Search

Almost all the nuclear genes of four Gramineae (maize, wheat, barley, rice) and pea are located in DNA fractions covering only a 1–2% GC range and representing between 10 and 25% of the different genomes. These DNA fractions comprise large gene-rich regions (collectively called the ‘gene space’) separated by vast gene-empty, repeated sequences. In contrast, in Arabidopsis thaliana , genes are distributed in DNA fractions covering an 8% GC range and representing 85% of the genome. Here, we investigated the integration of a transferred DNA (T-DNA) in the genomes of Arabidopsis and rice and found different patterns of integration, which are correlated with the different gene distributions. While T-DNA integrates essentially everywhere in the Arabidopsis genome, integration was detected only in the gene space, namely in the gene-rich, transcriptionally active, regions of the rice genome. The implications of these results for the integration of foreign DNA are discussed.

In Bacillus subtilis , the products of the pta and ackA genes, phosphotransacetylase and acetate kinase, play a crucial role in the production of acetate, one of the most abundant by-products of carbon metabolism in this gram-positive bacterium. Although these two enzymes are part of the same pathway, only mutants with inactivated ackA did not grow in the presence of glucose. Inactivation of pta had only a weak inhibitory effect on growth. In contrast to pta and ackA in Escherichia coli , the corresponding B. subtilis genes are not cotranscribed. Expression of the pta gene was increased in the presence of glucose, as has been reported for ackA . The effects of the predicted cis -acting catabolite response element (CRE) located upstream from the promoter and of the trans -acting proteins CcpA, HPr, Crh, and HPr kinase on the catabolite regulation of pta were investigated. As for ackA , glucose activation was abolished in ccpA and hprK mutants and in the ptsH1 crh double mutant. Footprinting experiments demonstrated an interaction between CcpA and the pta CRE sequence, which is almost identical to the proposed CRE consensus sequence. This interaction occurs only in the presence of Ser-46-phosphorylated HPr (HPrSer-P) or Ser-46-phosphorylated Crh (CrhSer-P) and fructose-1,6-bisphosphate (FBP). In addition to CcpA, carbon catabolite activation of the pta gene therefore requires at least two other cofactors, FBP and either HPr or Crh, phosphorylated at Ser-46 by the ATP-dependent Hpr kinase.

Genetic studies of Saccharomyces cerevisiae have identified many components acting to deliver specific proteins to their cellular locations. Genome analysis, however, has indicated that additional genes may also participate in such protein trafficking. The product of the yeast Yarrowia lipolytica TSR1 gene promotes the signal recognition particle-dependent translocation of secretory proteins through the endoplasmic reticulum. Here we describe the identification of a new gene family of proteins that is well conserved among different yeast species. The TSR1 genes encode polypeptides that share the same protein domain distribution and, like Tsr1p, may play an important role in the early steps of the signal recognition particle-dependent translocation pathway. We have identified five homologues of the TSR1 gene, four of them from the yeast Saccharomyces cerevisiae and the other from Hansenula polymorpha. We generated a null mutation in the S. cerevisiae YHC8 gene, the closest homologue to Y. lipolytica TSR1, and used different soluble (carboxypeptidase Y, alpha-factor, invertase) and membrane (dipeptidyl-aminopeptidase) secretory proteins to study its phenotype. A large accumulation of soluble protein precursors was detected in the mutant strain. Immunofluorescence experiments show that Yhc8p is localized in the endoplasmic reticulum. We propose that the YHC8 gene is a new and important component of the S. cerevisiae endoplasmic reticulum membrane and that it functions in protein translocation/insertion of secretory proteins through or into this compartment.

The gene corresponding to the lactococcal oligopeptidase PepF1 (formerly PepF [V. Monnet, M. Nardi, A. Chopin, M.-C. Chopin, and J.-C. Gripon, J. Biol. Chem. 269:32070-32076, 1994]) is located on the lactose-proteinase plasmid of Lactococcus lactis subsp. cremoris NCDO763. Use of the pepF1 gene as a probe with different strains showed that pepF1 is present on the chromosome of Lactococcus lactis subsp. lactis IL1403, whereas there is a second, homologous gene, pepF2, on the chromosome of strain NCDO763. From hybridization, PCR amplification, and sequencing experiments, we deduced that (i) pepF1 and pepF2 exhibit 80% identity and encode two proteins which are 84% identical and (ii) pepF2 is included in an operon composed of three open reading frames and is transcribed from two promoters. The protein, encoded by the gene located downstream of pepF2, shows significant homology with methyltransferases. Analysis of the sequences flanking pepF1 and pepF2 indicates that only a part of the pepF2 operon is present on the plasmid of strain NCDO763, while the operon is intact on the chromosome of strain IL1403. Traces of several recombination events are visible on the lactose-proteinase plasmid. This suggests that the duplication of pepF occurred by recombination from the chromosome of an L. lactis subsp. lactis strain followed by gene transfer. We discuss the possible functions of PepF and the role of its amplification.

