1. How Genes are Controlled Muse 2009

2. CONTROL OF GENE EXPRESSION

3. –Gene expression is the overall process of information flow from genes to proteins –Mainly controlled at the level of transcription –A gene that is “ turned on ” is being transcribed to produce mRNA that is translated to make its corresponding protein –Organisms respond to environmental changes by controlling gene expression 11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response to environmental changes

4. –An operon is a group of genes under coordinated control in bacteria –The lactose (lac) operon includes –Three adjacent genes for lactose-utilization enzymes –Promoter sequence where RNA polymerase binds –Operator sequence is where a repressor can bind and block RNA polymerase action

5. 11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response to environmental changes  Regulation of the lac operon –Regulatory gene codes for a repressor protein –In the absence of lactose, the repressor binds to the operator and prevents RNA polymerase action –Lactose inactivates the repressor, so the operator is unblocked

6. 11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response to environmental changes –Types of operon control –Inducible operon (lac operon) –Active repressor binds to the operator –Inducer (lactose) binds to and inactivates the repressor –Repressible operon (trp operon) –Repressor is initially inactive –Corepressor (tryptophan) binds to the repressor and makes it active –For many operons, activators enhance RNA polymerase binding to the promoter (ie NAC)

8. DNA RNA polymerase cannot attach to promoter Lactose-utilization genes Promoter Operator Regulatory gene OPERON Protein mRNA Inactive repressor Lactose Enzymes for lactose utilization RNA polymerase bound to promoter Operon turned on (lactose inactivates repressor) mRNA Active repressor Operon turned off (lactose absent) Protein

9. DNA RNA polymerase cannot attach to promoter Lactose-utilization genes Promoter Operator Regulatory gene OPERON mRNA Active repressor Operon turned off (lactose absent) Protein

10. DNA Protein Inactive repressor Lactose Enzymes for lactose utilization RNA polymerase bound to promoter Operon turned on (lactose inactivates repressor) mRNA

11. DNA Inactive repressor Active repressor Inactive repressor Active repressor Lactose Promoter Tryptophan OperatorGene lac operon trp operon

12. 11.2 Differentiation results from the expression of different combination genes –Differentiation involves cell specialization, in both structure and function –Differentiation is controlled by turning specific sets of genes on or off

13. 11.3 DNA packing in eukaryotic chromosomes helps regulate gene expression –Eukaryotic chromosomes undergo multiple levels of folding and coiling, called DNA packing –Nucleosomes are formed when DNA is wrapped around histone proteins –“Beads on a string” appearance –Each bead includes DNA plus 8 histone molecules –String is the linker DNA that connects nucleosomes –Tight helical fiber is a coiling of the nucleosome string –Supercoil is a coiling of the tight helical fiber –Metaphase chromosome represents the highest level of packing –DNA packing can prevent transcription

14. 11.3 DNA packing in eukaryotic chromosomes helps regulate gene expression Animation: DNA Packing

15. DNA double helix (2-nm diameter) “Beads on a string” Linker Histones Metaphase chromosome Tight helical fiber (30-nm diameter) Nucleosome (10-nm diameter) Supercoil (300-nm diameter) 700 nm

16. 11.4 In female mammals, one X chromosome is inactive in each somatic cell –X-chromosome inactivation –In female mammals, one of the two X chromosomes is highly compacted and transcriptionally inactive –Random inactivation of either the maternal or paternal chromosome –Occurs early in embryonic development and all cellular descendants have the same inactivated chromosome –Inactivated X chromosome is called a Barr body –Tortoiseshell fur coloration is due to inactivation of X chromosomes in heterozygous female cats

17. Two cell populations in adult X chromosomes Early embryo Allele for black fur Inactive X Black fur Allele for orange fur Orange fur Cell division and random X chromosome inactivation Active X Inactive X Active X

18. 11.5 Complex assemblies of proteins control eukaryotic transcription –Eukaryotic genes –Each gene has its own promoter and terminator –Are usually switched off and require activators to be turned on –Are controlled by interactions between numerous regulatory proteins and control sequences

19. 11.5 Complex assemblies of proteins control eukaryotic transcription –Regulatory proteins that bind to control sequences –Transcription factors promote RNA polymerase binding to the promoter –Activator proteins bind to DNA enhancers and interact with other transcription factors –Silencers are repressors that inhibit transcription –Control sequences –Promoter –Enhancer –Related genes located on different chromosomes can be controlled by similar enhancer sequences

20. 11.5 Complex assemblies of proteins control eukaryotic transcription Animation: Initiation of Transcription Regions on the DNA called enhancers control genes that may be quite distant. Enhancer binding proteins may have multiple controls built into them

21. Enhancers Other proteins DNA Transcription factors Activator proteins RNA polymerase Promoter Gene Bending of DNA Transcription

