Module 6: Mechanisms of genetic variation and microbial DNA technologies

Question 1

what is screening

Answer 1

process whereby individual cells (or clone of cells that have grown into a colony) are checked one by one to determine if they have the mutant phenotype
- replica plating

Question 2

what is selecting

Answer 2

subjecting a population of cells to an environment condition that only allows for the growth/survival of the desired mutant phenotype
- a very large number of cells can be tested by the selection and only selected mutants will growth the colony

Question 3

what is an auxotroph

Answer 3

• unable to grow on a minimal media that the wild type parent can
• can now be used as the parent or starting strain from which to select for revertant strains that have undergone further mutations that afford the cell to further undergo growth in minimal media

Question 4

what is proof reading

Answer 4

10-100 fold reduction in errors rates while copying the genome

Question 5

what is a mismatched pair

Answer 5

• next line of defence
• repairs trails behind the advancing replication fork, and scans the newly formed double helix for mispaired bases

Question 6

what is an excision repair

Answer 6

a repair pathway that detects distortions in the DNA double helix
- it is not specific to any type of error, it is an effective and versitile repair system
- it excises (removes) a short sequence of DNA from one of the strands

Question 7

what is a base exsision repair

Answer 7

• carried out by a family of enzymes that each individually recognize specific unnatural bases within the DNA double helix
• enzymes are glycosylases, they remove damaged base from the sugar phosphate backbone, leaving an abasic site

Question 8

what is direct repair

Answer 8

• other enzymes have the ability to recognize specific changes to bases in te duplex and reverse those changes without removing the affected base or region

Question 9

what is recombinational repair

Answer 9

• when a cell carries more than one copy of the chromosome (which is the case for regions that have been replicated, even haploid bacteria)

Question 10

what is the SOS response

Answer 10

• is high alert response that coordinates multiple repaire pathways in their responses following a relatively large degree of damage to the cells DNA
• it is regulated by a common repressor protein (LEXa ) which is inactivated when RecA protein is bound to excess single strand DNA arising from stalled replication and/or incomplete DNA repair

Question 11

what is horizontal gene transfer

Answer 11

transfer of bits of genetic information from a non parent cell is given
- this process is not as frequent as the exchange that occur during sexual reproduction, it is very powerful in that it brings new genetic information and tools from multiple different selection and evolutionary experiences, within the recipient species having had to proceed through the same selection

Question 12

what is transposable element

Answer 12

• ## small DNA molecule that carries the genes for transposition (hopping to a new location) and can move around between genomes

Question 13

what is transposons

Answer 13

• more complex transosable elements that carry more than just their recombinase gene
• many bacterial transposons have been found that carry antibiotic

Question 14

what is recombination

Answer 14

bacteria is characterized by joining DNA from one DNA molecule (the donor) to another DNA molecules (the recipient)

Question 15

what is homogous recombination

Answer 15

two dna molecules that are very similar in nucleotide sequence. the mechanism depends on a homology search by recombination specific enzymes
- this is also a mechanism of DNA repair in bacteria

Question 16

what is site specific recombination

Answer 16

• happens between two DNA molecules that do not have extensive areas of homology
• is catalyzed by enzymes called recombinases = transposition occurs

Question 17

what is conjugation

Answer 17

• bacteria does not undergo sexual reproduction, they have mating system
  a number of bacteria plasmids have been identified that have the ability to transfer a copy of their DNA from a donor cell to recipient cell

Question 18

what is transformation

Answer 18

• the ability of an organism to take up foreign DNA from its environment, and if it shares sufficient homology with its chromosome, recomdining that DNA into its genome

Question 19

what is competent

Answer 19

• when a cell has turned on its DNA uptake machinery

Question 20

what is natural compentency

Answer 20

• carry the potentialn to turn on the competent function,

Question 21

what is transduction

Answer 21

• transfer of genes between bacterial or archaeal cells by viruses
• major source of horizontal gene transfer in nature

Question 22

what is generalized transduction

Answer 22

• random packaging of host DNA by a virus
• has the ability to randomly transfer any gene from the chromosome
• viruses that facilitate generalized transduction are typically lytic phages that are dedicated to the lifestyle of an infect (replicate, package and lyse (destroy the cell) repeat)

Question 23

what is specialized transduction

Answer 23

• gene transfer carried out by viruses that are lysogenic phages

Question 24

what is a restrictive enzyme

Answer 24

cut or break any DNA within the cell that was unmethylated at those same specific DNA sequences
- DNA endonucleases

Question 25

how do you purify DNA

Answer 25

• amplification (replications of cells carrying only that clone)
• purification of the vector with the insert (creating a solution with many copies of the same DNA)
• cutting that DNA with a specific restrictive enzyme, or combination of restrictive enzymes
• separation of the created fragment based on their size
• collection of the fragment of interst for further analysis

Question 26

what is gel electrophoresis

Answer 26

separating DNA molecule based on size (length)
- typically agarose or polyacrylamide gel,
- the pores within the Gel allow for movement of the negatively charged DNA molecules towards the positive pole

Question 27

what is reverse transcriptase (RT)

Answer 27

does the reverse of transcription - it starts with an RNA template and makes a complementary DNA or cDNA copy

Question 28

what are cloning vectors

Answer 28

-small piece of self replicating DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted
- plasmid vectors, phage and other viral vectors, cosmids and artificial chromosomes

Question 29

what is a genomic libraries

Answer 29

• collections of ramdomly - fragmented pieces, each individually ligated into a copy of the cloning vector and trasnformed into the host cell