Class Notes

Genetic Transfer

• Genetic transfer is the mechanism by which DNA is transferred from a donar to a recipient.
• Once donar DNA is inside the recipient, crossing over can occur.
  • The result is a recombinant cell that has a genome different from either the donar or the recipient.
• In bacteria genetic transfer can happen three ways:
  1. Transformation
  2. Transduction
  3. Conjugation
• Remember that a recombination event must occur after transfer in order that the change in the genome be heritable(passed on to the next generation).

Transformation

• After death or cell lyses, some bacteria release their DNA into the environment.
• Other bacteria, generally of the same species, can come into contact with these fragments, take them up and incorporate them into their DNA by recombination.
  • This method of transfer is the process of transformation.
• Any DNA that is not integrated into he chromosome will be degraded.
• The genetically transformed cell is called a recombinant cell because it has a different genetic makeup than the donar and the recipient.
  • All of the descendants of the recombinant cell will be identical to it.
  • In this way, recombination can give rise to genetic diversity in the population.

Griffith's Experiment

• The transformation process was first demonstrated in 1928 by Frederick Griffith.
• Griffith experimented on Streptococcus pneumoniae, a bacteria that causes pneumonia in mammals.
• When he examined colonies of the bacteria on petri plates, he could tell that there were two different strains.
  • The colonies of one strain appeared smooth.
    • Later analysis revealed that this strain has a polysaccharide capsule and is virulent, that it, it causes pneumonia.
  • The colonies of the other strain appeared rough.
    • This strain has no capsules and is avirulent.
  • When Griffith injected living encapsulated cells into a mouse, the mouse died of pneumonia and the colonies of encapsulated cells were isolated from the blood of the mouse.
  • When living nonencapsulated cells were injected into a mouse, the mouse remained healthy and the colonies of nonencapsulated cells were isolated from the blood of the mouse.
  • Griffith then heat killed the encapsulated cells and injected them into a mouse.
    • The mouse remained healthy and no colonies were isolated.
    • The encapsulated cells lost the ability to cause the disease.
  • However, a combination of heat-killed encapsulated cells and living nonencapsulated cells did cause pneumonia and colonies of living encapsulated cells were isolated from the mouse.
    • How can a combination of these two strains cause pneumonia when either strand alone does not cause the disease?
    • If you guessed the process of transformation you are right!
      • The living nonencapsulated cells came into contact with DNA fragments of the dead capsulated cells.
      • The genes that code for thr capsule entered some of the living cells and a crossing over event occurred.
      • The recombinant cell now has the ability to form a capsule and cause pneumonia.
      • All of the recombinant's offspring have the same ability.
      • That is why the mouse developed pneumonia and died.

Transduction

• Another method of genetic transfer and recombination is transduction.
• This method involves the transfer of DNA from one bacterium to another with the use of a bacteriophage (phage).
  • A phage is a virus that infects bacteria.
  • The phage T4 and the phage lambda, for example, both infect E. coli.
• Because the phage reproductive system is important to understanding transduction, we will briefly review phage lifecycle.
• Phages are obligatory intracellular parasites and must invade a host cell in order to reproduce.
  • T4 multiplies by the lytic cycle which kills the host and lamba multiplies by the lysogenic cycle which does not cause the death of the host cell.
  • In lysogeny, the phage DNA remains latent in the host until it breaks out in a lytic cycle.
• General Steps Of The Lytic Cycle:
  1. Attachment of T4 to receptors on E. coli cell wall.
  2. Penetration of the cell wall by tail core. Inject DNA into host.
  3. E. coli DNA is hydrolyzed. Phage DNA directs biosynthesis of viral parts using the host cell's machinery.
  4. The phages mature as the parts are assembled.
  5. Lyses of E. coli and release of the new phages.

  Watch A T4 Virus Inject Its DNA Into A Bacteria.

• General Steps Of The Lysogenic Cycle:
  1. Phage attaches to E. coli and injects DNA.
  2. Phage circularizes and can enter either the lytic or the lysogenic cycle.
  3. The lytic cycle would occur as previously described.
  4. In the lysogenic cycle the circular phage DNA recombines with E. coli DNA and the phage DNA is now called prophage.
  5. E. coli undergoes cell division, copying prophage and passing to daughter.
     • With more divisions there are more cells with the prophage.
  6. The prophage may exit the chromosome and start a lytic cycle at any time.
• Now that you have reviewed phage lifecycles, we can discuss transduction.
• Transduction can be generalized or specialized.
• The Steps Of General Transduction:
  1. A phage attaches to cell wall of bacterium and injects DNA.
  2. The bacterial chromosome is broken down and biosynthesis of phage DNA and protein occurs.
  3. Sometimes bacterial DNA can be packaged into the virus instead of phage DNA.
     • This phage is defective (can't destroy another host cell) because it does not carry its own genetic material.
  4. The cell lyses, releasing viruses.
  5. The phage carrying bacterial DNA infects another cell.
  6. Crossing over between donor and recipient DNA can occur producing a recombinat cell.
• In generalized transduction, any bacterial genes can be transferred bacause the host's chromosome is broken down into fragments.
  • Whatever piece of bacterial DNA happens to get packaged within the phage is the genetic material that will be transferred between cells.
• In specialized transduction, on the other hand, only certain bacterial genes can be transferred.
  • These genes, as you will see, must exist on either side of the prophage.
  • Specialized transduction requires a phage that uses the lysogenic cycle for reproduction.
  • The Steps In Specialized Transduction:
    1. Remember that in the lysogenic cycle, phage DNA cn exist as a prophage integrated in the bacterial chromosome)
    2. Occasionally when the prophage exits it can take adjacent bacterial genes with it.
    3. The phage DNA directs synthesis of new phages.
       • The phage particles carry phage DNA and bacterial DNA.
    4. The cell lyses, releasing the phages.
    5. A phage carrying bacterial DNA infects another cell.
    6. The joined phage and bacterial DNA circularize.
    7. Along with the prophage, bacterial DNA integrayes with the recipient chromosome by a cross over event.
       • This forms a recombinant cell.

