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Bacteriology/Mycology > Lecture 3 - Genetics, Vaccines, etc. > Flashcards

Lecture 3 - Genetics, Vaccines, etc. Flashcards

1
Q

A transformant is a cell that has taken up _____ _____ by transformation.

A

recipient DNA

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2
Q

A “competent” bacteria is bacteria that can take up DNA ______ (i.e. without _______ _______).

A

naturally, artificial treatment

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3
Q

Homologous recombination is ______ or ____ regions of DNA ____ and _____ each other by ______ _____. This process is also known as _____ exchange or strand _______.

A

identical, similar, align, exchange, excision-repair, allelic, replacement

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4
Q

A transformasome is a compartment that has taken up ______-_______ _____.

A

double-stranded, DNA

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5
Q

Transfection is defined as uptake of _____ ____.

A

viral DNA

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6
Q

Complementation is defined as the ______ DNA expressed on a _______ (in _____) will ______ the mutation.

A

mutated, plasmid, trans, correct

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7
Q

Describe the mechanism of transformation.

A
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8
Q

Only _____-______ DNA can bind to cells; only _____-_____ DNA can be taken up.electroporation

A

double-stranded, single- stranded

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9
Q

Competence can be induced by ________ treatment or ________, and such cells can take up ________-________ and ______-______ DNA and linear and circular ________.

A

Ca++ , electroporation, double stranded, single stranded, plasmids

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10
Q

What is the significance of transformation?

A
  1. Transformation provided evidence that the transfer of DNA is responsible for pathogenic determinants aka genes.
  2. Transformation explained how bacteria can acquire other genes.
  3. Transformation helped develop artificial transformation schemes (e.g. electroporation), introduced “foreign genes” into both eukaryotic and prokaryotic cells, and allowed for the easy production of mutants which defined gene function (e.g. virulence determinants).
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11
Q

Label the image accordingly.

A
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12
Q

Transduction is defined as the process of transferring _____ from one bacterium to another by a ____. Describe the steps of this process:

A

DNA, phage
1. Virus infects cell
2. Replicates and makes more of itself
3. May take up some bacterial DNA too and incorporate that into virus head. So when this virus infects new bacterial cell, some bacterial DNA is transfected into new cell, it will outcompete other cells in the population.

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13
Q

Which part of the transduction process of the lytic phase? Which is the lysogenic phase?

A

top arrow = lytic phase
bottom arrow = lysogenic phase

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14
Q

Lysogeny is characterized by ?

A

viral DNA being incorporated into the host DNA. The host then carries this DNA with them for life (e.g. herpesvirus).

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15
Q

______ does not lyse, but _____ DNA integrates into and ______ in the host cell DNA. Often determined by large difference in the ____ _ ____ content of the bacterial DNA

A

Cell, phage, replicates, G+C

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16
Q

Plasmids = genes of _______ origin and often not necessary for _____; others contain genes encoding _______ _____.

A

unknown, growth, virulence factors

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17
Q

F-factor = ______ factor; genes promote ______ and _____ of the factor to _____ cells e.g. ______.

A

fertility, replication, transfer, recipient, conjugation

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18
Q

R-factor = ___ factor + _____ encoding ______ to specific ______

A

F, genes, resistance, antibiotics

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19
Q

If an F factor carries ________ _______ genes, that is an R factor.

A

antibiotic resistance

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20
Q

Describe the process of conjugation

A
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21
Q

F factor encodes a ____, can transfer F factor through ____. Some material from plasmid can be integrated into ______.

A

pilis, pillis, genome

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22
Q

R (_______)-factor: plasmid containing two types of genes:
1) _______ transfer factor = ____ encodes origin of _______, sex _____ genes.
2) __-______ = genes encoding antibiotic
resistance, _____ ____ resistance or other virulence
factors (______), _______ ______ (IS).

What are two examples of an R factor?

A

resistance, Resistance, RTF, replication, pilin, R-determinants, heavy metal, determinants, insertion sequences

RTF: tra genes, r determinant: antibiotic resistance genes and IS element.

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23
Q

Transposons are ________ genes that can move from one region of a _______ to another. They are designated as “Tn” elements, each of which contain a gene encoding a __________ which binds to ____ sites that mediates _______ and ________.

A

jumping, chromosome, transposase, excision, insertion.

24
Q

What are the net effects of Tn movement?

A
  • mutation resulting from insertion into middle of another gene
  • neutral event; insertion next to an intact gene
  • excision; can carry along a neighboring gene: virulence gene!
25
Q

Bacteria can obtain genes by ?

A

transformation, conjugation, or transduction

26
Q

Plasmids are extrachromosomal elements that can?

A

• facilitate gene transfer
- F factors including integrated ones (Hfr) contain genes
encoding pilin proteins that form the sex pilus - the
conjugal bridge through which the DNA passes.
- R factor = F factor and antibiotic (or other) resistance genes • express virulence determinants e.g. anthrax toxin

27
Q

Transposons are _____ genetic elements that can mediate their own _____, but also ________ genes encoding ______ and ________ ______.

A

mobile, transfer, neighboring, toxins, antibiotic resistance

28
Q

A live vaccine is _______ –> limited ______ in host.

A

attenuated, growth (does not cause disease in host).

29
Q

A killed vaccine is made up of _______ _______ ______ cells.

A

chemically, inactivated, whole

30
Q

An example of a subunit vaccine is?

A

Outer surface protein

31
Q

A live recombinant vaccine is a gene for _____ expressed in a _______. Example?

