Repetitive DNAs in the slug Milax nigricans: association of ribosomal (18S-28S and 5S rDNA) and (TTAGGG)n telomeric sequences in the slug M. nigricans (Mollusca: Gastropoda: Pulmonata).

Spermatocyte chromosomes of the slug Milax nigricans (Mollusca: Gastropoda: Pulmonata) were studied using silver staining (Ag-NOR) and fluorescent in situ hybridization (FISH) with four repetitive DNA probes [18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n]. Silver impregnation was inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) since no silver dots occurred on the chromosomes at spermatogonial metaphase and a diffuse silver stainability could be observed on the bivalents at metaphase-I. Unlike silver staining, single-colour rDNA FISH consistently mapped major ribosomal sites (18S-28S rDNA) on two small-sized chromosomes in spermatogonial cells and on the correspondent metaphase-I bivalent in spermatocytes. While telomeric (TTAGGG)n sequence hybridized to all chromosomes, (GATA)n probe localized abundant hybridization sites, dispersed throughout the genome. Simultaneous double-colour FISH demonstrated a close chromosomal association of 18S-28S rDNA, 5S rDNA and (TTAGGG)n.

Organometallic complexes with biological molecules. XVIII. Alkyltin(IV) cephalexinate complexes: synthesis, solid state and solution phase investigations.

Dialkyltin(IV) and trialkyltin(IV) complexes of the deacetoxycephalo-sporin-antibiotic cephalexin [7-(d-2-amino-2-phenylacetamido)-3-methyl-3-cephem-4-carboxylic acid] (Hceph) have been synthesized and investigated both in solid and solution phase. Analytical and thermogravimetric data supported the general formula Alk(2)SnOHceph(.)H(2)O and Alk(3)Snceph(.)H(2)O (Alk=Me, n-Bu), while structural information has been gained by FT-IR, (119)Sn Mössbauer and (1)H, (13)C, (119)Sn NMR data. In particular, IR results suggested polymeric structures both for Alk(2)SnOHceph(.)H(2)O and Alk(3)Snceph(.)H(2)O. Moreover, cephalexin appears to behave as monoanionic tridentate ligand coordinating the tin(IV) atom through ester-type carboxylate, as well as through beta-lactam carbonyl oxygen atoms and the amino nitrogen donor atoms in Alk(2)SnOHceph(.)H(2)O complexes. On the basis of (119)Sn Mössbauer spectroscopy it could be inferred that tin(IV) was hexacoordinated in such complexes in the solid state, showing skew trapezoidal configuration. As far as Alk(3)Sn(IV)ceph(.)H(2)O derivatives are concerned, cephalexin coordinated the Alk(3)Sn moiety through the carboxylate acting as a bridging bidentate monoanionic group. Again, (119)Sn Mössbauer spectroscopy led us to propose a trigonal configuration around the tin(IV) atom, with R(3)Sn equatorial disposition and bridging carboxylate oxygen atoms in the axial positions. The nature of the complexes in solution state was investigated by using (1)H, (13)C and (119)Sn NMR spectroscopy. Finally, the cytotoxic activity of organotin(IV) cephalexinate derivatives has been tested using two different chromosome-staining techniques Giemsa and CMA(3), towards spermatocyte chromosomes of the mussel Brachidontes pharaonis (Mollusca: Bivalvia). Colchicinized-like mitoses (c-mitoses) on slides obtained from animals exposed to organotin(IV) cephalexinate compounds, demonstrated the high mitotic spindle-inhibiting potentiality of these chemicals. Moreover, structural damages such as "chromosome achromatic lesions", "chromosome breakages" and "chromosome fragments" have been identified through a comparative analysis of spermatocyte chromosomes from untreated specimens (negative controls) and specimens treated with the organotin(IV) complexes.

Chromosome banding and molecular cytogenetic study of two Mediterranean trachinoid fish species (Teleostei: Trachinidae, Uranoscopidae).

The chromosomes of Echiichthys vipera (Trachinidae) and Uranoscopus scaber (Uranoscopidae) were analyzed by means of various banding methods and fluorescence in situ hybridization (FISH) with telomeric and major rDNA probes, respectively. The karyotype of E. vipera was composed of 48 acrocentric chromosomes and NOR sites, as revealed by all detection methods, were situated pericentromerically on a single pair of middle-sized chromosomes. Blocks of constitutive heterochromatin were present in the pericentromeric regions of all pairs of chromosomes. The karyotype of U. scaber showed three karyomorphs: 2n = 30 (18 m + 12 a/st [m = metacentric, a = acrocentric and st = subtelocentric]), 2n = 28 (20 m + 8 a/st), 2n = 27 (21 m + 6 a/st). NORs, as revealed by FISH, were situated pericentromerically on a single pair of middle-sized chromosomes in spite of Ag-positive signals in the centromeres of all pairs of chromosomes. Robertsonian fusions were hypothesized for observed variation due to invariable number of chromosome arms FN = 48.

