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Chapter 5

Chapter 5 notes (Exam II).docx

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University of Rochester
BIO 190
Elaine Sia

Chapter 5 notes Why are bacteria convenient as model organisms? They are fast-dividing, take up little space, convenient. They can be culture on liquid or solid surface as long as basic nutrients are provided. How does plating lead to colony growth? 7 Plating is done with a sterile spreader and once the cell mass is more than 10 it forms a colony. What are resistant mutants? Unlike wild types, thse can grow even in the presence of an inhibitor. How was conjugation demonstrated? Two cells that were auxotrophic for different nutrients were mixed and the mixture was plated and compared with controls of only A and B cells. Some of the mixture cells grew into colonies to produce prototrophs showing that some form of recombination of genes must have ocured between the two different types of cells. It waqs determined that physical contact was needed for wt to form and this was determined to be conjugation uunder an electron microscope. Do the two cells that are in conjugation act equally? It was determined later that one cell acts as the recipient and the other as a donor. What is the fertility factor? How does it lead to conjugation? It is the bacterial episome whose presence confers donor ability. It is an example of a small nonessential plasmid. It replicated independently of the host chromosome. The F plasmid directs synthesis of pili to intiate contact with the recipient and draw it closer. The F DNA then makes a single-stranded copy of itself via rolling circle replication. What is the difference between Hfr and F+? - Hfr produced 1000x the number of recombinants than normal F+ strain. HfrxF virtually none of the F- were converted into F+ or Hfr. How is Hfr created? It is created via the integration of the F factor into the genome of the F+ via recombination. There are homologous pairing regions derived from mobile elements called insertion sequences so that F is inserted into E.Coli chromosome. How does Hfr get transferred? DNA transfer begins in the middle of the F facto within the Hfr chromosome. Usually the Hfr chromosome breaks before it is completely transferred. As a result the F cell does not receive a full copy of the F factor. Since it does not receive a full copy it remains an F- cell giving the recipient new chromosomal genes. The exogenate transferred fragment from donor is converted into double helix via crossing over into the double stranded endogenate DNA in the exconjugant. Double crossover inserts donor DNA. The cell is now a recombinant and the exconjugant DNA is now lost. What are the key observations in the marker entry? Each donor allele appears at a specific time in the F recipients. The donoe alleles appear in a specific sequence. Later alleles are present in fewer recipient cells because the pilus starts breaking. Why does an F cell upon recombination with Hfr not become Hfr? The point O is the point at which F plasmid is transferred. However, the part of F that confers donor ability is tramitted as the last element of the linear chromosome but the transmission process would have stopped before getting that far. What are the different states that the fertility factor exist as? 1. The plasmid state: as a free cytoplasmic element F is easily transferred into the F recipients. 2. Integrated state: as a contiguous part of a circular chromosome, F is transmitted only very later in conjugation. How can bacterial chromosome be mapped? In broad-scale mapping, time in mins is used as units of map distance. In fine-scale recombinants: the presence of both exogenote and endogenote means that the bacteria is a merogenote—partial diploid. Even number of crossovers would only result in circular, intact chromosome. How can we calculate map distances between three loci? We need a trihibrid that has received all three donor markers: if an interrupted-matinmg experiment has revealed the position of the alleles then we need a merozygote with a stable exconjugant bearing the last donor allele (e.g. leu+). Since we selected for leu+, the other loci are the unselected markers. We need to sample which of the other markers were integrated. How is an F’ plasmid formed? Occasionally an F plasmid carrying bacterial genome cleanly excises from a Hfr strains genome via a reversal of recombination process. This is done by repairing of the homologous pairing regions on either side of the side. However, sometimes there is a faulty outlooping that produces F’, an F plasmid that contains chromosomal DNA. What happens to the recipients of F’ plasmids? The transferred lac+ is incorporated into the recipient’s main chromosome, which still retains lac- because F+lac+ exconjugants sometimes give rise to F- lac- daughter cells. What are R plasmids and how do they work? They are plasmids that provide multiple resistances and are carried in a transposon—unique segments of DNA that can move around by transposition. They are conjugative—effectively transmitted to a recipient cell during conjugation. Even if R plasmid is not conjugative, it can donate R alleles via transposition to conjugative plasmids, such that antibiotic resistance alleles cam spread rapidly throughout a population of bacteria. How does transformation differ from conjugation? In conjugation DNA is transferred from one living cell to another via close contact. However, in transformation isolated pieces of external DNA are taken up by a cell through th
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