Genetics Unit 4

12) How has RNA silencing been used as a potential gene therapy and what is the main problem that still needs to be overcome? (528-529)

By RNA interference: using short 21-25 nucleotide long sequences pieces called siRNA. They bind to RISC and block translation that leads to degradation of siRNA bound mRNAs The delivery of double stranded RNA or siRNA. An injected delivery through a plasmid possibly using a lentivirus vector

12. How do cells become cancerous? (lecture)

Cells can become cancerous because when any substance or event that damages DNA.

4. What is a biofactory? (380)

Combination of living organisms or cells to produce a product not normal for those cells.

10. What is a Southern blot? Why is it used? What are the steps in a Southern blot? (334)

DNA binding to a DNA probe, it is used to identify specific genes in a particular DNA sample, it is called blotting because blotting paper is used to transfer to filter. • DNA samples with restriction enzymes are loaded on agarose gel for electrophoresis • DNA is separated by electrophoresis • DNA-binding membrane, paper towels, and weight are placed on gel; buffer passes upward through sponge by capillary action, transferring DNA fragments to membrane. • The membrane is placed in heat-seated bag with a solution containing labeled probe; probe hybridizes with complementary sequences • Bound probe detected by film or probe signal captured with a digital camera.

5. What is an expression vector? Why are expression vectors important? (327)

Expression vectors are designed to ensure mRNA expression of a cloned gene with the purpose of producing many copies of the gene's encoded protein in the host cell. They allow the expression of inserted genes.

7. What is the difference between a genomic library and a cDNA library? How is each one generated? (328-329)

Genomic library: It is made taking organism's chromosomes and cutting with restriction enzymes to produce pieces that are cloned into lots of plasmids so the plasmids contain at least one copy all of DNA from that organism. cDNA library: contains DNA copies made from the mRNA molecules present in a cell population at a given time. Eukaryotic mRNA is isolated and mixed with oligo-dT to generate a new complementary DNA library.. • RNAse H cuts parts of the RNA-DNA hybrid making primers for DNA polymerase • A cDNA library can also be made using reverse transcriptase PCR (RT-PCR)

3) What is glyphosate and why is it important to agriculture? (508)

Glyphosate is the active ingredient in commercial herbicides like Roundup. It can treat fields, even while GM crops grow. It will reduce soil damage and there is less environmental impact compared to other mechanisms.

11) How has CRISPR/Cas been utilized for gene therapy and what are the advantages of the system? (527-528)

Has been adapted by using selective RNA molecules, which can combine with cas9 proteins to target defective genes. Cas9 will remove the defective gene. If you put in a gene replacement at the same time then a new gene will be able to fill in for the old one and cure the condition. *Advantages*: selective process: you can choose where the gene is put into the chromosome. -Guide RNA synthesis is inexpensive and less labor intensive than generating a fusion plasmid.

5) What are the 5 different types of genetic tests and how do they differ? (502)

. Diagnostic tests- to detect the presence or absence of a gene variation 2. Predictive tests- detects mutations and variants in patients with family history of a known disorder 3. Carrier tests- identify patients who carry a mutation that might be passed on to offspring 4. Preimplantation tests- done on early embryos to select embryos for implantation that do not carry a disease 5. Prenatal tests- detect potential genetic diseases in a fetus

3) What are the criteria for gene therapy? (519)

1. The gene or genes involved in causing a disease have been identified 2. The gene can be cloned or synthesized in a lab

7) What are the three types of small noncoding RNAs that mediate gene regulation in eukaryotes and how do they differ from each other? (478-479)

1. siRNAs - Small interfering RNAs protect cells from exogenous RNAs 2. miRNAs - MicroRNAs regulate gene expression 3. piRNAs - Piwi-interacting RNAs protect germ cells from mobile DNA elements

4. What is tumorigenesis and how is that related to carcinogens? (309)

A carcinogen is a cancer-causing agent, Tumorigenesis, which is the development of a malignant tumor that occurs in steps, on more genetic alterations. It appears to be the result of two or more genetic alterations that release the cells progressively from the controls that normally operate on proliferation and malignancy

7. What is synthetic biology, what is its goal and what successes have come out of it? (385)

A field of research that combines science and engineering to construct novel biological-bases systems and/or organisms that do not exist in nature. Goal: to find the minimum number of genes necessary for life, and in 2010 scientists reported the first functional genome.

