High chromosomal mobility of rDNA clusters in holocentric chromosomes of Triatominae, vectors of Chagas disease (Hemiptera-Reduviidae).

The subfamily Triatominae (Hemiptera-Reduviidae) includes more than 150 blood-sucking species, potential vectors of the protozoan Trypanosoma cruzi, causative agent of Chagas disease. A distinctive cytogenetic characteristic of this group is the presence of extremely stable chromosome numbers. Unexpectedly, the analyses of the chromosomal location of ribosomal gene clusters and other repetitive sequences place Triatominae as a significantly diverse hemipteran subfamily. Here, we advance the understanding of Triatominae chromosomal evolution through the analysis of the 45S rDNA cluster chromosomal location in 92 Triatominae species. We found the 45S rDNA clusters in one to four loci per haploid genome with different chromosomal patterns: On one or two autosomes, on one, two or three sex chromosomes, on the X chromosome plus one to three autosomes. The movement of 45S rDNA clusters is discussed in an evolutionary context. Our results illustrate that rDNA mobility has been relatively common in the past and in recent evolutionary history of the group. The high frequency of rDNA patterns involving autosomes and sex chromosomes among closely related species could affect genetic recombination and the viability of hybrid populations, which suggests that the mobility of rDNA clusters could be a driver of species diversification.

A step to the gigantic genome of the desert locust: chromosome sizes and repeated DNAs.

The desert locust (Schistocerca gregaria) has been used as material for numerous cytogenetic studies. Its genome size is estimated to be 8.55 Gb of DNA comprised in 11 autosomes and the X chromosome. Its X0/XX sex chromosome determinism therefore results in females having 24 chromosomes whereas males have 23. Surprisingly, little is known about the DNA content of this locust's huge chromosomes. Here, we use the Feulgen Image Analysis Densitometry and C-banding techniques to respectively estimate the DNA quantity and heterochromatin content of each chromosome. We also identify three satellite DNAs using both restriction endonucleases and next-generation sequencing. We then use fluorescent in situ hybridization to determine the chromosomal location of these satellite DNAs as well as that of six tandem repeat DNA gene families. The combination of the results obtained in this work allows distinguishing between the different chromosomes not only by size, but also by the kind of repetitive DNAs that they contain. The recent publication of the draft genome of the migratory locust (Locusta migratoria), the largest animal genome hitherto sequenced, invites for sequencing even larger genomes. S. gregaria is a pest that causes high economic losses. It is thus among the primary candidates for genome sequencing. But this species genome is about 50 % larger than that of L. migratoria, and although next-generation sequencing currently allows sequencing large genomes, sequencing it would mean a greater challenge. The chromosome sizes and markers provided here should not only help planning the sequencing project and guide the assembly but would also facilitate assigning assembled linkage groups to actual chromosomes.

Recombination, chromosome number and eusociality in the Hymenoptera.

Extraordinarily high rates of recombination have been observed in some eusocial species. The most popular explanation is that increased recombination increases genetic variation among workers, which in turn increases colony performance, for example by increasing parasite resistance. However, support for the generality of higher recombination rates among eusocial organisms remains weak, due to low sample size and a lack of phylogenetic independence of observations. Recombination rate, although difficult to measure directly, is correlated with chromosome number. As predicted, several authors have noted that chromosome numbers are higher among the eusocial species of Hymenoptera (ants, bees and wasps). Here, we present a formal comparative analysis of karyotype data from 1567 species of Hymenoptera. Contrary to earlier studies, we find no evidence for an absolute difference between chromosome number in eusocial and solitary species of Hymenoptera. However, we find support for an increased rate of chromosome number change in eusocial taxa. We show that among eusocial taxa colony size is able to explain some of the variation in chromosome number: intermediate-sized colonies have more chromosomes than those that are either very small or very large. However, we were unable to detect effects of a number of other colony characteristics predicted to affect recombination rate - including colony relatedness and caste number. Taken together, our results support the view that a eusocial lifestyle has led to variable selection pressure for increased recombination rates, but that identifying the factors contributing to this variable selection will require further theoretical and empirical effort.

