Husop Genomics 6 Biotechnology

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Biotechnology has been defined as

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the use of living things or parts of things to create or modify drugs and other substances, to modify food crops or other macroscopic organisms; or to adapt microorganisms to agricultural, medical, or other purposes.

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Bioechnologies include: (4)

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1) fermentation 2) baking (saccharyomyces cerevisiae) 3) selective breeding 4) antibiotics

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Nucleic acid therapeutics biotechnology includes:

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antisense nucleotides, siRNA, and DNA vaccines

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Age of Modern Medical Biotechnology began:

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1) 1982 – Human recombinant insulin (Humulin, E. Lilly) approved FDA 2) 1986 – Muromonab-CD3 (Orthoclone OKT3, Janssen), first monoclonal antibody approved by the FDA

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emphasis of pharmaceutica biotechnology is on (3)

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1) large molecule therapeutics 2) correcting gene mutation 3) replacement of damaged cells

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•During 2009, the FDA and European Union regulators approved ___ new recombinant proteins (valued at ____).

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20, $99 billion

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The USDA regulates _______(3)

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plants, plant pests, animal vaccines

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The EPA regulates _____(4)

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microbial/plant pesticides, microorganisms & animals that produce toxic compounds

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The FDA regulates____(4)

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food, human and animal drugs, human vaccines, transgenic animals

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the lead regulatory agency in the United States is the

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FDA

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CBER is:____They regulate_____

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–CBER: Center for Biologics Evaluation and Research regulates biotech products and other “biologics”

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CDER is:_____ They regulate_____

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–CDER: Center for Drug Evaluation and Research regulates traditional small molecule therapeutic agents.

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FDA function include (3)

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1) clinical development process 2) efficacy and safety 3) standards of manufacture, labeling, and distribution

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Application to the FDA for biotherapeutics is called ___ _____ _____

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Biologics License Application

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biopharmaceutical is concerned with (2)

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1) characterization of product 2) Contaminants not just restricted to small molecules

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Transformation of Bacteria was first performe using 2 strains of ______

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Diplococcus pneumoniae

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___ virulent strain that grew as _____ looking colonies on agar

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S, smooth

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_____ nonvirulent strain that produced ______ looking colonies on agar

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R, rough

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in the Griffth Experiment, heated S cells mixed with R cells resulted in _____ mice

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dead

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In the Griffth experiment, S cells contain a _____ _____ that can be transferred between cells

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Virulence Factor

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of the purified enzymes, only ______ treated smooth strains

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DNAse

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Chargaff’s Nucleotide Complementarity states (2)

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1.# of purines (A,G) = # of pyrimidines (C,T) 2.# of adenine = # of thymidine bases; and the # of guanine = # of cytodine bases.

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_____ _____ _____allowed Watson & Crick to propose the double helix model that explained these identities.

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Chargaff’s Nucleotide Complementarity

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in order to manipulate DNA a series of _____ _____ enzymes are required

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highly purified

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DNA manipulation enzymes (5)

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1) DNA nucleases (DNase) 2)Restriction nucleases
3) DNA ligase
4) DNA polymerase
5) Reverse transcriptase

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_____ is any enyzme that is able to cleave the phosphodiester bond in DNA.

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Deoxyribonuclease (DNase)

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Deoxyribonuclease (DNase) comes in a Variety of specificities w/ some cleaving only _____ _____ DNA

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single stranded

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DNA exonuclease – cleaves the _____ ___ ____ from the end of the DNA strand.

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terminal (3’) nucleotide

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DNA ______ – cleaves within the DNA strand.

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endonuclease

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Mammalian _______ DNase has low specificity.

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pancreatic

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_____ ______are bacterial derived DNA endonucleases that possess unique specificities

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Restriction Nucleases

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It is believed that restriction nucleases evolved to protect bacteria from the insertion of _____ ___ into the chromosomal DNA

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foreign DNA

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It was found that Restriction Nucleases could cleave DNA in a _____ number of _____ sequences.

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limited, specific

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Restriction nucleases recognize _____ in DNA seuences

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palindromes

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Which of the following have overlap cutting? Arthrobacter luteus, Haemophilus aegyptius, Bacilus amyloliquefaciens H, Escherichia coli RY14, Haemophilus influenzae Rd?

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The last 3

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T/F the restriction enzyme is named from the source organism it cleaves.

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true (ex. Alu for Arthrobacter luteus)

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_____ is required in mammalian cells to ligate the Okazaki fragments during DNA replication

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DNA Ligase

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Selected ligases are able to work on _____ ended DNA fragments, but _____ fragments are more generally ligated.

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blunt, overlapping

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Ligase forms a _______ bond, leading to a covalent bond, healing DNA cleavage points

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phosphodiester

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____ ______ is the enzyme used to form a DNA strand using a template strand of DNA.

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DNA Polymerase

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DNA Polymerase Always extends DNA strand in the _____ direction using a 3’-OH on a pre-existing primer piece of DNA.

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5’ → 3’

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•DNA polymerase requires a _____

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primer

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At least ___ types of eukaryotic DNA polymerase identified.

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15

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the primer strand of the polymerase reaction has a ____ for binding new bases

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hydroxyl group at carbon 3

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Prokaryotic DNA Polymerase 1 possesses three enzymatic activities:

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1) 5’ → 3’ polymerase activity 2) 3’ → 5’ exonuclease activity (proofreading)
3) 5’ → 3’ exonuclease activity (DNA repair)

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the Klenow fragment is cleaved from polymerase 1 by ______

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subtilicin

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Which enzymatic activity is the klenow fragment unable to perform that polymerase 1 can?

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the 5’ → 3’ exonuclease activity

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In 1970, _____ _____ was isolated from RNA viruses that caused neoplasms in animals.

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Reverse Transcriptase

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the RNA viruses that caused neoplasms in animals was ____ _____ _____

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Rous sarcoma virus

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Reverse Transcriptase is a ____ _____ DNA polymerase, meaning it uses _____ as a template for DNA synthesis.

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RNA dependent, RNA

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The enzyme from ____ _____ _____ _____widely used to convert RNA into DNA for manipulation

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Moloney-murine leukemia virus (MMLV)

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Using a series of highly purified enzymes, one can: (4)

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1) Convert RNA into complementary DNA
2) Trim RNA to a particular size 3) Attach it to another DNA molecule
4) Make many copies of it in vitro