Unit 7 Objectives
CHAPTER
11
How Genes Are Controlled
Control of Gene Expression
11.1
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Describe and compare the regulatory mechanisms of the lac operon, trp operon, and operons
using activators.
Lac operon:
o When lactose is plentiful in the intestine, E. Coli makes the enzymes necessary to absorb the sugar
and use it as an energy source.
o When lactose is not plentiful, E. coli does not waste its energy producing these enzymes.
o Regulatory gene: Information for making repressor protein
o Repressor protein + lactose: Allows RNA polymerase to transcribe genes
o Repressor protein without lactose: Keeps RNA polymerase from attaching to promoter and
transcribing genes
o RNA polymerase: Transcribes genes into mRNA for protein synthesis
o Promoter: Where RNA polymerase starts transcribing genes
o Operator: Repressor protein attaches here
o Operon genes: Information for making enzymes that use lactase
o Enzyme: Use lactose
Trp operon:
o Regulatory gene: Information for making repressor protein
o Repressor protein + tryptophan: Keeps RNA polymerase from attaching to promoter and
transcribing genes
o Repressor protein without tryptophan: Allows RNA polymerase to transcribe genes
o RNA polymerase: Transcribes genes into mRNA for protein synthesis
o Promoter: Where RNA polymerase starts transcribing genes
o Operator: Repressor protein attaches here
o Operon genes: Information for making enzymes that make tryptophan
o Enzymes: Make tryptophan
Operons using activators:
o Activators: proteins that turn operons on by binding to DNA
o Makes it easier for RNA polymerase to bind to the promoter, rather than by blocking RNA
polymerase, as repressors do.
o Help control a wide variety of operons.
11.2 Explain how selective gene expression yields a variety of cell types in multicellular eukaryotes.
During the repeated cell divisions that lead from a zygote to an adult in a multicellular organism, individual
cells must undergo differentiation – that is, they must become specialized in structure and function, with
each type of cell fulfilling a distinct role.
To perform its specialized role, each cell type must maintain a specific program of gene expression in which
some genes are expressed and others are not.
A cells function determines it’s genes that are active.
11.2 Explain how DNA is packaged into chromosomes. Explain how packing influences gene
expression.
DNA is packaged into chromosomes through an elaborate, multilevel system of packing – coiling and folding
– of the DNA in each chromosome.
Important part: the association of the DNA with small proteins called histones (which account for about half
the mass of eukaryotic chromosomes).
Frist level:
o Histones attach to the DNA double helix, and “bead like” nucleosome (consisting of DNA wound
around a protein core of eight histone molecules. Linkers (short stretches of DNA) are the “strings”
that join consecutive “beads” of nucleosomes.
Next level:
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o The beaded string is wrapped into a tight helical fiber.
o This fiber coils further into a thick supercoil with a diameter of about 300 ne.
o Looping and folding can further compact the DNA.
DNA packing can block gene expression by preventing RNA polymerase and other transcription proteins
from contraction the DNA.
The longer the inactivation genes, the higher levels of packing.
11.2 Explain how a cat’s tortoiseshell coat pattern is formed and why this pattern is only seen in
female cats.
It forms by the relevant fur-color gene being on the X chromosome, and the tortoiseshell phenotype requires
the presence of two different alleles, one for orange fur and one for black fur.
Only females can have both alleles because only they have two X chromosomes.
Orange patches are formed by populations of cells in which the X chromosome with the orange allele is
active; black patches have cells in which the X chromosome with the black allele is active.
This involves X inactivation
If a male cat had XXY chromosome, then could it have tortoiseshell coat pattern?
11.3 Explain how eukaryotic gene expression is controlled. Compare the eukaryotic gene expression
mechanisms to those of prokaryotes.
The eukaryotic gene expression is controlled by complex assemblies of proteins:
o The fine-tuning begins with the initiation of RNA synthesis – transcription.
o In prokaryotes and eukaryotes, the initiation of transcription (whether transcription starts or not) is
the most important stage for regulation gene expression.
o Most have individual promoters and others control sequences and are not clustered together as in
operons.
o The “default” state for most genes seems to be “off”.
o A typical animal or plant cell needs to turn on (transcribe) only a small percentage of its genes, those
required for the cell’s specialized structure and function.
o Housekeeping genes, those continually active in virtually all cells for routine activities such as
glycolysis, may be in an “on” state by default.
o Eukaryotic RNA polymerase requires the assistance of proteins called transcription factors.
o First step in initiating gene transcription is the binding of activator proteins to DNA control
sequences called enhancers.
o In contrast to the operators of prokaryotic operons, enhancers are usually far away on the
chromosome from the gene they help regulate.
