Quiz 3
structure of T4 phage and explain the life cycle of T4 phage
- Phage is absorded to bacterial host cell - Phage DNA is injected; host DNA is degraded - Phage DNA is replicated; phage protein components are synthesized - Mature phages are assembled - Host cell is lysed; phages are released
6-4
2 BACTERIAL CELLS MUST BE IN PHYSICAL CONTACT TO EXCHANGE GENETIC INFO The genetic exchange & recombination that produces auxotrophs must require cell to cell contact and not just something that is diffusible through the media because the presence of a filter that allowed movement of media but not cells across it prevents generation of the auxotrophs
Forty-eight plaques were counted on a plate when 0.1 ml of bacteriophage-infected bacteria from a 10−6 dilution of a 10‑ml culture was plated. The original bacteriophage density was __________.
48 × 10^7
Assume that one counted 67 plaques on a bacterial plate where 0.1ml of a 10-5 dilution of phage was added to bacterial culture. What is the initial concentration of the undiluted phage?
6.7 x 10^7 pfu/ml
How does recombination in bacteria occur?
A portion of DNA from one genome is replaced with homologous DNA from another strain of bacteria. (Recombination in bacteria does not involve an exchange of DNA between homologous chromosomes. Rather, it occurs only under special circumstances, such as conjugation or transduction between chromosomes of two different bacteria cells.)
bacteriophages
A virus that infects bacteria. Usually specific for a specific bacterial species
Where does the DNA break
At the origin
HFR x F- matings
BECAUSE THIS WOULD REQUIRE THE F- CELL TO RECIEVE THE ENTIRE HFR CHROMOSOME SINCE THAT IS WHERE THE F FACTOR IS LOCATED IN THE HFR CELL F- cells mated with Hfr cells remain F- because the site at which the F factor integrates becomes the origin (O). DNA next to the integrated F factor passes first through the pilus followed by the rest of the bacterial chromosome. The F factor is the last to pass and conjugate rarely lasts long enough for the whole chromosome to pass
Who can HFR mate with
Cannot mate with another Hfr or F+ bacteria; can only mate with F-
Two yeast strains are identified. As the result of different mutations, each in a separate gene, each strain is auxotrophic for compound Y, a compound required for growth. Which of the following results would be expected in a complementation test involving these two strains?
Cells would grow without supplementing compound Y.
does transformation need conjugation
Conjugation is not required but the bacteria must be in a state of competence. Some cells are naturally competent under certain conditions and other can be made that way in the lab. This process has nothing to do with conjugation and it doesn't matter if they are F+ or F-. The DNA just gets picked up and put through by receptors
how does F+ becomes Hfr
F+ becomes Hfr when the F factor is integrated into the bacterial chromosome
Donor
F+ cell
Which of the following matings are possible: F+ x F-; F+ x F+; F- x F- and F+ x F+?
F+ x F-
Recipient
F- cell
For two mutations, each occurring in a separate gene, mutants can complement each other only if the genes are not involved in the same pathway.
False 2 genes involved in a single pathway may act at different points in that pathway. As long as a single functional copy of each gene is present, the pathway can be completed.
Which of the following statements about mapping bacterial genes by conjugation is are true
For any two genes transferred from donor to recipient, all genes residing between them have also been transferred. It is necessary that all Hfr cells be absent from the population of cells recovered for genotyping. Two genes that are very close together may appear to be transferred at the same time.
Mapping bacterial genes by conjugations based on which of the following assumptions
Genes are transferred from donor to recipient in a linear fashion
How does Hfr become F'
Hfr becomes F' when the integrated copy of the F factor reverts to the free plasmid state (F+). When this happens frequently a few of the bacterial chromosome genes adjacent to the F factor are excised with it (not a clean cut of just the F factor of DNA
partial diploidy is seen in bacteria
If the F' cell (acts like F+) conjugates with the F- cell the F factor plasmid plus the chromosomal genes that ended up in the plasmid because of imprecise excision of the F factor from the chromosome will end up in the cytoplasm of the bacteria. This bacteria creates a partially diploid cell called a MEROZYGOTE
temperate phages
If the virus can either integrate or replicate in the cytoplasm
virulent phages
If they can only replicate in the cytoplasm
Interrupted mating
Interrupted mating occurs when conjugation ends before a copy of the entire genome of the donor cell has passes into the recipient cell (most conjugation will be interrupted that is why the recipient doesn't become F+)
Which of the following statements is true regarding the RecA protein?
It is involved in single-strand displacement. (This characteristic is known as competence. The RecA protein is not the DNA receptor on the bacterial cell surface.)
