High-frequency phenotypic switching in Candida albicans.

Most strains of Candida albicans are capable of switching spontaneously and at high frequencies between a number of phenotypes distinguished by colony morphology. Unlike switching in many other microbial pathogens, switching in C. albicans is pleiotropic, affecting several morphological and physiological parameters. Recently, the first phase-specific genes were identified and shown to be regulated at the level of gene transcription.

Functional analysis of the promoter of the phase-specific WH11 gene of Candida albicans.

Candida albicans WO-1 switches spontaneously, frequently, and reversibly between a hemispherical white and a flat gray (opaque) colony-forming phenotype. This transition affects a number of morphological and physiological parameters and involves the activation and deactivation of phase-specific genes. The WH11 gene is transcribed in the white but not the opaque phase. A chimeric WH11-firefly luciferase gene containing the 5' upstream region of WH11 was demonstrated to be under phase regulation regardless of the site of integration, and a series of promoter deletion constructs was used to delineate two white-phase-specific transcription activation domains. Gel retardation experiments with the individual distal or proximal domain and white-phase or opaque-phase protein extract demonstrated the formation of one distal white-phase-specific complex and two proximal white-phase-specific complexes. Specific subfragments were tested for their ability to compete with the entire domain in the formation of complexes with white-phase protein extract in order to map the proximal domain sequence involved in white-phase-specific complex formation. Our results indicate that white-phase-specific transcription of WH11 is positively regulated by trans-acting factors interacting with two cis-acting activation sequences in the WH11 promoter.

Transcription of the gene for a pepsinogen, PEP1, is regulated by white-opaque switching in Candida albicans.

Cells of Candida albicans WO-1 spontaneously switch between a white and opaque CFU, and this phase transition involves a dramatic change in cellular phenotype. By using a differential hybridization screen, an opaque-specific cDNA, Op1a, which represents the transcript of a gene regulated by switching, has been isolated. The gene for Op1a is transcribed by opaque but not by white cells. The nucleotide sequence of the Op1a cDNA reveals over 99% base homology with an acid protease gene of C. albicans, and the predicted amino acid sequence demonstrates that the product of this gene is a member of the family of pepsinogens, which possess a hydrophobic leader sequence for secretion and two catalytic aspartate domains. Southern blots of both genomic DNA digested with 14 different endonucleases and electrophoretically separated chromosomes were probed with the Op1a cDNA. No polymorphisms were detected in either case between white and opaque cells, suggesting that no genomic reorganization occurs in the proximity of the gene during the white-opaque transition. Although transcription of Op1a correlates with the high levels of extracellular protease activity in opaque cell cultures and the absence of activity in white cell cultures, stimulation of extracellular protease activity by addition of serum albumin is not accompanied by Op1a transcription in cultures of WO-1 white cells or cultures of two additional clinical isolates of C. albicans, suggesting that expression of one or more other protease genes is stimulated in these cases. The results demonstrate that transcription of the Op1a gene is under the rigid control of switching in strain WO-1.

Reporters for the analysis of gene regulation in fungi pathogenic to man.

In the past few years, highly sensitive gene reporters have been developed for the infectious fungi including gene reporters with altered codon usage. The tools are, therefore, now at hand for functionally characterizing the promoters of genes regulated by the bud-hypha transition, high frequency switching and cues from the cellular environment.

Retropseudogenes for human chromosomal protein HMG-17.

The human genome contains multiple copies of sequences homologous to the cDNA coding for non-histone chromosomal protein HMG-17. To study the mechanism of generation and dispersion of the HMG-17 multigene family a human genomic library was screened and 70 clones isolated and studied by Southern transfer and restriction site analysis. The results suggest that most of the clones contain unique sequences. Sequence analysis of two genomic clones indicates that they contain elements typical of processed retropseudogenes. Even though both sequences contained open reading frames the sequences lacked introns, were flanked by short, direct repeats and lacked elements associated with functional genes. The sequences of the two pseudogenes were 85% homologous to each other and each was 90% homologous to the human cDNA. Based on the sequence difference in the open reading frame between the pseudogenes and the cDNA it can be estimated that the sequences arose approximately ten million years ago from a common precursor. The present paper, which is the first study on genes coding for this nucleosomal binding protein, indicates that the HMG-17 multigene family is the largest known human retropseudogene family.

A single copy gene for chicken chromosomal protein HMG-14b has evolutionarily conserved features, has lost one of its introns and codes for a rapidly evolving protein.

