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Molecular Cell Biology > Complete review for molecular cell biology midterm 1 > Flashcards

Complete review for molecular cell biology midterm 1 Flashcards

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1
Q

Define complementation.

A

Mutation on two different genes that result in a wild-type phenotype. For example, given a particular pathway crossing two people who have mutations in genes involved in the pathway but on different genes leading to a heterozygote that has sufficient protein being made.

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2
Q

What are the steps of a genetic screen?

A

Mutagenize genes.
Cross to obtain individuals with mutations.
Cross(es) to make homozygous lines.
identify lines with phenotype of interest.
Complementation.
Clone and sequence gene.
Often use positive or negative selection and a screen for conditional mutants.

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3
Q

What are the two types of forward genetics?

A

Using a chemical mutagen or through insertional mutagenesis (TEs)

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4
Q

What are the different methods of reverse genetics?

A

HR, gene editing and use of RNAi for gene knockdown.

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5
Q

What is transgenics?

A

Introduction of genes into heterologous system. Use transformation or transfection or transduction.

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6
Q

How is DNA cloned?

A

PCR or with plasmids.

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7
Q

What are the two different DNA libraries that can be made?

A

genomic library and cDNA library.

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8
Q

What are the steps of PCR?

A

Heat to denature strands.
Cool to annealing temperature and add primers.
Then add DNA polymerase and nucleotides.

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9
Q

Draw the general protein structure including a peptide bond.

A

Refer to notes

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10
Q

What are the different proteins structure levels?

A

primary- sequence of amino acids
secondary- local 3D structure
tertiary- 3D structure of protein
quaternaty- 3D structure of a protein complex

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11
Q

What bonds allow rotation?

A

Bonds around alpha carbon, phi psi bonds.

phi - NH, psi - C=O

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12
Q

Describe the a-helix.

A

every amino acid is hydrogen bonded to the amino acid 4 peptide bonds away. (C=O and NH)
Hydrophilic backbone is sequestered within the helix.
Side chains are oriented outward.

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13
Q

Describe the B-sheet.

A

Two or more parallel or anti-parallel stretches of amino acids.
Hydrogen bonds form between adjacent amino acids. (C=O and NH)
Side chains oriented perpendicular to the sheet.

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14
Q

Describe the different forces and proteins involved in protein folding?

A

Proteins fold according to the conformation of lowest energy. This is accomplished through hydrophobic interactions, electrostatic interactions, hydrogen bonds and van der Waals forces as well as through the use of chaperone proteins.

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15
Q

What are protein domains and what are some basic characteristics?

A

Protein domains are modular units from which larger proteins are built.
Domains can be identified based on functional studies.
Protein domains have a particular function or ability.
Generally able to fold independently.
A given domain may be found on multiple different proteins.

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16
Q

How are different domains thought to be shared in the human genome?

A

Exon shuffling

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17
Q

What leads to antibody diversity?

A

Hypervariable loops.

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18
Q

How does the cell regulate the function of its enzymes?

A

Negative regulation in which the products of an enzymatic pathway when abundant serve as inhibitors of the pathway.

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19
Q

What are kinases?

A

Add phosphate to target amino acid residue.

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20
Q

What are phosphatases?

A

Remove phosphate from target amino acid residue.

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21
Q

Draw the Src protein kinase mechanism.

A

Refer to notes.

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22
Q

Draw the GTPase mechanism.

A

Refer to notes.

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23
Q

Draw the ubiquitination mechanism.

A

Refer to notes.

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24
Q

What does E1 do?

A

ubiquitin activating enzyme, transfers charged ubiquitin to E2.

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25
Q

What does E2 do?

A

Ubiquitin conjuguation enzyme, finds E3.

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26
Q

What is E3?

A

Diverse group of proteins, ubiquitin ligase.

Binds protein and E2 in specific site.

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27
Q

What is GEF?

A

Guanine exchange factor.

Promotes GTP bound state.

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28
Q

What is GAP?

A

GTPase activating protein.

Promotes GDP bound state.

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29
Q

Draw the kinase/phosphatase system.

A

Refer to notes.

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30
Q

What is an in vitro assay?

A

Reconstitute a specific cellular event in a test tube.

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31
Q

What are the different types of chromatography?

