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| Extended Lecture Outline |
Chapter 18: Gene Regulation And Genetic Engineering |
A. FUNDAMENTALS OF GENE REGULATION
18.1 In procaryotes, genes with related functions tend to be located together in blocks and regulated together.
a. Figure 18.1 shows a block of 11 genes, all which encode enzymes for histidine.
b. Cells have mechanisms that prevent the unnecessary production of components.
c. Bacteria have evolved regulatory mechanisms at two levels (Figure 18.2).
d. Feedback inhibition is one mechanism (Section 11.5), whereby the end product of a pathway inhibits enzymes that already exist in the cell.
e. Repression is another mechanism, where control mechanisms keep genes from being expressed.
f. Enzymes are regulated coordinately, so they remain proportional to one another.
g. Cells making unneeded proteins grow more slowly, and are at a selective disadvantage.
h. Transcriptional controls are favored over translational controls, due to the energy saved.
18.2 The regulation of genes for lactose metabolism is a classical model system.
a. Housekeeping proteins are those needed for essential metabolism.
b. E. coli cells use glucose as a preferred energy source, and have enzymes for glucose metabolism.
c. Lactose can also be used, and is broken down into glucose and galactose by b-Galactosidase.
d. b-Galactosidase is not made by the cells unless lactose is present, but is readily made in the presence of lactose (Figure 18.3).
e. b-Galactosidase is thus known as an inducible enzyme and lactose is an inducer.
f. Jacob and Monod found how the genes for lactose metabolism are regulated.
g. The lacZ gene encodes for the b-Galactosidase enzyme.
h. The lacY gene encodes for another protein that transports lactose across the cell membrane.
i. The lacA gene, located next to Y, has an unknown function, but is thought to be part of the regulatory mechanism.
j. These three genes are structural genes, as they encode structural, rather than regulatory, proteins.
k. Mutants called lacI make all three lac proteins due to a defective control mechanism.
l. Figure 18.4 shows an experiment by Pardee, Jacob, and Monod, to test alternative models for the action of the I gene.
m. In one model, I encodes a repressor protein that keeps the Z, Y, and A genes silent.
n. In the other model, I encodes an activator protein that combines with an inducer to turn on the three genes.
o. Evidence supports the first model, and suggests that a mutant repressor cannot turn the genes off and is thus recessive to a normal repressor.
p. Figure 18.5 shows another Pardee—Jacob—Monod experiment that gives results consistent with the repressor model.
q. The conclusion is that the I+ gene is dominant to the I- gene that encodes a defective repressor.
r. The repressor was found to be a protein with a specific DNA binding site known as an operator.
s. In the absence of an inducer, the protein binds to the operator, O, next to the Z gene (Figure 18.6), and regulates the gene.
t. Genes regulated by an operator constitute an operon; the Z-Y-A block is known as the lac operon.
u. The direction of transcription is called downstream, and the operator site is upstream of the genes it controls.
18.3 Genetic regulatory circuits fit the general model of communication and regulation.
a. Genetic regulation fits the general model, as outlined in Chapter 11, of a sensor (monitor) and effector.
b. Homeostasis is regulated by sensor proteins that sense and respond to certain cell conditions.
c. In the lac system, the repressor protein is both the sensor and the effector, as it turns on lac genes in response to signals from sensor.
d. The common genetic regulatory pattern is:
1. A regulatory protein combines with a signal ligand, changing its shape and thus its ability to bind to a control site on DNA.
2. In response, neighboring gene(s) are turned on or off, affecting transcription.
e. There are at least three variations of these regulatory mechanisms (Figure 18.7):
1. Negative repression, as in the lac system.
2. Positive repression (Section 18.5).
3. Positive activation (Section 18.4).
f. Regulatory proteins diffuse throughout the cell and bind to any control site they recognize.
g. Cells called merodiploids are made and used in regulatory experiments, as illustrated in Exercise 18.1.
18.4 Genes may also be regulated by positive mechanisms.
a. Englesberg discovered the arabinose sugar regulatory mechanism (Figure 18.8).
b. Arabinose binds to an activator, which then binds to an upstream initiator site, I, activating the genes and resulting in transcription.
c. This is an example of a positive activator protein.
18.5 Biosynthetic genes may be regulated by repressors.
a. A positive repressor only has an affinity for its operator when the appropriate ligand is bound to it (Figure 18.9).
b. Such repressors engage in feedback by responding to high concentrations of endproducts.
c. Repression thus stops synthesis of new proteins, rather than regulating the activity of existing proteins, as in end-product inhibition. (Section 11.5).
d. A regulon is a set of coordinately regulated operons, which may be located at various sites on the genome, but which are all encoded by a single repressor protein (Figure 18.10).
