Antisense repression in Cryptococcus neoformans as a laboratory tool and potential antifungal strategy

Jenifer M. Gorlach1, Henry C. McDade1, John R. Perfect1 and Gary M. Cox1

Departments of Medicine and Microbiology, Duke University Medical Center, Durham, NC, USA1

Author for correspondence: Gary M. Cox. Tel: +1 919 681 5055. Fax: +1 919 684 8902. e-mail: gary.cox{at}duke.edu


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Antisense repression was used as a method to alter gene function in the human-pathogenic fungus Cryptococcus neoformans. The calcineurin A gene (CNA1) and the laccase gene (LAC1) were targeted since disruption of these loci results in phenotypes that are easy to screen (temperature sensitivity and lack of melanin, respectively). Serotype D yeasts were transformed with a plasmid containing the CNA1 cDNA in an antisense orientation under the control of the inducible GAL7 promoter, and serotype A yeasts were transformed with a plasmid containing the LAC1 cDNA in an antisense orientation under the control of the constitutive actin promoter. The calcineurin transformants demonstrated a temperature-sensitive phenotype only when grown on galactose, and the laccase transformants had decreased melanin production. Northern blot analysis of the calcineurin antisense transformants confirmed that the inducible phenotype was associated with a decrease in the native CNA1 transcript levels. Furthermore, it was possible to modestly impair growth of C. neoformans at 37 °C by using a 30 bp antisense oligonucleotide targeting CNA1. Antisense repression is now available as a tool for molecular studies in this organism, and may be applicable to other human-pathogenic fungi that have less amenable genetic systems.

Keywords: calcineurin, antisense oligonucleotide, yeast, fungus, pathogenesis

Abbreviations: RACE, rapid amplification of cDNA ends; 5-FOA, 5-fluoroorotic acid


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Cryptococcus neoformans is one of the most significant pathogenic fungi to infect immunocompromised humans. The treatment of this infection, and of all fungal infections in general, is associated with high morbidity and mortality (Casadevall & Perfect, 1998 ). The hope is that basic research on the pathogenesis of fungal infections will identify targets for the development of improved treatment strategies. In this regard, C. neoformans has proven to be an excellent model system for molecular pathogenesis studies (Perfect, 1996 ).

Antisense repression is a means to alter gene function that works at the level of mRNA rather than at the level of the genomic locus (Inouye, 1988 ; Kuss & Cotter, 1999 ; Persidis, 1999 ). Antisense repression starts by having single-stranded RNA that is complementary to the target mRNA. The antisense RNA binds to the target mRNA to form an RNA–RNA duplex, and the target mRNA is inactivated, possibly as a result of degradation or inability to access the translational machinery (Liu & Carmichael, 1994 ; Kumar & Carmichael, 1998 ).

Antisense repression has several features that can be complementary to the standard methods of gene inactivation using targeted disruption. In conventional gene disruption strategies, especially in an organism like C. neoformans whose genome has many introns, the genomic locus must be cloned. Antisense repression does not require the isolation of the genomic locus, and gene inactivation can be accomplished by using relatively short pieces of coding sequence rather than entire genes. This allows for the use of techniques such as RACE (rapid amplification of cDNA ends) to quickly make constructs for mutagenesis. Another advantage is that antisense repression does not require homologous integration, and it may be especially useful for other fungal organisms in which rates of homologous recombination are very low. Another potential use for antisense repression is the simultaneous inactivation of multiple loci by using a single antisense molecule that targets a sequence common to all of the target mRNAs. For example, a single antisense transcript affected the expression of all three discoidin I genes in Dictyostelium, a feat that otherwise would have required three separate rounds of targeted disruption (Crowley et al., 1985 ). In addition, a single antisense oligonucleotide targeted against the multicopy gene encoding gp90 of Trypanosoma cruzi was able to inhibit function in vivo (Malaga & Yoshida, 2001 ). Genes essential for vegetative growth can be identified with antisense repression (Liu et al., 1992 ; Tentler et al., 1997 ), and this was also recently demonstrated in Candida albicans (DeBacker et al., 2001 ). Proving that genes are essential to an organism is extremely complicated since they cannot be easily identified using conventional gene knockout strategies. However, genes essential to the organism may be ideal targets for the development of new antifungal drugs since inhibition of function would confer fungicidal rather than fungistatic activities.

