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Gene Regulation and Cloning: Understanding Control of Gene Expression

This chapter explores the control of gene expression through gene regulation and the potential benefits and concerns of cloning. Topics include prokaryotic gene regulation, differentiation in eukaryotes, DNA packing in chromosomes, X chromosome inactivation, and eukaryotic transcription control.

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Gene Regulation and Cloning: Understanding Control of Gene Expression

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  1. Chapter 11 The Control of Gene Expression

  2. To Clone or Not to Clone? • A clone is an individual created by asexual reproduction and thus is genetically identical to a single parent • Cloning an animal using a transplanted nucleus shows that an adult somatic cell contains a complete genome • Cloning has potential benefits but evokes many concerns • Does not increase genetic diversity • May produce less healthy animals

  3. GENE REGULATION 11.1 Proteins interacting with DNA turn prokaryotic genes on or off in response to environmental changes • Gene regulation is the "turning on" and "turning off" of genes • Helps organisms respond to environmental changes • Gene expression is the process by which information flows from genes to protein • Early understanding of gene control came from studies of the bacterium Escherichia coli

  4. An operon is a cluster of genes with related functions, along with two control sequences • Promoter: A sequence of genes where the RNA polymerase attaches and initiates transcription • Operator: A sequence of genes between the operon and the promoter that acts as a switch for the binding of RNA polymerase • A repressor binds to the operator, stopping transcription • A regulatory gene, located outside the operon, codes for the repressor

  5. The lac operon contains the genes that code for the enzymes that metabolize lactose • Repressor is active when alone and inactive when bound to lactose • The trp operon allows bacteria to stop making tryptophan when it is already present • Repressor is inactive alone; must bind to the amino acid tryptophan to be active • A third type of operon uses activators, proteins that turn operons on by binding to DNA

  6. LE 11-1b OPERON Regulatory gene Promoter Operator Lactose-utilization genes DNA mRNA RNA polymerase cannot attach to promoter Active repressor Protein Operon turned off (lactose absent) DNA RNA polymerase bound to promoter mRNA Protein Inactive repressor Enzymes for lactose utilization Lactose Operon turned on (lactose inactivates repressor)

  7. LE 11-1c Promoter Operator Genes DNA Active repressor Active repressor Tryptophan Inactive repressor Inactive repressor Lactose lac operon trp operon

  8. 11.2 Differentiation yields a variety of cell types, each expressing a different combination of genes • Gene regulation is much more complex in eukaryotes than in prokaryotes • In multicellular eukaryotes, cells become specialized as a zygote develops into a mature organism • The particular genes that are active in each type of cell are the source of its particular function

  9. LE 11-2 Muscle cell Pancreas cells Blood cells

  10. 11.3 Differentiated cells may retain all of their genetic potential • Though differentiated cells express only a small percentage of their genes, they retain a complete set of genes • Allows for propagation of crop plants • In animal cells can lead to regeneration

  11. LE 11-3 Root of carrot plant Single cell Root cells cultured in nutrient medium Cell division in culture Plantlet Adult plant

  12. 11.4 DNA packing in eukaryotic chromosomes helps regulate gene expression • DNA can fit into a chromosome because of packing • DNA winds around clusters of histone proteins, forming a string of bead-like nucleosomes • The beaded fiber coils, supercoils, and further folds into chromosomes • DNA packing prevents gene expression most likely by preventing transcription proteins from contacting the DNA

  13. LE 11-4 DNA double helix (2-nm diameter) Histones Linker “Beads on a string” TEM Nucleosome (10-nm diameter) Tight helical fiber (30-nm diameter) Supercoil (300-nm diameter) TEM 700 nm Animation: DNA Packing Metaphase chromosome

  14. 11.5 In female mammals, one X chromosome is inactive in each cell • An extreme example of DNA packing is X chromosome inactivation in interphase cells of female mammals • In each cell line, the X chromosome from either parent may be inactivated • Leads to a random mosaic of expression of the two X chromosomes • Example: coat color in tortoiseshell cat

  15. LE 11-5 Early embryo Two cell populations in adult Cell division and random X chromosome inactivation Active X Orange fur X chromosomes Inactive X Inactive X Allele for orange fur Black fur Active X Allele for black fur

