Purification and characterization of an autoregulatory substance capable of regulating the morphological transition in Candida albicans

The yeast Candida albicans has a distinguishing feature, dimorphism, which is the ability to switch between two morphological forms: a budding yeast form and a multicellular invasive filamentous form. This ability has been postulated to contribute to the virulence of this organism. Studies on the morphological transition from a filamentous to a budding yeast form in C. albicans have shown that this organism excretes an autoregulatory substance into the culture medium. This substance was extracted and purified by normal-phase and reversed-phase HPLC. The autoregulatory substance was structurally identified as 3,7,11-trimethyl-2,6,10-dodecatrienoate (farnesoic acid) by NMR and mass spectrometry. Growth experiments suggest that this substance does not inhibit yeast cell growth but inhibits filamentous growth. These findings have implications for developmental signaling by the fungus and might have medicinal value in the development of antifungal therapies.

Gene discovery and gene function assignment in filamentous fungi

Filamentous fungi are a large group of diverse and economically important microorganisms. Large-scale gene disruption strategies developed in budding yeast are not applicable to these organisms because of their larger genomes and lower rate of targeted integration (TI) during transformation. We developed transposon-arrayed gene knockouts (TAGKO) to discover genes and simultaneously create gene disruption cassettes for subsequent transformation and mutant analysis. Transposons carrying a bacterial and fungal drug resistance marker are used to mutagenize individual cosmids or entire libraries in vitro. Cosmids are annotated by DNA sequence analysis at the transposon insertion sites, and cosmid inserts are liberated to direct insertional mutagenesis events in the genome. Based on saturation analysis of a cosmid insert and insertions in a fungal cosmid library, we show that TAGKO can be used to rapidly identify and mutate genes. We further show that insertions can create alterations in gene expression, and we have used this approach to investigate an amino acid oxidation pathway in two important fungal phytopathogens.

Nuclear waste forms for actinides

The disposition of actinides, most recently 239Pu from dismantled nuclear weapons, requires effective containment of waste generated by the nuclear fuel cycle. Because actinides (e.g., 239Pu and 237Np) are long-lived, they have a major impact on risk assessments of geologic repositories. Thus, demonstrable, long-term chemical and mechanical durability are essential properties of waste forms for the immobilization of actinides. Mineralogic and geologic studies provide excellent candidate phases for immobilization and a unique database that cannot be duplicated by a purely materials science approach. The “mineralogic approach” is illustrated by a discussion of zircon as a phase for the immobilization of excess weapons plutonium.

Congruences between modular forms: Raising the level and dropping Euler factors

We discuss the relationship among certain generalizations of results of Hida, Ribet, and Wiles on congruences between modular forms. Hida’s result accounts for congruences in terms of the value of an L-function, and Ribet’s result is related to the behavior of the period that appears there. Wiles’ theory leads to a class number formula relating the value of the L-function to the size of a Galois cohomology group. The behavior of the period is used to deduce that a formula at “nonminimal level” is obtained from one at “minimal level” by dropping Euler factors from the L-function.

A TRP homolog in Saccharomyces cerevisiae forms an intracellular Ca2+-permeable channel in the yeast vacuolar membrane

The molecular identification of ion channels in internal membranes has made scant progress compared with the study of plasma membrane ion channels. We investigated a prominent voltage-dependent, cation-selective, and calcium-activated vacuolar ion conductance of 320 pS (yeast vacuolar conductance, YVC1) in Saccharomyces cerevisiae. Here we report on a gene, the deduced product of which possesses significant homology to the ion channel of the transient receptor potential (TRP) family. By using a combination of gene deletion and re-expression with direct patch clamping of the yeast vacuolar membrane, we show that this yeast TRP-like gene is necessary for the YVC1 conductance. In physiological conditions, tens of micromolar cytoplasmic Ca2+ activates the YVC1 current carried by cations including Ca2+ across the vacuolar membrane. Immunodetection of a tagged YVC1 gene product indicates that YVC1 is primarily localized in the vacuole and not other intracellular membranes. Thus we have identified the YVC1 vacuolar/lysosomal cation-channel gene. This report has implications for the function of TRP channels in other organisms and the possible molecular identification of vacuolar/lysosomal ion channels in other eukaryotes.

Rad52 forms DNA repair and recombination centers during S phase

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA, enabling gene segment rearrangements without disturbing the coding frame (i.e., protein-segment shuffling). These properties may give an extensive evolutionary advantage to DNA.

Organization and expression of the double-stranded RNA genome of Helminthosporium victoriae 190S virus, a totivirus infecting a plant pathogenic filamentous fungus.

