Bacterial Physiology (Micr430) Lecture 13 Bacterial Genomics and Functional Genomics (from various sources)

Genome and Proteome Genome: The complete genetic information carried by an organism Proteome: Encompasses all the expressed proteins of an organism (protein array) Gene array: an orderly array of sequence tags each specific for one gene of a genome; (other names: gene chip, DNA microchip)

Genomics and Functional Genomics Genomics: The study of all of the nucleotide sequences, including structural genes, regulatory sequences, and noncoding DNA segments, in the chromosomes of an organism Functional genomics: the branch of genomics that determines the biological function of the genes and their products

Protein array

Gene Array or DNA microaray There are two main applications Identification of gene or mutation Determination of expression level of genes There are two formats: cDNA (500 – 5000 bp) molecules are spotted on the chip Array of oligonucleotides are synthesized and immobilized on the chip

Gene Array or DNA microarray 1) Probe (cDNA/oligo with known identity) 2) Chip fabrication (Putting probes on the chip) 3) Target (fluorecently labeled sample) 4) Assay 5) Readout 6) Informatics

DNA microarray applications Gene discovery Study of gene functions and expression Study relationship of genes Disease diagnostics – infectious diseases

Study of gene function Gene replacement (knock-out): also known as reverse genetics. The purpose is to remove (knock-out) most of one gene and see what happens to the phenotype of the organism. Suicide vector is used Transposon mutagenesis. Similar to above but is more random disruption of genes.

Study of gene function Antisense RNA expression. Random cloning and expression of short pieces of genomic DNA on a plasmid in an microorganism to elucidate the function of the genes Essential gene: a gene whose function is absolutely required for the growth and survival of a cell.

Conditional Antisense Inhibition of Protein Synthesis Inducible promoter Normal cell Antisense cell Plasmid DNA No protein Protein Antisense RNA X mRNA mRNA DNA DNA

Shotgun Antisense Expression Determines Essentiality of Genes Pathogen genome Millions of random DNA fragments Non essential gene blocked by antisense Essential gene No cell growth mRNA Essential Protein DNA

Ultra-Rapid Functional Genomics Identify >100 essential gene drug targets per month Antisense (+ inducer) No antisense (- inducer)

Traditional Tools Reporter gene: A gene whose product can be easily assayed Transcriptional gene fusions Translational gene fusions PCR DNA Mobility Shift

PCR

Gel Shift Assay