The intergenic spacer region of the rDNA in Haplopappus gracilis (Nutt.) Gray.

In this paper, we provide further information on the genome organisation of Haplopappus gracilis, one of the six angiosperms showing the lowest chromosome number, i.e. 2n = 4, by determining the nucleotide sequence of the intergenic spacer region of the ribosomal RNA genes and its cytological localization on metaphase chromosomes. DNA sequence analysis reveals the occurring of a product of 4,382 bp in length, characterised by the presence of four blocks of different repeated sequences. Our analysis also evidenced putative promoter regions with three transcription initiation sites for polymerase I, as previously reported in Artemisia absinthium, belonging to the same Asteraceae family. A fluorescent in situ hybridization with the intergenic spacer probe indicates the presence of rDNA genes only in the satellited chromosomes of H. gracilis; besides, differences in the signal intensity between homologous chromosomes were frequently observed, thus suggesting for these chromosome sites the presence of a variable number of rDNA gene copies, even if a divergent chromatin organisation in corresponding regions cannot be ruled out.

Cytological and molecular characterization of Vicia barbazitae Ten. & Guss.

Vicia barbazitae, a taxon belonging to section Vicia of subgenus Vicia, was recovered and analysed by cytological, karyological and molecular methods with the aim of both proposing a general characterisation of this species and studying the relationships among the species of section Vicia . Phylogenetic relationships among the species of the section Vicia and those of the sections Microcarinae, Wiggersia and Atossa were also analysed. Automated karyotype analysis has been determined after Feulgen's reaction; chromosome banding was performed by sequence-specific fluorochrome staining. Fluorescent chromosome banding showed CMA(+)/DAPI(-) NOR-associated heterochromatin in the satellite pair. Karyomorphological parameters, based on symmetry indices, the dendrogram of linkage distance constructed on 37 chromosome parameters, as well as the molecular data based on internal transcribed spacer sequences provided information about phylogenetic position of this species inside the section Vicia and among the species belonging to the sections Microcarinae, Wiggersia, Atossa and Vicia. From our karyological and molecular results, it emerges that V. barbazitae can be considered a natural member of section Vicia.

Cytology of Vicia species. X. Karyotype evolution and phylogenetic implication in Vicia species of the sections Atossa, Microcarinae, Wiggersia and Vicia.

Automated karyotype analyses and sequence of rDNA spacers have been analysed for the species belonging to sections Atossa, Microcarinae, Wiggersia and Vicia. Karyomorphological parameters, based on Rec, Syi and TF% indices, have been determined and evidenced that, in term of symmetry, the karyotype of Vicia lathyroides was the most asymmetric one. A multivariate analysis using 34 karyological parameters, in addition to the symmetry indices, has been carried out and the corresponding dendrogram of linkage distances showed six different groups. Molecular investigations on the inclusive group in study by employing ITS DNA sequences indicated a different pattern of relationships. The cladistic analysis combining the molecular data set with karyological parameters evidenced that the species of sections Vicia and Atossa join closely to each other in a paraphyletic group, which includes the monophyletic section Wiggersia. Therefore, our karyological and molecular data provide information about the phylogenetic position of the analysed species inside the subgenus Vicia and are discussed in relation to previous results obtained by morphology, isozymes and ribosomal genes analyses.

Cytological characterization of Vicia oroboides Wulfen in Jacq.

Vicia oroboides, a rare taxon belonging to section Atossa of subgenus Vicia, was recovered and analysed by means of cytological and karyological methods with the aim of both characterising this species and integrating our knowledge on phylogeny of subgenus Vicia. Automated karyotype analysis and nuclear DNA content have been determined after Feulgen's reaction; chromosome banding was performed by fluorochrome staining to evidence heterochromatic blocks along the chromosome complement. The chromosome number is in line with the values of the species of section Atossa; the GC- and AT-rich sites were identified by CMA and DAPI staining. Karyomorphological parameters, based on symmetry indices, provide information about the phylogenetic position of this species inside the subgenus Vicia. DNA content is reported for the first time.

Cytological investigation of Haplopappus gracilis (Nutt.) Gray: 5-methylcytosine-rich regions, fluorochrome banding and chromatin sensitivity to DNase I digestion.

