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Micro Genetics > Test 4 - Final > Flashcards

Test 4 - Final Flashcards

0
Q

A recipient cell containing donor DNA

A

Transformant

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1
Q

The direct uptake of exogenous genetic material

A

Transformation

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2
Q

Describe the two stages of transformation. (very unclear from notes)

A
  1. Competence - likely due to changes in the cell wall that increase the likelihood of DNA uptake by the cell. Influenced by type of media, stage of growth, degree of aeration and the secretion of competence factors (aka pheromones).
  2. DNA uptake - DNA enters the cell. Different for Gram + vs -. (see handout). Results of uptake: 1) Plasmid that circularizes. 2) ssDNA undergoes homologous recombination. 3) The DNA is degraded in the cell.
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3
Q

An culture that will readily uptake DNA.

A

competent cells

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4
Q

A competence system component that binds to the DNA and pulls it into the cell.

A

pseudopilus

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5
Q

A sequence on own DNA that codes for receptors for the uptake of exogenous DNA.

A

uptake sequences

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6
Q

Proteins that activate the rest of the cells to become competent

A

Competence factors (pheromones)

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7
Q

Two stages of DNA uptake. (Don’t know what each is though???)

A
  1. Brief, reversible binding stage

2. Longer, irreversible stage

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8
Q

Describe Gram negative transformation via a transformisome.

A

A transformasome forms as a buldge of cytoplasmic membrane through the cell wall and dsDNA is taken in. As the dsDNA enters the main cell area, nuclease cuts it into ssDNA. SSB or RecA will coat the ssDNA. At this point it can either circularize (using homologous recombination), or integrate into cell’s genome (using homologous recombination), or be degraded.

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9
Q

Gram positive vs Gram negative transformation

A

Gram negative - 2 methods, either via pseudopilus or via transformisome.

Gram positive - 1 method, competence factor binds DNA, pore forms in peptidoglycan and a pseudopilus pulls the DNA through the cell wall.

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10
Q

A set of genes that aid in the formation of a pseudopilus

A

Com genes

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11
Q

An enzyme that cuts dsDNA into ssDNA as it enters the cell during transformation.

A

nuclease

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12
Q

Describe Induced vs Natural transformation

A

Induced - Manipulation in the lab. Includes calcium ion induction, electroporation, protoplasting, and heat/cold shock.

Natural - Transformation found in nature. Increases genetic variability. The availability of “self” DNA aids in repair of own damaged DNA and reduces problems associated with foreign DNA.

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13
Q

Nucleotide sequences are exchanged between two similar molecules of DNA.

A

Homologous recombination

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14
Q

Describe why homologous recombination is beneficial.

A

Maintain cell viability by repairing damaged DNA.

Provides a basis for genetic diversity.

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15
Q

A group of proteins that act at the Holliday Junction of a crossing over event.

A

Ruv proteins

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16
Q

An event wherein two similar strands of DNA begin an exchange of sequences.

A

Crossing over

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17
Q

The outcome or the name of a completed crossing over event.

A

recombination event

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19
Q

Describe the 3 steps of reaction of RecA in the homologous recombination model.

A
  1. Presynaptic binding - coats ssDNA with RecA. Requires ATP. SSB removes secondary structure.
  2. Synapsis (conjunction) - RecA/ssDNA forms a complex with an elongated dsDNA along its major grove. The sequences move along until homologous sequences are lined up, (homologous allignment). 2 joints can be formed, paranemic or plectonemic.
  3. Postsynaptic strand exchange (heteroduplex extension) - RecA promotes displacement of dsDNA and replacement with the new strand. Unclear mechanism.
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19
Q

Describe what the RecBCD complex does.

A

Activates 3’ to 5’ exonuclease to eat away at one strand in preparation for crossing over event. Exonuclease stops at the chi sequence.
Activates 5’ to 3’ exonuclease and helicase to prepare the DS break end for homologous recombination. Exonuclease increases at the chi sequence to create overhang where RecA will attach in preparation for the actual Homologous Recombination event (Depicted in handout Figure 9.6)

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20
Q

proteins of a multifunctional complex that play a key role in homologous recombination model.

