Microbiology Lecture 19: Microbial Genetics

Front 1

Transfection involves the introduction of bacteriophage DNA into a cell. Describe the process.

Why is it a useful process?

Back 1

Treat the cells with CaCl2, infect them with DNA that has been purified from bacteriophage.

Useful because a large amount of DNA can be put into a bacteriophage head.

Front 2

What is a bacterial test to screen for carcinogens that cause mutations in bacteria?

It measures the reversion of histidine BLANK in Salmonella typhimurium to histidine BLANK.

Describe the test and one commerical area where it is used.

Back 2

Ames Test

auxotrophs to prototrophs

The test involves feeding suspected carcinogens to rats, removing their livers and providing the extract to his- Salmonella typhimurium with the idea that the carcinogen will cause a mutation in the bacterial strain to allow it to grow without requiring the addition of histidine to the medium. Used in comestic production testing.

Front 3

What is a mutation?

What is mutagensis?

What are mutants?

What is a mutagen?

Back 3

Mutation: inheritable change in the sequence of DNA; in Latin: to change

Mutagenesis: creating mutations

Mutants: Organisms that have undergone mutation, phenotypic change in the organism. The original form of the organism is the wild type.

Mutagen: a physical or chemical agent that changes the DNA of an organism and thus increases the frequency of mutations .

Front 4

What is the difference between spontaneous and induced mutations?

Back 4

Spontaneous mutation: can occur naturally, usually as the of errors in DNA replication.

Induced mutations: result of chemical or radiation impact on genes. Usually increases the mutation rate.

Front 5

Wild type bacteria are isolated from soil or water. What are some mutants of Pseudomonas aeruginosa?

Back 5

PAO1c-->mutagen-->PAO2 (serine) or PAO25 (arginine) (auxotrophs)

PAO7: histidine (his^-)

Front 6

--Base substitution--
1. BLANK mutations involve a single substitution in a base. If this occurs in the third position of the codon, it is a BLANK mutation. What the effect?

2. BLANK mutations are substitutions in either the #1 or #2, position of the codon. What is the effect?

3. BLANK mutation results in the formation of a nonsense codon. What is the effect?

Back 6

Point; silent; No effect on the amino acid coded by the affected area of the DNA.

Missense; Consequences vary from protein to protein.

Nonsense; this may result in premature termination of protein synthesis

Front 7

What do you call mutations where segments of the genome are removed?

What do you call mutations when one or more bases are added to DNA?

Back 7

Deletions

Insertions

Front 8

Transposons aka BLANK genes: encode BLANK resistance; transposons can be used to create BLANK.

Back 8

jumping

antibiotic

mutants (mutagenesis)

Front 9

What do you call single base deletions or insertions that could completely change the amino acid sequence of the protein starting at the point of the mutation?

Back 9

Reading frameshifts

Front 10

What is a reversible effect that occurs when mutated base is changed back into its original form?

What about a reversible effect that overcome or suppress the effects of the initial mutation without alteration in the original gene? Name an example and how anticodon-tRNA is involved

Back 10

Back mutation

Suppressor mutations

Ex: codon changed GCU to ACU, the new codon codes for threonine instead of alanine, however if the anticodon-tRNA is also changed, the effects of the original mutation are suppressed.

Front 11

What are mutagenic agents? Why does exposure leads to a higher incidence of cancer?

What is a mutagenic agent that causes the formation of pyrimidine dimers in chromosomes and distorts the helix, causing bulges?
What effect does it have?

Back 11

Mutagenic agents increase the rate of mutation; many are carcinogens

UV radiation; Interferes with DNA replication and transcription.

Front 12

What is a mutagenic agent that is a base analog, or chemical, which can substitute for thymine?

Why is this bad and what does it lead to?

Back 12

5-bromouracil in cancer drugs

This is bad since it will bind with guanine instead of adenine. The incorrect pairing leads to errors in replication.

Front 13

Mutagenic agents can cause transitions or transversions in DNA. Define both.

Back 13

Transitions: replacement of a purine by a different purine or a pyrimidine for a different pyrimidine.

Transversions: a purine replaced by a different pyrimidine.

Front 14

1. What is a mutagenic agent that is an alkylating agent that acts at the DNA replication fork causes crosslinking of the DNA strands and errors in replication?

2. This agent creates BLANK BLANK but is dangerous.

3. Best way to get microbes to...

4. You can also find mutants with this agent by...

Back 14

N-methyl-N’-nitro-N-nitrosoguanidine, (NTG)

stable mutants

produce mutants

designing media that requires the amino acid

Front 15

What is a mutagenic agent that deaminates DNA?