The complete nucleotide sequence, 5178 bp, of the totivirus Helminthosporium vicotoriae 190S virus (Hv190SV) double-stranded RNA, was determined. Computer-assisted sequence analysis revealed the presence of two large overlapping ORFs; the 5'-proximal large ORF (ORF1) codes for the coat protein (CP) with a predicted molecular mass of 81 kDa, and the 3'-proximal ORF (ORF2), which is in the -1 frame relative to ORF1, codes for an RNA-dependent RNA polymerase (RDRP). Unlike many other totiviruses, the overlap region between ORF1 and ORF2 lacks known structural information required for translational frameshifting. Using an antiserum to a C-terminal fragment of the RDRP, the product of ORF2 was identified as a minor virion-associated polypeptide of estimated molecular mass of 92 kDa. No CP-RDRP fusion protein with calculated molecular mass of 165 kDa was detected. The predicted start codon of the RDRP ORF (2605-AUG-2607) overlaps with the stop codon (2606-UGA-2608) of the CP ORF, suggesting RDRP is expressed by an internal initiation mechanism. Hv190SV is associated with a debilitating disease of its phytopathogenic fungal host. Knowledge of its genome organization and expression will be valuable for understanding its role in pathogenesis and for potential exploitation in the development of biocontrol measures.

Alternatively spliced forms in the cytoplasmic domain of the human growth hormone (GH) receptor regulate its ability to generate a soluble GH-binding protein.

The mechanism underlying the generation of soluble growth hormone binding protein (GHBP) probably differs among species. In rats and mice, it involves an alternatively spliced mRNA, whereas in rabbits, it involves limited proteolysis of the membrane-bound growth hormone receptor (GHR). In humans, this latter mechanism is favored, as no transcript coding for a soluble GHR has been detected so far. To test this hypothesis, we analyzed COS-7 cells transiently expressing the full-length human (h) GHR and observed specific GH-binding activity in the cell supernatants. Concomitantly, an alternatively spliced form in the cytoplasmic domain of GHR, hGHR-tr, was isolated from several human tissues. hGHR-tr is identical in sequence to hGHR, except for a 26-bp deletion leading to a stop codon at position 280, thereby truncating 97.5% of the intracellular domain of the receptor protein. When compared with hGHR, hGHR-tr showed a significantly increased capacity to generate a soluble GHBP. Interestingly, this alternative transcript is also expressed in liver from rabbits, mice, and rats, suggesting that, in these four species, proteolysis of the corresponding truncated transmembrane GHR is a common mechanism leading to GHBP generation. These findings support the hypothesis that GHBP may at least partly result from alternative splicing of the region encoding the intracellular domain and that the absence of a cytoplasmic domain may be involved in increased release of GHBP.

Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications.

The neuropeptide gonadotropin-releasing hormone (GnRH) is the major regulator of reproduction in vertebrates. Our goal was to determine whether GnRH could be isolated and identified by primary structure in a protochordate and to examine its location by immunocytochemistry. The primary structure of two novel decapeptides from the tunicate Chelyosoma productum (class Ascidiacea) was determined. Both show significant identity with vertebrate GnRH. Tunicate GnRH-I (pGlu-His-Trp-Ser-Asp-Tyr-Phe-Lys-Pro-Gly-NH2) has 60% of its residues conserved, compared with mammalian GnRH, whereas tunicate GnRH-II (pGlu-His-Trp-Ser-Leu-Cys-His-Ala-Pro-Gly-NH2) is unusual in that it was isolated as a disulfide-linked dimer. Numerous immunoreactive GnRH neurons lie within blood sinuses close to the gonoducts and gonads in both juveniles and adults, implying that the neuropeptide is released into the bloodstream. It is suggested that in ancestral chordates, before the evolution of the pituitary, the hormone was released into the bloodstream and acted directly on the gonads.

Multiple forms of complement C3 in trout that differ in binding to complement activators.

In all other species analyzed to date, the functionally active form of complement component C3 exists as the product of a single gene. We have now identified and characterized three functional C3 proteins (C3-1, C3-3, and C3-4) in trout that are the products of at least two distinct C3 genes. All three proteins are composed of an alpha-and a beta-chain and contain a thioester bond in the alpha-chain. However, they differ in their electrophoretic mobility, glycosylation, reactivity with monospecific C3 antibodies, and relative ability to bind to various surfaces (zymosan, Escherichia coli, erythrocytes). A comparison of the partial amino acid sequences of the three proteins showed that the amino acid sequence identity/similarity of C3-3 to C3-4 is 87/91%, while that of C3-3 and C3-4 to C3-1 is 51.5/65.5% and 60/73% respectively. Thus, trout possess multiple forms of functional C3 that represent the products of several distinct genes and differ in their ability to bind covalently to various complement activators.

A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage.

