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In Arabidopsis thaliana, root high-affinity nitrate (NO3–) uptake depends mainly on NRT2.1, 2.4, and 2.5, which are repressed by high NO3– supply at the transcript level. For NRT2.1, this regulation is due to the action of (i) feedback down-regulation by N metabolites and (ii) repression by NO3– itself mediated by the transceptor NRT1.1(NPF6.3). However, for NRT2.4 and NRT2.5, the signalling pathway(s) remain unknown as do the molecular elements involved. Here we show that unlike NRT2.1, NRT2.4 and NRT2.5 are not induced in an NO3– reductase mutant but are up-regulated following replacement of NO3– by ammonium (NH4+) as the N source. Moreover, increasing the NO3– concentration in a mixed nutrient solution with constant NH4+ concentration results in a gradual repression of NRT2.4 and NRT2.5, which is suppressed in an nrt1.1 mutant. This indicates that NRT2.4 and NRT2.5 are subjected to repression by NRT1.1-mediated NO3– sensing, and not to feedback repression by reduced N metabolites. We further show that key regulators of NRT2 transporters, such as HHO1, HRS1, PP2C, LBD39, BT1, and BT2, are also regulated by NRT1.1-mediated NO3– sensing, and that several of them are involved in NO3– repression of NRT2.1, NRT2.4, and NRT2.5. Finally, we provide evidence that it is the phosphorylated form of NRT1.1 at the T101 residue, which is most active in triggering the NRT1.1-mediated NO3– regulation of all these genes. Altogether, these data led us to propose a regulatory model for high-affinity NO3– uptake in Arabidopsis, highlighting several NO3– transduction cascades downstream of the phosphorylated form of the NRT1.1 transceptor.

Potassium (K+) plays crucial roles in many physiological, molecular and cellular processes in plants. Direct uptake of this nutrient by root cells has been extensively investigated, however, indirect uptake of K+mediated by the interactions of the roots with fungi in the frame of a mutualistic symbiosis, also called mycorrhizal nutrient uptake pathway, is much less known. We identified an ion channel in the arbuscular mycorrhizal (AM) fungusRhizophagus irregularis. This channel exhibits the canonical features of Shaker-like channel shared in other living kingdoms and is named RiSKC3. Transcriptionally expressed in hyphae and in arbuscules of colonized rice roots, RiSKC3 has been shown to be located in the plasma membrane. Voltage-clamp functional characterization inXenopusoocytes revealed that RiSKC3 is endowed with outwardly-rectifying voltage-gated activity with a high selectivity for K+over sodium ions. RiSKC3 may have a role in the AM K+pathway for rice nutrition in normal and salt stress conditions. The current working model proposes that K+ions taken up by peripheral hyphae ofR. irregularisare secreted towards the host root into periarbuscular space by RiSKC3.Significance StatementMutualistic symbiosis with arbuscular mycorrhizal (AM) fungi are beneficial for about 80% of land plants thanks to an exchange of nutrients. The AM pathway responsible for potassium (K+) nutrition of the plant is not known. Here we uncovered a key step of this phenomenon, by functionally characterizing the first transport system in the AM fungusRhizophagus irregularis, and we univocally demonstrated that RiSKC3 is an K+outwardly-rectifying voltage-gated Shaker-like channel.

International audience; Diazotrophic bacteria isolated from the rhizosphere of a wild wheat ancestor, grown from its refuge area in the Fertile Crescent, were found to be efficient Plant Growth-Promoting Rhizobacteria (PGPR), upon interaction with an elite wheat cultivar. In nitrogen-starved plants, they increased the amount of nitrogen in the seed crop (per plant) by about twofold. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar, and to analyze the exo-metabolomes and exo-proteomes. Altered root development was observed, with distinct responses depending on the strain, for instance, with respect to root hair development. A first conclusion from these results is that the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have undergone systematic deep reprogramming during domestication. Exo-metabolome analysis revealed a complex set of secondary metabolites, including nutrient ion chelators, cyclopeptides that could act as phytohormone mimetics, and quorum sensing molecules having inter-kingdom signaling properties. The exo-proteome-comprised strain-specific enzymes, and structural proteins belonging to outer-membrane vesicles, are likely to sequester metabolites in their lumen. Thus, the methodological processes we have developed to collect and analyze bacterial exudates have revealed that PGPRs constitutively exude a highly complex set of metabolites; this is likely to allow numerous mechanisms to simultaneously contribute to plant growth promotion, and thereby to also broaden the spectra of plant genotypes (species and accessions/cultivars) with which beneficial interactions can occur.

