Pleiotropic function of intersectin homologue Cin1 in Cryptococcus neoformans

The manifestation of virulence traits in Cryptococcus neoformans is thought to rely on intracellular transport, a process not fully explored in this pathogenic fungus. Through interaction cloning, we identified a multi‐modular protein, Cin1 (cryptococcal intersectin 1), whose domain structure is similar to that of the human endocytic protein ITSN1. Cin1 contains an N‐terminal EH domain, a central coiled‐coil region, a WH2 domain, two SH3 domains and a C‐terminal RhoGEF (DH)‐PH domain. Interestingly, alternative mRNA splicing resulted in two Cin1 isoforms, and Cin1 homologues are also restricted to basidiomycetous fungi. Disruption of the CIN1 gene had a pleiotropic effect on growth, normal cytokinesis, intracellular transports and the production of several virulence factors. Additionally, Cin1 interacts with cryptococcal Cdc42 and Wsp1 (a WASP homologue) proteins in vitro, suggesting a conserved role in the regulation of the actin cytoskeleton. However, deletion of RhoGEF or SH3 and RhoGEF domains did not result in any phenotypic changes, suggesting that functional redundancy exists in proteins containing similar domains or that the activities by other domains are necessary for Cin1 function. Our study presents the first evidence of a multi‐modular protein whose function in intracellular transport underlies the growth, differentiation and virulence of a pathogenic microorganism.     

Heterologous expression, isotopic-labeling and immuno-characterization of Cin1, a novel protein secreted by the phytopathogenic fungus Venturia inaequalis

The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel V. inaequalis gene, cin1, is strongly up-regulated in the early stages of infection. This gene codes for a 523 residue secreted protein, containing eight imperfect repeats of 60 amino acids. Cin1 was expressed in the methanolytic yeast Pichia pastoris using the pPICZ vector system. A protein of 57 kDa was secreted by these transformants and peptide fingerprinting indicated that it was the Cin1 protein product. Multiple angle laser light scattering confirmed the predicted mass of Cin1, showing it was not glycosylated by Pichia and was monomeric in solution. Through measurements of the hydrodynamic properties of Cin1, the experimental Stokes radius of Cin1 was calculated and corresponded to the theoretical value for a natively folded globular protein of size 57 kDa. The mobility of recombinant Cin1 on native PAGE was also consistent with that of a folded protein. To simplify future structural analyses, a two-domain truncated version, Cin1-2D, consisting of domains one and two, was also expressed using the same vector system. Both proteins were purified to homogeneity. Conditions for maximal (>98%) incorporation of ¹³C and ¹⁵N were determined. A mouse polyclonal antibody and three monoclonal antibodies (MAbs) were raised against the full-length version of Cin1. Analysis of the three MAbs using surface plasmon resonance indicated binding to distinct epitopes on the Cin1 protein. Western blots confirmed the different specificities of each MAb.     

Studies on transvection at the bithorax complex in Drosophila melanogaster

Summary We have studied the influence of some mutations in the bithorax complex on the observed synapsis dependent phenotype of the genotypes Cbx ¹Ubx¹/+ and bx ^34e/Ubx¹. The effect of these mutations is similar to that introduced by disruption of pairing or by the z ^a mutation. Among the bx mutations, we find that bx ⁸ behaves differently from most other bx mutations in its influence on the synapsis dependent phenotype. This observation induced us to map the position of bx ⁸ with respect to other bx mutations; we find that it maps between bx ^34e and bx ³. We show how some of the observations reported here can be fitted into a model of activation of the bithorax complex proposed by one of us.     

