The tomato Mi-1 gene confers resistance to both root-knot nematodes and potato aphids.

Mi-1, a Lycopersicon peruvianum gene conferring resistance to the agricultural pests, root-knot nematodes, and introgressed into tomato, has been cloned using a selective restriction fragment amplification based strategy. Complementation analysis of a susceptible tomato line with a 100 kb cosmid array yielded a single cosmid clone capable of conferring resistance both to the root-knot nematode Meloidogyne incognita and to an unrelated pathogen, the potato aphid Macrosiphum euphorbiae. This resistance was stable. The Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat-containing type of plant resistance genes.

Genome-wide expression analysis of plant cell cycle modulated genes.

Genome-wide expression analysis is rapidly becoming an essential tool for identifying and analysing genes involved in, or controlling, various biological processes ranging from development to responses to environmental cues. The control of cell division involves the temporal expression of different sets of genes, allowing the dividing cell to progress through the different phases of the cell cycle. A landmark study using DNA microarrays to follow the patterns of gene expression in synchronously dividing yeast cells has allowed the identification of several hundreds of genes that are involved in the cell cycle. Although DNA microarrays provide a convenient tool for genome-wide expression analysis, their use is limited to organisms for which the complete genome sequence or a large cDNA collection is available. For other organisms, including most plant species, DNA fragment analysis based methods, such as cDNA-AFLP, provide a more appropriate tool for genome-wide expression analysis. Furthermore, cDNA-AFLP exhibits properties that complement DNA microarrays and, hence, constitutes a useful tool for gene discovery.

High-level production of the EcoRI endonuclease under the control of the pL promoter of bacteriophage lambda.

Escherichia coli strains overproducing the EcoRI restriction endonuclease have been constructed, using lambda pL promoter expression vectors. In a first step we constructed endRI::lacZ gene fusions by fusing the N-terminal part of the endRI gene with a lacZ gene fragment, whereafter the hybrid gene was positioned randomly under the control of the pL promoter to optimize the level of expression. These plasmids direct the synthesis of large amounts of fusion protein approaching 30% of the total cellular protein content. In most cases the overproduced protein forms enzymatically inactive intracellular aggregates. The position of the promoter in front of the hybrid gene had little effect on the level of expression, except in fusions directly affecting the ribosome-binding site (RBS). In a second step, several of these promoter-gene configurations were used to reconstruct the intact endRI gene in appropriate hosts producing EcoRI methylase and cI-coded repressor. The levels of EcoRI endonuclease overproduction were similar to that obtained for the corresponding fusion protein, despite the fourfold difference in protein size. Intracellular precipitation was also observed with the overproduced EcoRI endonuclease.

Cloning and nucleotide sequence of different iso-IS231 elements and their structural association with the Tn4430 transposon in Bacillus thuringiensis.

A family of five repetitive sequences (RS) has been isolated from a plasmid DNA library of Bacillus thuringiensis strain berliner 1715. In a previous paper [Mahillon et al., EMBO J. 4(1985)3895-3899] one of these was shown to harbor all the features of an IS element (IS231). Further nucleotide sequence analysis revealed that two other RS, flanking the delta-endotoxin gene, are actually variants of IS231. Comparison of the nucleotide sequences surrounding the iso-IS231 elements showed a unique structural association between some of these elements and the transposon Tn4430. Although these IS231 elements have transposed into Tn4430, both these IS231 s and the transposon Tn4430 remain structurally intact.

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.

A standardized vector system for manipulation and enhanced expression of genes in Escherichia coli.

Different families of cloning and expression vectors were engineered on a standard plasmid. They contain several regulatory signals for transcription and/or translation initiation and termination. The plasmids in each series differ only in the number, type, and order of unique restriction cleavage sites clustered in front of a transcription terminator. The pLK30 plasmids are general cloning vectors and the corresponding pLK50 plasmids carry the lambda pL promoter. The pLK60 vectors carry the lambda pR promoter and translation initiation signals of the cro gene containing the Shine-Dalgarno sequence and initiation codon. The pLK70 series is similar to pLK60 except that additional 5'-translated cro sequences are included. The pLK80 plasmids have a lacZ gene fragment suitable for the construction of hybrid genes. The presence of translational stop signals in the pLK90 series facilitates the manipulation of genes truncated at the 3' end. This standardized pLK vector system offers great versatility in gene manipulation and in optimization of gene expression under the control of strong regulatable promoters. Measurement of expression levels under repressed conditions permits the identification of optimal promoter-gene configurations in constructions directing high-level expression.

Enhanced expression of cro-beta-galactosidase fusion proteins under the control of the PR promoter of bacteriophage lambda.

