1. Chapter 19 The Organization and Control of Eukaryotic Genomes Things to Know: 1.All those bold-faced words again 2.Figure 19.1: the basics of DNA structure 3.Spend some time thinking about these questions (and their answers): a)How does your genome get changed during your lifetime (pg 359) b)What controls the expression of eukaryotic genes (pgs 362 – 368) and how does this contrast with gene regulation in prokaryotes? c)Whazzzzzup with cancer at the molecular level?

2. Figure 19.0 Chromatin in a developing salamander ovum White portion: part of the chromatin of each sister chromatid around which the other chromatin is arranged. Red Loops are the actively transcribed regions

3. Figure 19.1 Levels of chromatin packing

4. Figure 19.x1a Chromatin

5. Figure 19.x1b Chromatin, detail

6. Repetitive DNA and other noncoding sequences Prokaryotic DNA 1.Codes for protein, rRNA and tRNA 2.The noncoding regions are regulatory regions such as the promoters 3.The coding sequence goes from start to finish without interruption

7. Repetitive DNA and other noncoding sequences Eukaryotic DNA 1.97% DOES NOT code for protein, rRNA and tRNA 2.Some regulatory sequences but most of the DNA is the introns. 3.Also lots of repetitive DNA, which is noncoding and is usually not within the genes.

8. Tandemly Repetitive DNA Definition: a short sequence that is repeated in a series.  Text’s example: GTTACGTTAC... Etc.  you could have hundreds of repeats of the GTTAC  these repeats are usually up to 10 bases long.  this can be composed of specific bases to give this area a different density so that you can cut the DNA, centrifuge it and spin it out.  originally known as satellite DNA because it was a “separate” or satellite band from the other DNA that was centrifuged.

9. Table 19.1 Types of Repetitive DNA

10. Tandemly Repetitive DNA  minisatellite and microsatellite DNA refers to how big the repeated segments are, 100 – 100,000 bps and 10 – 100 bps respectively.  the microsatellite DNA can be used in DNA fingerprinting.

11. Tandemly Repetitive DNA Genetic Disorders Caused by Tandem Repeats Fragile X: triplet (CGG) repeat about 100’s to 1000s of times (normal is 30); causes different degrees of mental retardation; the number of repeats increases each generation; causes the tip of the X chromosome to hang from the rest of the chromosome Huntington’s: CAG repeat; this repeat is translated producing a protein containing lots of glutamines. The more repeats, the more severe the disease. Regular Satellite DNA is at the telomeres and centromeres and may play a role in the basic structure of DNA.

12. Interspersed Repetitive DNA Alu Inserts or elements  These are repeats but they aren’t one right after another.  We will find out about some of your Alu inserts in a lab  Interspersed DNA makes up 20-40% of mammalian genome  An Alu insert is 300 bps long.  An Alu insert may be transcribed but has no known function yet.

13. Multigene Families Multigene Family: a group of identical or very similar genes  long repeating units since they are genes  a part of the group may be close or far apart  Example: genes for rRNA  there are three of these genes each coding for a different size rRNA.  they are transcribed together but there are hundreds of copies of this transcription unit throughout the genome.

14. Figure 19.2 Part of a family of identical genes for ribosomal RNA

15. Multigene Families (cont’d) Nonidentical Gene example: genes for hemoglobin  there are different forms of the alpha genes and different forms of the beta genes.  the different versions are transcribed at different times so they function at different times during mammalian development.  fetal Hb is different from adult Hb and has a higher affinity for oxygen. This is due to the difference in the types of alpha and beta strands.

16. Figure 19.3 The evolution of human  -globin and  -globin gene families

17. Multigene Families (cont’d) How do you get a family of similar/identical genes from a single gene?  gene duplications occurring over and over again.  Recombination can produce multiple copies  Mutations can create different forms of the same gene and the product still has a similar function.  Some of the mutations produce nonfunctioning products, and these are called pseudogenes.

18. Gene Amplification, loss or rearrangement can alter a cell’s genome Important Concepts 1.These processes do not occur in gametes therefore are not passed on to the offspring. 2.They do not contribute to evolution 3.They do occur in your somatic cells and therefore can change your phenotype through alterations in gene expression within cells and tissues.

19. Gene Amplification, (cont’d) Gene Amplification and Selective Gene Loss 1.At a special stage of develop the number of copies of a particular gene or gene family can change (increase). a) This is gene amplification b) Developing eggs of amphibians will produce bunches of copies of the gene that makes rRNA so more ribosomes can be made during early development and hence increase protein production. These extra rRNA genes are then later broken down. c) Cancer cells may also possess amplified genes that increase resistance to a drug that is being administered. Hence, treatment may be “incomplete.”