18.6 Alarmones regulate still larger blocks of genes.
a. Alarmones are ligands that trigger emergency responses by large sets of genes.
b. Guanosine tetraphosphate (ppGpp) is a nucleotide alarmone made when protein synthesis is halted in a cell; it stimulates operons for amino acid synthesis.
c. Cyclic AMP (cAMP, Figure 18.11) is an alarmone that is made from ATP when energy sources are low, and it binds to a cAMP acceptor protein (CAP) to turn on genes that supply energy.
d. The lac operon contains a binding site for binding active cAMP-CAP (Figure 18.12).
18.7 Promoters, the DNA sequences to which RNA polymerase binds, also regulate gene expression.
a. Some genes are regulated at the transcription level by the properties of their promoters.
b. Procaryote promoters are located just upstream of the genes to be transcribed (Figure 18.13).
c. The sequences of most promoters are represented by a consensus sequence (Figure 18.14), and the strongest promoters are most like the consensus sequence.
d. Procaryote promoters always have a TATA box, or a Pribnow box, as part of their consensus sequence (Figure 18.14).
e. Genes can be regulated when the affinity of RNA polymerase for various promoters is changed, as with the transcription of phage T4 genes (Figure 18.15).
f. In this case, RNA polymerase attaches to a set of early promoters, then middle promoters, and finally late promoters; each promoter changes the polymerase's affinity for a transcription site.
g. Post-transcriptional and translational regulatory mechanisms also exist.
18.8 Eucaryotic genes are regulated primarily by combinations of promoters and enhancers.
a. Yeasts are eucaryotes that live and regulate their genes in ways similar to procaryotes.
b. Most other eucaryotic organisms, with differentiated tissues, have evolved regulatory mechanisms not found in procaryotes.
c. Tissue-specific proteins, such as myoglobin, are found in eucaryotes in addition to housekeeping proteins, and regulate specific functions of their specialized cells.
d. Eucaryotic genes are regulated primarily by promoters and enhancers (Figure 18.16).
e. A typical eucaryotic promoter contains critical upstream promoter elements (UPEs).
f. A UPE appears to be a site to which specific regulatory proteins can bind.
g. Enhancers are regulatory sites than enhance transcription of a gene; they can lie either upstream or downstream of the gene.
h. Enhancers contain specific binding sites for stimulatory and inhibitory proteins.
i. These binding sites are generally located at a distance from the regulated gene, and the DNA has to bend in order to bring the two sequences together and effect regulation (Figure 18.17).
j. Silencers are enhancer-like elements that inhibit transcription.
18.9 Some genes are directly regulated by steroid hormones.
a. Figure 18.18 shows steroid hormones.
b. These don't bind to cell surface receptors (Section 41.1), but can pass through the cell membrane and bind to receptor proteins.
c. Steroid receptor proteins are specific for different hormones and have specific binding sites for enhancer elements (Figure 18.19).
d. Each steroid selectively affects a small set of genes in a particular tissue.
18.10 Chromosome structure may regulate gene transcription.
a. The normal eucaryotic chromosome, consisting of DNA and proteins, can have its expression regulated by opening or compacting.
b. Heterochromatin is the term for chromatin that is highly compacted; it contains few genes and may repress genes that are near it.
c. Euchromatin is the term for the rest of the uncompacted chromosome.
d. Figure 18.20 shows the position effect that results from inverting a segment of chromosome, as a nearby gene's activity is repressed.
e. Chapter 19 covers permanently heterochromatized X chromosomes that are never transcribed.
f. The lampbrush chromosomes in many oocytes (Figure 18.21) are an example of active DNA remaining uncoiled and producing the mRNA needed for early development of the embryo.
g. The salivary gland giant chromosomes of Drosophila (Figure 18.22) are an example of polytene chromosomes, which consist of many tandem DNA molecules that were replicated but not separated.
h. Polytene chromosomes appear in embryonic tissues that have to make large amounts of certain proteins.
i. The DNA in most polytene bands remains compact, but a few bands are opened into chromosomal puffs (Figure 18.23), each of which has been correlated one-to-one with specific proteins needed in the organism.
j. Ecdysone is a steroid hormone that activates molting in arthropods, and which is associated with puffing on polytene chromosomes (Figure 18.24).
B. APPLICATIONS TO RECOMBINANT-DNA WORK
18.11 Recombinant-DNA research employs a few basic methods.
a. Recombinant-DNA technology is reliant on several methods and processes.
1. High temperature and high pH both cause double-stranded nucleic acid molecules to separate in a process called melting.
2. Mixtures of single-stranded DNA and RNA can be made by adjusting pH to induce separation and then binding (annealing) of complementary sequences.
b. In 1983 Kary Mullis invented the polymerase chain reaction (PCR), which is outlined and illustrated in the text.
1. DNA replication must begin from a primer sequence, a short second strand attached to the template strand.
2. Primers of any desired length can now be chemically synthesized.
3. The PCR method is used to amplify a region called "target DNA."
4. At least short sequences at each end of the target region must be known, and complementary primer sequences are made from these.
