chapter 13 biology
18. What property of DNA allows for the recombination of DNA using "sticky ends" produced by restriction enzymes?
if you cut two pieces of DNA with the same restriction enzyme you can use this principle of complimentary base pairing to stick them together
23. IN what organism was CRISPR-Cas9 discovered? What does this system do in that organism?
•A bacterial adaptive immune response •Past exposure to a pathogen stimulates improved defense against future exposure •clustered regularly interspaced short palindromic repeats- repeated DNA sequences •"spacer" DNA identical to invading pathogen (phages) •Removing or adding spacers changes resistance Insert pieces of foreign DNA They use restriction enzymes to cut up invading DNA and it inserts little chunks of that foreign DNA into the genome of the bacteria in this CRISPER region •A mechanism for "RNA guided" destruction of invading DNA •CRISPR region plus cas genes (DNAases, RNAses, other proteins) •Invading DNA is cut in to pieces and placed in to the bacterial genome. •RNA is produced which find new invading DNA from a phage and cut it up. 1.Phage invades 2.Phage DNA inserted in to bacterial genome 3.RNA produced 4.RNA binds to CRISPR Complex 5.CRISPR-Cas9 Recognizes and cuts up new invading pha 6.Phage DNA
19. What properties if DNA structure allow for visualization in gel electrophoresis?
•DNA can be visualized in a clear gel •DNA fragments separate based on charge and size •Negatively charged fragments move towards a positive anode •Smaller fragments move more quickly The phosphate group has a lot of oxygen's and that makes the DNA molecule overall negatively charged so when you apply a current to the it, it pulls the DNA towards the positive anode in your electrophoresis chamber Use the charge to separate the DNA fragments
16. Why are mutations significant? Are they always good or always bad?
•Errors may occur after replication •Cigarette smoke, x-rays, chemicals, spontaneous changes •Mutations are errors that are passed on to the next generation. •Mutations can cause changes in populations that can affect the evolution of that population
15. What enzymes are involved in DNA proofreading and repair?
•Errors only occur 1/10 billion nucleotides, but errors are important for evolution and genetic variation. •DNA polymerases proofreads as it goes and makes corrections •Mismatch repair- enzymes remove any mismatches and polymerase replaces the incorrect base pairs.
6. How did Franklin's famous images of DNA (pictured in the chapter) show that helped Watson and Crick determine DAN's overall structure?
•James Watson and Francis Crick used Franklin's image and calculations along with models to deduce the 3D structure of DNA One knew how to use x-ray crystallography and was able to interpret Franklin's photos Franklin's found specific measurements of DNA and Watson and Crick turned it into a 3D model It confirmed that DNA was helical in shape also suggested the width of the helix and the spacing of the nitrogenous bases along it the photo implied that the helix was made up of two strands contrary to a three stranded model that Linus Pauling had proposed she confiremed the sugar phosphate backbones were on the outside of the DNA molecule X-ray indicated that the helix makes a full turn
20. Describe the purpose of polymerase chain reaction (PCR). What are a few uses for PCR?
•PCR can produce many copies of a specific target segment of DNA •"Primers" start DNA replication •Taq polymerase added nucleotides •Repeated cycles copy specific lengths of DNA PCR has many research and practical applications. It is routinely used in DNA cloning, medical diagnostics, and forensic analysis of DNA.
Transformation
A change in genotype and phenotype due to the assimilation of external DNA by a cell.
Bacteriophage
A virus that infects bacteria
22. What feature of the DNA molecule allows for DNA sequencing to happen? (You don't need to know the exact method, just understand what structural aspect allows for sequencing.)
Complementary base pairing
1. Be able to describe the structure of RNA and DNA. (refer to Ch 3 Nucleic acid for a review; Ch 3 information may be tested again)
DNA is made up of molecules called nucleotides. Each nucleotide contains a phosphate group, a sugar group and a nitrogen base. The four types of nitrogen bases are adenine (A), thymine (T), guanine (G) and cytosine (C). ... To fit inside cells, DNA is coiled tightly to form structures we call chromosomes RNA consists of ribose nucleotides (nitrogenous bases appended to a ribose sugar) attached by phosphodiester bonds, forming strands of varying lengths. The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil, which replaces thymine in DNA. •Nucleic acids(DNA and RNA) are made of nucleotides •Each nucleotide is made of: •Sugar (ribose or deoxyribose) •nitrogenous base (A, T or U, C, G) •phosphate group RNA has a hydroxyl group OH on the sugar while DNA has an H Uracil instead of thymine DNA deoxyribose sugar 3 prime carbon and 5 prime carbon •Nucleotides are joined by covalent bonds with nitrogenous base appendages •—OH group on the 3¢ carbon is joined to the 5' carbon phosphate next monomer •New monomers are added to the 3' end! •We say DNA has a 5' to 3' direction •Purines •adenine (A) guanine (G) Pyrimidines cytosine (C) thymine (T) (DNA ONLY) uracil (U) (RNA ONLY) •5 carbon ring-shaped sugar •DNA is deoxyribose •RNA it is ribose
7. What are they key features of DNA's structure as determined by Watson and Crick? How do they fit together to make a functional molecule?
