Genetics Ch.6
Partial Diploid?
A bacterium that has two copies of some genes but only one copy of genes. It is formed when a recipient cell receives an F plasmid from a donor.
What is a prophage and how is it formed?
A prophage is a bacteriophage genome that is part of the host cell chromosome. It is formed by integration of a bacteriophage chromosome into the host cell chromosome by site specific recombination.
How do bacteria propogate?
Bacteria propagate by binary fission: the chromosome replicates and a copy is distributed to each of the progeny cells. In a matter of hours a single cell can generate a colony of thousands of identical cells. Studies in the 1940s and 1950s described three mechanisms of gene transfer and recombination between bacteria
Characteristics of Bacterial Genomes
Bacterial genomes are usually composed of a single chromosome that carries mostly essential genes. Usually a quite small closed circular molecule of double-stranded DNA.
Cotransformation
Closely linked genes will often end up on the same fragment of transforming DNA and require only two crossover events. When three genes are examined, the order can often be determined by cotransformation frequency.
Genetic Transfer Occurs by Three Processes
Conjugation is the transfer of replicated DNA from a donor to a recipient. Transformation is the uptake of DNA from the environment. Transduction is the transfer of DNA from one bacterium to another by a viral vector
Transfer of Hfr genes
Conjugation occurs between Hfr and F- cell. The F factor is nicked by an enzyme, creating the origin of transfer of the chromosome (O). Chromosome transfer across conjugation begins; the Hfr chromosome rotates clockwise. Replication begins on both strands as chromosome transfer continues. The F factor is now on the end of the chromosome adjacent to the origin. Conjugation is usually interrupted before the chromosome transfer is complete. If crossing over occurs, then the DNA from the Hfr strain can be incorporated into the original chromosome of the F- cell.
Conjugation v. Transduction v. Transformation
Conjugation requires some genetic information (F+ cell/HFR/F') and physical contact between donor and recipient by sex pillus. Transduction is characterized by infection of the donor cell by a bacteriaphage exclusively involves homologous recombination. Transformation during virus lyosgenic cycle does not require genes in the donor or the help of phage rather the DNA is released from the donor cell due to cell lysis and enters the recipient cell via DNA transporters.
Which of the F factors can convert exoconjugants?!
F+ and F' can convert F- into donor cells. HFR can only transfer donor genes something F' is actually incapable of.
What is the the state of each F factor?
F+ means the the plasmid is present. F- means the plasmid is not present. Hfr means the F plasmid is part of the chromosome. F' means the F plasmid is present AND contains part of the chromosome.
Which of the F factors donate and receive?
F+, F', and HFR can all donate their plasmids. F- can only receive plasmids!
What features that make bacteria useful to Geneticists
Genome simplicity: fewer genes and fewer bases than other organisms. Uncomplicated genotypes: mutations can be observed directly because there is one copy of each gene (haploid). Rapid generation times: bacterial generation time can be measured in minutes. Large numbers of progeny: enormous numbers of progeny allow detection of rare events. Ease of propagation: bacterial culture is easy and inexpensive and takes up very little space. Numerous heritable differences: mutants are easily created, identified, isolated, and manipulated for study
Davis U-Tube experiment
Hayes proposed that the ability to act as a donor was hereditary and determined by a "fertility factor" (F factor). Donor cells possess an F factor and are called F+ cells whereas recipients, called F- cells, lack an F factor. Microbiologists now know that conjugation is controlled by genes carried on the F plasmid
How can a conjugation of two Hfr result from F- cell and Hfr.
Hfr contains F+ so if the WHOLE F- plasmid can recombine with Hfr it will make another Hfr cell!
Among the mechanisms of gene transfer in bacteria which one is capable of transferring the largest chromosome segment from donor to recipient? smallest?
Hfr is capable of the most because it can in theory transfer the entire chromosome. Transduction typically results in the least because it is limited to fragments of chromosome that can fit into the transducting particle.
Plasmid Replication High v. Low copy
High-copy-number" plasmids can replicate independently of the bacterial chromosome so that the number of plasmids per cell can increase rapidly (>50 or more per cell). "Low-copy-number" plasmids are present in one or two copies per bacterial cell and usually cannot replicate independently of the bacterial chromosome
How can you tell which F factor is donating?
If genotype does not change except for becoming a new donor than F+ was the perp. If both F- genotype changes and can donate than F' is responsible. If the genotype changes but does not become a donor than Hfr is responsible.
