ࡱ>  df[\]^_`abc 1bjbj .uuaP5GG111$222PS2'3L2ds5s5555DDDccccccc$=fhZdE12E/,C2E2EdGG55JKdZZZ2EG$5185cZ2EcZZ:_,k)$1"`5mPxd2K_ cad0d_R9iK 9i"`"`9i10bD>AD,ZmD$DDDDddXDDDd2E2E2E2E9iDDDDDDDDD : Molecular Genetics T.A. Blakelock High School Grade 12 University Preparation Biology    Student:______________________ Date:___________________ Teacher:______________________ Room:__________________ Period:_________ Unit 3 MOLECULAR GENETICS INTRODUCTION Pg 198 ARE YOU READY? Part I DNA, RNA and PROTEIN SYNTHESIS A] HISTORY OF DNA Figure 11, pg 215 NOTABLE SCIENTISTS: ____________________________ : Discovery that the Nucleus is the source of inheritance ______________________ and __________________ : Showed that DNA actually controls heredity ____________________________ : Crystallography Structure of DNA ____________________ and _____________________ : Mathematical modeling of DNA Structure B] DNA STRUCTURE 1] Parts of a nucleotide ____________________ _________________ ______________________  2] arrangement of nucleotides a] the __________ bonds to the last _______________ at carbon _____ and so the sugar end of DNA is called the _______ end b] the ______________ bonds to the next ___________ at carbon ____ and so the phosphate end of DNA is called the _____ end c] the two strands are arranged as shown below   Sections of the DNA with more ________________ bonds, are more stable. [_____ H-bonds vs. ______ H-bonds] 4. DNA shape the stands are arranged in a ________________________________ Each Human Cell contains approximately ___________________________ base pairs. These are arranged into roughly ______________________ different genes. In addition, up to __________________ does not code for any protein. In total, DNA from 1 cell could be _________________ long. ___________________ is very stable but ______________ is very easily __________________. C. DNA REPLICATION Introduction a. Before a cell _________________ it must first copy all its DNA b. It must do this while keeping the amount of ______________ to a minimum. c. Also, the DNA will have to be __________________ to allow access to the bases. [The bold numbers following the bold terms refer to the DNA picture of replication on the next page] 1. Separating the strands a] DNA helicase [1] breaks___________ between the strands at an origin of replication b] DNA topoisomerase [2] - releases _____________ by unwinding c] replication fork [3] - an area where the two strands are _________________ d] ssBs [4] - single-stranded binding proteins keep strands from_______________ 2. Building complementary strands a] starting -DNA primase [5] - creates a _____________ strand of RNA called an RNA-primer[6] at the exposed______end of the replication fork b] building -the enzyme DNA polymerase III [7]- adds complimentary bases that are _________________ moving along the old DNA in the ________ direction - the energy released by removing ______________ allows the bonding - the strand that is copied continuously __________ strand is called the leading strand [8] c] other strand DNA polymerase III only moves__________________ - so the lagging strand [9] has to be built_________________________ - these ____________________ pieces are called Okazaki fragments [10] d] finishing DNA polymerase I [11] removes the __________________________ & replaces them with the appropriate DNA bases DNA ligase [12] attaches __________________ fragments by bonding sugar and ___________________________ 3. Picture see next page  ___ DNA Helicase - ___ DNA Topoisomerase - ___ DNA Ligase - ___ DNA Polymerase I - ___ DNA Polymerase III - ___ Lagging strand - ___ Leading strand - ___ Okazaki fragments - ___ DNA Primase - ___ Replication Fork - ___ RNA primer ___ ssBs - D. RNA 5. RNA ribonucleic acid RNA Structure a] _________ sugar + phosphate + nitrogenous base but ____________ replaces Thymine b] __________ stranded molecule c] much _______________ than DNA 5 PsugarPsugarPsugarPsugarPsugarPsugar 3 l l l l l l A C G C U G RNA features unlike DNA, RNA can _______________ in the cytoplasm if properly prepared Types of RNA 1. mRNA messenger RNA - varies in _______________, but always much _______________ than DNA - carries the ______________ message for protein building from DNA to the _____________ 2. tRNA transfer RNA - very short, _________________ bases only - acts to transport the ________________________ to the mRNA at the ribosomes 