1. What is cancer? (307)

A large number of complex diseases that behave differently depending on the cell types from which they originate. They share two fundamental properties: proliferation (abnormal growth and division and metastasis.

9) What is RNA-induced transcriptional silencing and how does it function? (481-482)

A mechanism by which small noncoding RNAs direct a protein complex (RITS) to complimentary DNA sequences to methylate nearby histones and thus silence transcription from this locus in the genome. • Involved in heterochromatin formation Binds to nascent transcripts during centromeric transcription Catalyzes formation of dsRNA from single-stranded transcripts dsRNA substrate for Dicer; leads to siRNA and targets centromeric transcripts Recruits DMTs and H3K9 methylation → triggers heterochromatin formation

5. What is a mutator phenotype and how does that relate to cancer? (311)

A mutator phenotype is a high level of genomic instability. Cancer cells show higher normal rates of mutation and results from a loss of cells normal ability to repair DNA.

1) What are genetically modified organisms (GMOs) (507)

A plant or an animal whose genome carries a gene transferred from another species by recombinant DNA technology that is expressed to produce a gene product.

8. What is a probe? How are probes used to screen libraries? (329-330)

A probe is a DNA or RNA sequence that has been labeled in some way and is complimentary to a specific target. • Clones from a library are grown on a plate • A replica of the colonies is made by pressing nylon filter onto the plate • Cells on the replica are lysed so that DNA is exposed • The cloned filter is then exposed to a probe to screen the library • The probes then bind to the filter • The replica is exposed to X-ray film and the correct clone is identified • Cells containing the correct clone are amplified by grow in bacteria.

11. What is a dideoxy nucleotide? How is it used in DNA sequencing? How does DNA sequencing work (automated) (336-337)?

An altered nucleotide that have a 3' H instead of the 3' OH. DNA synthesis will terminate after this nucleotide. • Reaction components: DNA template, primer, DNA polymerase, dNTPs, small amount of ddNTps with fluorochromes -As DNA synthesis takes place the polymerase occasionally inserts a dideoxynucleotide instead of a deoxyribonucleotide -This terminates DNA synthesis -As the reaction proceeds the tube with ddATP will accumulate DNA molecules that terminate at all the different positions containing A -Other reactions with G, C, and T are separated in adjacent lanes This is now automated with fluorescent nucleotides used and run together

6. What is the purpose of apoptosis? What are the proteins that carry out apoptosis called? What do these proteins do? (313)

Apoptosis is known as programmed cell death, it normally occurs during development to eliminate certain cells that are defective and damaged. It is carried out by a series of proteases called caspases. Breaks cells into pieces like proteins, DNA and organelles.

9) What are the controversies about GM food? What do the advocates say and what do the critics say? (515-517)

Advocates: that they increase production, reduce pesticide use, preserve soils, and have the potential to feed growing populations -no adverse health affects -vast majority of toxicity tests in animals have shown no negative effects - Critics: that they are unsafe for humans and the environment -most are used as feed and not eaten directly -the process of many food ingredients removes almost all the proteins -not enough clinical trials -GMOs released into the environment have documented and potential consequences (herbicide and insecticide resistance) -some GM crops are spreading into non-GM crops by cross pollination

12. What are some of the ethical issues facing genetics (specifically GM foods, modified organisms, genetic testing, and discrimination) and why are they a problem? (396-399)

Are GMOs safe? Will GMO plants pass on their traits to weeds? Do they benefit our nutrition? Will growth factors from food pass onto humans and cause obesity? Genetic discrimination?

