Chapter 19 Biology 1 Pituitary Dwarfism a Pituitary gland structure at base of the brain that produces a protein that stimulated growth b Protein that stimulated growth called Human Growth Hormone c GH1 gene codes for HGH dwarfism might be caused by defect in this gene d ppl w defective GH1 gene have pituitary dwarfism type 1 the disease is an autosomal trait 2 Early Efforts to Treat Disease a Physicians began treating dwarfism with growth hormones from cadavers b the treatment was at first successful as long as it came from humans c the growth hormones from cadavers contained a prion protein thus causing degenerative brain diseases in ppl who got the hormone in teens and younger 3 Engineering a safe supply a 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 hormones b But how would they do this 4 Reverse Transcriptase to Produce cDNAs a Reverse Transcriptase Enzyme catalyzes synthesis of DNA from RNA thus producing a complementary DNA or cDNA b Researchers add a primer to single stranded cDNAs and use DNA polymerase to synthesize the second strand c They isolated mRNAs from pituitary gland cells and used reverse transcriptase to made cDNAs through transcription d Now they must make many identical copies of cDNAs 5 Using Plasmids in Cloning a You can clone a gene by inserting it into a small circular DNA molecule called a plasmid b If the plasmid could be inserted into a bacterial cell the plasmid would be replicated and passed to daughter cells c Allow the resulting recombinant bacterium to grow in a nutrient broth billions of copies of original cell w identical plasmid would result When plasmid used like this it is called a cloning vector d Now how to insert gene into a plasmid 6 Cutting and Pasting DNA a Researchers us restriction endonucleases bacterial enzyme that cuts DNA molecules at specific base sequences In bacteria these enzymes defend against invading viruses or infection b Most restiction endonuclease enzymes cut DNA at sites w palindromes 7 Inserting cDNAs into plasmids a Attach palindromic seq to ends of each cDNA b Cut recognition sites w each plasmid and end of each cDNA with restriction endonuclease called EcoRI c 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 pairing d Seal recombinant pieces of DNA together using DNA ligase 8 Transformation a Cells that take up DNA from environment and incorporate it into its genome are said to undergo transformation b chemical treatment electrical shock used to increase permeability of cell s plasma membrane which is essential for trans formation to occur 9 Producing a cDNA library a collection of sequences of cDNAs from certain cell type or tissue the library is called a cDNA library b Fragments of DNA that rep entire genome of organism is a genomic library c allow researchers to store information from cell type that is accessible 10 Screening a DNA library Figure 19 6 a Probe marked molecule that binds to molecule being searched for single stranded fragment that binds to target seq through complementary base pairing b Researchers used genetic code to predict DNA seq of GH1 thus infer mRNA codon and DNA seq that coded for each Amino Acid 11 Mass Producing Growth Hormone a Researchers used recombinant DNA techniques to transfer growth hormone cDNA to new plasmid the plasmid in question had promoter recognized by E coli s holoenzyme b recombinant plasmids then introduced to E coli cells reproduced and passed down the DNA c Thus producing safe human growth hormones Amplification of Fossil DNA 1 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 PCR a 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 reaction b If target DNA made single stranded the primers will bond to complementary seq DNA polymerase can then extend each strand in 5 3 direction c Denaturation primer bonding extension all constitute a single PCR cycle 3 Why PCR is so Valuable a 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 mixed c PCR can be used to determine disease in embryos and can even be used in forensics Dideoxy DNA Sequencing 1 Useful to determine a gene s base sequence once a gene has been cloned by PCR 2 Logic of Dideoxy Sequencing Sanger a First insight use dideoxyribonucleoside triphosphates ddNTPs along with deoxyribonucleoside triphosphates dNTPs b Unlike dNTPs ddNTPs lack a hydroxyl group on number 3 carbon c Four types of ddNTPs used in dideoxy sequencing adenine ddATP thymine ddTTP cytosine ddCTP guanine ddGTP 3 Realization of Sanger a if ddNTP was added to growing DNA strand synthesis would be terminated b 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 added 4 Next Generation Sequencing a 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 strand b This doesnt require any ddNTPs and are sometimes called pyrosequencing or sequencing by synthesis Finding Genes by Mapping 1 Signs of Huntington s Disease a Victims of it show symptoms bet ages of 35 to 45 b Individuals appear clumsy with tics and abnormal movements in the beginning c As disease progresses uncontrollable movements become more pronounced d personality and intelligence also compromised disease often misdiagnosed as schizophrenia e Illness may progress for 10 20 years and is eventually fatal f Trait for Huntington s due to single autosomal dominant allele 2 Using Mapping Markers a Biologists use a physical map of the genome which records the position of a gene in numbers of base pairs along a chromosome b Genetic map contains genetic markers easily identified genes or sequences that have known locations provides a marker on chromosome known relative to other markers c Genetic marker must be polymorphic
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