Antisense repression is not only useful as a laboratory tool, but has also been used in vivo as an antibiotic and to alter host gene function. There is currently an FDA-approved medication consisting of antisense oligonucleotides for the treatment of cytomegalovirus (CMV) retinitis (Perry & Balfour, 1999 ), and there are clinical trials in humans that are using antisense oligonucleotides to treat leukaemia and other malignancies (Kuss & Cotter, 1999 ). Furthermore, investigators have injected antisense oligonucleotides into experimental animals to alter gene expression of both the host (McKinley et al., 2000 ) and the pathogen (Chakraborty et al., 1999 ).

In this paper we describe the adaptation of antisense repression to the study of a human-pathogenic fungus, and the potential use of antisense oligonucleotides as antifungals. The availability of this technique should facilitate future molecular studies using Cryptococcus neoformans, and we also propose that antisense repression can be applied to other human-pathogenic fungi. Antisense repression has already proven itself to be valuable in the diploid Candida albicans, where a single transformation event has resulted in altered gene function (DeBacker et al., 2001 ). Antisense repression may also be valuable in molecular studies of other fungi such as Coccidioides immitis and Histoplasma capsulatum where genetic manipulations are much more technically difficult.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Cryptococcus neoformans strains and media.
JEC43 (ura5) and JEC50 (ade2) were derived from JEC21 (serotype D, mating type {alpha}) and were a generous gift from Jeff Edman. MCC1 (cna1::ADE2) was derived from JEC50 (Cruz et al., 2000 ) and was a generous gift from M. Cristina Cruz and Joseph Heitman. HMC6 (lac1::URA5) was derived from JEC21 (Rosas et al., 2000 ). Strain H99 is a wild-type, serotype A, mating type {alpha} strain, and H99R is a spontaneous ura5 auxotroph that resulted from plating H99 on 5-FOA medium. All strains were stored in glycerol stocks at -80 °C, and were maintained on YEPD agar (1% yeast extract, 2% peptone, 2% dextrose [glucose] and 1·8% agar). Induction of antisense vector was done on YEPGal (1% yeast extract, 2% peptone, 2% galactose). Minimal medium plates were made using 0·67% yeast nitrogen base without amino acids, 2% glucose and 1·8% agar. Dopamine medium was made as described by Rosas et al. (2000) .

Antisense vectors.
The antisense expression vectors (Figs 1 and 2) were constructed by first amplifying a 310 bp fragment containing the terminator sequence and stop codon from the C. neoformans strain H99 TRP1 (Perfect et al., 1992 ) using primers to introduce EcoRI and XbaI sites at the 5' and 3' ends, respectively (CTGAATTCTGAAGGCGGTAAGGGGTTAA and TGTCTAGAGCGACAGAAGAGAAATGGTGA; enzyme sites underlined). The amplicon was digested with EcoRI and XbaI, and cloned into pBluescript SK- (Stratagene). The resulting plasmid was digested with XbaI and SacII, and a fill-in reaction using Taq polymerase was performed. Into this blunt site was cloned a 2012 bp fragment containing the C. neoformans URA5 (Edman & Kwon-Chung, 1990 ), which was digested from pCnTEL (Edman, 1992 ) using ClaI and EcoRI, and blunt-ended using Taq polymerase. A 624 bp fragment containing the promoter and ATG start site from the C. neoformans strain JEC21 GAL7 (Wickes & Edman, 1995 ) was amplified using primers to introduce a KpnI site at the 5' end and NotI, XhoI, StuI and EcoRI sites at the 3' end (GAGGTACCATCACACATTTGTTCCGCCGGT and GAGAATTCAGGCCTCTCGAGAGGCGGCCGCCATTCTCAAGAGGGGATTGAGCGCTGATAA). The amplicon was digested with KpnI and EcoRI and ligated into the plasmid containing URA5 and the TRP1 terminator sequence to result in pJMM170 (Fig. 1a). An 860 bp fragment containing the promoter and ATG start site from the C. neoformans strain H99 actin gene (Cox et al., 1995 ) was amplified using primers to introduce the same sites on the ends as above (ATGGTACCACTCGCTATTGTCCAGGCT and GAGAATTCAGGCCTCTCGAGAGGCGGCCGCCATAGACATGTTGGGCGAGT). The amplicon was digested with KpnI and EcoRI and ligated into the plasmid containing URA5 and the TRP1 terminator sequence to result in pJMM180 (Fig. 2a). All ligations were confirmed with sequencing.



