K.GeneExTech - Gene Expression & Biotechnology...

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Unformatted text preview: Gene Expression & Biotechnology Molecular Genetics Genetic Expression • Replication – Precisely copying all the genetic information (DNA) – S-stage of cell cycle – Exact replicas passed to daughter cells • Gene Expression – – – – Using a specific bit of the genetic information Make a “working copy ” of the needed bit (gene) Take the working copy to the workshop (ribosome) Use the copied instructions to build a specific protein Feb 16, 2001 Nitrogen base determines type of nucleotide DNA & RNA are polymers of nucleotides One nucleotide: • Phosphate • Sugar • • • • – Deoxyribose (DNA) – Ribose (RNA) Adenine Guanine Cytosine Thymine – DNA only • Uracil • Nitrogenous base – RNA only RNA nucleotide DNA & RNA are nucleotide polymers Heyer The key to gene expression: complementary base pairing 1 Gene Expression & Biotechnology DNA Structure Complementary base pairing • Strand base sequences are Complimentary to each other • A pairs with T • C pairs with G DNA ’s complementary base sequence Replication is Semi-Conservative • Replication depends on base pairing • Old strands serve as templates • Both copies have old and new strands • Structure: What is a gene? – Short DNA sequence of nucleotides – One chromosome carries hundreds of genes • Function: Template model for DNA replication The Central Dogma of Biology – Order of nucleotides in DNA determines order of amino acids in protein – Each gene codes for a different protein Gene Expression • Copy the recipe from the master document (DNA gene) in the nucleus. • Use the copy of the recipe (mRNA) to produce the protein on ribosomes in the cytoplasm. Heyer 2 Gene Expression & Biotechnology Genes are Expressed as Proteins I.Transcription: DNA to RNA • 2 main stages 1) Transcription – DNA information copied to RNA – Occurs in the nucleus • A specific gene is “turned on ” • Making RNA strand from DNA gene • Starts at Promoter - DNA region before a gene • Ends at Terminator - DNA region at the end of a gene 2) Translation – RNA information used to construct a protein – Occurs in the cytoplasm The flow of genetic information Transcription of a gene RNA nucleotides can base pair with DNA nucleotides • RNA polymerase moves along DNA strand and builds RNA strand Fig. 7.7 Closer view of transcription II. Translation: the sequence of mRNA codons determines the sequence of amino acids in the polypeptide II. Translation • On the ribosome • tRNAs translate the sequence of 3-base nucleotide “words” (codons) into a sequence of amino acids in a polypeptide • NOTE: the mRNA is not “turned into” protein! • Codons (“ words”) are RNA nucleotide triplets • Each codon represents a specific amino acid Translation of RNA codons Heyer 3 Gene Expression & Biotechnology The Genetic Code Ribosomes: the site of translation Ribosomes are bound to ER and free in cytoplasm • • specific mRNA codons are associated with specific amino acids 64 codons , but only 20 aa’s – Many codons are redundant – Some are start/stop signals Fig. 7.12 Ribosome: Made of rRNA and protein Making lots of protein • Many copies of mRNA can be made from one gene • Many ribosomes can make protein from the same mRNA • Amplification of information allows rapid production of proteins A problem of origins: which came first? TERMINOLOGY Similar terms — often used interchangeably • Biotechnology: the manipulation of organisms to produce a product. – Fermentation, artificial breeding, pharmaceutical and nutritional supplements, and now… • Genetic engineering: the direct manipulation of an organism’s DNA. • Recombinant DNA: insertion of DNA from one source into