InArabidopsis thaliana, root high-affinity nitrate (NO3-) uptake depends mainly on NRT2.1, 2.4 and 2.5, which are repressed by high NO3-supply at the transcript level. ForNRT2.1, this regulation is due to the action of (i) feedback downregulation by N metabolites and (ii) repression by NO3-itself mediated by the transceptor NRT1.1(NPF6.3). However, forNRT2.4andNRT2.5the signaling pathway(s) remain unknown along with the molecular elements involved. Here we show that unlikeNRT2.1, NRT2.4andNRT2.5are not induced in a NO3-reductase mutant but are strongly upregulated following replacement of NO3-by ammonium (NH4+) as the N source. Moreover, increasing NO3-concentration in a mixed nutrient solution with constant NH4+concentration results in a gradual repression ofNRT2.4andNRT2.5, which is suppressed in anrt1.1mutant. This indicates thatNRT2.4andNRT2.5are subjected to repression by NRT1.1-mediated NO3-sensing, and not to feedback repression by reduced N metabolites. We further show that key regulators of NRT2s transporters, such as HHO1, HRS1, PP2C, LBD39, BT1 and BT2, are also regulated by NRT1.1-mediated NO3-sensing, and that several are involved in NO3-repression ofNRT2.1, 2.4and2.5. Finally, we provide evidence that it is the phosphorylated form of NRT1.1 at the T101 residue, which is most active in triggering the NRT1.1-mediated NO3-regulation of all these genes. Altogether, these data led to propose a regulatory model for high-affinity NO3-uptake inArabidopsis, highlighting several NO3-transduction cascades downstream the phosphorylated form of the NRT1.1 transceptor.One sentence summaryCharacterisation and identification of molecular elements involved in the signaling pathways repressing NRT2s transporters and root nitrate uptake in response to nitrate.

LCMS raw data from: Two diazotrophic bacteria, BPMP-PU-28 and BPMP-EL-40, isolated from the rhizosphere of a wild wheat ancestor (T. t. dicoccoides) grown in soil from its refuge area in the Fertile Crescent or from south of France, respectively, were found to be efficient Plant Growth Promoting Rhizobacteria (PGPR) upon interaction with an elite wheat cultivar (Anvergur), providing about 50% of the seed nitrogen content in plants grown under low assimilable nitrogen availability. A bacterial growth medium was developed to investigate the effects of bacterial exudates on root development in the elite cultivar and to analyze the exo-metabolomes and exo-proteomes of the two strains. Altered root development was observed, with distinctive responses according to the strain, BPMP-PU-28 also inducing a strong increase in root hair length and density. Thus, the ability of wheat to establish effective beneficial interactions with PGPRs does not appear to have been profoundly reprogrammed during domestication and breeding. Exo-metabolome analysis revealed a complex set of secondary metabolites including fatty acids potentially involved in volatile organic compounds (VOCs) metabolism, cyclopeptides that could act as phytohomone mimetics, quorum sensing molecules having inter-kingdom signaling properties and nutrient ion chelators. The exo-proteome comprised a set of strain specific enzymatic activities (e.g., proteases) and structural proteins belonging to outer-membrane vesicles likely to sequester metabolites, peptides and enzymes in their lumen. PGPR constitutively exude rich and complex metabolomes and proteomes, in absence of partner roots, which could allow numerous mechanisms to simultaneously contribute to plant growth promotion and thereby broaden the range of responsive plant species.

K+ uptake to root conducting cells is a prerequisite for its release to the xylem vessels and long-distance delivery to the aerial parts, though the molecular mechanism underpinning such process remains insufficiently understood. Here we report the discovery in rice root of an essential component involved in loading of K+ to the stelar tissues encoded by an unusual short Shaker K+ channel OsK2.2. OsK2.2 represents a novel type of short Shaker channels lacking the sequences immediately downstream of the putative cyclic nucleotide binding domain that present to the C-termini of all plant Shaker channels reported to date. In silico analysis suggests that such short Shaker channels are monocot-specific and especially found in the Poaceae species. Functional assessment in Xenopus oocytes reveals that OsK2.2 uses such innate C-terminal shortness as an intrinsic strategy for maintaining large capacity of K+ uptake to the cells. OsK2.2 predominantly localizes to the phloem and xylem peripheral cells of rice root vasculature. Disruption of OsK2.2 function causes approximately 30% lower K+ in both phloem and xylem sap and reduces K+ accumulation to the shoots. OsK2.2 strongly contributes to the grain yield of rice under field conditions. The essential contribution of such particular type of inward Shaker channel, operating in stelar tissues, to K+ transport between roots and shoots and to grain yield, here evidenced in rice, may be a common trait amongst Poaceae species.