The evolutionary origins and common features of the genes coding for the HMG-14/-17 family of chromosomal proteins have been studied by isolating and sequencing the chicken HMG-14b gene, the true homolog of the human and calf HMG-14 gene. Comparison of the structure of this gene to that of the human HMG-14 gene and to the human and chicken HMG-17 genes indicates that the HMG-14 and HMG-17 genes evolved from a common ancestor. We postulate that the ancestral gene consisted of six exons. In all genes the first exon codes for the entire 5' untranslated region and for the first four amino acids, which are invariant among all the known members of the HMG-14/-17 protein family. The last exon codes for ten to 16 amino acids and for the entire 3' untranslated region, which, for each gene, constitutes over 70% of the transcript. The DNA-binding domain of the proteins is encoded by two distinct exons. The genes are characterized by 5' regions that are highly enriched in G + C residues and have features characteristic of "housekeeping" genes. The HMG-17 genes are distinct from the HMG-14 in that the 5' regulatory region of the former has two TATA boxes while the HMG-14 genes have no such regulatory element. The chicken HMG-14b gene is a single-copy gene and produces a unique transcript. In this gene, exons II and III are fused and intron 2 is missing. The fusion of the two exons produced a codon for valine in a position that, among all HMG-14/-17 proteins, is unique to HMG-14b. The possible consequences of a valine insertion at the N-terminal end of the DNA-binding domains are discussed. The HMG-14 proteins evolve significantly faster than HMG-17, suggesting that the proteins are subject to different evolutionary pressure. However, certain amino acids are conserved among all the known members of the HMG-14/-17 protein family, suggesting that they are part of the functional domain of this family of chromosomal proteins.

A histone deacetylation inhibitor and mutant promote colony-type switching of the human pathogen Candida albicans.

Most strains of Candida albicans undergo high frequency phenotypic switching. Strain WO-1 undergoes the white-opaque transition, which involves changes in colony and cellular morphology, gene expression, and virulence. We have hypothesized that the switch event involves heritable changes in chromatin structure. To test this hypothesis, we transiently exposed cells to the histone deacetylase inhibitor trichostatin-A (TSA). Treatment promoted a dramatic increase in the frequency of switching from white to opaque, but not opaque to white. Targeted deletion of HDA1, which encodes a deacetylase sensitive to TSA, had the same selective effect. These results support the model that the acetylation of histones plays a selective role in regulating the switching process.

A white-specific gene in the white-opaque switching system of Candida albicans.

The Candida albicans strain WO-1 cells switch spontaneously, frequently, and reversibly between white and opaque colony-forming units which differ dramatically in their budding phenotypes. By screening a subtracted white cDNA library, the first white-specific cDNA, cWh11, was isolated and sequenced. This, in turn, was used to clone the Wh11 gene. Wh11 shows significant homology to the glucose/lipid-regulated GLP1 gene of Saccharomyces cerevisiae. Upon temperature-induced mass conversion from opaque to white colonies, transcription of Wh11 is abruptly activated at the second cell doubling, concomitant with commitment to the white phenotype. Wh11 exhibits the unique characteristics of being regulated not only by switching, but also by the bud-hypha transition.

Cloning of the chicken chromosomal protein HMG-14 cDNA reveals a unique protein with a conserved DNA binding domain.

The isolation and sequencing of a cDNA clone coding for the entire sequence of chicken chromosomal protein HMG-14 is described. The open reading frame constitutes only 25% of the transcript; the 5'-untranslated region is extremely rich in GC residues; and the 3'-untranslated region is highly enriched in AT residues. Comparison with other cDNAs coding for HMG-14 and HMG-17 reveals that the transcripts of genes coding for this family of chromosomal proteins have a characteristic structure. The deduced amino acid sequence is unique and different from all other known HMG-14 and -17 sequences. Analysis of amino acid position identity between the chicken HMG-14 and other HMG-14/-17 proteins revealed that the protein has 37% similarity to the HMG-17 group and 69% similarity to the HMG-14 group; therefore, the protein is classified as belonging to the HMG-14 group. Additional analysis leads to the conclusion that the chicken cDNA described here codes for the true homolog of calf and human HMG-14 protein. Comparison of all the known HMG-14 sequences reveals the DNA binding domain is conserved and contains the invariant dodecapeptide PKRRSARLSAKP. The HMG-14 proteins have a distinct charge distribution along the polypeptide chain: while the central region is positively charged the C-terminal domain is negatively charged.

Single copy gene for the chicken non-histone chromosomal protein HMG-17.

A chicken genomic library was screened with the human cDNA encoding the non-histone chromosomal protein HMG-17 and a 4565-base pair fragment containing the entire gene encoding this protein was isolated and characterized. Sequence analysis of the fragment revealed that from the start to end of transcription, the HMG-17 gene is 3293 base pairs long and is comprised of 6 exons ranging in size from 30 to 890 base pairs. Upstream of the putative cap site are both a CAAT box and a TATA box as well as several Sp1 binding sites. The gene has an extremely high content of G and C residues (75%) in a 1150-base pair fragment starting 500 base pairs from the putative cap site. This region satisfies the definition of an HpaII tiny fragment island. Southern analysis indicated that there is a single copy of this gene in chickens, whereas Northern analysis revealed that a single transcript is being synthesized from this gene. A comparison of the chicken and human cDNA and protein sequences and subsequent calculation of the evolutionary rates indicated that HMG-17 is a slowly evolving gene. The present article, which is the first study on the isolation and characterization of a complete gene coding for a high mobility group non-histone protein, indicates that the gene has features characteristic of housekeeping genes.