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Fig. 1. (a) Map of the relevant sites in the antisense vector pJMM170. PGAL7, GAL7 promoter; T, TRP1 terminator sequence; N, NotI; X, XhoI; S, StuI. (b) Partial map of the cloned CNA1 cDNA that was used in the antisense vectors. The left-pointing arrows represent the portions of the cDNA that were cloned in an antisense orientation into pJMM170 for each antisense vector. The approximate locations of CNA1 that were used to design antisense oligonucleotides CN1, CN2 and CN4 are also noted.

 


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Fig. 2. (a) Map of the relevant sites in the antisense vector pJMM180. PACT1, ACT1 promoter; T, TRP1 terminator sequence; N, NotI; X, XhoI; S, StuI. (b) The full-length LAC1 cDNA was amplified and cloned into a vector, from which it was digested using EcoRI and subcloned into pJMM180 to result in pLAC-full.

 
The full-length, 1920 bp CNA1 cDNA from C. neoformans strain B3501 was amplified using a pooled cDNA library as template. The amplicon was subcloned to plasmid and sequenced to confirm its identity. The cloned CNA1 cDNA was digested from the plasmid (pCR2.1, Invitrogen) using EcoRI, and subcloned to the EcoRI site of pJMM170. Several subclones were digested with XhoI to identify a plasmid where the CNA1 cDNA had been cloned into pJMM170 in an antisense orientation, and this plasmid was designated pCNA-full (Fig. 1b). A partial CNA1 cDNA fragment containing the first 1330 bp extending from the ATG start codon to a XhoI site was digested from the cloned CNA1 cDNA using XhoI and EcoRI, and subcloned to pJMM170 with the designation pCNA-5' (Fig. 1b). Another partial cDNA fragment containing the last 570 bp of CNA1 was amplified to introduce XhoI and EcoRI sites into the 3' and 5' ends, respectively, and the amplicon was ligated into pJMM170 with the designation pCNA-3' (Fig. 1b). The full-length, 1870 bp LAC1 cDNA from strain B3501 was amplified using a pooled cDNA library as template, and subcloned to plasmid for sequencing. The cloned LAC1 cDNA was digested from the plasmid (pCR2.1, Invitrogen) using EcoRI, and subcloned to the EcoRI site of pJMM180 (Fig. 2b). Several plasmids were screened using PCR to identify one containing the LAC1 cDNA in an antisense orientation behind the actin promoter (pLAC-full, Fig. 2b).

Transformation of strains JEC43 and H99R was performed using biolistic DNA delivery of intact plasmid as described by Davidson et al. (2000) . Transformants were selected on minimal medium at 30 °C.