22. 11.6 Eukaryotic RNA may be spliced in more than one way –Alternative RNA splicing –Production of different mRNAs from the same transcript –Results in production of more than one polypeptide from the same gene –Can involve removal of an exon with the introns on either side Animation: RNA Processing

23. 1 or Exons DNA RNA splicing RNA transcript mRNA 2 3 4 5 1 2 3 4 5 1 2 4 5 1 2 3 5

24. 11.7 Small RNAs play multiple roles in controlling gene expression –RNA interference (RNAi) –Prevents expression of a gene by interfering with translation of its RNA product –Involves binding of small, complementary RNAs to mRNA molecules –Leads to degradation of mRNA or inhibition of translation –MicroRNA –Single-stranded chain about 20 nucleotides long –Binds to protein complex –MicroRNA + protein complex binds to complementary mRNA to interfere with protein production

25. 11.7 Small RNAs play multiple roles in controlling gene expression Animation: Blocking Translation Animation: mRNA Degradation Anti-sense RNA and snRNAi

26. miRNA 1 Translation blocked OR mRNA degraded Target mRNA Protein miRNA- protein complex 2 3 4

27. 11.8 Translation and later stages of gene expression are also subject to regulation –Control of gene expression also occurs with –Breakdown of mRNA –Initiation of translation –Protein activation –Protein breakdown

28. Folding of polypeptide and formation of S—S linkages Initial polypeptide (inactive) Folded polypeptide (inactive) Active form of insulin Cleavage Disulfide bonds Quartenary structure

29. 11.9 Review: Multiple mechanisms regulate gene expression in eukaryotes –Many possible control points exist; a given gene may be subject to only a few of these –Chromosome changes (1) –DNA unpacking –Control of transcription (2) –Regulatory proteins and control sequences –Control of RNA processing –Addition of 5’ cap and 3’ poly-A tail (3) –Splicing (4) –Flow through nuclear envelope (5)

30. 11.9 Review: Multiple mechanisms regulate gene expression in eukaryotes –Many possible control points exist; a given gene may be subject to only a few of these –Breakdown of mRNA (6) –Control of translation (7) –Control after translation –Cleavage/modification/activation of proteins (8) –Breakdown of protein (9)

31. –Applying Your Knowledge For each of the following, determine whether an increase or decrease in the amount of gene product is expected –The mRNA fails to receive a poly-A tail during processing in the nucleus –The mRNA becomes more stable and lasts twice as long in the cell cytoplasm –The region of the chromatin containing the gene becomes tightly compacted –An enzyme required to cleave and activate the protein product is missing 11.9 Review: Multiple mechanisms regulate gene expression in eukaryotes

32. –Applying Your Knowledge For each of the following, determine whether an increase or decrease in the amount of gene product is expected –The mRNA fails to receive a poly-A tail during processing in the nucleus --------- –The mRNA becomes more stable and lasts twice as long in the cell cytoplasm ++++++ –The region of the chromatin containing the gene becomes tightly compacted -------- –An enzyme required to cleave and activate the protein product is missing +++++ feedback 11.9 Review: Multiple mechanisms regulate gene expression in eukaryotes

33. Animation: Protein Processing Animation: Protein Degradation

34. NUCLEUS DNA unpacking Other changes to DNA Addition of cap and tail Chromosome Gene RNA transcript Gene Transcription Intron Exon Splicing Cap mRNA in nucleus Tail Flow through nuclear envelope Broken- down mRNA CYTOPLASM Breakdown of mRNA Translation mRNA in cytoplasm Broken- down protein Cleavage / modification / activation Breakdown of protein Polypeptide Active protein

35. NUCLEUS DNA unpacking Other changes to DNA Addition of cap and tail Chromosome Gene RNA transcript Gene Transcription Intron Exon Splicing Cap mRNA in nucleus Tail Flow through nuclear envelope

36. Broken- down mRNA CYTOPLASM Breakdown of mRNA Translation mRNA in cytoplasm Broken- down protein Cleavage / modification / activation Breakdown of protein Polypeptide Active protein

37. 11.10 Cascades of gene expression direct the development of an animal –Role of gene expression in fruit fly development –Orientation from head to tail –Maternal mRNAs present in the egg are translated and influence formation of head to tail axis –Segmentation of the body –Protein products from one set of genes activate other sets of genes to divide the body into segments –Production of adult features –Homeotic genes are master control genes that determine the anatomy of the body, specifying structures that will develop in each segment

38. Animation: Development of Head-Tail Axis in Fruit Flies 11.10 Cascades of gene expression direct the development of an animal Animation: Cell Signaling

39. Head of a normal fruit fly Antenna Eye Head of a developmental mutant Leg

40. Egg cell within ovarian follicle Follicle cells “Head” mRNA Protein signal Egg cell Gene expression 1 Cascades of gene expression 2 Embryo Body segments Adult fly Gene expression 3 4

