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Drug Discovery (2037A) > Block 2 Exam Study > Flashcards

Block 2 Exam Study Flashcards

1
Q

How does chromosomal DNA of prokaryotes differ from that of eukaryotes in terms of structure?

A

Prokaryotic chromosomal DNA is not enclosed by a nuclear membrane. Instead, the prokaryotic genome exists as a single circular DNA molecule (chromosome) bound by proteins to form a nucleoid.

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2
Q

Discuss the differences in genome size and DNA content of prokaryotic vs eukaryotic genomes?

A

Prokaryotic genomes are smaller (less than 5 million base pairs) and have fewer genes (less than 5000 genes) than eukaryotic genomes, however, they have a higher % of genes per genome size.

Eukaryotic genomes are larger and have more genes than prokaryotic genomes, however, they have a lower % of genes per genome size.

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3
Q

True or false: Genome size is indicates complexity of organism?

A

False! Genome size does not necessarily indicate organism complexity.

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4
Q

Do eukaryotes or prokaryotes contain more non-coding DNA?

A

Eukaryotes contain more non-coding DNA than prokaryotes.

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5
Q

True or false? In eukaryotes, the percentage of protein-coding genes in the genome decrease as the genome grows larger.

A

True! In eukaryotes, the percentage of protein-coding genes in the genome decrease as the genome grows larger.

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6
Q

True or false? In prokaryotes, the number of protein-coding genes determines genome size diversity.

A

True! In prokaryotes, the number of protein-coding genes determines genome size diversity.

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7
Q

What are some factors that can account for the larger genome size of eukaryotes?

A
  1. Whole-genome duplications.
  2. Repetitive DNA sequences.
  3. Centromeres.
  4. Gene regulatory elements.
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8
Q

Can a prokaryote function without plasmids?

A

Yes, a prokaryote can function without plasmids.

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9
Q

What are plasmids?

A

Plasmids are small, circular, independently replicating, extrachromosomal DNA molecules.

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10
Q

How do prokaryotes fit their DNA into such a tiny space?

A

Prokaryotes are able to fit their DNA into such a tiny space by making use of DNA supercoiling.

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11
Q

Name the enzymes and the protein involved in DNA supercoiling:

A

Enzymes: DNA gyrase and DNA topoisomerase I.

HU protein, has a similar function to eukaryotic histones.

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12
Q

What is an operon?

A

An operon is a region of DNA that contains multiple co-regulated genes.

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13
Q

What do operons enable?

A

Operons enable the simultaneous regulation of a gene cluster as the genes share a common promotor and repressor.

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14
Q

What is the function of the CAP site?

A

The CAP site promotes the binding of the RNA polymerase to the operon.

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15
Q

What is the function of the repressor?

A

The repressor binds to the operator and blocks the RNA polymerase from binding to the genes.

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16
Q

What are co-repressors and inducers?

A

Co-repressors are small molecules that bind to repressors in order for them to function.

Inducers are small molecules that bind to activators in order for them to function.

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17
Q

How do eukaryotic and prokaryotic chromosomes differ?

A

Eukaryotic chromosomes are linear whilst prokaryotic chromosomes are circular.

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18
Q

What is extrachromosomal DNA?

A

Extrachromosomal DNA is DNA that is found outside of the nucleus.

Can be found in mitochondria and chloroplasts.

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19
Q

Discuss the structure of human mitochondrial DNA.

A

Human mitochondrial DNA is a single, circular, double-stranded DNA molecule.

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20
Q

In humans, what are the only non diploid cells (2n)?

A

Gametes are the only haploid (n) cells in humans.

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21
Q

What is a nucleosome?

A

A nucleosome is a structural unit of the eukaryotic chromosome, consisting of a length of DNA tightly coiled around a histone octamer.

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22
Q

How long is each chromosome?

What is the length of the whole diploid genome?

What is the diameter of the nucleus?

What do these measurements mean?

A

Chromosomes are 5cm long, the whole diploid genome is 2m long, and the diameter of the nucleus is 10 um.

Therefore, in order to fit inside the nucleus, the DNA is tightly wound around 8 histones to form a nucleosome.

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23
Q

What are the 4 histones involved in the histone octamer of the nucleosome?

A

2x H2A

2x H2B

2x H3

2x H4

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24
Q

What is the function of the H1 histone in DNA packaging?

A

H1 is an additional protein that winds 20 bp of DNA to form 2 full turns (rather than 1.65) of DNA around the octamer, creating the chromatosome.

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25
Q

What is the chromatosome?

A

Nucleosome + H1 = chromatosome.

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26
Q

What is the function of histone modifications when it comes to genome access?

