sepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.

In Aspergillus nidulans conidia, cytokinesis (septation) is delayed until three rounds of nuclear division have been completed. This has permitted the identification of essential genes that are involved in the coordination of cytokinesis with nuclear division. Conditional mutations in the sepB gene block septation but allow germinating spores to complete the first three rounds of nuclear division at restrictive temperature. sepB3 mutants demonstrate transient delays in M-phase, accumulate aneuploid nuclei and show defects in chromosome segregation. Molecular analysis of the sepB gene reveals that it is essential and possesses limited similarity to the CTF4 gene of Saccharomyces cerevisiae. Using temperature-shift analysis we show that sepB is required after the first nuclear division but before the onset of cytokinesis. A failure to execute the sepB function results in a block to nuclear division and leads to cell death at a time when wild-type cells would be undergoing cytokinesis. Finally, we demonstrate that sepB is also required for the uninucleate cell divisions of developing conidiophores. Our results suggest that sepB3 mutants accumulate specific nuclear defects that do not arrest mitosis, but block the initiation of septum formation. Thus, proper chromosome segregation and a functional sepB gene are required to initiate cytokinesis.     

The Aspergillus nidulans xprF gene encodes a hexokinase-like protein involved in the regulation of extracellular proteases

The extracellular proteases of Aspergillus nidulans are produced in response to limitation of carbon, nitrogen, or sulfur, even in the absence of exogenous protein. Mutations in the A. nidulans xprF and xprG genes have been shown to result in elevated levels of extracellular protease in response to carbon limitation. The xprF gene was isolated and sequence analysis indicates that it encodes a 615-amino-acid protein, which represents a new type of fungal hexokinase or hexokinase-like protein. In addition to their catalytic role, hexokinases are thought to be involved in triggering carbon catabolite repression. Sequence analysis of the xprF1 and xprF2 alleles showed that both alleles contain nonsense mutations. No loss of glucose or fructose phosphorylating activity was detected in xprF1 or xprF2 mutants. There are two possible explanations for this observation: (1) the xprF gene may encode a minor hexokinase or (2) the xprF gene may encode a protein with no hexose phosphorylating activity. Genetic evidence suggests that the xprF and xprG genes are involved in the same regulatory pathway. Support for this hypothesis was provided by the identification of a new class of xprG(-) mutation that suppresses the xprF1 mutation and results in a protease-deficient phenotype.     

Aspergillus nidulans swoK encodes an RNA binding protein that is important for cell polarity

The Aspergillus nidulans swoK1 mutant is defective in polarity maintenance when grown at restrictive temperature (38 degrees C). Upon germination, the mutant extends a primary germ tube that swells to an enlarged, non-uniform cell with pronounced wall thickenings. The mutant is fully restored to wild-type growth when transformed with a plasmid containing the AN5802.2 ORF as designated in The Broad Institute A. nidulans sequence database. Genetic mapping places swoK in the same region of chromosome I, as that occupied by An5802.2 on the physical map. swoK is predicted to encode a protein that contains an N-terminal RRM (RNA Recognition Motif) and a highly repetitive C-terminus with numerous RD/DR and RS/SR dipeptides. We hypothesize that SwoK participates in one of the known functions of SR proteins (those that contain SR/RS repeats): mRNA maturation through the spliceosome and or transport of mRNAs out of the nucleus to sites of protein translation.     

The Aspergillus nidulans creC gene involved in carbon catabolite repression encodes a WD40 repeat protein

Expression of many microbial genes required for the utilisation of less favoured carbon sources is carbon catabolite repressed in the presence of a preferred carbon source such as D-glucose. In Aspergillus nidulans, creC mutants show derepression in the presence of D-glucose of some, but not all, systems normally subject to carbon catabolite repression. These mutants also fail to grow on some carbon sources, and show minor morphological impairment and altered sensitivity to toxic compounds including molybdate and acriflavin. The pleiotropic nature of the phenotype suggests a role for the creC gene product in the carbon regulatory cascade. The creC gene was cloned and found to encode a protein which contains five WD40 motifs. The sequence changes in three mutant alleles were found to lead to production of truncated proteins which lack one or more of the WD40 repeats. The similarity of the phenotypes conferred by these alleles implies that these alleles represent loss of function alleles. Deletion analysis also showed that at least the most C-terminal WD40 motif is required for function. The CreC protein is highly conserved relative to the Schizosaccharomyces pombe protein Yde3 – whose function is unknown – and human and mouse DMR-N9, which may be associated with myotonic dystrophy. Received: 1 July 1999 / Accepted: 31 January 2000     

The Arabidopsis DIMINUTO/DWARF1 gene encodes a protein involved in steroid synthesis.