41. DNA microarrays test for the transcription of many genes at once –DNA microarray –Contains DNA sequences arranged on a grid –Used to test for transcription –mRNA from a specific cell type is isolated –Fluorescent cDNA is produced from the mRNA –cDNA is applied to the microarray –Unbound cDNA is washed off –Complementary cDNA is detected by fluorescence

42. cDNA Nonfluorescent spot Fluorescent spot Actual size (6,400 genes) Each well contains DNA from a particular gene DNA microarray mRNA isolated DNA of an expressed gene DNA of an unexpressed gene Reverse transcriptase and fluorescent DNA nucleotides 1 cDNA made from mRNA 2 cDNA applied to wells 3 Unbound cDNA rinsed away 4

43. Global transcription analysis

44. 11.12 Signal transduction pathways convert messages received at the cell surface to responses within the cell –Signal transduction pathway is a series of molecular changes that converts a signal at the cell ’ s surface to a response within the cell –Signal molecule is released by a signaling cell –Signal molecule binds to a receptor on the surface of a target cell

45. 11.12 Signal transduction pathways convert messages received at the cell surface to responses within the cell –Relay proteins are activated in a series of reactions –A transcription factor is activated and enters the nucleus –Specific genes are transcribed to initiate a cellular response Animation: Signal Transduction Pathways Animation: Overview of Cell Signaling

46. Signaling cell DNA Nucleus Transcription factor (activated) Signaling molecule Plasma membrane Receptor protein Relay proteins Transcription mRNA New protein Translation Target cell 2 1 3 4 5 6

47. CLONING OF PLANTS AND ANIMALS Hello Dolly!

48. 11.14 Plant cloning shows that differentiated cells may retain all of their genetic potential –Most differentiated cells retain a full set of genes, even though only a subset may be expressed –Evidence is available from –Plant cloning –A root cell can divide to form an adult plant –Animal limb regeneration –Remaining cells divide to form replacement structures –Involved dedifferentiation followed by redifferentiation into specialized cells

49. Root of carrot plant Root cells cultured in nutrient medium Cell division in culture Single cell Plantlet Adult plant

50. 11.15 Nuclear transplantation can be used to clone animals –Nuclear transplantation –Replacing the nucleus of an egg cell or zygote with a nucleus from an adult somatic cell –Early embryo (blastocyst) can be used in –Reproductive cloning –Implant embryo in surrogate mother for development –New animal is genetically identical to nuclear donor –Therapeutic cloning –Remove embryonic stem cells and grow in culture for medical treatments –Induce stem cells to differentiate

51. Remove nucleus from egg cell Implant blastocyst in surrogate mother Add somatic cell from adult donor Donor cell Remove embryonic stem cells from blastocyst and grow in culture Reproductive cloning Nucleus from donor cell Grow in culture to produce an early embryo (blastocyst) Therapeutic cloning Clone of donor is born Induce stem cells to form specialized cells

52. Remove nucleus from egg cell Add somatic cell from adult donor Donor cell Nucleus from donor cell Grow in culture to produce an early embryo (blastocyst)

53. Implant blastocyst in surrogate mother Remove embryonic stem cells from blastocyst and grow in culture Reproductive cloning Therapeutic cloning Clone of donor is born Induce stem cells to form specialized cells

54. Reproductive cloning has valuable applications, but human reproductive cloning raises ethical issues –Cloned animals can show differences from their parent due to a variety of influences during development –Reproductive cloning is used to produce animals with desirable traits –Agricultural products –Therapeutic agents –Restoring endangered animals –Human reproductive cloning raises ethical concerns

55. CAT1 CC

56. 11.17 CONNECTION: Therapeutic cloning can produce stem cells with great medical potential –Stem cells can be induced to give rise to differentiated cells –Embryonic stem cells can differentiate into a variety of types –Adult stem cells can give rise to many but not all types of cells –Therapeutic cloning can supply cells to treat human diseases –Research continues into ways to use and produce stem cells

57. Blood cells Different types of differentiated cells Nerve cells Adult stem cells in bone marrow Different culture conditions Cultured embryonic stem cells Heart muscle cells

58. THE GENETIC BASIS OF CANCER

59. 11.18 Cancer results from mutations in genes that control cell division –Mutations in two types of genes can cause cancer –Oncogenes –Proto-oncogenes normally promote cell division –Mutations to oncogenes enhance activity –Tumor-suppressor genes –Normally inhibit cell division –Mutations inactivate the genes and allow uncontrolled division to occur