A

Rec BCD protein complex

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21
Q

Describe some ways in which Homologous Recombination can be used as a repair mechanism to stalled or collapsed forks.

A

Collapsed fork - create a new replication fork by strand invasion homologous recombination. Forms a Holliday Junction that when resolved reforms the replication fork via resolving enzymes.

Stalled fork - formation of reverse replication to create a new strand so that new repaired piece of the strand can be spliced and to restart the fork via branch migration and template switching. (doesn’t make much sense)

Dinner fork - not used in DNA repair.

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22
Q

Holliday model vs Homologous Recombination model.
(do they both occur? it looks like one is a single strand invasion and the other is a double strand crossing over…maybe.)

A

Holliday - a junction is formed wherein the dsDNA molecules are held together by crossed over strands and Ruv proteins.

Homologous Recombination - Break two DNA molecules in the same region where they are similar and join one to the other in a crossing over event.

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23
Q

A region of complementary bases within a DNA molecule wherein each strand is contributed by one of two parent DNA molecules.

A

Heteroduplex

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24
Q

Describe how we know that certain enzymes play a role in Homologous Recombination and repair. (question he said in class was a good test question so I made a card.)

A

We can detect which enzymes get turned on then mutate the genes for those enzymes. If we do a deletion mutation which inactivates the enzyme, and no repair occurs, we will know that that specific enzyme must have been involved.

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25
Q

What 3 things were key to the discovery and emergence of the first recombinant DNA technology experiments? Describe why each is important.

A
  1. Plasmids - Found while trying to find reasons for antibiotic resistance.
  2. Ligase - Found while trying to figure out how DNA is replicated. Puts open SS ends back together after use of EcoR1.
  3. Restriction Endonucleases - Found while trying to figure out purpose of DNA methylation. EcoR1 creates open SS ends that can be put back together with ligase.
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26
Q

Side note - go to chapter 10 in the book. It is much more clear than his notes and has some color to the figures he used in his handouts. The book was more clear on homologous recombination.

A

Did you get your book out?…did you?… … … you didn’t.

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27
Q

What properties does a vector need in order to transfer genes?

A

Able to enter host
Able to replicate in host (ori region)
Selectable marker genes for identification (resistance)

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28
Q

A gene transfer vector that can accept large DNA fragments

A

λ phage

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29
Q

A gene transfer vector with unique promoters that aid in DNA sequencing.

A

M13 phage

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30
Q

A gene transfer vector that contains antibiotic resistance genes, an ori site, polylinkers, etc…
(give an example)

A

Plasmids. pBR322

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31
Q

Describe the method for the use of insertional inactivation for the identification of clones.

A
  1. Cut and anneal a plasmid with foreign DNA. Results in re-annealed original plasmid, or desired plasmid containing foreign DNA inserted into and inactivating the tetracycline resistant gene.
  2. Cause a transformation event.
  3. Grow cells on Ampicillin, all cells will grow.
  4. Replica plate cells on Tetracycline, cells containing foreign DNA will not grow due to insertional inactivation.
  5. Isolate colonies that grew on ampicillin but not on tetracycline. These contain the cloned, foreign DNA.
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32
Q

Describe the α-complementation system (aka Xgal or lacZ system).

A
  1. Ligate and anneal a lacZ-containing plasmid with foreign DNA. Either get re-annealed original plasmid, or plasmid with foreign DNA causing insertional inactivation of lacZ region.
  2. Plate on agar containing Ampicillin so only transformants will grow.
  3. Agar also contains IPTG and X-gal.
  4. Cells with functional lacZ - lacZ induced by IPTG to cleave Xgal, which makes the colony blue.
  5. Cells with inactivated lacZ will not be induced and white colonies will grow. These contain the desired, cloned, foreign DNA.
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33
Q

A sulfur analog of lactose

A

IPTG

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34
Q

A portion of genetic sequence inserted into a plasmid which contains unique restriction enzyme cutting sites for use in inserting foreign DNA.