Segregations of rice isozymes made it possible to ascertain the genetic control of 13 enzymes by 30 loci. In addition to numerous cases of independence, several linkages were observed. Got-2 and Enp-1 were localized on chromosome 3 and a linkage group composed of Pox-3, Pox-4, and Est-1 was identified. Inconsistencies of recombination rates between linked loci among different crosses were noted. Several cases of distorted segregations and pseudolinkages were recorded. The relationships between these results and the study of genome organization in cultivated rice are discussed.Key words: Oryza, isozyme, genetic analysis, segregation distortion, linkage.

Fluorescence in situ hybridization (FISH) was used to study the presence of alien chromatin in interspecific hy- brids and one introgressed line (S.288) derived from crosses between the cultivated species Coffea arabica and the diploid relatives C. canephora and C. liberica. In situ hybridization using genomic DNA from C. canephora and C. arabica as probes showed elevated cross hybridization along the hybrid genome, confirming the weak differentiation between parental genomes. According to our genomic in situ hybridization (GISH) data, the observed genomic resemblance between the modern C. canephora genome (C) and the C. canephora-derived subgenome of C. arabica (C a ) appears rather consider- able. Poor discrimination between C and C a chromosomes supports the idea of low structural modifications of both ge- nomes since the C. arabica speciation, at least in the frequency and distribution of repetitive sequences. GISH was also used to identify alien chromatin segments on chromosome spreads of a C. liberica-introgressed line of C. arabica. Further, use of GISH together with BAC-FISH analysis gave us additional valuable information about the physical localization of the C. liberica fragments carrying the SH3 factor involved in resistance to the coffee leaf rust. Overall, our results illustrate that FISH analysis is a complementary tool for molecular cytogenetic studies in coffee, providing rapid localization of ei- ther specific chromosomes or alien chromatin in introgressed genotypes derived from diploid species displaying substantial genomic differentiation from C. arabica. Resume´ : L'hybridation in situ fluorescente (« FISH ») a eteutilisee pour etudier la presence de chromatine introgressee chez des hybrides interspecifiques mais aussi chez une lignee cultivee, issues du croissement entre l'espece cultivee Coffea arabica et ses proches diploo¨des C. canephora et C. liberica, respectivement. L'hybridation in situ en utilisant l'ADN ge ´- nomique des especes C. canephora et C. arabica comme sondes, a montreune hybridation croisee importante chez les ge ´- nomes hybrides, ce qui confirme une etroite differentiation genomique entre les parents. Cette ressemblance entre le genome actuel de C. canephora et le sous-genome de C. arabica derivant de l'espece C. canephora (C a ), semble tresele- vee. De plus, la faible discrimination entre les chromosomes du type C et ceux du type C a , suggere des modifications structurales mineures des l'origine meme de l'espece C. arabica, au moins dans ce qui concerne la frequence et distribu- tion des sequences repetees. L'hybridation genomique in situ (« GISH ») a permis aussi l'identification de differents frag- ments d'introgression chez une ligneed eC. arabica introgressee par C. liberica, ce qui a conduit al'identification de quatre fragments d'introgression. De plus, l'analyse combineGISH plus BAC-FISH a donnedes informations importantes sur la localisation physique du fragment issu de C. liberica porteur du facteur SH 3d e resistance a la rouille des cafeiers. L'ensemble des resultats montre que la technique FISH represente un outil puissant pour l'etude cytogenetique des cafeiers permettant l'identification rapide de chromosomes mais aussi de l'ADN introgressechez les genotypes issus de croissements concernant des especes diploo¨des caracterisees par une differentiation genomique importante par rapport a C. arabica. Mots-cles: cafeier, BAC-FISH, GISH, hybridation interspecifique, introgression, evolution du genome. (Traduit par la Redaction)

International audience; A three-generation outbred pedigree of 186 individuals was used to identify the genomic regions involved in the variability of chemical and physical wood properties of Pinus pinaster. A total of 54 quantitative trait loci (QTLs) was detected, with an average of 2.4 QTLs per trait. Clusters of wood properties QTLs were found at several points in the genome, suggesting the existence of pleiotropic effects of a limited number of genes. The colocalizations observed in this study are in accordance with the genetic correlations previously reported in the literature. In addition, in an attempt to identify the genes underlying the QTLs, nine wood quality candidate genes involved in cell wall structure were localized on the genetic map. Only one of them, Korrigan, a gene encoding for a β 1-4 endo-glucanase known in Arabidopis thaliana to be involved in polysaccharide biosynthesis, co-localized with a wood quality QTL cluster involved in hemicellulose content and fibre characteristics. This finding is in accordance with results previously reported for this gene regarding its expression variability (transcriptome and proteome levels) and patterns of molecular evolution. The pertinence of this result will be tested in more rigorous designs in order to identify early selection predictors for wood quality.