Function of tubulin binding proteins in vivo

Overexpression of the beta-tubulin binding protein Rbl2p/cofactor A is lethal in yeast cells expressing a mutant alpha-tubulin, tub1-724, that produces unstable heterodimer. Here we use RBL2 overexpression to identify mutations in other genes that affect formation or stability of heterodimer. This approach identifies four genes-CIN1, CIN2, CIN4, and PAC2-as affecting heterodimer formation in vivo. The vertebrate homologues of two of these gene products-Cin1p/cofactor D and Pac2p/cofactor E-can catalyze exchange of tubulin polypeptides into preexisting heterodimer in vitro. Previous work suggests that both Cin2p or Cin4p act in concert with Cin1p in yeast, but no role for vertebrate homologues of either has been reported in the in vitro reaction. Results presented here demonstrate that these proteins can promote heterodimer formation in vivo. RBL2 overexpression in cin1 and pac2 mutant cells causes microtubule disassembly and enhanced formation of Rbl2p-beta-tubulin complex, as it does in the alpha-tubulin mutant that produces weakened heterodimer. Significantly, excess Cin1p/cofactor D suppresses the conditional phenotypes of that mutant alpha-tubulin. Although none of the four genes is essential for viability under normal conditions, they become essential under conditions where the levels of dissociated tubulin polypeptides increase. Therefore, these proteins may provide a salvage pathway for dissociated tubulin heterodimers and so rescue cells from the deleterious effects of free beta-tubulin.     

A structure-function analysis of NOD, a kinesin-like protein from Drosopbila melanogaster

We have analyzed a collection of 12 mutations in the Drosophila melanogaster nod locus, which encodes a kinesin-like protein involved in female meiotic chromosome segregation. The kinesin-like domain is at the N-terminus of the protein, while the C-terminal portion of the protein is unique. Four of the mutations are missense and affect highly conserved domains of the kinesin-like portion of the predicted protein, and thus demonstrate that the sequence conservation is biologically relevant. Surprisingly, two other mutations, which behave genetically as null alleles, are the result of mutations in the last exon of the nod gene. Thus, these two mutations affect the most C-terminal residues in the unique portion of the predicted protein. Based on these mutations, we suggest that this part of the protein may also be essential for wild-type function. The mutations were induced by either gamma-rays or ethyl methanesulfonate (EMS). All of the gamma-ray induced mutations were small or large chromosomal rearrangements, while all of the EMS mutations were G A transitions. These findings are consistent with the biochemical basis of the mode of action of each mutagen.     

Birth and Death of Genes and Functions in the β-Esterase Cluster of <Emphasis Type="Italic">Drosophila</Emphasis>

Here we analyze the molecular evolution of the β-esterase gene cluster in the Drosophila genus using the recently released genome sequences of 12 Drosophila species. Molecular evolution in this small cluster is noteworthy because it contains contrasting examples of the types and stages of loss of gene function. Specifically, missing orthologs, pseudogenes, and null alleles are all inferred. Phylogenetic analyses also suggest a minimum of 9 gene gain–loss events; however, the exact number and age of these events is confounded by interparalog recombination. A previous enigma, in which allozyme loci were mapped to β-esterase genes that lacked catalytically essential amino acids, was resolved through the identification of neighbouring genes that contain the canonical catalytic residues and thus presumably encode the mapped allozymes. The originally identified genes are evolving with selective constraint, suggesting that they have a “noncatalytic” function. Curiously, 3 of the 4 paralogous β-esterase genes in the D. ananassae genome sequence have single inactivating (frame-shift or nonsense) mutations. To determine whether these putatively inactivating mutations were fixed, we sequenced other D. ananassae alleles of these four loci. We did not find any of the 3 inactivating mutations of the sequenced strain in 12 other strains; however, other inactivating mutations were observed in the same 3 genes. This is reminiscent of the high frequency of null alleles observed in one of the β-esterase genes (Est7/EstP) of D. melanogaster. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cloning of a genomic fragment carrying the insertion element Cin 1 of Zea mays

Summary Genomic clones from a Northern Flint Line of Zea mays are shown to carry a 700 bp insertion, Cin 1. The Cin 1 insert is not present in the homologous, unique DNA fragment of a midwestern maize line. However, Cin 1 is present in multiple copies in both lines.Restriction enzyme analysis of the cloned DNAs and their corresponding genomic regions suggest that no alterations have occurred during the in vitro cloning procedure.     

Second-site modifiers of the split mutation of Notch define genes involved in neurogenesis in Drosophila melanogaster

Summary We have searched for dominant modifiers, i.e., enhancers and suppressors, of the compound eye phenotype of split, a recessive viable allele of Notch. Among the spl modifiers found, we have detected mutations in loci whose functions were previously known to cooperate with Notch in embryonic neurogenesis, such as daughterless, master mind, Delta and Hairless. In addition, other spl modifier mutations have been found in loci that were not previously known to interact with Notch, such as scabrous, glass, roughened eye, and several other genes that have not yet been assigned to known loci. The phenotypes associated with mutations in some of these latter loci suggest the participation of the corresponding genes in embryonic neurogenesis. We show that in some cases the observed interactions are due to genetic haplo-insufficent expression of the genes, whereas allele-specific interactions with spl are observed in master mind and Delta alleles. From this observation, we propose a direct functional association between the proteins encoded by Notch, Delta and master mind.     

