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.

A physical amplified fragment-length polymorphism map of Arabidopsis.

We have positioned amplified fragment-length polymorphism (AFLP) markers directly on the genome sequence of a complex organism, Arabidopsis, by combining gel-based AFLP analysis with in silico restriction fragment analysis using the published genome sequence. For placement of the markers, we used information on restriction fragment size, four selective nucleotides, and the rough genetic position of the markers as deduced from the analysis of a limited number of Columbia (Col)/Landsberg (Ler) recombinant inbred lines. This approach allows for exact physical positioning of markers as opposed to the statistical localization resulting from traditional genetic mapping procedures. In addition, it is fast because no extensive segregation analysis is needed. In principle, the method can be applied to all organisms for which a complete or nearly complete genome sequence is available. We have located 1,267 AFLP Col/Ler markers resulting from 256 SacI+2, MseI+2 primer combinations to a physical position on the Arabidopsis genome. The positioning was verified by sequence analysis of 70 markers and by segregation analysis of two leaf-form mutants. Approximately 50% of the mapped Col/Ler AFLP markers can be used for segregation analysis in Col/C24, Col/Wassilewskija, or Col/Cape Verde Islands crosses. We present data on one such cross: the localization of a viviparous-like mutant segregating in a Col/C24 cross.

Partial purification and identification of GDP-mannose 3",5"-epimerase of Arabidopsis thaliana, a key enzyme of the plant vitamin C pathway.

The first step in the biosynthetic pathway of vitamin C in plants is the formation, at the level of sugar nucleotide, of l-galactosyl residues, catalyzed by a largely unknown GDP-d-mannose 3",5"-epimerase. By using combined conventional biochemical and mass spectrometry methods, we obtained a highly purified preparation of GDP-d-mannose 3",5"-epimerase from an Arabidopsis thaliana cell suspension. The native enzyme is an 84-kDa dimer, composed of two apparently identical subunits. In-gel tryptic digestion of the enzyme subunit, followed by peptide sequencing and a blast search, led to the identification of the epimerase gene. The closest homolog of the plant epimerase is the BlmG gene product of Streptomyces sp., a putative NDP-d-mannose 5"-epimerase. The plant GDP-d-mannose 3",5"-epimerase is, to our knowledge, a novel member of the extended short-chain dehydrogenase/reductase family. The enzyme was cloned and expressed in Escherichia coli cells.

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.

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.

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.

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.

AFLP-based fine mapping of the Mlo gene to a 30-kb DNA segment of the barley genome.

Resistance of barley (Hordeum vulgare) to the powdery mildew fungus Erysiphe graminis f.sp. hordei is conferred by several dominant genes, but also by recessive alleles of the Mlo locus mapping on the long arm of chromosome 4. In addition, this single-factor-mediated resistance is active against all known physiological races of the parasite. Thus the mechanism underlying mlo-mediated resistance should differ substantially from that mediated by the dominant genes. A positional cloning strategy to isolate the Mlo gene from the barley genome, the size of which is almost double the size of the human genome, has been designed. The AFLP technique was employed to identify markers tightly linked to the Mlo locus and to produce a local high-resolution genetic map. The use of this high-volume marker technology allowed the rapid screening of approximately 250,000 loci for linkage to Mlo. A large number of Mlo-linked AFLP markers were identified, one of which cosegregated with Mlo on the basis of more than 4000 meiotic events. A four-genome-equivalent barley YAC library (average insert size 480 kb) was constructed and screened with this cosegregating marker. Four YACs containing this marker were isolated and subsequent characterization by AFLP-based physical mapping allowed the physical delimitation of the Mlo locus to a DNA segment of 30 kb.

Evaluation of the DNA fingerprinting method AFLP as an new tool in bacterial taxonomy.

