The Cryptococcus neoformans STE12alpha gene: a putative Saccharomyces cerevisiae STE12 homologue that is mating type specific.

Cryptococcus neoformans possesses two mating types, MATalpha and MATa. Alpha-Cells are more virulent than a-cells and are also, unlike a-cells, capable of producing extensive hyphae in the haploid phase. The molecular analysis of hyphae production in C. neoformans has resulted in the identification of a gene which displays substantial similarity to other fungal STE12 genes, including the presence of a highly conserved homeodomain. Overexpression of the C. neoformans gene resulted in poor growth, altered morphology and the presence of hyphal projections, phenotypes reported in similar studies of the Saccharomyces cerevisiae STE12 gene. Overexpression was also found to induce MFalpha, a pheromone, and CNLAC1, a confirmed C. neoformans virulence gene. The C. neoformans STE12alpha gene, however, has one striking difference from other fungal STE12 genes; it is found only in alpha-cells. The existence of STE12alpha in C. neoformans suggests that this fungus has elements of a conserved MAP kinase cascade, which may be organized in a novel manner.

Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type.

Cryptococcus neoformans is a major opportunistic fungal pathogen in AIDS and other immunosuppressed patients. We have shown that wild-type haploid C. neoformans can develop an extensive hyphal phase under appropriate conditions. Hyphae produced under these conditions are monokaryotic, possess unfused clamp connections, and develop basidia with viable basidiospores. The ability to undergo this transition is determined by the presence of the alpha-mating type locus and is independent of serotype. The association of the hyphal phase with the alpha-mating type may explain the preponderance of this mating type in the environment and the nature of the infectious propagule of C. neoformans.

Trypanosoma brucei dihydrofolate reductase-thymidylate synthase: gene isolation and expression and characterization of the enzyme.

The gene encoding the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) of Trypanosoma brucei brucei has been isolated and expressed in Escherichia coli, and the enzyme has been purified and characterized. The coding sequence of the DHFR-TS is 1581 nt, encoding a 527-amino-acid protein of 58,505 Da. The gene was expressed under control of the trc promoter in pKK233-2. The resulting expression plasmid conferred trimethoprim resistance to E. coli DH5 alpha and complemented the TS deficiency in chi 2913recA cells indicating the presence of active DHFR and TS. DHFR-TS was purified by methotrexate-Sepharose chromatography. In addition to the full-length enzyme, the purified enzyme contained 31 and 31.5-kDa forms of the enzyme that cross-reacted with anti-L. major DHFR-TS antibodies; one was truncated at the N- and C termini, and the other at only the C terminus. Despite the presence of sufficient TS for complementation, TS activity was not detectable in the crude extract or in the final purified enzyme preparation. Although the majority of the enzyme appears to be full length, it is possible that the TS domain has been degraded by one of more residues, which would inactivate the ability to synthesize thymidylate. Kinetic analysis of DHFR yielded kcat and Km values similar to those of related enzymes. The T. brucei DHFR has Ki values for antimicrobial antifolates pyrimethamine and trimethoprim which are significantly lower than the closely related T. cruzi or L. major DHFRs or than human DHFR.

Cloning, expression and characterization of thymidylate synthase from Cryptococcus neoformans.

The thymidylate synthase (TS)-encoding gene from Cryptococcus neoformans (Cn) has been isolated from cDNA and genomic libraries. The 1127-bp gene contains three introns and a 951-bp open reading frame encoding a 35,844-Da protein. The cDNA clones lack 324 bp of the 5' coding region of the gene. The complete coding sequence was assembled as an expression cassette in pUC19 using parts of the coding sequence from the cDNA and genomic DNA and completing the sequence using synthetic DNA. Production of active TS from Cn (CnTS) was first demonstrated by complementation of a thymine(Thy)-requiring Escherichia coli strain. The expression cassette was subsequently subcloned into the T7 polymerase vector pET15-b. In this construct, CnTS is produced as approximately 10% of the total soluble protein in E. coli. Homogeneous enzyme was obtained at a 36% yield after consecutive chromatography on DEAE-cellulose, Q-Sepharose, phenyl-Sepharose and Affi-Gel Blue. Steady-state kinetic analysis showed that the Km values for dUMP and CH2H4.folate were 2.7 +/- 0.5 microM and 38.2 +/- 2.5 microM, respectively, and the kcat was 5.1 s-1. The enzyme was stable upon storage at -80 degrees C in Tris.HCl pH 7.4 and thiol.