International audience; Nucleotide diversity in eight genes related to wood formation was investigated in two pine species, Pinus pinaster and P. radiata. The nucleotide diversity patterns observed and their properties were compared between the two species according to the specific characteristics of the samples analysed. A lower diversity was observed in P. radiata compared with P. pinaster. In particular, for two genes (Pp1, a glycin-rich protein homolog and CesA3, a cellulose synthase) the magnitude of the reduction of diversity potentially indicates the action of nonneutral factors. For both, particular patterns of nucleotide diversity were observed in P. pinaster (high genetic differentiation for Pp1 and close to zero differentiation associated with positive Tajima's D-value for CesA3). In addition, KORRIGAN, a gene involved in cellulose-hemicellulose assembly, demonstrated a negative Tajima's D-value in P. radiata accompanied by a high genetic differentiation in P. pinaster. The consistency of the results obtained at the nucleotide level, together with the physiological roles of the genes analysed, indicate their potential susceptibility to artificial and/or natural selection

Direct detection of fluorescent in situ hybridization signals on R-banded chromosomes stained with propidium iodide is a rapid and efficient method for constructing cytogenetic maps for species with R-banded standard karyotypes. In this paper, our aim is to establish an R-banded rabbit karyotype nomenclature that is in total agreement with the 1981 G-banded standard nomenclature. For this purpose, we have produced new GTG- and RBG-banded mid-metaphase karyotypes and an updated version of ideograms of R-banded rabbit chromosomes. In addition, to confirm correlations between G- and R-banded chromosomes, we have defined a set of 23 rabbit BAC clones, each containing a specific gene, one marker gene per rabbit chromosome, and we have localized precisely each BAC clone by FISH on both G- and R-banded chromosomes.

A review of the present and future of rice genomics is presented. Rice is a model species for cereals as well as a very important crop. Its genome has been the focus of many mapping experiments associated with QTL localization. These genetic maps now serve as a background for physical mapping, genome sequencing and gene discovery. Recent progress are reviewed. The next step in rice genomics is functional genomics with the determination of the function of the genes. The most straightforward approaches are discussed.

The purpose of this study was to increase the number of genes assigned by in situ hybridization to equine chromosomes and thus the number of links for comparative mapping with other species. Forty-four new sequences were added to the horse cytogenetic map by FISH mapping of BAC clones containing genes (35) or ESTs (9). Three approaches were developed: use of horse BAC clones screened with (i) horse EST primers, (ii) interspecific consensus intraexonic primers, and (iii) use of goat BAC containing genes previously localized on goat chromosomes. Present data suggest that the second approach is the most promising. A total of 46 segments containing one or several genes could be compared, among which 40 loci could be included in 16 synteny groups between human and horse, displaying one ordered segment and several breaking points along chromosomes. All single BAC localizations confirm the most recent mapping data. Twenty-six out of 31 chromosomes now contain a gene mapped by in situ hybridization, and 14 new arm-to-arm segment homologies were revealed.

Summary: The XPR2 gene from Yarrowia lipolytica encodes an inducible alkaline extracellular protease. Its complex regulation involves pH, carbon, nitrogen and peptones. Two previously identified upstream activating sequence (UAS) regions were analysed in a reporter system, outside the XPR2 context. Fragments from the UAS regions were inserted upstream of a minimal LEU2 promoter directing the expression of a reporter gene. The activity of the hybrid promoters was assessed following integration into the Y. lipolytica genome. This study confirmed the presence of two UASs composed of several interacting elements. Within the distal UAS (UAS1), a TUF/RAP1 binding site exhibited a UAS activity, which was enhanced by the presence of two adjacent repeats, overlapping sites similar to the CAR1 upstream repressing sequence from Saccharomyces cerevisiae. Within the proximal UAS (UAS2), the UAS activity required the interaction of both an ABF1-like binding site and a decameric repeat, containing Aspergillus nidulans PacC site consensus sequences. This decameric repeat was able to mediate repression due to carbon and/or nitrogen sources as well as pH-dependent activation. A study in the context of trans-regulatory mutations in the Y. lipolytica RIM101 gene showed that the PacC-like sites, potential binding sites for YIRim101p, were implicated in the derepression of UAS2-driven expression at neutral-alkaline pH. The in vivo response of the PacC-like decamers to external pH was dependent on the status of the pH-regulated activator YIRim101p, which is homologous to the A. nidulans PacC regulator. The carbon/nitrogen regulation imposed on the decamers was shown to be independent of YIRim101p and to override its effects.

