1. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Chapter 19 Eukaryotic Genomes

2. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Eukaryotes  DNA-protein complex,  chromatin – More complex structural levels than prokaryotes Figure 19.1

3. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Both prokaryotes and eukaryotes – Must alter patterns of gene expression in response to changes in environment

4. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Chromatin structure based on successive levels of DNA packing Eukaryotic DNA – Combined w/ protein Eukaryotic chromosomes – Contain an enormous amount of DNA relative to their condensed length

5. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Nucleosomes, or “Beads on a String” Basic packing unit DNA wrapped around histone protein Figure 19.2 a 2 nm 10 nm DNA double helix Histone tails His- tones Linker DNA (“string”) Nucleosome (“bead”) Histone H1 (a) Nucleosomes (10-nm fiber)

6. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Nucleosome 30 nm (b) 30-nm fiber Next level of packing – Forms 30-nm chromatin fiber Figure 19.2 b

7. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The 30-nm fiber, in turn – Forms looped domains, making up a 300-nm fiber Figure 19.2 c Protein scaffold 300 nm (c) Looped domains (300-nm fiber) Loops Scaffold

8. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Mitotic chromosome – Looped domains coil and fold forming the metaphase chromosome Figure 19.2 d 700 nm 1,400 nm (d) Metaphase chromosome

9. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Interphase cells – Most chromatin is highly extended (euchromatin)

10. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Gene expression can be regulated at any stage, but the key step is transcription All organisms – regulate which genes are expressed at any given time During development of a multicellular organism  cell specialization in form and function (cell differentiation)

11. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Each cell of a multicellular eukaryote – Expresses only a fraction of its genes In each type of differentiated cell – Unique subset of genes is expressed

12. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Many key stages of gene expression (regulation) In eukaryotic cells Figure 19.3 Signal NUCLEUS Chromatin Chromatin modification: DNA unpacking involving histone acetylation and DNA demethlation Gene DNA Gene available for transcription RNA Exon Transcription Primary transcript RNA processing Transport to cytoplasm Intron Cap mRNA in nucleus Tail CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypetide Cleavage Chemical modification Transport to cellular destination Active protein Degradation of protein Degraded protein

13. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Genes within highly packed heterochromatin usually not expressed

14. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Histone acetylation – Loosens chromatin structure  enhances transcription Figure 19.4 b (b) Acetylation of histone tails promotes loose chromatin structure that permits transcription Unacetylated histones Acetylated histones

15. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Addition of methyl groups to DNA bases – Associated w/ reduced transcription

16. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Multiple control elements – Noncoding DNA that regulate transcription Figure 19.5 Enhancer (distal control elements) Proximal control elements DNA Upstream Promoter Exon IntronExon Intron Poly-A signal sequence Exon Termination region Transcription Downstream Poly-A signal ExonIntron Exon IntronExon Primary RNA transcript (pre-mRNA) 5 Intron RNA RNA processing: Cap and tail added; introns excised and exons spliced together Coding segment P P P G mRNA 5 Cap 5 UTR (untranslated region) Start codon Stop codon 3 UTR (untranslated region) Poly-A tail Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Cleared 3 end of primary transport

17. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Alternate RNA Processing Different mRNA molecules produced from same primary transcript, depending on which RNA segments are treated as exons and which as introns Figure 19.8 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Exons DNA Primary RNA transcript mRNA RNA splicing or

18. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings mRNA Degradation Life span of mRNA molecules in the cytoplasm – Important in protein synthesis

19. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings RNA interference (RNAi) by single-stranded microRNAs (miRNAs) –  degradation of mRNA or block its translation Figure 19.9 5 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Degradation of mRNA OR Blockage of translation Target mRNA miRNA Protein complex Dicer Hydrogen bond The micro- RNA (miRNA) precursor folds back on itself, held together by hydrogen bonds. 1 2 An enzyme called Dicer moves along the double- stranded RNA, cutting it into shorter segments. 2 One strand of each short double- stranded RNA is degraded; the other strand (miRNA) then associates with a complex of proteins. 3 The bound miRNA can base-pair with any target mRNA that contains the complementary sequence. 4 The miRNA-protein complex prevents gene expression either by degrading the target mRNA or by blocking its translation. 5

20. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Proteasomes – Giant protein complexes that degrade molecules Figure 19.10 Chromatin changes Transcription RNA processing mRNA degradation Translation Protein processing and degradation Ubiquitin Protein to be degraded Ubiquinated protein Proteasome and ubiquitin to be recycled Protein fragments (peptides) Protein entering a proteasome Multiple ubiquitin mol- ecules are attached to a protein by enzymes in the cytosol. 1 The ubiquitin-tagged protein is recognized by a proteasome, which unfolds the protein and sequesters it within a central cavity. 2 Enzymatic components of the proteasome cut the protein into small peptides, which can be further degraded by other enzymes in the cytosol. 3

21. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Cancer results from genetic changes that affect cell cycle control The gene regulation systems that go wrong during cancer are same systems found in embryonic development

22. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Oncogenes – Cancer-causing genes Proto-oncogenes – Normal genes that code for proteins that stimulate normal cell growth and division

23. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings DNA change  proto-oncogene excessively active  oncogene  excessive cell division  cancer Figure 19.11 Proto-oncogene DNA Translocation or transposition : gene moved to new locus, under new controls Gene amplification: multiple copies of the gene Point mutation within a control element Point mutation within the gene Oncogene Normal growth-stimulating protein in excess Hyperactive or degradation- resistant protein Normal growth-stimulating protein in excess Normal growth-stimulating protein in excess New promoter

24. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Tumor-suppressor genes – Code f/ proteins that inhibit abnormal cell division

25. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings p53 gene encodes a tumor-suppressor protein – (cell cycle–inhibiting proteins) – ‘Guardian angel’ of the genome Figure 19.12b UV light DNA Defective or missing transcription factor, such as p53, cannot activate transcription MUTATION Protein that inhibits the cell cycle pathway, DNA damage is an intracellular signal that is passed via protein kinases and leads to activation of p53. Activated p53 promotes transcription of the gene for a protein that inhibits the cell cycle. The resulting suppression of cell division ensures that the damaged DNA is not replicated. Mutations causing deficiencies in any pathway component can contribute to the development of cancer. (b) Cell cycle–inhibiting pathway. In this 1 3 2 Protein kinases 2 3 Active form of p53 DNA damage in genome 1

26. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Mutations that knock out the p53 gene  excessive cell growth and cancer Figure 19.12c EFFECTS OF MUTATIONS Protein overexpressed Cell cycle overstimulated Increased cell division Cell cycle not inhibited Protein absent Effects of mutations. Increased cell division, possibly leading to cancer, can result if the cell cycle is overstimulated, as in (a), or not inhibited when it normally would be, as in (b). (c)

27. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Normal cells are converted to cancer cells – By the accumulation of multiple mutations affecting proto-oncogenes and tumor- suppressor genes

28. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings A multistep model for the development of colorectal cancer Figure 19.13 Colon Colon wall Normal colon epithelial cells Small benign growth (polyp) Larger benign growth (adenoma) Malignant tumor (carcinoma) 2 Activation of ras oncogene 3 Loss of tumor- suppressor gene DCC 4 Loss of tumor-suppressor gene p53 5 Additional mutations 1 Loss of tumor- suppressor gene APC (or other)

29. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Certain viruses – Promote cancer by integration of viral DNA into a cell’s genome

30. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Inheritance of a mutant oncogene  increased risk of developing cancer

31. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Noncoding DNA sequences Bulk of eukaryotic genomes – In the past called “junk DNA” Evidence is accumulating – noncoding DNA plays important roles in the cell

32. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Genomes of eukaryotes (v. prokaryotic) – Larger – Longer genes – Much greater amount of noncoding DNA

33. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Most of the 98.5% that does not code for proteins  rRNAs, or tRNAs Figure 19.14 Exons (regions of genes coding for protein, rRNA, tRNA) (1.5%) Repetitive DNA that includes transposable elements and related sequences (44%) Introns and regulatory sequences (24%) Unique noncoding DNA (15%) Repetitive DNA unrelated to transposable elements (about 15%) Alu elements (10%) Simple sequence DNA (3%) Large-segment duplications (5-6%)

34. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Transposable Elements and Related Sequences Wandering DNA segments – Barbara McClintock’s breeding experiments with Indian corn Figure 19.15

35. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Transposon New copy of transposon Transposon is copied DNA of genome Insertion Mobile transposon (a) Transposon movement (“copy-and-paste” mechanism) Retrotransposon New copy of retrotransposon DNA of genome RNA Reverse transcriptase (b) Retrotransposon movement Insertion Movement of Transposons and Retrotransposons Transposons: move by means of a DNA intermediate Retrotransposons: move by means of an RNA intermediate Figure 19.16a, b

36. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Multiple copies of transposable elements – scattered throughout genome In humans and other primates – are called Alu elements

37. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Other Repetitive DNA Simple sequence DNA – Copies of tandemly repeated short sequences – Common in centromeres and telomeres (structural roles in chromosome)

38. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Genes and Multigene Families Most eukaryotic genes – present in one copy per haploid set of chromosomes The rest of the genome – Occurs in multigene families, collections of identical or very similar genes

39. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings e.g. related families of genes that encode globins Figure 19.17b The human  -globin and  -globin gene families  -Globin Heme Hemoglobin  -Globin  -Globin gene family  -Globin gene family Chromosome 16Chromosome 11 Embryo Fetus and adult Embryo FetusAdult  GG AA        22  11 22 11 

40. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Duplications, rearrangements, and mutations of DNA contribute to genome evolution The basis of change at the genomic level is mutation which underlies much of genome evolution

41. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Duplication of Chromosome Sets Accidents in cell division –  extra copies of all or part of a genome, which may then diverge if one set accumulates sequence changes

42. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Unequal crossing over –  one chromosome with a deletion and another with a duplication Figure 19.18 Nonsister chromatids Transposable element Gene Incorrect pairing of two homologues during meiosis Crossover and

43. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Genes for various globin proteins – Evolved from one common ancestral globin gene, which duplicated and diverged Figure 19.19 Ancestral globin gene          22  11 22 11   GG AA    -Globin gene family on chromosome 16  -Globin gene family on chromosome 11   Evolutionary time Duplication of ancestral gene Mutation in both copies Transposition to different chromosomes Further duplications and mutations

44. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Similarity in the amino acid sequences of the various globin proteins – Supports this model of gene duplication and mutation Table 19.1

45. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings The copies of some duplicated genes – Have diverged so much during evolutionary time that the functions of their proteins are now substantially different

46. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Rearrangements of Parts of Genes: Exon Duplication and Exon Shuffling

47. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings Exon shuffling – Mixing and matching of different exons either within a gene or between two nonallelic genes Figure 19.20 EGF Epidermal growth factor gene with multiple EGF exons (green) F FF F Fibronectin gene with multiple “finger” exons (orange) Exon shuffling Exon duplication Exon shuffling K FEGFK K Plasminogen gene with a “kfingle” exon (blue) Portions of ancestral genesTPA gene as it exists today

48. Copyright © 2005 Pearson Education, Inc. publishing as Benjamin Cummings How Transposable Elements Contribute to Genome Evolution Movement of transposable elements – Can generates new sequence combinations that are beneficial to the organism Some mechanisms – Alter functions of genes or their patterns of expression and regulation