Select topics from Chapter 15

© 2014 Pearson Education, Inc.

Select topics from Chapter 15


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


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


Figure 15.2

Regulation of gene expression

Precursor

trpE gene

(a) Regulation of enzyme activity

Feedback inhibition

Enzyme 1

Enzyme 2

Enzyme 3

Tryptophan

(b) Regulation of enzyme production

trpD gene

trpC gene

trpB gene

trpA gene


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


§  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


§  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


§  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


Figure 15.3

Promoter Promoter

trp operon

Genes of operon DNA

Regulatory gene

mRNA

Protein

RNA polymerase

Inactive repressor

Operator Start codon Stop codon

mRNA 5ʹ

trpE trpD trpC trpB trpA

E D C B A

(a) Tryptophan absent, repressor inactive, operon on

DNA

mRNA

Protein Active repressor

No RNA made

Tryptophan (corepressor)

(b) Tryptophan present, repressor active, operon off

Polypeptide subunits that make up enzymes for tryptophan synthesis

5ʹ

3ʹ

trpR


Figure 15.3a

(a) Tryptophan absent, repressor inactive, operon on

Polypeptide subunits that make up enzymes for tryptophan synthesis

Protein Inactive repressor

mRNA

5ʹ

3ʹ

E D C B A

Promoter

DNA Regulatory gene

RNA polymerase

Promoter

trp operon

Genes of operon

Operator Start codon Stop codon

mRNA 5ʹ

trpE trpD trpC trpB trpA trpR


Figure 15.3b

DNA

mRNA

Protein Active repressor

No RNA made

Tryptophan (corepressor)

(b) Tryptophan present, repressor active, operon off


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


§  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


§  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


Figure 15.4

DNA

mRNA

Promoter Operator

Regulatory gene

No RNA made

RNA polymerase

3ʹ

5ʹ

IacZ

Active repressor Protein

(a) Lactose absent, repressor active, operon off

IacZ IacY IacA IacI DNA

mRNA

Protein

RNA polymerase

Inactive repressor

Allolactose (inducer)

(b) Lactose present, repressor inactive, operon on

mRNA 5ʹ 3ʹ

5ʹ

lac operon

lacI

Permease Transacetylase β-Galactosidase


Figure 15.4a

DNA

Promoter Operator

Regulatory gene

No RNA made

IacZ lacI

mRNA RNA polymerase

3ʹ

5ʹ

Active repressor Protein

(a) Lactose absent, repressor active, operon off


Figure 15.4b

IacZ IacY IacA IacI

DNA lac operon

Permease Transacetylase β-Galactosidase

mRNA

Protein

RNA polymerase

mRNA 5ʹ 3ʹ

5ʹ

Inactive repressor

Allolactose (inducer)

(b) Lactose present, repressor inactive, operon on


§  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


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


§ 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


Figure 15.5

DNA

Promoter

Operator IacZ lacI

CAP-binding site

cAMP

Inactive CAP

Active CAP

RNA polymerase binds and transcribes

Allolactose

Inactive lac repressor

(a) Lactose present, glucose scarce (cAMP level high):

Promoter

abundant lac mRNA synthesized

DNA

Operator IacZ lacI

CAP-binding site RNA polymerase less likely to bind

(b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized

Inactive CAP



Figure 15.5a

DNA

Promoter

IacZ lacI Operator RNA

polymerase binds and transcribes

Active CAP

Inactive CAP

Allolactose


CAP-binding site

cAMP

(a) Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized


Figure 15.5b

Promoter

DNA Operator

IacZ lacI

CAP-binding site

Inactive CAP Inactive lac

repressor

RNA polymerase less likely to bind

little lac mRNA synthesized (b) Lactose present, glucose present (cAMP level low):


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


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


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


§  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

DNA Methylation



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


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


§  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

Enhancers and Specific Transcription Factors


§  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 protein-protein interactions that result in transcription of a given gene


Figure 15.10-3

DNA

Enhancer Distal control element

Activators Promoter Gene

TATA box

DNA- bending protein

Group of mediator proteins

General transcription factors

RNA polymerase II

RNA polymerase II

RNA synthesis Transcription initiation complex


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


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


Figure 15.12

DNA

Primary RNA transcript

mRNA or

Exons

Troponin T gene

RNA splicing

1 2 3 4 5

1 2 3 5 1 2 4 5

1 2 3 4 5


Select topics from Chapter 16


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


A Genetic Program for Embryonic Development

§  The transformation from zygote to adult results from cell division, cell differentiation, and morphogenesis


§  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


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


Figure 16.3

(a) Cytoplasmic determinants in the egg (b) Induction by nearby cells Unfertilized egg Early embryo

(32 cells) Sperm

Fertilization

Nucleus

Two-celled embryo

Mitotic cell division

Zygote (fertilized egg)

Molecules of two different cytoplasmic determinants Signal

transduction pathway

Signaling molecule (inducer)

Signal receptor

NUCLEUS


§  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


Sequential Regulation of Gene Expression During Cellular Differentiation

§  Determination commits a cell irreversibly to its final fate

§  Determination precedes differentiation


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


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


§  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


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


Figure 16.7a

0.5 mm

Head Thorax Abdomen

Dorsal

Ventral

Posterior Anterior Right

Left

(a) Adult

BODY AXES


§  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


Figure 16.7b-5

Larval stage

(b) Development from egg to larva

Segmented embryo

Body segments

Fertilized egg

Unfertilized egg

Egg developing within ovarian follicle

Hatching 0.1 mm

Embryonic development

Egg shell Depleted

nurse cells Fertilization Laying of egg

Egg

Nurse cell

Follicle cell Nucleus 1

2

3

4

5


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


Figure 16.8

Wild type

Eye

Mutant


§  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


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 egg-polarity genes because they control orientation of the egg and consequently the fly