DNA molecules are formed by two polynucleotides spiraling around an imaginary axis Twisted ladder shape called a DOUBLE HELIX Antiparallel: In the DNA double helix, the two backbones run in opposite directions ØEach strand runs 5' to 3' in opposite directions does this so the nitrogenous bases can pair correctly and gives it polarity ØThis is REALLY important for replication and use of one strand as a template. Complimentary: Antiparallel strands are held together by complimentary base pairs.(Hydrogen bonding) This creates a regular width across the molecule One Purine binds to one Pyrimidine Adenine always binds with thymine Guanine always binds with Cytosine ØThis is REALLY important for replication and use of one strand as a template. •Uniform width due to complimentary base pairing. •Uniform turns in the helix produce a regular structure Purine+ purine= too wide Pyrimidine+Pyrimidine= too narrow Purine + Pyrimidine = uniform
11. Know the major enzymes involved in DNA replication.
DNA replication requires other enzymes in addition to DNA polymerase, including DNA primase, DNA helicase, DNA ligase, and topoisomerase. Helicase (unwinds the DNA double helix) Primase (lays down RNA primers) DNA polymerase III (main DNA synthesis enzyme) DNA polymerase I (replaces RNA primers with DNA) Ligase (fills in the gaps) DNA replication starts at origins of replication(short stretches of DNA that have specific sequence of nucleotides) then proteins that initiate DNA replication recognize this sequence and attach to the DNA and start to separate the two strands and make a replication fork(a y shaped region where the parental strand is being unwound) Helicase does the untwist the double helix at the replication forks, separating the two parental strands and making them available as template strands then single stranded binding proteins bind to the unpaired DNA strands keeping them from re-pairing Topoisomers help relive the strain caused by the unwinding of the double helix Primase synthesizes RNA primers using the parental DNA as a template Primer: Inital nucleotide chain that is produced during DNA sythesis is actually a short stretch of RNA Primase: primer is synthesized by this. Starts a complementary RNA chain with single RNA nucleotide and adds RNA nucleotides one at a time
21. Why might someone want to sequence a DNA molecule? Give a list of some specific uses for DNA sequencing.
DNA sequencing is the process used to determine the order of nucleotides in a specific DNA molecule. This information is useful for researchers in understanding the type of genetic information that is carried in the DNA, which may affect its function in the body essentially PCR with labeled nucleotide read by a computer program that will tell you the sequence of the base pairs •Complementary base pairing can be exploited to determine the gene's complete nucleotide sequence •Primers begin replication •Labeled base pairs are added as a computer monitors each base pair that is added. •In each tube, one labeled nucleotide is added which will STOP the DNA sentence, producing sequences of all lengths that can be analyzed to determine the whole sequence.
4. What were Chargaff's rules? How did they help Watson and Crick determine DNA's overall structure?
Erwin Chargaff- 1952 Analyzed DNA from several species. Found: DNA composition varied by species Sea urchin 32.8% of DNA was Adenine E. coli 24.7% of DNA was Adenine This evidence of molecular diversity among species which has been presumed absent from DNA, made it a more credible candidate for the genetic material For each species %A = %T %C = %G 1). DNA base composition varies between species 2). for each species the percentage of A and T bases are roughly equal as are those of G and C bases With Frankilins x ray they determined the structure, but Cargoff helped with the chemistry side of the structure Watson and crick took into account chargoffs ratios and explained them A pairs with T and C pairs with G so that A=T and G=C
3. Describe the Hershey/Chase experiment. What key observation did they use to determine whether DNA or proteins were the genetic material?
Hershey and Chase (1952) •T2 phage (E.coli virus) •Does the bacteriophage pass on DNA or proteins? •Labeled radioactively labeled DNA or protein to determinewhat T2 was passing on. studying the T2 phage specifically infecting E coli(E coli is the model organism) they wanted to know if the phage was passing on DNA or proteins as the infectious genetic material wanted to find out which of those(proteins or DNA) was causing the infection and causing the production of new viruses They did 2 parallel experiments •Injected cells with phages who had: •Proteins labeled with radioactive sulfur OR •DNA labeled with radioactive phosphorous First cell they used radioactively labeled proteins allowed virus to attack to E coli cell then agitated it using a blender able to knock the viruses off the cell but not burst the cells then they separated the cells from the liquid with the virus in it and found that all of the protein was in the solution around the cell(found the protein was not in the cell) Did the same thing but with DNA found that the radioactive DNA was still in the portion where they expected the cells to be This told them that DNA was the genetic material DNA was the molecule that those proteins where injecting into the cell that was causing the viral infection DNA entered the host cells while proteins did not