Molecular cloning of mouse pancreatic islet R-cadherin: differential expression in endocrine and exocrine tissue.

A search for novel pancreatic islet cadherins was undertaken using the polymerase chain reaction with mouse beta TC3 cell line cDNA and degenerate primers based on conserved C-terminal sequences in neural (N), epithelial, and placental cadherin (CAD). A hitherto uncharacterized rodent sequence was detected which was then cloned from a mouse insulinoma cDNA library and shown to be the mouse equivalent of chicken retina CAD (R-CAD). The similarity of the mouse and chicken sequences was remarkable (eight nonconservative changes in the 747 amino acids of the mature protein sequence; 95% overall identity), indicating strong conservation of function. Mouse R-CAD was also closely homologous to N-CAD (72% identity), including those regions of N-CAD implicated in the cadherin-cadherin interaction and Ca2+ binding. In vitro translation of the cDNA indicated that mouse R-CAD enters the secretory pathway and undergoes posttranslational glycosylation and proteolytic cleavage. R-CAD mRNA was distributed widely in mouse tissues with high levels present in brain, skeletal muscle, and thymus. In the pancreas, R-CAD and N-CAD showed endocrine cell specificity and a differential expression in beta- and non-beta-cells. Messenger RNA expression was evident during early pancreatic development at a time when the first pluripotent hormone-producing cells differentiate to attain their adult phenotype and become organized in islet-like clusters. The presence of R-CAD and N-CAD in islets is consistent with the neurone-like properties of this tissue. Differences in CAD expression might explain the segregation of exocrine and endocrine cells during development of the pancreas and the characteristic morphological distribution of the different endocrine cells within the islet.

Purification and characterization of recombinant Pneumocystis carinii thymidylate synthase.

Catalytically active Pneumocystis carinii thymidylate synthase is expressed to the extent of about 4% of the soluble protein in Escherichia coli chi 2913 harboring plasmid pUETS-1.8 (U. Edman, J. C. Edman, B. Lundgren, and D. V. Santi, Proc. Natl. Acad. Sci. USA 86, 6503-6507, 1989). Ion-exchange, affinity, hydrophobic, and reactive dye agarose chromatography steps were explored to devise a large-scale purification protocol for P. carinii thymidylate synthase. Sequential DE52, Q-Sepharose, phenyl-Sepharose, and Cibacron Blue F3GA chromatography yielded enzyme that was homogeneous by SDS-PAGE in a yield of over 50%. The sequence of the first 10 amino acid residues of the purified protein was in accord with that predicted from the DNA sequence. Isoelectric focusing gave a pI of 6.2. Kinetic analysis of the purified enzyme revealed that the Km values were 4.7 +/- 1.3 microM for dUMP and 15.7 +/- 4.3 microM for 5,10-methylenetetrahydrofolate, similar to those of many other thymidylate synthases; the kcat of the most active preparation was 0.8 s-1. The enzyme is stable for at least 2 months when stored at -80 degrees C in the presence of 40% glycerol, Tris-HCl, and thiol.

Characterization of an immuno-dominant variable surface antigen from pathogenic and nonpathogenic Entamoeba histolytica.

