Deposition of collagen III and alterations in basement membrane integrity as candidate prognostic markers in prostate cancer.

The extracellular matrix surrounding the tumor undergoes changes in its organization during the metastasis process. The present study aims to quantify total collagen, collagen I (Col I) and collagen III (Col III), analyze the alignment of collagen fibers and assess the basement membrane integrity in samples from patients with metastatic and non-metastatic prostate cancer. Tissue samples from 60 patients were classified into groups based on prognostic parameters: better prognosis (n = 20), worse prognosis without metastasis (n = 23) and metastatic (n = 17). Picrosirius red with further analysis under polarizing microscope was used to quantify (with validation using immunohistochemistry) and analyze collagen alignment, and Periodic Acid Schiff staining was used to analyze the basement membrane integrity. The Col I/Col III ratio was found to be higher in the metastatic group than in the groups with better prognosis (p = 0.012) and worse prognosis without metastasis (p = 0.018). Basement membrane integrity constitution in malignant tumor tissue differed from that of adjacent non-tumor tissue (p < 0.001). Moreover, the worsening in the tumor tissue integrity was positively correlated with worse prognostic parameters. All in all, absence of Col III and basement membrane integrity might be indicators of poor prognosis in prostate cancer.

Evolutionary features of microspore and pollen grain development in Cyperaceae.

KEY MESSAGE: Asymmetric meiosis leading to the release of pollen grains as pseudomonads is a synapomorphy in Cyperaceae, but differences in microspore development are relevant in the family's evolutionary history. Cyperaceae members present atypical microsporogenesis, in which one meiotic product is functional while the other three degenerate, culminating in pseudomonad pollen formation. Differences during development, such as pseudomonad shape and degenerative microspore positioning, are seen throughout the family, but no phylogenetic interpretation has been made regarding these variances thus far. In this study, we analyzed the early- and late-diverging sedge genera Hypolytrum and Eleocharis, respectively, while comparing them to data available in the literature and conducting an ancestral character reconstruction for pseudomonad traits. Light microscopy results show that pseudomonad development in Hypolytrum is homologous to several other sedge genera, presenting apical degenerative microspores. However, pseudomonads are round and centrally arranged in the anther locule in this case, which consists of a pleisiomorphic trait for the family. The basal positioning of degenerative microspores is restricted to Rhynchospora, consisting of an apomorphic trait for this genus. Despite these differences, ultrastructural analysis of Eleocharis pseudomonad revealed shared features with other genera studied, which include variations in chromatin condensation and cytoplasmic turnover in functional cells. These common features seem related to the different cellular fates seen during microspore development and further corroborate the synapomorphic status of pseudomonads in sedges.

Abundance of distal repetitive DNA sequences in Capsicum L. (Solanaceae) chromosomes.

Chili peppers (Solanaceae family) have great commercial value. They are commercialized in natura and used as spices and for ornamental and medicinal purposes. Although three whole genomes have been published, limited information about satellite DNA sequences, their composition, and genomic distribution has been provided. Here, we exploited the noncoding repetitive fraction, represented by satellite sequences, that tends to accumulate in blocks along chromosomes, especially near the chromosome ends of peppers. Two satellite DNA sequences were identified (CDR-1 and CDR-2), characterized and mapped in silico in three Capsicum genomes (C. annuum, C. chinense, and C. baccatum) using data from the published high-coverage sequencing and repeats finding bioinformatic tools. Localization using FISH in the chromosomes of these species and in two others (C. frutescens and C. chacoense), totaling five species, showed signals adjacent to the rDNA sites. A sequence comparison with existing Solanaceae repeats showed that CDR-1 and CDR-2 have different origins but without homology to rDNA sequences. Satellites occupied subterminal chromosomal regions, sometimes collocated with or adjacent to 35S rDNA sequences. Our results expand knowledge about the diversity of subterminal regions of Capsicum chromosomes, showing different amounts and distributions within and between karyotypes. In addition, these sequences may be useful for future phylogenetic studies.