Haplopappus gracilis (Nutt.) Gray, one of the five known higher plants with a chromosome number of 2n = 4, was studied from a cytological point of view. The chromosome complement of this species was characterized by means of automated karyotype analysis. Moreover, the DNA methylation pattern and fluorochrome banding were determined and compared with cytological data present in the literature. DNA methylation distribution along metaphase chromosomes involved all chromosome territories evidenced by C-banding. Other methylated bands correlated positively with aceto-orcein-positive heterochromatic portions and/or with late replicating bands and/or fluorochrome bands. Some methylated bands showed differences between homologous chromosomes. These bands belonged partly to certain heterochromatic domains and partly to intercalary sites not defined by other standard banding techniques. Differences between the homologues were also indicated by our DNA content data obtained after DNase I digestion.

Cytological and molecular characterization of Vicia esdraelonensis Warb. & Eig: a rare taxon.

Vicia esdraelonensis, a rare taxon belonging to section Hypechusa of subgenus Vicia, was recovered and analyzed by cytological, karyological, and molecular methods, with the aim of both characterizing this species and furthering our knowledge of the phylogeny of subgenus Vicia. Automated karyotype analysis, nuclear DNA content, and chromatin organization were determined by the Feulgen reaction, as well as chromosome banding after double staining with 4',6-diamidino-2-phenylindole (DAPI) and chromomycin A3. The chromosome number and the nuclear DNA content were in agreement with the values of the species of section Hypechusa. The GC- and AT-rich preferential sites were determined by chromomycin A3 and DAPI staining. Karyomorphological parameters indicated that V. esdraelonensis is in an intermediate position in the spatial representation of the species of section Hypechusa on the basis of symmetry indices, as well as in the dendrogram of linkage distance constructed on 37 chromosome parameters. Molecular data based on internal transcribed spacer sequences show that V. esdraelonensis can doubtlessly be included in section Hypechusa and document its closeness to V. noeana. A cladistic analysis combining the molecular data set with karyological characters is also reported. Karyological, cytological, and molecular data allow characterization of the V. esdraelonensis genome and provide information about the phylogenetic position of this species within the Hyrcanicae series of section Hypechusa.

Nuclear DNA contents, rDNAs, and karyotype evolution in subgenus Vicia: III. The heterogeneous section Hypechusa.

Nuclear DNA contents, automated karyotype analyses, and sequences of internal transcribed spacers from ribosomal genes have been determined in the species belonging to section Hypechusa of the subgenus Vicia. Karyomorphological results and phylogenetic data generated from the comparison of rDNA (genes coding for rRNA) sequences showed that sect. Hypechusa is not monophyletic; however, some monophyletic units are apparent (one including Vicia galeata, V. hyrcanica, V. noeana, and V. tigridis, another including V. assyriaca, V. hybrida, V. melanops, V. mollis, and V. sericocarpa), which partly correspond to morphology-based infrasectional groups. The relationships among these species and the species in sections Faba, Narbonensis, Bithynicae, and Peregrinae have been also investigated.

Microsatellite markers are powerful tools for discriminating among olive cultivars and assigning them to geographically defined populations.

Twelve simple sequence repeat (SSR) loci were used to differentiate among 118 cultivars sampled in several countries of the Mediterranean basin and to analyze the genetic structure of olive cultivar gene pools. The markers were found to have high discrimination power. On average, with a single assay it was possible to discriminate 96% of the pairwise comparisons and, with a combination of 3 loci, virtually all cultivars were distinguished. The SSR markers were also tested for their ability to assign cultivars to their geographic population of origin. A selection of 6 loci was found to maximize assignment accuracy, correctly reallocating up to 75.4% of cultivars to their population of origin. Because of the confusion surrounding the origin of most olive cultivars, their molecular identification and ascertainment of origin will be extremely useful for germplasm management and breeding.

Nuclear DNA contents, rDNAs, and karyotype evolution in Vicia subgenus Vicia: II. Section Peregrinae.

Nuclear DNA contents, automated karyotype analyses, and sequences of rDNA spacers have been determined for the species of Vicia belonging to sect. Peregrinae, as well as for V. mollis. The phylogenetic data generated from the comparison of rDNA sequences and karyomorphological results would both indicate that Vicia mollis is a sister group to sect. Peregrinae. The relationships among the species belonging to the Peregrinae section and species enclosed in sections Faba, Narbonensis, and Bithynicae have been also investigated: a clade including V. mollis and sect. Peregrinae is a sister group to a clade including V. bithynica and sect. Narbonensis. With our choice of outgroup, Vicia faba (including subsp. paucijuga) is external to the above mentioned inclusive group.

