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molecular2 Flashcards

1
Q

Initiation codon AUG interacts with tRNA.

A

in eukaryotes: methionyl trna, in bacteria: n-fmeth trna

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2
Q

Position of AUG at start: ____ at middle:_____

A

initiator, reg methionine

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3
Q

Shine Dalgarno is ____ of AUG, and functions to ___

A

upstream, attract ribosomes. it is 5’ UTR

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4
Q

Eukaryotes dont have a ribosome binding site (T/F)

A

True, so they have special cap on 5’ end of mRNA

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5
Q

Initiation includes

A

mRNA, tRNA, Ribosome

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6
Q

Elongation starts

A

once 2nd codon is used

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7
Q

5’ UTR

A

untranslated region before the initiator. These can hybridize with 16s

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8
Q

3’ UTR

A

downstream of the stop codon

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9
Q

P site

A

after initiaiton, aminoacyl tRNA binds to this site on the ribosome

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10
Q

Elongation adds __ one at a time to initiating amino acid

A

AA’s

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11
Q

First elongation step

A

binding second aminoacyl tRNA to the A site. This requires: EF-Tu, and energy from GTP

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12
Q

E site

A

exit, where tRNA exits ribosome.

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13
Q

Translation occurs through ____

A

elongation factor G

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14
Q

Peptidyl transferase

A

makes peptide bond with carboxyl and amino

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15
Q

Stop codons are:

A

UAG, UAA, UGA. these do not code for amino acids.

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16
Q

Release factors

A

recognize stop codons, and stop translation, releases polypeptide chain.

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17
Q

Peptidyl transferase cuts ____ when theres ____

A

polypeptide chain, stop codons

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18
Q

In between initiation and termination codons, is the

A

open reading frame

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19
Q

Transcription ___ primer dependent, replication ____ primer dependent.

A

isnt, is

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20
Q

Semiconservative replication

A

produces new DNA w each daughter double helix having 1 parental strand and 1 new strand

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21
Q

Without complete replication there is no ______

A

cell division.

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22
Q

Conservative replication

A

2 parental strands stay together, along with 2 new strands.

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23
Q

Dispersive replication

A

DNA is fragmented with both

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24
Q

Mutations can only occur during ______

A

replication, if DNA polymerase leaves a mistake behind

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25
Q

Mutation

A

silent mutation- occurs in 3rd base of codon, but makes same AA. Often changes are conservative like this, doesnt change the AA.

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26
Q

Sickle cell occurs when

A

Single base change of B-globin (hemoglobin). This causes substitution of wrong AA, distorting RBCs in low O2.

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27
Q

A change in a gene can change the protein product of a gene (T/F)

A

True

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28
Q

Sickle cell codon changes:

A

GAG (glutamate) changed to GUG (valine)

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29
Q

Molecular Cloning

A

combining a foreign gene in a plasmid. combines 2 DNA’s and makes it self replicating, which allows large production of target gene. Also, links eukaryotic genes to small bacterial/phage DNA, and inserts recomb. molec into bacterial hosts.

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30
Q

In between Nitrogenous bases are ______

A

covalent phosphodiester bonds.

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31
Q

Restriction endonucleases

A

-enzymes. usually a dimer
-made gene cloning possible
-discovered in E coli
-cut at sites within foreign DNA instead of chewing from ends, specifically cutting at the sugar phosphate backbones (at recognition sites)
-ligase recombines the cut ends by finding OH groups.

32
Q

The frequency of cuts from restriction endonuclease is ____ frequent as recognition sequences are longer

A

less

33
Q

Restriction modification

A

prevents restriction endonuclease from cutting host DNA. The mod is paired with methylases, they recognize and methylate the same sites. Methylation protects DNA

34
Q

Boyer and Cohen experiment using restriction endonuclease

A

used EcoR1 to cut 2 plasmids, converting them to linear with the same sticky ends. DNA ligase joined the two pieces with covalent bonds. The enzymes, ligase, foreign DNA, and DNA being used, transform to desired bacteria.

35
Q

Vectors

A

DNA carriers, allowing replication of recomb DNA. Carries foreign DNA to bacteria. Usually with 1 vector+ 1 piece of foreign DNA. Vector does not have origin of replication. Vectors can be plasmids or phages.

36
Q

Multiple Cloning site

A

cloning sites clustered together. allows one to cut the vector and foreign gene with 2 different restriction enzymes. Uses directional cloning to know orientation for insertion.

37
Q

Antibiotic resistance genes

A

allow for selection of bacteria that have a copy of the vector.

38
Q

LacZ must have

A

plasmid transformation

39
Q

Yeast artifical chromosomes and bacterial artificial chromosomes are used for

A

cloning huge amounts of DNA

40
Q

Eukaryotic vectors

A

vectors designed for cloning genes to euk cells, or Ti-plasmid vector to carry genes to plant cells

41
Q

Identifying clones with Probes

A

probes identify desired clone. The two types are polynucleotides and antibodies.

42
Q

Factors supporting strand seperation

A

high temp, high organic solvent, low salt.

43
Q

cDNA

A

complementary DNA or copy DNA that is a DNA copy of RNA. cDNA library is a set of clones with as many as possible of mRNAs in that cell type at a given time.

44
Q

For successful cloning…..

A

synthesis of cDNA from mRNA template using reverse transcriptase- RNA dependent DNA polymerase.

45
Q

Nick translation

A

removes DNA ahead of nick, synthesizes DNA behind nick. Result is moving the nick in 5’ to 3’ direction. The enzyyme used is E coli DNA polymerase 1.

