Erratum to: Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

Unfortunately, the original version of this article [1] contained an error. Figure 1 in the original article, corresponded to the first coinertia analysis that was carried out with no data on the procyclin PE repeats for the T. brucei brucei strains. After including these data, the coinertia analysis was modified both in the directionality of the arrows in the Y Hyperspace and in the biplot generated by the interaction of the two coinertia axes. The modified coinertia analysis is included in Fig. 1.

Molecular characterization and classification of Trypanosoma spp. Venezuelan isolates based on microsatellite markers and kinetoplast maxicircle genes.

BACKGROUND: Livestock trypanosomoses, caused by three species of the Trypanozoon subgenus, Trypanosoma brucei brucei, T. evansi and T. equiperdum is widely distributed throughout the world and constitutes an important limitation for the production of animal protein. T. evansi and T. equiperdum are morphologically indistinguishable parasites that evolved from a common ancestor but acquired important biological differences, including host range, mode of transmission, distribution, clinical symptoms and pathogenicity. At a molecular level, T. evansi is characterized by the complete loss of the maxicircles of the kinetoplastic DNA, while T. equiperdum has retained maxicircle fragments similar to those present in T. brucei. T. evansi causes the disease known as Surra, Derrengadera or "mal de cadeiras", while T. equiperdum is the etiological agent of dourine or "mal du coit", characterized by venereal transmission and white patches in the genitalia. METHODS: Nine Venezuelan Trypanosoma spp. isolates, from horse, donkey or capybara were genotyped and classified using microsatellite analyses and maxicircle genes. The variables from the microsatellite data and the Procyclin PE repeats matrices were combined using the Hill-Smith method and compared to a group of T. evansi, T. equiperdum and T. brucei reference strains from South America, Asia and Africa using Coinertia analysis. Four maxicircle genes (cytb, cox1, a6 and nd8) were amplified by PCRfrom TeAp-N/D1 and TeGu-N/D1, the two Venezuelan isolates that grouped with the T. equiperdum STIB841/OVI strain. These maxicircle sequences were analyzed by nucleotide BLAST and aligned toorthologous genes from the Trypanozoon subgenus by MUSCLE tools. Phylogenetic trees were constructed using Maximum Parsimony (MP) and Maximum Likelihood (ML) with the MEGA5.1® software. RESULTS: We characterized microsatellite markers and Procyclin PE repeats of nine Venezuelan Trypanosoma spp. isolates with various degrees of virulence in a mouse model, and compared them to a panel of T. evansi and T. equiperdum reference strains. Coinertia analysis of the combined repeats and previously reported T. brucei brucei microsatellite genotypes revealed three distinct groups. Seven of the Venezuelan isolates grouped with globally distributed T. evansi strains, while TeAp-N/D1 and TeGu-N/D1 strains clustered in a separate group with the T. equiperdum STIB841/OVI strain isolated in South Africa. A third group included T. brucei brucei, two strains previously classified as T. evansi (GX and TC) and one as T. equiperdum (BoTat-1.1). Four maxicircle genes, Cytochrome b, Cythocrome Oxidase subunit 1, ATP synthase subunit 6 and NADH dehydrogenase subunit 8, were identified in the two Venezuelan strains clustering with the T. equiperdum STIB841/OVI strain. Phylogenetic analysis of the cox1 gene sequences further separated these two Venezuelan T. equiperdum strains: TeAp-N/D1 grouped with T. equiperdum strain STIB818 and T. brucei brucei, and TeGu-N/D1 with the T. equiperdum STIB841/OVI strain. CONCLUSION: Based on the Coinertia analysis and maxicircle gene sequence phylogeny, TeAp-N/D1 and TeGu-N/D1 constitute the first confirmed T. equiperdum strains described from Latin America.

Mosaic maternal uniparental isodisomy for chromosome 7q21-qter.

Uniparental disomy (UPD) for several human chromosomes is associated with clinical abnormalities. We report the case of a 2-year-old boy with severe intrauterine and post-natal growth retardation (IUGR/PNGR) and highly variable sweat chloride concentrations. The patient was identified as heterozygous for the F508del mutation of the CFTR (cystic fibrosis transmembrane conductance regulator) gene. Unexpectedly, the signal corresponding to the maternally inherited F508del allele appeared much more intense than the paternally derived wild allele. Molecular analysis including polymorphic marker studies, microsatellites and single-nucleotide polymorphisms subsequently showed that the boy was a carrier of a de novo mosaic maternal isodisomy of a chromosome 7 segment while there was a biparental inheritance of the rest of the chromosome. This is the first report of a mosaic partial UPD7. The matUPD7 segment at 7q21-qter extends for 72.7 Mb. The karyotype (550 bands) of our patient was normal, and fluorescence in situ hybridization with probes mapping around the CFTR gene allowed us to rule out a partial duplication. The detection of this chromosomal rearrangement confirms the hypothesis that the 7q31-qter segment is a candidate for the localization of human imprinted genes involved in the control of IUGR and PNGR. It also emphasizes the importance of searching for UPD7 in severe, isolated and unexplained IUGR and PNGR.