Chromosome analysis and FISH mapping of ribosomal DNA (rDNA), telomeric (TTAGGG)n and (GATA)n repeats in the leech Haemopis sanguisuga (L.) (Annelida: Hirudinea).

In the present paper the chromosome complement (n = 13; 2n = 26) of the common leech Haemopis sanguisuga (L.) (Annelida: Hirudinea: Hirudinidae) was analyzed using banding techniques and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal clusters (18S-28S rDNA) in the pericentromeric region of one large metacentric chromosome pair; this region, which consisted of heterochromatin rich in GC base pairs, was preferentially stained by silver nitrate (Ag-NOR). The (TTAGGG)n telomeric probe was hybridized with the termini of nearly all chromosomes, whereas the (GATA)n probe did not label any chromosome areas.

rDNA (18S-28S and 5S) colocalization and linkage between ribosomal genes and (TTAGGG)(n) telomeric sequence in the earthworm, Octodrilus complanatus (Annelida: Oligochaeta: Lumbricidae), revealed by single- and double-color FISH.

Spermatogonial and metaphase I chromosomes of the lumbricid earthworm Octodrilus complanatus (Annelida: Oligochaeta) were examined using fluorescent in situ hybridization (FISH) with three repetitive DNA probes-5S rDNA, 18S-26S rDNA, and (TTAGGG)(n). Single-color FISH consistently mapped one chromosome pair per spread using either 5S rDNA or 18S-26S rDNA as probes. Simultaneous (18S-26S)-5S and (18S-26S)-(TTAGGG)(n) FISH demonstrated that repeated units of the two ribosomal families were overlapped and closely associated with telomeric sequences.

Organometallic complexes with biological molecules. XVII. Triorganotin(IV) complexes with amoxicillin and ampicillin.

Novel triorganotin(IV) complexes of two beta-lactamic antibiotics, 6-[D-(-)-beta-amino-p-hydroxyphenyl-acetamido]penicillin (=amoxicillin) and 6-[D-(-)-alpha-aminobenzyl]penicillin (=ampicillin), have been synthesized and investigated both in solid and solution states. The complexes corresponded to the general formula R(3)Sn(IV)antib*H(2)O (R=Me, n-Bu, Ph; antib=amox=amoxicillinate or amp=ampicillinate). Structural investigations about configuration in the solid state have been carried out by interpreting experimental IR and 119Sn Mössbauer data. In particular, IR results suggested polymeric structures both for R(3)Sn(IV)amox.H(2)O and R(3)Sn(IV)amp*H(2)O. Moreover, both antibiotics appear to behave as monoanionic bidentate ligands coordinating the tin(IV) atom through ester-type carboxylate, as well as through the beta-lactamic carbonyl. Evidence that in none of these compounds water molecules were involved in coordination, was provided by thermogravimetric investigations. On the basis of 119Sn Mössbauer spectroscopy it can be inferred that tin(IV) was pentacoordinate in all of the complexes in the solid state, showing an equatorial R(3)Sn(IV) trigonal bipyramidal (tbp) configuration. The nature of the complexes in solution state was investigated by using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy, while an 119Sn spectrum was obtained for n-Bu(3)Sn(IV)amp*H(2)O. Although 1H- and 13C-NMR measurements suggested that in dimethyl sulfoxide (DMSO)-d(6) solution the polymeric structure collapsed, due to a solvolysis process of the beta-lactamic carbonyl bonding to the organometallic moiety, the complexes have been shown to maintain the same trigonal bipyramidal configuration at tin(IV) atom by the coordination of a DMSO molecule. Cytotoxic activity of these novel semisynthetic antibiotic derivatives has been tested towards spermatocyte chromosomes of the mussel Brachidontes pharaonis (Mollusca: Bivalvia) using two different chromosome-staining techniques such as Giemsa and CMA(3). The occurrence of typical colchicinized-like (c-like) mitoses on slides obtained from animals exposed to organotin compounds, directly confirmed the high mitotic spindle-inhibiting potency of these chemicals. In addition, by comparative analysis of spermatocyte chromosomes from untreated specimens (negative controls) and specimens treated with the triorganotin(IV) complexes, structural damages such as 'achromatic lesions' and 'chromosome breakages' have been identified.

FISH mapping of 18S-28S and 5S ribosomal DNA, (GATA)n and (TTAGGG)n telomeric repeats in the periwinkle Melarhaphe neritoides (Prosobranchia, Gastropoda, Caenogastropoda).