8) What are some of the current successful gene therapy trials? (524-525)

Patients with LCA, causes blindness, targets RPE65 and metabolizes retinol. Over two dozen trials are occurring Hemophilia B which is caused by a deficiency in clotting factor IX Beta-thalassemia involving the beta-globin gene Metachromatic leukodystrophy

10) What is the difference between a zinc-finger nuclease and TALENs in targeted therapy? (526-527)

Are both used to cut out bad sequences and insert new ones. ZFN: a class of DNA-binding domains that bind to a cutter.. They have a characteristic pattern of cysteine and histidine residues that complex with zinc ions, throwing intermediate amino acid residues into a series of loops or fingers. Talens: (transcription activator-like effector nucleases) adding a DNA-binding motif from a transcription factor to a restriction enzyme. Cleave as dimers Each TALEs binds to a specific nucleotide Meant to combine with certain integrases to cut out defective sequences and replace them

2) Why is pharmacogenomics needed? What are the problems with current drug therapy? (498)

Because drugs will usually only be effective in about 50% of all patients who take them. It is needed because doctors usually switch patients from one drug to another until they find an effective one. A lot of money and time may be wasted, and it could be dangerous to those. It works to target drugs to specific patients who will benefit.

14) What are the three different types of gene therapy, which is the only one we currently use and what are the ethical concerns about the two others? (529)

Somatic cell therapy: what we use. Use somatic cells as targets Germ-line therapy: germ cells are targeted. Future generation will be affected without consistent Enhancement therapy: the enhancement of some desired trait. Should we work to enhance abilities such as height, intelligence, athletic ability.

2. What does it mean when a cancer has undergone metastasis? How is that different from a benign tumor (308)

If the cancer spreads and grows out of control into other parts of the body is has metastasized. If it is benign, it only loses genetic control of growth that is non-cancerous.

5) What is the difference between adaptive and innate viral defense mechanisms in prokaryotes? (477)

Innate: a nonspecific response viral defense mechanism. Block bacteriophage adsorption Block DNA insertion Inducing suicide in infected cells Produce Restriction enzymes Destroy bacteriophage DNA Adaptive: previous infection by a pathogen provides immunity to the cell and its descendants. It is dependent upon CRISPR CRISPR's unique spacer sequences are identical to bacteriophage sequences. Phage DNA fragment insertion into CRISPR loci is required for adaptive immunity. Cas proteins have DNase, RNase, and helicase domains

8) What is a Ti plasmid and how is it used to introduce genes in plants? (512)

Integrates a segment of its DNA known as transfer DNA into the plant genome. It integrates into a random location

2. What is biopharmaceuticals? How are they typically generated? (379)

Is the most successful use of recombinant DNA technology, they are done by introducing human genes into bacteria. Cloned into a plasmid and introduced into a bacterial host.

8. How can restriction enzyme analysis be used? What is a restriction fragment length polymorphism (387-388)?

It can be used to examine sickle-cell anemia. A single nucleotide substitution eliminates a cutting site in the B-globin gene, it will alter the pattern of restriction fragments seen on southern blots. restriction fragment length polymorphism: variation in length of DNA fragments generated by restriction endonuclease. These variations are caused by mutations that create or abolish cutting sites for restriction enzymes. They are inherited in a codominant fashion and are extremely useful as genetic markers.

7) What is a gene gun and how does it work? (512-514)

It is a physical method of introducing DNA into cells. It is a heavy metal particle coated with DNA that fires at a high speed.

14. What is the significance of the Genetic information nondiscrimination act? (397)

It prohibited improper use of genetic information in health insurance and employment.

4. What is a polylinker/multiple cloning site? Why are polylinker sites used in recombinant DNA technology? (lecture)

Location in a plasmid with lots of recognition sites for restriction enzymes. Useful because you can clone lots of different sequences into it.