another. • Transgenics: producing an organism with foreign DNA inserted into its genome. Heyer 4 Gene Expression & Biotechnology TOOLS & TECHNIQUES • • • • • • Restriction Digests RFLP — “genetic fingerprinting” Molecular Probes Recombinant DNA Gene Cloning Transgenics RESTRICTION ENZYMES RESTRICTION ENZYMES • Bacteria produce special enzymes to chop up viral DNA. • Biotechnologist use these “restriction enzymes” to cut DNA in specific places (restriction sites). • Many restriction enzymes cut the DNA polymer in a staggered pattern that produce “sticky” single-stranded ends to the DNA fragments. RESTRICTION FRAGMENT ANALYSIS DNA Technology as a diagnostic tool • Everyone’s DNA is unique • Closer the relationship the more similar the DNA • Restriction Fragment Length Polymorphisms – RFLPs – “Ruff-lips” RESTRICTION DIGEST Æ RESTRICTION FRAGMENTS RESTRICTION FRAGMENT LENGTH POLYMORPHISM • Electrophoresis of fragments Heyer 5 Gene Expression & Biotechnology USES OF RESTRICTION FRAGMENT ANALYSIS USES OF RESTRICTION FRAGMENT ANALYSIS • Missing persons • Criminology USES OF RESTRICTION FRAGMENT ANALYSIS USES OF RESTRICTION FRAGMENT ANALYSIS • Medicine – Inborn errors of • Criminology • Missing persons metabolism • Carriers • Prenatal testing • Paternity – Pro-virus DNA TRANSGENICS RECOMBINANT DNA TECHNOLOGY • Set of techniques for combining genes – – – – In a test tube Different sources of DNA Same species Different species • Transferring genes – Into cells – Where they can be replicated The bad news is they’re all dark meat... Heyer 6 Gene Expression & Biotechnology GENES FROM ONE CELL CAN BE INSERTED INTO ANOTHER CELL “Genetic Engineering” Cut and Paste: ¸Restriction digest ¸Anneal sticky ends ¸DNA ligase ¸Voila! ¯ PLASMIDS AND BACTERIAL SEX • Most bacteria can exchange some genetic material (DNA) by means of plasmids • Small circular DNA molecule Recombinant DNA PLASMIDS CAN BE USED TO CUSTOMIZE BACTERIA PLASMIDS CAN BE USED TO CUSTOMIZE BACTERIA Transgenics: transferring DNA from one organism into another PHARMACEUTICAL BIOTECHNOLOGY Transgenic Bacteria • Protein production – Insulin – Growth hormone – Erythropoietin • Vaccines – Hepatitis B TRANSFORMING EUKARYOTES WITH RECOMBINANT DNA Transgenic Plants, Fungi, & Animals • Agrobacterium Ti plasmid – Natural pathogen of broad-leaf plants – Ti plasmid inserts into plant chromosome • Microparticle accelerator “gene gun” – DNA fragments coated onto gold or tungsten particles – Particle blasted by gas pressure burst through tissue, leaving trail of DNA residue in cells • Microfiber “gene whiskers” – DNA fragments coated onto microscopic needles – Needles and cells suspended and shaken; impaled cells take up DNA from needles • Electroporation – Rapid electrical pulses induce cellular pores to open allowing small fragments of DNA to enter Heyer 7 Gene Expression & Biotechnology AGRICULTURAL BIOTECHNOLOGY AGRICULTURAL BIOTECHNOLOGY soybean Top U.S. GMO* Crops corn PHARMACEUTICAL AGRICULTURAL BIOTECHNOLOGY “Pharming” * Genetically Modified Organism Some proposed benefits of GMO crops: • Intrinsic pesticide (bacterial insect pathogens) • Herbiside resistance • Enhanced productivity • Enhanced shelf life ( FlaverSaver® tomatoes) • Frost resistance cotton TRANSGENIC RESEARCH • Mice that are susceptible to human cancers or viruses – Test therapies GENE MICROINJECTION AND ANIMAL CLONING • Microinjection is labor intensive • Cloning embryos is slow, expensive, and produces few recombinant subjects • Thus, use only for gene products with huge potential profits to justify the expense and effort. Heyer BIOTECHNOLOGY RISKS • Risks – Health / Culture – Environment – Corporate patent monopolies 8 ...
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