3. rRNA ribosomal RNA - varies in length, but ______________ than mRNA - binds the large and small __________________ to form a functional _________________ E. TRANSCRIPTION the process of making mRNA 1. Initiation a. ______________________ binds to a specific region of the DNA that is _______ rich and so easy to separate b. the DNA is _________________ and the two strands are _________________ 2. Elongation a. only the ___________________ DNA strand is used ( _________________ strand b. the unused strand [5 to 3] is called the_________________ strand c. free floating nucleotides A, U, C & G are __________________ to their complimentary bases by ______________________ d. RNA polymerase moves along the DNA _________________& _______________ as it goes while the DNA winds back up behind it e. the ____________ hangs free off the side of the DNA strand attached only at a few base pairs  3. Termination a. RNA polymerase encounters a sequence of bases at the end of the gene called a ___________________________________ b. this results in RNA polymerase and the _______________ being released from the DNA 4. Post-transcriptional modifications a. a ______________ is added to the mRNA to protect it from cytoplasmic enzymes b. a ________________ of 200 or so adenines is added by poly-A-polymerase to the 3 end c. this results in the _____________________ being ready for release from the nucleus d. in eukaryotes__________________ remove all the non-coding regions [_____________] and ____________ the coding regions [_________________] to form the ____________________________________     F. PROTEIN SYNTHESIS [or DNA translation] converting the RNA message into protein 1. The code mRNA a. the message on mRNA is written in 3-letter ____________ called ______________ b. there are ________ or ____________ possible codons c. pg 248 shows the codons you will have to learn the ______________ but NOT memorize this with the exception of the __________ and the ___________ codons d. notice that UUU & UUC both code for _____ and CUU, CUC, CUA & CUG all code for_____ this indicates that the __________ letter is the least important in the code e. only two amino acids have a unique code UGG ( _______ and AUG ( _________ f. all proteins must start with ________ as AUG is the __________code; this may be removed later g. there is__________ amino acid that is coded by UAA, UAG or UGA and so the protein breaks here and these are called _______________codes 2. The ribosomes [rRNA] a. the rRNA holds together the ____________ subunit [60s] and the___________ subunit [40s] to form the active ribosome [___________] b. the _________ [CAP region] adheres to the active ribosome c. the ribosome will move the _________ through it starting at the _________ end and going toward the _________ end 3. The tRNA a. each tRNA has a_________________________ structure [pg 251] b. at the base there is an _______________ which is the compliment of the _____________ on the mRNA eg the mRNA has the codon ________ for met ( the tRNAmet has the anticodon ______ c. at the 3 end of the tRNA is the _______________that holds the amino acid d. it requires 1 ATP to attach each amino acid to the correct tRNA to make an ________________ e. there are ______ possible tRNA for the 20 aa, therefore a cell needs ____ enzymes for this 4. Protein Synthesis STEPS IN PROTEIN SYNTHESIS                                       E-site P-site A-site      From the piece of DNA below, make the complimentary strand of DNA, transcribe the appropriate strand, package it for the cytoplasm and then translate it as described in the steps below. In row A; make the complimentary DNA strand; In row C, transcribe the DNA into RNA; In column 1 and 28, place the appropriate 3 or 5; In column 2 & 27, package the RNA in row C; In row D, underline the words in the RNA; In row E, translate the RNA into amino acids using the 3-letter codes for them 12345678910111213141516171819202122232425262 728AB3GGTACGCTACCGCAAGGACTCTCG5CDE  G. GENE CONTROL MECHANISMS 1. Genes ON or OFF? a. each cell in a multicellular organism has the_______________ genetic information b. yet each cell makes a ____________ set of proteins & these may change over timeHOW? c. therefore, most genes in most cells are _________________ most of the time 2. Types of control pg 255 Table 1 3. Transcriptional control parts a. promoter a sequence of DNA that is rich in _____&_____ to