Term
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Definition
| enzymes that cut sequences of DNA |
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Term
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Definition
| cuts off terminal base pairs. nibb les off ends, enzyme specific and have to bump into right end and chop off |
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Term
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Definition
| enzymes that cut into specific base parts that form a recognition site. can chop in middle. look for binding or restriction sites and cut specific to that pattern |
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Term
| What determines where endonuclease cuts |
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Definition
| restriction site pattern (palindrome) |
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Term
| Where do restriction enzymes come from |
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Definition
| found in nature as part of the way bacteria defend themselves against viruses |
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Term
| What role do restriction enzymes play in nature |
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Definition
| bacteria's defense against viruses |
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Term
| what is a restriction site |
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Definition
| specific pattern of bases recognized by a nuclease |
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Term
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Definition
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Term
| What types of bonds must be broken so EcoRI can cut into DNA duplex |
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Definition
| phosphodiester bonds and hydrogen bonds |
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Term
| What types of bonds must be broken so EcoRV can cut DNA duplex |
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Definition
| phosphodiester but NOT hydrogen bonds |
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Term
| why are there no sticky ends when EcoRV cuts DNA |
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Definition
| no H-bonds are cut so nothing tries to anneal back together |
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Term
| What is meant by "DNA molecules can have a fingerprint" |
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Definition
| nucleases can be used to create a pattern of cut DNA molecules from restriction sites |
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Term
| How many fragments will result from cutting a piece of linear DNA that has 10 restriction sites for a certain restriction enzyme? |
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Definition
1 + number of sites = number of resulting fragments
so, 10+1= 11 fragments |
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Term
| if the sequence at one of the 10 restriction sites changes because of mutation, how many fragments will you now get using this enzyme |
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Definition
| if this occurs you only get 9 restriction sites that will be recognized so only 10 fragments should form |
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Term
| What is agarose gel electrophoresis |
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Definition
| Tool used in DNA fingerprinting |
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Term
| How does agarose gel electrophoresis work |
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Definition
Since nucleic acids are charged molecules, when placed in a constant electric field the acids will separate by size
- largest molecules will move the slowest
pattern that comes about is the "finger print"
- is a logarithmic scale |
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Term
| What is restriction fragment length polymorphism |
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Definition
| change in the length of pieces of a strand of DNA with the use of a restriction enzyme, due to a mutation |
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Term
| What does restriction fragment length polymorphism assume |
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Definition
every identical DNA molecule will have the same "finger prints"
if one DNA is missing a restriction site, a fragment will not occur like it should. |
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Term
| What analytical method is used to study the fragments |
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Definition
| you can count how many fragments occur, and the size of each |
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Term
| What is meant by mapping DNA with restriction enzymes |
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Definition
| you can find out how DNA is linked together by using different restriction enzymes to find the pattern |
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Term
| Letters on diagram stand for: (silde 8) |
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Definition
| different fragment lengths. |
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Term
| If there are 3 A restriction sites how many fragments |
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Definition
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Term
| If there are 3 A restriction sites and 1 B how many fragments |
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Definition
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Term
| What gets recombined in recombinant DNA |
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Definition
| Fragments of DNA are combined |
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Term
| What property must the restriction enzymes share when 2 diff. DNA molecules are recombined |
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Definition
| they must share the same kind of restriction site that is cleaved |
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Term
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Definition
a special ring of bacterial chromosome that can have new DNA spliced into it
- it's naturally occuring circular pieces of DNA, have add-ons that can improve (but not necessary) life. |
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Term
| What key sequence regions must a plasmid have to function as a recombinant DNA vector |
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Definition
-ORI (origin of replication): sequence of DNA from origional plasmid, recognized by bacteria that it is a plasmid and allows bacteria to replicate it
- Polylinker (place to put recombinant gene): place to put foreign gene
- Bacterial selection gene (amp^R): way of selecting which bacteria that has taken up plasmid, antibiotic will kill cells that didn't take up the plasmid |