Polycomb-group (PcG) proteins are major chromatin complexes that regulate gene expression, mainly described as repressors keeping genes in a transcriptionally silent state during development. Recent studies have nonetheless suggested that PcG proteins might have additional functions, including targeting active genes or acting independently of gene expression regulation. However, the reasons for the implication of PcG proteins and their associated chromatin marks on active genes are still largely unknown. Here, we report that combining mutations for CURLY LEAF (CLF) and LIKE HETEROCHROMATIN PROTEIN1 (LHP1), two Arabidopsis PcG proteins, results in deregulation of expression of active genes that are targeted by PcG proteins or enriched in associated chromatin marks. We show that this deregulation is associated with accumulation of small RNAs corresponding to massive degradation of active gene transcripts. We demonstrate that transcriptionally active genes and especially those targeted by PcG proteins are prone to RNA degradation, even though deregulation of RNA degradation following the loss of function of PcG proteins is not likely to be mediated by a PcG protein-mediated chromatin environment. Therefore, we conclude that PcG protein function is essential to maintain an accurate level of RNA degradation to ensure accurate gene expression.

Potassium (K+) acquisition, translocation and cellular homeostasis are mediated by various membrane transport systems in all organisms. We identified and described an ion channel in the ectomycorrhizal fungus Hebeloma cylindrosporum (HcSKC) that harbors features of animal voltage-dependent Shaker-like K+ channels, and investigated its role in both free-living hyphae and symbiotic conditions. RNAi lines affected in the expression of HcSKC were produced and used for in vitro mycorrhizal assays with the maritime pine as host plant, under standard or low K+ conditions. The adaptation of H. cylindrosporum to the downregulation of HcSKC was analyzed by qRT-PCR analyses for other K+-related transport proteins: the transporters HcTrk1, HcTrk2, and HcHAK, and the ion channels HcTOK1, HcTOK2.1, and HcTOK2.2. Downregulated HcSKC transformants displayed greater K+ contents at standard K+ only. In such conditions, plants inoculated with these transgenic lines were impaired in K+ nutrition. Taken together, these results support the hypothesis that the reduced expression of HcSKC modifies the pool of fungal K+ available for the plant and/or affects its symbiotic transfer to the roots. Our study reveals that the maintenance of K+ transport in H. cylindrosporum, through the regulation of HcSKC expression, is required for the K+ nutrition of the host plant.

International audience; In Arabidopsis thaliana, NRT2.1 codes for a main component of the root nitrate high-affinity transport system. Previous studies revealed that post-translational regulation of NRT2.1 plays an important role in the control of root nitrate uptake and that one mechanism could correspond to NRT2.1 C-terminus processing. To further investigate this hypothesis, we produced transgenic plants with truncated forms of NRT2.1. It revealed an essential sequence for NRT2.1 activity, located between the residues 494-513. Using a phospho-proteomic approach, we found that this sequence contains one phosphorylation site, at serine 501, which can inactivate NRT2.1 function when mimicking the constitutive phosphorylation of this residue in transgenic plants. This phenotype could neither be explained by changes in abundance of NRT2.1 and NAR2.1, a partner protein of NRT2.1, nor by a lack of interaction between these two proteins. Finally, the relative level of serine 501 phosphorylation was found to be increased by ammonium nitrate in wild-type plants, leading to the inactivation of NRT2.1 and to a decrease in high affinity nitrate transport into roots. Altogether, these observations reveal a new and essential mechanism for the regulation of NRT2.1 activity.

InArabidopsis thaliana,NRT2.1codes for a main component of the root nitrate high-affinity transport system. Previous studies revealed that post-translational regulation of NRT2.1 plays an important role in the control of root nitrate uptake and that one mechanism could correspond to NRT2.1 C-terminus processing. To further investigate this hypothesis, we produced transgenic plants with truncated forms of NRT2.1. It revealed an essential sequence for NRT2.1 activity, located between the residues 494-513. Using a phospho-proteomic approach, we found that this sequence contains one phosphorylation site, at serine 501, which can inactivate NRT2.1 function when mimicking the constitutive phosphorylation of this residue in transgenic plants. This phenotype could neither be explained by changes in abundance of NRT2.1 and NAR2.1, a partner protein of NRT2.1, nor by a lack of interaction between these two proteins. Finally, the relative level of serine 501 phosphorylation was found to be modulated by nitrate in wildtype plants. Altogether, these observations allowed us to propose a model for a new and essential mechanism for the regulation of NRT2.1 activity.

In Arabidopsis thaliana, the High-Affinity Transport System (HATS) for root NO3- uptake depends mainly on four NRT2 transporters, namely NRT2.1, NRT2.2, NRT2.4 and NRT2.5. The HATS is the target of many regulations to coordinate nitrogen (N) acquisition with the N status of the plant and with carbon (C) assimilation through photosynthesis. At the molecular level, C and N signaling pathways have been shown to control gene expression of the NRT2 transporters. Although several regulators of these transporters have been identified in response to either N or C signals, the response of NRT2 genes expression to the interaction of these signals has never been specifically investigated and the underlying molecular mechanisms remain largely unknown. To address this question we used an original systems biology approach to model a regulatory gene network targeting NRT2.1, NRT2.2, NRT2.4 and NRT2.5 in response to N/C signals. Our systems analysis of the data highlighted the potential role of three putative transcription factors, TGA3, MYC1 and bHLH093. Functional analysis of mutants combined with yeast one hybrid experiments confirmed that all 3 transcription factors are regulators of NRT2.4 or NRT2.5 in response to N or C signals.One sentence summaryIdentification of three transcription factors involved in the regulation of NRT2s transporters using a systems biology approach and NRT2.1 as target gene in response to combinations of N/C treatments