Molecular characterisation of mitochondrial and cytosolic trypanothione-dependent tryparedoxin peroxidases in Trypanosoma brucei.

In trypanosomatids, removal of hydrogen peroxide and other aryl and alkyl peroxides is achieved by the NADPH-dependent trypanothione peroxidase system, whose components are trypanothione reductase (TRYR), trypanothione, tryparedoxin (TRYX) and tryparedoxin peroxidase (TRYP). Here, we report the cloning of a multi-copy tryparedoxin peroxidase gene (TRYP1) from Trypanosoma brucei brucei encoding a protein with two catalytic VCP motifs similar to the cytosolic TRYP from Crithidia fasciculata. In addition, we characterise a novel single copy gene encoding a second tryparedoxin peroxidase (TRYP2). TRYP2 shows 51% similarity to TRYP1, possesses a putative mitochondrial import sequence at its N-terminus and has a variant IPC motif replacing the second VCP motif implicated in catalysis in other 2-Cys peroxiredoxins. TRYP1 and TRYP2 were expressed in Escherichia coli, and the purified recombinant proteins shown to utilise hydrogen peroxide in the presence of NADPH, trypanothione, TRYR and TRYX from T. brucei, similar to the C. fasciculata cytoplasmic system. Western blots showed that TRYX, TRYP1 and TRYP2 are expressed in both bloodstream and procyclic forms of the life cycle. To determine the precise localisation of TRYX, TRYP1 and TRYP2 in the parasite, polyclonal antibodies to purified recombinant TRYX and TRYP1 and monoclonal antibody to TRYP2 were generated in mice. In-situ immunofluorescence and immunoelectron microscopy revealed a colocalisation of TRYX and TRYP1 in the cytosol, whereas TRYP2 was principally localised in the mitochondrion.

Conservation of metacyclic variant surface glycoprotein expression sites among different trypanosome isolates.

We identified in a Trypanosoma brucei brucei strain (AnTat 1) an expression site for a metacyclic variant surface glycoprotein (MVSG) gene (MVSG) that was previously characterized in a T. b. rhodesiense strain (WRATat 1.1). The 3.4 kb sequences of the two expression sites are 99.6% identical, with no differences in the sequence of the 1.5 kb MVSG. Two other MVSGs in the WRATat 1.1 genome are not present in the AnTat 1 genome. In addition, five other T. b. brucei and T. b. rhodesiense strains, isolated in the same geographic region as the two former strains, do not contain any of these three MVSGs. Two of these five strains, however, appear to possess a very similar MVSG expression site, but with different MVSGs in it. Thus, the presence of the same MVSG in the same expression site in two different isolates is unusual and may be the result of genetic exchange in the field between T. b. brucei and T. b. rhodesiense isolates. Analysis of other African trypanosome strains for the presence of the three WRATat 1.1 MVSG expression sites demonstrated that the expression sites' promoter sequences are much more likely to be present than are specific MVSGs, suggesting that loss of MVSGs is the result of replacement by other VSGs. The promoter region of the MVSG expression site active in the WRATat 1.1 MVAT7 variant was found to be highly conserved among T. b. brucei, T. b. rhodesiense and T. b. gambiense group 2 isolates, whereas it does not occur in the T. b. gambiense group 1 isolates tested. A phylogenetic analysis of this promoter region sequence shows that the T. b. gambiense group 2 isolates form a monophyletic clade well separated from the T. b. brucei/T. b. rhodesiense isolates. Thus, whilst the T. b. brucei, T. b. rhodesiense and T. b. gambiense group 2 isolates are closely related but heterogenous, molecular tools may be developed to distinguish T. b. gambiense group 2 isolates from the others.

Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers.

Genetic variation of microsatellite loci is a widely used method for linkage analysis, individual identification or inter-population studies. Here we analyse a repeated DNA coding sequence and eleven new microsatellites identified within the Trypanosoma (Trypanozoon) brucei genome. Ninety-seven isolates belonging to the five species and subspecies Trypanosoma evansi, T. equiperdum, T. brucei brucei, T. b. rhodesiense and T. b. gambiense were compared regarding the genetic patterns of these markers. The results reveal a great heterogeneity of the genotypes related to the repeated coding sequence and five microsatellites, some of which show a high degree of polymorphism. This allows us to define group-specific genotypes or alleles; in particular, we show that one specific pattern clearly segregates the human pathogen T. b. gambiense group 1.

Characterization of Trypanozoon isolates using a repeated coding sequence and microsatellite markers.