A

Histone modifications alter the structure of the nucleosome, allowing for gene expression, gene repair, genome replication, and genome repression.

Histone modifications include acetylation, methylation, phosphorylation, and ubiquitation.

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27
Q

What is nucleosome remodeling?

A

Nucleosome remodeling refers to the repositioning of a nucleosome along a strand of DNA in order to facilitate access to different regions of a chromosome.

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28
Q

What are the three types of nucleosome remodeling?

A
  1. Repositioning: The structure of the nucleosome is altered but the position is not.
  2. Sliding: The nucleosome slides along the DNA in cis.
  3. Transfer or trans-placement: The nucleosome is transferred to another DNA strand or to an adjacent part of the same DNA.
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29
Q

What is a clone?

A

A clone is a copied DNA fragment.

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30
Q

Distinguish between the enzymes endonuclease and exonuclease?

A

Endonuclease enzymes cleave DNA in the middle of a region/sequence, whilst exonuclease enzymes cleave DNA at the end of a fragment.

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31
Q

Restriction enzymes are a type of…?

A

Restriction enzymes are a type of endonuclease.

32
Q

What is DNA ligase?

A

DNA ligase is a type of enzyme that can join together ends of DNA.

33
Q

What is recombinant DNA technology used for?

A

Recombinant DNA technology is used to isolate, replicate, and analyze genes

34
Q

What is the recognition site?

A

The recognition site is the sequence of bases that are identified by restriction enzymes for cutting DNA at a specific site.

35
Q

What is a palindrome?

A

A palindrome is a symmetrical sequence of DNA that reads the same on both strands in the 5’ to 3’ direction.

-Posses a 2-fold rotational system that enables them to form cruciform structures to which restriction enzymes and other proteins can bind.

36
Q

Why are we interested in type 2 restriction enzymes?

A

We are interested in type 2 restriction enzymes as they cleave DNA at specific positions close to or within a recognition sequence.

37
Q

What is RFLP (restriction fragment length polymorphism)?

A

RFLPs are the differences among individuals in the lengths of their DNA cut by restriction enzymes. (Differences in DNA fingerprint)

38
Q

What are some useful applications of RFLP analysis?

A

Paternity testing.

Forensics.

Detection of certain genetic diseases.

Genotyping.

39
Q

What is PCR (polymerase chain reaction)?

A

PCR is a technique used to amplify specific regions of DNA.

40
Q

What is restriction mapping?

A

Restriction mapping is a technique used to identify the position of restriction sites on a cloned fragment of DNA.

Establishes the number of, order of, and distance between restriction enzyme cleavage sites of a cloned fragment of DNA

41
Q

How is restriction mapping achieved?

A

Restriction mapping is achieved by cutting DNA with different restriction enzymes and separating DNA fragments by size with gel electrophoresis.

42
Q

What is molecular cloning?

A

Molecular cloning is a technique used to create many copies of a DNA fragment or protein.

43
Q

What are inserts?

A

inserts are genes of interest that we wish to clone and study further.

44
Q

What are vectors?

A

vectors are carrier DNA molecules capable of independent replication.

45
Q

Plasmids used for molecular cloning must have?

A

Plasmids used for molecular cloning must have an origin of replication and a multiple cloning site.

46
Q

What is the multiple cloning site?

A

The multiple cloning site is a restriction site for commonly used restriction enzymes.

47
Q

What is non-directional cloning?

A

Non-directional cloning refers to when the DNA fragment can be inserted into the vector in any orientation.

Eg, TA cloning, blunt-end cloning, and single restriction digests.

48
Q

What is directional cloning?

A

Directional cloning is when the DNA fragment ligates into the vector in a specific orientation.

Eg. Double digest.

49
Q

Discuss TA cloning:

A
  • Taq DNA polymerase leaves 3’ A overhangs.
  • Use of vector containing 3’ T overhangs.
  • Non-directional cloning.
  • Can only be used when products are amplified with Taq DNA polymerase.
50
Q

Discuss blunt-end cloning:

A

Insert blunt ends ligate to vector blunt ends.

  • Non-directional cloning.
  • Used when you are unable to find appropriate restriction enzyme sites on the insert DNA.
  • Use a high fidelity enzyme that does not leave overhangs.
51
Q

What is transformation and what are the two methods with which to achieve it?

A

Transformation is the insertion of a recombinant DNA fragment into a bacterial host.

  • Heat shock: CaCl makes cell walls permeable, and a brief heat shock pulses DNA into the cell.
  • Electroporation: A brief high intensity electricity shock pulses DNA into the cell.
52
Q

How does positive selection work?