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Term
| Why is it very important that the E. coli bacteria we use to make many copies of the recombinant plasmid are unable to grow in the animal intestine? |
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Definition
- we don't want to make a threat against humanity
- developing SAFE Ecoli strain- helps up recombinant DNA tech. for 10 years
the strain that's used can not survive outside of the lab, can't live in the intestines of vertebrates |
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Term
| What happens when a 2',3'-dideoxy analog of the natural deoxyribonucleotides gets incorporated as a DNA strand is synthesized |
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Definition
analog can still be attached to the previous nucleotide OH group
-- now have a broken 3' end- terminates the chain because it can't attach anymore nucleotides (H is at 3') |
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Term
| If each of the 4 analogs (of the natural deoxyribonucleotides) has a different fluorescent color attached to the analog, what property will terminated DNA fragment have |
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Definition
| the terminates DNA fragment will have a different color depending on the analog. look where it terminates and look at color to see which analog it is. |
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Term
| What technique is used to obtain large amts of DNA for study |
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Definition
| Polymerase Chain reaction (PCR) |
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Term
| If you want to study a very specific target, what info do you have to get before you can do PCR? |
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Definition
- must find the 5' and 3' ends of the target
- have to know some of the flanking sequence on the 5' and 3' ends in order to get the whole sequence |
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Term
| What do primers do in PCR |
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Definition
| provide the necessary binding site and starting point for DNA polymerase; primer sequences are contained in the amplicon and make up the extreme 5' and 3' ends |
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Term
| What relationship do the primers have to the target of PCR |
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Definition
| mark the 5' and 3' flanks of the target |
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Term
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Definition
| molecule produced in the PCR process, in huge numbers, that contains exact copy of the target |
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Term
| Why do you have to heat the DNA temp in PCR |
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Definition
| because you must melt the DNA so the target source becomes available |
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Term
| What property of the DNA polymerase enzyme is essential |
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Definition
| it does not get destroyed in high heat environment |
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Term
| What is the natural source of the DNA polymerase enzyme used in PCR? |
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Definition
| taq DNA polymerase, Bacterium Thermus aquaticus lives in geyser pools at 73 degrees C (163 degrees F) and survives boiling |
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Term
| What would happen in PCR if you forgot to add the primers |
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Definition
| the 2 strand would re-anneal |
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Term
| What would happen if you only added 1 primer instead of 2 |
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Definition
the amplicon would not form
extension will occur through LOW EFFICIENCY!! |
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Term
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Definition
| any change in the DNA sequence |
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Term
| Are mutations always bad? |
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Definition
| No, a mutation can be advantageous, neutral or disadvantageous |
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Term
| If a mutation gets inherited, where must the mutation have appeared |
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Definition
| it must have appeared din one of the original germ cells, the sperm or the egg |
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Term
| How common is single base change mutation |
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Definition
| 10^-8 base changes/ replication of haploid gene. (.0000001) or 1 in 10000000 |
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Term
| how common is a deletion or duplication mutation |
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Definition
| 10^-5 gametes have a duplication or deletion change= MORE COMMON, MOST NOT FIXABLE BY REPAIR MECHANISMS (1 in 10000 |
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Term
| If a mutation occurs in a somatic cell during embryonic developments, what could happen |
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Definition
THIS COULD BE BAD
- if mutation occurs w/any process of celldivision in the begining of the development it could destroy embryonic development, dysfunctional tissue or no formation of tissue
- must protect embryo from mutagens! |
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Term
| What process do the mutations involving large scale deletions or duplications of DNA occur |
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Definition
| Meiosis- homologous recombination- "crossing over" |
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Term
| What can happen to gene copy numbers when Meosis- homologous recombination or crossing over occurs |
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Definition
| if there are regions of duplication that already exist, the systems that line them up can get confused, result is broken attempt for a cross over that can end w/a deletion or a duplication |
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Term
| What are the 4 general categories of mutation agents |
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Definition
1) natural/industrial chemicals (pharmaceutics)
2) Ionization radiation (UVB light)
3) Retroviruses Transponsons (HIV/AIDS)
4) Mistakes in DNA replication (random and non random) |
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Term
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Definition
| Replication is proofread to make sure that the complementary base pair is on the other side |
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Term
| why is it mutagenic if DNA repair fails |
