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R Kahmann

Publications and source records attributed to R Kahmann.

At least 55 records · Page 3Linked to original sources
PubMed1995

Multiallelic recognition: nonself-dependent dimerization of the bE and bW homeodomain proteins in Ustilago maydis.

In the plant pathogenic fungus Ustilago maydis, sexual and pathogenic development are controlled by the multiallelic b mating-type locus. The b locus encodes a pair of unrelated homeodomain proteins termed bE and bW, with allelic differences clustering in the N-terminal domains of both polypeptides. Only combinations of bE and bW of different allelic origin are active. We have investigated the underlying molecular mechanism for this intracellular self/nonself recognition phenomenon. By using the two-hybrid system, we were able to show that bE and bW dimerize only if they are derived from different alleles. Dimerization involves the N-terminal variable domains. Different point mutants of bE2 were isolated that function in combination with bW2. The majority of such bE2 mutant polypeptides were also able to form heterodimers with bW2 in the two-hybrid system. Nonself-dependent dimerization of bE and bW was supported with a biochemical interaction assay with immobilized proteins. Our results suggest a model for self/nonself recognition in which variable cohesive contacts direct dimerization.

Alleles↗
PubMed1995

FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter.

The Escherichia coli DNA binding protein FIS activates stable RNA promoters during outgrowth of cells from stationary phase. The upstream activating sequences (UASs) of these promoters contain three highly conserved FIS binding sites positioned in helical register. Neither the apparent requirement for three sites nor the mechanism of FIS-mediated activation has been established. We demonstrate here that on saturation of its three binding sites in the UAS, FIS forms a specific nucleoprotein complex which 'traps' RNA polymerase (RNAP) at the promoter of the tyrT operon. This effect is abolished by a change in helical phasing between FIS sites II and III, which impaires cooperative interactions between DNA-bound FIS dimers. The sigma 70 subunit of RNAP stimulates the formation of higher order FIS complexes, a property that is indicative of protein-protein interactions. We propose that after initiation of transcription, the released sigma 70 subunit may be recaptured by the FIS nucleoprotein 'trap' and recycled in successive rounds of holoenzyme assembly. Such a mechanism could overcome transient limitations on the availability of sigma 70 or core polymerase after a prolonged stationary phase.

Base Sequence↗
PubMed1995

Control of mating and development in Ustilago maydis.

In the fungus Ustilago maydis, the ability to distinguish between partners that are of the same or of different mating type is controlled by two mating-type loci. One locus allows extracellular recognition though a pheromone-based system. After cell fusion, the other mating-type locus, which exists in multiple alleles, determines intracellular recognition. Each allele encodes a pair of homeodomain proteins that are active only in pairwise combinations in which the two partners originate from different alleles of the locus. Recent discoveries suggest that the underlying molecular recognition mechanism is the ability to form heterodimers. Whereas the proteins in all different allelic combinations interact, it is a specific feature of proteins from the same allele not to interact. This suggests the existence of a code for protein-protein recognition.

Alleles↗
PubMed1995

Filament-specific expression of a cellulase gene in the dimorphic fungus Ustilago maydis.

The phytopathogenic fungus Ustilago maydis exists in a yeast-like haploid form and as a filamentous dikaryon. Only the dikaryon can infect corn plants. We have isolated a gene, egl1, that is not expressed in haploid cells but strongly induced in the filament. Molecular and biochemical analyses revealed that egl1 encodes a cellulase. By immunogold labelling, secreted protein could be detected at the hyphal tip. Mutants deleted for egl1 are viable and are affected neither in filament formation nor in pathogenic development under the conditions tested.

Amino Acid Sequence↗
PubMed1994

Pheromones trigger filamentous growth in Ustilago maydis.

Cell recognition and mating in the smut fungus Ustilago maydis have been proposed to involve specific pheromones and pheromone receptors. The respective structural genes are located in the a mating type locus that exists in the alleles a1 and a2. We demonstrate that binding of pheromone to the receptor can induce a morphological switch from yeast-like to filamentous growth in certain strains. Using this as biological assay we were able to purify both the a1 and a2 pheromone. The structure of the secreted pheromones was determined to be 13 amino acids for a1 and nine amino acids for a2. Both pheromones are post-translationally modified by farnesylation and carboxyl methyl esterification of the C-terminal cysteine. An unmodified a1 peptide exhibits dramatically reduced activity. The pheromone alone is able to induce characteristic conjugation tubes in cells of opposite mating type and confers mating competence; even cells of the same mating type undergo fusion. We discuss the role of pheromones in initiating filamentous growth and pathogenic development.