Control of K+ and Na+ transport plays a central role in plant adaptation to salinity. In the halophyte Hordeum maritimum, we have characterized a transporter gene, named HmHKT2;1, whose homolog HvHKT2;1 in cultivated barley, Hordeum vulgare, was known to give rise to increased salt tolerance when overexpressed. The encoded protein is strictly identical in two H. maritimum ecotypes, from two biotopes (Tunisian sebkhas) affected by different levels of salinity. These two ecotypes were found to display distinctive responses to salt stress in terms of biomass production, Na+ contents, K+ contents and K+ absorption efficiency. Electrophysiological analysis of HmHKT2;1 in Xenopus oocytes revealed distinctive properties when compared with HvHKT2;1 and other transporters from the same group, especially a much higher affinity for both Na+ and K+, and an Na+–K+ symporter behavior in a very broad range of Na+ and K+ concentrations, due to reduced K+ blockage of the transport pathway. Domain swapping experiments identified the region including the fifth transmembrane segment and the adjacent extracellular loop as playing a major role in the determination of the affinity for Na+ and the level of K+ blockage in these HKT2;1 transporters. The analysis (quantitative reverse transcription-PCR; qRT-PCR) of HmHKT2;1 expression in the two ecotypes submitted to saline conditions revealed that the levels of HmHKT2;1 transcripts were maintained constant in the most salt-tolerant ecotype whereas they decreased in the less tolerant one. Both the unique functional properties of HmHKT2;1 and the regulation of the expression of the encoding gene could contribute to H. maritimum adaptation to salinity.

PRC2 is a major regulator of gene expression in eukaryotes. It catalyzes the repressive chromatin mark H3K27me3, which leads to very low expression of target genes. NRT2.1, which encodes a key root nitrate transporter in Arabidopsis, is targeted by H3K27me3, but the function of PRC2 on NRT2.1 remains unclear. Here, we demonstrate that PRC2 directly targets and down-regulates NRT2.1, but in a context of very high transcription, in nutritional conditions where this gene is one of the most highly expressed genes in the transcriptome. Indeed, the mutation of CLF, which encodes a PRC2 subunit, leads to a loss of H3K27me3 at NRT2.1 and results, exclusively under permissive conditions for NRT2.1, in a further increase in NRT2.1 expression, and specifically in tissues where NRT2.1 is normally expressed. Therefore, our data indicates that PRC2 tempers the hyperactivity of NRT2.1 in a context of very strong transcription. This reveals an original function of PRC2 in the control of the expression of a highly expressed gene in Arabidopsis.

Plant defensins are recognized for their antifungal properties. However, a few type 1 defensins (PDF1s) were identified for their cellular zinc (Zn) tolerance properties after a study of the metal extremophile Arabidopsis halleri. In order to investigate whether different paralogues would display specialized functions, the A. halleri PDF1 family was characterized at the functional and genomic levels. Eleven PDF1s were isolated from A. halleri. Their ability to provide Zn tolerance in yeast cells, their activity against Fusarium oxysporum f. sp. melonii, and their level of expression in planta were compared with those of the seven A. thaliana PDF1s. The genomic organization of the PDF1 family was comparatively analysed within the Arabidopsis genus. AhPDF1s and AtPDF1s were able to confer Zn tolerance and AhPDF1s also displayed antifungal activity. PDF1 transcripts were constitutively more abundant in A. halleri than in A. thaliana. Within the Arabidopsis genus, the PDF1 family is evolutionarily dynamic, in terms of gain and loss of gene copy. Arabidopsis halleri PDF1s display no superior abilities to provide Zn tolerance. A constitutive increase in AhPDF1 transcript accumulation is proposed to be an evolutionary innovation co-opting the promiscuous PDF1 protein for its contribution to Zn tolerance in A. halleri.

