CHAPTER 19 THE ORGANIZATION AND CONTROL OF EUKARYOTIC GENOMES

1. CHAPTER 19THE ORGANIZATION AND CONTROL OF EUKARYOTIC GENOMES • => Larger than prokaryotes • Not all 25,000 genes are active in all cells

2. Junk DNA anyone? • Prokaryotes- most of the DNA in a genome codes for protein (or tRNA and rRNA), with a small amount of noncoding DNA, primarily regulators. • Eukaryotes - most of the DNA (about 97% in humans) does not code for protein or RNA. • Only 25,000 genes in humans - 3% of total DNA in cell (YIKES!) • Rest of it (97%) - junk????? (noncoding DNA) 96% similar to humans)

3. “Noncoding” DNA and what it does in the eukaryotic genome 1) Some noncoding regions are regulatory sequences (these are promotors and enhancers that can increase binding of RNA polymerase to DNA). 2) Other are introns. 3) Finally, even more of it consists of repetitive DNA, present in many copies in the genome.

4. REPPPPETITIVEDNA IN EUKARYOTES GTTACGTTACGTTAC….repeated 10 to 10 million times (Satellite DNA) • Know 2 types of Repetitive DNA: 1)TANDEMLY REPETITIVE DNA (AKA SATELLITE DNA - *3 types) - 10 to 15% of DNA 2) INTERSPERSED REPETITIVE DNA - 25 - 40% of DNA

5. REPPPPETITIVEDNA IN EUKARYOTES • 1) SATELLITE DNA/TANDEMLY REPETITIVE DNA • These sequences (1 to 10 base pairs) are repeated up to a million times in series. • GTTACGTTACGTTAC…. • 3 types: a) Regular Satellite -100,000 - 10 mill b) Minisatellite -100 -100,000 repeats c) Microsatellite - 10 to 100 repeats - Very important for forensics - helps figure out uniqueness of a person’s DNA

6. A number of genetic disorders are caused by abnormally long stretches of tandemly repeated nucleotide triplets within the affected gene. • CAG Repeat

8. CAG Repeat Size Median Age at Onset * (years) (95% confidence interval) 39 66 (72-59) 40 59 (61-56) 41 54 (56-52) 42 49 (50-48) 43 44 (45-42) 44 42 (43-40) 45 37 (39-36) 46 36 (37-35) 47 33 (35-31) 48 32 (34-30) 49 28 (32-25) 50 27 (30-24)

10. You know that antisocial neighbor - May be a microsatellite problem!

11. Satellite DNA plays a structural role at telomeres and centromeres. This is important! You don’t want non-repetitive DNA in telomeres because?

12. CSI Lab on this coming up- Microsatellites – more repeats but really short! • Are only 1-10 nucleotides long and are repeated only 10-100 times in the genome • Used in DNA fingerprinting (forensics)

13. What, more “junk”? • (2) About 25-40% of most mammalian genomes consists of interspersed repetitive DNA. -One common family of interspersed repetitive sequences, Alu elements, is transcribed into RNA molecules with unknown roles in the cell. -Alu sequences may help alternate RNA splicing -Transposons are interspersed repetitive DNA Table 19.1 bottom

14. Out of 25,000 genes what gets expressed depends upon: • Type of cell: Not all genes are expressed in all cells (epigenetics controls it) • Development period: During embryonic development certain genes may be expressed that are not expressed in adults (and viceversa)

15. Gene families - collection of genes that may be identical/nonidentical

16. Gene families have evolved by duplication of ancestral genes • Most genes are present as a single copy per haploid set of chromosomes • Multigene families exist as a collection of identical or very similar genes (exceptions). • These likely evolved from a single ancestral gene. • The members of multigene families may be clustered or dispersed in the genome.

17. Identical genes are multigene families that are clustered tandemly.

18. Pseudogenes- DNA segments that have sequences similar to real genes but that do not yield functional proteins - remnants of evolution or? • Evolution - first duplicate a gene and then mutate the copy; result : original copy is still there, mutated gene - could make a new protein = new function (natural selection acts on it) • Nonidentical genes have diverged since their initial duplication event.

19. Did you know your genome changes continually in your lifetime? 1) Rare mutations (between 1/106 and 1/105 nucleotides ) 2) Gene amplification – selective DNA replication of some genes to increase protein expression (ex. after chemotherapy) 3) Transposons/ Retrotransposons- (Jumping genes) 50% - Corn 10% Human (Not inherited) 4)Gene rearrangement Transposon moved into the purple color gene destroying its activity

20. Altering genomes during your lifetime? continued • Rare mutations • Gene amplification - temporary increase (selective loss also possible) in number of gene copies • Transposons and retrotransposons • Gene rearrangement in Immunoglobin genes Fig. 19.5

21. B lymphocytes (WBC) produce immunoglobins, or antibodies, that specifically recognize and combat viruses, bacteria, and other invaders. • Millions of types of Antibodies can be produced depending on what the infectious agent is - how? • Immunoglobins have constant and variable region • 100s of gene segments code for the variable region of the antibody. • DNA segments are put together to create an endless combination of constant and variable regions - gene rearrangement occurs in your lifetime!

22. TRANSLATION TRANSCRIPTION Promotor RNA Polymerase makes premRNA using the ‘elves’ - transcription factors (proteins) Many protein ‘factors’ are involved in translation as well

23. How is gene expression controlled? • That is = if/what protein is made? How can you control this? Levels of control –goals….. • 1) Changing DNA physically =>’ mRNA making’ affected • 2) Changing access to DNA Promotor • 3) If mRNA is made; How long mRNA hangs around; change which protein is made from one mRNA - (splicing); don’t use the mRNA • 4) Change/destroy the protein after its made

24. How is gene expression controlled in you? (IMPORTANT) • When is the gene active (on or off)? That is what protein is made? How can you control this? • Gene expression control = which genes are “on” • Levels of control – • 1) chromatin (DNA) packing and chromatin modification - change access sites on DNA for RNA Polymerase so that its binding decreases/increases (epigenetics - layer of control above the genome - NOVA Video) • 2) Transcription - when DNA makes mRNA • 3) Post-transcriptional - RNA processing, translation • 4) Post-translational - various alterations to the protein product.