46
Q

cDNA: sticky ends

A

cDNA doesnt have sticky ends cleaved w restriction enzymes. Sticky ends will be made using terminal deoxy nucleotidyl transferase with one dNTP.

47
Q

PCR

A

used to amplify DNA, creates a dna fragment for cloning. created by dr Kary Mullis. no need to add fresh DNA polymerase after each replication. Taq polymerase is used for PCR because its an enzyme that can be used at high temps.

48
Q

PCR temps

A

boiling 99 deg to seperate ds DNA, 52-64 deg for primers/annealing, 72 deg for DNA polymerase to synthesize.

49
Q

RT PCR

A

quantifies amplification of DNA as it occurs. As the strands separate, they anneal to forward and reverse primers, and to reporter probe.

50
Q

Reporter probe

A

the 3rd primer. fluorescent tagged oligonucleotide that is complementary to part of a DNA strand, flores tag at 5’ end, flores quench at 3’ end. As PCR goes on from the forward primer, the 5’ tag is separated from the 3’ tag, allowing 5’ tag to fluoresce. can be quantitated.

51
Q

Flourescence in RT PCR increases with _____

A

incorporation into DNA product.

52
Q

Quencher of RT PCR

A

keeps surroundings dormant, stops from fluorescing. DNA polymerase separates the florescent from the quencher, allowing the F to fluoresce.

53
Q

Quencher of RT PCR

A

keeps surroundings dormant, stops from fluorescing. DNA polymerase separates the florescent from the quencher, allowing the F to fluoresce.

54
Q

Expression vectors

A

express genes. Used to put a foreign DNA into bacterium to replicate. These can make protein products of the cloned genes.

55
Q

Bacterial expression vectors

A

Two required elements: a strong promoter (host RNA polymerase must be able to recognize), and a ribosome binding site by the initiating codon.

56
Q

Inducible expression vectors

A

allows to express a toxic gene on the host cell

57
Q

lac promoter

A

inducible, stays off till stimulated by inducer IPTG. Repression can be leaky, expression will still occur. To stop leak, use plasmid carrying its own lac1 repressor, this will stop RNA polymerase from binding.

58
Q

Most vectors express _____

A

fusion proteins. not natural product of gene.

59
Q

Oligo histidine expression vector

A

encodes 6 His. high affinity for metal ions like nickel . purified by nickel affinity chromatography.

60
Q

His-tag can be removed by _____

A

enterokinase- which cuts histidine off desired protein. Result is eluted.

61
Q

Bacterial expression Cons

A

-eukaryotic proteins in bacteria may be seen as foreign and destroyed.
-post -translational mods are different in bacteria.
-bacteria environment may not fold protein correctly.
-cloned euk proteins could be useless.
*Using yeast is safer

62
Q

E coli can replicate both bacterial and euk cells (T/F)

A

True

63
Q

Ti plasmid

A

used to transfer genes to plants. DNA stays in interstitial space.
-bacterial vector promoters and replication origins arent recognized by plant cells.
-plasmids with T DNA are used. (transfer DNA)
-Ti (tumor inducing) comes from bacteria that cause crown galls- plant tumors.
-crown galls occur if Ti is successful

64
Q

Ti plasmid process

A

bacterium infects plant, transfers Ti plasmid to host, T DNA (may also contain gene of interest) integrates into plant DNA, T DNA synthesizes unusual organic acids (opines).

65
Q

Encoded in tDNA:

A

Oxin, cytokinen, and opine

66
Q

Gel electrophoresis

A

used to separate nucleic acids and proteins.

67
Q

DNA gel electrophoresis

A

melted agarose is used with comb, electric current is run through gel at a neutral pH. DNA runs from neg to positive. DNA is negatively charged (because of phosophates). It will distribute DNA by their size, large to top, small to bottom, and stained with florescent dye.

68
Q

Large DNA for gel electrophoresis is ____

A

fragile

69
Q

Pulsed Field Gel Electrophoresis

A

used for DNA> 1kb. long pulses of current with short pulses in opp direction, allows to handle friction.

70
Q

Protein Gel electrophoresis

A

Made vertically between 2 plates. polyacrylamide gel electro. performed with sodium dodecyl sulfate (SDS). small proteins move faster to anode. If mix is very complex, use also the isoelectric focusing gel- proteins electrophoresed through gel. Contains ampholytes for pH gradient. negative protein moves to isoelectric point. proteins are separated based on isoelectric points.

71
Q

SDS

A

denatures protein, masks charge, provides negative charge

72
Q

Ion exchange chromatography

A

used with pockets of charge. uses a resin to separate substances by charge. sample is loaded to column with resin.

73
Q

Anion exchange chromatography

A

uses pos charged diethylaminoethyl groups (DEAE), that’ll mind to neg charged AA’s. Passes a soln of gradual ionic strength to column. uses salt gradient

74
Q

Cation Exchange chromatography

A

negatively charged phosphocellulose resin attracts pos charged AA to seperat3e pos substances. Most proteins have net negative, but can still bond if they have significant pos charge

75
Q

Gel filtration chromatography

A

protein size used as a basis of physical separation, uses porous whiffle ball type filtration. small molecules only will enter holes, so they will come out last because they follow path of holes, where as larger will go around and come out.

76
Q

Affinity chromatography

A

enables purification of a biomolecule w respect to its biological function or individual chemical structure. can purify a certain molecule from a mixed sample. molecule will bind to a column resin coupled to affinity reagent. molecule of interest is retained.

Flash Cards (21 decks)

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