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Definition
| Id FNA is not checked than the mutated strand will serve as a template for a mutated complimentary strand |
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Term
| What is the risk if DNA repair mechanisms are damaged |
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Definition
| many mistakes could happen and eventually cause harm or cancer |
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Term
| Do human cells maintain a high level or low level of proofreading correction fidelity |
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Definition
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Term
| why do microorganisms sometimes allow their DNA repair to be sloppy |
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Definition
| if their environment all of the sudden becomes less inhabitable, the bacteria lets mistakes happen in hopes that one will be beneficial to survive in that new environment |
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Term
| What can be the consequence of a base change in a codon of a gene that codes for a protein |
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Definition
| if another amino acid is substituted, it may not fold correctly and funciton would be bad |
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Term
| what are nonsense mutations |
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Definition
| a single base change that is disastrous. goes from one sequence (UAU) to a nonsense one like GAU or AAU or CAU all which result in a coding for a different protein- |
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Term
| what is a silent mutation |
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Definition
a single base change that does NOT result in coding for a different protein
(UAU)--> Tyr
(UAC)--> Tyr |
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Term
| What are the 2 types of single base mutation |
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Definition
transition: change from one member of one class to another member of that same class, chemistry straight forward (purine to purine and so on)
Transversions: change from one type to the otehr, chemistry is complex (one class to another) ( purine changes to pyrimidine) |
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Term
| which is more common and why (transition or transversion) |
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Definition
| transitions b/c change chemistry w/in same class, easier to do |
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Term
| What info must you have in order to speak about an INDEL mutation |
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Definition
| MUST know original strand! |
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Term
| What ist hte reading frame of mRNA molecule |
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Definition
| codons btwn the start and stop codons |
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Term
| what determines the reading frame |
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Definition
| start condon initiates the frame and stop codon ends it |
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Term
| What is a frame shift errro |
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Definition
| when a base is inserted or deleted, ribosome will read every 3 codons and make amino acid and whole sequence will be thrown off if something is added or deleted |
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Term
| What is the most common cause of a C to T transtion mutation |
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Definition
| nitrite catalyses this type of acidic oxidative deamination |
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Term
| What conditions favor the transtion of C to T |
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Definition
| slightly acidic environment |
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Term
| How can oxidants cause a transversion mutation |
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Definition
- Superoxide (O2) and hydroxyl radical (OH) can oxidize guanine into 8-oxoguaning
- 8-oxoguanine can base pair with an A or a C
- a transversion mutation will then occur in the next round of DNA replication and replace GC pair with a TA pair... this is BAD..
- chronic inflammation can lead to cancer because too much superoxide (from excessive inflammation) causes oxidation of guanine and leads to cancer
- tobacco smokers have high excess of oxoguanine (causes cancer)
--> this mutation can still be corrected by proofreading |
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Term
| What are thyrmidine dimers and what causes their formation |
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Definition
thymidine dimers are 2 thymidine nucleotides that have binded to eachother
- UVB rays cause the combination |
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Term
| unless repaired what disease can result from thymidine dimers |
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Definition
| loss of cell division control and CANCER |
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Term
| what is excision repair of DNA and which type of mutation can be repaired by excision repair |
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Definition
| Type of point mutation repair- repairs thymine dimer |
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Term
| if the enzymes involved in excision repair are not functioning properly, what could that do to your health |
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Definition
| more susceptible to skin cancer |
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Term
| Why are alkylating agents useful for treating skin cancer |
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Definition
| they STOP DNA replication and STOP cancer from growing |
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Term
| what can happen if drugs alkylate the DNA of normal cells |
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Definition
| have potential to cause cancer in healthy cells |
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Term
| Why was the discovery of reverse transcriptase necessary before human genes could be expressed in bacteria |
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Definition
bacteria can't splice introns, so gene expression would quit at the first random STOP codon encountered in an intron
- bacteria would transcribe everything giving bits of proteins (from introns) that should be there in the final proteins |
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Term
| What is the product of reverse transcriptase |
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Definition
| cDNA: DNA molecule that is complementary to the RNA template |
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Term