12. Know the steps in both leading strand and lagging strand synthesis?
Lagging: built at the same time of the leading strand it happens slightly behind the leading strand, but is happening at the same time built in fragments: when replication bubble opens up DNA polymerase is working on adding to a free 3 prime end so as the replication bubble opens up we make more room for new pieces of DNA but they are not being made because they are starting farther down the molecule and moving away from the replication fork, so what happens with the lagging strand is that each time you open up the molecule a little bit more you have room to start a new fragment Okasaki fragments: have to be joined by a molecule called DNA lygase Leading: a Piece of DNA produced in one single long piece on the leading strand That piece is going to grow from your primer towards the replication fork so as DNA helicase moves the piece follows it At a replication fork, both strands are synthesized in a 5′ → 3′ direction. The leading strand is synthesized continuously, whereas the lagging strand is synthesized in short pieces termed Okazaki fragments. as the fork opens up we get another primer followed by DNA polymerase which adds nucleotides until it runs into another primer DNA ligase come in and link up the fragments last thing to be done is remove those primers RNA is replaced with DNA in the final step DNA ploymerase I: work as a proofreading enzyme •Errors only occur 1/10 billion nucleotides, but errors are important for evolution and genetic variation. •DNA polymerases proofreads as it goes and makes corrections •Mismatch repair- enzymes remove any mismatches and polymerase replaces the incorrect base pairs. •Errors may occur after replication •Cigarette smoke, x-rays, chemicals, spontaneous changes •Mutations are errors that are passed on to the next generation. •Mutations can cause changes in populations that can affect the evolution of that population
24. In the laboratory, how is CRISPR-Cas9 used? What is significant about this process?
Study the function of a specific gene Repair mutated genes Editing gene sequences Bacterial immune response to foreign DNA •CRISPR-Cas9 system •Is used to identify specific sequences •DNA is cut •Cell repairs cut gene by replacing mutations •Knockout genes with missense nucleotides •Repair mutations with correct sequence
13. How do leading and lagging strand synthesis differ? WHY do they differ?
The main difference between leading and lagging strand is that the leading strand is the DNA strand, which grows continuously during DNA replication whereas lagging strand is the DNA strand, which grows discontinuously by forming short segments known as Okazaki fragments
DNA replication
The process in which DNA makes a duplicate copy of itself.
2. What are bacteriophages? Why are phages a good mechanism for studying DNA?
The viruses that infect bacteria are called bacteriophages, and certain bacteriophages have been studied in detail in the lab viruses are much more simple than cells
9. There are three possible models for DNA replication. Although our book does not explain the experiment that shows that semi-conservative replication is the way in which DNA is replicated, how does this method differ from the other two? In your opinion, why does this process make the most sense? (Think about additional steps in the process needed to produce the other two molecules besides just the DNA duplication.
There were three models for how organisms might replicate their DNA: semi-conservative, conservative, and dispersive. The importance of the semi conservative model is that it makes sure that you have copies of the DNA that are identical to each other. Otherwise you wouldn't be able to make an exact copy of the DNA. This type of replication works thanks to DNA base pairing.
Virus
a little more than DNA or sometimes RNA, enclosed by a protective coat, which is often simply protein
17. What are restriction enzymes and why do you use them?
•Restriction enzymes- cut DNA at very specific and precise locations •Naturally in bacteria as a defense mechanism •Isolated and use in molecule biology laboratories •Recognize specific restriction sites and cut DNA in to restriction fragments •"Sticky ends" can be used to insert foreign pieces of DNA restriction enzyme: a group of enzymes that were originally isolated from bacteria They were actaully a bacterial immune system that recognizes infection by viruses or other foreign peices of DNA will cut at very specific locations natural defense mechanism used to break down DNA
8. What feature(s) of DNA's structure lends itself to replication? What model did Watson and Crick propose for a model of replication?
•Watson and Crick •Based on their structure, they proposed a mechanism for replication!!! •Each strand behaves as a template! because of complimentary base pairing each strand can behave as a template for the other, so if you use one of the parents strands you are able to make an exact copy of that parent strand that can eventually be passed on to the next generation semi-conservative model of DNA replication: the parental DNA double helix is pulled apart and each one is now available to be a template each one is used as a template to make a new copy or a second strand onto the parental strand, so the new strands that form are a combination of parental strand DNA and new DNA
5. What research were Rosalind Franklin and Maurice Wilkins performing?
•Wilkins and Franklin (1952) •X-ray crystallography images show a diffraction pattern that can be used to determine the shape of a macro molecule They were trying to figure out the structure of DNA used a technique called crystallography: shine a x-ray at a crystal and the x-ray beams are scattered onto a piece of film, you can interpret diffraction pattern from the x-ray beam and infer the shape/ chemical structure of a molecule Franklin knew based on some of her images that DNA was a double helix