A

Positive selection works by using a lethal gene, therefore only plasmids containing the disrupted gene will grow, indicating recombinant DNA.

53
Q

What is a lethal gene?

A

A lethal gene is a gene encoding a restriction enzyme that will cleave host DNA.

54
Q

What is capillary electrophoresis?

A

Capillary electrophoresis is a method used to separate negatively charged DNA fragments based on size and electrophoretic mobility. Larger DNA fragments will pass through the capillary tube slower as they have lower electrophoretic mobility.

55
Q

What is thermostability?

A

Thermostability refers to the stability of enzymes at high temperatures.

56
Q

What is processivity?

A

Processivity refers to the number of DNA nucleotides a polymerase can add before disassociating from a template strand.

57
Q

What is polymerase fidelity?

A

Polymerase fidelity refers to the ability of a polymerase to add the correct nucleotides onto the growing polynucleotide chain.

58
Q

What is polymerase specificity?

A

Polymerase specificity refers to the ability of the polymerase to amplify the correct DNA fragment of interest.

59
Q

What are the five characteristics of DNA polymerases that regulate their capabilities in a PCR reaction?

A

Five characteristics of DNA polymerases that regulate their capabilities in a PCR reaction include:

  • thermostability
  • processivity
  • speed
  • fidelity
  • specificity
60
Q

What are primers?

A

Primers are short, artificially produced oligonucleotides that are designed to be complementary to the target DNA fragment.

61
Q

Discuss the forward and reverse primers involved in PCR.

A

The forward primer is the exact same sequence as the first 20-25 nucleotides of the antisense strand of the fragment that you wish to amplify.

The reverse primer binds to the sense strand and it is the reverse complement of the last 20-25 nucleotides. eg. ATTG = CAAT

62
Q

What is innate immunity?

A

Innate immunity refers to an internal system that can be used to defend against foreign pathogens.

63
Q

What is adaptive immunity?

A

Adaptive immunity refers to a kind of immunity that can change when exposed to new pathogens.

64
Q

Is CRISP a form of innate or adaptive immunity?

A

CRISPR is a form of adaptive immunity.

65
Q

What is CRISPR?

A

CRISPR is a genomic locus in bacteria that contains CLUSTERED, REGULARLY INTERSPACED, SHORT, PALINDROMIC REPEATS.

66
Q

What are CRISPR spacer sequences?

A

CRISPR spacer fragments are segments of DNA that are identical to fragments of bacteriophage genomes, thus allowing them to serve as a molecular memory of previous viral attacks.

67
Q

What is the mechanism of CRISPR?

A

RNA guided destruction of invading DNA.

68
Q

What are Cas genes?

A

Cas genes encode Cas proteins that function as DNases and RNases.

69
Q

What is the CRISPR-Cas mechanism of adaptive immunity in prokaryotes?

A
  1. Spacer acquisition.
  2. crRNA biogenesis.
  3. Target interference.
70
Q

What are protospacers?

A

Protospacers are smaller fragments of invading phage DNA.

71
Q

Discuss the process of spacer acquisition.

A

Spacer acquisition:

Invading phage DNA is cleaved into protospacers, which are then inserted into CRISPR loci to become new spacers.

72
Q

Describe the process of crRNA biogenesis.

A

crRNA biogenesis:

CRISPR loci are transcribed, starting at the promoter within the leader, into long RNA transcripts. These RNA transcripts are then processed into crRNAs (CRISPR-derived RNAs), each containing a single spacer flanked on both sides by repeat sequences.

73
Q

Describe the process of target interference:

A

Target interference:

Mature crRNAs associate with Cas nucleases, or nuclease complexes, and recruit them to complementary sequences in invading phage DNA. Cas nucleases then cleave the viral DNA, thus neutralizing infection.

74
Q

Which types require multi-subunit protein complexes and which type requires only Cas9?

A

Type II = Only Cas9

Type I and Type III = multi-subunit protein complexes.

75
Q

Cas9 plays a role in…?

A

Cas9 plays a role in spacer acquisition and crRNA biogenesis.

76
Q

Cas9 has 2 nuclease domains that cleave the target DNA sequence, what are they and what do they cleave?

A
  1. HNH domain cleaves the viral strand that is complementary to the crRNA.
  2. RuvC domain cleaves the non-complimentary strand.
77
Q

Cas9 will only cleave DNA if the target is adjacent to what?

A

Cas9 will only cleave DNA if the target is adjacent to a PAM sequence.

PAM = protospacer adjacent motif (5’ - NGG - 3’)

Drug Discovery (2037A) (4 decks)

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