Satellite DNA in insects: a review.

The study of insect satellite DNAs (satDNAs) indicates the evolutionary conservation of certain features despite their sequence heterogeneity. Such features can include total length, monomer length, motifs, particular regions and/or secondary and tertiary structures. satDNAs may act as protein-binding sites, structural domains or sites for epigenetic modifications. The selective constraints in the evolution of satDNAs may be due to the satDNA sequence interaction with specific proteins important in heterochromatin formation and possible a role in controlling gene expression. The transcription of satDNA has been described in vertebrates, invertebrates and plants. In insects, differential satDNA expression has been observed in different cells, developmental stages, sex and caste of the individuals. These transcription differences may suggest their involvement in gene-regulation processes. In addition, the satDNA or its transcripts appear to be involved in heterochromatin formation and in chromatin-elimination processes. The importance of transposable elements to insect satDNA is shown by their presence as a constituent of satDNA in several species of insects (including possible active elements). In addition, they may be involved in the formation of centromeres and telomeres and in the homogenization and expansion of satDNA.

A new approach using tissue alkaline phosphatase histochemistry to identify Crohn's disease.

In the present study, we investigate intestinal alkaline phosphatase activity in mucosal biopsies in patients with inflammatory bowel disease. Crohn's disease influences the alkaline phosphatase activity in the intestine, increasing its activity. We present a histochemistry-based method for alkaline phosphatase that is useful for the identification of Crohn's disease and the differentiation of ulcerative colitis.

Potential role of the IL-33/ST2 axis in celiac disease.

The IL-33/ST2 axis has been implicated in the pathogenesis of several tissue-specific autoimmune diseases. Celiac disease (CD) is the only autoimmune disease in which both the major genetic factors (HLA-DQ2/DQ8) and etiologic ones (dietary gluten) for susceptibility are known. We have measured serum levels and determined intestinal tissue expression of IL-33 and its receptor soluble ST2 in patients with CD to investigate their association with disease activity. Serum and tissue levels of both IL-33 and sST2 were significantly higher in patients with CD compared with those in control patients without CD. We show that toxic peptides extracted from barley and wheat gliadin significantly stimulate the production of IL-33 and ST2 in cultured peripheral blood mononuclear cell from celiac patients, strongly implicating the IL-33/ST2 axis in the pathogenesis of CD. The higher levels of IL-33 and its receptor ST2 in tissue and serum reflect an active inflammatory state and may represent a potential biomarker for disease activity. A better understanding of IL-33/ST2 release, mode of action, and regulation will be crucial to develop therapeutics that target the IL-33/ST2 pathway to treat CD.Cellular & Molecular Immunology advance online publication, 7 September 2015; doi:10.1038/cmi.2015.85.

Isolation and characterization of two families of satellite DNA with repetitive units of 135 bp and 2.5 kb in the ant Monomorium subopacum (Hymenoptera, Formicidae).

Analyzing the satellite DNA in the ant species Monomorium subopacum we found two unrelated families of satellite DNA. Because these satellite DNA families were isolated using the two enzymes HaeIII and EcoRI we called the two families HaeIII and EcoRI family, respectively. The HaeIII family proved to be organized in a 135-bp basic unit repeat, the EcoRI family in a 2.5-kb basic unit repeat. The latter represents perhaps the longest satellite DNA isolated up to now in insects. The HaeIII family apparently comprises about 10% of the total genomic DNA whereas the EcoRI family represents only about 1-2%. A comparative analysis of the two satellite DNA sequences showed no homology between the two families although both sequences possessed long A and T stretches. Eight of the 34 chromosomes showed hybridization with the HaeIII family and hybridization signals are visible in six chromosomes with the EcoRI family. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that the HaeIII family is only slightly curved. However, the unit of the EcoRI satellite DNA family has curvature, especially the first 1000 bp of the monomeric repeat, in which this DNA is AT rich and has numerous A and T stretches. There are also internal inverted subrepeats in each family. The sequences of satellite DNA families found in Monomorium subopacum are different from the sequences of other satellite DNAs cloned in insects, including other species of ants.