  16. 11.6 Complex assemblies of proteins control eukaryotic transcription • A variety of regulatory proteins interact with DNA and with each other to turn eukaryotic genes on or off • In contrast to bacteria • Each eukaryotic gene has its own promoter and control sequences • Activators are more important than repressors

  17. Eukaryotic RNA polymerase needs the assistance of transcription factors • The binding of activators to enhancers initiates transcription • Silencers inhibit the start of transcription • Coordinated gene expression in eukaryotes seems to depend on the association of specific enhancers with groups of genes Animation: Initiation of Transcription

  18. LE 11-6 Enhancers Promoter Gene DNA Activator proteins Transcription factors Other proteins RNA polymerase Bending of DNA Transcription

  19. 11.7 Eukaryotic RNA may be spliced in more than one way • After transcription, splicing removes noncoding introns • Alternative splicing may generate two or more types of mRNA from the same transcript Exons DNA RNA transcript RNA splicing or mRNA Animation: RNA Processing

  20. 11.8 Translation and later stages of gene expression are also subject to regulation • After eukaryotic mRNA is processed and transported to the cytoplasm, there are additional opportunities for regulation • Breakdown of mRNA: The lifetime of an mRNA molecule helps determine how much protein is made • Initiation of translation: A great many proteins control the start of polypeptide synthesis

  21. Protein activation: After translation, polypeptides may be cut into smaller, active products • Protein breakdown: Rapid selective breakdown of proteins allows the cell to respond to environmental changes SH Folding of polypeptide and formation of S—S linkages SH S-S S-S Cleavage SH S-S SH S-S SH S-S HS S-S Initial polypeptide (inactive) Folded polypeptide (inactive) Active form of insulin

  22. 11.9 Review: Multiple mechanisms regulate gene expression in eukaryotes • Cellular differentiation results from selective turning on or off of genes at multiple control points • In nucleus • DNA unpacking and other changes • Transcription • Addition of cap and tail • Splicing

  23. In cytoplasm • Breakdown of mRNA • Translation • Cleavage/modification/activation • Breakdown of protein • Each differentiated cell still retains its full genetic potential

  24. LE 11-9 NUCLEUS Chromosome DNA unpacking Other changes to DNA Gene Gene Transcription Exon RNA transcript Intron Addition of cap and tail Splicing Tail mRNA in nucleus Cap Flow through nuclear envelope mRNA in cytoplasm CYTOPLASM Breakdown of mRNA Broken- down mRNA Translation Polypeptide Cleavage / modification / activation Active protein Breakdown of protein Broken- down protein

  25. ANIMAL CLONING 11.10 Nuclear transplantation can be used to clone animals • Nuclear transplantation • Nucleus of a somatic cell is transplanted into a surrogate egg stripped of nucleus • Cell divides to the blastocyst stage • Reproductive cloning • Blastocycst is implanted into uterus • Live animal is born

  26. Therapeutic cloning • Embryonic stem cells are harvested from blastocyst • These cells give rise to all the specialized cells of the body Donor cell Nucleus from donor cell Implant blastocyst in surrogate mother Clone of donor is born (reproductive cloning) Add somatic cell from adult donor Grow in culture to produce an early embryo (blastocyst) Remove nucleus from egg cell Remove embryonic stem cells from blastocyst and grow in culture Induce stem cells to form specialized cells (therapeutic cloning)

  27. CONNECTION 11.11 Reproductive cloning has valuable applications, but human reproductive cloning raises ethical issues • Reproductive cloning of nonhuman mammals is useful in research, agriculture, and medicine • There are many obstacles, both practical and ethical, to human cloning • Research continues in the absence of consensus

  28. CONNECTION 11.12 Therapeutic cloning can produce stem cells with great medical potential • In culture, embryonic stem cells • Can give rise to all cell types in the body • Must be obtained from human embryos • Adult stem cells • Can give rise to many, but perhaps not all, cell types • Are present in adult tissues and, thus, are less controversial than embryonic cells

  29. LE 11-12 Blood cells Adult stem cells in bone marrow Nerve cells Cultured embryonic stem cells Heart muscle cells Different culture conditions Different types of differentiated cells

  30. THE GENETIC CONTROL OF EMBRYONIC DEVELOPMENT 11.13 Cascades of gene expression and cell-to-cell signaling direct the development of an animal • Studies of mutant fruit flies led to early understanding of gene expression and embryonic development • Before fertilization, communication between the egg and adjacent cells determines body polarity • A cascade of gene expression controls development of an animal from a fertilized egg • Master control homeotic genes regulate batteries of genes that shape anatomical parts