Lytic v. Lysogenic
Lysis=Infects.Release progeny. Lyso=Integrates chromosome to prophage. Dormant state. transduction. Hfr.
Types of medium used
Minimal Media contains a carbon source, essential elements like N and P, vitamins, and other ions and nutrients. Complete Media contains everything necessary for bacteria to grow and reproduce.
Viruses
Must have a host cell to reproduce. More genetically related to host cell than other viruses
Two colonies leu- and leu+ are cultured and put on two plates. One plate is a medium with leucine the other is without it. On which plate would each colony grow?
ONLY LEU+ will grow on the minimal medium. BOTH will grow on the complete media.
Explain the importance of each: origin of transfer, conjugation pillus, homologous recombine, relaxosome, relaxase, T strand DNA, and pillin.
Origin=site where binding, cleavage and replication occur. Pillus=Physical tunnel for F+ to transfer plasmid to F-. Homo=Hfr must maintain this process for stable transfer. Relaxosome=protien that binds to F origin and initiates transfer by cleavage and provides a coupling protien. Relaxase=Bindings to coupling protein to feed DNA into the pillus. T strand=strand of F origin DNA that is cleaved and bound by relaxase.. Pollin=protein that assembles into conjugation pilus.
Bacterial DNA transfer experiment by Lederberg and Tatum in 1946
Proposal: one auxotrophic strain was transferring some of its prototrophic alleles to the other auxotrophic strain. They hypothesized that physical contact of the cells was necessary for this transfer
Prototrophic v. Auxotrophic
Prototrophic bacteria can use these nutrients to make all compounds necessary for growth v. Auxotrophs lack an enzyme necessary to metabolize one or more of the nutrients in minimal media, so can't grow unless the media is supplemented with what they need.
Plasmids
Small double-stranded circular DNA molecules containing non-essential genes that are easily modified and are used in a variety of recombinant DNA applications. They can integrate into the bacterial chromosome, to form an episome.
Types of plasmids:
The F (fertility) plasmid contains genes that promote its own transfer from donors to recipients. An R (resistance) plasmid carries antibiotic resistance genes that can be transferred to recipient cells
How does one determine the The order of gene transfer and time of first appearance of each in exconjugants?
The distance of the gene from the origin of transfer (oriT). Genes closest to oriT will transfer earlier and be incorporated more frequently into recipients
Exoconjugant cell
The exconjugant cell is produced by the conjugation; it is the recipient cell with its genetic information modified by receiving DNA from the donor cell
How do viruses reproduce?
Transduction. Lytic: A virus attaches to a host bacterial cell. Injects DNA, replicates, takes up the bacteria DNA, then lyses to release contents. The process is repeated on subsequent bacteria cells. Lysogenic:crossover
(-)F--->(+)F<----->Hfr-->F'
Transfer of F+ plasmid from a F+ to F- through a sex pillus results in a newly formed F+ cell. The plasmid then gets integrated into the host chromosome and converts and F+ to Hfr. Excision of the F plasmid can convert Hfr back to F+. Excision of F plasmid plus host DNA results in F'.
Bacteriophage
Virus that infects bacteria. Particles are less than 1% the size of the bacteria they attack. They are composed of an icosahedral head, hollow protein sheath, and sometimes a set of tail fibers. The head contains a single chromosome from 5,000 to 100,000 base pairs; replication and gene expression require enzymes and factors of the host cell
Detail the process of conjugation
cytoplasmic bridge known as the pilus forms. Dna then replicates and transfers one cell to another. A crossover in the recipient leads to the creation of a recombined chromosome.
Rate of cotransformation is inversely related to
distances between genes. Genes with the lowest cotransformation frequency should be the flanking genes. Cotransformation frequency will be highest for the closest pair, allowing placement of the middle gene
If crossing over occurs, then the DNA
from the Hfr strain can be incorporated into the original chromosome of the F- cell.
F factor position (O) determines
initial point of transfer. It can be oriented either direction in the bacterial chromosome
Specialized transduction
is carried out only by temperate phages and the only genes transferred are those close to the integration site
Interrupted mating
is the cessation of conjugation by breaking the conjugation tube. Hfr chromosome isn't completely transferred to the recipient cell. Experiments that test for gene transfer at timed intervals (time-of-entry mapping) are used to determine the distances between genes.
Cell is considered competent if
it can take up DNA (Any kind!) through envelope
Time to transfer a gene indicates
its relative position on the chromosome
How do we study bacteria?
sterile medium is inoculated with bacteria where they then begin to grow and divide.