A

Ion exchange chromatography-separates based on charge.
Gel-filtration chromatography- separates based on hydrodynamic volume.
Affinity chromatography-separates based on binding ability to a matrix.

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32
Q

What are the steps of IP?

A

Grind up cells in extraction buffer, centrifuge and obtain total cell extract.
Add antibody against specific protein.
Add beads coupled to protein A.
Centrifuge and keep pellet.

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33
Q

What are the steps of Co-IP?

A

Identical to IP except use a milder buffer to preserve protein-protein interactions.

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34
Q

How are proteins separated by SDS-PAGE?

A

SDS coats amino acids with a negative charge. Separates based on charge through porous gels.

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35
Q

What are the steps of mass spectrometry?

A

Proteins are broken up into peptides.
Go through mass spec.
Mass of peptide determined by computer.
Then computer searches protein database for predicted proteins that have those amino acids.

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36
Q

What are the steps of tandem mass spec?

A

First step is to break up the protein into peptides, goes through MS1 then broken up again into even smaller peptides.
Goes through MS2.
Computer can determine the sequence of amino acids and if they were altered.

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37
Q

What is GST pulldown?

A

Start with fusion protein of gene X and GST.

Make cell extract, run down glutathione beads, can identify interacting proteins.

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38
Q

What are the steps of DNA cloning?

A

Take expression vector cut with restriction enzymes, insert protein coding gene of interest. Add into cell. Get protein.

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39
Q

What is X-ray crystallography?

A 
Purify protein.
Has to be highly soluble in water.
Dehydrate protein to form crystals.
Subject to X-Rays.
Identify crystal structure.
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40
Q

Why are proteins made into crystals?

A

Proteins all arranged in same way.

41
Q

What is NMR?

A

Doesn’t require crystallization but can only be used for smaller proteins.
Not as precise.

42
Q

What is a blastp search?

A

Identify related proteins in same or other species.

43
Q

What is domain prediction?

A

Identify putative domains based on sequence similarity to characterized domains. (putative = thought to be)

44
Q

What are the steps to generate antibodies?

A

Purified proteins or in vitro synthesis of short peptide sequence (epitope of POI) *generally the short peptide sequence doesn’t work.
Injected into rabbit, mouse, other.
Wait for antibodies to be generated and take serum.
Purify antibodies of interest from other antibodies from serum through affinity purification.

45
Q

What are the steps to generate monoclonal antibodies?

A

Immunize mouse.
Remove spleen and harvest antibody secreting B-cells.
Fuse these cells to myeloma cells to make hybridoma cells.
Identify cell that makes antibody of interest (from all hybridoma lines)
supernatant of this cell contains pure mono-specific antibodies.

46
Q

What is co-labelling?

A

Antibody made against an antigen then the addition of a fluorescent secondary antibody.

47
Q

Why do the primary antibodies in double labeling have to be produced in different animals?

A

So two different secondary antibodies bind to the two different primary antibodies.

48
Q

How is GFP used in miscroscopy?

A

Fusion protein.

49
Q

What is immunogold electron microscopy?

A

Gold particles are attached to secondary antibodies and due to their high density they make black dots for the electron microscope.

50
Q

What are the three mechanisms that ensure accuracy of DNA replication?

A

Tightening of DNA polymerase, 3’ to 5’ exonuclease activity of DNA polymerase and mismatch repair.

51
Q

Describe all three Okazaki experiments, including reagents and steps and readouts.

A

refer to notes.

52
Q

What are the components of the DNA replication machinery?

A

MCM helicase, DNA topoisomerase, RPA, PCNA, DNA polymerase.

53
Q

Draw the looping of the lagging strand to bring it closer to the leading strand.

A

Refer to notes.

54
Q

What is MSH2 and MLH1?

A

Protein complex that scans newly synthesized DNA for mismatches and can make another nick on lagging strand to remove and repair it. Part of mismatch repair.

55
Q

Draw the mismatch repair mechanism.

A

refer to notes.

56
Q

Draw the DNA topoisomerase mechanism.

A

refer to notes.

57
Q

Draw and explain the two methods to find eukaryotic origins of replication.

A

Refer to notes, silver grains and microarray.

58
Q

What is ORC?

A

Recruits cdc6 and cdt1 and is bound to origin constitutively.

59
Q

What do CDt1 and cdc6 do?

A

Recruited by ORC and then recruit mcm helicase.

