrates into plant cell chromosomes, 25 bp repeats and vir genes are essential

Genome alterations and New Products of Biotechnology PLANTS: T DNA encodes enzymes that convert plant metabolites into 2 classes of compounds that benefit the bacterium Plant growth hormones - Unusual amino acids - stimulate crown gall tumor growth bacterial food source, only metabolized by bacterium only Diverts plant resources by converting them to a form that benefits only bacterium

Genome alterations and New Products of Biotechnology PLANTS: Rare example of DNA transfer from prok to euk is a natural genetic engineering process - researchers use this to transfer recombinant DNA to plant (instead of T DNA) Vir genes on plasmid (a) aid in transfer of foreign DNA in plasmid (b) into plant genome

Genome alterations and New Products of Biotechnology PLANTS: SUCCESS!! Luciferase gene from fireflies introduced into cells of a tobacco plant

Genome alterations and New Products of Biotechnology PLANTS: SUCCESS!! Production of crop plants that are resistant to herbicides, plants viruses, insects Other benefits - increased yields & less need for use of chemicals Expresses a gene for a protein toxin derived from Bacillus thuringiensis This toxin kills larvae of some moth species while being harmless to humans

Genome alterations and New Products of Biotechnology PLANTS: development of soybeans that are resistant to general herbicide glyphosate (Round. Up) One treatment can last all year Worries: evolution of glyphosate-resistant weeds, and escape of difficult-to-control recombinant plants

Genome alterations and New Products of Biotechnology ANIMAL CELLS: want to be able to introduce foreign DNA into animal cells to study structure & function of genome, proteins, and to generate animals with new traits Use of animal tissues (difficult to maintain & manipulate) so use cell lines/tissue culture NO PLASMID-LIKE vector for introducing DNA into animal cells SO it is necessary to integrate DNA into host-cell chromosome CHALLENGES: efficient delivery of DNA to cell nucleus, integration into chromosome without disrupting any critical genes

Genome alterations and New Products of Biotechnology ANIMAL CELLS: Introduction of DNA into cells: electroporation - very inefficient microinjection - inject DNA straight into nucleus using a fine needle - small number of cells treated takes a lot of skill liposomes - small vesicles with a lipid bilayer, recombinant DNA inside viral vectors - effective mechanisms for introducing foreign DNA into cells, integrate into host chromosome, retroviruses & adenoviruses modified to serve as viral vectors into introduce foreign DNA into cells

Genome alterations and New Products of Biotechnology ANIMAL CELLS: Introduction of DNA into cells: retroviral vectors - special regions required: • LTR for integration through homologous recombination • for packaging into viral particles also need “helper virus” to provide genes to produce viral particle

Genome alterations and New Products of Biotechnology ANIMAL CELLS: Introduction of DNA into cells: adenoviral vectors - lack mechanism for integrating DNA into chromosome so recombinant DNA expressed from this vector is short-lived - OK for transient gene expression PROBLEMS WITH TRANSFORMATION OF ANY ANIMAL CELLS: Random integration Nonhomologous recombination occurs frequently If disrupt essential genes - cell functions/protein altered New evidence that integration events can sometimes activate genes that stimulate cell division (CANCER ENSUES!) Site of integration can have an effect on expression of gene (No way to control this)

Genome alterations and New Products of Biotechnology ANIMAL CELLS: Despite challenges, transformation of animal cells used to study chromosome structure & function, regulation & gene expression Microinjection of DNA into nuclei of fertilized mouse eggs, those in the germline that are affected can be identified by testing their offspring Careful breeding results in a TRANSGENIC mouse line where all mice are homozygous for new gene(s) Used to introduce gene for human growth hormone, under control of an inducible promoter Fed a diet with inducer Lots of trangenic mice have been made already - also make “knockout mice”, where a particular gene has been inactivated - can establish function of gene

Genome alterations and New Products of Biotechnology DISCOVERY OF NEW PHARMACEUTICALS Hypertension, congestive heart failure, hypercholesterolemia, & obesity are treated by a pharm drug that alters human physiology Proteomics will help to identify future drug targets Example: most potent vasoconstrictor - peptide hormone urotensin II Use of proteomics, etc. helped identify its target in the cell - GPR 14, an “orphan” receptor initially Also identify new agents to treat human pathogenic diseases ID enzymatic targets in microbial pathogens