which RNA polymerase _______ this is located ________________ from the TAC start signal on the DNA b. operator a sequence of DNA beside the promoter to which a ______________________ binds if the repressor protein does _____________ to the operator, the RNA polymerase can______________ to the promoter and so ________ transcription occurs c. repressor protein binds to the ________________ unless it first binds to the ______________ binding to the inducer changes the shape of the _____________ and now it ___________________ binds to the operator therefore the RNA polymerase can bind to the _________________ and so now ________________________ can occur d. inducer a molecule that binds to the repressor and___________________ it from stopping transcription 4. trp operon in bacteria [see pg 257] a. trp is____________ in the environment and cells need it so normally the gene to make trp is ________________ b. if trp is ________________, it binds to the repressor protein and changes its ________________ so that it does bind to the promoter and blocks the RNA polymerase from binding which turns the gene ________________ c. because it helps bind, trp is called a ________________________ 5. lac operon in bacteria a. lactose is a ___________ sugar [food source] in the environment so the lac genes to breakdown lactose are usually ____________________ b. if lactose is present, it is the _______________ that binds to the repressor protein which now does NOT bind to the ___________________ c. when _______________ is present, the gene is ON   Part II: Manipulating DNA A. Isolating a specific DNA Segment a. This would allow researchers to __________________ DNA samples b. It may allow researchers to ________________ DNA from one organism to another. Why would we want to do this? B. Cutting the DNA Segment: _________________________________ a. Enzymes that cut _______________ at a specific recognition site b. These recognition sites are ______________________________  c. eg.  d. the restriction endonucleases often cut the DNA unevenly [as shown above] and leave _______________________ which are short pieces of ssDNA that will be able to __________________to similar ssDNA pieces e. generally a piece of DNA will have a __________________ for any one restriction enzyme every _____________________ bp or so, spaced at random along the DNA [eg 2000, 9000, 1400, 16000 23000] f. the restriction enzymes are named after the bacteria that they are extracted from FactorEcoR1Hind IIIGenusspeciesstrainisolate C. Methylases a. These enzymes add _____________ groups to the nitrogenase bases b. A methylated base will __________________ be recognized by a restriction enzyme & the DNA will NOT be _________________ c. This allows researchers to ______________ sections of DNA before using the restriction enzymes DNA Ligase d. This enzyme attaches ssDNA _______________________________________ e. After, the sticky ends form _______________________________ to hold the bases together eg.  D. Gel Electrophoresis a. a procedure for _____________ the cut pieces of DNA to allow researchers to select the one they want b. a gel is a _____________________ substance made of starch, agarose or polyacrylamide c. electrophoresis refers to separation based on _________________________ d. ______________ pieces of DNA will move further than large pieces of DNA separation by________ e. so that researchers can find the DNA on the gel ___________________________ is added this chemical fits into the twists of DNA without altering it and __________________ under UV light f. DNA pieces can now be cut, separated & isolated from the gel and checked for the ___________ gene Using DNA fingerprinting [which does not involve _________________________] 1. Paternity tests the childs DNA has to match both __________________ and _________________  2. Criminal investigations eg. ________________ where the victim and the ___________ are both sampled eg. ________________ where matching the _____________ is the key 6. Polymerase chain reactions make notes from web presentation D. Increasing the DNA ( Polymerase Chain Reactions [PCR] [Refer to Youtube -NOT ON POWERPOINT] Why do researchers do this? They wish to amplify _______ copies of DNA to ____________ copies. What section of DNA is amplified? Only a _______________ section of the DNA is amplified. It is chosen because it is highly ____________________ and is called the ___________ DNA. The idea is that if we look at __________________ of these sections, any one person should have a ________________ pattern. Procedure: Start with the sample of DNA and add: 1. _____________________: short [20 nucleotide] pieces of DNA of known sequence that will bond to both the _____________ and the _________________ strands. 