Mutations in the <Emphasis Type="Italic">S</Emphasis> gene region of hepatitis B virus genotype <Emphasis Type="Italic">D</Emphasis> in Turkish patients

The S gene region of the hepatitis B virus (HBV) is responsible for the expression of surface antigens and includes the ‘a’-determinant region. Thus, mutation(s) in this region would afford HBV variants a distinct survival advantage, permitting the mutant virus to escape from the immune system. The aim of this study was to search for mutations of the S gene region in different patient groups infected with genotype D variants of HBV, and to analyse the biological significance of these mutations. Moreover, we investigated S gene mutation inductance among family members. Forty HBV-DNA-positive patients were determined among 132 hepatitis B surface antigen (HbsAg) carriers by the first stage of seminested PCR. Genotypes and subtypes were established by sequencing of the amplified S gene regions. Variants were compared with original sequences of these serotypes, and mutations were identified. All variants were designated as genotype D and subtype ayw3. Ten kinds of point mutations were identified within the S region. The highest rates of mutation were found in chronic hepatitis patients and their family members. The amino acid mutations 125 (M → T) and 127 (T → P) were found on the first loop of ‘a’-determinant. The other consequence was mutation inductance in a family member. We found some mutations in the S gene region known to be stable and observed that some of these mutations affected S gene expression.     

A new gene (madI) involved in the phototropic response of Phycomyces

Summary Only eight genes are known to be involved in the phototropic response of Phycomyces (madA-H). Mutants affected in these genes have played a major role in the analysis of photosensory transduction processes in this system. A set of new mutants isolated by Alvarez et al. (1989) that are unable to bend towards dim unilateral blue light were studied by complementation and recombination. Two of these mutants have mutations in madE, one has a mutation in madF and one is a double madE madF mutant. The three remaining mutants tested did not complement each other and showed positive complementation with strains carrying mutations in the genes madA, madB, and madC, indicating that they carried mutations in a new gene designated madI. Recombination analysis showed that madI is unlinked to madA, madB and madC.     

Role of the central dinucleotide at the crossover sites for the selection of quasi sites in DNA inversion mediated by the site-specific Cin recombinase of phage P1

Summary The crossover sites for Cin-mediated inversion consist of imperfect 12 bp inverted repeats with non-palindomic dinucleotides at the center of symmetry. Inversion is believed to occur in vivo between the homologous central 2 bp crossover sequences at the inversely repeated crossover sites through introduction of 2 bp staggered cuts and subsequent reciprocal strand exchanges. The site-specific Cin recombinase acts not only on the normal crossover sites but also, less efficiently, on quasi crossover sites which have some homology with the normal sites. We identified 15 new quasi sites including 4 sites within the cin structural gene. Homology at the 2 bp crossover sequences between recombining sites favors selection as quasi crossover sites. The Cin enzyme can occasionally mediate inversion between nonidentical crossover sequences and such recombinations often result in localized mutations including base pair substitutions and deletions within the 2 bp crossover sequences. These mutations are explained as the consequences of heteroduplex molecules formed between the staggered dinucleotides and either tubsequent resolution by DNA replication or subsequent mismatch repair. Occasional utilization of quasi crossover sites and localized mutagenesis at the crossover sequences in enzyme-mediated inversion processes would be one of the mechanisms contributing to genetic diversity.     

