2014 Pearson Education, Inc. Select topics from Chapter 15

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1 Select topics from Chapter 15

2 Overview: Differential Expression of Genes Prokaryotes and eukaryotes alter gene expression in response to their changing environment Multicellular eukaryotes also develop and maintain multiple cell types Gene expression is often regulated at the transcription stage, but control at other stages is important, too

3 Concept 15.1: Bacteria often respond to environmental change by regulating transcription Natural selection has favored bacteria that produce only the gene products needed by the cell A cell can regulate the production of enzymes by feedback inhibition or by gene regulation Gene expression in bacteria is controlled by a mechanism described as the operon model

4 Figure 15.2 Feedback inhibition Precursor trpe gene Enzyme 1 trpd gene Regulation of gene expression Enzyme 2 trpc gene Enzyme 3 trpb gene trpa gene Tryptophan (a) Regulation of enzyme activity (b) Regulation of enzyme production

5 Operons: The Basic Concept A group of functionally related genes can be coordinately controlled by a single on-off switch The regulatory switch is a segment of DNA called an operator usually positioned within the promoter An operon is the entire stretch of DNA that includes the operator, the promoter, and the genes that they control

6 The operon can be switched off by a protein repressor The repressor prevents gene transcription by binding to the operator and blocking RNA polymerase The repressor is the product of a separate regulatory gene

7 The repressor can be in an active or inactive form, depending on the presence of other molecules A corepressor is a molecule that cooperates with a repressor protein to switch an operon off For example, E. coli can synthesize the amino acid tryptophan

8 By default the trp operon is on and the genes for tryptophan synthesis are transcribed When tryptophan is present, it binds to the trp repressor protein, which then turns the operon off The repressor is active only in the presence of its corepressor tryptophan; thus the trp operon is turned off (repressed) if tryptophan levels are high

9 Figure 15.3 DNA Promoter Regulatory gene mrna 5ʹ trpr 3ʹ Promoter Operator RNA polymerase Start codon mrna 5ʹ trp operon Genes of operon trpe trpd trpc trpb trpa Stop codon E D C B A Protein Inactive repressor (a) Tryptophan absent, repressor inactive, operon on Polypeptide subunits that make up enzymes for tryptophan synthesis DNA No RNA made mrna Protein Tryptophan (corepressor) Active repressor (b) Tryptophan present, repressor active, operon off

10 Figure 15.3a DNA Promoter mrna 5ʹ Protein trpr Regulatory gene 3ʹ RNA Operator Stop codon polymerase Start codon mrna 5ʹ Inactive repressor Promoter trp operon Genes of operon (a) Tryptophan absent, repressor inactive, operon on trpe trpd trpc trpb trpa E D C B A Polypeptide subunits that make up enzymes for tryptophan synthesis

11 Figure 15.3b DNA No RNA made mrna Protein Tryptophan (corepressor) Active repressor (b) Tryptophan present, repressor active, operon off

12 Repressible and Inducible Operons: Two Types of Negative Gene Regulation A repressible operon is one that is usually on; binding of a repressor to the operator shuts off transcription The trp operon is a repressible operon An inducible operon is one that is usually off; a molecule called an inducer inactivates the repressor and turns on transcription

13 The lac operon is an inducible operon and contains genes that code for enzymes used in the hydrolysis and metabolism of lactose By itself, the lac repressor is active and switches the lac operon off A molecule called an inducer inactivates the repressor to turn the lac operon on

14 For the lac operon, the inducer is allolactose, formed from lactose that enters the cell Enzymes of the lactose pathway are called inducible enzymes Analogously, the enzymes for tryptophan synthesis are said to be repressible enzymes Video: lac Repressor Model

15 Figure 15.4 Regulatory gene Promoter Operator DNA mrna 5ʹ laci 3ʹ RNA polymerase IacZ No RNA made Protein Active repressor (a) Lactose absent, repressor active, operon off lac operon DNA IacI IacZ IacY IacA mrna 5ʹ 3ʹ RNA polymerase mrna 5ʹ Protein β-galactosidase Permease Transacetylase Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on

16 Figure 15.4a DNA mrna Regulatory gene 5ʹ laci Promoter 3ʹ RNA polymerase Operator IacZ No RNA made Protein Active repressor (a) Lactose absent, repressor active, operon off

17 Figure 15.4b DNA lac operon IacI IacZ IacY IacA mrna 5ʹ Protein 3ʹ RNA polymerase mrna 5ʹ β-galactosidase Permease Transacetylase Allolactose (inducer) Inactive repressor (b) Lactose present, repressor inactive, operon on