Diversity of the repetitive DNA fraction in Cestrum, the genus with the largest genomes within Solanaceae.

BACKGROUND: Cestrum species present large genomes (2 C = ~ 24 pg), a high occurrence of B chromosomes and great diversity in heterochromatin bands. Despite this diversity, karyotypes maintain the chromosome number 2n = 16 (except when they present B chromosomes), and a relative similarity in chromosome morphology and symmetry. To deepen our knowledge of the Cestrum genome composition, low-coverage sequencing data of C. strigilatum and C. elegans were compared, including cytogenomic analyses of seven species. METHODS AND RESULTS: Bioinformatics analyses showed retrotransposons comprising more than 70% of the repetitive fraction, followed by DNA transposons (~ 17%), but FISH assays using retrotransposon probes revealed inconspicuous and scattered signals. The four satellite DNA families here analyzed represented approximately 2.48% of the C. strigilatum dataset, and these sequences were used as probes in FISH assays. Hybridization signals were colocalized with all AT- and GC-rich sequences associated with heterochromatin, including AT-rich Cold-Sensitive Regions (CSRs). Although satellite probes hybridized in almost all tested species, a satDNA family named CsSat49 was highlighted because it predominates in centromeric regions. CONCLUSIONS: Data suggest that the satDNA fraction is conserved in the genus, although there is variation in the number of FISH signals between karyotypes. Except to the absence of FISH signals with probes CsSat1 and CsSat72 in two species, the other satellites occurred in species of different phylogenetic clades. Some satDNA sequences have been detected in the B chromosomes, indicating that they are rich in preexisting sequences in the chromosomes of the A complement. This comparative study provides an important advance in the knowledge on genome organization and heterochromatin composition in Cestrum, especially on the distribution of satellite fractions between species and their importance for the B chromosome composition.

A comparative approach to understanding the ovule, seed, and fruit development in Bulbostylis (Cyperaceae: Cyperoideae: Abildgaardieae).

In the present work, we study the ovule, seed, and fruit development in six Bulbostylis species in order to characterize the genus in a comparative approach and to identify the characteristics that can be used in taxonomy and phylogeny. Flowers and fruits at different developmental stages were analyzed using LM and SEM after processing according to standard techniques. The species studied have the following: anatropous and bitegmic ovules, weak crassinucellar ovules, obturator of integumentary origin, monosporic embryo sac of the Polygonum type, nuclear endosperm, hypostase formation, seed coat formed by tanniferous endotegmen and exotesta, and Bulbostylis-type embryo. On the other hand, the pericarp development constitutes the main variation within Bulbostylis since the cells of the exocarp may or may not present starch grains, and their inner periclinal walls may be slightly or deeply concave depending on the degree of development of the mesocarp sclereids. In a taxonomic context, the results herein obtained are in conflict with studies which suggest infrageneric groupings based on fruit micromorphology, and also with the relationship among the Bulbostylis species based on molecular analysis. This work contributes to a better understanding of the reproductive anatomy and embryology in Bulbostylis, and reveals the first insights about the origin of multiple embryos in Cyperaceae. Given the frequent presence of polyembryony in Bulbostylis, and the poor mention of this condition in the family, this work highlights an aspect in the anatomy of Cyperaceae that must be re-explored.

An 82 bp tandem repeat family typical of 3' non-coding end of Gypsy/TAT LTR retrotransposons is conserved in Coffea spp. pericentromeres.