60. 11.18 Cancer results from mutations in genes that control cell division –Oncogenes –Promote cancer when present in a single copy –Can be viral genes inserted into host chromosomes (src, ras) –Can be mutated versions of proto-oncogenes, normal genes that promote cell division and differentiation –Converting a proto-oncogene to an oncogene can occur by –Mutation causing increased protein activity –Increased number of gene copies causing more protein to be produced –Change in location putting the gene under control of new promoter for increased transcription

61. 11.18 Cancer results from mutations in genes that control cell division –Tumor-suppressor genes –Promote cancer when both copies are mutated

62. Mutation within the gene Hyperactive growth- stimulating protein in normal amount Proto-oncogene DNA Multiple copies of the gene Gene moved to new DNA locus, under new controls Oncogene New promoter Normal growth- stimulating protein in excess Normal growth- stimulating protein in excess

63. Mutated tumor-suppressor gene Tumor-suppressor gene Defective, nonfunctioning protein Normal growth- inhibiting protein Cell division under control Cell division not under control

64. 11.19 Multiple genetic changes underlie the development of cancer –Four or more somatic mutations are usually required to produce a cancer cell –One possible scenario for colorectal cancer includes –Activation of an oncogene increases cell division –Inactivation of tumor suppressor gene causes formation of a benign tumor –Additional mutations lead to a malignant tumor

65. 1 Colon wall Cellular changes: DNA changes: Oncogene activated Increased cell division Tumor-suppressor gene inactivated Growth of polyp Second tumor- suppressor gene inactivated Growth of malignant tumor (carcinoma) 2 3

66. Chromosomes 1 mutation Normal cell 4 mutations 3 mutations 2 mutations Malignant cell

67. 11.20 Faulty proteins can interfere with normal signal transduction pathways –Path producing a product that stimulates cell division Product of ras proto-oncogene relays a signal when growth hormone binds to receptor Product of ras oncogene relays the signal in the absence of hormone binding, leading to uncontrolled growth

68. 11.20 Faulty proteins can interfere with normal signal transduction pathways –Path producing a product that inhibits cell division –Product of p53 tumor-suppressor gene is a transcription factor –p53 transcription factor normally activates genes for factors that stop cell division –In the absence of functional p53, cell division continues because the inhibitory protein is not produced

69. Growth factor Protein that Stimulates cell division Translation Nucleus DNA Target cell Normal product of ras gene Receptor Relay proteins Transcription factor (activated) Hyperactive relay protein (product of ras oncogene) issues signals on its own Transcription

70. Growth-inhibiting factor Protein that inhibits cell division Translation Normal product of p53 gene Receptor Relay proteins Transcription factor (activated) Nonfunctional transcription factor (product of faulty p53 tumor-suppressor gene) cannot trigger transcription Transcription Protein absent (cell division not inhibited)

71. Lifestyle choices can reduce the risk of cancer –Carcinogens are cancer-causing agents that damage DNA and promote cell division –X-rays and ultraviolet radiation –Tobacco –Healthy lifestyle choices –Avoiding carcinogens –Avoiding fat and including foods with fiber and antioxidants –Regular medical checkups

73. Wrap up and Review Bacteria arrange multiple genes into operons and control promoters with repressors and activators. Eukaryotes have multiple ways to control genes.

74. Regulatory gene Promoter DNA Gene 1 Operator A typical operon Gene 2 Gene 3 Encodes repressor that in active form attaches to operator RNA polymerase binding site Switches operon on or off

75. Nucleus from donor cell Early embryo resulting from nuclear trans- plantation Surrogate mother Clone of donor Whole Organism Cloning

76. Nucleus from donor cell Early embryo resulting from nuclear trans- plantation Embryonic stem cells in culture Specialized cells Therapeutic cloning

77. Gene regulation (a) prokaryotic genes often grouped into in eukaryotes may involve when abnormal may lead to can produce is a normal gene that can be mutated to an oncogene can cause (c) (g)(f) multiple mRNAs per gene transcription female mammals (e) (d) (b) operons are switched on/off by in active form binds to controlled by protein called occurs in are proteins that promote

78. § Explain how prokaryotic gene control occurs in the operon § Describe the control points in expression of a eukaryotic gene § Describe DNA packing and explain how it is related to gene expression § Explain how alternative RNA splicing and microRNAs affect gene expression § Compare and contrast the control mechanisms for prokaryotic and eukaryotic genes You should now be able to

79. § Distinguish between terms in the following groups: promoter — operator; oncogene — tumor suppressor gene; reproductive cloning — therapeutic cloning § Define the following terms: Barr body, carcinogen, DNA microarray, homeotic gene; stem cell; X-chromosome inactivation § Describe the process of signal transduction, explain how it relates to yeast mating, and explain how it is disrupted in cancer development You should now be able to

80. § Explain how cascades of gene expression affect development § Compare and contrast techniques of plant and animal cloning § Describe the types of mutations that can lead to cancer § Identify lifestyle choices that can reduce cancer risk You should now be able to