Genetic variation of microsatellite loci is a widely used method for linkage analysis, individual identification or inter-population studies. Here we analyse a repeated DNA coding sequence and eleven new microsatellites identified within the Trypanosoma (Trypanozoon) brucei genome. Ninety-seven isolates belonging to the five species and subspecies Trypanosoma evansi, T. equiperdum, T. brucei brucei, T. b. rhodesiense and T. b. gambiense were compared regarding the genetic patterns of these markers. The results reveal a great heterogeneity of the genotypes related to the repeated coding sequence and five microsatellites, some of which show a high degree of polymorphism. This allows us to define group-specific genotypes or alleles; in particular, we show that one specific pattern clearly segregates the human pathogen T. b. gambiense group I.

Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition.

In Trypanosoma brucei, we have cloned a gene approximately 5 kb downstream of the glucose transporter gene cluster, containing a variable number of 102 bp repeats. This gene encodes a protein with no homologues in the data bases. Antibodies raised against the 34 amino acids repeated motif recognized proteins ranging from 145 to 270 kDa, depending on strains, in both bloodstream and procyclic forms of T. brucei. A correlation was established between the apparent molecular mass of the detected proteins and the number of 34 amino acid repeats which varies from 3 to 40. We have called this protein the flagellum transition zone component (FTZC) due to its localization to the proximal region of the axoneme, within the transition zone. FTZC is the only reported example of a trypanosomal protein present in the transition zone. To determine the role of FTZC we developed a new strategy of gene inactivation based on conditional expression of double-stranded RNA. In the presence of tetracycline, expression of the double-stranded RNA, we observed a complete disappearance of FTZC in the EATRO 1125 and EATRO 427 strains of T. hrucei. Molecular ablation of FTZC does not generate any obvious phenotype such as, lethality, modification of growth rate or cellular shape, in the growth conditions used.

Analysis of the polymorphism of the tumour necrosis factor (TNF) gene and promoter and of circulating TNF-alpha levels in heart-transplant patients suffering or not suffering from severe rejection.

Plasma TNF-alpha levels are generally higher in heart-graft patients who experience a rejection episode than in those who do not. Because the TNF gene and its promoter are polymorphic, we studied the relationships between genetic variability at the TNF locus, the occurrence of graft rejection and TNF-alpha plasma levels in 62 heart-transplant patients in order to investigate inter-individual differences in plasma TNF-alpha levels after allogeneic stimulation. TNF-alpha was immunoenzymatically measured in blood specimens collected on the same day as endomyocardial biopsy. After PCR amplification of DNA, NcoI and AspHI polymorphisms were characterized by their restriction profiles, TNFa microsatellites by electrophoretic separation on acrylamide and the promoter region by sequencing. Plasma levels and molecular genetic results were compared to the grade of heart graft rejection established according to pathological criteria. In our study, allograft rejection was associated neither with NcoI or AspHI polymorphism nor with nucleotide changes in the TNF-A promote. We observed low TNF-alpha levels in n1/n1 homozygous patients and in subjects with G-->A at position--308 of the promoter sequence. Concerning the polymorphism of the TNFa microsatellite, our results might suggest an association with graft rejection but we have to be very careful in drawing conclusions because of the small size of the sample.

Compound heterozygote for both rare apolipoprotein E1 (Gly127-->Asp, Arg158-->Cys) and E3(Cys112-->Arg, Arg251-->Gly) alleles in a multigeneration pedigree with hyperlipoproteinaemia.

1. A French multigeneration pedigree with hyperlipoproteinaemia was investigated for the transmission of the rare apolipoprotein E1(Gly127-->Asp, Arg158-->Cys) variant. The proband, a 46-year-old male carrying the rare apoE1 variant, presented a severe type III hyperlipoproteinaemia like his three brothers and his sister. 2. ApoE phenotyping and genotyping showed a discrepancy in the second allele carried by the proband's wife and two of her children, thus suggesting another apoE gene mutation. Cloning and sequencing of the entire exon 4 demonstrated a point mutation at codon 251, leading to an apoE3(Cys112-->Arg, Arg251-->Gly) allele. The proband's wife was normolipaemic and heterozygous for this rare isoform and the common apoE3 protein. The rare apoE3(Cys112-->Arg, Arg251-->Gly) allele has been transmitted to her two daughters. The first, aged 19, was normolipaemic and heterozygous for this allele and the common apoE2 allele. The second, carrying both the rare isoforms apoE1(Gly127-->Asp, Arg158-->Cys) and apoE3(Cys112-->Arg, Arg251-->Gly), presented a hypertriglyceridaemia at the age of 10. 3. The exploration of apoE status associated with plasma lipid levels and lipoprotein profiles in this three-generation pedigree made it possible to describe a compound heterozygote for two mutated alleles, one mutation being located in the N-terminal domain of the apoE protein and the other arising in the C-terminal domain.