5. Taq polymerase (from bacteria that can live at high temperatures) is used in a mixture with the DNA target region and the four nucleoside triphosphates needed to make new DNA.
6. A temperature cycler put tubes of the mixture through a cycle of melting, annealing, and replication, and the Taq polymerase extends the primer and replicates the DNA.
c. Nucleic acid fragments can be separated in electrophoresis gels of agarose (Figure 18.25); the sizes of the fragments are measured by using standards of known size.
d. Edward Southern invented Southern blotting (Figure 18.26), where fragments of DNA carrying particular genes can be found.
1. Fragments of DNA that were cut with enzymes and separated on a gel are transferred to a nitrocellulose filter.
2. The corresponding messenger RNA is extracted from a cell making the protein of interest, and is made radioactive.
3. The RNA is known as a probe, a specific nucleic acid used to find complementary nucleic acids.
4. The nitrocellulose filter is sealed with a solution containing the labeled probe, then dried and X-rayed; only the few bands containing parts of the gene of interest are radioactive.
e. Many recombinant methods require the use of the mRNA for a specific protein.
1. The techniques for finding these mRNAs are complex and are not covered in this text.
2. The RNA itself is not always useful, as it cannot be cloned.
3. Reverse transcriptase (Figure 18.34) is an enzyme that can make a DNA molecule with the sequence of the RNA of interest.
4. Complementary DNA (cDNA) copies of useful RNA molecules are made by such enzymes (Figure 18.27).
18.12 Foreign genes can be cloned in plasmids that permit their expression, and can be used for research and practical applications.
a. To find a gene of interest for cloning, a method similar to Southern blotting (Figure 18.28) is used.
b. Bacteria carrying a gene library are found on Petri plates, their DNA is probed, and clones carrying the target DNA are found.
c. An expression vector is a vector that places the gene into a manipulative form and allows the gene to be transcribed.
d. The pUC and pET vectors are expression vectors (Figure 18.29).
e. A typical expression vector carries the lacI gene, the lac promoter region, and the lacZ gene.
f. The b-Galactosidase gene is a reporter gene, one that can be detected easily and used to show whether a cell carries the desired DNA.
g. A polylinker is carried by the plasmid at the start of the lacZ gene; this contains the sites cut by endonucleases, and allows the selection of the best restriction enzyme for cloning the target gene.
h. After cloning (Figure 18.30), different bacteria can be distinguished by plating with the antibiotic, the lac inducer, and X-Gal, a white substrate that turns blue when hydrolyzed.
i. Bacterial cloning systems are useful for cheap and easy production of large amounts of protein, such as human growth hormone and insulin.
j. Bacteria lack the complex apparatus for expressing typical eucaryotic genes, however, so a eucaryotic system is needed for studying these genes.
k. Once a gene has been cloned and expressed, the challenge is therefore to find a transgenic organism that will illustrate the effects of the gene.
18.13 Genes can be cloned in plants by means of a natural bacteria—plant —plasmid system.
a. Plants, vital to agriculture, are desirable targets for genetic engineering.
b. The techniques for getting DNA across the plant cell wall, and expressed, have been varied.
c. The crown gall bacterium Agrobacterium tumifaciens (Figure 18.31) causes galls due to a tumor inducing (Ti) plasmid.
d. The plasmid carries important genes in a region called T-DNA, which, once transferred into a plant cell, inserts itself at random in the chromosomes and transforms the cell, causing tumor growth.
e. Ti derivatives called disarmed vectors have been developed to transform plant cells.
f. Modified Ti plasmids can transform plant cells in tissue culture, or even in germinating seeds.
g. Section 21.1 covers growing plants from single cells.
h. Figure 18.32 shows the results of transferring a firefly gene into tobacco, an experiment that proved that many kinds of genes can be transferred and expressed in plant cells.
i. Certain desirable plant qualities, such as resistance to herbicides or to insect pests, have been conferred upon plants using genetic engineering.
j. Figure 18.33 shows the strategy used to produce tomatoes that do not ripen until they are chemically treated at their destination for sale.
k. Section 39.9 covers tomato ripening.
18.14 Animal cloning has been limited by unknown physiological problems.
a. Viral vectors have been used to transform animal cells, but pose the danger of unknown or unintended results.
b. Retroviruses (Figure 18.34) are RNA viruses that are copied into a DNA copy once inside a cell.
c. Microinjection (Figure 18.35) of DNA into embryos has been successful and also is more controllable than viral vectors.
d. A transgenic "Supermouse" was produced (Figure 18.36) by cloning genes for rat growth hormone into a mouse's liver cells under the control of a promoter.
e. Transgenic sheep and cows have also been produced, but have health problems associated with abnormal growth rates.
f. The use of transgenic animals to fight or cure human diseases is addressed in Chapter 19.
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