2. _____________________: the basic building blocks of DNA 3. _____________________: an enzyme from a thermophilic bacteria that lives in _______________ and so is adapted to very warm temperatures. Cycles: a. heat to _____oC: The heat _____________ the DNA breaking ___________ bonds. b. cool to _____oC: To allow the primers to _____________ to complimentary sequences on both of the strands. c. heat to _______oC: To allows Taq polymerase to _______________ DNA by adding complimentary bases. Results: one cycle ( _______ copies two cycles ( _______ copies three cycles ( _______ copies [general formula ________________________] twenty-five cycles ( __________________ copies The amplified sample is now run on a ____________ to separate the DNA pieces based on size and to establish the _____________ identity of the sample. GENETIC ENGINEERING A. Overview 1. _________________ the DNA segment with the desired gene 2. _________________ the DNA segment from the genome 3. __________________________ to place the desired gene in a new organism B. Finding the DNA segment 1. Based on starting with a __________________ a. -make a synthetic ___________________ based on the amino acid sequence of the protein b -allow a radioactively labeled mRNA to __________________ to heated, single-stranded DNA [ssDNA] c. the labeled mRNA will stick to piece of DNA with the_______________ gene 2. Based on starting with _________________ a. find a cell that is active in synthesis of the protein so that its making lots of ____________ b. isolate the mRNA from the cytoplasm and label it radioactively then allow it to __________________ to heated ssDNA as above                  6 if the piece of labeled mRNA anneals to piece 6, 7 as well as 8 it may be because of exons and so a different restriction enzyme would have to be used   STEPS TO SOLVING LOCATION of GENE PROBLEM STEP 1 Write the segment numbers of the digested DNA on the samples of the gel grid, based on relative lengths of the segments. STEP 2 Based on step 1, write the segment letters on the digested DNA grid corresponding to the few samples for which protein synthesis data was obtained. STEP 3 Identify the segment of DNA that produces a normal protein. This segment contains the gene, and likely more DNA on both ends, the next steps will allow you to eliminate the excess DNA that is not part of the gene. STEP 4 Any segment that produces a smaller, not useful, protein is missing the stop codon at the end of the gene. STEP 5 Any segment that produces no protein until a promoter is added, has the start codon, but is missing the promoter that is found just upstream from the gene. STEP 6 The final segment of DNA that is actually needed should include the shortest segment that contains the stop codon and the start codon plus a tiny upstream segment. A] Samples of identical DNA digested by restriction enzymes  B] Gel electrophoresis samples EcoR IHind IIISol I Band C made a normal protein. Band F made a smaller protein that is not useful. No Sol I band made a protein, but when a promoter was added, band M did make a normal protein. On the grid at the top, show where the gene is located, showing your calculations on the gel grid, the digested DNA and on a separate page if necessary. eg. D. Vectors to get the gene into other cells 1. Plasmids a. these are small circular pieces of _____________ found in bacteria b. bacteria have restriction enzymes to allow them to cut their own large ______________________ and integrate ___________________ into it c. most _____________________ resistance genes occur on plasmids d. there are genetically engineered plasmids that have known restriction enzyme __________________ and known antibiotic ________________________ e. the _______________ [segment of DNA selected from the electrophoresis procedure and annealing with the mRNA] is now placed into the _____________________ using the restriction enzymes f. the plasmid is now placed into bacteria and if the bacteria ____________________ the plasmid 1( it will have the _________________________ 