We investigated the usefulness of a novel DNA fingerprinting technique, AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate bacterial strains at the subgeneric level. In totals, 147 bacterial strains were subjected to AFLP fingerprinting: 36 Xanthomonas strains, including 23 pathovars of Xanthomonas axonopodis and six pathovars of Xanthomonas vasicola, one strain of Stenotrophomonas, 90 genotypically characterized strains comprising all 14 hybridization groups currently described in the genus Aeromonas, and four strains of each of the genera Clostridium, Bacillus, Acinetobacter, Pseudomonas and Vibrio. Depending on the genus, total genomic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were ligated to restriction halfsite-specific adaptors. These adaptors served as primer-binding sites allowing the fragments to be amplified by selective PCR primers that extend beyond the adaptor and restriction site sequences. Following electrophoretic separation on 5% (w/v) polyacrylamide/8.3 M urea, amplified products could be visualized by autoradiography because one of the selective primers was radioactively labelled. The resulting banding patterns, containing approximately 30-50 visualized PCR products in the size range 80-550 bp, were captured by a high-resolution densitoscanner and further processed for computer-assisted analysis to determine band-based similarity coefficients. This study reveals extensive evidence for the applicability of AFLP in bacterial taxonomy through comparison of the newly obtained data with results previously obtained by well-established genotypic and chemotaxonomic methods such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to characterize strains at the infrasubspecific level), highlighting the potential of this novel fingerprinting method in epidemiological and evolutionary studies.

Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development.

Using a highly synchronous in vitro tuberization system, in combination with an amplified restriction fragment polymorphism (AFLP)-derived technique for RNA fingerprinting (cDNA-AFLP), transcriptional changes at and around the time point of potato tuberization have been analyzed. The targeted expression analysis of a specific transcript coding for the major potato storage protein, patatin and a second transcript, coding for ADP-glucose pyrophosphorylase, a key gene in the starch biosynthetic pathway is described. This paper confirms that kinetics of expression revealed by cDNA-AFLP analysis are comparable to those found in Northern analysis. Furthermore, this paper reports the isolation and analysis of two tuber-specific transcript-derived fragments (TDFs) coding for the lipoxygenase enzyme, which are differentially induced around the time point of tuber formation. Analysis of the two lox TDFs demonstrates that it is possible to dissect the expression modalities of individual transcripts, not independently detectable by Northern analysis. Finally, it is shown that using cDNA-AFLP, rapid and simple verification of band identity may be achieved. The results indicate that cDNA-AFLP is a broadly applicable technology for identifying developmentally regulated genes.

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.

Identification of amplified restriction fragment polymorphism (AFLP) markers tightly linked to the tomato Cf-9 gene for resistance to Cladosporium fulvum.

Using the technique of amplified restriction fragment polymorphism (AFLP) analysis, and bulked segregant pools from F2 progeny of the cross Lycopersicon esculentum (Cf9) x L. pennellii, approximately 42,000 AFLP loci for tight linkage to the tomato Cf-9 gene for resistance to Cladosporium fulvum have been screened. Analysis of F2 recombinants identified three markers which co-segregated with Cf-9. The Cf-9 gene has recently been isolated by transposon tagging using the maize transposon Dissociation (Ds). Analysis of plasmid clones containing Cf-9 shows that two of these markers are located on opposite sides of the gene separated by 15.5 kbp of intervening DNA. AFLP analysis provides a rapid and efficient technique for detecting large numbers of DNA markers and should expedite plant gene isolation by positional cloning and the construction of high-density molecular linkage maps of plant genomes.

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.

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.

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii.

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.

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.

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.

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.

Structural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715.

A plasmid-encoded crystal protein gene (bt2) has been cloned from Bacillus thuringiensis berliner 1715. In Escherichia coli, it directs the synthesis of the 130-kDa protein (Bt2) which is toxic to larvae of Pieris brassicae and Manduca sexta. Comparison of the deduced amino acid sequence of this Bt2 protein with the B. thuringiensis kurstaki HD1 Dipel, B. thuringiensis kurstaki HD73 and B. thuringiensis sotto crystal protein sequences suggests that homologous recombination between the different genes has occurred during evolution. Treatment of the Bt2 protein with trypsin or chymotrypsin yields a 60-kDa protease-resistant and fully toxic polypeptide. The minimal portion of the Bt2 protein required for toxicity has been determined by analysing the polypeptides produced by deletion derivatives of the bt2 gene. It coincides with the 60-kDa protease-resistant Bt2 fragment and it starts between amino acids 29 and 35 at the N-terminus and terminates between positions 599 and 607 at the C-terminus.