Coffea spp. chromosomes are very small and accumulate a variety of repetitive DNA families around the centromeres. However, the proximal regions of Coffea chromosomes remain poorly understood, especially regarding the nature and organisation of the sequences. Taking advantage of the genome sequences of C. arabica (2n = 44), C. canephora, and C. eugenioides (C. arabica progenitors with 2n = 22) and good coverage genome sequencing of dozens of other wild Coffea spp., repetitive DNA sequences were identified, and the genomes were compared to decipher particularities of pericentromeric structures. The searches revealed a short tandem repeat (82 bp length) typical of Gypsy/TAT LTR retrotransposons, named Coffea_sat11. This repeat organises clusters with fragments of other transposable elements, comprising regions of non-coding RNA production. Cytogenomic analyses showed that Coffea_sat11 extends from the pericentromeres towards the middle of the chromosomal arms. This arrangement was observed in the allotetraploid C. arabica chromosomes, as well as in its progenitors. This study improves our understanding of the role of the Gypsy/TAT LTR retrotransposon lineage in the organisation of Coffea pericentromeres, as well as the conservation of Coffea_sat11 within the genus. The relationships between fragments of other transposable elements and the functional aspects of these sequences on the pericentromere chromatin were also evaluated. Highlights: A scattered short tandem repeat, typical of Gypsy/TAT LTR retrotransposons, associated with several fragments of other transposable elements, accumulates in the pericentromeres of Coffea chromosomes. This arrangement is preserved in all clades of the genus and appears to have a strong regulatory role in the organisation of chromatin around centromeres.

Ultrastructural analysis of Rhynchospora ovules: The first record of Cyperaceae megagametophyte on transmission electron microscope.

Microsporogenesis and microgametogenesis are unusual in sedges (Cyperaceae), the third largest monocotyledonous family, as three microspores are aborted in favor of a single functional microspore. However, studies using light microscopy show that megasporogenesis and megagametogenesis occur normally. Nevertheless, the lack of ultrastructural details limits our knowledge of female gametophyte development in this family. Given the importance of morphological studies of reproductive structures, ovules and megagametophytes of Rhynchospora pubera were analyzed under transmission electron microscopy for the first time. Overall, ovules presented features similar to those described for the family, but ultrastructural details revealed an absence of a clear boundary between the egg cell and the central cell cytoplasm. Most interestingly, antipodal and nucellar cells showed several signs of vacuolar cell death, which suggest that programmed autolysis in sporogenous and gametophytic tissue is common in gametophyte development in the Cyperaceae. This may be related to the reproductive success of this family.

Genome relationships and LTR-retrotransposon diversity in three cultivated Capsicum L. (Solanaceae) species.

BACKGROUND: Plant genomes are rich in repetitive sequences, and transposable elements (TEs) are the most accumulated of them. This mobile fraction can be distinguished as Class I (retrotransposons) and Class II (transposons). Retrotransposons that are transposed using an intermediate RNA and that accumulate in a "copy-and-paste" manner were screened in three genomes of peppers (Solanaceae). The present study aimed to understand the genome relationships among Capsicum annuum, C. chinense, and C. baccatum, based on a comparative analysis of the function, diversity and chromosome distribution of TE lineages in the Capsicum karyotypes. Due to the great commercial importance of pepper in natura, as a spice or as an ornamental plant, these genomes have been widely sequenced, and all of the assemblies are available in the SolGenomics group. These sequences were used to compare all repetitive fractions from a cytogenomic point of view. RESULTS: The qualification and quantification of LTR-retrotransposons (LTR-RT) families were contrasted with molecular cytogenetic data, and the results showed a strong genome similarity between C. annuum and C. chinense as compared to C. baccatum. The Gypsy superfamily is more abundant than Copia, especially for Tekay/Del lineage members, including a high representation in C. annuum and C. chinense. On the other hand, C. baccatum accumulates more Athila/Tat sequences. The FISH results showed retrotransposons differentially scattered along chromosomes, except for CRM lineage sequences, which mainly have a proximal accumulation associated with heterochromatin bands. CONCLUSIONS: The results confirm a close genomic relationship between C. annuum and C. chinense in comparison to C. baccatum. Centromeric GC-rich bands may be associated with the accumulation regions of CRM elements, whereas terminal and subterminal AT- and GC-rich bands do not correspond to the accumulation of the retrotransposons in the three Capsicum species tested.