20. Gene Amplification, (cont’d) Rearrangements in the Genome 1.Introduction  Some genes can be shuffled around and have their location (loci) changed.  This occurs in somatic cells ( not gametes) 2.Transposons and Retrotransposons  Definition: a transposon is simply a segment of DNA that moves throughout the genome.  It moves into the middle of a coding region of a gene  It therefore interferes with normal gene transcription

21. Figure 19.4 The effect of a transposon on flower color The lighter color of this morning glory flower is due to the placement of a genetic element, a transposon, into the gene that codes for the purple flower color. The production of the purple color was altered. Why wasn’t the entire flower affected?

22. Gene Amplification, (cont’d) 2.Transposons and Retrotransposons (cont’d)  If the insertion into the gene occurs where transcription is regulated, this could increase or decrease mRNA production.  Sometimes the transposon carries a gene. If it inserts downstream from the promoter, the transposon gene gets transcribed, mRNA is made, new proteins, new characteristics. Barbara McClintock demonstrated these kind of mobile genetic elements with corn kernels that are yellow, reddish, purple, etc.

23. Figure 19.x2 Transposons in corn

24. Figure 19.x3 Transposons in corn

25. Gene Amplification, (cont’d) 2.Transposons and Retrotransposons (cont’d)  To make it even more complicated... Retrotransposons: these are pieces of DNA but they were made from the mRNA of a transposon. mRNA DNA by reverse transcriptase  Alu inserts are retrotransposons (but no rev. transcriptase)  The enzyme reverse transcriptase catalyzes the reaction of mRNA to DNA and is part of the genetic code of the retrotransposon.

26. Figure 19.5 Retrotransposon movement

27. Gene Amplification, (cont’d) 3.Immunoglobulin Genes a)Some of the basics of the immune system  Antibodies are also called immunoglobulins  Antibodies are proteins  Antibodies are made by special cells called B lymphocytes or just “B cells”  B cells are a type of white blood cell  Each B cell can make a different type of antibody and therefore attack a specific kind of antigen  An antigen is a foreign “invader”: virus, bacteria, fungi, protein.

28. Gene Amplification, (cont’d) 3.Immunoglobulin Genes b)Antibody Structure  Two chains held together by disulfide bonds  Each chain has a constant region (C) or same series of amino acids  Then there is a variable (V) region that gives the antibody its specificity to a particular antigen.  It’s the variable region that recognizes the specific antigen that is in your body.

29. Figure 43.15a,b The structure of a typical antibody molecule

30. Figure 43.15c Antibody molecule

31. Figure 43.14 Epitopes (antigenic determinants)

32. Gene Amplification, (cont’d) 3.Immunoglobulin Genes (cont’d) c)How are these different antibodies made?  One set of genes undergoes permanent rearrangement of its DNA segments when cells differentiate when you are a embryo.  This places the antibody genes in different orders.  As a cell becomes specialized (differentiates) the antibody genes are moved around from different DNA regions in the embryonic cell

33. Figure 19.6 DNA rearrangement in the maturation of an immunoglobulin (antibody) gene 1.Undifferentiated cell J is a junction region (?) 1.Now the gene V3 has been moved and genes V2 and J are next to an intron. V2 is a variable region, this is a sequence of proteins that gives the Ab its specificity so it can bind to specific antigens. 3.Processing and translation occur and you have an antibody with a specific “end” or variable region that recognizes V2 type of antigens.

34. The Control of Gene Expression Each cell of a multicellular eukaryote expresses only a small fraction of its genes.  Continual turning on and off in response to signals from the environment.  Cellular differentiation requires only certain genes to be expressed  A typical human cell expresses only 3-5% of its genes at a given time so something must be regulating this activity.  Control can occur anywhere from the unwinding of the packed chromatin to translation.

35. The Control of Gene Expression (cont’d) Chromatin modifications affect the availability of genes for transcription 1.Introduction a)The physical condition (state) of the DNA near the gene of interest can control its availability for transcription and thus expression. i.The highly condensed heterochromatin is usually not expressed because the transcription factors can’t reach those genes. ii.If a gene is near a nucleosome is it more or less likely to be transcribed? What about its proximity to the chromosome scaffolding?