Antisense oligonucleotides having phosphorothioate substitutions were obtained unpurified from the manufacturer (Operon) with the following sequences: CN1, ataCCACATCTTGAGCGACCACAAATAaagC; CN2, gggATGGGGCGTACAATTGAATTGTCggaT; CN4, cgcCAAGCAGTGAGTGGCGGACCTCCAatgG (phosphorothioate-substituted nucleotides in lower case; approximate locations of each oligonucleotide within CNA1 shown in Fig. 1b). Oligonucleotides were suspended in TE buffer, aliquoted and frozen at -20 °C. Yeast experiments using oligonucleotides were done by diluting OligofectAMINE reagent (Life Technologies) 1:5 with RPMI (pH 7·0) medium. Ten microlitres of the diluted reagent was mixed with various concentrations of oligonucleotides and left to incubate at room temperature for 20 min. The oligonucleotide/OligofectAMINE mixtures were then placed into 96-well plates, and 10 µl of a suspension of 106 c.f.u. of strain H99 cryptococci ml-1 was added to each well. The total volume of each well was adjusted to 100 µl with RPMI, and the plates were incubated for 24 h at 37 °C with shaking. Growth was assessed using duplicate quantitative cultures. Each experiment was repeated three separate times and the data were averaged.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
Transformation with the antisense construct
In order to adapt antisense repression for use in C. neoformans, we targeted the CNA1 and LAC1 genes. Previous studies have shown that cna1 and lac1 mutants of C. neoformans generated via targeted gene disruption are unable to grow at 37 °C (Cruz et al., 2000 ; Odom et al., 1997 ) and lack melanin (Rosas et al., 2000 ; Salas et al., 1996 ), respectively. Both a full-length CNA1 cDNA and partial cDNA fragments were placed in an antisense orientation (3' to 5') in the antisense vector pJMM170, where transcription is under the control of the inducible GAL7 promoter (Fig. 1). The full-length LAC1 cDNA was placed in an antisense orientation in pJMM180, where transcription is controlled by the actin promoter (Fig. 2). The antisense constructs were used to transform yeast so as to integrate the vector into the genome in a random, ectopic fashion. Southern blots of selected pCNA-full and pCNA-5' transformants demonstrated that the native CNA1 was intact, and that the antisense construct appeared to integrate randomly with multiple, tandem repeats (data not shown).

Demonstration of antisense effect
The calcineurin transformants were screened for phenotype on glucose- and galactose-containing media at both 30 °C and 37  °C (Fig. 3). A cna1 strain (MCC1), wild-type and an ‘empty vector’ transformant (transformed with pJMM170 alone) were used as controls. The cna1 strain demonstrated the expected phenotype of no growth at 37 °C on both YEPD and YEPGal, whereas the empty vector control was able to grow on both media at both temperatures (Fig. 3). Ten independent transformants containing the full-length CNA1 antisense construct (pCNA-full) were able to grow on both YEPD and YEPGal at 30 °C and YEPD at 37 °C (data for five of these are shown in Fig. 3). However, the antisense transformants were not viable under conditions of induction (YEPGal) at 37 °C (Fig. 3).



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Fig. 3. Transformants containing the CNA1 antisense vector displayed an inducible phenotype identical to the cna1 knockout strain. Serial 1:10 dilutions of each strain were plated onto YEPGal (Gal, galactose) or YEPD (Glc, glucose) and grown at 30 °C and 37 °C. WT, wild-type strain JEC21; cna1, calcineurin A mutant generated by targeted gene disruption (strain MCC1); control, ‘empty vector’ control strain generated using pJMM170; 1–5, five independent strains generated with the CNA1 antisense construct pCNA-full.

 
In order to see if partial cDNA fragments could be used in antisense repression, transformants were made using the constructs pCNA-5' and pCNA-3' (Fig. 1b). Six transformants resulting from both plasmids were found to have phenotypes identical to transformants resulting from the construct containing the full-length cDNA (growth on all conditions except on YEPGal at 37 °C; data not shown).

The laccase antisense construct was used to transform the serotype A strain H99R. Twelve randomly selected transformants were screened on dopamine agar with H99 (wild-type), a lac1 strain (HCM6) and an ‘empty vector’ transformant as controls (Fig. 4). Eleven of the twelve antisense transformants had the albino phenotype of the lac1 strain, and one appeared to make melanin near wild-type levels (Fig. 4).



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Fig. 4. Transformants containing the antisense vector targeting LAC1 displayed an albino phenotype identical to the lac1 knockout strain. All strains were spotted onto dopamine agar, and 11 of the 12 strains generated with the pLAC-full construct (strains 1–4, 5–8, 9–12) were identical to strain HMC6 (lac1). WT, wild-type strain H99; control, ‘empty vector’ control strain generated using pJMM180.

