During 2007-2008, a 'knock in'construct was designed to introduce epitope taged coding DNA into endogenous loci by homologous recombination. To construct a knock-in of the Flag-epitope tag, a knock-in cassette was designed. The cassette contains sequences that encode a triple Flag epitope tag (3XFlag) adjacent to a URA5 gene flanked by two direct repeats of 120 nucleotides. For targeted knock-in of 3XFlag, sequences homologous to the C-terminus 5'and 3'regions flanking the C-terminus of the target locus were inserted into the two respective cloning sites flanking the knock-in cassette. A ura strain wasw then transformed with the targeting construct and selected for uracil prototrophs. The targeted clones were identified by genomic PCR and then the URA5 gene was excised by selecting on 5-fluorouracil containing medium. This strategy to Flag-tag the C terminus was first used for a protein that functions in cAMP pathway. This strain tagged with 3XFlag was re-used and transformed with a two HA epitope tag (2XHA) construct of another gene which was similar to the 3XFlag construct except that the 3XFlag was replaced with 2XHA. The resulting epitope-tagged proteins were readily detectable by western blot as well as by immunoprecipitation using commercially available anti-Flag and anti-HA. Moreover,the first HA-tagged protein was immunocoprecipitated with the flag-tagged second protein showing the protein-protein interaction between the two proteins. These data indicate that our targeting method is feasible for multiple loci and that 3XFlag and 2XHA can serve as universal epitopes for several antibody-based applications. The same principle can be used to tag many other proteins with different epitopes in the same strain. The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available. During 2009-2010, we have constructed drug marker-tagged isogenic pairs in serotype A as well as in serotype D strains. These strains are needed for the study of mixed infection in animal model in which mating type dependent difference in tissue invasion can be analyzed. A 2 kb genomic region between CNBC5600 and CNBC6510 from chromosome 3 of strain B-3501A was amplified by PCR and either the NAT or the NEO selectable marker was separately cloned into in this region. The resulting construct was each separately transformed into strains B-3501A and B-3501B. Transformants containing a single integration on chromosome 3 were screened by PCR and confirmed by Southern blot analysis. Four strains were saved for subsequent usage;C1421 (B3501A-NAT), C1419 (B3501A-NEO), C1423 (B3501B-NAT), and C1420 (B3501B-NEO). C. neoformans is notorious for not behaving like other yeasts in many aspect. One of such pattern is found in cell cycle and a method for synchronization has not been achieved. In many studies , cell cycle synchronization is required to resolve the questions on biological phenomenon that is associated with cell cycle. During 2009-2010, we were able to use a sucrose gradient to isolate cells enriched in G1 phase (>95% purity), but these cells did not exit the G1 phase in synchronized fashion. We used chemicals that have previously been used to achieve synchronization in other organisms, such as hydoxyurea, nocodazole, benomyl, azetidine 2-carboxylic acid and methylmethane sulfonate, but without success. Mutants of cell cycle genes known to cause cell cycle arrest in other systems were also generated in our laboratory. We have constructed a temperature sensitive allele of CDC28 and an ATP-analog sensitive allele of CDC15. However, both mutants failed to cause specific arrest in the cell cycle at restricted conditions. We are currently focusing on pheromone-mediated cell cycle arrest and are testing the G1 exit pattern in different strains in order to achieve synchronization of the cell cycle.
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