14-Base pair polymorphism of human leukocyte antigen-G as genetic determinant in heart transplantation and cyclosporine therapy monitoring.

The 14-base pair (bp) polymorphism within the HLA-G gene has been investigated in heart transplant patients for the first time. The 14-bp polymorphism is associated with HLA-G mRNA stability and the patterns of alternative isoforms splicing, and therefore may influence the functionality of the HLA-G molecule. In heart transplantation, the highest production of soluble HLA-G was related to the -14/-14-bp genotype in the pre- and post-transplantation periods. Our study findings showed that the 14-bp polymorphism of the HLA-G gene influenced the expression of soluble HLA-G in heart transplantation and accordingly resulted in low rejection rates, being a possible marker of genetic variability associated with heart transplantation. In addition, the 14-bp polymorphism of the HLA-G gene is related to the absorber status of cyclosporine of each individual patient, and is useful for determining the oral dose of cyclosporine to manage patients (to adjust immunosuppressive protocols) so as to minimize the risk of a low or high immunosuppression and the side effects in the early stages of heart transplantation.

Tryptophan metabolism and indoleamine 2,3-dioxygenase expression in coeliac disease.

We have investigated the possible role of the metabolism of tryptophan and activity of the enzyme indoleamine 2,3-dioxygenase (IDO) in the immune regulation of coeliac disease (CD). Serum concentrations of tryptophan and its metabolites kinurenines were determined by high performance liquid chromatography in 24 patients with CD, seven patients with Crohn's disease and five healthy patients. We detected an increase of kynurenine (4.2 micromol/l +/- 0.27 versus 2.6 micromol/l +/- 0.54, P < 0002) and of the kynurenine/tryptophan ratio in supernatants of coeliac patients (11.5 micromol/l +/- 1.01 versus 6.5 micromol/l +/- 1.57, P < 0005) in comparison with healthy patients, respectively, and we found no differences with Crohn's disease patients. Immunohistochemistry analysis of intestinal biopsies from CD patients showed an increased expression of IDO, interferon-gamma, interleukin-10 and transforming growth factor-beta. Our data suggest that a mechanism(s) dependent on tryptophan catabolism might regulate the immune responses in CD.

Effects of restriction endonucleases on nucleolar organizing regions in the ant Tapinoma nigerrimum.

The effects of some restriction endonucleases (REs) on the nucleolar organizing regions and on the genes for ribosomal RNA (rDNA) were analyzed using the nucleolar organizing region of the chromosome 6 of Tapinoma nigerrimum as an experimental model, since, in accordance with previous studies, the genes for ribosomal RNA seem to be present only in this chromosome. In situ non-digestion of the nucleolar organizing region was observed when EcoRI and HindIII were used. However, very evident digestion and partial digestion respectively were observed when HaeIII and Tru9I were used. Southern blot analysis realized on naked DNA digested with the same REs and using rDNA of Drosophila melanogaster as probe showed that there are target sequences for these enzymes in the rDNA. In accordance with the results obtained, the rDNA is poor in EcoRI and HindIII sequences, contains moderate amounts of Tru9I sequences and is rich in HaeIII sequences. All the data obtained suggest that in the nucleolar organizing region of Tapinoma nigerrimum, the major, if not the only, limiting factor affecting in situ digestion by the REs used is the presence and frequency of their specific restriction targets. Consequently, extraction of DNA from this chromosome region depends on the size of the fragments originated.

G-banding and chromosome condensation in the ant, Tapinoma nigerrimum.