 
Confirmation of phenotype with Northern analysis
Northern analysis was performed using a transformant containing the pCNA-5' construct. To avoid cross-reactivity between the antisense and native transcripts, the blot was probed with a labelled CNA1 fragment from the 3' end of the gene that was not included in the pCNA-5' construct. The blot established that the amount of CNA1 transcript was markedly decreased in the transformant when grown under the conditions of galactose induction (Fig. 5). An empty vector control strain demonstrated no significant differences in CNA1 expression in yeast grown on glucose as compared to galactose medium (Fig. 5).



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Fig. 5. Northern blot demonstrating decreased levels of native CNA1 transcript in an antisense strain generated with pCNA-5' (Anti 1) only when the cells are grown with galactose (Gal) and not with glucose (Glu). Total RNA from JEC21 (WT) and an ‘empty vector’ strain (Control) were included. The blot was stripped and reprobed with actin, which confirmed equivalent loading of RNA into each well (data not shown).

 
Use of antisense oligonucleotides
Three 30 bp antisense oligonucleotides that are complementary to various portions of the CNA1 coding sequence were synthesized with the terminal three residues of each oligonucleotide having a phosphorothioate-substituted sugar in order to provide resistance to nuclease degradation (Fig. 1b). The choices of the sequences targeted by the oligonucleotides were based on low levels of predicted secondary structure in the CNA1 mRNA, and the chosen sequences were conserved in both the H99 and B3501 strains. The oligonucleotides were mixed with wild-type C. neoformans strain H99 at different concentrations, and grown in RPMI medium at 37 °C. A T7 primer was used as a control oligonucleotide. None of the calcineurin antisense oligonucleotides appeared to have significant effects on growth at a concentration of 4 µg ml-1 as compared to both the no-oligonucleotide control and the T7 control (data not shown). At the 40 µg ml-1 concentration, only the CN4 oligonucleotide had a significant impact on high-temperature growth (Table 1), although the decrease in cell number compared to the two control groups was relatively modest. The growth rates were not affected by the same concentration of the CN4 oligonucleotide when the yeasts were grown at 30 °C (Table 1). Higher concentrations of oligonucleotides were used, but growth rates for both the T7 and calcineurin antisense groups were similarly decreased in the 400 µg ml-1 assays (Table 1).


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Table 1. Effects of oligonucleotides on the growth of strain H99

 
Conclusions
This is the first adaptation of antisense repression to the study of Cryptococcus neoformans. We constructed antisense vectors containing a multicloning site that allows for cDNA fragments from either standard libraries or RACE protocols to be directly ligated into the vector in an antisense orientation behind the promoter. One of the vectors has an inducible GAL7 promoter for use in serotype D strains, and the other has an actin promoter for constitutive expression in serotype A strains. Transformation using the antisense vectors and biolistic DNA delivery results in integration into the genome, often with multimeric repeats. Both the integration into the genome and the repeats work to the advantage of antisense repression since the antisense transcripts are made in high copy number in the nucleus so as to have the best chance of associating with the target mRNA.

Using the antisense vector to target CNA1 in a serotype D strain and LAC1 in a serotype A strain, we were able to demonstrate that the function of these genes could be altered by antisense repression. This was demonstrated by the fact that transformants containing the antisense vector had the expected phenotype of the corresponding knockout mutants. In the case of the calcineurin antisense mutants, the antisense phenotype was only observed under the conditions of induction of the antisense transcript. Furthermore, the level of CNA1 mRNA in an antisense transformant was markedly decreased only when the cells were grown under the conditions of induction, suggesting that the native mRNA was destroyed by the antisense transcripts. The use of an inducible promoter not only helps with demonstrating that antisense repression is responsible for the observed phenotypes, but also provides for the screening of essential genes. Antisense repression seems broadly applicable for use in C. neoformans since we have demonstrated success by targeting different genes in both of the main serotypes used in molecular studies of this yeast.

