Potassium and sodium transport in non-animal cells: the Trk/Ktr/HKT transporter family.

Bacterial Trk and Ktr, fungal Trk and plant HKT form a family of membrane transporters permeable to K(+) and/or Na(+) and characterized by a common structure probably derived from an ancestral K(+) channel subunit. This transporter family, specific of non-animal cells, displays a large diversity in terms of ionic permeability, affinity and energetic coupling (H(+)-K(+) or Na(+)-K(+) symport, K(+) or Na(+) uniport), which might reflect a high need for adaptation in organisms living in fluctuating or dilute environments. Trk/Ktr/HKT transporters are involved in diverse functions, from K(+) or Na(+) uptake to membrane potential control, adaptation to osmotic or salt stress, or Na(+) recirculation from shoots to roots in plants. Structural analyses of bacterial Ktr point to multimeric structures physically interacting with regulatory subunits. Elucidation of Trk/Ktr/HKT protein structures along with characterization of mutated transporters could highlight functional and evolutionary relationships between ion channels and transporters displaying channel-like features.

Characterization and repeat analysis of the compact genome of the freshwater pufferfish Tetraodon nigroviridis.

Tetraodon nigroviridis is a freshwater pufferfish 20-30 million years distant from Fugu rubripes. The genome of both tetraodontiforms is compact, mostly because intergenic and intronic sequences are reduced in size compared to other vertebrate genomes. The previously uncharacterized Tetraodon genome is described here together with a detailed analysis of its repeat content and organization. We report the sequencing of 46 megabases of bacterial artificial chromosome (BAC) end sequences, which represents a random DNA sample equivalent to 13% of the genome. The sequence and location of rRNA gene clusters, centromeric and subtelocentric satellite sequences have been determined. Minisatellites and microsatellites have been cataloged and notable differences were observed in comparison with microsatellites from Fugu. The genome contains homologies to all known families of transposable elements, including Ty3-gypsy, Ty1-copia, Line retrotransposons, DNA transposons, and retroviruses, although their overall abundance is <1%. This structural analysis is an important prerequisite to sequencing the Tetraodon genome.

Estimate of human gene number provided by genome-wide analysis using Tetraodon nigroviridis DNA sequence.

The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome.

Linkage analysis of candidate myelin genes in familial multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. A complex genetic etiology is thought to underlie susceptibility to this disease. The present study was designed to analyze whether differences in genes that encode myelin proteins influence susceptibility to MS. We performed linkage analysis of MS to markers in chromosomal regions that include the genes encoding myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMGP), and myelin oligodendrocyte glycoprotein (MOG) in a well-characterized population of 65 multiplex MS families consisting of 399 total individuals, 169 affected with MS and 102 affected sibpairs. Physical mapping data permitted placement of MAG and PLP genes on the Genethon genetic map; all other genes were mapped on the Genethon genetic map by linkage analysis. For each gene, at least one marker within the gene and/or two tightly linked flanking markers were analyzed. Marker data analysis employed a combination of genetic trait model-dependent (parametric) and model-independent linkage methods. Results indicate that MAG, MBP, OMGP, and PLP genes do not have a significant genetic effect on susceptibility to MS in this population. As MOG resides within the MHC, a potential role of the MOG gene could not be excluded.

A fine integrated map of the SPG4 locus excludes an expanded CAG repeat in chromosome 2p-linked autosomal dominant spastic paraplegia.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous disorder characterized by progressive spasticity of the lower limbs. A major locus (SPG4) causing AD-HSP in about 40% of the families was mapped to chromosome 2p. The analysis of six SPG4-linked AD-HSP families using the RED procedure previously showed the expansion of a CAG repeat in affected individuals. To identify the gene responsible for this form of HSP, we have constructed a 3.5-Mb YAC contig flanked by loci D2S400 and D2S367, have subcloned five of these YACs spanning the candidate region into cosmids, and screened these cosmid libraries for the presence of CAG repeat sequences. Four CAG repeats have been identified but none of them is expanded in 26 patients from 13 SPG4-linked AD-HSP families. A gene map comprising 21 transcripts was established using expressed sequence tags (ESTs) assigned previously to this region of 2p21-p22 with radiation hybrid panels GeneBridge 4 and G3. Full-length cDNAs corresponding to the 14 ESTs mapping to the SPG4 interval flanked by loci D2S352 and D2S2347 were isolated and sequenced. None contains a CAG repeat in its coding sequence. Finally, we have assembled a BAC contig composed of 37 clones that were also screened for the presence of CAG repeats; this failed to detect additional repeats to those identified on YACs.

