Arabidopsis thaliana is a small flowering plant that serves as the major model system in plant molecular genetics. The efforts of many scientists have produced genetic maps that provide extensive coverage of the genome (http://genome-www. stanford.edu/Arabidopsis/maps.html). Recently, detailed YAC, BAC, P1 and cosmid-based physical maps (that is, representations of genomic regions as sets of overlapping clones of corresponding libraries) have been established that extend over wide genomic areas ranging from several hundreds of kilobases to entire chromosomes. These maps provide an entry to gain deeper insight into the A. thaliana genome structure. A. thaliana has been chosen as the subject of the first large-scale project intended to determine the full genome sequence of a plant. This sequencing project, together with the increasing interest in map-based gene cloning, has highlighted the requirement for a complete and accurate physical map of this plant species. To supply the scientific community with a high-quality resource, we present here a complete physical map of A. thaliana using essentially the IGF BAC library. The map consists of 27 contigs that cover the entire genome, except for the presumptive centromeric regions, nucleolar organization regions (NOR) and telomeric areas. This is the first reported map of a complex organism based entirely on BAC clones and it represents the most homogeneous and complete physical map established to date for any plant genome. Furthermore, the analysis performed here serves as a model for an efficient physical mapping procedure using BAC clones that can be applied to other complex genomes.
A gene library of genomic DNA from the hydrogen uptake (Hup)-positive strain 128C53 of Rhizobium leguminosarum was constructed by using the broad-host-range mobilizable cosmid vector pLAFR1. The resulting recombinant cosmids contained insert DNA averaging 21 kilobase pairs (kb) in length. Two clones from the above gene library were identified by colony hybridization with DNA sequences from plasmid pHUl containing hup genes of Bradyrhizobium japonicum. The corresponding recombinant cosmids, pAL618 and pAL704, were isolated, and a region of about 28 kb containing the sequences homologous to B. japonicum hup-specific DNA was physically mapped. Further hybridization analysis with three fragments from pHUl (5.9-kb HindIll, 2.9-kb EcoRI, and 5.0-kb EcoRI) showed that the overall arrangement of the R. keguminosarum hup-specific region closely parallels that of B. japonwum. The presence of functional hup genes within the isolated cosmid DNA was demonstrated by site-directed TnS mutagenesis of the 128C53 genome and analysis of the Hup phenotype of the TnS insertion strains in symbiosis with peas. Transposon TnS insertions at six different sites spanning 11 kb of pAL618 completely suppressed the hydrogenase activity of the pea bacteroids.Certain strains of Bradyrhizobium japonicum and Rhizobium leguminosarum (Hup+ strains) induce, in symbiosis with soybeans and peas, respectively, the synthesis of an H2 uptake system which catalyzes the recycling of H2 generated by the nitrogenase complex as an obligate by-product of the nitrogen fixation process (11,13). This H2 uptake system has been studied in detail in free-living and symbiotic cells of Hup+ strains of B. japonicum (13,22,26). The first component of the system is a membrane-bound hydrogenase which contains nickel and two polypeptide subunits. Hydrogen is activated by the hydrogenase and oxidized with 02 to water through an electron transport chain, some of whose components may be specifically involved in the H2 oxidation. The oxidation of H2 in legume nodules has been shown to increase N2 fixation and legume productivity (13,14).Hydrogen uptake (hup) genes are not expressed in normal cultures of rhizobia. However, derepression of hup genes in free-living B. japonicum has been shown under certain culture conditions including low-carbon medium and an atmosphere with H2, C02, and low 02 (27)
Received 17 D e c e m b e r 1990; a c c e p t e d 8 J a n u a r y 1991
A bacterial artificial chromosome (BAC) library has been established for Arabidopsis thaliana (ecotype Col-0) covering about seven haploid nuclear genome equivalents. This library, called the Institut für Genbiologische Forschung (IGF) BAC library, consists of 10,752 recombinant clones carrying inserts (generated by partial EcoRI digestion) of an average size of about 100 kb in a modified BAC vector, pBeloBAC-Kan. Hybridization with organellar DNA and nuclear repetitive DNA elements revealed the presence of 1.1% clones with mitochondrial DNA, 0.2% clones with plastid DNA, 3.2% clones with the 180 bp paracentromeric repeat, 1.6% clones with 5S rDNA, and 10.8% clones with the 18S-25S rDNA repeat. With its extensive genome coverage, its rather uniformly sized inserts (80 kb < 85% < 120 kb) and low contamination with organellar DNA, this library provides an excellent resource for A. thaliana genomic mapping, map-based gene cloning, and genome sequencing.
The mitochondrial iron-sulfur protein (also termed Rieske iron-sulfur protein) of cytochrome c reductase was purified from potato tubers and identified with heterologous antibodies. The sequences of the N-terminus of this 25 kDa protein and of an internal peptide were determined to design oligonucleotide mixtures for screening a cDNA library. One class of cDNA clones containing an open reading frame of 265 amino acids was isolated. The encoded protein contains the peptide sequences of the 25 kDa protein and shares about 50% sequence identity with the Rieske iron-sulfur proteins from fungi and around 43% with those from mammals. In vitro transcription and translation of the cDNA reveals that the iron-sulfur protein is made as a larger precursor of 30 kDa which is processed by the cytochrome c reductase/processing peptidase complex from potato. The processing product obtained after in vitro processing has the same size as the mature protein imported into isolated mitochondria. The presequence, which targets the protein to the organelle, is 53 amino acids long and has molecular features different from those found in presequences of fungal iron-sulfur proteins, which are processed in two steps. Our results indicate that, unlike in yeast and Neurospora, the presequence of the iron-sulfur protein from potato is removed by a single processing enzyme in one step.
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