IGS sequences in Cestrum present AT- and GC-rich conserved domains, with strong regulatory potential for 5S rDNA.

The 35S and 5S ribosomal DNA (rDNA) organized in thousands of copies in genomes, have been widely used in numerous comparative cytogenetic studies. Nevertheless, several questions related to the diversity and organization of regulatory motifs in 5S rDNA remain to be addressed. The 5S rDNA unit is composed of a conserved 120 bp length coding region and an intergenic spacer (IGS) containing potential regulatory motifs (Poly-T, AT-rich and GC-rich) differing in number, redundancy and position along the IGS. The Cestrum species (Solanaceae) have large genomes (about 10 pg/1C) and conserved 2n = 16 karyotypes. Strikingly, these genomes show high diversity of heterochromatin distribution, variability in 35S rDNA loci and the occurrence of B chromosomes. However, the 5S rDNA loci are highly conserved in the proximal region of chromosome 8. Comparison of seventy-one IGS sequences in plants revealed several conserved motifs with potential regulatory function. The AT- and GC-rich domains appeared highly conserved in Cestrum chromosomes. The 5S genic and the GC-rich IGS probe produced FISH signals in both A (pair 8) and B chromosomes. The GC-rich domain presented a strong potential for regulation because it may be associated with CpG islands organization, as well as to hairpin and loop organization. Another interesting aspect was the ability of AT- and GC-rich motifs to produce non-heterochromatic CMA/DAPI signals. While the length of the 5S rDNA IGS region varied in size between the Cestrum species, the individual sequence motifs seem to be conserved suggesting their regulatory function. The most striking feature was the conserved GC-rich domain in Cestrum, which is recognized as a signature trait of the proximal region of chromosome pair 8.

Action of multi-enzyme complex on protein extraction to obtain a protein concentrate from okara.

The objective of this study was to optimize the extraction of protein by applying a multi-enzymatic pretreatment to okara, a byproduct from soymilk processing. The multi-enzyme complex Viscozyme, containing a variety of carbohydrases, was used to hydrolyze the okara cell walls and facilitate extraction of proteins. Enzyme-assisted extraction was carried out under different temperatures (37-53 °C), enzyme concentrations (1.5-4%) and pH values (5.5-6.5) according to a central composite rotatable design. After extraction, the protein was concentrated by isoelectric precipitation. The optimal conditions for maximum protein content and recovery in protein concentrate were 53 °C, pH 6.2 and 4% of enzyme concentration. Under these conditions, protein content of 56% (dry weight basis) and a recovery of 28% were obtained, representing an increase of 17 and 86%, respectively, compared to the sample with no enzymatic pretreatment. The multi-enzyme complex Viscozyme hydrolyzed the structural cell wall polysaccharides, improving extraction and obtaining protein concentrate from the okara. An electrophoretic profile of the protein concentrate showed two distinct bands, corresponding to the acidic and basic subunits of the protein glycinin. There were no limiting amino acids in the protein concentrate, which had a greater content of arginine.

Cestrum strigilatum (Ruiz & Pavón, 1799) B chromosome shares repetitive DNA sequences with A chromosomes of different Cestrum (Linnaeus, 1753) species.

