Linkage, Gene Mapping and Sequencing

Prokaryote Mapping

• Interrupted bacterial conjugation can provide a time map of the donor chromosome

• Bacteria doing conjugation has allowed for time mapping

Eukaryote Mapping

Linked genes that are close together on the chromosome can be separated by crossing over to produce recombinants whose frequency of occurrence enables mapping

Bacterial Gene (Horizontal) Transfer Methods

1. Conjugation

2. Transduction

3. Transformation

Horizontal gene transfer can happen between bacteria and eukaryotes

Conjugation

Transfer of DNA from one bacterium to another by cell-to-cell contact

Transduction

Virus-mediated transfer of DNA between bacteria

Transformation

Uptake of exogenous DNA from the surrounding environment

Advantages in using Bacteria and Viruses in Genetics

• Rapid reproduction

• Many progenies produced

• No ethical issues

• Small genome

• Haploid genome allows all mutations to be expressed directly

• Sexual reproduction simplifies the isolation of genetically pure strains

• Growth in laboratory is easy and requires little space

• Medical importance

• Can be genetically engineered to produce substances of commercial value

Plasmids

• A bacterial plasmid replicates independently of its bacterial chromosome

• Used at defence, transferring genes between bacteria

1. Replication begins at the ori (origin of replication) site

2. Strands separate and replication takes place in both directions

3. Daughter plasmids separate

Episomes

• A plasmid that is replicating

• Can integrate into bacterial chromosome

F pili

• The F pili attaches to a receptor site (OMP A protein) on the recipient cell (F-)

• The F pili doesn’t bind to donor cells (F+) due to presence of TRA T protein, which prevents interaction with OMP A and TRA S

• TRA S protein prevents DNA transfer

• The coupling is stabilised by TRA N and G proteins

• The pilus contracts so that the two cells come into close contact through formation of a conjugation tube

F factor

• F pili strains have a plasmid that promotes conjugation - the F factor

• Promotes the transfer of genes within bacterial populations

• Large (99.2 kBp)

• 34 tra genes that are involved in transfer process

• Pilus contracts so 2 cells come into close contact through conjugation tube (sex pilus) formation

F+ x F- matings

• F cells are converted to F+ ones

• Few bacterial genes are mobilised

Hfr x F- matings

• Plasmid integrates with bacterial chromosome

• F cells remain F- ones

• All bacterial genes are mobilised as plasmid is now in the chromosome

• It’s all transcribed and translated

Hfr Transfer Steps

1. F plasmid integrates into chromosomes by recombination

2. Cells join by a conjugation pilus

3. Portion of F plasmid partially moves into a recipient cell trailing a strand of donor’s DNA

4. Conjugation ends with pieces of F plasmid cell and donor DNA in recipient cell

5. Cells synthesise complementary DNA strands

6. Donor DNA and recipient DNA recombine making a recombinant F- cell

Interrupted Mating

• Hfr crosses were used to genetically map the E. coli chromosome using the interrupted mating technique

• Crosses are set up and samples are taken at time intervals, which are homogenised to break the conjugation tube so that further mating is prevented

• The samples are then plated to select for the genes that have been transferred

• Media selects recombinants, not parental types

• These colonies are then characterised for the other genes

• Amplify the gene with PCR/grow it in a colony (with bacteria)

• Genetic map of E. coli is measured in minutes rather than recombination units because it has been constructed using the interrupted mating technique

• The F plasmid can integrate at a number of sites in the chromosome, so that there are many Hfr strains

• Each of these strains will transfer genes in a specific direction, depending on its orientation in the chromosome

  • The map was constructed by piecing together the transfer times for genes mobilised by all these Hfr strains

Linked Genes

• Close together on the same chromosome

• Don’t assort independently

• Discovered by Bateson, Saunders, and Punnet (1905)

• Didn’t find 9:3:3:1 ratio expected by the F2 generation

• Each crossover leads to half recombinant and half non-recombinant gametes

• Therefore, the total % of recombinant gametes is always half the % of meiosis (where crossing over happens)

• The maximum proportion of recombinant gametes is 50%

No Crossing Over

1. Homologous chromosomes pair in prophase I

2. If no crossing over takes place, each gamete receives a nonrecombinant chromosome with an original combination of alleles

Crossing Over

1. A crossover may take place in prophase I

2. In this case, half of the resulting gametes will have an unchanged chromosome (nonrecombinant) and half will have recombinant chromosomes

Double Crossover

1. Single crossover will switch the alleles on homologous chromosomes

2. A second crossover will reverse the effects of the first, restoring the original parental combination of alleles

3. It produces only nonrecombinant gametes, although parts of the chromosomes have recombined

Creating a Genetic Map (pre-genomic revolution)

• The map unit is called Morgan

• One map unit = one centimorgan = 1% probability that two alleles swap at each generation

• Can require having a marker to obtain heterozygous genotypes (in the case of no clear phenotype)

• Double crossing-overs can happen and bias the map

• Calculating relative distance on genetic map by add up recombinant

Recombination Frequency

= (no. of recombinant progeny/total no. of progeny) x 100%,

Limitations of Genetic Maps

• Genetic distance is not strictly equivalent to physical distance

• Recombination rates vary along the genome

Other Techniques to Physically Map the Gene

• FISH (fluorescence in-situ hybridisation)

• If probe is available

How the Genomic Revolution has Simplified Genetic Mapping

• High throughput technologies allow for the generation of scaffolds

• Even entire genomes (prokaryotes) bringing the researcher much closer to a sequencing a full chromosome to its full length

Power of Next Generation Sequencing Technologies

• Massive amounts of data

• Information collected across the whole genome

• More precise for downstream applications

• Less expensive

GWAS (Genome Wide Scans of Association)

Cohorts with 100,000 or even million individuals

QTL (Quantitative Trait Analysis)

Identify regions of the genome that discriminate the progeny for a phenotype