Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Among the four loci causing AD-HSP identified so far, the SPG4 locus at chromosome 2p2-1p22 has been shown to account for 40-50% of all AD-HSP families. Using a positional cloning strategy based on obtaining sequence of the entire SPG4 interval, we identified a candidate gene encoding a new member of the AAA protein family, which we named spastin. Sequence analysis of this gene in seven SPG4-linked pedigrees revealed several DNA modifications, including missense, nonsense and splice-site mutations. Both SPG4 and its mouse orthologue were shown to be expressed early and ubiquitously in fetal and adult tissues. The sequence homologies and putative subcellular localization of spastin suggest that this ATPase is involved in the assembly or function of nuclear protein complexes.

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We report the construction of a tiling path of around 650 clones covering more than 99% of human chromosome 14. Clone overlap information to assemble the map was derived by comparing fully sequenced clones with a database of clone end sequences (sequence tag connector strategy). We selected homogeneously distributed seed points using an auxiliary high-resolution radiation hybrid map comprising 1,895 distinct positions. The high long-range continuity and low redundancy of the tiling path indicates that the sequence tag connector approach compares favourably with alternative mapping strategies.

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Autosomal dominant familial spastic paraplegia (AD-FSP) is a genetically heterogeneous neurodegenerative disorder characterized by progressive spasticity of the lower limbs. Three loci on chromosome 14q (SPG3), 2p (SPG4), and 15q (SPG6) were shown to be responsible for AD-FSP. Analysis of recombination events in three SPG3-linked families allowed us to narrow the critical interval from 9 to 5 cM. An ∼5-Mb YAC contig comprising 32 clones and 90 STSs was built from D14S301 to D14S991, encompassing this region of 14q21. Fifty-six ESTs assigned previously to this region with radiation hybrid (RH) panels Genebridge 4 and G3 were precisely localized on the YAC contig. The 90 STSs positioned on the contig were tested on the TNG RH panel to compare our YAC-based map with an RH map at a high level of resolution. Comparison between our map and the whole genome mapping data on this interval of chromosome 14q is discussed.

The approximately 10 kbp region encompassing nprB and argJ at 102" on the Bacillus subtilis chromosome was sequenced, revealing 12 ORFs, four known genes (argJ, argC, ipi and nprB) and two genes, yitV and yitS, whose products respectively display significant homology with L-gulono-y-lactone oxidase of rat and dihydrofolate reductase of Staphy/ococcus aureus. The data also indicated that nprB mapped to a different position than previously published.Keywords : Bacillus subtilis, genome sequencing, 102' region, argJ, nprB Within the European programme for sequencing the Bacillus subtilis genome, a 60 kbp DNA fragment located between nprB (95") and arg] (102") was originally assigned to our group. Since, as described in this paper, nprB was, in fact, found to be located approximately 8 kb from arg], the assignment was redefined and the region to be sequenced was extended from arg] (102") to addAB (89"). In this first communication, we describe our cloning and sequencing strategy and we report the sequencing and gene features of the 10336 bp region from nprB to arg].For this study, we employed a chromosome walking strategy using the integrative plasmid pDIA5304 (Glaser et al., 1993), into which we inserted a 1 kb fragment from arg] obtained from pUL710. This allowed us, after digestion of the chromosomal DNA with an appropriate restriction enzyme ( S a d ) , to pick up a fragment of more than 10 kbp adjacent to argJ. The recombinant plasmid, pOMG3001, was propagated in Escherichia coli TP611, a mutant strain (pcnB) which facilitates cloning of B. subtilis DNA by reducing the copy number of ColEl derivatives (Xu et al., 1993). The integrity of the clone was verified by Southern hybridization. For shotgun analysis, pOMG3001 was subjected to a nebulization procedure and 1-1-5 kb fragments were subcloned into pUCl8. After identification and selection by Southern hybridization, the fragments corresponding to the insert were sequenced. The sequencing reactions were performed using the Taq Dye Primer Cycle Sequencing kit and the Taq Dye Deoxy Terminator Cycle Sequencing kit, followed by analysis with a 373A DNA sequencer Abbreviation: SD, Shine-Dalgarno.The EMBL accession number for the nucleotide sequence reported in this paper is 279580.from Applied Biosystems. The sequences were compiled using the Autoassembler program (Perkin Elmer) and analysed for possible ORFs (GENEMARK). The predicted amino acid sequence of the products of each ORF was used to search for similarity to sequences reported previously in databases using BLAST/FASTA.From the sequence data, all six possible reading frames were screened with the DNA Strider program. Based on starting codons ATG, GTG or TTG preceded by a putative Shine-Dalgarno (SD) sequence, 12 putative ORFs were detected. The direction of transcription and translation of all except orf2 ( yitS) and orf. ( yitU), was, like the majority of genes on the B. subtilis chromosome, 0