Genome plasticity in Festuca arundinacea: direct response to temperature changes by redundancy modulation of interspersed DNA repeats.

The response of the genome of Festuca arundinacea seedlings to changes in the temperature at which they were grown was investigated. Fifteen repeated sequences in the nuclear DNA were isolated and hybridized to the genomic DNA of seedlings grown at 10 degrees C or 30 degrees C. The redundancies of sequences recognized by four probes ( FaA5, FaH8, FaH13 and FaH14), were found to differ significantly in the two DNAs. DNA sequences recognized by FaH8, FaH13 and FaH14 were more represented in the genome of the 30 degrees C-raised seedlings than in the genome of the 10 degrees C-raised seedlings (76.5 x 10(3), 1.9 x 10(3), and 111.8 x 10(3) copies per haploid, 1C genome vs 62.7 x 10(3), 1.3 x 10(3), and 80.8 x 10(3) copies, respectively). In contrast, FaA5-related sequences were more represented in the genome of seedlings grown at the lower temperature (15.5 x 10(3) vs 10.2 x 10(3) copies, respectively). Southern-blot hybridization of these repeats to digested genomic DNA produced patterns which indicated that the probe sequences were part of longer repeated sequences having a limited degree of structural heterogeneity. These patterns were partly different when the probes were hybridized to the DNA from seedlings grown at 10 degrees C or 30 degrees C. In situ hybridization showed that the DNA sequences recognized by each probe were scattered along the length of all the chromosomes, with preferential location of FaA5- and FaH13-related sequences at given, mainly centromeric, regions of certain chromosomes. These findings suggest that redundancy modulations of interspersed repeated sequences allow direct responses of the genome of F. arundinacea to changes in environmental temperature.

Nucleotide sequence of the internal transcribed spacers of ribosomal DNA in Picea abies Karst.

The nucleotide sequences of the internal transcribed spacers (ITS1 and ITS2) of ribosomal DNA from Picea abies are reported. Two types of ITS1 of 2784 bp and 3271 bp long exist, whereas only one ITS2 type 238 bp long is present in this species. The shorter ITS1 is characterized by three shorter subrepeats: ssr1, ssr2 and ssr3, 221 bp, 227 bp and 226 bp long respectively. Between the ssr1 and ssr2 sub-repeats are inserted three longer sub-repeats: LSR1, LSR2 and LSR3, 480 bp, 480 bp and 581 bp long respectively. The similarity between the three ssr range from 66% to 79% and between the three LSR range from 65% to 96%. At the end of the LSR3 a microsatellite of 14 CT elements is present. The longer ITS1 type is due to a duplication of the LSR1, most probably obtained by unequal crossing-over and it has an identity of 97.3% with the shorter ITS1 type.

A family of dispersed repeats in the genome of Vicia faba: structure, chromosomal organization, redundancy modulation, and evolution.

A family of repeated DNA sequences of about 1200 bp in length and bordered by well-conserved, 18 bp inverted repeats (VfB family) was found in the nuclear genome of Vicia faba. The structure, chromosomal organization, redundancy modulation and evolution of these sequences were investigated. They are enriched in A+T base pairs (about 40% G+C) and lack any obvious internally repeated motif. A 64%-73% nucleotide sequence identity was found when pairwise comparisons between VfB sequences were carried out (average 69%). Direct repeats were not found to flank the inverted repeats that border these DNA sequences. The results obtained by hybridizing VfB repeats to Southern blots of V. faba genomic DNA digested with EcoRI indicated that these DNA elements are interspersed in the genome. The appearance of bands in these Southern blots and comparison of the structure of the sequences that flank different VfB elements showed that these repeats might be part of other, longer repeated DNA sequences. A high degree of dispersion throughout the genome was confirmed by cytological hybridization, which showed VfB sequences to be scattered along the length of all chromosomes and to be absent or rare only at heterochromatic chromosomal regions. These sequences contribute to intraspecific alterations of genomic size. Indeed, dot-blot hybridizations proved that their redundancy, which is positively correlated with the overall amount of nuclear DNA in each accession, varies between V. faba land races (27x10(3)-230x10(3) copies per 1C DNA). Southern blot hybridization of VfB repeats to restriction endonuclease-digested genomic DNAs of V. faba, V. narbonensis, V. sativa, Phaseolus coccineus, Populus deltoides, and Triticum durum revealed nucleotide sequence homology of these DNA elements, whatever the stringency conditions, only to the DNAs of Vicia species, and to a reduced extent to the DNAs of V. narbonensis and V. sativa compared with that of V. faba. It is concluded that VfB repeats might be descended from mobile DNA elements and contribute to change genomic size and organization during evolution.

