BIO 344- Quiz12GENOMESSome relatively simple Eukaryotes such as insects and amphibians have much larger genomes than do humans. Explain how this is possible.What are some purposes of studying entire genomes and sequences?1.Large scale examination of gene expressionTRANSGENICSTransgenic miceTransgenic plantsDirect DNA transferBIO 344- Quiz12GENOMESGenomics is the study of the structure and functions of whole genomes. Sequencing of genomes has allowed the scientific community to create the so-called “tree of life”. 1. What are the three main “kingdoms” in this tree?Archea, prokarya, eukarya2.Eukaryotes have 3 separate genomes within an individual cell. Name them.Nuclear, mitochondrial, chloroplast (plant cells)3.Describe the chloroplast genome in terms of copy number, structure, and number/function of genes it contains.Circular, multiple copies/chloroplast (20-100)160,000 bp; ~125 genes (for photosynthetic and genetic functions)Some relatively simple Eukaryotes such as insects and amphibians have much larger genomes than do humans. Explain how this is possible.Repeated DNATransposons-selfish DNAs that copy themselves and move to other parts of the genomeFrequency and size of intronsGenetic redundancy or gene familiesOther intergenic DNAWhat are some purposes of studying entire genomes and sequences?1.Large scale examination of gene expression2.Predict function of poorly understood genes. If you know the sequence you can predict toprotein which can help predict the function of the organism. The example that we saw in class was Lyme disease.3.Identify drug targets. Use sequence data to find unique genes that can be targeted to kill the virus or bacteria4.Identify gene linked to a disease through comparative population studiesTRANSGENICSTransgenic bacteria, plants, and animals are organisms that contain genes from sources foreign to their own origin. There are several reasons that a scientist might want to make atransgenic organism. Can you name a few of these?1. Transgenic bacteria can act as protein producing factories2. Curing disease. Gene therapy allows you to replace a bad gene with a good copy.3. Improve agriculture- An example would be to produce a pesticide-resistant crop4. Study gene function and regulation. Knockout organism can show altered phenotypesTransgenic miceMaking a transgenic or knockout mouse can be a difficult task. Two major steps must occur. First, you must get the interrupted gene into a germ line cell. Second, you have to get the interrupted gene (via the germ line cell) into a mouse, ultimately making an adult mouse homozygous for the interrupted gene.1. Describe the characteristics of the knockout (KO) plasmid that must be used carry the gene of interest. tk1 and tk2 genes- genes that cause cells to be sensitive to ganglocyclovir. Because these genes flank the region where our truncated gene in placed, they will only go into the genome by non-homolougous recombination (NHR). This means that cells that uptake the KO plasmid by NHR will be killed.NeoR- Neomycin resistance. This gene allows selection by resistance to neomycin resistance. Because this gene is placed inside of the coding region of the gene that you are knocking out, it is actually what causes the expressed protein to be truncated. 2. What type of cells are originally transformed with the KO plasmid?Embryonic stem cells3.How are cells that contain the interrupted gene selected?Ganglocyclovir will kill cells that took up the gene by HNR. Neomycin will kill any other cells that did not take up the plasmid. This leaves only cells that took up the truncated gene by homologous recombination4.What then happens to these cells with the interrupted gene?These cells are injected into a blastocyst and the blastocyst is then implanted into a female mouse5.Finally, how do you get mice that are homozygotes (i.e. true knockouts)?See figures 5.39, 5.40 for crossesTransgenic plantsIn order to make a transgenic plant one can use direct gene transfer, which is very inefficient, or Argobacterium-mediated transfer. Answer the following questions about Agrobacterium-mediated transfer.1.What is A. tumefaciens and what special feature does it have that has allowed it to become useful for genetic engineering?It is a bacteria that infects plants (tobacco). It has an extrachromosomal element that leaves the bacteria and enter the plant cell during infection. One can engineer genes of interest onto this DNA and these genes will be transferred to the plant cell (and ultimately integrated into the genome) during infection 2. Describe the Tumor inducing (Ti) plasmid.200 Kb, but only 30 Kb (called T-DNA is transferred). It has genes for opine metabolism and genes for virulence3.Make sure that you know the functions of the virulence (vir) genes. (See page 4 of the notes on this lecture).These genes function to get the T-DNA into the plant cell and integrated into the genome.4.Why must a binary vector (co-transformation) scheme be used to make a transgenic plant.The Ti plasmid is too large. One plasmid contains the virulence genes alone. The other contains the T-DNA with your gene of interest (along with a selectable marker).5.Describe the steps involved in making a transgenic plant (for example a tobacco plant with a pesticide resistant gene) using the leaf disc transformation method with Agrobacterium.Cut leaf, sterilize, incubate with Agrobacterium with both plasmidsculturekill agrobacteriuminduce shoot and root formation with hormonesplant in soil after a few weeksget seeds and check for presence of geneDirect DNA transferWhat are the 4 methods of direct DNA transfer.1. Chemical2. Microinjection3. Electroporation4. Biolistics1.Chemically-induced gene transformation is the oldest and still most reliable for animal cells because they have no cell walls. What are the two chemical methods that work well that we discussed in class? Which of these work better?1. Lipid fusion with vescicles2. bind DNA to polycationsthis will neutralize the negative charge on the DNA and allow it to be endocytosedboth of these used together work the best!!2.Electroporation is also a method of direct gene transfer. It too is only effective on animal cells or plant protoplasts (i.e. cells without walls). How does this process work and what are its drawbacks?Hit cells + DNA with electricity to disrupt cell membranemakes hole in membraneDNA entersMain drawback is that voltage will