Evidence for a recent horizontal transmission and spatial spread of Wolbachia from endemic Rhagoletis cerasi (Diptera: Tephritidae) to invasive Rhagoletis cingulata in Europe

The widespread occurrence of Wolbachia in arthropods and nematodes suggests that this intracellular, maternally inherited endosymbiont has the ability to cross species boundaries. However, direct evidence for such a horizontal transmission of Wolbachia in nature is scarce. Here, we compare the well‐characterized Wolbachia infection of the European cherry fruit fly, Rhagoletis cerasi, with that of the North American eastern cherry fruit fly, Rhagoletis cingulata, recently introduced to Europe. Molecular genetic analysis of Wolbachia based on multilocus sequence typing and the Wolbachia surface protein wsp showed that all R. cingulata individuals are infected with wCin2 identical to wCer2 in R. cerasi. In contrast, wCin1, a strain identical to wCer1 in R. cerasi, was present in several European populations of R. cingulata, but not in any individual from the United States. Surveys of R. cingulata from Germany and Hungary indicated that in some populations, the frequency of wCin1 increased significantly in just a few years with at least two independent horizontal transmission events. This is corroborated by the analysis of the mitochondrial cytochrome oxidase II gene that showed association of wCin1 with two distinct haplotypes in Germany, one of which is also infected with wCin1 in Hungary. In summary, our study provides strong evidence for a very recent inter‐specific Wolbachia transmission with a subsequent spatial spread in field populations.     

Structure, dynamics and domain organization of the repeat protein Cin1 from the apple scab fungus

Venturia inaequalis is a hemi-biotrophic fungus that causes scab disease of apple. A recently-identified gene from this fungus, cin1 (cellophane-induced 1), is up-regulated over 1000-fold in planta and considerably on cellophane membranes, and encodes a cysteine-rich secreted protein of 523 residues with eight imperfect tandem repeats of ~60 amino acids. The Cin1 sequence has no homology to known proteins and appears to be genus-specific; however, Cin1 repeats and other repeat domains may be structurally similar. An NMR-derived structure of the first two repeat domains of Cin1 (Cin1-D1D2) and a low-resolution model of the full-length protein (Cin1-FL) using SAXS data were determined. The structure of Cin1-D1D2 reveals that each domain comprises a core helix-loop-helix (HLH) motif as part of a three-helix bundle, and is stabilized by two intra-domain disulfide bonds. Cin1-D1D2 adopts a unique protein fold as DALI and PDBeFOLD analysis identified no structural homology. A (15)N backbone NMR dynamic analysis of Cin1-D1D2 showed that a short stretch of the inter-domain linker has large amplitude motions that give rise to reciprocal domain-domain mobility. This observation was supported by SAXS data modeling, where the scattering length density envelope remains thick at the domain-domain boundary, indicative of inter-domain dynamics. Cin1-FL SAXS data models a loosely-packed arrangement of domains, rather than the canonical parallel packing of adjacent HLH repeats observed in α-solenoid repeat proteins. Together, these data suggest that the repeat domains of Cin1 display a "beads-on-a-string" organization with inherent inter-domain flexibility that is likely to facilitate interactions with target ligands.     

The calnexin-independent state does not compensate for all calnexin functions in Schizosaccharomyces pombe

In the yeast Schizosaccharomyces pombe, the molecular chaperone calnexin (Cnx1p) has been shown to be essential for viability. However, we recently reported that, under certain circumstances, S. pombe cells are able to survive in the absence of calnexin/Cnx1p, indicating that an inducible pathway can complement the calnexin/Cnx1p essential function(s). This calnexin-independent state (Cin) is transmitted by a nonchromosomal proteinaceous element exhibiting several prion-like properties. To assess to what extent the Cin state compensates for the absence of calnexin/Cnx1p, the Cin strain was further characterized. Cin cells exhibited cell-wall defects, sensitivity to heat shock, as well as higher secretion levels of a model glycoprotein. Together, these results indicate that the Cin state does not compensate for all calnexin/Cnx1p functions. Reintroduction of plasmid-borne cnx1(+) partially rescued most but not all of the phenotypes displayed by Cin cells. Interestingly, Cin cells in stationary phase exhibited increased levels of caspase activation, and this phenotype was not suppressed by the reintroduction of cnx1(+), suggesting that cells in the Cin state are subjected to a stress other than the absence of calnexin/Cnx1p.     

