C18orf2, a novel, highly conserved intronless gene within intron 5 of the GNAL gene on chromosome 18p11

We have characterized a novel intronless human gene (C18orf2) which is embedded in intron 5 of the G-protein gene (GNAL) on chromosome 18p11. This gene codes for a 199 amino acid polypeptide with a predicted molecular weight of 22.1 kDa. It is highly homologous to a number of predicted developmental proteins in organisms ranging from yeasts to Drosophila. C18orf2 mRNA was found to be expressed in various tissues.     

Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco

To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.     

Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein

We have studied the domain structure of the A1 heterogeneous nuclear ribonucleoprotein using both partial proteolysis and photochemical cross-linking to oligodeoxynucleotides. Both the intact A1 protein and its proteolytic fragment, the UP1 protein, can be cleaved by Staphylococcus aureus V-8 protease to produce two polypeptides of 92 amino acids. These two polypeptides correspond to the internal repeat sequence previously noted by us to occur in UP1. The two polypeptides can be purified via single-stranded DNA cellulose chromatography and independently cross-linked to [32P]p(dT)8, indicating that each domain can bind to single-stranded nucleic acids. Purification and sequencing of A1 tryptic peptides that had been cross-linked to oligothymidylic acid revealed that 4 phenylalanine residues, phenylalanines 16, 58, 107, and 149 are the sites of covalent adduct formation, with phenylalanine 16 being the major site of cross-linking. These phenylalanine residues are internally homologous when the repeat sequences in A1 are aligned, that is, phenylalanines 16 and 107 occupy analogous positions in the 91-residue repeat, as do phenylalanines 58 and 149. An examination of the primary structures of a variety of eucaryotic RNA-binding proteins with sequence homology to A1 reveals that the cross-linked phenylalanines in A1 are highly conserved among all of these proteins. Our results provide the first experimental evidence that conserved residues in the 90-amino acid repeating domains shared by A1 and other single-stranded nucleic acid binding-proteins form part of an RNA-binding pocket.     

Molecular characterization of GmFOX2, an evolutionarily highly conserved gene from the mycorrhizal fungus Glomus mosseae, down-regulated during interaction with rhizobacteria.

Arbuscular mycorrhizal (AM) fungi form the most wide-spread symbiosis of the plant kingdom. More than 80% of vascular plants are susceptible to colonization by the zygomycetous fungi from the order Glomales, and profit significantly by the nutrient exchange between plant and fungus. However, knowledge of the biology of these fungi still remains elusive because of their obligate biotrophism and, up to now, unculturability. The molecular mechanisms underlying the pre-symbiotic stages and the cell-to-cell communication between AM fungi and other soil microorganisms are, particularly, unknown. Here, we study these aspects by means of a molecular approach to monitor changes in the gene expression of the fungus Glomus mosseae (BEG12) in response to the rhizobacterium Bacillus subtilis NR1. The bacterium was found to induce specific increases in mycelial growth as well as changes in expression of GmFOX2, a highly conserved gene encoding a multifunctional protein of the peroxisomal beta-oxidation. We determined the gene structure and studied its expression in response to rhizobacteria at two time points. The results show that the fungus is able to change its gene expression in response to stimuli other than the plant.     

Essential domains of the PRP21 splicing factor are implicated in the binding to PRP9 and PRP11 proteins and are conserved through evolution.

The yeast Prp9p, Prp11p, Prp21p proteins form a multimolecular complex identified as the SF3a splicing factor in higher eukaryotes. This factor is required for the assembly of the prespliceosome. Prp21p interacts with both Prp9p and Prp11p, but the molecular basis of these interactions is unknown. Prp21p, its human homologue, and the so-called SWAP proteins share a tandemly repeated motif, the surp module. Given the evolutionary conservation and the role of SWAP proteins as splicing regulators, it has been proposed that surp motifs are essential for interactions between Prp21p and other splicing factors. In order to characterize functional domains of Prp21p and to identify potential additional functions of this protein, we isolated a series of heat-sensitive prp21 mutants. Our results indicate that prp21 heat-sensitive mutations are associated with defects in the interaction with Prp9p, but not with Prp11p. Interestingly, most heat-sensitive point mutants associate a strong splicing defect with a pre-mRNA nuclear export phenotype, as does the prp9-1 heat-sensitive mutant. Deletion analyses led to the definition of domains required for viability. These domains are responsible for the interaction with Prp9p and Prp11p and are conserved through evolution. They do not include the most conserved surp1 module, suggesting that the conservation of this motif in two families of proteins may reflect a still unknown function dispensable in yeast under standard conditions.     

A stationary-phase gene in Saccharomyces cerevisiae is a member of a novel, highly conserved gene family.

The regulation of cellular growth and proliferation in response to environmental cues is critical for development and the maintenance of viability in all organisms. In unicellular organisms, such as the budding yeast Saccharomyces cerevisiae, growth and proliferation are regulated by nutrient availability. We have described changes in the pattern of protein synthesis during the growth of S. cerevisiae cells to stationary phase (E. K. Fuge, E. L. Braun, and M. Werner-Washburne, J. Bacteriol. 176:5802-5813, 1994) and noted a protein, which we designated Snz1p (p35), that shows increased synthesis after entry into stationary phase. We report here the identification of the SNZ1 gene, which encodes this protein. We detected increased SNZ1 mRNA accumulation almost 2 days after glucose exhaustion, significantly later than that of mRNAs encoded by other postexponential genes. SNZ1-related sequences were detected in phylogenetically diverse organisms by sequence comparisons and low-stringency hybridization. Multiple SNZ1-related sequences were detected in some organisms, including S. cerevisiae. Snz1p was found to be among the most evolutionarily conserved proteins currently identified, indicating that we have identified a novel, highly conserved protein involved in growth arrest in S. cerevisiae. The broad phylogenetic distribution, the regulation of the SNZ1 mRNA and protein in S. cerevisiae, and identification of a Snz protein modified during sporulation in the gram-positive bacterium Bacillus subtilis support the hypothesis that Snz proteins are part of an ancient response that occurs during nutrient limitation and growth arrest.