International audience; Root hairs are involved in water and nutrient uptake, and thereby in plant autotrophy. In legumes, they also play a crucial role in establishment of rhizobial symbiosis. To obtain a holistic view of Medicago truncatula genes expressed in root hairs and of their regulation during the first hours of the engagement in rhizobial symbiotic interaction, a high throughput RNA sequencing on isolated root hairs from roots challenged or not with lipochitooligosaccharides Nod factors (NF) for 4 or 20 h was carried out. This provided a repertoire of genes displaying expression in root hairs, responding or not to NF, and specific or not to legumes. In analyzing the transcriptome dataset, special attention was paid to pumps, transporters, or channels active at the plasma membrane, to other proteins likely to play a role in nutrient ion uptake, NF electrical and calcium signaling, control of the redox status or the dynamic reprogramming of root hair transcriptome induced by NF treatment, and to the identification of papilionoid legume-specific genes expressed in root hairs. About 10% of the root hair expressed genes were significantly up- or down-regulated by NF treatment, suggesting their involvement in remodeling plant functions to allow establishment of the symbiotic relationship. For instance, NF-induced changes in expression of genes encoding plasma membrane transport systems or disease response proteins indicate that root hairs reduce their involvement in nutrient ion absorption and adapt their immune system in order to engage in the symbiotic interaction. It also appears that the redox status of root hair cells is tuned in response to NF perception. In addition, 1176 genes that could be considered as "papilionoid legume-specific" were identified in the M. truncatula root hair transcriptome, from which 141 were found to possess an ortholog in every of the six legume genomes that we considered, suggesting their involvement in essential functions specific to legumes. This transcriptome provides a valuable resource to investigate root hair biology in legumes and the roles that these cells play in rhizobial symbiosis establishment. These results could also contribute to the long-term objective of transferring this symbiotic capacity to non-legume plants.

International audience; Revealing the over-represented functions in a particular gene list is a particular important post-genomic challenge. Genome-wide investigations generate gene lists from which meaningful information has to be extracted. This information is key to orient in planta investigations and to define the biological significance of genome-wide reprogramming. Here we describe the development and use of the GeneCloud algorithm, which retrieves the over-represented semantic terms in the description of a gene list.

International audience; Cloning and characterizations of plant K(+) transport systems aside from Arabidopsis have been increasing over the past decade, favored by the availability of more and more plant genome sequences. Information now available enables the comparison of some of these systems between species. In this review, we focus on three families of plant K(+) transport systems that are active at the plasma membrane: the Shaker K(+) channel family, comprised of voltage-gated channels that dominate the plasma membrane conductance to K(+) in most environmental conditions, and two families of transporters, the HAK/KUP/KT K(+) transporter family, which includes some high-affinity transporters, and the HKT K(+) and/or Na(+) transporter family, in which K(+)-permeable members seem to be present in monocots only. The three families are briefly described, giving insights into the structure of their members and on functional properties and their roles in Arabidopsis or rice. The structure of the three families is then compared between plant species through phylogenic analyses. Within clusters of ortologues/paralogues, similarities and differences in terms of expression pattern, functional properties and, when known, regulatory interacting partners, are highlighted. The question of the physiological significance of highlighted differences is also addressed.

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.

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.

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) an SH-3 domain found in a variety of cytoskeletal and signaling proteins. Northern blot analysis indicated that themore » 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 17p13 and 19p13.2- 19p1.3.3, respectively. 45 refs., 5 figs., 2 tabs.« less

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/.

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.

Zinc (Zn) is essential for the optimal growth of plants but is toxic if present in excess, so Zn homeostasis needs to be finely tuned. Understanding Zn homeostasis mechanisms in plants will help in the development of innovative approaches for the phytoremediation of Zn-contaminated sites. In this study, Zn tolerance quantitative trait loci (QTL) were identified by analyzing differences in the Bay-0 and Shahdara accessions of Arabidopsis thaliana. Fine-scale mapping showed that a variant of the Fe homeostasis-related FERRIC REDUCTASE DEFECTIVE3 (FRD3) gene, which encodes a multidrug and toxin efflux (MATE) transporter, is responsible for reduced Zn tolerance in A. thaliana. Allelic variation in FRD3 revealed which amino acids are necessary for FRD3 function. In addition, the results of allele-specific expression assays in F1 individuals provide evidence for the existence of at least one putative metal-responsive cis-regulatory element. Our results suggest that FRD3 works as a multimer and is involved in loading Zn into xylem. Cross-homeostasis between Fe and Zn therefore appears to be important for Zn tolerance in A. thaliana with FRD3 acting as an essential regulator., Author Summary Plants are adapted to soils in which the amounts of different nutrients vary widely, like Zn-deficient or Zn-contaminated soils. Exploring the molecular bases of plant adaptation to Zn-contaminated soils is important in determining strategies for phytoremediation. Here, we describe the mapping and characterization of a QTL for Zn tolerance in A. thaliana that underlies the natural variation of the root response to excess Zn. This physiological variation is controlled by different alleles of the AtFRD3 gene, which codes for a citrate transporter that uploads citrate into the xylem sap, hence playing a role in Fe homeostasis. In the Zn-sensitive accession Shahdara, the expression of AtFRD3 is drastically reduced and the protein encoded is unable to efflux citrate in vitro. Less Fe and Zn are found in Shahdara root exudates, and less Fe and Zn are translocated from root to shoot when Zn is in excess. We deduce that a fine-tuned Fe and Zn homeostasis is crucial for Zn tolerance in A. thaliana. Finally, as a range of alleles were identified, some rare, it was possible to define a sequence motif that is a putative metal-responsive cis-element and demonstrate that two amino acids are essential for the function of the FRD3 transporter.