Similar:
o Both employ regulatory proteins – activators and repressors – that bind to specific segments of DNA
and either promote or block the binding of RNA polymerase, turning the transcription of genes on or
off.
11.4 Describe the process and significance of alternative DNA splicing.
Process:
o RNA processing includes the addition of a cap and a tail, as well as the removal of any introns –
noncoding DNA segments that interrupt the genetic message – and the splicing together of the
remaining exons.
o Some think that the splicing process may help control the flow of mRNA from nucleus to cytoplasm
because until splicing is completed, the RNA is attached to the molecules of the splicing machinery
and cannot pass through the nuclear pores.
o In some cases, the cell can carry out splicing in more than one way, generating different mRNA
molecules from the same RNA transcript this is called alternative RNA splicing,
o With this sort of alternative RNA splicing, an organism can produce more than one type of
polypeptide from a single gene.
Significance:
o One interesting example of two-way splicing is found in the fruit fly, where the differences between
males and females are largely due to different patterns of RNA splicing.
o Included among the many instances already known in one gene whose transcript can be spliced to
encode seven alternative versions of a protein, each of which is made in a different type of cell.
11.6 Explain how mRNA breakdown, initiation of translation, protein activation, and protein
breakdown regulate gene expression.
 mRNA breakdown regulates gene expression by being broken down in the cytoplasm by enzymes, and the
timing of this event is an important factor regulating the amounts of various proteins that are produced in the
cell.
o Because short-lived mRNAs only have very short lifetimes; they are typically degraded by enzymes
within a few minutes after their synthesis. This is one reason bacteria can change their protein
production so quickly in response to environmental changes.
 Initiation of translation regulate gene expression by:
o The process of translating an mRNA into a polypeptide also offers opportunities for regulation.
o Among the molecules involved in translation are a great many proteins that control the start of
polypeptide synthesis.
o By controlling the start of protein synthesis, cells can avoid wasting energy if the needed
components are currently unavailable.
 Protein activation regulates gene expression by:
o After translation is complete, some polypeptides require alterations before they become functional.
o Post-translational control mechanisms in eukaryotes often involve the cleavage (cutting) of a
polypeptide to yield a smaller final product that is the active protein, able to carry out a specific
function in the organism.
o After translation is completed, the polypeptide folds up, and covalent bonds form between the sulfur
(S) atoms of sulfur-containing amino acids.
o This combination of two shorter polypeptides is the form of insulin that functions as a hormone.
o By controlling the timing of such protein modifications, the rate of insulin synthesis can be finetuned.
 Protein breakdown regulates gene expression by:
o The final control mechanism operating after translation is the selective breakdown of proteins.
o The lifetimes of many other proteins are closely regulated.
o Some of the proteins that trigger metabolic changes in cells are broken down within a few minutes
or hours.
o This regulation allows a cell to adjust the kinds and amounts of its proteins in response to changes in
its environment.
o Also enables the cell to maintain its proteins in prime working order.
o Indeed, when proteins are damaged, they are usually broken down right away and replaced by new
ones that function properly.
11.7 Explain how the control of gene expression in eukaryotic cells is analogous to the control of
water moving through the series of pipes that carry water from a local water supply to a home or
business.
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11.9 Explain how DNA microarrays can be used to study gene activity and treat
disease.
 A researcher collects all of the mRNA transcribed from genes in a particular type of cell at the current
moment.
 Isolate all of the mRNA from the cell,
 Tag them with a flourecent die
 Expose to a microary where some of them will stick and the rest will wash away
 The computer can then tell
 Focus on what a microarray is and what we use it for
Cloning of Plants and Animals
11.12 Describe experiments that demonstrate that differentiated cells retain all of their genes.
 When cells from a carrot are transferred to a culture medium, a single cell can begin dividing and eventually
grow into an adult plant, a genetic replica of the parent plant.
 This is asexual reproduction, and such an organism who does the above process is called a clone.
 This shows that one cell can hold the blue prints to grow an extra of any part of the organism without losing
any of its genes in the process.