Species of Cestrum (Linnaeus, 1753) have shown large diversity in the accumulation and distribution of repetitive DNA families, and B chromosomes have been described in seven species. Some types of repetitive DNA were identified in A and B chromosomes in species of this plant group, such as AT-rich SSR, 35S and 5S rDNA, C-Giemsa and C-CMA/DAPI bands and retrotransposons. To increase our understanding of the relationships of A and B chromosomes, the B of C. strigilatum Ruiz & Pavón, 1799 was microdissected, amplified and hybridized in situ against chromosomes of this species, and in six other species of this genus. FISH signals were observed in whole the B of C. strigilatum, including stretches of A chromosomes, as well as in some A chromosomes of all tested species. A strong FISH signal was seen adjacent to the 5S rDNA in the proximal region of pair 8 of all species and, due to this, we have searched for 5S rDNA fragments in the microdissected B chromosome. PCR and sequencing data evidenced 5S rDNA deletion along evolutionary pathways of the B of C. strigilatum. Although A and B chromosomes displayed redundancy in the repetitive DNA families in different species, the B of C. strigilatum seemed to differ from those Bs of other Cestrum species by the loss of rDNA fractions. A possible origin of Bs in Cestrum was discussed.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae).

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Heterochromatin base pair composition and diversification in holocentric chromosomes of kissing bugs (Hemiptera, Reduviidae)

The subfamily Triatominae (Hemiptera, Reduviidae) includes 150 species of blood-sucking insects, vectors of Chagas disease or American trypanosomiasis. Karyotypic information reveals a striking stability in the number of autosomes. However, this group shows substantial variability in genome size, the amount and distribution of C-heterochromatin, and the chromosome positions of 45S rDNA clusters. Here, we analysed the karyotypes of 41 species from six different genera with C-fluorescence banding in order to evaluate the base-pair richness of heterochromatic regions. Our results show a high heterogeneity in the fluorescent staining of the heterochromatin in both autosomes and sex chromosomes, never reported before within an insect subfamily with holocentric chromosomes. This technique allows a clear discrimination of the heterochromatic regions classified as similar by C-banding, constituting a new chromosome marker with taxonomic and evolutionary significance. The diverse fluorescent patterns are likely due to the amplification of different repeated sequences, reflecting an unusual dynamic rearrangement in the genomes of this subfamily. Further, we discuss the evolution of these repeated sequences in both autosomes and sex chromosomes in species of Triatominae.

Chromosomal distribution and evolution of abundant retrotransposons in plants: gypsy elements in diploid and polyploid Brachiaria forage grasses.

Like other eukaryotes, the nuclear genome of plants consists of DNA with a small proportion of low-copy DNA (genes and regulatory sequences) and very abundant DNA sequence motifs that are repeated thousands up to millions of times in the genomes including transposable elements (TEs) and satellite DNA. Retrotransposons, one class of TEs, are sequences that amplify via an RNA intermediate and reinsert into the genome, are often the major fraction of a genome. Here, we put research on retrotransposons into the larger context of plant repetitive DNA and genome behaviour, showing features of genome evolution in a grass genus, Brachiaria, in relation to other plant species. We show the contrasting amplification of different retroelement fractions across the genome with characteristics for various families and domains. The genus Brachiaria includes both diploid and polyploid species, with similar chromosome types and chromosome basic numbers x = 6, 7, 8 and 9. The polyploids reproduce asexually and are apomictic, but there are also sexual species. Cytogenetic studies and flow cytometry indicate a large variation in DNA content (C-value), chromosome sizes and genome organization. In order to evaluate the role of transposable elements in the genome and karyotype organization of species of Brachiaria, we searched for sequences similar to conserved regions of TEs in RNAseq reads library produced in Brachiaria decumbens. Of the 9649 TE-like contigs, 4454 corresponded to LTR-retrotransposons, and of these, 79.5 % were similar to members of the gypsy superfamily. Sequences of conserved protein domains of gypsy were used to design primers for producing the probes. The probes were used in FISH against chromosomes of accesses of B. decumbens, Brachiaria brizantha, Brachiaria ruziziensis and Brachiaria humidicola. Probes showed hybridization signals predominantly in proximal regions, especially those for retrotransposons of the clades CRM and Athila, while elements of Del and Tat exhibited dispersed signals, in addition to those proximal signals. These results show that the proximal region of Brachiaria chromosomes is a hotspot for retrotransposon insertion, particularly for the gypsy family. The combination of high-throughput sequencing and a chromosome-centric cytogenetic approach allows the abundance, organization and nature of transposable elements to be characterized in unprecedented detail. By their amplification and dispersal, retrotransposons can affect gene expression; they can lead to rapid diversification of chromosomes between species and, hence, are useful for studies of genome evolution and speciation in the Brachiaria genus. Centromeric regions can be identified and mapped, and retrotransposon markers can also assisting breeders in the developing and exploiting interspecific hybrids.