18 Inducible enzymes usually function in catabolic pathways; their synthesis is induced by a chemical signal Repressible enzymes usually function in anabolic pathways; their synthesis is repressed by high levels of the end product Regulation of the trp and lac operons involves negative control of genes because operons are switched off by the active form of the repressor

19 Positive Gene Regulation E. coli will preferentially use glucose when it is present in the environment When glucose is scarce, CAP (catabolite activator protein) acts as an activator of transcription CAP is activated by binding with cyclic AMP (camp) Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

20 When glucose levels increase, CAP detaches from the lac operon, and transcription proceeds at a very low rate, even if lactose is present CAP helps regulate other operons that encode enzymes used in catabolic pathways

21 Figure 15.5 Promoter DNA laci CAP-binding site camp Active CAP IacZ RNA Operator polymerase binds and transcribes Inactive CAP Allolactose (a) Lactose present, glucose scarce (camp level high): abundant lac mrna synthesized Promoter Inactive lac repressor DNA Inactive CAP laci CAP-binding site IacZ Operator RNA polymerase less likely to bind Inactive lac repressor (b) Lactose present, glucose present (camp level low): little lac mrna synthesized

22 Figure 15.5a Promoter DNA laci CAP-binding site camp Active CAP IacZ RNA Operator polymerase binds and transcribes Inactive CAP Allolactose Inactive lac repressor (a) Lactose present, glucose scarce (camp level high): abundant lac mrna synthesized

23 Figure 15.5b Promoter DNA laci CAP-binding site Inactive CAP IacZ Operator RNA polymerase less likely to bind Inactive lac repressor (b) Lactose present, glucose present (camp level low): little lac mrna synthesized

24 Concept 15.2: Eukaryotic gene expression is regulated at many stages All organisms must regulate which genes are expressed at any given time In multicellular organisms regulation of gene expression is essential for cell specialization

25 Differential Gene Expression Almost all the cells in an organism are genetically identical Differences between cell types result from differential gene expression, the expression of different genes by cells with the same genome Abnormalities in gene expression can lead to diseases, including cancer Gene expression is regulated at many stages

26 Regulation of Transcription Initiation Chromatin-modifying enzymes provide initial control of gene expression by making a region of DNA either more or less able to bind the transcription machinery

27 DNA Methylation DNA methylation is the addition of methyl groups to certain bases in DNA, usually cytosine Individual genes are usually more heavily methylated in cells where they are not expressed Once methylated, genes usually remain so through successive cell divisions After replication, enzymes methylate the correct daughter strand so that the methylation pattern is inherited

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29 Organization of a Typical Eukaryotic Gene Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that serve as binding sites for transcription factors that help regulate transcription Control elements and the transcription factors they bind are critical for the precise regulation of gene expression in different cell types

30 The Roles of Transcription Factors To initiate transcription, eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors are essential for the transcription of all protein-coding genes In eukaryotes, high levels of transcription of particular genes depend on interaction between control elements and specific transcription factors

31 Enhancers and Specific Transcription Factors Proximal control elements are located close to the promoter Distal control elements, groupings of which are called enhancers, may be far away from a gene or even located in an intron

32 An activator is a protein that binds to an enhancer and stimulates transcription of a gene Activators have two domains, one that binds DNA and a second that activates transcription Bound activators facilitate a sequence of proteinprotein interactions that result in transcription of a given gene

33 Figure DNA Enhancer Activators Distal control element Promoter TATA box Gene DNAbending protein General transcription factors Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

34 Mechanisms of Post-Transcriptional Regulation Transcription alone does not account for gene expression Regulatory mechanisms can operate at various stages after transcription Such mechanisms allow a cell to fine-tune gene expression rapidly in response to environmental changes

35 RNA Processing In alternative RNA splicing, different mrna molecules are produced from the same primary transcript, depending on which RNA segments are treated as exons and which as introns

36 Figure Exons DNA Troponin T gene Primary RNA transcript RNA splicing mrna or

37 Select topics from Chapter 16

38 Different genes are expressed in different cells A fertilized egg gives rise to many different cell types Cell types are organized successively into tissues, organs, organ systems, and the whole organism Gene expression orchestrates the developmental programs of animals

39 A Genetic Program for Embryonic Development The transformation from zygote to adult results from cell division, cell differentiation, and morphogenesis

40 Cell differentiation is the process by which cells become specialized in structure and function The physical processes that give an organism its shape constitute morphogenesis Differential gene expression results from genes being regulated differently in each cell type Materials in the egg can set up gene regulation that is carried out as cells divide