5) What is golden rice and why was it developed? (511)

Most GM crops are not used directly but humans but for animal feed. But some have been developed for humans such as golden rice. It synthesized modest levels of beta-carotene and is done by adding genes to rice, which is missing two genes. It was developed for a vitamin A deficiency.

4) What are Bt crops? (509)

Most widely used GM insect-resistant crops. Bacillus thuringienis is a group of dwelling bacterial stains that produce crystal proteins that are toxic to certain species of insects.

5. What is a subunit vaccine? How is a subunit vaccine made and used? (381-382)

One or more surface proteins from the virus or bacteria, but not the entire virus or bacteria. Often the surface protein is produced through recombinant DNA technology by cloning and expressing the genes encoding the protein to be used for the vaccine.

9. What is the normal function of the p53 gene? How can p53 be mutated to cause cancer? (315)

P53 gene is the most frequently mutates gene in human cancers. It will normally encode a nuclear protein that acts as a transcription factor that represses or stimulates transcription of more than 50 different genes; helps respond to DNA damage. Cells lacking the p53 gene are unable to arrest at cell-cycle checkpoints or to enter apoptosis, they move unchecked through the cell cycle, regardless of the condition of the cell's DNA. Cells lacking the p53 gene have high mutation rates and accumulate the types of mutations that lead to cancer.

7. What is the difference between a proto-oncogene, an oncogene, and a tumor suppressor? Where does each type of gene come from? (314)

Proto-oncogene: genes whose products are important for normal cell growth and division Oncogene: results from a mutated proto-oncogene, a proto-oncogene that has undergone a gain of function mutation. They confer a dominant cancer phenotype. Tumor-suppressor genes: whose products normally regulate cell-cycle checkpoints and initiate the process of apoptosis. Results in a loss of function and checkpoints are missed.

2) What are ribozymes and how have they contributed to the RNA world hypothesis? (475)

RNA acting as a biological catalyst. They led to the RNA world hypothesis It determined that RNA could be self-replicating, and that RNA was a precursor to cellular life. Only 3 ribozymes are capable of "multiple turnover". It was predicted that self-replicating RNAs did exist but were usurped by DNA

1) What is a non-coding RNA and what types exist? (474)

RNA's that do not encode for polypeptides. • snRNAs • microRNAs • sRNAs

10) What are long noncoding RNAs and how do they affect transcriptional activity in two different ways? (482-483)

RNAs that are longer than 200 nucleotides and do not encode for polypeptides. Have various functions including epigenetic modifications of DNA and regulation of the activity of transcription factors. 1. Repress transcription such as inactivation of the X chromosome. 2. Regulate the activity of transcription factor proteins. Androgen receptor binds to testosterone. It is regulated by two lncRNAs.

8. Explain the normal function of the ras gene. Explain how ras can be mutated to cause cancer (314)

Ras genes are some of the most frequently mutated in human tumors. It can encode for a signal transduction molecule that are associated with cell membrane and regulate growth and division. Mutations that cause the Ras gene to become an oncogene will prevent the ras protein from hydrolyzing GTP to GDP and freeze the ras gene into in on conformation which causes it to constantly divide.

1. What is recombinant DNA technology? (322)

Recombinant DNA is DNA created by joining together pieces of DNA from different sources. The methods used to clone the DNA is called recombination DNA technology. *Steps*: DNA to be cloned is purified from cells or tissues Restriction enzymes are used to generate specific fragments Fragments produced are joined to other DNA molecules that serve as vectors Makes a recombinant DNA molecule Recombinant DNA molecule is transferred into a host cell where it replicates, producing many clones As host cells replicate, cloned DNA molecules are passed on the progeny Cloned DNA can be recovered from host cells and purified for use in research or commercial applications

5) What are the three different viral vectors used for gene therapy and describe the advantages and disadvantages to each (520-522)

