genes. outline genes: definitions molecular genetics - methodology genome content molecular...

Genes

Outline

Genes: definitions Molecular genetics - methodology Genome Content Molecular structure of mRNA-coding genes Genetics Gene regulation

GeneticsMolecular biology

Arrays IssuesGenetic and misexpression approaches

Gene Definitions

Gene Molecular definition: stretch of DNA that encodes:

Functional RNAs - tRNA, rRNAFunctional proteins - mRNA

All sequences necessary for proper function (genetic) – includes regulatory elements and transcription unit

Generally excludes other types of genomic sequencesCentromeres, telomeres, origins of DNA replication,

transposons

Genetic definition: element required for proper organismal function

Molecular Genetics

Genetics [mutant phenotype]

Molecular Biology [gene: sequence, expression-arrays]

Biochemistry [activities, interactions]

Cell biology [structure, dynamics]

Genomic Content

Calf Thymus DNA sheared to a size of ~300 bp, denatured, and reannealed

3 classes:Highly repetitive – 10% DNA - anneals very rapidlyMiddle repetitive – 30% DNA - C0t1/2 = 0.04

Non-repetitive (unique) – 60% DNA - C0t1/2 = 4000

Highly Repetitive Simple Sequence DNA

Clusters of tandemly-linked 5-10 bp repeats Can have > 106 copies/genome Not transcribed

Drosophila virilissatellite DNAs> 95% each satelliteconsists of predominantsequence

Intermediately Repetitive DNA – Mobile Elements

Repetitive elements interspersed among unique DNA

Most are transposons – mobile DNA Many are no longer able to transpose Dispersed throughout the genome Different classes Transpose as DNA or RNA intermediates

Unique DNA Repeat

Unique DNA-Coding Sequence Genes

Slow kinetic class corresponds mainly to protein-coding genes

Average gene size (transcribed region only)/organismE. coli 1.2 kbYeast 1.7 kbDrosophila 11.3 kbHuman 27.0 kb

As complexity increases, so does gene size

Overview of Gene Expression-1

Regulatoryregion

Transcription unit

DNA > ACGT

RNA > ACGU

Transport tocytoplasm

Nucleus

Overview of Gene Expression-2

aa1 = methionine

Protein - myoglobin

DNA and Clones

Genomic or chromosomal DNA – genomic clones (exons, introns, spacer, etc.)

Transcription unit – entire region of gene transcribed (exons + introns)

mRNA – cDNA clones (exonic sequences)ESTs – expressed sequence tags

Oligonucleotides – small stretches of DNA (~20-50 nt)

Human Genome Project: Gene Number

Size 3,200 Mb Predicted gene number

Celera – 39,114 Public consortium – 29,691 Refseq (known genes) – 11,015

Non-identity ~64% novel genes don’t overlap > 80% novel genes expressed

Indicates they are real Estimate ~50,000 genes

Estimate ~ 64 kb/gene Transcribed region = 27 kb Spacer DNA = 37 kb

Repeats + control elements

Human – large number of transcripts/gene exist because of alternative splicing

Completed Genomic Sequencing Projects

Human – disease genes Drosophila – model system for animal development

and gene control Strength - genetics

Nematode - model system for development and behavior Strength - genetics Fly and human more related than worm-human

Arabidopsis – weed: model plant genetic system Crop plants – rice, maize Yeast – typical eukaryotic cell E. coli Many pathogenic bacteria - disease

Genome Projects in Progress

Multiple Humans – SNPs : disease genes and predispositions

Mouse – model system to study human/mammalian gene functionStrength – knockout mutants

Zebrafish - model vertebrate genetic systemStrength – large-scale genetic screens

Crop plants – poplar, apple, tomato + pests Additional Drosophila species

Identify gene control regions

Drosophila

Drosophila genome = 180 MbSequenced 120 Mb euchromatic region60 Mb heterochromatic region unsequenced (few

genes)

Annotation – 13,601 predicted genesGenie – predicts ORFs/exonsCompare to Expressed Sequence Tags (ESTs-cDNAs)Blast searches – sequence identity to known genes

Complications of Gene Prediction by Computer: Cranky Example

RT-PCR of embryonic RNA

33 kb

Exons

Genie CG12561 1-3CG14554

CG14553 1-3CG14552 1-4

EST-LP05454-3'

EST-LP05454-5'6 7 8 9542 31

Drosophila Gene Functions

14,113 predicted transcripts with different coding sequences

Biochemical function Process2,081 Transcription factors 2,274 Metabolism2,422 Enzymes 530 Cell communication665 Transporters 486 Development622 Signal transduction 201 Physiology303 Structural proteins 118 Sensation &

behavior216 Cell adhesion 8,884 Unknown7,576 Unknown