We report here the cloning and functional analysis of a novel homologue of the mitochondrial carriers predominantly expressed in the central nervous system and referred to as BMCP1 (brain mitochondrialcarrier protein-1). The predicted amino acid sequence of this novel mitochondrial carrier indicates a level of identity of 39, 31, or 30%, toward the mitochondrial oxoglutarate carrier, phosphate carrier, or adenine nucleotide translocator, respectively, and a level of identity of 34, 38, or 39% with the mitochondrial uncoupling proteins UCP1, UCP2, or UCP3, respectively. Northern analysis of mouse, rat, or human tissues demonstrated that mRNA of this novel gene is mainly expressed in brain, although it is 10–30-fold less expressed in other tissues.In situ hybridization analysis of brain showed it is particularly abundant in cortex, hippocampus, thalamus, amygdala, and hypothalamus. Chromosomal mapping indicates that BMCP1 is located on chromosome X of mice and at Xq24 in man. Expression of the protein in yeast strongly impaired growth rate. Analysis of respiration of total recombinant yeast or yeast spheroplasts and in particular of the relationship between respiratory rate and membrane potential of yeast spheroplasts revealed a marked uncoupling activity of respiration, suggesting that although BMCP1 sequence is more distant from the uncoupling proteins (UCPs), this protein could be a fourth member of the UCP family.

International audience; Proline 40 in Escherichia coli thioredoxin is located close to the redox active site (Cys32-Cys35) within the alpha2 helix. The conservation of this residue among most of the thioredoxins suggests that it could play an important role in the structure and/or function of this protein. We have substituted Pro40 for Ala by using site-directed mutagenesis and expressed the mutant P40A in E.coli. The effects of the mutation on the biophysical and biological properties of thioredoxin have been analyzed and compared with molecular dynamics simulations. Modeling predicted that the replacement of Pro40 by Ala induced a displacement of the active site which exposes Trp31 to the solvent and opens a cleft located between helices alpha2 and alpha3. The solvation free energy (SFE) calculation also indicated that P40A became more hydrophobic as W31 became more accessible. These predictions were totally in agreement with the experimental results. The mutant P40A exhibited chromatographic behavior and fluorescence properties very different from those of the wild-type (WT) protein, in relationship with the displacement of W31. The determination of the free energy of unfolding of P40A showed that the mutant was globally destabilized by 2.9 kcal/mol. However, the effect of the mutation on the transition curve was highly unusual as the midpoint of the unfolding transition increased, indicating that some local structures were actually stabilized by the mutation. Despite these structural modifications, neither the ability of the protein to reduce a chloroplastic enzyme nor its reactivity with the bacterial reductase decreased. The only functional difference was the higher stability of P40A in light activation of NADP-malate dehydrogenase under air, which suggests that the mutant was less rapidly re-oxidized than WT. Therefore, it can be concluded that Pro40 is not essential for maintaining the redox function of thioredoxin but rather is required for the stability of the protein.

Fatty acid synthase activity has been shown to be regulated mainly at the transcriptional level under both dietary and hormonal influences. As a first step towards elucidating the factors involved, we isolated and characterized chicken genomic clones encompassing the 5' part of the chicken fatty acid synthase gene and its flanking region. The entire region of the cloned DNA spans 30 kb, and the first three exons of the gene were mapped to a 6.3-kb genomic fragment. The transcription initiation site was determined after subcloning the cDNA which encodes the 5' end of the mRNA. The first exon, which was 129 bp long, was located approximately 5.3 kb upstream of the second exon, which contained the start codon. In the 5' flanking region, putative TATA and CAAT boxes were located 30 and 92 bp, respectively, upstream of the transcription initiation site. The 5' flanking region contained numerous sequences corresponding to consensus binding sites for transcription factors. Various lengths of flanking sequences extending up to 1028 bp upstream of the transcription initiation site and containing 100 bp of the first exon were linked to the bacterial chloramphenicol acetyltransferase gene; in this study, these constructs were analyzed in transient transfection assays in human hepatoma cells. The proximal 125-bp sequence upstream of the transcription start site was shown to be a basal promoter. The cloning and characterization of the chicken fatty-acid synthase gene provides some further insight into the regulation of fatty acid synthesis in birds as compared to mammals.

The genes coding for the insulin-like growth factor-1 receptor (IGF1R) and a porcine endothelium-derived 21-residue vasoconstrictor peptide (EDN) were localized in the pig by means of radioactive in situ hybridization. IGF1R was mapped to chromosome region 1q1.7→q2.1 and EDN to 7p1.3→p1.2. The results are discussed in relation to the position of their homologous loci in man.