Why do Hfr bacterial cells exhibit high levels of gene transfer?
Rather than existing as an isolated plasmid, the F factor is incorporated into the bacterial chromosome. (Hfr cells can be considered F+ cells, but they undergo recombination at a much higher frequency than normal F+ cells.)
Which of the following statements is true regarding genetic recombination in bacteriophages?
Recombination can occur within a gene.
Figure 6-3
THE CONJUGATION OCCURRED BETWEEN THE 2 AUXOTROPHS, MAKING THEM PROTOTROPHS The prototrophs that arose after 2 auxotrophic strains with multiple but nonoverlapping mutations is essential nutrition genes arose through some type of genetic exchange & recombination between the 2 mutant strains. production of prototrophs as a result of genetic recombination between 2 auxotrophic strains; neither auxotrophic strain will grow on minimal medium, but prototrophs do, suggesting that genetic recombination has occurred; physical contact between cells of the 2 strains is essential to genetic recombination
In F+ x F- matings, recombination frequency between bacterial DNA is extremely low. However, in Hfr x F- matings, the recombination frequency is 1000 times greater. Why?
THE F FACTOR CAN DIRECT A MORE COMPREHENSIBLE TRANSFER TO PART OF THE DONOR CHROMOSOME TO A RECIENT CELL BECAUSE IT IS A PART OF THE CHROMOSOME You only get recombination of chromosomal DNA when the F factor integrates into the bacterial chromosome. In a cross between F+ and F- the F factor integrates spontaneously into the chromosome with a low frequency converting the F+ cell to Hfr. Then recombination can occur. However if you start with a bacteria that has the F factor already integrated into the genome, recombination will be seen much more frequently
What item is transferred last
The F factor
How is a merozygote formed?
The F factor and several adjacent genes are excised from the chromosome of an Hfr cell and transferred to an F− strain.
Why most of the time F- cells remain F- after mating with Hfr strain
The F factor goes last and the transfer of genes are usually interrupted before it gets to the end
In an Hfr mating with an F− cell, why doesn't the recipient cell always become an Hfr or F+ cell?
The F factor is the last part of the chromosome to be transferred
6-5
The F factor is usually on a plasmid separate from the chromosome. During conjugation one strand of the double stranded plasmid is nicked. The strands are unraveled and one strand moved through the pilus into the F- recipient. DNA synthesis adds back the missing strand to each copy and a complete plasmid is then found in each cell. The F+ cell stays F+ but F- → F+
Read the experiments, observations and conclusions of these experiments. Explain the role P22 phage played in producing prototrophs only in LA22 side.
The P22 phage was an integrated prophage in the chromosome of the LA-22 bacteria. The P22 phage went into a lytic cycle. They removed themselves from the chromosome and started replicating like crazy and ended up lysing the cell. The P22 phage crossed the filter infected and lysed some of the LA-2 bacteria. In this process some of the LA-2 DNA got packaged into the phage head. In a rare case the DNA that was packaged contained the wild type phe and trp genes. Some of those phage went back to the LA-22 side and infected those bacteria. These bacteria will now act as prototrophs because the genes they needed from the LA-2 bacteria are now in there cytoplasm
Which of the following statements about mapping bacterial genes by conjugation is NOT true?
The closer a gene to the Hfr origin, the more likely it will be transferred to the recipient during conjugation.
What results would be expected from a plaque assay if the dilution factor of the phage‑infected culture were too low?
The entire lawn of bacteria would be lysed. (The concentration of phage‑infected bacteria increases as the dilution factor decreases. If the plate had only a few plaques, then the dilution factor was not too low.)
What determines which gene is going to be transferred first?
The first gene to go in is the one next to the integration site
Intragenic recombination and rII locus: Benzer had a large number of rII mutants of T4 phage. These mutant phages have the same phenotype: they produce plaques that are different from the plaques produced by rII+ i.e. wild type phages. In addition, unlike the wild type phages the mutant phages can infect E. coli k12(λ) but cannot lyse the. Benzer took two different rII mutant phages at a time and did mixed infection experiment. In many cases he recovered wild type phages (see Figure 6-19). This way he examined thousands of pairs of rII mutants. He noticed that the frequency of the recovery of wild type phages depended on the pair of rII mutants he used. For some pairs frequency was much higher than other pairs. Why? Read pages 153 - 154.