Lamellar ichthyosis: further narrowing, physical and expression mapping of the chromosome 2 candidate locus.

Lamellar ichthyosis (LI) is an autosomal recessive genodermatosis which has been shown to be both clinically and genetically heterogeneous. Keratinocyte transglutaminase (or transglutaminase 1: TGM1) has been demonstrated to be the disease-causing gene in some families, whilst in others, a second unidentified LI gene was mapped to chromosome 2q33-35 (ICR2B locus). In this study, we present a physical map that encompasses the ICR2B locus, including the mapping of new microsatellite markers. Based on this new map, genotyping additional families highly suggests a reduction in size of the candidate interval. The final interval is covered by a single yeast artificial chromosome (937-H-3) which is 2.2Mb in length. Fine mapping of potential candidate transcripts was also focused on this region.

Domain-structured N1,N2-derivatized hydrazines as inhibitors of ribonucleoside diphosphate reductase: redox-cycling considerations.

Eight analogues of 1-[5-halogenosalicylidene]-2-[2'-pyridinoyl]hydrazine and -[2'-pyridyl]hydrazine, four of 1-[pyridoxylidene]-2-[2'-pyridinoyl]hydrazine, seven of 1-[pyridoxylidene]-2-[2'-pyridyl]hydrazine, and one each of 1, 2-bis[pyridoxylidene]diaminoethane and bis[pyridoxylidenehydrazino]phthalazine were synthesized. Their solutions in DMF were assayed for activity against the metalloenzyme ribonucleoside diphosphate reductase (RdR), prepared from a subcutaneously growing murine tumor (sarcoma 180) implanted in B6D2F3 male mice. The 14C-labeled CDP reductase was assayed by the modified method of Takeda and Weber, in which [14C]cytidine was separated from deoxycytidine by thin-layer chromatography (TLC) on cellulose foil. Distribution of radioactivity was assessed with an automatic TLC linear analyzer. Of the 31 compounds tested, 13 were essentially inactive, 7 were highly active against RdR, and the remaining 20 were slightly more active than hydroxyurea (used as a reference compound). The mechanism of inhibition is discussed in terms of three alternative pathways, initiated by sequestration of iron embedded in the R1 subunit of the metalloenzyme to form a C-centered chelate radical (via redox cycling). Alternatively, the latter could either reduce the tyrosyl radical or intercept radicals generated in the reduction process.

Quality assessment of whole genome mapping data in the refined familial spastic paraplegia interval on chromosome 14q.

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 approximately 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.

A physical map of 30,000 human genes.

A map of 30,181 human gene-based markers was assembled and integrated with the current genetic map by radiation hybrid mapping. The new gene map contains nearly twice as many genes as the previous release, includes most genes that encode proteins of known function, and is twofold to threefold more accurate than the previous version. A redesigned, more informative and functional World Wide Web site (www.ncbi.nlm.nih.gov/genemap) provides the mapping information and associated data and annotations. This resource constitutes an important infrastructure and tool for the study of complex genetic traits, the positional cloning of disease genes, the cross-referencing of mammalian genomes, and validated human transcribed sequences for large-scale studies of gene expression.

A transcriptional Map of the FMF region.

Familial Mediterranean fever (FMF) is a recessively inherited disorder characterized by attacks of fever and serositis, which affects primarily non-Ashkenazi Jews, Armenians, Turks, and Arabs. We present here a transcriptional map covering the FMF locus that we constructed in the course of the positional cloning of the gene responsible for this disease. This map was established from a contig constructed with YAC, BAC, and cosmid clones and covers about 500 kb of 16p13.3. It contains nine transcriptional units corresponding to known genes or to genes belonging to known gene families, 23 gene fragments characterized by partial sequences, and an endogenous retrovirus sequence. It thus considerably increases the number of genes in this interval and improves our knowledge concerning some of the genes or gene families present in this region. Data accumulated in this region were also used in a comparative study of different methods of exon detection.