 A good indication that differentiation need not impair an animal cell’s genetic potential is the natural process
of regeneration, the regrowth of lost body parts.
11.13 Explain how nuclear transplantation can be used to clone animals.
 Nuclear transplantation can be used to clone animals by:
o Involves replacing the nucleus of an egg cell or a zygote with the nucleus of an adult somatic cell.
o The recipient cell may then begin to divide.
o About 5 days later, repeated cell divisions have formed a blastocyst, a hollow ball of about 100
cells.
o The blastocyst may be used for different purposes.
11.14 Describe some of the practical applications of reproductive cloning.
 Describe some of the practical usages of reproductive cloning
 Bringing back endangered species
 Instead of having to reengineer organisms whenever we need a new one, we can just clone them
11.15 Describe the process and goals of therapeutic cloning.
 Process:
o Using retroviruses as vectors to introduce extra copies of these genes into the skin cells.
o Goes through differentiation where the cultured embryonic stem cells can evolve into different
forms of cells under certain conditions specific to certain cells.
o So you take the embryonic stem cell, put it in a condition for nerve cells (for example), and you get
nerve cells?
 Goals:
o Ultimate aim: to supply cells for the repair of damaged or diseased organs.
CHAPTER
12
DNA Technology and Genomics
Gene Cloning
12.1 Explain how plasmids are used in gene cloning.
 Isolate two kinds of DNA: a bacterial plasmid that will serve as the vector, or gene carrier, and the DNA
containing the gene of interest along with other unwanted genes.
 An enzyme is chosen that cuts the plasmid in only one place.
 The other DNA, which is typically much longer in sequence, may be cut into many fragments, one of each
caring the desired gene.
 The cut DNA from both sources – the plasmid and target gene – are mixed. The single-stranded ends of the
plasmid base-pair with the complementary ends of the target DNA fragment.
 The enzyme DNA ligase joins the two DNA molecules by covalent bonds. This enzyme is a “DNA pasting”
enzyme that catalyzes the formation of covalent bonds between adjacent nucleotides, joining the strands.
 The recombinant plasmid containing the targeted gene is mixed with a culture of bacteria. Under the right
conditions, a bacterium takes up the plasmid DNA by transformation.
 This recombinant bacterium then reproduces to form a clone of cells, a group of identical cells descended
from a single ancestral cell, each carrying a copy of the desired gene. This step is the actual gene cloning.
 Gene cloning can be used to produce a variety of desirable products. Copies of the gene itself can be the
immediate product, to be used in further genetic engineering projects.
12.2 Explain how restriction enzymes are used to “cut and paste” DNA into
plasmids.
 In nature, these enzymes protect bacterial cells against intruding DNA from other organisms or viruses.
 They chop up the foreign DNA, a process that restricts the ability of the invader to do harm to the bacterium.
 The bacterial cell’s own DNA is protected from restriction enzymes through chemical modification by other
enzymes.
 The DNA sequence recognized by a particular restriction enzyme is called a restriction site.
 Then the restriction enzyme cuts both strands of the DNA at specific points within the sequence.
 A restriction enzyme cuts a DNA molecule in a precise, reproducible way.
 Steps:
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A restriction enzyme cuts the DNA into fragments
A DNA fragment from another source is added
Two (or more) fragments stick together by base pairing
DNA ligase pastes the strands together
Result = Recombinant DNA molecule
Genetically Modified Organisms
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12.6 Explain how different organisms are used to mass-produce proteins of human interest.
By transferring the gene for a desired protein into a bacterium, yeast, or other kind of cell that is easy to grow
in culture, a genetic engineer can produce large quantities of proteins that are otherwise difficult to obtain.
Bacteria are often the best organisms for manufacturing a protein product.
o The plasmids and phages available for use as gene-cloning vectors and the fact that bacteria can be
grown rapidly and cheaply in large tanks.
o Can be engineered to produce large amounts of particular proteins and, in some cases, to secrete the
proteins directly into their growth medium, simplifying the task of collecting and purifying the
products.
Sometimes necessary to use eukaryotic cells to produce a protein product.
o Yeast is often the first choice of eukaryotic cells to produce protein because it is easy to grow
 Can take up foreign DNA and integrate it into their genomes
 Have plasmids that can be used as gene vectors, and yeast is often better than bacteria at
synthesizing and secreting eukaryotic proteins.