It has been suggested that recombination and shuffling between exons has been a key feature in the evolution of proteins. We propose that this strategy could also be used for the artificial evolution of proteins in bacteria. As a first step, we illustrate the use of a self-splicing group I intron with inserted lox-Cre recombination site to assemble a very large combinatorial repertoire (> 10(11) members) of peptides from two different exons. Each exon comprised a repertoire of 10 random amino acids residues; after splicing, the repertoires were joined together through a central five-residue spacer to give a combinatorial repertoire of 25-residue peptides. The repertoire was displayed on filamentous bacteriophage by fusion to the pIII phage coat protein and selected by binding to several proteins, including beta-glucuronidase. One of the peptides selected against beta-glucuronidase was chemically synthesized and shown to inhibit the enzymatic activity (inhibition constant: 17 nM); by further exon shuffling, an improved inhibitor was isolated (inhibition constant: 7 nM). Not only does this approach provide the means for making very large peptide repertoires, but we anticipate that by introducing constraints in the sequences of the peptides and of the linker, it may be possible to evolve small folded peptides and proteins.

Induction of mucosal immune responses against a heterologous antigen fused to filamentous hemagglutinin after intranasal immunization with recombinant Bordetella pertussis.

Live vaccine vectors are usually very effective and generally elicit immune responses of higher magnitude and longer duration than nonliving vectors. Consequently, much attention has been turned to the engineering of oral pathogens for the delivery of foreign antigens to the gut-associated lymphoid tissues. However, no bacterial vector has yet been designed to specifically take advantage of the nasal route of mucosal vaccination. Herein we describe a genetic system for the expression of heterologous antigens fused to the filamentous hemagglutinin (FHA) in Bordetella pertussis. The Schistosoma mansoni glutathione S-transferase (Sm28GST) fused to FHA was detected at the cell surface and in the culture supernatants of recombinant B. pertussis. The mouse colonization capacity and autoagglutination of the recombinant microorganism were indistinguishable from those of the wild-type strain. In addition, and in contrast to the wild-type strain, a single intranasal administration of the recombinant strain induced both IgA and IgG antibodies against Sm28GST and against FHA in the bronchoalveolar lavage fluids. No anti-Sm28GST antibodies were detected in the serum, strongly suggesting that the observed immune response was of mucosal origin. This demonstrates, to our knowledge, for the first time that recombinant respiratory pathogens can induce mucosal immune responses against heterologous antigens, and this may constitute a first step toward the development of combined live vaccines administrable via the respiratory route.

Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae.

RAS2val19, a dominant activated form of Saccharomyces cerevisiae Ras2, stimulates both filamentous growth and expression of a transcriptional reporter FG(TyA)::lacZ but does not induce the mating pathway reporter FUS1::lacZ. This induction depends upon elements of the conserved mitogen-activated protein kinase (MAPK) pathway that is required for both filamentous growth and mating, two distinct morphogenetic events. Full induction requires Ste20 (homolog of mammalian p65PAK protein kinases), Ste11 [an MEK kinase (MEKK) or MAPK kinase (MEK) kinase], Ste7 (MEK or MAPK kinase), and the transcription factor Ste12. Moreover, the Rho family protein Cdc42, a conserved morphogenetic G protein, is also a potent regulator of filamentous growth and FG(TyA)::lacZ expression in S. cerevisiae. Stimulation of both filamentous growth and FG(TyA)::lacZ by Cdc42 depends upon Ste20. In addition, dominant negative CDC42Ala118 blocks RAS2val19 activation, placing Cdc42 downstream of Ras2. Our results suggest that filamentous growth in budding yeast is regulated by an evolutionarily conserved signaling pathway that controls cell morphology.

Molecular basis for dysfunction of some mutant forms of methylmalonyl-CoA mutase: deductions from the structure of methionine synthase.

Inherited defects in the gene for methylmalonyl-CoA mutase (EC 5.4.99.2) result in the mut forms of methylmalonic aciduria. mut- mutations lead to the absence of detectable mutase activity and are not corrected by excess cobalamin, whereas mut- mutations exhibit residual activity when exposed to excess cobalamin. Many of the mutations that cause methylmalonic aciduria in humans affect residues in the C-terminal region of the methylmalonyl-CoA mutase. This portion of the methylmalonyl-CoA mutase sequence can be aligned with regions in other B12 (cobalamin)-dependent enzymes, including the C-terminal portion of the cobalamin-binding region of methionine synthase. The alignments allow the mutations of human methylmalonyl-CoA mutase to be mapped onto the structure of the cobalamin-binding fragment of methionine synthase from Escherichia coli (EC 2.1.1.13), which has recently been determined by x-ray crystallography. In this structure, the dimethylbenzimidazole ligand to the cobalt in free cobalamin has been displaced by a histidine ligand, and the dimethylbenzimidazole nucleotide "tail" is thrust into a deep hydrophobic pocket in the protein. Previously identified mut0 and mut- mutations (Gly-623 --> Arg, Gly-626 --> Cys, and Gly-648 --> Asp) of the mutase are predicted to interfere with the structure and/or stability of the loop that carries His-627, the presumed lower axial ligand to the cobalt of adenosylcobalamin. Two mutants that lead to severe impairment (mut0) are Gly-630 --> Glu and Gly-703 --> Arg, which map to the binding site for the dimethylbenzimidazole nucleotide substituent of adenosylcobalamin. The substitution of larger residues for glycine is predicted to block the binding of adenosylcobalamin.