36. The Control of Gene Expression (cont’d) Chromatin modifications affect the availability of genes for transcription The Control of Gene Expression (cont’d) Chromatin modifications affect the availability of genes for transcription 2.DNA Methylation a)What is it? b)Inactivated X chromosomes such as in Barr bodies are highly methylated. c)If you compare the same genes in different tissues, genes that are being actively transcribed are not methylated. d)Removing the methyl groups makes the genes active. e)Methylation is involved in long-term inactivation of genes during cellular differentiation of embryonic development.

37. The Control of Gene Expression (cont’d) Chromatin modifications affect the availability of genes for transcription The Control of Gene Expression (cont’d) Chromatin modifications affect the availability of genes for transcription 3.Histone Acetylation a)What is it? The attachment of –COCH 3 groups to certain amino acids of the histones. b)This acetylation causes the histones to change shape, not bind to the DNA so tightly and therefore be more available to transcription factors for transcription.

38. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. 1.Organization of a Typical Eukaryotic Gene a) See next slide b)Presence of introns c)A transcription initiation complex forms at the promoter which is upstream from the gene. RNA polymerase is a part of this and it then begins transcription. d)Editing, G’cap at the 5’ end and a poly A tail at the 3’ end. e)Control elements are present: these bind transcription factors.

39. Figure 19.8 A eukaryotic gene and its transcript

40. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. 2.The Role of Transcription Factors a)Ts factors recognize TATA boxes b)These TATA boxes are within the promoter c)Control Elements increase the efficiency of transcription. What are control elements? i.Enhancers: may be far away from the promoter, upstream or downstream or within an intron. ii.DNA bending brings these distant areas with the enhancers close to the promoter iii.Interaction of these enhancers with Ts factors forms the initiation complex on the promoter.

41. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. 2.The Role of Transcription Factors iv.Activator: a complex of Ts factor and enhancer v.Repressors: this could be DNA methylation d)Transcription factors... i.Possesses a DNA binding domain to which it binds to the DNA ii.It also possesses a site to bind to another Ts factor

42. Figure 19.9 A model for enhancer action

43. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. The Control of Gene Expression (cont’d) Transcription initiation is controlled by proteins that interact with DNA and with each other. 3.Coordinately Controlled Genes a)This has to do with a bunch of genes that must be coordinated together to be turned on or off. i.In prokaryotes: all related genes are located in an operon, one right after another. They share the same promoter. ii.In eukaryotes, related genes can be scattered all over the genome, different promoters. b)There could be a main control element or a collection of them that get bound by Ts factors at the same time and thus all these related genes get transcribed simultaneously. i.Steroids turn on different genes at the same time.

44. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. Post-transcriptional mechanisms play supporting roles in the control of gene expression 1.Introduction a)Proteins need to be functional to represent gene expression b)RNA processing does not always occur the same way with the same pre-mRNA transcript. i.Alternative RNA-splicing: this means that different RNA molecules are produced from the same pre- mRNA transcript by changing which segments are treated as exons and which as introns.

45. Figure 19.11 Alternative RNA splicing

46. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. 2.Regulation of mRNA Degradation a)Prokaryotic mRNA is degraded quickly (few minutes). This affords the bacterium a quick adaptation to environmental changes. b)Eukaryotic mRNA lasts hours, days, weeks c)Degradation begins with the shortening of the poly A tail and then the G’cap. d)Nucleases, enzymes that eat up nucleic acids, then digest the mRNA.

47. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. 3.Control of Translation a)Stop the beginning so ribosomes can’t bind to the mRNA. This can be blocked by regulatory proteins that bind to the 5’ end of the mRNA i.These can be temporary blocks so the mRNA is simply “stored” for later such as in an ovum that stores bunches of mRNA waiting for fertilization and then BAM they all become active.

48. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. 3.Control of Translation (cont’d) b)Global control of translation: all mRNA’s are kept “silent” i.Hemoglobin is made of a Heme or iron containing group and polypeptides. If the Heme group is in short supply, the translation of all Hb polypeptides are turned off. ii.Egg Cells: mRNA’s are in waiting for fertilization iii.Plants and algae: mRNA’s are stored during periods of darkness and then the light triggers translation.

49. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. The Control of Gene Expression (cont’d) Post-transcriptional mechanisms play supporting roles in the control of gene expression. 4.Protein Processing and Degradation a)Modifications to the translated proteins can be inactivated i.Sugars are not added so proteins never make it to the cell’s surface. ii.Cystic Fibrosis: a chloride ion channel protein never makes it to the cell membrane and is eventually degraded. 5.Proteasomes: degrade “tagged for destruction” proteins. The tag is a molecule called ubiquitin.