A 125-kD surface antigen of Entamoeba histolytica is recognized by 73% of immune sera from patients with amoebic liver abscesses. Using pooled human immune sera a cDNA clone (lambda cM17) encoding this antigen (M17) has been isolated from a lambda gt11 expression library of the virulent stain E. histolytica HM1:IMSS. Monospecific antibodies, purified by binding to phage lysate of lambda cM17, and mAb FA7 reacted exclusively with the 125-kD antigen by Western blot analysis. Surface binding and cap formation are observed with patient sera, purified monospecific antiserum, and mAb FA7. Corresponding genomic clones (pBSgM17-1/2/3) were isolated by hybridization with the cDNA clone. These contained an open-reading frame of 3345 bp, which is in good agreement with the mRNA size of approximately 3.0 kb as revealed by Northern hybridization with lambda cM17. The inferred amino acid sequence predicts a 125,513 dalton protein that contains 17 potential N-linked glycosylation sites and is unusually rich in tyrosine and asparagine residues. A distinctly hydrophobic NH2-terminal region may serve as membrane anchor or signal sequence. In contrast to conservation of an immunodominant epitope recognized in pathogenic and nonpathogenic strains by monoclonal FA7 and human immune sera, amplification and sequence analysis of a 1,4000-bp fragment of this gene from a fresh nonpathogenic isolate by use of the PCR demonstrate regions of significant sequence divergence in this antigen. A 1% sequence variability among different isolates of the pathogenic strain HM1:IMSS and a 12-13% variability between pathogenic and nonpathogenic strains are revealed by comparison to published partial amino acid sequences (Tannich, E., R.D. Horstmann, J. Knobloch, and H.H. Arnold. 1989. Proc. Natl. Acad. Sci. USA. 86:5118). Some restriction enzymes were found that allowed PCR diagnosis of nonpathogenic and pathogenic isolates with the exclusion of E. histolytica-like Laredo, suggesting that a detailed study of nonpathogenic and pathogenic isolates in relation to the M17 antigen sequence will provide a basis of differentiating isolates.

Isolation and expression of the Pneumocystis carinii dihydrofolate reductase gene.

Pneumocystis carinii dihydrofolate reductase (DHFR; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) cDNA sequences have been isolated by their ability to confer trimethoprim resistance to Escherichia coli. Consistent with the recent conclusion that P. carinii is a member of the Fungi, sequence analysis and chromosomal localization show that DHFR is neither physically nor genetically linked to thymidylate synthase. Expression of recombinant P. carinii DHFR in heterologous hosts provides an abundant source of the enzyme that may form a basis for the development of new therapies for this enigmatic pathogen. Studies with the recombinant enzyme show that trimethoprim is a very poor inhibitor of P. carinii DHFR and, in fact, is a more potent inhibitor of human DHFR.

Isolation and expression of the Pneumocystis carinii thymidylate synthase gene.

The thymidylate synthase (TS) gene from Pneumocystis carinii has been isolated from complementary and genomic DNA libraries and expressed in Escherichia coli. The coding sequence of TS is 891 nucleotides, encoding a 297-amino acid protein of Mr 34,269. The deduced amino acid sequence is similar to TS from other organisms and is most closely related to the enzyme from Saccharomyces cerevisiae with 65% identity. TS is found on a 330-kilobase-pair chromosome in P. carinii. While TS and dihydrofolate reductase reside on a single polypeptide chain in all protozoa studied to date, TS is not linked to dihydrofolate reductase in P. carinii. The TS gene shows the presence of four small intervening sequences, some of which interrupt the coding sequence in highly ordered structural regions of the protein. Heterologous expression of P. carinii TS in E. coli was accomplished by cloning the coding sequence into plasmid vectors under control of the lac and tac promoters. These constructs direct the synthesis of catalytically active enzyme to the extent of 2% of total soluble protein.

Developmental regulation of tandem promoters for the major outer membrane protein gene of Chlamydia trachomatis.

Chlamydia trachomatis has a biphasic developmental cycle which is characterized by qualitative and quantitative changes in protein expression. The molecular mechanisms that mediate these changes are unknown. Evidence for transcriptional regulation of the chlamydial major outer membrane protein gene (omp1) was found by Northern hybridization of RNA isolated sequentially during the chlamydial developmental cycle. Early in the growth cycle a single transcript was detected, which was followed hours later in the cycle by an additional transcript. Mapping of the initiating nucleotide for each transcript suggested that this gene is regulated by differential transcription from tandem promoters.

Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding.

Activation of Ki-ras2 gene in human colon and lung carcinomas by two different point mutations.

Kirsten (Ki)-ras cDNA clones were prepared from human lung and colon carcinoma cell lines expressing an activated c-Ki-ras2 gene. DNA sequence analysis and transfection studies indicate that different point mutations at the same codon can activate the gene; that most human c-Ki-ras2 mRNA uses sequences from a fourth coding exon distinct from that of its viral counterpart; and that at least one cell line is functionally homozygous for the activated gene.