Chapter 19 Biology1. Pituitary Dwarfisma. Pituitary gland-structure at base of the brain-that produces a protein that stimulated growthb. Protein that stimulated growth called Human Growth Hormonec. GH1 gene codes for HGH-dwarfism might be caused by defect in this gened. ppl w. defective GH1 gene have pituitary dwarfism, type 1-the disease is an autosomal trait2. Early Efforts to Treat Diseasea. Physicians began treating dwarfism with growth hormones from cadaversb. the treatment was at first successful-as long as it came from humansc. the growth hormones from cadavers contained a prion protein-thus causing degenerative brain diseases in ppl who got the hormone in teens and younger3. Engineering a safe supplya. physicians had to find the GH1 gene and insert it into E. Choli-it would then produce large quantities of recombitant cells-which would produce useful growth hormonesb. But how would they do this?4. Reverse Transcriptase to Produce cDNAsa. Reverse Transcriptase Enzyme-catalyzes synthesis of DNA from RNA-thus producing a complementary DNA or cDNAb. Researchers add a primer to single stranded cDNAs-and use DNA polymerase to synthesize the second strandc. They isolated mRNAs from pituitary gland cells-and used reverse transcriptase to made cDNAs through transcriptiond. Now they must make many identical copies of cDNAs5. Using Plasmids in Cloninga. You can clone a gene-by inserting it into a small circular DNA molecule called a plasmidb. If the plasmid could be inserted into a bacterial cell-the plasmid would be replicated and passed to daughter cellsc. Allow the resulting recombinant bacterium to grow in a nutrient broth-billions of copies of original cell w. identical plasmid would resultWhen plasmid used like this it is called a cloning vectord. Now how to insert gene into a plasmid...6. Cutting and Pasting DNAa. Researchers us restriction endonucleases -bacterial enzyme that cuts DNA molecules at specific base sequences-In bacteria these enzymes defend against invading viruses or infectionb. Most restiction endonuclease enzymes cut DNA at sites w. palindromes7. Inserting cDNAs into plasmidsa. Attach palindromic seq. to ends of each cDNAb. Cut recognition sites w. each plasmid and end of each cDNA-with restriction endonuclease called EcoRIc. Resulting DNA fragments from cuts have sticky ends-allow one DNA fragment to pair up and form H-bonds w. other fragment-bind to each other through complementary base pairingd. Seal recombinant pieces of DNA together using DNA ligase8. Transformationa. Cells that take up DNA from environment and incorporate it into its genome-are said to undergo transformationb. chemical treatment, electrical shock used-to increase permeability of cell's plasma membrane-which is essential for trans formation to occur9. Producing a cDNA librarya. collection of sequences of cDNAs from certain cell type or tissue-the library is called a cDNA libraryb. Fragments of DNA that rep. entire genome of organism is a genomic libraryc. allow researchers to store information from cell type that is accessible 10. Screening a DNA library Figure 19.6a. Probe-marked molecule that binds to molecule being searched for-single-stranded fragment that binds to target seq. -through complementary base pairingb. Researchers used genetic code to predict DNA seq. of GH1-thus infer mRNA codon and DNA seq. that coded for each Amino Acid11. Mass-Producing Growth Hormonea. Researchers used recombinant DNA techniques-to transfer growth hormone cDNA to new plasmid-the plasmid in question had promoter recognized by E coli's holoenzymeb. recombinant plasmids then introduced to E. coli cells-reproduced and passed down the DNAc. Thus producing safe human growth hormonesAmplification of Fossil DNA1. The Polymerase Chain Reaction (PCR)a. In vitro DNA synthesis reaction-DNA polymerase replicates section of DNA continuously to amplify number of copies of seq.2. Requirements of PCRa. Sequence information required-need to start by synthesizing single-stranded DNA-that matches seq. on either side of gene of interest-these short segment strands serve as primers for the synthesis reactionb. If target DNA made single stranded the primers will bond to complementary seq.-DNA polymerase can then extend each strand in 5-3 directionc. Denaturation, primer bonding, extension all constitute a single PCR cycle3. Why PCR is so Valuablea. DNA taken from neanderthals-to compare same DNA segment to humans'b. Used PCR to produce a lot of the DNA to study it-found that the genes bet. humans and them are different in that segment-so modern humans and neanderthals never mixedc. PCR can be used to determine disease in embryos-and can even be used in forensicsDideoxy DNA Sequencing1. Useful to determine a gene's base sequence-once a gene has been cloned by PCR2. Logic of Dideoxy Sequencing Sangera. First insight: use dideoxyribonucleoside triphosphates (ddNTPs)-along with deoxyribonucleoside triphosphates (dNTPs)b. Unlike dNTPs, ddNTPs lack a hydroxyl group on number 3 carbonc. Four types of ddNTPs used in dideoxy sequencing-adenine (ddATP), thymine (ddTTP), cytosine (ddCTP), guanine (ddGTP)3. Realization of Sangera. if ddNTP was added to growing DNA strand-synthesis would be terminatedb. This is bec. no hydroxyl group was longer available on 3 carbon-to link to the 5 carbon on an incoming dNTP monomer-so as a result DNA polymerization stops once ddNTP added4. Next Generation Sequencinga. Newer methods of sequencing individuals-based on detecting the pyrophosphate molecule-that is released after a DNA polymerase adds a dNTP to a growing DNA strandb. This doesnt require any ddNTPs-and are sometimes called pyrosequencing or sequencing-by-synthesisFinding Genes by Mapping1. Signs of Huntington's Diseasea. Victims of it show symptoms bet. ages of 35 to 45b. Individuals appear clumsy with tics and abnormal movements in the beginningc. As disease progresses uncontrollable movements become more pronouncedd. personality and intelligence also compromised-disease often misdiagnosed as schizophreniae. Illness may progress for 10-20 years and is eventually fatalf. Trait for Huntington's due to single autosomal dominant allele2. Using Mapping Markersa. Biologists use a physical map of the genome-which records the position of a gene in numbers of base pairs along a chromosomeb. Genetic map contains genetic markers-easily identified genes or sequences that have known locations-provides a marker on chromosome known relative to
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