Hybrid plasmids carrying cro-lacZ gene fusions have been constructed by joining DNA segments carrying the PR promoter and the start of the cro gene of bacteriophage lambda to the lacZ gene fragment carried by plasmid pLG400 . Plasmids in which the translational reading frames of the cro and lacZ genes are joined in-register (type I) direct the synthesis of elevated levels of cro-beta-galactosidase fusion protein amounting to 30% of the total cellular protein, while plasmids in which the genes are fused out-of-register (type II) produce a low level of beta-galactosidase protein. Sequence rearrangements downstream of the cro initiator AUG were found to influence the efficiency of translation, and have been correlated with alterations in the RNA secondary structure of the ribosome-binding site. Plasmids which direct the synthesis of high levels of beta-galactosidase are conditionally lethal and can only be propagated when the PR promoter is repressed. Deletion of sequences downstream of the lacZ gene restored viability, indicating that this region of the plasmid encodes a function which inhibits the growth of the cells. The different applications of these plasmids for expression of cloned genes are discussed.

The ral gene of phage lambda. I. Identification of a non-essential gene that modulates restriction and modification in E. coli.

Host controlled restriction in Escherichia coli can be relieved by pre-infecting restricting cells with modified lambda helper phages. This process, in which intact unmodified phage genomes are allowed to escape restriction attack, is mediated by a newly identified lambda function called ral. The ral gene has been located by deletion mapping between cIII and N. Efficient expression of the ral gene requires the product of the regulator gene N. Polyacrylamide gel analysis of the lambda proteins specified by the cIII-N region failed to reveal the product of the ral gene, but demonstrated that protein Ea10 is encoded by a gene located immediately to the left of ral. From these results the map order cIII-Ea10-ral-TL1-N was deduced. Ral specifically alleviates restriction in E. coli K and E. coli B, but does not affect restriction systems EcoRI, EcoRII and EcoP1. In addition, ral enhances the modification activity of the EcoK and EcoB restriction enzymes: we observed that efficient modification of progeny phages obtained by propagating unmodified lambda phages in r-m+ hosts, is dependent upon the presence of ral. We thus conclude that the ral gene product acts by modulating the restriction and modification activities of the type I restriction systems in E. coli, and the possible mechanisms will be discussed.

The ral gene of phage lambda. II. Isolation and characterization of ral deficient mutants.

The lambda ral function modulates the restriction and modification activities of the Escherichia coli K12 and B restriction enzymes (Zabeau et al., 1980). In order to further analyse this function, ral deficient mutants have been isolated, using a method which exploits the property of the strong mutagen N-methyl-N'-nitro-N-nitrosoguanidine (N.G.) to induce multiple closely linked mutations. Hence, mutagenized phages carrying mutations in one locus were frequently found to contain additional mutations in adjacent loci. This very efficient mutagenesis procedure enabled us to isolate 27 independent Ral deficient mutants. Seven mutants were found to affect the ral gene directly and were located between the genes N anc cIII. Detailed mapping of two of these mutants showed that the lambda ral gene is located at position 70.6-70.9% on the physical map. The isolation and characterization of these mutants further supports the conclusion that ral is a gene different from the N gene, and demonstrates that the ral gene product is responsible for both counteracting restriction and enhancing modification.

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI.

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.

Overproduction of the EcoR V endonuclease and methylase.

Strains overproducing the EcoR V endonuclease and methylase have been obtained by inserting each of the two genes in expression vectors containing the lambda PL promoter. The methylase is overproduced to a level reaching 5-10% of the total cellular proteins, which represents a 50-100 fold increase. A 30 fold overproduction of endonuclease was achieved by randomly positioning the EndRV gene downstream of the lambda PL promoter. The situation in the endonuclease overproducing clone resembles that encountered in maxi-cells. The strains described here allowed a quick purification of both enzymes in sufficient amounts for crystallisation attempts.

Characterization of the genes coding for the Eco RV restriction and modification system of Escherichia coli.

A plasmid encoding the recently described Eco RV restriction and modification system has been isolated and characterized. This plasmid, pLB1 , is 6.2 kb long and carries only the Eco RV genes. A subclone of 3 kb has been inserted in pBR322. The relative positions of the endonuclease and the methylase genes were determined by the construction of a set of overlapping deletions generated by Bal31 resection. The DNA sequence of a 2.2 kb fragment containing the two genes was determined. The two genes are transcribed divergently from a 310 bp region and the assignment of the coding region has been confirmed by direct aminoacid sequence analysis. Possible mechanisms of regulation of the endonuclease gene expression at the translational level are proposed and discussed.

A high-resolution map of the vicinity of the R1 locus on chromosome V of potato based on RFLP and AFLP markers.