Retroviral vectors: created by removing replication and disease-causing genes and replace them with a cloned human gene • Virus packages DNA and once infected, the RNA is reverse transcribed into DNA which enters the nucleus and integrates into host cell chromosomes • Can provide long-term expression of genes • Can produce severe toxicity to due insertional mutations Adenovirus: • Can carry large therapeutic genes • Humans can have an immune response • Adeno-associated virus is now widely used as a vector • -non-pathogenic • -does not integrate into host cell genome • DNA forms episomes and this therapy must be repeated Lentivirus vectors: an active area of gene therapy • A retrovirus that can accept large pieces of genetic material • Can infect non-diving cells; others can not • Not possible to control where it integrates; HIV is example; does not gravitate towards gene-regulatory regions

3. What is the role of DNA ligase in recombinant DNA? (324)

Scientists take two pieces of DNA with the same recognition sequence and cut with the enzyme. They mix them together; the ends will anneal together. DNA ligase is used to seal the gaps between the gaps between the two strands of DNA.

6. What is selective breeding and how does it differ from agricultural biotechnology? (382-383)

Selective breeding: breeding plants to reinforce that they already have to select the best ones. Agriculture biotechnology: working to add in traits that the plant may not have and therefore couldn't be selected for.

6) What is SCID, how was it the first successful gene therapy trial, and how was the therapy performed? (522-523)

Severe combined immunodeficiency which is no functional immune system and people normally die from minor infections. It is caused by the lack of ADA o First began with taking her white blood cells o Mixed with a retroviral vector carrying a copy of ADA o WBC were grown in the lab to make sure the ADA gene was expressed o Injected the altered WBC into her bloodstream o Repeated treatments were needed o Still needed supplements of ADA enzyme to live normally

9. What is an allele-specific oligonucleotide? What are they used for? (388)

Short single-stranded fragments of DNA to identify alleles that differ by as little as a single nucleotide. • They will detect changes that don't change restriction sites because not all DNA changes will add a restriction site • Only hybridize when complimentary, use either radioactivity or fluorescence to detect if hybridization occurs • ASO and PCR analysis are now able to screen for many disorders such as sickle-cell anemia.

10. What is a DNA microarray? How are DNA microarrays made? How can a gene expression microarray be used to generate a gene expression profile? (389-390)

Small solid supports such as glass, or membrane on which fragments of known DNA are deposited. • Each spot is a field; typically, a million identical DNA molecules • DNA samples are hybridized onto the field Gene expression microarrays are probes that are either cDNA or synthetic oligos that represent coding regions of genes • Two cell types; normal and cancer • Normal is green dye and cancer is red dye • Mix them together • Examine the pattern; if red then strongest in cancer. If green, then strongest in green. If yellow, in both Date from gene expression is collected and examined according to expression pattern which gives the profiles.

2) What is translational medicine? (519)

Taking a scientific discovery and translating the finding into an effective therapy a. Moving from the bench to the bedside

13. What are direct-to consumer (DTC) genetic tests? (397)

Tests that can be bought to analyze different results of your genetics. Usually done by giving saliva, blood, etc.

1. What is genetic engineering? How is genetic engineering different from biotechnology? (378)

The alteration of the genetic makeup of living organisms, using recombinant DNA technology. Biotechnology covers the use of living organisms or products derived from them, to carry out a wide range

1) What is gene therapy? (519)

The delivery of therapeutic sequences (DNA or RNA) to treat or correct genetic disease conditions.

3) What is the drug Herceptin, what does it treat and how was it a success of personalized medicine? (498-499)

The drug Herceptin was developed using recombinant DNA technology to generate an antibody to the HER-2 gene. It treats breast cancer and the HER-2 gene by inhibiting the signal capacity of the gene and may flag the system for destruction. It will only act on breast cancer cells. It was of the first success stories of pharmacogenomics.