A

antigen, microbe, rabies capside protein in vaccine

32
Q

A DNA vaccine is a gene encoding antigen expressed _____ in ______ host.

A

directly, immunized

33
Q

What are the limitations of a live, attenuated vaccine?

A

Can cause mild symptoms of disease, induces a longer Th1 response.

34
Q

What are the limitations of a killed vaccine?

A

Not very effective, too many antigens, and tend to stimulate only a short-lived Th2 response

35
Q

What are the limitations of a subunit vaccine?

A

useful but often expensive & need to
be mixed with adjuvants to be effective

36
Q

What are the limitations of a live recombinant vaccine?

A

regulatory hurdles & public negative to use of recombinant

37
Q

What are the limitations of a DNA vaccine?

A

still needs refinement and way to avoid
gene silencing by host

38
Q

What are the advantages of using a DNA based test?

A

High sensitivity
- Can theoretically detect the presence of a single
organism • High specificity
- Can detect specific genotypes - Can determine drug resistance - Can predict/confirm virulence
• Speed
- Quicker than traditional culturing for certain
organisms

39
Q

Leading uses for DNA based tests?

A

Culture identity & confirmation
• Low numbers or difficult to isolate and grow
- e.g. Mycobacterium, fungi, viruses
• Organisms present in small volume specimen
- Intra-ocular fluid
- Forensic samples (e.g. swab of sample)
• Molecular epidemiology
- To identify point sources for hospital and
community-based outbreaks
Zoonotic & highly infectious agents
- minimize hazards during handling
- e.g. Brucella melitensis, Franciscella
tularensis, Coccidiodes immitis • Non-viable organisms
- Organisms tied up in immune complexes

40
Q

What are the disadvantages of using a DNA based test ?

A

Expensive - supplies & equipment • So specific that must have good clinical data to
support infection by that organism before testing is
initiated. • Will miss new organisms unless sequencing is
done (not practical in a clinical setting). • May be a problem with mixed culture infections –
would have to assay for all organisms causing the
infection symptoms.

41
Q

What is one way to make a vaccine?

A

Change an already made vaccine

42
Q

What made the vaccine for brucella? Who cannot receive this vaccine?

A

The vaccine for brucella was made when the bacteria it lost its O antigen and it was not virulent unless the animal is pregnant ( can not be given to pregnant animals)

43
Q

How were they able to vaccinate for anthrax?

A

Can take this protective PAG gene and put it into the vaccine already made for brucella and can be used to protect against 2 diseases.

44
Q

What is PAG ? What does it encode for?

A

PAg is a gene that codes for PA. PA is a part of anthrax toxin that antibodies neutralize.

45
Q

What is PCR? Is it a diagnostic test?

A

It is not a diagnostic test but an amplification tool.
Can amplify the DNA even if its not viable.
• can detect a really small quantity of DNA

46
Q

Why was PCR very manual previously? What has changed that has allowed it to be more automated?

A

• Used to be very manual because when the Denaturation would take place at 94 degrees Celsius the DNA polymerase would be destroyed.
• They now use a Thermophile DNA polymerase it can withstand those temperatures and it is a lot more automated.

47
Q

What causes brucellosis?

A

Causative agent: gram negative,
cocco-bacillus, genus Brucella.

48
Q

What does brucellosis cause?

A

Disease: brucellosis = abortion in livestock
caused by Brucella abortus

49
Q

What diagnostics can be used to diagnose the brucellosis?

A

Diagnosis: indirect - serum serology, antibodies to O-side chain
direct – culture, identification

50
Q

What is the isssue with brucellosis which make it difficult to test for?

A
  • all 8 Brucella species grow very slow, 3-7 days
  • biochemical phenotyping requires multiple tests - vaccine strain RB51can cause abortion IF
    given to pregnant cow!
51
Q

What is a real time PCR?

A

Real time PCR is very good for diagnostics and is very sensitive.

52
Q

How does Real Time PCR work ( TAC band)

A

• TAC band
• you have a reporter and a quencher. The reporter is fluorescent and the quencher inhibits it. When they are on the same DNA the quencher inhibits the fluorescence. Once it amplifies a specific
piece of DNA, the DNA polymerase is a 5 prime nuclease and it chews up the probe and releases the reporter from the quencher. Now the reporter fluoresces and will give off a signal, but only if the DNA was amplified and the probe/primer was very specific to that section of DNA

53
Q

What can be indicated by the number of cycles it takes for a result in a real time PCR?

A

The number of cycles it takes gives you an idea of how much DNA was there

54
Q

What are the advantages of Real Time PCR?

A

Fast, automated, can do alot of tests at once. But expensive.

55
Q

What is the best diagnostic method we have currently for most bacteria?

A

MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time Of Flight) Mass Spectrometry

56
Q

Why is MALDI-TOF system considered the best diagnostic tool out there right now?

A

• VERY SENSITIVE. Can give information down to species level. And if it is one of the ones it doesnt know which is only like .1 % of the time, it will say it cant identify it rather than give the wrong one.

57
Q

What is PCR? What is Real Time PCR? What is MALDI- TOF?

A

PCR- amplification not diagnostic. Real time PCR is diagnostic. IF DNA you are looking for is there it will tell you right away
• Malditof is the fastest, most accurate, and least expensive diagnostic method available.
• Protein is based on RNA in the sample. Which is a byproduct of the DNA.

Bacteriology/Mycology (40 decks)

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