MTOC formation during mitotic exit in fission yeast.

Microtubules polymerise from nucleation templates containing gamma tubulin. These templates are generally concentrated in discrete structures called microtubule organising centres (MTOCs). In Schizosaccharomyces pombe, an equatorial MTOC (EMTOC) forms mid-way through anaphase B and then disassembles during the final stages of cell separation. We show that the EMTOC was generated by recruiting gamma tubulin to the equatorial F-actin ring before it constricted to cleave the cell in two during cytokinesis. The EMTOC was not a continuous ring. It had a variable structure ranging from a horseshoe to a number of short bars. EMTOC integrity depended upon the integrity of the F-actin but not the microtubule cytoskeleton. EMTOC assembly required the activity of both the septation-inducing network (SIN) that regulates the onset of cytokinesis and the anaphase-promoting complex. Activation of the SIN in interphase cells induced F-actin ring formation and contraction and the synthesis of the primary septum but did not promote EMTOC assembly. In contrast, overproduction of the polo-like kinase, Plo1, which also induced multiple rounds of septation in interphase cells, induced EMTOC formation. Thus, the network governing EMTOC formation shared many of the regulatory elements that control cytokinesis but was more complex and revealed an additional function for Plo1 during mitotic exit.

HLA class II typing of whole genome amplified mouth swab DNA.

Postal collection of mouth swabs provides a cheap and convenient means of DNA sampling but hitherto has not provided sufficient genetic material for HLA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP). This study examined the feasibility of collecting mouth swabs from a test population by post, amplifying the DNA by whole genome amplification and genotyping for selected HLA class II alleles. We optimised a strategy for whole genome amplification or primer extension preamplification using a random 15 base pair primer which resulted in a 1,000-fold increase in DNA template. The amplified DNA was of sufficient quality for analysis of selected HLA Class II alleles by PCR-SSP and PCR using sequence-specific oligonucleotide probes. To test the reliability of our data, blood DNA from 30 individuals in 10 families, previously tested for all DRB1 alleles in a routine diagnostic laboratory, was then tested in our laboratory for DRB1 *03 and *04 following whole genome amplification. Further whole genome amplified product from another 10 families was tested for DRB1 *03, *04 in our laboratory and then tested for all DRB1 alleles in a routine diagnostic laboratory. One repeat typing was required to achieve 100% concordance between laboratories. Amplification of whole genome amplified DNA by PCR-SSP was then extended successfully to low-resolution HLA DRB1, DQA1, DQB1 and DPB1 typing. Mouth swab collection by post, followed by whole genome amplification of DNA provides an effective strategy for genetic analysis of large cohorts. We have optimised conditions for HLA class II typing on whole genome amplified DNA collected by mouth swab, but this method could potentially be applied to low concentrations of DNA from other sources.