41 Cytoplasmic Determinants and Inductive Signals An egg s cytoplasm contains RNA, proteins, and other substances that are distributed unevenly in the unfertilized egg Cytoplasmic determinants are maternal substances in the egg that influence early development As the zygote divides by mitosis, the resulting cells contain different cytoplasmic determinants, which lead to different gene expression

42 Figure 16.3 (a) Cytoplasmic determinants in the egg Unfertilized egg Sperm Nucleus (b) Induction by nearby cells Early embryo (32 cells) Fertilization Zygote (fertilized egg) Mitotic cell division Molecules of two different cytoplasmic determinants Signal transduction pathway NUCLEUS Two-celled embryo Signal receptor Signaling molecule (inducer)

43 The other major source of developmental information is the environment around the cell, especially signals from nearby embryonic cells In the process called induction, signal molecules from embryonic cells cause transcriptional changes in nearby target cells Thus, interactions between cells induce differentiation of specialized cell types

44 Sequential Regulation of Gene Expression During Cellular Differentiation Determination commits a cell irreversibly to its final fate Determination precedes differentiation

45 Differentiation of Cell Types Today, determination is understood in terms of molecular changes, the expression of genes for tissue-specific proteins The first evidence of differentiation is the production of mrnas for these proteins Eventually, differentiation is observed as changes in cellular structure

46 Pattern Formation: Setting Up the Body Plan Pattern formation is the development of a spatial organization of tissues and organs In animals, pattern formation begins with the establishment of the major axes Positional information, the molecular cues that control pattern formation, tells a cell its location relative to the body axes and to neighboring cells

47 Pattern formation has been extensively studied in the fruit fly Drosophila melanogaster Combining anatomical, genetic, and biochemical approaches, researchers have discovered developmental principles common to many other species, including humans

48 The Life Cycle of Drosophila Fruit flies and other arthropods have a modular structure, composed of an ordered series of segments In Drosophila, cytoplasmic determinants in the unfertilized egg determine the axes before fertilization

49 Figure 16.7a Head Thorax Abdomen 0.5 mm BODY AXES (a) Adult Anterior Left Dorsal Ventral Right Posterior

50 The Drosophila eggs develop in the female s ovary, surrounded by ovarian cells called nurse cells and follicle cells After fertilization, embryonic development results in a segmented larva, which goes through three stages Eventually the larva forms a cocoon within which it metamorphoses into an adult fly

51 Figure 16.7b-5 1 Egg developing within ovarian follicle Follicle cell Nucleus Egg Nurse cell 2 Unfertilized egg Depleted nurse cells Egg shell Fertilization Laying of egg 3 Fertilized egg Embryonic development 4 Segmented embryo Body segments 0.1 mm Hatching 5 Larval stage (b) Development from egg to larva

52 Genetic Analysis of Early Development: Scientific Inquiry Edward B. Lewis, Christiane Nüsslein-Volhard, and Eric Wieschaus won a Nobel Prize in 1995 for decoding pattern formation in Drosophila Lewis discovered the homeotic genes, which control pattern formation in late embryo, larva, and adult stages

53 Figure 16.8 Wild type Mutant Eye

54 Nüsslein-Volhard and Wieschaus studied segment formation They created mutants, conducted breeding experiments, and looked for the corresponding genes Many of the identified mutations were embryonic lethals, causing death during embryogenesis They found 120 genes essential for normal segmentation

55 Axis Establishment Maternal effect genes encode cytoplasmic determinants that initially establish the axes of the body of Drosophila These maternal effect genes are also called eggpolarity genes because they control orientation of the egg and consequently the fly

56 Bicoid: A Morphogen Determining Head Structures One maternal effect gene, the bicoid gene, affects the front half of the body An embryo whose mother has no functional bicoid gene lacks the front half of its body and has duplicate posterior structures at both ends

57 Figure 16.9 Head Tail T1 T2 T3 A1 A2 A3 A4 Wild-type larva A5 A8 A7 A6 250 µm Tail Tail A8 A7 A6 A7 Mutant larva (bicoid) A8

58 This phenotype suggested that the product of the mother s bicoid gene is concentrated at the future anterior end and is required for setting up the anterior end of the fly This hypothesis is an example of the morphogen gradient hypothesis; gradients of substances called morphogens establish an embryo s axes and other features