Amino Acid Sequence↗
PubMed1994

Control of mating and development in Ustilago maydis.

In Ustilago maydis the a and b mating type loci control pathogenicity as well as sexual development. We review the function of these loci in controlling the cell fusion step, the switch from yeast-like to filamentous growth and subsequent pathogenic development. Our special emphasis will be the role of pheromones and pheromone signaling in these processes.

Alleles↗
PubMed1993

Analysis of strand exchange and DNA binding of enhancer-independent Gin recombinase mutants.

The Gin recombination system of phage Mu mediates inversion of the DNA sequence between two sites (gix). In addition to Gin protein and gix sites, recombination requires an enhancer bound by the host factor FIS. We analyzed mutants of Gin that function in the absence of the enhancer and FIS and mediate deletion and intermolecular fusion in addition to inversion. The linking number changes caused by inversion imply that mutant Gin alone can form the same synaptic complex and can use the same strand exchange mechanism as the complete wild-type system. However, the linking number changes also reveal that unlike wild-type Gin, mutant Gin can recombine through more than one synaptic complex and can relax DNA in the absence of synapsis. This expanded repertoire allows mutant Gin to mediate DNA rearrangements not performed by wild-type Gin. Because mutant Gin, but not wild-type Gin, unwinds gix site DNA upon binding, we postulate that FIS and the enhancer function with (-) supercoiling to promote this unwinding with wild-type Gin. The analysis of the topological changes during DNA fusion shows that both the parallel gix site configuration and the right-handed rotation of the sites during exchange of wild-type Gin are a result of the (-) supercoiling of the substrate and the number of entrapped supercoils in the synaptic complex.

Bacteriophage mu↗
PubMed1992

A two-component regulatory system for self/non-self recognition in Ustilago maydis.

In U. maydis the multiallelic b locus controls sexual and pathogenic development. In the b locus a gene coding for a regulatory protein had been identified, and it was suggested that the interaction of two b polypeptides specified by different alleles programs sexual development in this fungus. We now demonstrate the existence of a second regulatory gene in the b locus. We term this gene bW and refer to the former as the bE gene. Both genes exist in many alleles. Although unrelated in primary sequence, both genes are similar in their overall organization. The gene products display allele-specific variability in their N-terminal domains, show a high degree of sequence conservation in the C-terminal domains, and contain a homeodomain-related motif. Genetic evidence is provided to show that the pair of bE and bW polypeptides encoded by different b alleles is the key regulatory species.

Alleles↗
PubMed1992

The a mating type locus of U. maydis specifies cell signaling components.

The a mating type locus of the phytopathogenic fungus U. maydis controls fusion of haploid cells and filamentous growth of the dikaryotic mycelium. The a locus exists in two alleles, termed a1 and a2, which are defined by nonhomologous DNA regions comprising 4.5 kb for a1 and 8 kb for a2, flanked by identical sequences. Based on functional assays, mutants, and sequencing, we demonstrate that the mating type in each allele is determined by a set of two genes. One encodes a precursor for a lipopeptide mating factor, and the other specifies the receptor for the pheromone secreted by cells of opposite mating type. Thus, U. maydis employs a novel strategy to determine its mating type by providing the primary determinants of cell-cell recognition directly from the mating type locus.

Alleles↗
PubMed1992

The E.coli fis promoter is subject to stringent control and autoregulation.

The DNA binding protein FIS is involved in processes like site specific DNA inversion, lambda excision and stimulation of stable RNA synthesis in Escherichia coli. The amount of FIS protein is subject to dramatic changes during growth. We demonstrate that fis is part of an operon with one ORF of unknown function preceding the fis gene. Regulation of fis synthesis occurs at the transcriptional level. Within 15 min after nutritional upshift a large burst of fis mRNA is produced which levels off when cells begin to grow. By mutational analysis using promoter-lacZ fusions we demonstrate that the fis promoter is autoregulated by FIS. Growth phase regulation of the fis promoter depends on the presence of a GC motif downstream of the -10 region. We show that the fis promoter is subject to stringent control and discuss this unusual feature with respect to the known and putative functions FIS serves in E. coli.

Amino Acid Sequence↗
PubMed1991

The N-terminal part of the E.coli DNA binding protein FIS is essential for stimulating site-specific DNA inversion but is not required for specific DNA binding.