What is a mutagenic agent that distorts the helix and causes frameshift mutation?

Back 15

Nitrous acid

acridine dye

Front 16

Why is UV not a useful mutagenic agent?

Why are X-rays and gamma rays not very useful for creating mutants?

Back 16

Microbes can fix the bulges caused by UV damage

They usually lethal for microbes.

Front 17

Excision repair (nucleotide excision) is a DNA repair mechanisms in bacteria that...

For example, a T-T dimers from UV radiation is cut out on both sides by BLANK endonuclease. The excision results in release of...

The ssDNA gap is filled by...

Back 17

corrects damage that causes distortion in the DNA helix.

UvrABC; a 12 nucleotide ssDNA segment containing the damaged bases.

polymerase I and DNA ligase joining the fragments.

Front 18

What is another example of DNA excision repair endonuclease (besides UvrABC)?

Give a four step summary of what it does.

Back 18

UvrABCD

UvrABCD endonucleases cut damaged DNA and release damaged bases

UvrD: helicase

ssDNA gap is replaced with helicase and DNA poly I

Ligase does the final DNA joining

Front 19

What is a DNA repair mechanism in bacteria where a light activated enzyme, BLANK, cleaves the covalent linkages between damaged pyrimidines? What gene encodes it?


This repair does not...

Back 19

Photoreactivation; photolyase; gene phrB

require removal and replacement as in excision repair.

Front 20

What is a DNA repair mechanism in bacteria where bases are removed by enzymes called BLANK that detect and remove the unnatural base?

The resulting hole is called an...

DNA polymerase I BLANK the bases, DNA ligase BLANK the fragments.

Back 20

Base excision; glycosylases

AP site, (apyrimidinic or apurinic) depending on the base removed

removes; joins

Front 21

What is a DNA repair mechanism where if the DNA damage is very severe(i.e. large gaps in the helix), 17 genes become involved in excision repair of DNA damage?

Expression of these genes is controlled by...

Back 21

SOS response

the lexA and recA genes.

Front 22

What is a method used to detect auxotrophs from the prototrophs (wild type strain)?

Provide a short summary

Back 22

Replica Plating

1. One media containing everything (draw circle)
2. Media 2 lacks serine (draw another circle)
3. Short streaks on both plates; ex: growing on plate 1, and no growth on plate 2; this indicates it is probably a serine mutant
4. Used to figure out the molecule that is lacking.

Front 23

What are the three methods of transfer of genetic material in bacteria?

Briefly describe each and give an example of its use.

Back 23

1. Transformation: insertion of naked DNA from a donor cell into a recipient cell.
Ex: different genera can take one strain and incorporate it into their own; this is bad because it leads to antibiotic resistance.

2. Transduction: bacterial virus (phage) is the vector of the transfer of the DNA material
Ex: how diptheria works (cornebacterium)

3. Conjugation: bacterial DNA transfer that involves contact between a donor and recipient cell.
Ex: mating between PAO2 and PAO25 (conjugation) on one plate and look for transconjugatants.

Front 24

Transformation was first seen by BLANK in 1928 in...

Natural transformation involving DNA occurs in some bacteria like...

Cells are transformable usually in late BLANK phase of growth and only a BLANK portion transferred. When the recipient is in the proper physiological state to take up the DNA it is said to be BLANK

This physiological state usually relates to...

Back 24

Griffith; S. pneumoniae

streptococcus, bacillus, acinetobacter (soil), and haemophilus (pneuomonia); (first two are G+, last two G-)

log; small; competent

a surface factor on the recipient, low MW protein.

Front 25

BLANK transformation is when a cell lyses and a closely related recipient takes up the DNA when it is competent.

Back 25

Natural transformation

Front 26

Take a deep breath, and summarize artificial transformation.

Back 26

DNA is extracted from a cell chemically, and purified and concentrated.

The log phase-recipient cells are chemically treated with CaCl2 to make the recipient cells competent.

The DNA is taken up by the recipient cells:
1. In dsDNA in Gram + bacteria is degraded to yield ssDNA transported into the cells.

2. In Gram – bacteria, dsDNA fragments are transported into the cell but only ssDNA fragments are incorporated into the chromosome.

Front 27

Don't memorize this, but review the cloning experiment example.

Back 27

Cloning experiment: clone anticancer drug; say its made by a gram negative bacterium's chromosome. Isolate that protein that stops cell growth.

Take organism and remove and purify its chromosome; cut the chromosome with endonuclease; then sew it together with vector, pair with ligase, and transform this DNA into recipent and check for production of anticancer compound.

Then patent it and buy a Lexus lolz

Insulin utilize the transformation technique?