Srchi24, a chitinase homolog lacking an essential glutamic acid residue for hydrolytic activity, is induced during nodule development on Sesbania rostrata.

The interaction between the tropical legume Sesbania rostrata and the bacterium Azorhizobium caulinodans results in the formation of nodules on both stem and roots. Stem nodulation was used as a model system to isolate early markers by differential display. One of them, Srchi24 is a novel early nodulin whose transcript level increased already 4 h after inoculation. This enhancement depended on Nod factor-producing bacteria. Srchi24 transcript levels were induced also by exogenous cytokinins. In situ hybridization and immunolocalization experiments showed that Srchi24 transcripts and proteins were present in the outermost cortical cell layers of the developing nodules. Sequence analyses revealed that Srchi24 is similar to class III chitinases, but lacks an important catalytic glutamate residue. A fusion between a maltose-binding protein and Srchi24 had no detectable hydrolytic activity. A function in nodulation is proposed for the Srchi24 protein.

Evidence for an ancient chromosomal duplication in Arabidopsis thaliana by sequencing and analyzing a 400-kb contig at the APETALA2 locus on chromosome 4.

As part of the European Scientists Sequencing Arabidopsis program, a contiguous region (396607 bp) located on chromosome 4 around the APETALA2 gene was sequenced. Analysis of the sequence and comparison to public databases predicts 103 genes in this area, which represents a gene density of one gene per 3.85 kb. Almost half of the genes show no significant homology to known database entries. In addition, the first 45 kb of the contig, which covers 11 genes, is similar to a region on chromosome 2, as far as coding sequences are concerned. This observation indicates that ancient duplications of large pieces of DNA have occurred in Arabidopsis.

Sequence analysis of a 40-kb Arabidopsis thaliana genomic region located at the top of chromosome 1.

As a contribution to the European Scientists Sequencing Arabidopsis (BIOTECH ESSA) project, a contig of almost 40kb has been sequenced at the extreme top of chromosome 1, around the Arabidopsis thaliana gene coding for a member of the 1-aminocyclopropane-1-carboxylate synthesis gene family. The region contains, besides the ACS1 gene itself, 10 putative genes, all new for Arabidopsis. Among these are three genes encoding kinases, a late embryogenesis-abundant protein, a MADS box-containing protein, a dehydrogenase, and a Myb-related transcription factor. In addition, six cDNAs have been sequenced that correspond to this region.

Sequence analysis of a 24-kb contiguous genomic region at the Arabidopsis thaliana PFL locus on chromosome 1.

As part of the European Union program of European Scientist Sequencing Arabidopsis (ESSA), the DNA sequence of a 24.053-bp insert of cosmid clone CC17J13 was determined. The cosmid is located on chromosome 1 at the PFL locus (position 30 cM). Analysis of the sequence and comparison to public databases predicts seven genes in this area, thus approximately one gene every 3.3 kb. Three cDNAs corresponding to genes in this region were also sequenced. The homologies and/or possible functions of the (putative) genes are discussed. Proteins encoded by genes in this region include a polyadenylate-binding protein (PAB-3) and a GTP-binding protein (Rab7) as well as a novel protein, possibly involved in double-stranded RNA unwinding and apoptosis. Intriguingly, the gene encoding the PAB-3 protein, which is very specifically expressed, is flanked by putative matrix attachment regions.

A group 3 LEA cDNA of rice, responsive to abscisic acid, but not to jasmonic acid, shows variety-specific differences in salt stress response.

A cDNA clone oslea3, encoding a group 3 late-embryogenesis abundant (LEA) protein was isolated from roots of rice seedlings (Oryza sativa L.). The encoded OSLEA3 protein has previously been found to accumulate to higher levels in roots of two salt-tolerant compared to a salt-sensitive rice variety in response to abscisic acid (ABA) [Moons et al., 1995. Plant Physiol. 107, 177-186]. The OSLEA3 protein (Mr 20.5, pI 6.5) characteristically contains ten imperfect 11-mer amino acid repeats. Exogenous application of ABA and exposure to salt shock (150 mM NaCl) rapidly induces a de novo, abundant oslea3 transcript accumulation in seedling roots, whereas application of jasmonic acid (9 microM) does not induce oslea3 expression. The stress-induced oslea3 transcript gradually declined upon prolonged salt shock, as wilting-induced damage became irreversible. oslea3 expression was compared for the salt-tolerant variety Pokkali and the salt-sensitive cultivar Taichung N1. Higher maximal mRNA levels were found in roots of the tolerant variety, also declining less rapidly upon sustained salt shock, concomitant with a delayed drop in shoot water content. DNA blot analysis indicated the existence of a small oslea3 gene family in rice with an equal gene number in both ecotypes. The results suggest that a differential regulation of oslea3 expression is an aspect of the varietal differences in salt stress tolerance.

LambdaZLG6: a phage lambda vector for high-efficiency cloning and surface expression of cDNA libraries on filamentous phage.

We describe a vector, lambdaZLG6, combining the high efficiency of cDNA library cloning in bacteriophage lambda with filamentous phage display of cDNA-encoded products. The cDNAs are expressed as fusions to the 3' end of M13 gene VI. The lambdaZLG library is converted to a pZLG6-cDNA phagemid library by in vivo mass excision. Helper phage infection generates a library of phagemid particles displaying the cDNA-encoded products and containing the corresponding nucleotide sequences within.

Surface expression and ligand-based selection of cDNAs fused to filamentous phage gene VI.

We describe a novel phage display system that affords the surface expression and hence affinity selection of cDNAs. The strategy is based on a new approach to functionally display proteins on filamentous phage through the attachment to the C-terminus of the minor coat protein VI. The utility of the method was evaluated using a cDNA library derived from the parasite Ancylostoma caninum. cDNA sequences were fused in each of the three reading frames to the 3'-end of the M13 gene VI expressed by a phagemid vector. Phages rescued from this cDNA expression library were subjected to biopanning against two serine proteases, trypsin and the human coagulation factor Xa. This led to the identification of cDNAs encoding novel members of two different families of serine protease inhibitors. The authenticity of the cDNA selected with trypsin as the target was demonstrated by purifying the encoded potent Kunitz-type inhibitor from an Ancylostoma caninum extract. The rapid isolation of specific cDNAs with the protein VI monovalent display system should facilitate the search for novel biologically important ligands.