10. What is the function of the RB1 gene? How does mutation of RB1 contribute to cancer? (315)

The loss or mutation of the RB1 tumor suppressor gene contributes to the development of many cancers. This gene gives off a product, pRB, that is a tumor suppressor that controls the G1/S cell cycle checkpoint. When RB1 is mutated, pRB is inactivated and the cell cycle is not regulated.

1) What is pharmacogenomics? (497)

The study of how an individual's entire genetic makeup determines how the body responds to drugs. This takes into account many aspects of drug metabolism and how genetic traits affect these aspects.

2. What is a restriction enzyme? How does a restriction enzyme work? (323)

They are DNA cutting enzymes. They will bind to DNA and recognizes a specific nucleotide sequence, a recognition sequence, and cuts both strands which produces restriction fragments.

9. What is the purpose of the Polymerase Chain Reaction (PCR)? What are the steps of a PCR reaction? How do those steps work? (331-332)

They are a rapid method of cloning specific DNA molecules. • Denature DNA (92-95 C) • Anneal primers (45-65 C) • Extend primers (65-75); product of the first cycles double the number of DNA molecules • Repeat the first two steps • Then repeat step 3 Millions of copies of DNA can be generated

11. How can microarray analysis be used to perform genome wide association studies? (393)

They have been used to identify genes that may influence disease risk. It is done by analyzing the genomes of thousands of unrelated individuals with a particular disease. Then it is compared to those without the disease.

12. What are gene knockouts, what is their purpose and how do you create a knockout organism? (339-340)

They were designed to disrupt or eliminate a gene's function A targeting vector is constructed from DNA with a similar sequence to the gene of interest and flanking DNA Targeting vector is introduced into embryonic stem cells (ES) DNA from vector recombines with DNA around gene of interest Cells are selected by different selection agents Microinject the ES cells into a blastocyst Then test mice for absence of a copy of gene Breed mice until you have a knockout

9) What is gene targeting? (526)

Transgenic technique used to create and introduce a specifically altered gene into an organism. Often involves the induction of a specific mutation in a cloned gene that is then introduced into the gamete of a gamete involving fertilization. Organism produced is bred to produce gene knockouts

2) What is the difference between a transgenic organism and a cisgenic? (507)

Transgenic: when genes are transferred between unrelated species. Cisgenic: used to describe transfer of genes within the same species.

3. What are cancer stem cells? (309)

Tumor-forming cells in a cancer that can rise to all cell types in a particular form of cancer. They have properties of normal stem cells which are: self-renewal, and the ability to differentiate into multiple cell types.

3. What is a fusion protein? (380)

Two protein parts fused together while retaining function.

11. How can a virus contribute to cancer? (317)

Viruses can infect cells and cause cancer, most are retroviruses, known as acute transforming retroviruses. They will use reverse transcriptase to generate a DNA copy of its genome and integrate into host chromosomes as a provirus. None of these have been found in humans. However, RNA and DNA viruses contribute to human cancers. Cancers are causes through the deregulation of parts of the cell cycle or DNA repair.

4) What are adverse drug reactions and why are they such a problem? (500)

When people have serious side effects from pharmaceutical drugs. It is a problem because they are causing harm as well as the cost of them are estimates to be 136 billion annually.

4) What are the two broad approaches for delivering gene therapy and how do they differ? (519-520)

a. Ex vivo gene therapy i. Cells from a person with a condition are removed and then treated by adding normal copies of a gene or a DNA or RNA that will inhibit expression of a defective gene 1. Cells are then transplanted back into the person b. In vivo gene therapy i. Does not involve removal of a patient's cells ii. Therapeutic DNA is introduced directly into the affected cells of the body 1. Tough to do because need to ensure delivery to only intended tissues and not all tissues

6) What are the two approaches to making genetically modified plants and how do they differ? (512)

a. The biolistic method i. Physical method of introducing DNA into cells 1. Uses a device called a gene gun a. heavy metal particle coated with DNA fired at high speed b. Agrobacterium tumefaciens -mediated transformation i. Much more improved transformation rate 1. Uses a Ti plasmid a. Ti plasmid integrates a segment of its DNA known as transfer DNA (T-DNA) into the plant genome, scientists mediate transformation by removing T-DNA segment and replacing it with cloned gene they want to use.