60
Q

At the end of G1, what is made in DNA replication?

A

Mature pre-replication complex (pre-RC)

61
Q

How is DNA replication initiated?

A

By two kinases, cdk2-cyclin A which phosphorylates the initiator proteins and by DDK which phosphorylates mcm helicase to stimulate its activity.

62
Q

What occurs at the termination of S phase.

A

DNA replication.

63
Q

Draw DNA replication through the phases of the cell cycle.

A

refer to notes.

64
Q

What does mcm do?

A

helicase that opens up dsDNA.

65
Q

What does topoisomerase do?

A

relieve torsional strain.

66
Q

What does RPA do?

A

ss binding proteins that stabilize ssDNA

67
Q

What does PCNA do?

A

With help of clamp loader, keeps DNA polymerase from falling off DNA.

68
Q

What are sheltrins?

A

Complex that prevents NHEJ and chromosome fusion. Bind to telomere sequence.

69
Q

Draw the telomerase mechanism.

A

refer to notes.

70
Q

Draw the rad mutant experiment.

A

refer to notes.

71
Q

Describe XP.

A

Disease in which individuals are extremely sensitive to sunlight. Most of the mutated genes are identified rad genes. High rate of cancer in these individuals. Cannot use NER so use translesion DNA polymerase.

72
Q

Draw the NER mechanism.

A

refer to notes.

73
Q

What does XPC-Rad23 do?

A

scan DNA and recognize pyrimidine dimers.

74
Q

What does XPG do?

A

Nuclease used in the NER pathway that causes excision of the damaged strand.

75
Q

Describe the translesion repair pathway.

A

Dimer causes stalling for DNA and RNA polymerase. Translesion DNA polymerase is therefore recruited, gives best guess of base to bypass lesion. Does not have exonuclease activity.

76
Q

Draw the TCR pathway.

A

refer to notes.

77
Q

What is CS?

A

UV sensitivity, most UV-induced thymine dimers can still be repaired. CS cells show failure to resume transcription after UV radiation. CS cells lose the ability to preferentially repair DNA damage that occurs within genes.

78
Q

What is CSB?

A

identifies stalled RNA polymerase and recruits repair factors like XPG.

79
Q

Draw the NHEJ mechanism.

A

refer to notes.

80
Q

Draw the HR mechanism.

A

refer to notes.

81
Q

Draw the HR rescue of a broken replication fork.

A

refer to notes.

82
Q

Draw the meiotic recombination mechanism.

A

refer to notes.

83
Q

Draw the two site-specific recombination mechanisms.

A

refer to notes.

84
Q

What are the two ways to study a knockout using the cre/lox system.

A

Inducible expression or tissue-specific.

85
Q

What does TF2D do?

A

Binds to TATA box.

86
Q

What does TF2B do?

A

Bind to BRE sequence.

87
Q

What do both TF2B/D do?

A

Bring RNA polymerase to the promoter.

88
Q

What does TF2H do?

A

Helicase activity and kinase activity. Phosphorylates Ser-5 to allow for release of TFs needed for promoter recognition and to start elongation.

89
Q

What are the activator and mediator for?

A

Activator binds to enhancer region upstream, is gene-specififc TFs and mediator is a scaffold for association with other proteins, links general TFs with activators, chromating modifyign factors and histone modifying factors.

90
Q

How does the 5’ of the RNA transcript get capped?

A

Phosphorylation of Ser-5 by TF2H causes recruitment of capping protein that hops onto 5’ end and adds 7-methyl guanosine cap.

91
Q

Draw the splicing mechanism.

A

refer to notes.

92
Q

What does E1 do?

A

binds to donor site.

93
Q

What does E2 do?

A

Bind to Adenine branch site.

94
Q

What does E4-6 do?

A

mediate Adenine branch attack of 5’ site.

95
Q

What does EJC do?

A

Recruited by spliceosome before falling off. Marks splicing spot and together with 5’ cap recruits nuclear export receptor.

96
Q

Draw the polyadenylation mechanism.

A

refer to notes.

97
Q

What is done in the nucleus?

A

Noncoding RNAs are synthesized and processed in the nucleus.

98
Q

What is the nucleolus?

A

Ribosome producing factory. Region of nucleus containing rRNA genes and tRNA genes. Processing of non-coding RNAs occurs here.

Molecular Cell Biology (9 decks)

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