The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21-GP179 interval. Two of those could not be separated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants.

PCR-based fingerprinting using AFLPs as a tool for studying genetic relationships in Lactuca spp.

AFLP markers were evaluated for determining the phylogenetic relationships Lactuca spp. Genetic distances based on AFLP data were estimated for 44 morphologically diverse lines of cultivated L. sativa and 13 accessions of the wild species L. serriola, L. saligna, L. virosa, L. perennis, and L. indica. The same genotypes were analyzed as in a previous study that had utilized RFLP markers. The phenetic tree based on AFLP data was consistent with known taxonomic relationships and similar to a tree developed with RFLP data. The genetic distance matrices derived from AFLP and RFLP data were compared using least squares regression analysis and, for the cultivar data, by principal component analysis. There was also a positive linear relationship between distance estimates based on AFLP data and kinship coefficients calculated from pedigree data. AFLPs represent reliable PCR-based markers for studies of genetic relationships at a variety of taxonomic levels.

Nucleotide sequence and structural organization of an insertion sequence element (IS231) from Bacillus thuringiensis strain berliner 1715.

This paper describes the structural organization of a repetitive DNA sequence isolated from plasmids of Bacillus thuringiensis strain berliner 1715. DNA sequence analysis of this repetitive sequence (RS) revealed all the characteristic features of an insertion sequence (IS). This 1656-bp element is delineated by two 20-bp inverted repeats which are flanked by two 11-bp direct repeats. A long open reading frame spans almost the entire sequence and is preceded by potential transcriptional and translational signals. A structural homology was observed between this RS and the Escherichia coli IS4 element in the length of their direct repeats, the sequence of their inverted repeats and the sequence of their putative transposases. These data strongly suggest that this sequence is an authentic insertion sequence. Therefore the name IS231 is proposed for this first Bacillus IS element. Further relationships with other known IS sequences were also found and are discussed.

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.

Effect of polyamines and basic proteins on cleavage of DNA by restriction endonucleases.

We have investigated the effect of the polyamines spermine, spermidine, and putrescine and the prokaryotic histone-like proteins NS1 and NS2 on the restriction endonuclease EcoRI catalyzed cleavage of plasmid and bacteriophage DNAs. At low concentrations of spermine and spermidine, the rate of DNA cleavage by EcoRI is increased, while high concentrations of spermine as well as of spermidine are inhibitory. These phenomena are also observed with other restriction endonucleases. They are, therefore, probably due to the interaction of the polyamines with the DNA. Putrescine does not have such an effect within the concentration range investigated. Remarkably, low concentrations of spermine and spermidine very efficiently suppress EcoRI activity. An inhibition of the EcoRI-catalyzed cleavage of DNA is also observed with NS1 and NS2, an effect that can be mimicked with other basic proteins that interact with DNA. The results are discussed in terms of the mechanism of restriction in vivo.

Quantitative cDNA-AFLP analysis for genome-wide expression studies.

An improved cDNA-AFLP method for genome-wide expression analysis has been developed. We demonstrate that this method is an efficient tool for quantitative transcript profiling and a valid alternative to microarrays. Unique transcript tags, generated from reverse-transcribed messenger RNA by restriction enzymes, were screened through a series of selective PCR amplifications. Based on in silico analysis, an enzyme combination was chosen that ensures that at least 60% of all the mRNAs were represented by an informative sequence tag. The sensitivity and specificity of the method allows one to detect poorly expressed genes and distinguish between homologous sequences. Accurate gene expression profiles were determined by quantitative analysis of band intensities, and subtle differences in transcriptional activity were revealed. A detailed screen for cell cycle-modulated genes in tobacco demonstrates the usefulness of the technology for genome-wide expression analysis.

An improved FORTRAN 77 recombinant DNA database management system with graphic extensions in GKS.

We have improved an existing clone database management system written in FORTRAN 77 and adapted it to our software environment. Improvements are that the database can be interrogated for any type of information, not just keywords. Also, recombinant DNA constructions can be represented in a simplified 'shorthand', whereafter a program assembles the full nucleotide sequence from the contributing fragments, which may be obtained from nucleotide sequence databases. Another improvement is the replacement of the database manager by programs, running in batch to maintain the databank and verify its consistency automatically. Finally, graphic extensions are written in Graphical Kernel System, to draw linear and circular restriction maps of recombinants. Besides restriction sites, recombinant features can be presented from the feature lines of recombinant database entries, or from the feature tables of nucleotide databases. The clone database management system is fully integrated into the sequence analysis software package from the Pasteur Institute, Paris, and is made accessible through the same menu. As a result, recombinant DNA sequences can directly be analysed by the sequence analysis programs.