We have identified the function of the Arabidopsis DIMINUTO/DWARF1 (DIM/DWF1) gene by analyzing the dim mutant, a severe dwarf with greatly reduced fertility. Both the mutant phenotype and gene expression could be rescued by the addition of exogenous brassinolide. Analysis of endogenous sterols demonstrated that dim accumulates 24-methylenecholesterol but is deficient in campesterol, an early precursor of brassinolide. In addition, we show that dim is deficient in brassinosteroids as well. Feeding experiments using deuterium-labeled 24-methylenecholesterol and 24-methyldesmosterol confirmed that DIM/DWF1 is involved in both the isomerization and reduction of the Delta24(28) bond. This conversion is not required in cholesterol biosynthesis in animals but is a key step in the biosynthesis of plant sterols. Transient expression of a green fluorescent protein-DIM/DWF1 fusion protein and biochemical experiments showed that DIM/DWF1 is an integral membrane protein that most probably is associated with the endoplasmic reticulum.     

The cytokinesis gene KEULE encodes a Sec1 protein that binds the syntaxin KNOLLE

KEULE is required for cytokinesis in Arabidopsis thaliana. We have positionally cloned the KEULE gene and shown that it encodes a Sec1 protein. KEULE is expressed throughout the plant, yet appears enriched in dividing tissues. Cytokinesis-defective mutant sectors were observed in all somatic tissues upon transformation of wild-type plants with a KEULE-green fluorescent protein gene fusion, suggesting that KEULE is required not only during embryogenesis, but at all stages of the plant's life cycle. KEULE is characteristic of a Sec1 protein in that it appears to exist in two forms: soluble or peripherally associated with membranes. More importantly, KEULE binds the cytokinesis-specific syntaxin KNOLLE. Sec1 proteins are key regulators of vesicle trafficking, capable of integrating a large number of intra- and/or intercellular signals. As a cytokinesis-related Sec1 protein, KEULE appears to represent a novel link between cell cycle progression and the membrane fusion apparatus.     

The gene frpB2 of Helicobacter pylori encodes an hemoglobin-binding protein involved in iron acquisition

Human hemoglobin (Hb) is a metalloprotein used by pathogens as a source of iron during invasive process. It can support the Helicobacter pylori growth and several proteins are induced during iron starvation. However, the identity of those proteins remains unknown. In this work, by in silico analysis we identified FrpB2 in H. pylori genome. This protein was annotated as an iron-regulated outer membrane protein. Multiple amino acid alignment showed the motifs necessary for Hb-binding. We demonstrate the ability of FrpB2 to bind Hb by overlay experiments. In addition, the overexpression of this gene allowed the cell growth in media without free iron but supplemented with Hb. All these results support the idea that frpB2 is a gene of H. pylori involved in iron acquisition when Hb is used as a sole iron source.     

Isolation of a gene involved in 1,3-beta-glucan synthesis in Aspergillus nidulans and purification of the corresponding protein.