The rll region had 2 genes in it. He made many crosses and ended up with 2 groups that could produce wild type progeny when they were crossed. This is because in one group all mutations were in gene 1 and in the second group all mutations were in group 2. To recover a wild type phage you must cross two mutants in which the mutations are in different genes
what is cotransformation
The size of DNA that can get through can be long enough to encode a few genes. So if a single transformation event moves 2 or more genes into the cell this is called cotransformation of genes A, B, etc
How these Hfr strains differ from one another
They differ by where the F factor was integrated in the chromosome (remember the origin (O) is right next to the integration site
Which of the following statements regarding Hfr bacteria is FALSE?
They frequently convert recipient bacteria to F+ cells.
Transformation and how does it work
Transformation is when small pieces of DNA floating around in the environment are taken up by a bacterium. Regions of homology between the DNA that got taken up and the chromosome of the bacterium can recombine
Which of the following statements best describes lysogeny?
Viral DNA is integrated into the bacterial DNA and passed on harmlessly with each round of replication and cell division.
What is a merozygote?
a partially diploid bacterial cell (A merozygote forms when an Hfr cell's plasmid DNA is excised from the chromosomal DNA, carrying with it some of the bacteria's chromosomal DNA. This cell is now an F′ cell, and it can act like an F+ cell and pass this plasmid on to an F- cell. The new F+ cell will be diploid for the chromosomal region on the plasmid and in its own genome.)
All of the following are required in complementation testing in yeast except _______. - two or more mutations that affect a single phenotype - diploid cells - mutations that are recessive - an auxotrophic organism
an auxotrophic organism
When a bacterial cell is transformed, after one round of cell division only one of the two resulting cells contains new genetic material because __________.
at transformation, a heteroduplex is formed that alters only one strand of the DNA, leaving the other unchanged
A cotransduction experiment was conducted to determine the order of three bacterial genes: a, b, c. A triple auxotroph was transduced with bacteriophages isolated from a prototrophic strain. The following types of cells were recovered: a+ b c; a+ b+ c; a b+ c+; a b c+. Which gene is in the middle?
b
prototrophs
bacteria that can synthesize all molecules required for life
Fertility factor (F factor)
bacteria with the f factor can establish connections with other bacteria and donate DNA; bacteria without the f factor can only receive DNA, and can't initiate the exchange
Which of the following elements is NOT involved in conjugation?
bacteriophages
F factor
confers the ability to donate parts of F+ cell's chromosome
Process used to transfer F factor from the donor to the recipient
conjugation
Bacterial cells are always haploid, unless __________.
conjugation occurs with an F′ cell
complete media
extensively supplemented
how is genetic information transferred during conjugation?
from one bacterium to another
needed in the media to grow his-E. Coli
histidine
When nutrients become limiting, what happens to a population of bacteria?
it enters stationary phase
F pilus (sex pilus)
on bacterial cells possessing an F factor, a filament-like projection that plays a role in conjugation (allows the cell to cell contact)
auxotrophs
one or more organic molecules cannot be made must be added as supplement in media
F- cells
receive the donor chromosome material and recombine it with part of their own chromosome
minimal media
requires that bacteria synthesize everything it needs for life; containing only the essential nutrients needed to support the growth & reproduction of wild-type strains of an organism; usually comprised of inorganic components that include a carbon & nitrogen source
F+ cells
serve as donors of parts of their chromosomes
HFR
strains of bacteria exhibiting a high frequency of recombination; these strains have a chromosomally integrated F factor that is able to mobilize & transfer part of the chromosome to a recipient F- cell (high frequency recombination)
A cell must be competent in order to __________.
take up DNA from the surrounding medium
Conjugation
temporary fusion of 2 single-celled organisms for the sexual transfer of genetic material
F factor in HFR
the F factor integrates into the chromosome at different points and its position determines the O site
Which of the following statements about the T4 lytic cycle is FALSE?
the phage DNA is injected into the host and integrates into the bacterial chromosome.
In an interrupted mating experiment, the purpose of the planting cells on a selective medium is_____________
to ensure that only recombinant genotypes are recovered
An E. coli nutritional mutant is described as an auxotroph, which requires minimal medium with supplementation of an organic component or inorganic ion for growth.
true (An auxotroph is a mutant bacterium that has lost its ability to synthesize one or more organic components, so those components must be added to compensate for the mutation. Wild-type bacteria, described as prototrophs, do not require this supplementation.)
Prophage
viral DNA integrated into the bacterial chromosome
lysogeny
when the viral DNA that enters the bacterium is integrated into the bacterial chromosome instead of replicating in the cytoplasm (replication of the DNA in the cytoplasm is what's going to happen if the event results in lysis of the cell)