The cells of choice for making some gene products come from mammals.
o Many proteins that are normally secreted are glycoproteins, proteins with chains of sugars attached.
o Because only mammalian cells can attach the sugars correctly, mammalian cells must be used for
making these products.
Recently, pharmaceutical researchers have been exploring the mass production of gene products by whole
animals or plants rather than cultured cells.
o Using recombinant DNA technology to insert genes for desired human proteins into other mammals,
where the protein encoded by the recombinant gene may be secreted in the animal’s milk.
o Genetically engineered animals are difficult and costly to produce.
o Starts by injecting the desired DNA into a large number of embryos, which are then implanted into
surrogate mothers.
o With luck, one or a few recombinant animals may result; success rates for such procedures are very
low.
o Once a recombinant organism is successfully produced, it may be cloned.
o The result can be a genetically identical herd – a grazing pharmaceutical “factory” or “pharm”
animals that produce otherwise rare biological substances for medical use.
12.7 Explain how DNA technology has helped to produce insulin, growth hormone, and vaccines.
Produce insulin:
o Developing bacteria that synthesize and secrete the human form of insulin.
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Produce growth hormone:
o Made an artificial gene for HGH by joining a human DNA fragment to a chemically synthesized
piece of DNA; using this gene, they were able to produce HGH in E. coli.
o HGH from recombinant bacteria is now widely used.
Produce vaccines:
o A vaccine is a harmless variant (mutant) or derivative of a pathogen – usually a bacterium or virus –
that is used to stimulate the immune system to mount a lasting defense against that pathogen.
o Use genetically engineered cells or organisms to produce large amounts of a protein molecule that is
found on the pathogens outside surface.
o Another way to use DNA technology in vaccine development is to make a harmless artificial mutant
of the pathogen by altering one or more of its genes.
 When a harmless mutant is used as a so-called “live vaccine,” it multiplies in the body and
may trigger a strong immune response. Artificial-mutant vaccines may cause fewer side
effects than vaccines that have traditionally been made from natural mutants.
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Another method employs a virus related to the one that causes smallpox.
 Smallpox was once a dreaded human disease, but it was eradicated worldwide in the 1970s
by widespread vaccination with a harmless variant of the smallpox virus.
12.8 Explain how genetically modified (GM) organisms are transforming agriculture.
 Genetically modified organisms (organisms that have acquired one or more genes by artificial means) are
transforming agriculture by:
o If the newly acquired gene is from another organism, typically of another species, the recombinant
organism is called a transgenic organism.
o The most common vector used to introduce new genes into plant cells is a plasmid from the soil
bacterium called the Ti plasmid
 With the help of a restriction enzyme and DNA ligase, the gene for the desired trait is
inserted into a modified version of the plasmid
 Then the recombinant plasmid is put into a plant cell, where the DNA carrying the new gene
integrates into the plant chromosome
 Finally, the recombinant cell is cultured and grown into a plant
o In addition to agricultural applications, genetic engineers are now creating plants that make human
proteins for medical use.
 A recently developed transgenic rice strain harbors genes for milk proteins that can be used
in rehydration formulas to treat infant diarrhea, a serious problem in developing countries.
 Using modified corn to treat cystic fibrosis
 Safflower to treat diabetes
 Duckweed to treat hepatitis.
 No plant-made drugs intended for use by humans have yet to be approved or sold.
o Agricultural researchers are also producing transgenic animals.
 To do this, scientists remove egg cells from a female and fertilize them.
 They then inject a previously cloned gene directly into the nuclei of the fertilized eggs.
 Some of the cells integrate the foreign DNA into their genomes.
 The engineered embryos are then surgically implanted in a surrogate mother.
 If an embryo develops successfully, the result is an animal containing a gene from a third
“parent,” which may even be of another species.
o The goals in creating a transgenic animal are often the same as the goals of traditional breeding – for
instance, to make sheep with better quality whole or a cow that will mature in less time.
o To date, the vast majority of the GM organisms that contribute to our food supply are not animals,
but crop plants.
DNA Profiling
12.11 Describe the basic steps of DNA profiling.
 The basic steps of DNA profiling:
o First, DNA samples are isolated from the crime scene, suspects, victims, or stored evidence
o Next, selected markers from each DNA sample are amplified (copied many times), producing an
adequate supply for testing
o Finally, the amplified DNA markers are compared, proving which samples were derived from the
same individual.
