1. Genes

2. Outline  Genes: definitions  Molecular genetics - methodology  Genome Content  Molecular structure of mRNA-coding genes  Genetics  Gene regulation  Genetics  Molecular biology  Arrays  Issues  Genetic and misexpression approaches

3. Gene Definitions  Gene  Molecular definition: stretch of DNA that encodes:  Functional RNAs - tRNA, rRNA  Functional proteins - mRNA  All sequences necessary for proper function (genetic) – includes regulatory elements and transcription unit  Generally excludes other types of genomic sequences  Centromeres, telomeres, origins of DNA replication, transposons  Genetic definition: element required for proper organismal function

4. Molecular Genetics  Genetics [mutant phenotype]  Molecular Biology [gene: sequence, expression-arrays]  Biochemistry [activities, interactions]  Cell biology [structure, dynamics]

5. Genomic Content  Calf Thymus DNA sheared to a size of ~300 bp, denatured, and reannealed  3 classes:  Highly repetitive – 10% DNA - anneals very rapidly  Middle repetitive – 30% DNA - C 0 t 1/2 = 0.04  Non-repetitive (unique) – 60% DNA - C 0 t 1/2 = 4000

6. Highly Repetitive Simple Sequence DNA  Clusters of tandemly-linked 5-10 bp repeats  Can have > 10 6 copies/genome  Not transcribed Drosophila virilis satellite DNAs > 95% each satellite consists of predominant sequence

7. 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

8. Unique DNA-Coding Sequence Genes  Slow kinetic class corresponds mainly to protein-coding genes  Average gene size (transcribed region only)/organism  E. coli 1.2 kb  Yeast 1.7 kb  Drosophila 11.3 kb  Human 27.0 kb  As complexity increases, so does gene size

9. Overview of Gene Expression-1 Regulatory region Transcription unit DNA > ACGT RNA > ACGU Transport to cytoplasm Nucleus

10. Overview of Gene Expression-2 aa1 = methionine Protein - myoglobin

11. 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)

12. 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

13. 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

14. Genome Projects in Progress  Multiple Humans – SNPs : disease genes and predispositions  Mouse – model system to study human/mammalian gene function  Strength – knockout mutants  Zebrafish - model vertebrate genetic system  Strength – large-scale genetic screens  Crop plants – poplar, apple, tomato + pests  Additional Drosophila species  Identify gene control regions

15. Drosophila  Drosophila genome = 180 Mb  Sequenced 120 Mb euchromatic region  60 Mb heterochromatic region unsequenced (few genes)  Annotation – 13,601 predicted genes  Genie – predicts ORFs/exons  Compare to Expressed Sequence Tags (ESTs-cDNAs)  Blast searches – sequence identity to known genes

16. Complications of Gene Prediction by Computer: Cranky Example  RT-PCR of embryonic RNA  33 kb Exons Genie CG12561 1-3 CG14554 CG14553 1-3 CG14552 1-4 EST-LP05454-3' EST-LP05454-5' 6 7 8 9 5 4 2 3 1

17. Drosophila Gene Functions  14,113 predicted transcripts with different coding sequences  Biochemical function Process  2,081 Transcription factors 2,274 Metabolism  2,422 Enzymes 530 Cell communication  665 Transporters 486 Development  622 Signal transduction 201 Physiology  303 Structural proteins 118 Sensation & behavior  216 Cell adhesion 8,884 Unknown  7,576 Unknown