Comparison Of CNLAC1 Between Wild Type(Mel+)and Mutant Type(Mel-)of Cryptococcus Neoformans

Cryptococcus neoformans is an encapsulated yeast which causes cryptococcosis,a disease typified by an initial pulmonary infection which can disseminate to cause a life threatening meningoencepha- litis.Infections caused by the fungal pathogen Cryptococcus neofor- mans have been increasing steadily over the past 10 years because of the onset of AIDS and the expanded use of immunosuppressive drugs.Research into the potential virulence factors of this yeast has recently attracted particular attention. Melanin production is a major virulence factor for Cryptococcus neoformans. The phenoloxidase enzyme system contributes to virulence by protecting C.neoformans against nitrogen~- and oxygen~+ derived oxidative antimicrobial molecules produced by immune effector cells. In order to characterize the events involved in melanin synthesis,an enzyme having diphenol oxidase activity was purified and its gene was cloned.The eenzyme was purified as a glycosylated 75-Kda protein whichmigrated at 66Kda after deglycosylation.The cloned gene of Cneoformans laccase (CNLACl) contained l4 intrOns ranging from 52 to340 bases long.We compare Cmptococcus neOformans between wild tyPes(MeI+) andmutants(Mel-).First a naturally occutring Mel-(CCCCl0l48) variant ofCryPtOcoccus neOfOrmans was obtained from the CmptOcoccus Laborato-ry,Department of dermatol ogy, Ch angzheng Hospi ta attached to theSecond Military Medical University,Shanghai.We investigae variationsbetween mutan(Mel-)(CCCCl0l48) and wild type(MeI+)(CCCCl0297).Based on the CNLACl DNA sequence of CneOformans,oligonucleOtideprimers were synthesized and used to amPlify DNA by PCR.Analysiswas cwhed ou.No deleton was foUnd in wild tyPe.HOwever,CNLACldeletion(about 450bp)in the mutants was present(between 17l5bp and26l7bp).We subclone the PCR DNA product into the vector PGEM-T forsequence. The sequence was found be different between wild type(Mel+)and mutant(mel-). An exPression vector has been constrUcted .They he1pus study the biochemical and molecular of the mutan in CNLACl .Theyalso migh provide us a target gene to prognosis and treatrnent.