Saccharomyces Cerevisiae Pac2 Functions with Cin1, 2 and 4 in a Pathway Leading to Normal Microtubule Stability

The products of the Saccharomyces cerevisiae CIN1, CIN2 and CIN4 genes participate in a nonessential pathway required for normal microtubule function. In this article, we demonstrate that the product of PAC2 also functions in this pathway. PAC2 deletion mutants displayed phenotypes and genetic interactions similar to those caused by cin1Δ, cin2Δ and cin4Δ. These include cold-sensitive microtubule structures and sensitivity to the microtubule depolymerizing agent benomyl. Involvement in a common functional pathway is indicated by the observation that all double mutant combinations are viable and no more affected than any single mutant. In addition, extra copies of CIN1 were found to suppress the benomyl sensitivity of pac2Δ, cin2Δ and cin4Δ, but not that caused by other mutations that affect microtubule function. Cin1p and Pac2p were found to be related in sequence to mammalian proteins that aid in the folding of β-tubulin into an assembly-competent state. Alleles of CIN1 were identified that could suppress the benomyl sensitivity of cin4-4 in a highly specific fashion. Our findings suggest that the guanine nucleotide-binding Cin4p interacts with Cin1p and regulates its tubulin folding activity.     

Nuclear genes required for post-translational steps in the biogenesis of the chloroplast cytochrome b6f complex in maize

Nuclear genes essential for the biogenesis of the chloroplast cytochrome b ₆ f complex were identified by mutations that cause the specific loss of the complex. We describe four transposon-induced maize mutants that lack cytochrome b ₆ f proteins but contain normal levels of other photosynthetic complexes. The four mutations define two nuclear genes. To identify the step at which each mutation blocks protein accumulation, mRNAs encoding each subunit were examined by Northern hybridization analysis and the rates of subunit synthesis were examined in pulse-labeling experiments. In each mutant the mRNAs encoding the known subunits of the complex were normal in size and abundance and the major subunits were synthesized at normal rates. Thus, these mutations block the biogenesis of the cytochrome b ₆ f complex at a post-translational step. The two nuclear genes identified by these mutations may encode previously unknown subunits, be involved in prosthetic group synthesis or attachment, or facilitate assembly of the complex. These mutations were also used to provide evidence for the authenticity of a proposed fifth subunit of the complex and to demonstrate a role for the cytochrome b ₆ f complex in protecting photosystem 11 from light-induced degradation.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Isolation and characterization of recessive sporeless mutants in the basidiomycete Coprinus cinereus

Summary Twenty-four sporeless mutants were isolated from an Amut Bmut strain (mutant in the incompatibility factors) of the basidiomycete Coprinus cinereus. All the sporeless mutations were recessive to the wild type. These mutants and a previously isolated recessive sporeless strain, N2-7 (Kanda and Ishikawa 1986) were crossed with a wildtype strain. An F1 random spore analysis indicated that sporulation deficiencies in these mutants were caused by single nuclear gene mutations. These mutations were all complementary to each other, thus twenty-five sporulation genes were identified. Five of them were linked to the A incompatibility factor. Cytological observations classified these mutants into the following four types according to the stage of the blockage: (1) meiosis stopped at meta-anaphase I; (2) meiosis was completed, but further basidial development did not occur; (3) basidial development stopped at the sterigma stage; (4) basidial development stopped at the prespore stage.     

Effects of Polycomb Group Mutations on the Expression of Ultrabithorax in the Drosophila Visceral Mesoderm

The Polycomb group (PcG) genes encode repressors of many developmental regulatory genes including homeotic genes and are known to act by modifying chromatin structure through complex formation. We describe how Ultrabithorax (Ubx) expression is affected by the PcG mutants in the visceral mesoderm. Mutant embryos of the genes extra sex combs (esc), Polycomb (Pc), additional sex combs (Asx) and pleiohomeotic (pho) were examined. In each mutation, Ubx was ectopically expressed outside of their normal domains along the anterior-posterior axis in the visceral mesoderm, which is consistent with the effect of PcG proteins repressing the homeotic genes in other tissues. All of these four PcG mutations exhibit complete or partial lack of midgut constriction. However, two thirds of esc mutant embryos did not show Ubx expression in parasegment 7 (PS7). Even in the embryos showing ectopic Ubx expression, the level of Ubx expression in the PcG mutations was weaker than that in normal embryos. We suggest that in PcG mutations the ectopic Ubx expression is caused by lack of PcG repressor proteins, while the weaker or lack of Ubx expression is due to the repression of Ubx by Abd-B protein which is ectopically expressed in PcG mutations as well.