  31. LE 11-13a Eye Antenna Leg Head of a normal fruit fly Head of a developmental mutant

  32. LE 11-13b Egg cell within ovarian follicle Egg cell Egg protein signaling follicle cells Follicle cells Gene expression in follicle cells Follicle cell protein signaling egg cell Localization of “head” mRNA “Head” mRNA Fertilization and mitosis Translation of “head” mRNA Embryo Gradient of regulatory protein Gene expression Gradient of certain other proteins Gene expression Body segments 0.1 mm Gene expression Larva Adult fly Head end Tail end 0.5 mm

  33. 11.14 Signal transduction pathways convert messages received at the cell surface to responses within the cell • Signal transduction pathway • Signaling cell secretes signal molecules • Signal molecules bind to receptors on target cell's plasma membrane • Cascade of events leads to the activation of a specific transcription factor

  34. Transcription factor triggers transcription of a specific gene • Translation of the mRNA produces a protein

  35. Signaling cell Signal molecule Plasma membrane Receptor protein Target cell Relay proteins Transcription factor (activated) Nucleus DNA Transcription mRNA Animation: Overview of Cell Signaling New protein Animation: Signal Transduction Pathways Animation: Cell Signaling Translation

  36. 11.15 Key developmental genes are ancient • Homeotic genes contain nucleotide sequences called homeoboxes • Regulate gene expression during development • Similarity of homeoboxes among organisms suggests a very early evolutionary origin

  37. LE 11-15 Fly chromosome Mouse chromosomes Mouse chromosomes Fruit fly embryo (10 hours) Mouse embryo (12 days) Adult fruit fly Adult mouse

  38. THE GENETIC BASIS OF CANCER 11.16 Cancer results from mutations in genes that control cell division • An oncogene can cause cancer when present in a single copy in a cell • A cell can acquire an oncogene from • A virus • A mutation in a proto-oncogene, a normal gene with the potential to become an oncogene

  39. LE 11-16a Proto-oncogene DNA Gene moved to new DNA locus, under new controls Mutation within the gene Multiple copies of the gene New promoter Oncogene Hyperactive growth- stimulating protein in normal amount Normal growth- stimulating protein in excess Normal growth- stimulating protein in excess

  40. Tumor-suppressor genes • Normally code for proteins that inhibit cell division • When inactivated by mutation, can lead to uncontrolled cell division and tumors

  41. LE 11-16b Tumor-suppressor gene Mutated tumor-suppressor gene Normal growth- inhibiting protein Defective, nonfunctioning protein Cell division under control Cell division not under control

  42. 11.17 Oncogene proteins and faulty tumor-suppressor proteins can interfere with normal signal transduction pathways • Stimulatory signal-transduction pathway • Stimulates cell division in response to growth factor • Can be stimulated by oncogene proteins that produce hyperactive relay proteins

  43. LE 11-17a Growth factor Receptor Target cell Hyperactive relay protein (product of ras oncogene) issues signals on its own Normal product of ras gene Relay proteins Transcription factor (activated) DNA Nucleus Transcription Translation Protein that stimulates cell division

  44. Inhibitory signal-transduction pathway • Inhibits cell division in response to growth-inhibiting factor • Faulty tumor-suppressor genes may produce proteins that fail to inhibit cell division

  45. LE 11-17b Growth-inhibiting factor Receptor Relay proteins Nonfunctional transcription factor (product of faulty p53 tumor-suppressor gene) cannot trigger transcription Normal product of p53 gene Transcription factor (activated) Transcription Translation Protein that inhibits cell division Protein absent (cell division not inhibited)

  46. 11.18 Multiple genetic changes underlie the development of cancer • Cancers result from a series of genetic changes in a cell linage • More than one somatic mutation is necessary • Accumulation of mutations over time leads to uncontrolled cell division • Example: Colon cancer develops in a stepwise fashion

  47. LE 11-18a Colon wall Increased cell division Growth of polyp Growth of malignant tumor (carcinoma) Cellular changes: Tumor-suppressor gene inactivated Second tumor- suppressor gene inactivated DNA changes: Oncogene activated

  48. LE 11-18b Chromosomes 1 mutation 2 mutations 3 mutations 4 mutations Normal cell Malignant cell

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