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.

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.

present address : T. Sahr : UP Biologie des Bactéries Intracellulaires, Institut Pasteur, Paris, France; T. Adam : UMR Plante Microbe Environnement, Dijon, France; International audience; Methylation of biologically active molecules is achieved by methyltransferases (MTases). MTases can act on proteins through N- or O-carboxylmethylation reactions. Methylation of lysine and glutamic acid residues was recently described on the N-terminal tail of AtPIP2;1, a plasma membrane aquaporin of plants. In this study, we combine a bioinformatic and a biochemical screen and identify two MTases of Arabidopsis thaliana, SDG7 (At2g44150) and OMTF3 (At3g61990), as acting on the N-terminal tail of AtPIP2;1, at Lys3 and Glu6, respectively. Confocal microscopy imaging showed the two enzymes to be associated with the endoplasmic reticulum. An in vitro assay using various AtPIP2;1 N-terminal peptides as a bait allowed characterization of the enzymatic properties of recombinant SDG7 and OMTF3. The two enzymes showed minimal apparent K(m) values for their substrates, S-adenosylmethionine and peptide, in the range of 5-8 and 2-9 M, respectively. SDG7 was shown to almost exclusively mono- or di-methylate Lys3. In contrast, OMTF3 specifically methylated Glu6, this methylation being dependent on the methylation profile of the neighboring Lys3 residue. In conclusion, this study allows the characterization of the first MTases able to methylate plant transmembrane proteins and provides the first identification of a glutamate-MTase in eukaryotes.

International audience; 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.

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers AB061311 (OsHKT2;1 mRNA), AJ491816 (OsHKT1;1 mRNA), and AJ491818 (OsHKT1;3 mRNA).; International audience; Plant growth under low K(+) availability or salt stress requires tight control of K(+) and Na(+) uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K(+) Transporters), permeable either to K(+) and Na(+) or to Na(+) only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na(+) transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na(+) only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na(+)-K(+) symport, Na(+) uniport, or inhibited states, depending on external Na(+) and K(+) concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K(+) and Na(+) accumulation in monocots.

Animal cytosolic ACO (aconitase) and bacteria ACO are able to switch to RNA-binding proteins [IRPs (iron-regulatory proteins)], thereby playing a key role in the regulation of iron homoeostasis. In the model plant Arabidopsis thaliana, we have identified three IRP1 homologues, named ACO1–3. To determine whether or not they may encode functional IRP proteins and regulate iron homoeostasis in plants, we have isolated loss-of-function mutants in the three genes. The aco1-1 and aco3-1 mutants show a clear decrease in cytosolic ACO activity. However, none of the mutants is affected in respect of the accumulation of the ferritin transcript or protein in response to iron excess. cis-acting elements potentially able to bind to the IRP have been searched for in silico in the Arabidopsis genome. They appear to be very rare sequences, found in the 5′-UTR (5′-untranslated region) or 3′-UTR of a few genes unrelated to iron metabolism. They are therefore unlikely to play a functional role in the regulation of iron homoeostasis. Taken together, our results demonstrate that, in plants, the cytosolic ACO is not converted into an IRP and does not regulate iron homoeostasis. In contrast with animals, the RNA binding activity of plant ACO, if any, would be more likely to be attributable to a structural element, rather than to a canonical sequence.

This is the first report of a systematic study of genes expressed by means of expressed sequence tag (EST) analysis in oil palm, a species of the Arecales order, a phylogenetically key clade of monocotyledons that is not widely represented in the sequence databases. Five different cDNA libraries were generated from male and female inflorescences, shoot apices and zygotic embryos and unidirectional systematic sequencing was performed. A total of 2411 valid EST sequences were thus obtained. Cluster analysis enabled the identification of 209 groups of related sequences and 1874 singletons. Putative functions were assigned to 1252 of the set of 2083 non-redundant ESTs obtained. The EST database described here is a first step towards gene discovery and cDNA array-based expression analysis in oil palm. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

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 Genethon 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.

Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3 - or NH4NO3 as N source.

Summary • In an attempt to advance the understanding of ectomycorrhizal symbiosis, which improves mineral nutrition and resistance to abiotic and biotic stresses of most woody plants from temperate and boreal regions, we produced a cDNA library of the ectomycorrhizal fungus Hebeloma cylindrosporum in a yeast expression vector. The library was used for functional complementation cloning and for sequencing of 4036 expressed sequence tags (ESTs). • Expression of the library in a yeast ade2 and trp1 mutant strain allowed cloning of two Hebeloma genes, HcADE2 and HcTRP1 by functional complementation. This experiment highlighted both the efficiency of the complementation strategy and the quality of the library. • All sequence data available on Hebeloma (4596) were analysed and contiged. The whole EST resource is accessible via a web interface, which allows searching and navigation through clusters and tentative contigs (TCs). • Analysis of the database revealed a low level of counterparts already identified in other ectomycorrhizal fungi. For example, it allowed identification of a large set of membrane transporters, such as phosphate, potassium, sulphate or micronutrient transporters. Thus, this EST resource provides a valuable tool for research on ectomycorrhiza and symbiotic interactions.