A

Multiple polylinker molecule

35
Q

Describe the power of metagenomics and the use of PCR in microorganism identification.

A

Plating a sample of mud from the Great Salt Lake and run the Xgal lab protocol on it. It will return plasmids with foreign 16s sequences inserted into it. If you take the same mud sample and harvest all of the DNA from it, run a PCR, and send it off to be sequenced, you can potentially receive hundreds of 16s sequences that did not grow on the plate. This is precisely how we know that there are so many microbes out there that cannot be grown in the lab but that do exist.

36
Q

A type of vector, engineered in E. coli, used for transferring genes into more complex eukaryotic cells such as yeast.

A

Shuttle Vector

37
Q

Uses a reporter gene connected to an inducible promoter site to examine the promoter’s regulation of that gene.

A

Expression Vector

we used Xgal as the inducible promoter and IPTG to promote it. Blue colonies showed expression of the gene

38
Q

Different organisms and enzymes that cut the same site but may generate a different cut.

A

Isoschizomers

39
Q

Cuts at palindromic sequences

A

Type II Restriction Endonuclease

40
Q

Method to determine where a RE cuts DNA relative to other RE.

A

Restriction Mapping

41
Q

joins blunt ended DNA fragments

A

T4 phage ligase

42
Q

Method of joining blunt end DNA into a plasmid form by adding multiple poly A and poly T tails to the separate strands then joining the tails together.

A

Homopolymer tailing

43
Q

The use of a T4 ligase to join two blunt ends of DNA together.

A

Blunt End Ligation

44
Q

Used to ensure palindromic sequences are inserted during blunt end ligation. (So we can cut the plasmid again once it is made)

A

Linker or Polylinker

45
Q

DNA created by reverse transcriptase activity

A

cDNA

46
Q

Describe the process of cDNA creation.

A
  1. Primer annealed to mRNA
  2. DNA copy made with reverse transcriptase
  3. Alkali treatment removes mRNA
  4. DNA polymerase extends hook back into complementary strand
  5. Treat with S1 nuclease to remove hook
47
Q

A list of cDNA within a cell composed of coding regions that are searchable. Used in looking for genes or proteins of interest.

A

cDNA library

48
Q

Describe ways to detect recombinant molecules

A

Insertional inactivation - Inactivate one of 2 resistance genes and grow on two different antibiotic media to see which has the genes inserted.

Cosmids - plasmids that cary the COS site from λ phage. (read handout). The power of this is that the camel DNA used contains both COS sites, one on either side of the desired sequence. It will be cut at both COS sites and the sequence EXACTLY fits into the phage, and NOTHING else.

Another method dealing with cohesive ends that doesn’t make sense to me.

49
Q

Pulled glass pipette method used to directly insert DNA into cells.

A

Microinjection

50
Q

Artificial membranes which fuse to host cell membrane, used in transfer of DNA into host cell.

A

Liposomes

51
Q

Method used to shoot DNA into a cell with gas and microprojectiles made of gold covered in charged silicon and DNA.

A

Gene gun

52
Q

Used to detect protein production by the use of radioactive antibodies.

A

Radioactive antibody test

53
Q

Discovered PCR

A

Kary Mullis (mullet?)

54
Q

What is required for a PCR reaction?

A

Primers
Thermocycler
dNTPs
Taq Polymerase - harvested from hot pots extremophiles

55
Q

Name 5 uses of PCR.

A 
Make copies of genes for 16s rRNA sequencing
Identify rare genes
Site directed mutagenesis
Environmental surveying
DNA sequencing technology
56
Q

Alteration of an embryo so that when it fully matures, the animal creates a desired protein product. (ex. goat making insulin)

A

Pharming

57
Q

The replacement of bad genes in a eukaryotic host, usually human.