59 The bicoid mrna is highly concentrated at the anterior end of the embryo After the egg is fertilized, the mrna is translated into Bicoid protein, which diffuses from the anterior end The result is a gradient of Bicoid protein Injection of bicoid mrna into various regions of an embryo results in the formation of anterior structures at the site of injection

60 Figure Results Anterior end 100 µm Bicoid mrna in mature unfertilized egg Fertilization, translation of bicoid mrna Bicoid protein in early embryo Bicoid mrna in mature unfertilized egg Bicoid protein in early embryo

61 The bicoid research is important for three reasons It identified a specific protein required for some early steps in pattern formation It increased understanding of the mother s role in embryo development It demonstrated a key developmental principle that a gradient of molecules can determine polarity and position in the embryo

62 Concept 16.2: Cloning organisms showed that differentiated cells could be reprogrammed and ultimately led to the production of stem cells In organismal cloning one or more organisms develop from a single cell without meiosis or fertilization The cloned individuals are genetically identical to the parent that donated the single cell The current interest in organismal cloning arises mainly from its potential to generate stem cells

63 Cloning Plants and Animals F. C. Steward and his students first cloned whole carrot plants in the 1950s Single differentiated cells from the root incubated in culture medium were able to grow into complete adult plants This work showed that differentiation is not necessarily irreversible Cells that can give rise to all the specialized cell types in the organism are called totipotent

64 In cloning of animals, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell, called nuclear transplantation Experiments with frog embryos showed that a transplanted nucleus can often support normal development of the egg The older the donor nucleus, the lower the percentage of normally developing tadpoles John Gurdon concluded from this work that nuclear potential is restricted as development and differentiation proceeds

65 Figure Experiment Frog embryo Frog egg cell Frog tadpole UV Less differentiated cell Fully differentiated (intestinal) cell Results Donor nucleus transplanted Enucleated egg cell Egg with donor nucleus activated to begin development Donor nucleus transplanted Most develop into tadpoles. Most stop developing before tadpole stage.

66 Reproductive Cloning of Mammals In 1997, Scottish researchers announced the birth of Dolly, a lamb cloned from an adult sheep by nuclear transplantation from a differentiated cell Dolly s premature death in 2003, and her arthritis, led to speculation that her cells were not as healthy as those of a normal sheep, possibly reflecting incomplete reprogramming of the original transplanted nucleus

67 Figure Technique Mammary cell donor Egg cell donor Cultured mammary cells 3 Cell cycle arrested, causing cells to dedifferentiate 1 2 Cells fused Egg cell from ovary Nucleus removed 4 5 Grown in culture Implanted in uterus of a third sheep Nucleus from mammary cell Early embryo Surrogate mother 6 Results Embryonic development Lamb ( Dolly ) genetically identical to mammary cell donor

68 Figure 16.12a Technique Mammary cell donor Egg cell donor Cultured mammary cells 3 Cell cycle arrested, causing cells to dedifferentiate 1 2 Cells fused Egg cell from ovary Nucleus removed Nucleus from mammary cell

69 Figure 16.12b Technique 4 Grown in culture Nucleus from mammary cell 5 Implanted in uterus of a third sheep Early embryo Surrogate mother 6 Results Embryonic development Lamb ( Dolly ) genetically identical to mammary cell donor

70 Faulty Gene Regulation in Cloned Animals In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth Many cloned animals exhibit defects Epigenetic changes must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

71 Stem Cells of Animals A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely and differentiate into specialized cells of one or more types Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types The adult body also has stem cells, which replace nonreproducing specialized cells

72 Figure Stem cell Cell division Stem cell and Precursor cell Fat cells or Bone cells or White blood cells

73 Figure Embryonic stem cells Adult stem cells Cells that can generate all embryonic cell types Cells that generate a limited number of cell types Cultured stem cells Different culture conditions Different Liver cells Nerve cells Blood cells types of differentiated cells

74 ES cells are pluripotent, capable of differentiating into many cell types Researchers are able to reprogram fully differentiated cells to act like ES cells using retroviruses Cells transformed this way are called ips, or induced pluripotent stem cells

75 Cells of patients suffering from certain diseases can be reprogrammed into ips cells for use in testing potential treatments In the field of regenerative medicine, a patient s own cells might be reprogrammed into ips cells to potentially replace the nonfunctional (diseased) cells

76 Concept 16.3: Abnormal regulation of genes that affect the cell cycle can lead to cancer The gene regulation systems that go wrong during cancer are the same systems involved in embryonic development