Genetic linkage of Meleda disease to chromosome 8qter.

Meleda disease (mal de Meleda) MIM *248300 is an autosomal recessive disorder, clinically characterised by transgressive palmoplantar keratoderma, hyperhidrosis and perioral erythema. It was first described on the Adriatic island of Meleda, where it was relatively common. The prevalence in the general population is estimated to be 1 in 100,000. Linkage analysis of two large consanguineous families from Algeria, including 10 affected individuals, showed strong evidence for localisation of Meleda disease to chromosome 8qter with a maximum two-point lod score for D8S1751 of 8.21 at theta = 0. Analysis of homozygosity regions and recombination events places the gene in a region of at least 3 cM, telomeric to D8S1727. A common haplotype was observed in the two families, suggesting a founder effect.

Cloning of the genes encoding two murine and human cochlear unconventional type I myosins.

Several lines of evidence indicate a crucial role for unconventional myosins in the function of the sensory hair cells of the inner ear. We report here the characterization of the cDNAs encoding two unconventional type I myosins from a mouse cochlear cDNA library. The first cDNA encodes a putative protein named Myo1c, which is likely to be the murine orthologue of the bullfrog myosin I beta and which may be involved in the gating of the mechanotransduction channel of the sensory hair cells. This myosin belongs to the group of short-tailed myosins I, with its tail ending shortly after a polybasic, TH-1-like domain. The second cDNA encodes a novel type I myosin Myo1f which displays three regions: a head domain with the conserved ATP- and actin-binding sites, a neck domain with a single IQ motif, and a tail domain with the tripartite structure initially described in protozoan myosins I. The tail of Myo1f includes (1) a TH-1 region rich in basic residues, which may interact with anionic membrane phospholipids; (2) a TH-2 proline-rich region, expected to contain an ATP-insensitive actin-binding site; and (3) a SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that the genes encoding Myo1c and Myo1f display a widespread tissue expression in the adult mouse. Myo1c and Myo1f were mapped by in situ hybridization to the chromosomal regions 11D-11E and 17B-17C, respectively. The human orthologuous genes MYO1C and MYO1F were also characterized, and mapped to the human chromosomal regions 17p18 and 19p13.2-19p13.3, respectively.

A gene map of the human genome.

The human genome is thought to harbor 50,000 to 100,000 genes, of which about half have been sampled to date in the form of expressed sequence tags. An international consortium was organized to develop and map gene-based sequence tagged site markers on a set of two radiation hybrid panels and a yeast artificial chromosome library. More than 16,000 human genes have been mapped relative to a framework map that contains about 1000 polymorphic genetic markers. The gene map unifies the existing genetic and physical maps with the nucleotide and protein sequence databases in a fashion that should speed the discovery of genes underlying inherited human disease. The integrated resource is available through a site on the World Wide Web at http://www.ncbi.nlm.nih.gov/SCIENCE96/.

A radiation hybrid map of the human genome.

We have developed a panel of whole-genome radiation hybrids by fusing irradiated diploid human fibroblasts with recipient hamster cells. This panel of 168 cell lines has been typed with microsatellite markers of known genetic location. Of 711 AFM genetic markers 404 were selected to construct a robust framework map that spans all the autosomes and the X chromosome. To demonstrate the utility of the panel, 374 expressed sequence tags (ESTs) previously assigned to chromosomes 1, 2, 14 and 16 were localized on this map. All of these ESTs could be positioned by pairwise linkage to one of the framework markers with a LOD score of greater than 8. The whole genome radiation hybrid panel described here has been used as the starting material for the Genebridge4 panel that is being made widely available for genome mapping projects.

A comprehensive genetic map of the human genome based on 5,264 microsatellites.

The great increase in successful linkage studies in a number of higher eukaryotes during recent years has essentially resulted from major improvements in reference genetic linkage maps, which at present consist of short tandem repeat polymorphisms of simple sequences or microsatellites. We report here the last version of the Généthon human linkage map. This map consists of 5,264 short tandem (AC/TG)n repeat polymorphisms with a mean heterozygosity of 70%. The map spans a sex-averaged genetic distance of 3,699 cM and comprises 2,335 positions, of which 2,032 could be ordered with an odds ratio of at least 1,000:1 against alternative orders. The average interval size is 1.6 cM; 59% of the map is covered by intervals of 2 cM at most and 1% remains in intervals above 10 cM.

