Genetics Ch. 6-7 Lab

Gene Transfer Mechanism in Bacteria: Transformation

Transfer of cell-free or “naked” DNA from one cell to another

• Transfers fragments of chromosomal DNA after lysis and partial degradation of donor chromosomal DNA

Gene Transfer Mechanism in Bacteria: Transduction

Transfer of genes from one cell to another by a bacteriophage

• Transfers fragments of donor DNA by incorporation into defective phage particles

• Transfers smallest amount of chromosomal DNA since fragments have to fit into the transducing particle

• A form of recombination in bacteria

Gene Transfer Mechanism in Bacteria: Conjugation

Transfer of genes between cells that are in physical contact w/ one another

• Transfers chromosomal DNA from Hfr and F’ donor to F- recipients

• Transforms the largest amount of donor chromosomal DNA

  • the donor retains a copy of the transferred gene

• Transfers DNA horizontally to cells in the same generation

• A form of recombination in bacteria

• Transfers genes for drug resistance

• Transfers genes for enzymes and adherence molecules

F- strains are conjugated by …

By F+ to F+

A donor is effectively F+ if …

It initiates F factor mediated conjugation because only cells that have the F factor are able to do that

• An F+ cell (containing the F factor) conjugates w/ an F- cell to make it into F+ while the F+ donor stays F+

Hfr strain

• A bacterium w/ a conjugative plasmid integrated into the chromosomal DNA

• Can act as donors b/c have F factor integrated into chromosome, so when initiating conjugation, attempts to transfer its entire chromosome/host chromosome

• In mapping experiments, are conjugated to F- strains → F factor is excised imprecisely, carrying some adjacent chromosomal DNA w/ it

  • distance between 2 genes is determined by comparing their times of entry during a conjugation experiment

    • genetic distance in minutes = difference in entry times

Cotransduction frequency

• Fraction of transductants where both genes were transferred together

  • Large value = closer together

  • Small value = farther apart

  • If two genes are very close together on chromosome, are more likely to fit into the same phage head

Meaning of + and -

• + means can synthesize it (i.e. met+ = can synthesize methionine)

• - means cannot synthesize it (i.e. met- = cannot synthesize methionine

Complete media vs Minimal media

• Complete: Medium contains all the required compounds for bacterial growth and reproduction

• Minimal: contains only carbon and nitrogen source

Phototrophic bacteria

• Only grow on minimal media

• Can produce all necessary compounds required for their growth and reproduction utilizing the carbon source provided on the minimal media (i.e. ala+ pro+ lac+)

Auxotrophic Bacteria

• Can’t grow on minimal media b/c have mutation on some amino acid biosynthetic pathway gene → only grows in complete media or on right supplemented minimal media

• i.e. ala+ pro- lac+ can only grow on complete media or minimal media supplemented w/ amino acid proline

Purines

• Adenine and Guanine (have 2 bonds between)

• 2 rings

Pyrimidines

• Cytosine and Thymine (have 3 bonds between)

• 1 ring

The melting temp of DNA increases as the …

G+C content increases

Phosphodiester bond

• The bond that joins one nucleotide to another in the DNA strand

• Covalent

• Formation between alpha phosphate of incoming nucleotide triphosphate and 3’ hydroxyl group of the last nucleotide added to strand → catalyzed by DNA polymerase

Hydrogen bonds

• The bond that joins one strand of DNA duplex to the other strand

• Non covalent

Complementary

Is used to describe the pattern of base pairing between one DNA strand and its partner in a duplex

Antiparallel

• Is used to describe the polarity of two DNA strands in a duplex

• Means DNA strands run in opposite direction (i.e. one strand of DNA goes from 5’ to 3’ and the other strand is opposite in direction going 3’ to 5’)

Topoisomerase

Works at the region ahead of the replication fork to prevent supercoiling

Helicase

Opens up the DNA at the replication fork

Single-strand binding protein

Coats the DNA around the replication fork to prevent rewinding of the DNA

RNA Primase

• Synthesizes RNA primers complementary to the DNA strand

• Creates Okazaki fragment primers

• Note: RNA has no T, has U instead, but % A doesn’t equal % U

DNA Polymerase

Can proofread replicating DNA, delete incorrect bases, excise them, and correctly replace them

DNA Polymerase III

• Extends the primers, adding on to the 3’ end to make the bulk of the new DNA

• Polymerase activity

  • 5’ → 3’

  • Replication, proofreading, editing

• Exonuclease activity

  • 3’ → 5’

• If it could add bases in the 3’ → 5’ direction in E.coli, there would be no need for Okazaki fragments

• Most important enzyme/main enzyme for DNA replication

DNA Polymerase I

• Removes RNA primers and replace them w/ DNA

  • aka does primer removal and gap filling in the completion of an Okazaki fragment

• Polymerase activity

  • 5’ → 3’

  • Filling in gap after removal RNA primer, DNA repair, removal of RNA primers

• Exonuclease activity

  • 5’ → 3’, 3’ → 5’

DNA Polymerase II

• Polymerase Activity

  • 5’ → 3’

  • DNA repair

• Exonuclease activity

  • 3’ → 5’

Ligase

• Seal gaps between DNA fragments

• Okazaki fragments (which are formed on lagging strand) are joined by this

  • Okazaki fragments: short DNA segments formed on the discontinuously replicated strand that contains both DNA and RNA nucleotides

• The final covalent bond made in the completion of an Okazaki fragment, including primer removal and gap filling

Prokaryotic chromosomes vs Eukaryotic chromosomes

• Prokaryotic chromosomes have a single origin of replication

• Eukaryotic chromosomes have multiple origins of replication

Why do leading and lagging strands exist?

Because there is no known enzyme that replicates in the 3' - 5’ direction

The Hershey and Chase experiment with P32 and S35 demonstrated that …

Phage DNA enters the host cell

When genes are found on different chromosomes or far apart on the same chromosome …

They assort independently and are said to be unlinked

• Common types of gametes = parental configuration

• Rare types of gametes = recombinant configurations

When genes are closer together on the same chromosome …

They are linked

• alleles/gene versions will be inherited as a unit more frequently

Gene interference

A measure of the independence of crossovers from each other

• If they aren’t independent → crossover in one region does affect the likelihood of there being a crossover in an adjacent region

• If a crossover in one region does affect a crossover in another region, but interaction is called interference

Interference value explained

If I = 0.49, that means only 0.49 of expected double-crossover progeny were not observed