13) What are six of the main questions that still need to be answered about gene therapy? (529)

a. What is the proper route for delivery in different kinds of disorders? b. What percentage of cells in an organ or tissue need to express a therapeutic gene to alleviate the effects of a disorder? c. What amount of a therapeutic gene product must be produced to provide lasting improvement? d. Will it be possible to use gene therapy to treat disorders involving multiple genes? e. Can expression or the timing of expression of therapeutic genes be controlled in a patient so they may be turned on or off when needed? f. Will targeted gene delivery approaches become more widely used for gene therapy trials?

11) How can mRNAs regulate translational activity? (483-485)

mRNAs are localized to discrete locations and localized translation control creates asymmetrical protein distribution in cells . Defines cellular regions with distinct functions mRNAs associate with RBPs (RNA-binding proteins) Which influence splicing, nuclear export, localization, stability, degradation, and translation

7) What are the five main drawbacks to viral gene therapy and describe them (524)

o Integration of retroviral genomes only occur if host cells are replicating their DNA; most are not dividing o Infection of large amounts of most viral vectors (adenovirus) can cause an adverse immune response o Insertion of viral genomes can activate or mutate an essential gene o AAV vectors cannot carry DNA sequences larger than 5 kb, and retroviruses larger than 10 kb o Possibility that fully infections virus could be created if inactivated vector were to recombine with another unaltered viral genome in host cells.

6) What are the many hurdles for personalized medicine? (504-506)

o Sequencing needs to be faster o Need to use electronic health records o Tests need to be reimbursed by insurance companies o Need to train doctors differently o Ethical issues such as being available to everyone

8) How does siRNAs function and how does this differ from how miRNAs function? (479-480)

siRNA: match up to mRNA exactly cause mRNA to degrade miRNA: prohibit translation of mRNA, do not match up exactly

6) How does CRISPR/Cas system work to provide adaptive immunity to viruses? (477)

• Acquisition: integration of phage DNA into CRISPR loci • crRNA biogenesis: transcription of CRISPR loci into CRISPR-derived RNAs • Interference: targeting and cleavage of phage DNA sequences complimentary to crRNAs -need cas proteins -differentiates between foreign and normal DNA

6. What are the steps in DNA cloning? (326)

• DNA is isolated and treated with a restriction enzyme • Fragments are linked to plasmids that have been cut with the same restriction enzyme, creating a collection of recombinant vectors • The recombinant vectors are transferred into E. Coli host cell. • The bacteria are plated on nutrient medium, where they form colonies, and are screened to identify those that have taken up the recombinant plasmid

4) What are the roles of small noncoding RNAs in prokaryotes? (476)

• Functions are still being regulated • Between 50-500 nucleotides • they are involved in gene regulation • respond to changes in environment • bacterial sRNAs have positive and negative regulation.

3) How do scientists perform in vitro evolution of ribozymes? (475)

• Start with a large collection of lab-synthesized RNAs of varying sequences and they identify the ones of catalytic activity. • Found RNA and DNA phosphorylation, C-C bond formation and RNA polymerase activity

10) What are some of the future developments for GM crops? (517-518)

•Research is continuing on ways to fortify stable crops with nutrients to address diet problems in poor countries -adding genes to bananas to provide resistance to panama disease and an increase in beta-carotene and iron -plants engineered to resist draught, low temperature, harsh conditions -inserting genes in precise location rather than random •More ways to protect plants from insects and disease -gene that produces an alarm signal to aphids -transfer from one resistant plant to another Will likely include animals -Atlantic salmon to receive approval soon -transgenic chickens that protect them from bird flu