A method for isolation of protoplasts from dermatophytes.

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.

Expression of human chromosomal proteins HMG-14 and HMG-17 in Saccharomyces cerevisiae.

The cDNAs coding for human chromosomal proteins HMG-14 and HMG-17 were cloned into yeast expression vector pBM150, under the control of the Gal10 promoter. Northern analysis of transformed yeast cells revealed that both cDNAs were efficiently transcribed. Western analysis indicated that the mRNAs were translated into authentic proteins. Expression of human HMG proteins in yeast cell did not produce detectable phenotypic changes, as measured by the growth rate of the yeast cells under a variety of conditions. The antibiotic resistance of the transfected cells was similar to that of control cells, suggesting that the presence of HMG did not affect the expression of actively transcribed genes. However, examination of the protein profile on two-dimensional polyacrylamide gel electrophoresis revealed differences between control and HMG-transfected cells.

The frequency of integrative transformation at phase-specific genes of Candida albicans correlates with their transcriptional state.

The phase transition between the white and opaque phenotypes in the switching system of Candida albicans strain WO-1 is accompanied by the differential expression of the white-specific gene WH11 and the opaque-specific gene PEP1. The frequency of integrative transformation at the white-specific gene locus WH11 is between 4.5 and 7.0 times more frequent in white than in opaque spheroplasts, and the frequency of disruptive transformation at the opaque-specific gene locus PEP1 is 30.5 times more frequent in opaque spheroplasts than in white spheroplasts. In contrast, the frequencies of integrative transformation at the constitutively expressed loci ADE2 and EF1 alpha 2 are similar in the white and opaque phases. Therefore, the frequency of integration of linear plasmid DNA containing sequences of phase-specific genes correlates with the transcriptional state of the targeted locus.

Coordinate regulation of two opaque-phase-specific genes during white-opaque switching in Candida albicans.

Cells of Candida albicans WO-1 switch spontaneously and frequently between a white and an opaque CFU. Recently, an opaque-phase-specific cDNA, PEP1, was cloned and was demonstrated to code for a pepsinogen. By using a differential hybridization screen, a second opaque-phase-specific cDNA, Op4, has been isolated and its corresponding gene has been cloned. Op4 is coordinately regulated with PEP1 but resides on a different chromosome. During temperature-induced mass conversion from opaque to white, transcription of PEP1 and Op4 is immediately inhibited by the increase in temperature, but transcription of both genes can be rapidly reestablished by a downshift in temperature prior to phenotypic commitment. However, the capacity to rapidly induce both PEP1 and Op4 is lost coincidentally with the second semisynchronous round of cell division and phenotypic commitment during mass conversion. Op4 shows no significant base or amino acid sequence homology with a known gene or protein, respectively. However, the deduced Op4 protein exhibits several interesting characteristics, including a hydrophobic amino terminus with 26 amino acids, a pI of 10.73 for the last 100 amino acids, two serine repeats adjacent to alanine repeats, and the potential for alpha-helical conformation within the alanine-rich sequences. No genomic reorganization was evident in the proximity of Op4 during transcriptional activation and deactivation accompanying the white-opaque transition.

Chromosomal protein HMG-14. Complete human cDNA sequence and evidence for a multigene family.

The isolation and sequencing of cDNA clone coding for the entire sequence of human nonhistone chromosomal protein HMG-14 is described. Sequence analysis reveals that the open reading frame constitutes only 25% of the transcript, that the 5'-untranslated region is extremely rich in GC residues (75%), and that the 3'-untranslated region is highly enriched in AT residues. The amino acid sequence, deduced from the reading frame, is 94% homologous to the calf thymus protein suggesting evolutionary constraints on the conformation of the protein. The human genome contains 60-90 HMG-14 gene copy equivalents which, as suggested by Southern analysis, are not tandemly arranged. Northern analysis of RNA isolated from several sources reveals that a single-sized mRNA codes for this protein. Southern analysis reveals that cross-hybridizing sequences are present in the genome of several different species indicating that the evolutionary origin of this gene was over 350 million years ago. The overall features of the human HMG-14 cDNA are very similar to those of the human HMG-17 cDNA, and the number of gene equivalents present in the human genome is similar for the two proteins. However, their nucleotide sequence is significantly different indicating that the multigene family coding for HMG-14 is distinct from that coding for HMG-17.

The WH11 gene of Candida albicans is regulated in two distinct developmental programs through the same transcription activation sequences.

