1. ©1999 Timothy G. Standish Job 38:36 36Who hath put wisdom in the inward parts? or who hath given understanding to the heart?

2. ©1999 Timothy G. Standish The Eukaryotic Genome Timothy G. Standish, Ph. D.

3. ©1999 Timothy G. Standish Eukaryotes Have Large Complex Genomes The human genome is about 3 x 10 9 base pairs or ≈ 1 m of DNA That’s a lot more than a typical bacterial genome E. coli has 4.3 x 10 6 bases in its genome Because humans are diploid, each nucleus contains 6 x 10 9 base pairs or ≈ 2 m of DNA That is a lot to pack into a little nucleus!

4. ©1999 Timothy G. Standish Only a Subset of Genes is Expressed at any Given Time It takes lots of energy to express genes Thus it would be wasteful to express all genes all the time By differential expression of genes, cells can respond to changes in the environment Differential expression, allows cells to specialize in multicelled organisms. Differential expression also allows organisms to develop over time.

5. ©1999 Timothy G. Standish Eukaryotic DNA Must be Packaged Eukaryotic DNA exhibits many levels of packaging The fundamental unit is the nucleosome, DNA wound around histone proteins Nucleosomes arrange themselves together to form higher and higher levels of packaging.

6. ©1999 Timothy G. Standish A T T A G C C G G C TATA T A G C C G G C T A A T Packaging DNA Histone proteins Histone octomer B DNA Helix 2 nm

7. ©1999 Timothy G. Standish A T T A G C C G G C TATA T A G C C G G C T A A T Packaging DNA Histone proteins B DNA Helix Histone octomer 2 nm

8. ©1999 Timothy G. Standish A T T A G C C G G C TATA T A G C C G G C T A A T Packaging DNA Histone proteins Histone octomer Nucleosome 11 nm B DNA Helix 2 nm

9. ©1999 Timothy G. Standish Packaging DNA A T T A G C C G G C T A A T

10. ©1999 Timothy G. Standish Packaging DNA A T T A G C C G G C T A A T

11. ©1999 Timothy G. Standish Packaging DNA A T T A G C C G G C T A A T Protein scaffold 11 nm “Beads on a string” 30 nm Tight helical fiber Looped Domains 200 nm

12. ©1999 Timothy G. Standish Packaging DNA G C A T Protein scaffold Metaphase Chromosome 700 nm 11 nm 30 nm 200 nm 2 nm Looped Domains Nucleosomes B DNA Helix Tight helical fiber

13. ©1999 Timothy G. Standish Highly Packaged DNA Cannot be Expressed The most highly packaged form of DNA is “heterochromatin” Heterochromatin cannot be transcribed, therefore expression of genes is prevented Chromosome puffs on some insect chromosomes illustrate where active gene expression is going on

14. ©1999 Timothy G. Standish DNA Cytoplasm Nucleus G AAAAAA Export Degradation etc. G AAAAAA Control of Gene Expression G AAAAAA RNA Processing mRNA RNA Transcription Nuclear pores Ribosome Translation Packaging Modification Transportation Degradation

15. ©1999 Timothy G. Standish Logical Expression Control Points DNA packaging Transcription RNA processing mRNA export mRNA masking/unmasking and/or modification mRNA degradation Translation Protein modification Protein transport Protein degradation Increasing cost The logical place to control expression is before the gene is transcribed

16. ©1999 Timothy G. Standish A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site 3’5’ RNA Transcript Introns Exon 2Exon 3 Int. 2 Exon 1 Int. 1 3’ Untranslated Region 5’ Untranslated Region Exons

17. ©1999 Timothy G. Standish 5’ DNA 3’ Enhancers EnhancerTranscribed Region 3’ 5’ TF 3’ 5’ TF 5’ RNA Pol. RNA Pol. Many bases Promoter

18. ©1999 Timothy G. Standish Eukaryotic mRNA Protein Coding Region 3’ Untranslated Region5’ Untranslated Region Exon 2Exon 3Exon 1 AAAAA G 3’5’ 3’ Poly A Tail5’ Cap RNA processing achieves three things: Removal of introns Addition of a 5’ cap Addition of a 3’ tail This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm

19. ©1999 Timothy G. Standish “Junk” DNA It is common for only a small portion of a eukaryotic cell’s DNA to code for proteins In humans, only about 3 % of DNA actually codes for the about 100,000 proteins; 50,000 in older estimates, 150,000 in more recent estimates Non-coding DNA was once called “junk” DNA as it was thought to be the molecular debris left over from the process of evolution We now know that much non-coding DNA plays important roles like regulating expression and maintaining the integrity of chromosomes

20. ©1999 Timothy G. Standish The Globin Gene Family Globin genes code for the protein portion of hemoglobin In adults, hemoglobin is made up of an iron-containing heme molecule surrounded by 4 globin proteins: 2  globins and 2  globins During development, different globin genes are expressed which alter the oxygen affinity of embryonic and fetal hemoglobin Fe    

21. ©1999 Timothy G. Standish Model For Evolution Of The Globin Gene Family Ancestral Globin gene Duplication   Duplication and Mutation   Chromosome 16Chromosome 11   Transposition Mutation                 Duplication and Mutation AdultEmbryoFetusEmbryoFetus and Adult Pseudogenes (  ) resemble genes, but may lack introns and, along with other differences, typically have stop codons coming soon after the start codons.

22. ©1999 Timothy G. Standish Antibody Diversity Results From Differential Splicing Humans produce antibodies to many millions of different antigens The human genome codes for less than 200,000 genes Antibodies are proteins, so how are many millions of different antibodies produced by so few genes? The answer lies in differential splicing of DNA

23. ©1999 Timothy G. Standish SS Light Chain Light Chain SS Antibody Structure Constant V V V V Antigen binding site Heavy Chains

24. ©1999 Timothy G. Standish Antigen Binding Variable Light Variable Heavy Antigen 1 Antigen 3 Antigen 2

25. ©1999 Timothy G. Standish An Antibody “Gene” DNA coding for antibodies are made up of many exons referred to as genes Different exons are spliced together to make the many different antibodies V2V2 V4V4 V1V1 V3V3 IntronJ2J2 J3J3 J1J1 Constant V2V2 J2J2 V1V1 V3V3 IntronConstant Random splicing of DNA as cell differentiates J2J2 V3V3 Constant Translation produces a light chain with a variable region at one end J2J2 V3V3 IntronConstant Transcription J2J2 V3V3 Constant RNA Processing

26. ©1999 Timothy G. Standish Classes of Immunoglogulins IgG - A monomer - Most abundant antibody in blood. IgG easily leaves the circulatory system to fight infection and crosses the placenta conferring passive immunity to a fetus. IgD - A monomer - Found on the surface of B cells probably allowing recognition of antigens thus triggering differentiation into plasma and memory B cells IgE - A monomer - The least common antibody. The tails attach to mast cells and basophils. When antigens bind, they signal release of histamine. IgA - A dimer - Produced by cells in the mucus membranes to prevent attachment of pathogens. IgA is also found in many body secretions including milk. IgM - A pentamer - First antibody to appear following exposure to an antigen. Because it declines rapidly in the blood, high IgM levels indicate a current infection.

27. ©1999 Timothy G. Standish Cancer Regulation of cell division is vital in multi- celled organisms Cancer can be defined as uncontrolled division of cells As regulation of cells is achieved through genes expressed in those cells, mutation of those genes can result in the loss of regulation and consequently cancer

28. ©1999 Timothy G. Standish