FIS protein is involved in several different cellular processes stimulating site-specific recombination in phages Mu and lambda as well as transcription of stable RNA operons in E.coli. We have performed a mutational analysis of fis and provide genetic and biochemical evidence that a truncated version of FIS lacking the N-terminal region is sufficient for specific DNA binding and for stimulating lambda excision. These mutants also retain their ability to autoregulate fis gene expression. Such mutant proteins, however, cannot stimulate the enhancer dependent DNA inversion reaction.

Amino Acid Sequence↗
PubMed1991

Translational stimulation: RNA sequence and structure requirements for binding of Com protein.

Translation of the bacteriophage Mu mom gene is positively regulated by the phage Com protein. We report here that purified Com protein specifically stimulates mom gene expression in vitro. Furthermore, Com is shown to bind a site in the mom translational initiation region (TIR) in a sequence-specific manner. In vitro RNA footprint experiments have been used to define the Com-binding site and to study mRNA secondary structure in the mom TIR. Com binding is shown to correlate with a conformational change in the mom TIR both in vivo and in vitro. The role of secondary structure was further examined by testing the effects of mutations in the TIR on translation and stimulation. The results support a model for translational stimulation in which Com binding induces a conformational change in the mom mRNA, thereby enhancing ribosome binding.

Bacteriophage mu↗
PubMed1991

Three-dimensional structure of the E. coli DNA-binding protein FIS.

The factor for inversion stimulation, FIS, is involved in several cellular processes, including site-specific recombination and transcriptional activation. In the reactions catalysed by the DNA invertases Gin, Hin and Cin, FIS stimulates recombination by binding to an enhancer sequence. Within the enhancer, two FIS dimers (each 2 x 98 amino acids) bind to two 15-base-pair consensus sequences and induce bending of the DNA. Current models propose that the enhancer-FIS complex organizes a specific synapse, either through direct interactions with Gin, or by modelling the substrate into a configuration suitable for recombination. Using X-ray analysis at 2.0 A resolution, we now show that FIS is composed of four alpha helices tightly intertwined to form a globular dimer with two protruding helix-turn-helix motifs. The 24 N-terminal amino acids are so poorly defined in the electron density map as to make interpretation doubtful, indicating that they might act as 'feelers' suitable for DNA or protein (invertase) recognition. We infer from model building that DNA has to bend for tight binding to FIS.

Amino Acid Sequence↗
PubMed1991

The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts.

A mutant Gin recombinase of the phage Mu DNA inversion system was successfully expressed in Arabidopsis thaliana and tobacco protoplasts. Site-specific recombination was monitored both physically and biologically with the help of a recombination assay system in which expression of a beta-glucuronidase (gus) gene requires Gin-mediated recombination. We demonstrate that the wild-type Gin protein is not able to promote recombination in plant protoplasts, presumably because plant cells do not contain a protein that can substitute for the Escherichia coli FIS protein needed for full activity of wild-type Gin in E. coli. A FIS-independent Gin mutant protein on the other hand was efficient in promoting recombination on recombination substrates introduced transiently and on substrates stably integrated into the plant genome. We discuss the various advantages this system can provide for genetic manipulation of plant cells.

Bacteriophage mu↗
PubMed1991

Lumbosacral kyphosis, tethered cord, and diplomyelia. A unique spinal dysraphic condition.

Four patients are described with a triad of neuroectodermal abnormalities consisting of lumbosacral kyphosis, tethered cord, and dyplomyelia. Of utmost importance is the recognition of the association between this type of kyphosis and the underlying spinal cord pathology and the progressive nature of the deformity. The patient presenting with lumbosacral kyphos and the presence of sacral hypoplasia should have a neuroradiographic evaluation (magnetic resonance imaging scanning or enhanced computed tomography) to rule out the presence of a tethered cord or other neural abnormalities. Early surgical intervention for release of the tether and fusion should be carried out to prevent neurologic deterioration and curve progression.

Diagnostic Imaging↗
PubMed1990

Processive recombination by the phage Mu Gin system: implications for the mechanisms of DNA strand exchange, DNA site alignment, and enhancer action.

The Gin DNA invertase of bacteriophage Mu carries out processive recombination in which multiple rounds of exchange follow synaptic complex formation. The stereostructure of the knotted products determined by electron microscopy establishes critical features of site synapsis and DNA exchange. Surprisingly, the invertase knots substrates with directly repeated sites as well as those with inverted sites. The results suggest that the Gin synaptic complex contains three mutually perpendicular dyads; one is the axis of site rotation during exchange, and they cause inverted and direct site substrates to form a similar synaptic complex. The extensive knotting by Gin has implications for the energetics of recombination and shows that the enhancer for recombination is required only at an early stage, and thus may normally operate in a hit-and-run fashion.

Bacteriophage mu↗