Bacterial Physiology (Micr430)

Lecture 13Bacterial 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

Proteinarray

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

Antisense cellAntisense cell

Noprotein

XXAntisense RNAAntisense RNA

Inducible promoterInducible promoter

mRNAmRNA

Normal cellNormal cell

Protein

mRNAmRNA

Plasmid DNA

DNADNA

Shotgun Antisense Expression Determines Essentiality of Genes

Shotgun Antisense Expression Determines Essentiality of Genes

Non essential geneblocked by antisenseNon essential geneblocked by antisense

Essential geneblocked by antisenseEssential geneblocked by antisense

Millions of random DNA fragmentsMillions of random DNA fragments

No cell growthNo cell growthmRNAmRNA

Essential Protein

DNA

Pathogen genome

Ultra-Rapid Functional GenomicsUltra-Rapid Functional Genomics

Identify >100 essential gene drug targets per monthIdentify >100 essential gene drug targets per monthAntisense

(+ inducer)Antisense(+ inducer)

No 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 ShiftAssay