An STS-based map of the human genome.

A physical map has been constructed of the human genome containing 15,086 sequence-tagged sites (STSs), with an average spacing of 199 kilobases. The project involved assembly of a radiation hybrid map of the human genome containing 6193 loci and incorporated a genetic linkage map of the human genome containing 5264 loci. This information was combined with the results of STS-content screening of 10,850 loci against a yeast artificial chromosome library to produce an integrated map, anchored by the radiation hybrid and genetic maps. The map provides radiation hybrid coverage of 99 percent and physical coverage of 94 percent of the human genome. The map also represents an early step in an international project to generate a transcript map of the human genome, with more than 3235 expressed sequences localized. The STSs in the map provide a scaffold for initiating large-scale sequencing of the human genome.

Lenograstim prevents morbidity from intensive induction chemotherapy in the treatment of inflammatory breast cancer.

PURPOSE: To compare the efficacy and safety of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) versus its inert vehicle in patients with unilateral nonmetastatic inflammatory breast cancer treated with fluorouracil, epirubicin, and cyclophosphamide high-dose (FEC-HD) neoadjuvant chemotherapy. PATIENTS AND METHODS: One hundred twenty patients have been enrolled by nine French centers in this double-blind, parallel-group, vehicle-controlled study to compare at each cycle subcutaneous lenograstim (5 micrograms/kg/d) with placebo given from day 6 to day 15 after the induction chemotherapy (day 1 to day 4, fluorouracil 750 mg/m2 continuous intravenous [IV] infusion; day 2 to day 4, epirubicin 35 mg/m2 and cyclophosphamide 400 mg/m2 both IV push). Four cycles were planned every 3 weeks before locoregional treatment. Patients with febrile neutropenia remained blinded for the subsequent cycles. RESULTS: Lenograstim significantly reduced the duration of neutropenia at less than 0.5 x 10(9)/L and less than 1 x 10(9)/L to a median duration of 2 and 3 days, respectively, as compared with 5 and 7 days in the placebo group. This translated into a statistically significant reduced incidence of microbiologically documented infections, and a decreased need for rehospitalizations for infectious events and antibiotic use. Clinical objective tumor response rate observed after four cycles was 89.6% and 93%, respectively, in the placebo and treated groups. Mild transient bone and injection-site pain, myelemia, and hyperleukocytosis were the most frequently reported adverse events associated with lenograstim. CONCLUSION: Lenograstim is safe and effective to reduce morbidity associated with FEC-HD neoadjuvant chemotherapy in inflammatory breast cancer. Response rate is not affected.

The 1993-94 Généthon human genetic linkage map.

In 1992, we described a second-generation genetic linkage map of the human genome. Using 1,267 new microsatellite markers, we now present a new genetic linkage map containing a total of 2,066 (AC)n short tandem repeats, 60% of which show a heterozygosity of over 0.7. Statistical linkage analysis based on the genotyping of eight large CEPH families placed these markers in the 23 linkage groups. The map includes 1,266 intervals and spans a total distance of 3690 centiMorgans (cM). A total of 1,041 markers could be ordered with odds ratios greater than 1000:1. About 56% of this map is at a distance of 1 cM or less from one of its markers.

Assignment of 112 microsatellite markers to 23 chromosome 11 subregions delineated by somatic hybrids: comparison with the genetic map.

Using a panel of 25 somatic cell hybrids, we have regionally localized 112 microsatellite markers generated by Généthon and assigned to chromosome 11. A genetic map of 74 of them was produced using linkage analysis of the eight largest CEPH (Centre d'Etude du Polymorphisme Humain) families. They could be ordered on chromosome 11 with an average distance of 2.1 cM. The tight correlation observed between the genetic order and the physical assignment of these microsatellites reinforces the genetic map data. These newly localized markers identified by the PCR method using a standardized protocol represent useful tools for mapping YAC clones and establishing YAC contigs and for studying genetic diseases or cancers associated with specific genes and/or germinal/somatic rearrangements of chromosome 11.