Origin and distribution of AT-rich repetitive DNA families in Triatoma infestans (Heteroptera).

Triatoma infestans, one of the most important vectors of Trypanosoma cruzi, is very interesting model, because it shows large interpopulation variation in the amount and distribution of heterochromatin. This polymorphism involved the three large pairs up to almost all autosomal pairs, including the sex chromosomes. To understand the dynamics of heterochromatin variation in T. infestans, we isolated the AT-rich satDNA portion of this insect using reassociation kinetics (C0t), followed by cloning, sequencing and FISH. After chromosome localization, immunolabeling with anti-5-methylcytosine, anti-H4K5ac and anti-H3K9me2 antibodies was performed to determine the functional characteristics of heterochromatin. The results allowed us to reorganize the karyotype of T. infestans in accordance with the distribution of the families of repetitive DNA using seven different markers. We found that two arrays with lengths of 79 and 33bp have a strong relationship with transposable element sequences, suggesting that these two families of satDNA probably originated from Polintons. The results also allowed us to identify at least four chromosome rearrangements involved in the amplification/dispersion of AT-rich satDNA of T. infestans. These data should be very useful in new studies including those examining the cytogenomic and population aspects of this very important species of insect.

Karyotype diversity among predatory Reduviidae (Heteroptera).

Species of infraorder Cimicomorpha of Heteroptera exhibit holokinetic chromosomes with inverted meiosis for sex chromosomes and high variation in chromosome number. The family Reduviidae, which belongs to this infraorder, is also recognized by high variability of heterochromatic bands and chromosome location of 18S rDNA loci. We studied here five species of Reduviidae (Harpactorinae) with predator habit, which are especially interesting because individuals are found solitary and dispersed in nature. These species showed striking variation in chromosome number (including sex chromosome systems), inter-chromosomal asymmetry, different number and chromosome location of 18S rDNA loci, dissimilar location and quantity of autosomal C-heterochromatin, and different types of repetitive DNA by fluorochrome banding, probably associated with occurrence of different chromosome rearrangements. Terminal chromosome location of C-heterochromatin seems to reinforce the model of equilocal dispersion of repetitive DNA families based in the "bouquet configuration".

The conservation of number and location of 18S sites indicates the relative stability of rDNA in species of Pentatomomorpha (Heteroptera).

Fluorescent in situ hybridization (FISH) with rDNA probes has been used for comparative cytogenetics studies in different groups of organisms. Although heteropterans are a large suborder within Hemiptera, studies using rDNA are limited to the infraorder Cimicomorpha, in which rDNA sites are present in the autosomes or sex chromosomes. We isolated and sequenced a conserved 18S rDNA region of Antiteuchus tripterus (Pentatomidae) and used it as a probe against chromosomes of 25 species belonging to five different families of Pentatomomorpha. The clone pAt05, with a length of 736 bp, exhibited a conserved stretch of 590 bp. FISH analysis with the probe pAt05 always demonstrated hybridization signals in sub-terminal positions, except for Euschistus heros. Apparently, there is a tendency for 18S rDNA sites to locate in autosomes, except for Leptoglossus gonagra and Euryophthalmus rufipennis, which showed signals in the m- and sex chromosomes, respectively. Although FISH has produced evidence that rearrangements are involved in rDNA repositioning, whether in different autosomes or between sex and m-chromosomes, we have no conclusive evidence of what were the pathways of these rearrangements based on the evolutionary history of the species studied here. Nevertheless, the diversity in the number of species analyzed here showed a tendency of 18S rDNA to remain among the autosomes.