50. Figure 19.12 Degradation of a protein by a proteasome

51. Figure 19-12x Proteasomes

52. Figure 19.7 Opportunities for the control of gene expression in eukaryotic cells

53. Molecular Biology of Cancer Cancer results from genetic changes that affect the cell cycle 1.Many genes regulate the cell cycle so any mutation in these genes may cause cells to lose the control of cell division resulting in cancerous growth. 2.Oncogenes: cancer causing genes first found in retroviruses 3.Proto-oncogene: normal gene found in humans and other animals that coded for proteins regulating cell growth and division that can become an oncogene.

54. Cancer results from genetic changes that affect the cell cycle 4.How does a proto-oncogene that is functioning normally and in a healthy fashion become an oncogene? What are the genetic changes? a)A gene can move to a new location and become under the control of a new promoter that is more active and thus more of the gene’s product is produced which stimulates the cell cycle. b)A gene can be duplicated (amplified) in a cell and therefore there are bunches of copies of this gene in a cell and they are all being transcribed, making their gene product that affects the cell cycle. c)Point mutations can occur in a gene and this mutation could alter the protein product and this “abnormal” or unexpected gene product affects the cell cycle putting the cell cycle regulation out of control and thus a cell is now dividing bunches and bunches of times (malignancy) Molecular Biology of Cancer

55. Cancer results from genetic changes that affect the cell cycle 4.How does a proto-oncogene that is functioning normally and in a healthy fashion become an oncogene? What are the genetic changes? i.A mutation could affect a tumor suppressor gene. Normally these genes produce proteins that prevent uncontrolled cell division but if there is a mutation then this suppressor product is not made and thus the cell can now divide out of control. a.These tumor suppressor genes can normally:  Repair damaged DNA which if allowed to accumulate can cause cancer.  Maintain the need for cells to adhere to other cells and to the extra cellular matrix (something that cancer cells do not need to do)  Act in cell-signaling pathways to keep cells from dividing. Molecular Biology of Cancer

56. Figure 19.13 Genetic changes that can turn proto-ocogenes into oncogenes

57. Oncogene proteins and faulty tumor-suppressor proteins interfere with normal signaling pathways. 1.ras Gene a)This gene is mutated in about 30% of human cancers. b)It is a component of a signal transduction pathway that affects the DNA c)The product of the ras gene is the Ras protein and it is a G protein which again is a protein, that requires GTP, and gets a signal from a membrane bound receptor, a tyrosine-kinase receptor. d)Normally, a growth factor is produced that stimulates the cell cycle but an oncogene protein, a hyperactive version of the Ras protein causes the stimulation of the tyrosine-kinase receptors without the growth factor being around. Molecular Biology of Cancer

58. Figure 19.14 Signaling pathways that regulate cell growth (Layer 3)

59. Oncogene proteins and faulty tumor-suppressor proteins interfere with normal signaling pathways. 2.p53 gene a)This gene gets activated when the cell’s DNA is damaged. b)The p53 gene makes a p53 protein which is a transcription factor c)This transcription factor can affect lots of different genes i.It can affect a gene called p21 that makes a protein that shuts down the cell cycle so the DNA can be repaired. ii.It can turn on DNA repair genes iii.It can stimulate “suicide genes” that produces a protein that causes apoptosis. Molecular Biology of Cancer

60. Figure 19.14 Signaling pathways that regulate cell growth (Layer 3)

61. Figure 19.15 A multi-step model for the development of colorectal cancer The accumulation of more than one mutation can cause cancer and thus the older you are the more likely you will get cancer. The tumor polyp genes may be benign initially and then become malignant and more mutations then destroy the tumor-suppressor genes.

62. Multiple mutations underlie the development of cancer 1.Many changes occur for a cell to become cancerous a)One active oncogene is usually present b)Mutation(s) in tumor-suppressor genes. c)Telomerase gene is activated so the cell keeps dividing Molecular Biology of Cancer 2.Some viruses can cause cancer. a)15% of world wide cancers are caused by viruses b)Some forms of leukemia are caused by retroviruses (viruses that contain RNA as their nucleic acid) c)Hepatitis viruses can cause liver cancer. d)Wart viruses can cause cervical cancer e)This happens by the injected RNA representing an oncogene f)Viral DNA could also be injected and disrupt a tumor sup. gene.

63. Multiple mutations underlie the development of cancer Molecular Biology of Cancer 3.Breast Cancer a)BRCA1 and BRCA2 are two identified genes. b)Strong genetic, inherited link. c)Mutations in either gene increase risk for breast and ovarian cancer. d)BRCA1 and 2 are tumor suppressor genes e)What their gene products due may still be unknown. They may be DNA repair proteins.