2 ( it will resist the ________________________ g. researchers screen _________________ of bacteria to find the few that have integrated the plasmid h. however, without the _______________________ region, the target gene will not work in the bacteria i. these plasmids may also be used to infect _________ and integrate the target gene into the _______ 2. Viruses a. a virus is used as a ___________________ instead of a plasmid b. the virus is modified to remove all of the ___________________ genes, if possible c. these must be _______________ viruses that undergo a _____________________ cycle d. the virus is then used to infect ____________________ cells and introduce the target gene into the __________________________ cell      PAGE \* MERGEFORMAT 2 O II HOPOH I OH ____ end l basesugar l P l basesugar l P l basesugar l P l ____ end _____ end l P l sugarbase l P l sugarbase l P l sugarbase l _____ end pyrimadine ___________ ___________ purine ___________ ___________ O OH CH2 5 3 3 5 3 12 11 10 9 8 7 7 7 6 5 4 2 1 DNA Replication 3 5 5 3 3 5 5 3 5 genes are_________ three b-galactosidase genes lacA lacY lacZ OPERATOR PROMOTER genes are ______________ three b-galactosidase genes lacA lacY lacZ OPERATOR PROMOTER EXON INTRON EXON INTRON EXON INTRON EXON INTRON EXON INTRON EXON INTRON UAC Met G C C T A A T C G T C A C T G C A A C T 3  C G C A T T G C G T A A C G5 5G C G T A A C G C A T T G C3 G A C G G A T T A G C A G T G A C G T T G T G C A C C A C G T G HOH2C OH OH O O II HOPOH I O combined to form ______________ UAC CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC E-site P-site A-site Val CAU Gln GUC Gly CCG Ile UAG Pro GGU Met UAC CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC 4. The tRNA with ____________ attached, randomly enters the _____________ on the ribosome. Because its ________________ matches the _____________ in the P-site, this tRNA is held in place. P-site A-site CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC Val CAU Gln GUC Gly CCG Ile UAG Met UAC Pro GGU _______ subunit of ribosome 2. The mRNA ____________ the nucleus and encounters the _______________ of the ribosome. 3. The________________ attaches the large subunit to the small subunit that is attached to the mRNA. The tRNAs are ____________ by the attachment of an amino acid, by the use of an _____________. CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC ______ subunit of ribosome UAC CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC 1. The mRNA, rRNA and tRNA are ____________ from DNA in the ___________. They randomly drift around in the _______________where the large and small subunits of the _____________ are also drifting. 3. Hydrogen bonding - adenine forms _____ hydrogen bonds with thymine - cytosine forms _____ hydrogen bonds with guanine Since A T and C G are the usual bondings, if the amount of A does not equal T or the amount of C does not equal G, then the DNA is not double stranded Mary Bob Larry Child L M N O P F G H I J K A B C D E 1 2 3 4 5 12 13 14 15 16 6 7 8 9 10 11 EcoR I Hind III Sol I exon 1 exon 2 exon 3 segment segment segment 6 7 8 Locating a complete gene on a segment of DNA 5 the mRNA ________ to the piece with the target gene sequence 3 heat the DNA to make it ________ + 4 add a piece of _______ mRNA for the desired gene 2 1 cut with ______________ enzymes separated after being run on the _____________gel 8 5 6 3 9 10 2 1 4 7 1 2 3 4 5 6 7 8 9 10 protected DNA after being cut and first 2 segments ligated CH3 CH3 protected DNA [red] cut with restriction enzymes unprotected DNA cut with restriction enzymes uncut DNA Hind III EcoR1 Sal 1 A A G C T T T T C G A A G A A T T C C T T A A G Met-Pro-Ile-Gly-Gln-Val CAU CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC UAG 6. The tRNA that brought Met now is ____________ after Met is _________ to the Pro. The ______ is now moved through the ribosome until the ___________ with Met-Pro is in the ________. The tRNA for the next code now fits into the _______ P-site A-site UAC Ile UAG Gln GUC Gly CCG Val CAU Met-Pro GGU CC GAG CUC AUG CCA AUC GGC CAG GUA UAG GGG ACC 5. The tRNA for the next amino acid _______________ moves into the ______________ P-site A-site Val CAU Gln GUC Gly CCG Ile UAG Pro GGU GGU CCG GUC 7. The processes of step 5 & step 6 are repeated until one of the ________ codes is in the _____________. 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