77 Types of Genes Associated with Cancer Cancer research led to the discovery of cancercausing genes called oncogenes in certain types of viruses The normal version of such genes, called protooncogenes, code for proteins that stimulate normal cell growth and division An oncogene arises from a genetic change leading to either an increase in the amount or the activity of the protein product of the gene

78 Figure Proto-oncogene Proto-oncogene Proto-oncogene Translocation or transposition: gene moved to new locus, under new controls Gene amplification: multiple copies of the gene within a control element Point mutation: within the gene New Oncogene promoter Oncogene Oncogene Normal growthstimulating protein in excess Normal growthstimulating protein in excess Normal growthstimulating protein in excess Hyperactive or degradationresistant protein

79 Figure 16.16a Proto-oncogene Translocation or transposition: gene moved to new locus, under new controls New Oncogene promoter Normal growthstimulating protein in excess

80 Figure 16.16b Proto-oncogene Gene amplification: multiple copies of the gene Normal growthstimulating protein in excess

81 Figure 16.16c Proto-oncogene Point mutation: within a control element within the gene Oncogene Oncogene Normal growthstimulating protein in excess Hyperactive or degradationresistant protein

82 Proto-oncogenes can be converted to oncogenes by Movement of the oncogene to a position near an active promoter, which may increase transcription Amplification, increasing the number of copies of a proto-oncogene Point mutations in the proto-oncogene or its control elements, causing an increase in gene expression

83 Tumor-suppressor genes encode proteins that help prevent uncontrolled cell growth Mutations that decrease protein products of tumorsuppressor genes may contribute to cancer onset Tumor-suppressor proteins Repair damaged DNA Control cell adhesion Inhibit the cell cycle

84 Interference with Cell-Signaling Pathways Mutations in the ras proto-oncogene and p53 tumor-suppressor gene are common in human cancers Mutations in the ras gene can lead to production of a hyperactive Ras protein and increased cell division

85 Figure Growth factor 3 G protein 2 Receptor Ras GTP 5 NUCLEUS 6 Transcription factor (activator) Protein that stimulates the cell cycle 4 Protein kinases MUTATION Ras GTP Ras protein active with or without growth factor. NUCLEUS Transcription factor (activator) Overexpression of protein

86 Suppression of the cell cycle can be important in the case of damage to a cell s DNA; p53 prevents a cell from passing on mutations due to DNA damage Mutations in the p53 gene prevent suppression of the cell cycle

87 Figure Protein kinases UV light NUCLEUS Protein that inhibits the cell cycle 1 DNA damage in genome 3 Active form of p53 UV light DNA damage in genome MUTATION Defective or missing transcription factor Inhibitory protein absent

88 The Multistep Model of Cancer Development Multiple somatic mutations are generally needed for full-fledged cancer; thus the incidence increases with age The multistep path to cancer is well supported by studies of human colorectal cancer, one of the best-understood types of cancer The first sign of colorectal cancer is often a polyp, a small benign growth in the colon lining

89 About half a dozen changes must occur at the DNA level for a cell to become fully cancerous These changes generally include at least one active oncogene and the mutation or loss of several tumorsuppressor genes

90 Figure Colon 1 Loss of tumor- 2 Activation of suppressor gene ras oncogene APC (or other) 4 Loss of tumor-suppressor gene p53 Normal colon epithelial cells Colon wall Small benign growth (polyp) 3 Loss of tumorsuppressor gene DCC Larger benign growth (adenoma) 5 Additional mutations Malignant tumor (carcinoma)

91 Figure 16.19a 1 Loss of tumorsuppressor gene APC (or other) Normal colon epithelial cells Colon wall Small benign growth (polyp) 2 Activation of ras oncogene 4 Loss of tumor-suppressor gene p53 3 Loss of tumor-suppressor gene DCC Larger benign growth (adenoma) 5 Additional mutations Malignant tumor (carcinoma)

92 Inherited Predisposition and Other Factors Contributing to Cancer Individuals can inherit oncogenes or mutant alleles of tumor-suppressor genes Inherited mutations in the tumor-suppressor gene adenomatous polyposis coli (APC) are common in individuals with colorectal cancer Mutations in the BRCA1 or BRCA2 gene are found in at least half of inherited breast cancers, and tests using DNA sequencing can detect these mutations

93 DNA breakage can contribute to cancer, thus the risk of cancer can be lowered by minimizing exposure to agents that damage DNA, such as ultraviolet radiation and chemicals found in cigarette smoke Also, viruses play a role in about 15% of human cancers by donating an oncogene to a cell, disrupting a tumor-suppressor gene, or converting a proto-oncogene into an oncogene