Conjugation

• A third mechanism by which genetic transfer takes place is conjugation.
• This mechanism requires the presence of a special plasmid called the F plasmid.
• Therefore, we will briefly review plamid structure before continuing.
  • Plasmids are small, circular pieces of DNA that are separate and replicate indepentently from the bacterial chromosome.
  • Plasmids contain only a few genes that are usually not needed for growth and reproduction of the cell.
  • However, in stressful situations, plasmids can be crucial for survial.
  • The F plasmid, for example, facilites conjugation.
    • This can give a bacterium new genes that may help it survive in a changing environment.
  • Some plasmids can integrate reversibly into the bacterial chromosome.
    • An integrated plasmid is called an episome.
• Bacteria that have a F plasmid are referred to as as F+ or male.
  • Those that do not have an F plasmid are F- of female.
• The F plasmid consists of 25 genes that mostly code for production of sex pilli.
• A conjugation event occurs when the male cell extends his sex pili and one attaches to the female.
  • This attached pilus is a temporary cytoplasmic bridge through which a replicating F plasmid is transferred from the male to the female.
  • When transfer is complete, the result is two male cells.
• The F plasmid can behave as an episome.
  • When the F+ plasmid is integrated within the bacterial chromosome, the cell is called an Hfr cell (high frequency of recombination cell).
  • The F plasmid always insetrs at the same spot for a bacterial species.
• The Hfr cell still behaves as a F+ cell, transferring F genes to a F-cell, but now it can take some of the bacterial chromosome with it.
• Replication of the Hfr chromosome begins at a fixed point within the F episome and the chromosome is transferred to the female as it replicates.
• Movement of the bacteria usually disrupts conjugation before the entire chromosome, including the tail of the F episome can be transferred.
  • Therefore, the recipient remains F- because the F plasmid is not entirely transferred.
• A cross over event can occur between homologous genes on the Hfr fragment and the F- DNA.
• Pieces of DNA not recombined will be degraded or lost in cell division.
• Now the recombinant genome can be passed on to future generations.

  Watch DNA Travel Through The Conjugation Tube.

Plasmids

• Plasmids are genetic elements that can also provides a mechanism for genetic change.
• Plasmids, as we discussed previously, are small, circular pieces of DNA that exist and replicate separately from the bacterial chromosome.
• We have already seen the importance of the F plasmid for conjugation, but other plasmids of equal importance can also be found in bacteria.
• One such plasmid is the R plasmid.
• Resistance or R plasmids carry genes that confer resistance to certain antibiotics. A R plasmid usually has two types of genes:
  1. R-determinant: resistance genes that code for enzymes that inactivate certain drugs
  2. RTF (Resistance Transfer Factor): genes for plasmid replication and conjugation.
• Without resistance genes for a particular antibiotic, a bacterium is sensitive to that antibiotic and probably destroyed by it.
• But the presence of resistance genes, on the other hand, allows for their transcription and translation into enzymes that make the drug inactive.
• Resistance is a serious problem. The widespread use of antibiotics in medicine and agriculture has lead to an increasingnumber of resistant strain pathogens.
• These bacteria survive in the presence of the antibiotic and pass the resistance genes on to future generations.
• R plasmids can also be transferred by conjugation from one bacterial cell to another, further increasing numbers in the resistant population.

  Read The Paper To Learn More About Antibiotic Resistance

Transposons

• Transposons (Transposable Genetic Elements) are pieces of DNA that can move from one location on the chromosome another, from plasmid to chromosome or vice versa or from one plasmid to another.
• The simplest transposon is an insertion sequence.
  • An insertion sequence contains only one gene that codes frotransposase, the enzyme that catalyzes transposition.
  • The transposase gene is flanked by two DNA sequences called inverted repeats because that two regions are upside-down and backward to each other.
• Transposase binds to these regions and cuts DNA to remove the gene.
• Yhe transposon can enter a number of locations.
  • When it invades a gene it usually inactivates the gene by interrupting the coding sequence and the protein that the gene codes for.
  • Luckil, transposition occurs rarely and is comparable to spontaneous mutation rates in bacteria.
• Complex transposons consist of one or more genes between two insertion sequences.
• The gene, coding for antibiotic resistance, for example, is carried along with the transposon as it inserts elsewhere.
• It could insert in a plasmid and be passed on to other bacteria by conjugation.