12.12 Explain how PCR is used to amplify DNA sequences. Polymerase Chain Reaction (PRC) cycle
 The reaction mixture is heated to separate the strands on the DNA double helices.
 The strands are cooled. As they cool, primer molecules hydrogen-bond to their target sequences on the DNA.
 A heat-stable DNA polymerase builds new DNA strands by extending the primers in the 5’  3’ direction.
 Cycle 1 yields two molecules
 Cycle 2 yields four molecules
 Cycle 3 yields eight molecules
12.13 Explain how gel electrophoresis is used to sort DNA and proteins.
 Shorter pieces go farther and faster that the longer.
 A DNA sample from each source is placed in a separate well (or hole) at one end of a flat, rectangular gel.
 A negatively charged electrode from a power supply is attached near the end of the gel containing the DNA,
and a positive electrode is attached near the other end.
 Because all nucleic acid molecules carry negative charges on their phosphate groups, the DNA molecules all
travel through the gel toward the positive pole.
 However, longer DNA fragments are held back by the thicket of polymer fibers within the gel, so they move
more slowly than the shorter fragments.
 Over time, shorter molecules move farther through the gel than longer fragments.
 Thus, gel electrophoresis separates DNA fragments by length, with shorter molecules migrating toward the
bottom faster than longer molecules.
 When the current is turned off, a series of bands is left in each “lane” of the gel.
 Each band is a collection of DNA fragments of the same length.
 The bands can be made visible by staining, by exposure onto photographic film (if the DNA is radioactively
labeled), or by measuring fluorescence (if the DNA is labeled with a fluorescent dye).
DNA Fingerprinting and Electrophoresis
 Isolate DNA
 Cut DNA with restriction enzymes
 Separate DNA fragments according to size by electrophoresis
 Stain the DNA to see results
 The gel is made up of microscopic fibers
 Small pieces can move through gel pretty quickly, and the longer pieces get caught up on the fibrous pieces.
Electrophoresis: overviews
 DNA fragments are repelled by the electric current because they are negatively charged
 Smallest fragments of DNA migrate farthest, largest fragment get caught up in the gel.
 DNA is visualized with staining.
Electrophoresis: a closer look
 Isolate DNA
 Cut DNA with restriction enzymes to create restriction fragments
 Separate restriction fragments
 According to size by electrophoresis
DNA fingerprinting – an example
 DNA ladder: used as a reference point when comparing a sample of DNA
Practice Free Response Question
Prompt:
Explain how bacteria can be altered to make genetically engineered products. (3pt. max)
Answer:
By separating the plasmid of the DNA and inserting the DNA of the desired trait/s, you can alter the genetic
structure of the bacteria. You cut it at a restricted enzyme, insert the DNA and then the end pieces
should connect together
Rubric
 Isolating DNA/gene; using restriction enzyme; making cDNA, etc.
 Preparing recombinant vector: cutting vector using restriction enzyme; splicing sticky ends (with ligase).
 Delivering vector: transformation with recombinant plasmid, heat shock, virus/retrovirus, etc.
 Testing product or selecting for strain
 Proliferation of reproducing cells protein purification
 Examples of products of modified bacteria are insulin, growth hormone, gene amplification, waste
decomposition enzymes, etc.
Microarry
 Want to decide which genes are turned “on” and which are turned “off”
 You die the strands of DNA with a fluorescent die
 cDNA is ]
Operons
 a bunch of genes that make proteins and then an area that acts as a switch (operator)
 if a repressor is bound to the operator, it turns off the who operon
 Promoter is where the RNA polymerase binds
Lac operon
 When lactose is present, operon is on to produce the enzyme to brake the lactose down
Trp operon
 When trp is present, operon is off
 When tryptophan is used up (its one of the amino acids that’s needed to make proteins) operon is turned on to
make tryptophan
Reproductive vs. Therapeutic cloning
 Reproductive is creating entirely new organisms
 Therapeutic is creating entirely new cells
Bar Body
 To turn off a chromosome, it is coiled up and methylated so that it is coiled up
 Highly methylated X chromosome that
 X chromosome that is tightly packed and just sits there
PCR
 Artificial DNA replication
 Amplifies one segment of DNA
Genes are turned on and turned off by coiling the DNA around the histones. Tight coils doesn’t allow translation,
lose allows translation.