A

Gene therapy

58
Q

A short DNA tag, added to an ORF, recognized by an antibody probe once it has been transcribed.

A

Epitope tag

59
Q

Describe the Sanger Method of DNA sequencing.

A

Use of PCR to create numerous segments of entire genome. Altered nucleotides (ddNTP) are mixed in with normal nucleotides which terminate the sequences at varying locations when incorporated. Done in 4 separate tubes with one ddNTP per tube. Various PCR sequences are compared and a reading is made by reading the terminal base of each of the PCR products. Doing this with all 4 tubes, and comparing chain lengths and their terminal ends, tells you the entire sequence.

60
Q

Describe pyrosequencing

A

Short sequences done in parallel at the same time. Uses pyrophosphate and is read as PCR is occuring. Very fast but like putting a puzzle together at the end. (Find more info about this to clarify)

61
Q

What is it called where they run the entire genome through a capillary tube with florescent dyes attached to each different type of base. It is ran past a laser and read by the computer.

A

Is it capillary electrophoresis?

62
Q

A group of overlapping clones representing a region of DNA, used in assembling of sequenced data.

A

Contigs

63
Q

How do you tell where a gene is on a sequence?

A

Look for an ORF that has a start codon, or a ribosome binding site, and a stop codon. In Eukaryotes, search for exon/intron boundaries.

64
Q

Describe the two methods for entire genome sequencing.

A
  1. Clone by clone approach - create thousands of clones and sequence portions of the genome from each of the clones. Very slow but reliable.
  2. Whole genome shotgun - generate random pieces of DNA and trying to piece them back together. Very fast but difficult to assemble.
65
Q

The use of computational tools to acquire, analyze, store, and access DNA and protein sequences

A

Bioinformatics

66
Q

The total complement of genetic information of a cell or virus

A

Genome

67
Q

total set of proteins encoded by a genome or the total protein complement of an organism.

A

Proteome

68
Q

Every protein made under specific conditions

A

Translatome

69
Q

The complement of all RNA produced by an organism under a specific set of conditions

A

Transcriptome

70
Q

Oligonucleotide for each gene put on a solid matrix, hybridization between mRNA and the specific nucleotide.

A

Microarrays (gene chips)

71
Q

Uses of microarrays (gene chips)

A

Global gene expression
Comparison of genes in closely related organisms
Assess microbial diversity
Assess metabolic methods

72
Q

Total complement of small molecules and metabolic intermediates produced by a cell or organism, determined by mass spec.

A

Metabolome

73
Q

Total set of interactions between proteins or other macromolecules in an organism, network diagrams of proteins with interacting proteins connected.

A

Interactome

74
Q

total complement of organisms present in a particular environment, can reveal uncultured or unculturable organisms.

A

Metagenome

75
Q

Area of study that maps, sequences, analyzes, and compares genomes.

A

Genomics

76
Q

Area of genome-wide study of the structure, function, and regulation of the proteins of an organism

A

Proteomics

77
Q

Genomic analysis of pooled DNA or RNA from an environmental sample containing organisms that have not been isolated.

A

Metagenomics

78
Q

The determination of gene expression by an organism under a specific set of conditions.

A

Transcriptomics

79
Q

Open reading frame

A

ORF

80
Q

putting the DNA sequences in the correct order

A

Genome assembly

81
Q

process of identifying genes and functional regions on the genome

A

Annotation

82
Q

comparison of sequence data to all known sequences

A

BLAST search

83
Q

collection of prokaryotic cells associated with another organism, often in reference to humans.

A

Microbiome

84
Q

One electrophoresis direction separates proteins by pH, another direction of electrophoresis separates proteins by size.

A

2D gel electrophoresis

85
Q

A homologous recombination joint wherein DNA is not entwined

A

Paranemic (unstable)

86
Q

A homologous recombination joint that is double helical with free a free end joined

A

Plectonemic (stable)

Micro Genetics (2 decks)

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