We were able to target CNA1 using antisense repression by using both whole and partial cDNA sequences as template for the antisense transcripts. Because one potential use of antisense repression is to screen cDNA libraries for phenotypes of interest, it was important to verify that partial cDNA sequences, especially from the 3' portion of the gene, can be used in antisense repression. It is difficult to predict how small an antisense transcript can be and still disrupt gene function since the efficiency of binding to the target mRNA is dependent on multiple different factors including copy number of the native mRNA and secondary structure. It is also likely that genes differ widely in the number of mRNA copies that are necessary for wild-type function. Antisense repression probably cannot completely abolish all native mRNAs, and some genes may have wild-type function with only very few copies of mRNA present, whereas other genes may require many copies. It appears that the C. neoformans CNA1 may be an example of the latter possibility. The Northern blots of transformants grown under the conditions of induction show that the levels of the native CNA1 mRNA were very low, but still detectable. However, the transformants were still unable to grow at high temperatures, suggesting that high levels of message are needed for wild-type expression of this particular phenotype.

In order to test whether very small pieces of DNA could also be used in antisense repression in C. neoformans, we used 30 bp oligonucleotides complementary to various parts of CNA1. We used sequences that were predicted to have low secondary structure so as to select for parts of the target mRNA that are more likely to bind the antisense oligonucleotide. The sugars of the terminal three nucleotides had phosphorothioate substitutions in order to increase resistance to nucleases, and thus increase stability within the cell. Only one of the three oligonucleotides appeared to have any significant effect on growth at 37 °C, and the associated growth defect was relatively modest. There did appear to be non-specific toxicities associated with high concentrations of oligonucleotides, as demonstrated by the yeast exposed to 400 µg ml-1 of the T7 oligonucleotide. However, the growth defect at 37 °C associated with the CN4 oligonucleotide does not appear to be a non-specific toxicity since yeast grown with the same concentration of the CN4 oligonucleotide did not have a growth defect at 30 °C. The fact that the CN1 and CN2 oligonucleotides did not appear to affect growth as compared to both the T7 and no-oligonucleotide controls also supports the absence of non-specific toxicities at the 40 µg ml-1 concentration. Therefore, we have demonstrated that a biological effect on growth can be achieved in this pathogenic yeast with antisense oligonucleotides.

There are two possible reasons why the antisense effect using the CN4 oligonucleotide was so much less pronounced than when using the integrated antisense vectors. The much smaller sizes of the oligonucleotides as compared to the antisense transcripts are likely to result in less effective targeting of the native mRNA. Furthermore, the introduced antisense oligonucleotides need to be taken up by the yeasts, whereas the antisense transcripts are produced in high numbers within the nucleus. We used the OligofectAMINE reagent with the oligonucleotides to form transformation complexes in an effort to increase delivery to the yeasts since this has apparently worked with a variety of cultured eukaryotic cells (unpublished data provided by the manufacturer). However, it seems clear that in order to optimize antisense oligonucleotides for use as antifungal agents, there will have to be technological advances in the delivery of oligonucleotides to yeast. There will also have to be careful mapping of selected genes in order to identify short sequences that can be targeted with high efficiency. Ideally, the targeted sequences would be conserved across a wide range of fungi so that the oligonucleotide would have a broad spectrum of action. It is possible that antisense oligonucleotides may be useful as adjuncts to standard antifungals in the treatment of infections.


   ACKNOWLEDGEMENTS
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ABSTRACT
INTRODUCTION
METHODS
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This work was done as part of the Duke University Mycology Research Unit (DUMRU), and was supported by Public Health Service grants AI01334 (G.M.C.) and AI28388 (J.R.P.) from the National Institute of Allergy and Infectious Diseases. G.M.C. was a recipient of the Burroughs Wellcome Fund New Investigator Award in Molecular Pathogenic Mycology. We would like to acknowledge Joseph Heitman for his support and helpful suggestions, and Garrett Heinrich for technical assistance. Portions of this work were presented at the Fourth International Conference on Cryptococcus and Cryptococcosis, September 1999.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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Received 4 May 2001; revised 5 September 2001; accepted 11 September 2001.