25. Fig. 19.7

26. 1a) Level of packing is one way that gene expression is regulated. • Densely packed areas are inactivated. (Heterochromatin) • Loosely packed areas are being actively transcribed. (Euchromatin) - - during mitosis - during Interphase

27. Chromatin structure is based on successive levels of DNA packing INTERPHASE • Interphase - chromatin fibers highly extended • Mitosis - chromatin coils and condenses to form short, thick chromosomes. MITOSIS

28. Which stage do you see ‘beads on a string’? (Interphase) Are genes active? - Yes transcribed into mRNA! • Histone proteins are responsible for the first level of DNA packaging. • Their positively charged amino acids bind tightly to negatively charged DNA. Beads on a string = a nucleosome, in which DNA winds around a core of histone proteins

29. Next level of packing - ‘30 nm solenoid fiber’ – nucleosome fiber • Has (DNA + HISTONES) with 6 nucleosomes per turn Which stage do you see ‘30 nm fiber’? (Mitosis) Are genes active? - Yes transcribed into mRNA!

30. The 30 nm fiber forms looped domains attached to a scaffold of nonhistone proteins. Which stage do you see ‘looped domains’? (Mitosis) Are genes active? -No

32. 1b) Chromatin modifications (epigenetics) • Chemical modifications of DNA bases: • A) DNA methylation is the attachment by specific enzymes of methyl groups (-CH3) Inactive DNA is highly methylated compared to DNA that is actively transcribed. • Genomic imprinting is related to DNA methylation

33. DNA Methylation - add a methyl group to make DNA less accessibleto RNA Polymerase

34. 1b) Chromatin modifications • B) Histone acetylation (addition of an acetyl group -COCH3) and deacetylation • Acetylated histones grip DNA less tightly = ? • More access to RNA Polymerase! SO,….

35. Epigenetics - DNA methylation and histone acetylation may be responsible for a lot of traits that are not just related to whether you have the gene/not. Example: If your gene is methylated you may never express the trait!

36. 2) Control of Transcription – very important - to make or not make mRNA Control elements - noncoding DNA segments that regulate transcription by binding transcription factors that are needed for RNA Polymerase binding. (TATA Box -Promotor, Activators in bacteria - ‘Enhancers’ in Eukaryotes, Repressors in bacteria - ‘Silencers’ in Eukaryotes)

37. How can a DNA control element 100s of basepairs upstream of a gene regulate the access to RNA Polymerase? • Bending of DNA enables transcription factors, activators (like steroid hormones), bound to enhancers to contact the complex at the promoter. Mostly positive gene regulation in eukaryotes! Fig. 19.9

38. The hundreds of eukaryotic transcription factors follow only a few basic structural principles. • Each protein generally has a DNA-binding domain that binds to DNA and a protein-binding domain that recognizes other transcription factors. Fig. 19.10

39. 3) Post-transcriptional mechanisms - so mRNA is made, what next? • A) RNA processing – alternative splicing - controls which protein is made from one mRNA - mix-n-match introns/exons

40. 3) Post-transcriptional mechanisms • B) Life span of a mRNA molecule • Prokaryotic mRNA molecules degraded by enzymes after only a few minutes. • Eukaryotic mRNAs endure typically for hours or even days or weeks. G L T AAAAA 5’ Cap Leader Trailer Poly A tail

41. 3) Post-transcriptional mechanisms • C) Translation - can be blocked by regulatory proteins that bind to 5’ leader region of mRNA. (prevents attachment of mRNA to ribosomes) • Protein factors required to initiate translation = simultaneous control of translation of allthe mRNA in a cell. G L T AAAAA 5’ Cap Leader Trailer Poly A tail

42. 4) Post-translational mechanisms • Processing of polypeptides to yield functional proteins. • This may include cleavage, chemical modifications, and transport to the appropriate destination. • Regulation may occur at any of these steps.

43. The cell limits the lifetimes of normal proteins by selective degradation. • Proteins intended for degradation are marked by the attachment of ubiquitin proteins. • Giant proteosomes recognize the ubiquitin and degrade the tagged protein. Fig. 19.12

44. CANCER REVIEW- read on your own - use these animations

45. Cancer results from genetic changes that affect the cell cycle • Cell cycle CONTROL events don’t work • Spontaneous mutations or environmental influences (carcinogens) • Cancer-causing genes – oncogenes (retroviruses), proto-oncogenes (in other organisms). • What happens when proto-oncogenes/oncogenes are turned ‘ON’? (Ras gene) • Cell will divide without stopping

46. Malignant cells often have significant changes in chromosomes

47. Fig. 19.13

48. Are there genes that prevent cancer? • Tumor-suppressor genes -normal products inhibit cell division, repair DNA, control adhesion (p53). • Mutations to these tumor suppressor genes = cancer

49. Oncogene proteins and faulty tumor-suppressor proteins

50. The p53 gene, named for its 53,000-dalton protein product, is often called the “guardian angel of the genome”. • Damage to the cell’s DNA acts as a signal that leads to expression of the p53 gene. • The p53 protein is a transcription factor for several genes. • It can activate the p21 gene, which halts the cell cycle. • It can turn on genes involved in DNA repair. • When DNA damage is irreparable, the p53 protein can activate “suicide genes” whose protein products cause cell death by apoptosis.