Candida albicans strain WO-1 undergoes two developmental programs, the bud-hypha transition and high-frequency phenotypic switching in the form of the white-opaque transition. The WH11 gene is expressed in the white budding phase but is inactive in the white hyphal phase and in the opaque budding phase. WH11 expression, therefore, is regulated in the two developmental programs. Through fusions between deletion derivatives of the WH11 promoter and the newly developed Renilla reniformis luciferase, the WH11 promoter has been characterized in the two developmental programs. Three transcription activation sequences, two strong and one weak, are necessary for the full expression of WH11 in the white budding phase, but no negative regulatory sequences were revealed as playing a role in either the white hyphal phase or the opaque budding phase. These results suggest that regulation is solely through activation in the white budding phase and the same mechanism, therefore, is involved in regulating the differential expression of WH11 in the alternative white and opaque phases of switching and the budding and hyphal phases of dimorphism.

Misexpression of the white-phase-specific gene WH11 in the opaque phase of Candida albicans affects switching and virulence.

Candida albicans WO-1 switches between a white- and an opaque-colony-forming phenotype. The gene WH11 is expressed differentially in the white phase. The WH11 open reading frame was inserted downstream of the promoter of the opaque-phase-specific gene OP4 in the transforming vector pCWOP16, and resulting transformants were demonstrated to misexpress WH11 in the opaque phase. Misexpression had no effect on the ability to switch from the white to the opaque or the opaque to the white phase, and it had no effect on the genesis of the unique opaque-phase cellular phenotype, even though the Wh11 protein was distributed throughout the cytoplasm in a manner similar to that observed for the endogenous gene product in the white phase. Misexpression did, however, increase the frequency of the opaque-to-white transition 330-fold and markedly increased the virulence of cells in the opaque phase in a mouse tail injection model.

Cell cycle regulated synthesis of an abundant transcript for human chromosomal protein HMG-17.

The abundance and cell cycle dependent expression of the mRNA for human nonhistone protein HMG-17 were studied in synchronized HeLa cells. Slot blot analysis indicates that the HMG-17 mRNA is a very abundant message, significantly more so than histone or actin mRNA. RNA prepared from tissue culture cells contains higher amounts of HMG-17 transcripts than RNA prepared from liver suggesting a correlation between the rate of cell division and HMG-17 mRNA levels. HMG-17 mRNA is present in the cells throughout the cell cycle however there is a significant increase in the mRNA levels late in S phase suggesting that the protein is deposited on chromatin after nucleosome assembly. Synthesis of the HMG-17 transcript is not coupled to DNA replication suggesting that the cell cycle related expression during late S phase is regulated in a different manner from that of the nucleosomal histones.

Chromosomal protein HMG-14. Identification, characterization, and chromosome localization of a functional gene from the large human multigene family.

The human HMG-14 multigene family is one of the largest retropseudogene families known. To identify and isolate a functional human HMG-14 gene, genomic clones, selected with the cDNA, were screened with a set of 6 oligonucleotides. A single genomic clone was isolated suggesting that the human genome contains few, and perhaps only one, functional genes. An 8882-base pair (bp) genomic clone containing the complete, 6804-bp-long human gene together with 850 bp 5' to the start of transcription and 1228 bp 3' to the end of transcription was sequenced. The gene is comprised of 6 exons ranging in size from 30 to 839 bp, two of which code for the entire DNA binding site of the protein, and has several features typical of "housekeeping" genes. Using human-rodent somatic cell hybrids, the HMG-14 gene was localized to human chromosome 21. A restriction fragment length polymorphism, useful for further analysis and mapping, has been detected. The present article, which describes the first isolation and characterization of a gene coding for chromosomal protein HMG-14, indicates that genes coding for HMG-14 and HMG-17 may share several distinctive characteristics. Comparison with the human and chicken HMG-17 genes reveals that all contain 6 exons, that all have exons of similar size, that all have 5' regions highly enriched in GC residues and that all have features typical of housekeeping genes.

Development of DNA probes for fingerprinting Aspergillus fumigatus.

Several different DNA fragments containing nonribosomal repetitive sequences have been isolated from the genome of Aspergillus fumigatus and tested as potential DNA fingerprinting probes. Eight of these clones generate 19 or more bands when hybridized to EcoRI-digested DNA of a reference strain in Southern blots, and they fall into four families. Individual clones from two families were tested and were found to generate complex Southern blot hybridization patterns which are stable within a single strain over many generations, which vary among unrelated strains, and which are amenable to computer-assisted analyses involving large numbers of strains in epidemiological studies. Clones from three of the families clustered a majority of test strains in a similar fashion in individual dendrograms based on similarity coefficients computed from band positions in Southern blot hybridization patterns. These clones therefore fulfill the major requisites for effective DNA fingerprinting probes.