The nucleotide sequence of Saccharomyces cerevisiae chromosome IV.

The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome IV has been determined. Apart from chromosome XII, which contains the 1-2 Mb rDNA cluster, chromosome IV is the longest S. cerevisiae chromosome. It was split into three parts, which were sequenced by a consortium from the European Community, the Sanger Centre, and groups from St Louis and Stanford in the United States. The sequence of 1,531,974 base pairs contains 796 predicted or known genes, 318 (39.9%) of which have been previously identified. Of the 478 new genes, 225 (28.3%) are homologous to previously identified genes and 253 (32%) have unknown functions or correspond to spurious open reading frames (ORFs). On average there is one gene approximately every two kilobases. Superimposed on alternating regional variations in G+C composition, there is a large central domain with a lower G+C content that contains all the yeast transposon (Ty) elements and most of the tRNA genes. Chromosome IV shares with chromosomes II, V, XII, XIII and XV some long clustered duplications which partly explain its origin.

Genotypic evolution of HIV-1 isolates from patients after a switch of therapy from zidovudine to didanosine.

The existence of zidovudine (ZDV)-resistant and didanosine (ddI)-resistant human immunodeficiency-1 (HIV-1) variants mutated in the reverse transcriptase (RT) gene has been previously demonstrated. In this study, we tried to follow up the genotypic changes in the RT after the switch of therapy from ZDV to ddI. We studied HIV-1 isolates from 11 patients undergoing ddI therapy. Genotypic data were obtained with differential polymerase chain reaction (PCR) and with direct sequencing after PCR. The prevalence of ZDV resistance-related mutations showed a very slow decrease, particularly when patients had been treated with ZDV for a long time. The appearance of a mutation at codon 74 seemed to be independent of the presence or absence of ZDV resistance-related mutations. The broad genotypic heterogeneity of the isolates and the complexity of the evolution in one patient's isolates plead for large sequencing studies of the RT genome in new therapeutic approaches.

[Value of the monitoring of curarisation during prolonged mivacurium induced neuromuscular block]. / Intérêt du monitorage de la curarisation lors d'un bloc neuromusculaire prolongé par mivacurium.

A case of neuromuscular blockade of about 200 min of duration, in a 9-year-old boy from mivacurium 0.15 mg.kg-1 is reported. The diagnosis was delayed, after onset of the first signs of recovery, due to the lack of monitoring of neuromuscular transmission. The neuromuscular blockade was reversed with neostigmine 0.04 mg.kg-1. Complete reversal required fifty minutes. The presence of an abnormal genetic variant of pseudocholinesterases was demonstrated by the measurements of pseudocholinesterase activity and dibucaine number. The importance of monitoring of neuromuscular transmission for diagnosis and treatment of mivacurium-induced neuromuscular blockade is underlined.

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

The complete sequence of a 6794 bp segment located on the right arm of chromosome II of Saccharomyces cerevisiae. Finding of a putative dUTPase in a yeast.

The DNA sequence of a 6794 bp fragment located at about 100 kb from the right telomere of chromosome II from Saccharomyces cerevisiae has been determined. Sequence analysis reveals five open reading frames. One is the ARO4 gene encoding the 3-deoxy-D-arabinoheptulosonate 7-phosphate synthase. Another presents strong homology with the S5 ribosomal protein from bacteria. The open reading frame YBR1705 shows significant homology with dUTPase, suggesting for the first time the existence of such an enzyme in S. cerevisiae.

The complete sequence of a 19,482 bp segment located on the right arm of chromosome II from Saccharomyces cerevisiae.

We report here the sequence of a 19,482 bp DNA segment of chromosome II of Saccharomyces cerevisiae. The fragment contains 16 open reading frames (ORFs) covering 74% of the sequence. Four predicted products present homology with known proteins. The ORF YBR1732 exhibits a strong homology to serine hydroxymethyl transferase; the best score is 53.1% identity in 458 amino acids overlap with the serine hydroxymethyl transferase from rabbit liver. YBR1724, which shows homology with riboflavin synthase of Bacillus subtilis, is probably the RIB5 gene implied in riboflavine synthesis and mapped in this region. YBR1733 is homologous to rab protein and YBR1728 is presumably a GTPase activating protein.

The complete sequence of a 10.8kb fragment to the right of the chromosome III centromere of Saccharomyces cerevisiae.

The complete nucleotide sequence of the D10H fragment (10850 bp) was determined. The D10H fragment is located on the right arm of chromosome III near the centromere and contains the SUF2 gene. Six open reading frames (ORFs) larger than 300 bp were found. One of them is the CIT2 gene encoding the cytoplasmic citrate synthase. The others are new putative genes and show no significant similarity with any known gene. In addition two tRNA genes (Asn and Pro) and a solo delta element were identified. Two ORFs were disrupted; no peculiar phenotype was observed.