§ 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

Bicoid: A Morphogen Determining Head Structures


Figure 16.9

Head Tail

Tail Tail

Wild-type larva

Mutant larva (bicoid)

T1 T2 T3 A2 A3 A1 A4

A8

A5 A6 A7

A8 A6 A7 A8 A7

250 µm


§  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


§  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


Figure 16.10

Anterior end 100 µm

Bicoid protein in early embryo

Results

Bicoid protein in early embryo

Bicoid mRNA in mature unfertilized egg

Bicoid mRNA in mature unfertilized egg

Fertilization, translation of bicoid mRNA


§  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


§  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

Concept 16.2: Cloning organisms showed that differentiated cells could be reprogrammed and ultimately led to the production of stem cells


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


§  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


Figure 16.11

Frog embryo

Less differentiated cell

Results

Enucleated egg cell

Donor nucleus transplanted

Experiment Frog egg cell Frog tadpole

Donor nucleus transplanted

Fully differentiated (intestinal) cell

Egg with donor nucleus activated to begin

development

Most develop into tadpoles.

Most stop developing before tadpole stage.

UV


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


Figure 16.12

Grown in culture

Results

Cell cycle arrested, causing cells to dedifferentiate

Implanted in uterus of a third sheep

Cultured mammary cells


Technique Mammary cell donor

Egg cell donor

Egg cell from ovary

Nucleus removed

Nucleus from mammary cell

Surrogate mother

Cells fused

Early embryo

Lamb (“Dolly”) genetically identical to mammary cell donor

1 2

3

4

5

6


Figure 16.12a

Cell cycle arrested, causing cells to dedifferentiate

Cultured mammary cells

Technique Mammary cell donor

Egg cell donor

Egg cell from ovary

Nucleus removed


Cells fused

1 2

3


Figure 16.12b

Grown in culture

Results

Implanted in uterus of a third sheep



Surrogate mother

Early embryo

Lamb (“Dolly”) genetically identical to mammary cell donor

4

5

6

Technique


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


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


Figure 16.14

Stem cell

Stem cell Precursor cell

Fat cells

Cell division

and

or or Bone cells White blood cells


Figure 16.15

Cultured stem cells

Embryonic stem cells

Liver cells Nerve cells Blood cells

Adult stem cells

Different culture conditions

Different types of differentiated cells

Cells that can generate all embryonic cell types

Cells that generate a limited number of cell types


§  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


§  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


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


Types of Genes Associated with Cancer

§  Cancer research led to the discovery of cancer-causing genes called oncogenes in certain types of viruses

§  The normal version of such genes, called proto-oncogenes, 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


Figure 16.16

within a control element

Proto-oncogene

Gene amplification: multiple copies of the gene

Proto-oncogene Proto-oncogene

Point mutation:

within the gene

Translocation or transposition: gene moved to new locus, under new controls

Normal growth- stimulating protein in excess

New promoter

Oncogene Oncogene Oncogene



Hyperactive or degradation- resistant protein


Figure 16.16a

Proto-oncogene

Translocation or transposition: gene moved to new locus, under new controls


New promoter

Oncogene


Figure 16.16b

Gene amplification: multiple copies of the gene

Proto-oncogene



Figure 16.16c

within a control element

Proto-oncogene

Point mutation:

within the gene

Oncogene Oncogene


Hyperactive or degradation- resistant protein


§  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


§  Tumor-suppressor genes encode proteins that help prevent uncontrolled cell growth

§ Mutations that decrease protein products of tumor-suppressor genes may contribute to cancer onset

§  Tumor-suppressor proteins

§ Repair damaged DNA

§ Control cell adhesion §  Inhibit the cell cycle


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


Figure 16.17

Growth factor

G protein

Receptor Protein kinases

NUCLEUS Transcription factor (activator)

Protein that stimulates the cell cycle

NUCLEUS Transcription factor (activator)

Overexpression of protein

MUTATION

Ras protein active with or without growth factor.

GTP

Ras

GTP

Ras

1

2

3

4

5 6


§  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


Figure 16.18

Protein kinases

NUCLEUS

DNA damage in genome

Defective or missing transcription factor

MUTATION UV light

1

2

3

Inhibitory protein absent

DNA damage in genome

UV light

Active form of p53

Protein that inhibits the cell cycle


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


§  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 tumor-suppressor genes


Figure 16.19

Colon

Loss of tumor- suppressor gene APC (or other)

Loss of tumor-suppressor gene p53

Activation of ras oncogene

Colon wall Normal colon epithelial cells

Small benign growth (polyp)

Loss of tumor- suppressor gene DCC

Malignant tumor (carcinoma)

Larger benign growth (adenoma)

Additional mutations

1 2

3

4

5


Figure 16.19a

Loss of tumor- suppressor gene APC (or other)

Loss of tumor-suppressor gene p53

Activation of ras oncogene

Colon wall Normal colon epithelial cells

Small benign growth (polyp)

Loss of tumor-suppressor gene DCC Malignant tumor

(carcinoma) Larger benign growth (adenoma)

Additional mutations

1

2

3

4

5


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


§  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

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Select topics from Chapter 15