Well-defined G-bands were obtained on metaphase chromosomes from Tapinoma nigerrimum using trypsin and warm 2 x SSC in sequence. The G-banded pattern allowed the identification of all chromosomes. Evidence for asynchronous condensation of the chromosomes of this species is provided. Different banding patterns were obtained when metaphase chromosomes were stained with DA/DAPI alone and with DA/DAPI after a standard G-banding procedure. The G-banding phenomenon is discussed using the result obtained.

Characterization and chromosome location of satellite DNA in the leaf beetle Chrysolina americana (Coleoptera, Chrysomelidae).

This paper is the first record of the satellite DNA of the specialized phytophagous genus Chrysolina. The satellite DNA of Chrysolina americana is organized in a tandem repeat of monomers 189 bp long, has a A + T content of 59.6% and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation sensitive enzymes suggests that this repetitive DNA is undermethylated. In siti hybridization with a biotinylated probe of the satellite DNA showed the pericentromeric localization of these sequences in all meiotic bivalents. The presence of this repetitive DNA in other species of the genus was also tested by Southern analysis. The results showed that this satellite DNA sequence is specific to the C. americana genome and has not been found in three other species of Chrysolina with a different choice of host plants than in the former.

Analysis of the nucleolar organizing regions in the ant Tapinoma nigerrimum (Hymenoptera, Formicidae).

This study analyses the NORs of Tapinoma nigerrimum, a species that, as known from previous studies, has various chromosomes which carry a NOR site. The analysis was made by a combination of three methods: silver nitrate staining, in situ hybridization with fluorescein-or digoxigenin-labelled probes, and staining with the CG-specific fluorochrome chromomycin A3. The silver staining technique showed an Ag-positive region on chromosome 6 and on various other chromosomes. However, the application of in situ hybridization techniques showed only one positive signal in the proximal region of the short arm of chromosome 6 of T. nigerrimum. Similar results were observed by CMA banding. The absence of rDNA genes or the presence of only a small number of these, not detectable with the above probes, might explain the absence of hybridization signal in the remaining chromosomes.

Satellite DNA in the ant Messor structor (Hymenoptera, Formicidae).

This paper is the first record of the satellite DNA of Formicidae. The satellite DNA of the ant Messor structor is organized in a tandem repeat of monomers of 79 bp. Like satellite DNAs of other insects, it is AT rich and presents direct and inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes strongly suggests that this DNA is undermethylated. The presence of this repetitive DNA in other species of the genus Messor is also tested.

Patterns of DNase I sensitivity in the chromosomes of the ant Tapinoma nigerrimum (Hymenoptera, Formicidae).

We have analysed the patterns of DNase I/nick translation in the chromosomes of Tapinoma nigerrimum. The results show a non-uniform DNase I sensitivity in different chromosome domains. The hypersensitivity appears to be specially concentrated at both the NOR and the distal regions. The resemblance to and differences from the situation in other animal species, in which active genes are DNase I hypersensitive, are discussed.

Conservation of (TTAGG)(n) telomeric sequences among ants (Hymenoptera, Formicidae).

To determine the telomere sequence in Tapinoma nigerrimum, we carried out in situ hybridization using TTAGGG and TTAGG repeat polymerase chain reaction (PCR)-generated probes. No hybridization signals were found when TTAGGG was used as a probe. However, strong signals were observed at the end of the chromosomes with the TTAGG probe. Southern blot analysis carried out on genomic DNA using TTAGG as a probe showed a strong hybridization signal even under highly stringent conditions. Similar results were obtained in Southern blot analysis carried out on genomic DNA of 19 species of ants belonging to three different subfamilies. In accordance with all the results shown in this article, the TTAGG repeat seems to be the major component of the telomere sequence in the majority of ant species.

A new repetitive DNA sequence family in the olive (Olea europaea L.).