QT interval prolongation and risk of life-threatening arrhythmias during toxoplasmosis prophylaxis with spiramycin in neonates.

We recently reported two cases of QT interval prolongation and cardiac arrest in newborns receiving antibiotic therapy with spiramycin, a macrolide agent extensively used for toxoplasmosis prophylaxis. In this study we assessed the effects of this drug on ventricular repolarization and on the potential risk of lethal arrhythmias in eight newborn infants in whom toxoplasmosis prophylaxis after birth was necessary. Electrocardiograms (ECGs) and echocardiograms were recorded during spiramycin therapy (350,000 i.u./kg/ day) and after its withdrawal. In a control group of eight healthy newborns matched for age and sex, no differences were found between two ECGs analogously recorded. The QT interval corrected for heart rate (QTc) was longer during spiramycin therapy than after drug withdrawal (448 +/- 32 msec vs 412 +/- 10 msec, +9%, p = 0.021). QTc dispersion, expressed as the difference between the longest and the shortest value in 12 different leads (QTcmax-min), was also higher during spiramycin therapy (60 +/- 32 msec vs 34 +/- 8 msec, +76%, p = 0.021), mainly because of a major lengthening of the longest QTc (QTcmax). QTc and QTc dispersion were markedly increased in the two newborns who experienced cardiac arrest after beginning treatment compared with the six neonates who had no drug-induced symptoms. During therapy seven of eight newborns had a rare abnormality in the thickening of the left ventricular posterior wall similar to that observed in patients with congenital long QT syndrome. This abnormality disappeared after drug withdrawal. Thus antibiotic therapy with spiramycin in the neonatal period may induce QT interval prolongation and increase QT dispersion. When this effect on ventricular repolarization is more marked, it may favor the occurrence of torsades des pointes and lead to cardiac arrest.

Chromatin characterization by banding techniques, in situ hybridization, and nuclear DNA content in Cicer L. (Leguminosae).

The karyotypes of three accessions, one each from three annual species of the genus Cicer, namely Cicer arietinum, Cicer reticulation, and Cicer echinospermum, were examined and compared using C-banding, the fluorochromes chromomycin A3, DAPI, and Hoechst 33258, in situ hybridization of the 18S-5.8S-25S and 5S rDNA sequences, and silver staining. The nuclear DNA content of the three species and the amount of heterochromatin were also determined. The results suggest an evolutionary pathway in which C. reticulatum is the ancestral species from which both C. arietinum and C. echinospermum are derived with the loss of one pair of satellites; subsequently, C. echinospermum further differentiated by the accumulation of chromosomal rearrangement(s) that gave rise to a hybrid sterility barrier. Key words : Cicer, C-banding, fluorochromes, Ag staining, rRNA genes.

[The laryngeal mask in pediatric anesthesia]. / La maschera laringea in anestesia pediatrica.

We carried out a perspective study in order to assess the ease of insertion, the type and the incidence of perioperative complications connected with the use of the Laryngeal Mask Airway (LMA). We examined 300 consecutive patients, M/F 261/39, average age 4.2 yrs. (range 0.1-16), ASA I-II, who underwent surgical operations of short or average length not involving the pleural, the oropharyngeal or the peritoneum cavity. The choice about anesthesia was left to the discretion of the anesthesiologist. In 27 cases the position of the LM was controlled through a flexible fiberoptics. In 269 patients (89.6%) the LMA was correctly positioned during the first attempt. In 27 patients (9%), 2 or more attempts were necessary, and in 4 patients (1.4%) it was not possible to set the LMA. No differences of statistical significance were noticed between the different size of LMA, with regards to the facility of insertion. The control through fiberoptics showed a correct position, from an anatomical point of view, in 11 patients (41%), whereas in 13 patients (48%) some signs of partial obstruction were noticed (epiglottis interposing between the opening of LMA and larynx) and in 3 patients (11%) vocal cords are not visible. The following complications took place: laryngeal spasm on induction (2.3%), cough or movements on positioning (2.3%), hypoxia (4.3%), obstruction (1%), laryngeal spasm on awakening (1.7%), trauma (5%) and vomiting (0.3%). No connections were found between the size of LMA and total complications. Nevertheless, cough or movement during positioning and laryngeal spasm on awakening were significantly more frequent with LMA n. 3. In our experience, the LMA proved to be effectual and safe in the control of the airway during elective operations in pediatric surgery.

