09_23_tree.of.life.jpgWe’re all related!There are three main domains of life, bacteria, archaea, and eukaryotes,all of which derived from a common ancestor.01_02_DNA to RNA.jpgAll cells, whether prokaryotic oreukaryotic,use this system:We’re all related!All three domains of life do the central dogma the same way. Thismeans that you can put a gene from one organism into another organism.Pax6 is a mouse gene that is responsible for eye formation. A mousethat lacks functional Pax6 is eyeless.08_25_eye.on.leg.jpgMetazoan genes can be readily interchanged!If you express the Pax6 mouse gene in fruitflies in the leg region of thefly, the fly will grow an eye on its leg. Thus mouse Pax6 works in flies.You can express genes incells that are fusions of the geneyou are interested in to another gene. We will talk about how andwhy one does this in the cellbiology lecture.If you purify your protein ofinterest from cells, you can askwhich other proteins are associatedwith your protein when you purified it. This tells you which proteins bind to your protein ofinterest in the cell, which may help you determine the function of yourprotein of interest.10_32_expressionvector.jpgYou can use this scheme to introduceand express any gene from oneorganism in anyother organism.01_11_E. Coli.jpgAre there problems with expressing eukaryotic genesin bacteria?07_11_pores.nuc.envl.jpgBacteria don’t have a nucleus, but remember that eukaryotesdo, so their mRNAs are made in the nucleus and then exportedto the cytoplasm.07_20_Pro_v_Eucar.jpgEukaryotes do splicingbut bacteria don’t, so ifyou want to put a eukaryotic gene intoa bacterium, you have to use an altered formof the gene that hasits introns cut out already. 07_13_Eucar_v_bact.jpgEukaryotes do splicing but bacteria don’t, so if you want to put aeukaryotic gene into a bacterium, you have to use an altered formof the gene that has its introns cut out already.This is a picture of protein production in Saccharomyces kluyveri -- to make large quantities of your protein, you have to grow huge vats of yeast.Since there are problems expressing eukaryotic genes in bacteria,some people express them in yeast.Most extracellular proteins of metazoa are glycosylated(i.e., have sugars covalently attached). Yeast don’t do this the same wayas animals.To solve this problem, some people make human proteins in CHO (Chinese hamster ovary cells) instead of in yeast or bacteria.15_11_ER.jpgThe organelle called the Rough Endoplasmic Reticulum is the organellewhere proteins that get secreted are glycosylated and prepared for secretion.15_29_Secretory_vesicl.jpgThis is a picture of an aggregate of proteins getting secreted from a cell.CHO cellsIf you want to produceyour protein in CHOcells, you have to growvats of CHO cells.Why mightyou want tomake largequantities ofa protein?Oneexample isthat youmight wantto make lotsof HGH(humangrowthhormone). These girls are sisters. The girl on the left lacked growth hormone. In this picture she was 18cm shorter than her sister, despite being one and a half years older.HGH can be given to childrenwho don’t make theirown HGH so that thosechildren can growtaller than theywould if they weren’t treated.HGH is made by the pituitary.Currently we don’t have thetechnology to treat this girlby putting a normal copy of the gene for HGH into herpituitary cells, although thatwould theoretically also workto treat her.This is the 3D structure of Human growth hormoneSomeathletes takeHGH to tryto growlarger.Reverse the Aging Process and Be Young Again with (HGH) Human Growth Hormone"Discover the Natural Fountain of Youth, scientists have discovered the hormone that controls aging and it is accessible to you."An ad:Reverse the Aging Process and Be Young Again with (HGH) Human Growth Hormone"Discover the Natural Fountain of Youth, scientists have discovered the hormone that controls aging and it is accessible to you."An ad:Caution: The most common side e!ect with this supplement is shortnessof breath and a tingling sensation after administration. This is harmless and will pass within a few minutes. This product is not recommendedfor anyone with respiratory problems or individuals diagnosed withasthma.!!!!!!18_29_myostatin_gene.jpgThis is what a bull would look like if it were givenlots of HGH.Another protein produced by recombinant DNA technology is Epo.This is the 3D structure of Erythropoietin (EPO).Low oxygentension(hypoxia)kidneyBonemarrowEpoerythropoiesisredblood cellsEpo is made in your kidney whenever Oxygen is low. Epo is sensedby cells in your bone marrow, and induces you to make more RBCs(red blood cells).This shows how RBCs (erythrocytes) are only one of the many kindsof blood cells in your blood. Most of the blood cells at the top ofthe chart are different kinds of white blood cells. Epo only stimulates the step on the lower right, in which more RBCs are made.Some athletes take Epo to try to increase their oxygen levels sothat they can run or bike for longer periods of time. However toomany RBCs makes your blood thick, and causes clotting that islethal.Epo is being used currently to treat patients with anemia (who donot make enough RBCs) or patients who can’t make their own Epo, often because of kidney malfunction.(marketed asNeupogen)G-CSF is another protein thatis being made by recombinantDNA technology. G-CSF stimulates production of white blood cells. Peoplewho undergo chemotherapyto treat cancer are often given G-CSF that has beenmade using recombinantDNA technology, so that they can have their immunesystems restored by increasing their numbersof white blood cells backto normal levels.Drugs used to be smallmolecules, but now many“drugs” being produced arenormal human proteins, likeinsulin, Epo, and G-CSF.Figure 6.7b The Biology of Cancer (© Garland Science 2007)Scientists can also make lots of a protein using recombinantDNA technology so as to study the protein in lab and, forinstance, find small molecule drugs that inhibit the function ofthat protein.Figure 16.11a The Biology of Cancer (© Garland Science 2007)This is a picture of a small molecule drug fitting into a pocket in the 3Dstructure of the protein. This drug inhibits the function of this protein.Figure 16.11b The Biology of Cancer (© Garland Science 2007)This is a picture of a small molecule from green tea fitting into a pocket in the 3D structure of the protein. There are tricks one can use to get DNA into plant cells, just like thereare
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