The human genome is in the process of being completely sequenced, and an attempt is being made in parallel to systematically identify functionally relevant sequences. Current gene identification methods are based on software predictions or comparisons with expressed sequence tags and still lack accuracy and completeness. Comparisons between the human genomic sequence and the complete sequence of another vertebrate should be a useful complement to rapidly and accurately reveal regions of functional interest. Indeed, two vertebrate genomes that are evolutionarily distant should only show strong conservation of sequences of functional importance (protein coding regions; tRNA; rRNA) while other segments submitted to random mutations will show much less similarity. It has been amply demonstrated that the genomic sequence of a tetraodontiform such as Fugu rubripes is a powerful yet efficient tool to reveal such coding regions (Elgar 1996; Elgar et al. 1999). We have chosen Tetraodon nigroviridis to develop such comparative analyses on a genome scale (Roest Crollius et al. 2000) because of its widespread availability and trivial and inexpensive maintenance in the laboratory. It was also reasoned that studying a species related to Fugu but distant by 20–30 million years (Crnogorac-Jurcevic et al. 1997) would enable the identification of functionally important sequences that appeared after the human/teleostean divergence. We have initiated a random sequencing approach of the Tetraodon genome based on bacterial artificial chromosome (BAC) end templates and have generated 46 Mb of DNA or 13% of the genome. The average read length is 1 kb, which contributes to making this approach a very fast and cost-effective method of genome scanning. BAC end sequencing provides an added advantage by physically linking two sequences over a relatively short distance (75–200 kb), allowing direct comparisons between linked sequences in Tetraodon and other genomes. It also represents an ideal genomic resource for long-range physical mapping, as well as an STC resource (Mahairas et al. 1999) to assist shotgun sequencing in specific regions. This Tetraodon genome sample was exploited in combination with fluorescence in situ hybridization experiments, to decipher the organization of repeat sequences. This study serves several purposes. First, repeat sequences occur naturally in multiple copies in the genome either in tandem or in dispersed distribution, and therefore can seriously hamper clustering studies or sequence assemblies. In any case such sequences must be identified and eliminated, generally by masking, during sequence comparison procedures to avoid the formation of unwanted repeat alignments. Second, major satellite and rRNA gene clusters form heterochromatic blocks in the genome that are easily recognizable cytogenetically. These blocks can serve as useful markers when the chromosome formula is difficult to establish, as is the case in pufferfishes (Barat and Khuda-Bukhsh 1984; Miyaki et al. 1995; Grutzner et al. 2000; Fischer et al. 2000). Finally, repeat sequences are important elements of the genome from an evolutionary point of view (Charlesworth et al. 1994). They can contribute an important fraction of the DNA in a genome, between 50% in some mammalian species. In addition, repeat sequences and in particular transposable elements, can influence chromosome evolution by promoting chromosome breakage, deletions, inversions and amplifications (Lim and Simmons 1994; Dimitri et al. 1997; O'Neill et al. 1998). Transposable elements and tandem repeats are closely associated in heterochromatic regions of the genomes of many distant eukaryotes such as Drosophila (Pimpinelli et al. 1995) and plants (Presting et al. 1998), a situation that further supports the structural role of such repeats in genome evolution (Dimitri and Junakovic 1999). It is therefore of particular interest to investigate repeat distribution in Tetraodon considering its unusual evolution which positions it today as the smallest known vertebrate genome. We have identified the major satellite sequences, which are localized in the centromeres and acrocentric arms. The complete sequence of rRNA genes has been determined and their cluster localized on a small heteromorphic chromosome. The detection of minisatellite sequences essentially reveals their paucity in the genome. A comprehensive cataloguing of microsatellites compared with Fugu, shows that this genome is particularly rich in polyA stretches. We have found homologies to transposable elements (TEs) belonging to all major families, although their overall abundance is low compared to other eukaryotes. Globally, the genome contains 6.17% of repeated sequence. Taken together, these results represent a structural basis on which new studies focused on genome organization, evolution, and coding potential can be initiated.

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.

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 (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 2.2 Mb in length. Fine mapping of potential candidate transcripts was also focused on this region.

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 prevalance 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 Θ = 0. Analysis of homozygosity regions and recombination events places the 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.