Saccharomyces cerevisiae has two highly homologous genes, FKS1 and FKS2, which encode interchangeable putative catalytic subunits of 1,3-beta-glucan synthase (GS), an enzyme that synthesizes an essential polymer of the fungal cell wall. To determine if GS in Aspergillus species is similar, an FKS homolog, fksA, was cloned from Aspergillus nidulans by cross-hybridization, and the corresponding protein was purified. Sequence analysis revealed a 5,716-nucleotide coding region interrupted by two 56-bp introns. The fksA gene encodes a predicted peptide of 229 kDa, FksAp, that shows a remarkable degree of conservation in size, charge, amino acid identity, and predicted membrane topology with the S. cerevisiae FKS proteins (Fksps). FksAp exhibits 64 and 65% identity to Fks1p and Fks2p, respectively, and 79% similarity. Hydropathy analysis of FksAp suggests an integral membrane protein with 16 transmembrane helices that coincide with the transmembrane helices of the Saccharomyces Fksps. The sizes of the nontransmembrane domains are strikingly similar to those of Fks1p. The region of FksAp most homologous to the Saccharomyces FKS polypeptides is a large hydrophilic domain of 578 amino acids that is predicted to be cytoplasmic. This domain is 86% identical to the corresponding region of Fks1p and is a good candidate for the location of the catalytic site. Antibodies raised against a peptide derived from the FksAp sequence recognize a protein of approximately 200 kDa in crude membranes and detergent-solubilized active extracts. This protein is enriched approximately 300-fold in GS purified by product entrapment. Purified anti-FksAp immunoglobulin G immunodepletes nearly all of the GS activity in crude or purified extracts when Staphylococcus aureus cells are used to precipitate the antibodies, although it does not inhibit enzymatic activity when added to extracts. The purified GS is inhibited by echinocandins with a sensitivity equal to that displayed by whole cells. Thus, the product of fksA is important for the activity of highly purified preparations of GS, either as the catalytic subunit itself or as an associated copurifying subunit that mediates susceptibility of enzymatic activity to echinocandin inhibition.     

The Arabidopsis HINKEL gene encodes a kinesin-related protein involved in cytokinesis and is expressed in a cell cycle-dependent manner

Plant cytokinesis starts in the center of the division plane, with vesicle fusion generating a new membrane compartment, the cell plate, that subsequently expands laterally by continuous fusion of newly arriving vesicles to its margin. Targeted delivery of vesicles is assisted by the dynamic reorganization of a plant-specific cytoskeletal array, the phragmoplast, from a solid cylinder into an expanding ring-shaped structure. This lateral translocation is brought about by depolymerization of microtubules in the center, giving way to the expanding cell plate, and polymerization of microtubules along the edge. Whereas several components are known to mediate cytokinetic vesicle fusion [8-10], no gene function involved in phragmoplast dynamics has been identified by mutation. Mutations in the Arabidopsis HINKEL gene cause cytokinesis defects, such as enlarged cells with incomplete cell walls and multiple nuclei. Proper targeting of the cytokinesis-specific syntaxin KNOLLE [8] and lateral expansion of the phragmoplast are not affected. However, the phragmoplast microtubules appear to persist in the center, where vesicle fusion should result in cell plate formation. Molecular analysis reveals that the HINKEL gene encodes a plant-specific kinesin-related protein with a putative N-terminal motor domain and is expressed in a cell cycle-dependent manner similar to the KNOLLE gene. Our results suggest that HINKEL plays a role in the reorganization of phragmoplast microtubules during cell plate formation.     

The Aspergillus nidulans uvsB gene encodes an ATM-related kinase required for multiple facets of the DNA damage response

In Aspergillus nidulans, uvsB and uvsD belong to the same epistasis group of DNA repair mutants. Recent observations suggest that these genes are likely to control cell cycle checkpoint responses to DNA damage and incomplete replication. Consistent with this notion, we show here that UVSB is a member of the conserved family of ATM-related kinases. Phenotypic characterization of uvsB mutants shows that they possess defects in additional aspects of the DNA damage response besides checkpoint control, including inhibition of septum formation, regulation of gene expression, and induced mutagenesis. The musN227 mutation partially suppresses the poor growth and DNA damage sensitivity of uvsB mutants. Although musN227 partially suppresses several uvsB defects, it does not restore checkpoint function to uvsB mutants. Notably, the failure of uvsB mutants to restrain septum formation in the presence of DNA damage is suppressed by the musN227 mutation. We propose that UVSB functions as the central regulator of the A. nidulans DNA damage response, whereas MUSN promotes recovery by modulating a subset of the response.