| How do retroviruses use reverse transcriptase |
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Definition
| Use it to integrate cDNA into host DNA to replicate teh virus proteins |
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Term
| What threat does retrovial life cycle pose for us |
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Definition
| a favorite site for cDNA to insert is lethal for the host cell, the infected cell is destined to die and could disrupt the defense mechanisms in our bodies |
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Term
| What is a loss of function mutation |
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Definition
| when a gene loses an ability to perfom some function |
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Term
| under what circumstances is a loss-of-function mutation recessive |
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Definition
"recessive":when one copy of an allele is bad but the other copy is sufficient enough to compensate
-"dose of one" is good enough |
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Term
| When is loss-of-function mutation dominant |
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Definition
| when "dose of 2" is needed for proper function |
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Term
| what is a gain-of-function mutation |
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Definition
| a gene gains the ability to perform another role |
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Term
| what is the new function gained in sickle cell trait |
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Definition
| ability of the beta subunit to polymerize and block malaria |
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Term
| why is sickle cell disease recessive, but malaria resistance caused by sickle cell trait is dominant |
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Definition
-b/c the wild type allele provides subunits that don't polymerize, so most of the time, hemoglobin doesn't polymerize
- resistance is dominant b/c enough hemoglobin polymerized to protect from malaria |
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Term
| What is the difference in health status between sickle cell homozygotes and hetrerozygotes |
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Definition
- homozygotes: have sickle cell anemia symptoms all of the time, very sick
- heterozygotes- will have syptoms of the disease only in low oxygen environments (and resistant to malaria) |
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Term
| Why is hydroxyurea used to treat homozygous sickle cell anemia |
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Definition
hydroxy urea can be used b/c it causes activation of gene from unclear MOA it stimulates another gene of hemoglobin
--> causes one of the embryonic subunit genes (gama units) to activate causing a new hemoglobin molecule w/o chance of sickle cell |
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Term
| what are the gamma proteins |
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Definition
- the gamma proteins homologs of the beta subunits of hemoglobin that is not susceptible to sickle cell formation
- fetal gamma shows regular functions, homozygous for sickle cell naturally do this |
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Term
| What conditions stimulate the polymerization of sickle cell hemoglobin |
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Definition
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Term
| why does having 1 normal and 1 sickle cell allele confer resistance to malaria |
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Definition
| a dose of one is needed to have resistance |
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Term
| why does the frequencey of the sickle cell allele in human populations co-distribute w/the presence of endemic malaria |
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Definition
| the people who developed or inherited this mutation survived and the people who didn't died |
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Term
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Definition
| the ends of chromosomal molecules |
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Term
| what happens to the telomeres when the chromosome is replicated |
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Definition
| they don't get replicated like normal, but when the telomerases they recreate them at the ends. only get replaced in cells that have telomerases... if they don't have it then they don't get replaced... each time it's replicated it gets shorter and shorter and then dies. |
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Term
| why is a problem that cancer cells often express the telomerase genes |
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Definition
| they can reproduce continuously w/o need of stem cells |
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Term
| a gene has 1 copy in the haploid genome, and 2 alleles(A and B) exist for this gene. Both A and B have an allele frequence of 50% in a certain group of people. can you precisely predict which combo of alleles any individual from that group will have |
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Definition
NO= jut gives stats of what might be in population
- as allele frequency increases, statistically there is a higher chance that at least 1 person will be carrying that allele |
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Term
| What is the selection force when bacteria are exposed to antibiotics |
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Definition
| - bacteria wants mutation so it can survive antibiotics |
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Term
| how does bacterial gene expression change to deal with the threat |
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Definition
| lowers proof reading ability to generate mutations |
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Term
| why does the allele frequency of the resistance musttion sweep to 100% in the threatened bacterial population |
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Definition
| the cell that developed mutation has a growth advantage and others will die off |
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Term
| what happens to resistance allele frequency when a population that has fixed the resistance allele moves into an antibiotic-free environment |
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Definition
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Term
| What happens to resistance allele frequency when a population that has fixed the resisitance allele moves into an antibiotic- free environment |
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Definition
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