Familial spastic paraplegia (FSP) is a heterogeneous group of degenerative disorders of the central motor system characterized by progressive spasticity of the lower limbs. FSP has been classified (Sutherland 1975) according to the mode of inheritance and whether spasticity occurs in isolation (pure FSP) or with additional symptoms (complicated FSP). In pure FSP, inheritance is most commonly autosomal dominant (AD-FSP) although both pure and complicated FSP may be transmitted as autosomal dominant, autosomal recessive, or X chromosome-linked traits. Pure AD-FSP is genetically heterogeneous and three AD-FSP-causing loci have been identified to date: SPG3 on chromosome 14q (Hazan et al. 1993; Gispert et al. 1995), SPG4 on chromosome 2p (Hazan et al. 1994; Hentati et al. 1994), and SPG6 on chromosome 15q (Fink et al. 1995). Known AD-FSP loci have been excluded in ∼50% of AD-FSP families (Hereditary Spastic Paraplegia Working Group 1996), providing evidence for the existence of at least a fourth locus. Linkage to chromosome 2p has been observed in the majority of the AD-FSP pedigrees analyzed so far (Hazan et al. 1994; Dube et al. 1995; Lennon et al. 1995; Hereditary Spastic Paraplegia Working Group 1996), whereas linkage to chromosome 15q has only been detected in one large North American kindred (Fink et al. 1995). Linkage to chromosome 14q has been reported in five AD-FSP pedigrees, including one French (Hazan et al. 1993), one German (Gispert et al. 1995), one Tibetan (Huang et al. 1997), and two North American (Hentati et al. 1994; Lennon et al. 1995) families. Recombinant analysis and linkage data first placed SPG3 within a 15-cM interval between markers D14S266 and D14S66 (Hazan et al. 1993). The SPG3 candidate region was then reduced to a 7-cM interval flanked by loci D14S288 and D14S281 (Gispert et al. 1995). The distance between D14S288 and D14S281 was then re-evaluated at 9 cM in the last version of the Genethon map (Dib et al. 1996). Here, we report linkage to chromosome 14q in an additional French family and present the refined genetic map of the SPG3 candidate interval using microsatellite markers from a published set (Dib et al. 1996). Subsequent studies of recombination events in two pedigrees from France and one from Germany allowed us to narrow the SPG3 region to 5 cM between markers D14S259 and D14S1018. As a first step toward the isolation of one of the genes responsible for AD-FSP, we constructed an ∼5-Mb YAC contig from D14S301 to D14S991 consisting of 32 clones. To evaluate the whole genome mapping data in this particular region of 14q21, our physical map was compared with the Whitehead/MIT contig WC14.1 (Hudson et al. 1995), which revealed some discrepancies in both STS order and YAC/STS content. To establish a transcript map, 104 ESTs that had been localized within or near the SPG3 interval (Schuler et al. 1996) with two radiation hybrid (RH) panels, Genebridge 4 (Gyapay et al. 1996) and G3 (Stewart et al. 1997), were tested by PCR on the 32 YAC clones of the contig: 56 ESTs including 10 known genes were mapped to the YAC contig between D14S301 and D14S991; 24 of them belong to the restricted SPG3 interval flanked by loci D14S259 and D14S1018. To compare the YAC physical map of the critical region with an RH map with a higher level of resolution, the 90 STSs positioned on the SPG3 YAC contig were tested on a third RH panel, the TNG panel (Lunetta et al. 1996) which could be used to order markers at 50-kb resolution. In this paper we integrate all of the RH and YAC data into an accurate high-resolution physical and transcript map of the SPG3 interval that represents a powerful tool for identification of the gene responsible for this form of FSP.

NIDDM and obesity are complex metabolic disorders with a significant genetic component (1). The two conditions are frequently associated and share many metabolic abnormalities suggesting that they may also share some susceptibility genes. However, their multifactorial nature makes it difficult to dissect the genetic determinants. Despite numerous studies, only a few candidate genes accounting for a minor part of the genetic background have been implicated, usually in particular populations. Hormone-sensitive lipase (HSL), which plays a critical role in the control of energy homeostasis by catalyzing the hydrolysis of adipose tissue triglycerides into free fatty acids, appears as a good candidate gene. Indeed, this enzyme may be involved in the loss of insulin sensitivity since there is increasing evidence that free fatty acids are involved in the development of insulin resistance (2). The adipose tissue isoform of HSL is encoded by a single gene composed of nine exons (3). The gene locus symbolized LIPE has been assigned to chromosome 19ql3.1-13.2 (4). However, no genetic mapping studies have been carried out for LIPE to date. This report describes the characterization of a novel polymorphism in the HSL gene coupled with its genetic mapping. Moreover, we report association studies of this novel microsatellite with NIDDM and obesity within a Caucasian population. First, we characterized a (CA)21 repeat, named HSIi6[CA]n, found within HSL intron six after sequencing of genomic clone hHSLrllll (3). Polymorphism of this repeat was

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.