Two families of repeated DNA sequences were cloned from Olea europaea ssp sativa cv. "Picual". The first repetitive DNA is organized in a tandem repeat of monomers of 178 bp. Sequencing of several clones showed that it is relatively A-T rich (54.49%) and possesses short direct and inverted subrepeats as well as some palindromic sequences. Comparison between the monomers revealed heterogeneity of the sequence primary structure. This repetitive DNA is present in several cultivars of olive cultivates. Comparison of sequences with other repetitive DNAs described in Olea europaea has been carried out. No significant similarity was found. All the obtained results suggest that this repetitive DNA described here is a new family of repetitive DNA. The second repetitive DNA is organized in a tandem repeat of monomers of 78 bp. This second family of repetitive DNA showed significant similarity with other repetitive DNAs previously described in Olea europaea. Their existence in new cultivars of olive is shown.

Genomic organization and transcription of satellite DNA in the ant Aphaenogaster subterranea (Hymenoptera, Formicidae).

A satellite DNA family (APSU) was isolated and characterized in the ant Aphaenogaster subterranea. This satellite DNA is organized in tandem repeats of 162 bp and is relatively AT rich (51.9%). Sequence analysis showed a high level of homogeneity between monomers. Loss of satellite DNA has been detected in queens in relation to workers, because the amount of satellite DNA in queens is about 25% of the amount found in workers. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this DNA is not methylated. Analysis of the electrophoretic mobility of satellite DNA on non-denaturing polyacrylamide showed that this satellite DNA is only very lightly curved. Their possible transcription was analyzed using reverse transcription and polymerase chain reaction (RT-PCR). The satellite DNA is transcribed on the two DNA strands at the same level in worker and queen pupae, as well as in worker adults.

Satellite DNA in the elm leaf beetle, Xanthogaleruca luteola (Coleoptera, Chrysomelidae): characterization, interpopulation analysis, and chromosome location.

In this paper the satellite DNA (stDNA) of the phytophagous beetle Xanthogaleruca luteola is analyzed. It is organized in a tandem repeat of 149-bp-long monomers, has an AT content of 59%, and presents inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this repetitive DNA is not methylated. Analysis of the electrophoretic mobility of stDNA on non-denaturing polyacrylamide gels showed that this stDNA is not curved. In situ hybridization with a biotinylated probe of the stDNA revealed a pericentromeric localization of these sequences in the majority of the meiotic bivalents. We have studied the stDNA of X. luteola from two populations with very distinct geographical origins. The sequence and phylogenetic analysis of monomers from these two populations showed that the repetitive element is conserved within the species. Putative gene conversion tracts are identified when the different monomers of the same population are compared. These results could indicate the existence of processes of homogenization that would extend these mutations to all the satellite repeats.

Expression of HLA-G in inflammatory bowel disease provides a potential way to distinguish between ulcerative colitis and Crohn's disease.

In addition to being involved in nutrient uptake, the epithelial mucosa constitute the first line of defense against microbial pathogens. A direct consequence of this physiological function is a very complex network of immunological interactions that lead to a strong control of the mucosal immune balance. The dysfunction of immunological tolerance is likely to be a cause of inflammatory bowel disease (IBD), ulcerative colitis (UC) and Crohn's disease (CD). HLA-G is a non-classical major histocompatibility complex (HLA) class I molecule, which is highly expressed by human cytotrophoblast cells. These cells play a role in immune tolerance by protecting trophoblasts from being killed by uterine NK cells. Because of the deregulation of immune system activity in IBD, as well as the immunoregulatory role of HLA-G, we have analyzed the expression of HLA-G in intestinal biopsies of patients with UC and CD. Our study shows that the differential expression of HLA-G provides a potential way to distinguish between UC and CD. Although the reason for this differential expression is unclear, it might involve a different mechanism of immune regulation. In addition, we demonstrate that in the lamina propria of the colon of patients with UC, IL-10 is strongly expressed. In conclusion, the presence of HLA-G on the surface of intestinal epithelial cell in patients with UC lends support to the notion that this molecule may serve as a regulator of mucosal immune responses to antigens of undefined origin. Thus, this different pattern of HLA-G expression may help to differentiate between the immunopathogenesis of CD and UC.