Distribution of 5-methylcytosine-rich regions in the metaphase chromosomes of Vicia faba.

The DNA methylation pattern of Vicia faba metaphase chromosomes was examined with a specific monoclonal antibody. 5-methylcytosine (5-mC) residues are present in different chromosomal sites, and are particularly abundant in telomeric and/or subtelomeric regions and in certain intercalary bands. Chromosomal localization of methylated regions enables a better knowledge of the lengthwise differentiation of this chromosome complement. Our results also indicate that there may be differences in monoclonal antibody binding between corresponding regions of homologous chromosomes in V. faba. This behaviour is detectable in specific regions with different frequencies. The data support results previously obtained for Allium cepa metaphase chromosomes using the same monoclonal antibody.

Genomic relationships between Dasypyrum villosum (L.) Candargy and D. hordeaceum (Cosson et Durieu) Candargy.

The origin and genomic constitution of the tetraploid perennial species Dasypyrum hordeaceum (2n = 4x = 28) and its phylogenetic relationships with the annual diploid Dasypyrum villosum (2n = 2x = 14) have been investigated by comparing the two genomes using different methods. There is no apparent homology between the conventional or Giemsa C-banded karyotypes of the two Dasypyrum species, nor can the karyotype of D. hordeaceum be split up into two similar sets. Polymorphism within several chromosome pairs was observed in both karyotypes. Cytophotometric determinations of the Feulgen-DNA absorptions showed that the genome size of D. hordeaceum was twice as large as that of D. villosum. Both the cross D. villosum x D. hordeaceum (crossability rate 12.1%) and the reciprocal cross (crossability rate 50.7%) produced plump seeds. Only those from the former cross germinated, producing sterile plants with a phenotype that was intermediate between those of the parents. In these hybrids (2n = 21), an average of 13.77 chromosomes per cell paired at meiotic metaphase I. Trivalents were only rarely observed. Through dot-blot hybridizations, a highly repeated DNA sequence of D. villosum was found not to be represented in the genome of D. hordeaceum. By contrast, very similar restriction patterns were observed when a low-repeated DNA sequence or different single-copy sequences of D. villosum or two sequences in the plastidial DNA of rice were hybridized to Southern blots of the genomic DNAs of the two Dasypyrum species digested with different restriction endonucleases. By analyzing glutamic-oxaloacetic-transaminase, superoxide dismutase, alcohol dehydrogenase, and esterase isozyme systems, it was shown that both Dasypyrum species shared the same phenotypes, which differed from those found in hexaploid wheat. In situ hybridizations using DNA sequences encoding gliadins showed that these genes were located close to the centromere of three pairs of D. villosum chromosomes and that they had the same locations in six pairs of D. hordeaceum chromosomes. We conclude that the autoploid origin of D. hordeaceum from D. villosum, which cannot be defended on the basis of chromosomal traits, is suggested by the other findings obtained by comparing the two genomes. Key words : Dasypyrum hordeaceum, Dasypyrum villosum, phylogenetic relationships.

FokI DNA repeats in the genome of Vicia faba: species specificity, structure, redundancy modulation, and nuclear organization.

Tandemly repeated DNA sequences about 60 bp in length, which may be isolated by digestion with FokI restriction endonuclease, were studied by means of molecular and cytological hybridization in Vicia faba and other Vicia species. The results obtained can be summarized as follows: (i) FokI repeats are almost species specific to V. faba, since they hybridize to a minimum extent to genomic DNA of only two out of five related species; (ii) these tandemly repeated elements display variability in structure even within one and the same array, where different repeats may share not more than 71% homology; (iii) their redundancy in the genome of V. faba is remarkably high and varies largely between land races (copy numbers per haploid, 1C, genome range from 21.51 x 10(6) to 5.39 x 10(6)); (iv) FokI repeats are clustered in differing amounts in each subtelocentric pair of the chromosome complement and are missing or present in a nondetectable amount in the submetacentric pair; (vi) chromosome regions that bear these repeats associate closely to varying degrees in interphase nuclei. These results are discussed in relation to possible functional roles that tandemly repeated DNA sequences such as the FokI elements might play.