Asymmetric cytokinesis guide the development of pseudomonads in Rhynchospora pubera (Cyperaceae).

The late stages of microsporogenesis in the family Cyperaceae are marked by the formation of an asymmetrical tetrad, degeneration of three of the four nuclei resulting from meiosis and the formation of pseudomonads. In order to understand the cytological changes involved in the development of pseudomonads, a combination of 11 different techniques (conventional staining, cytochemistry procedures, immunofluorescence, FISH and transmission electron microscopy: TEM) were used to study the later stages of microsporogenesis in Rhynchospora pubera. The results demonstrated the occurrence of two cytoplasmic domains in the pseudomonads, one functional and the other degenerative, which are physically and asymmetrically separated by cell plate with an endomembrane system rich in polysaccharides. Other changes associated with endomembrane behaviour were observed, such as a large number of lipid droplets, vacuoles containing electron-dense material and concentric layers of endoplasmic reticulum. Concomitant with the isolation of degenerative nuclei, the tapetal cells also showed evidence of degeneration, indicating that both tissues under programmed cell death (PCD), as indicated by immunofluorescence and TEM procedures. The results are significant because they associate cellular polarisation and asymmetry with different cytoplasmic domains, and hence open new possibilities for studying cellular compartmentalisation and PCD.

FISH using a gag-like fragment probe reveals a common Ty3-gypsy-like retrotransposon in genome of Coffea species.

The genus Coffea possesses about 100 species, and the most economically important are Coffea canephora and Coffea arabica. The latter is predominantly self-compatible with 2n = 4x = 44, while the others of the genus are diploid with 2n = 2x = 22 and mostly self-incompatible. Studies using molecular markers have been useful to detect differences between genomes in Coffea; however, molecular and cytogenetic studies have produced only limited information on the karyotypes organization. We used DOP-PCR to isolate repetitive elements from genome of Coffea arabica var. typica. The pCa06 clone, containing a fragment of 775 bp length, was characterized by sequencing and used as a probe in chromosomes of C. arabica and six other species: C. canephora, Coffea eugenioides, Coffea kapakata, Coffea liberica var. dewevrei, Coffea racemosa, and Coffea stenophylla. This insert shows similarities with a gag protein of the Ty3-gypsy-like super-family. Dot blot and FISH analyses demonstrated that pCa06 is differentially accumulated between species and chromosomes. Signals appeared scattered and clustered on the chromosomes and were also associated with heterochromatic regions. While the literature shows that there is a high karyotype similarity between Coffea species, our results point out differences in the accumulation and dispersion of this Ty3-gypsy-like retrotransposon during karyotype differentiation of Coffea.

Cytogenetic characterization reveals differences in nuclear organization of cystic cells among Brazilian species of Triatoma (Heteroptera, Reduviidae).

Cytogenetic studies in triatomines have described the occurrence of holokinetic chromosomes, heterochromatin distribution and the location of rDNA (ribosomal DNA) sites, but few aspects of nuclear organization in this group have been discussed. We have focused on ultrastructural and cytogenetic features and differences in cystic cells of seminiferous tubules between five species of Triatoma. Cystic cells showed evidence of polyploidy events and heterochromatic blocks appeared predominantly in the central region of the nuclei. Cytogenetic analyses showed that there was variation in chromocenter number between species, and that the central regions were AT-rich [DAPI+ (4',6-diamidino-2-phenylindole+)], whereas the periphery was CG-rich (CMA+). Another characteristic was the distribution of 45S rDNA, which differed according to the chromosomal location of this sequence. In all we have compared aspects of nuclear organization, polyploidy, heterochromatin, rDNA site distribution and methylation levels, as well as the relationships between five species of Triatoma from a cystic cell perspective.