Spermatocyte chromosomes of Melarhaphe neritoides (Mollusca, Prosobranchia, Caenogastropoda) were studied using fluorescent in situ hybridization (FISH) with four repetitive DNA probes (18S rDNA, 5S rDNA, (TTAGGG)n and (GATA)n). Single-colour FISH consistently mapped one chromosome pair per spread using either 18S or 5S rDNA as probes. The telomeric sequence (TTAGGG)n hybridized with termini of all chromosomes whereas the (GATA)n probe did not label any areas. Simultaneous 18S-5S rDNA and 18S-(TTAGGG)n FISH demonstrated that repeated units of the three multicopy families are closely associated on the same chromosome pair.

Chromosome analysis using different staining techniques and fluorescent in situ hybridization in Cerithium vulgatum (Gastropoda: Cerithiidae).

In the present paper one population of the large" subtidal mollusc Cerithium vulgatum Bruguière, 1792 (Gastropoda: Cerithiidae) from the Northwestern coast of Sicily was investigated from a karyological point of view. The chromosome complement was Giemsa stained, conventionally karyotyped in 18 homomorphic chromosome pairs (10 bi-armed and 8 mono-armed), and subsequently analysed using silver, CMA3 and DAPI staining, and fluorescent in situ hybridization (FISH) with three repetitive DNA probes [ribosomal DNA (rDNA), (TTAGGG)n and (GATA)n]. FISH with the rDNA probe consistently mapped major ribosomal sites (18S-28S rDNA) in the terminal region of the short arms of one small sized mono-armed chromosome pair. Ribosomal DNA was transciptionally active as indicated by its preferential impregnation with silver nitrate (Ag-NOR) and did not contain a high amount of GC base pairs as suggested by the lack of a bright CMA3 fluorescence. The (TTAGGG)n telomeric probe was hybridized to the termini of nearly all chromosomes, thus demonstrating that, in C. tulgatum, this sequence has been conserved during the genomic evolution. The finding of the telomeric hexanucleotide in six species belonging to the three high taxa of Gastropoda supports the notion that this sequence is widespread within this class. The (GATA)n probe did not label any chromosome regions except for a minute terminal area of a single bivalent at pachytene stage.

Physical mapping of rDNA genes, (TTAGGG)n telomeric sequence and other karyological features in two earthworms of the family Lumbricidae (Annelida: Oligochaeta).

A cytogenetical study was carried out on the chromosomes and nuclear DNA amounts of the terrestrial earthworms Octodrilus complanatus and Eisenia foetida (Annelida: Oligochaeta: Lumbricidae). Chromosomes were studied using Giemsa staining, banding methods and fluorescent in situ hybridization (FISH) with two repetitive DNA probes [rDNA and (TTAGGG)n]. rDNA FISH and silver staining consistently identified one chromosome pair per spread in both species. The telomeric sequence (TTAGGG)n hybridized with termini of all the chromosomes in both earthworms. Flow cytometry DNA assays showed that O. complanatus and E. foetida had different nuclear DNA contents (2C value=1.72 and=1.40 pg, respectively) but very similar base composition in their genomes.

Karyotype analysis, banding, and fluorescent in situ hybridization in the scarab beetle Gymnopleurus sturmi McLeay (Coleoptera Scarabaeoidea : Scarabaeidae).

Conventional staining, differential banding, and in situ hybridization with both ribosomal and telomeric probes to mitotic chromosomes of Gymnopleurus sturmi (Scarabaeoidea : Scarabaeidae) are described. The karyotype is distinguished by a pericentric inversion polymorphism in chromosome 3, which is either acrocentric or subtelocentric. Silver staining (Ag-NOR) and chromomycin A3 (CMA3), failed to study the detection of nucleolar organizer regions (NORs), due to the extensive silver and CMA3 stainability of all GC-rich heterochromatin. Fluorescent in situ hybridization (FISH) using a Paracentrotus lividus (Echinodermata) rDNA probe mapped the ribosomal RNA genes (rDNA). FISH with the all-human telomeric sequences (TTAGGG)n revealed a lack of homology between the telomeric probe and the telomeres of G. sturmi. This suggests that the telomeric hesanucleotide (TTAGGG)n is not so conserved within eukaryotes as it has been hypothesized.

NOR regions of polychaete worms of the genus Ophryotrocha studied by chromosome banding techniques and FISH.

This article reports the results of cytogenetic analyses carried out on 10 species of polychaete worms belonging to the genus Ophryotrocha (Dorvilleidae). Nucleolar organizer regions (NORs) were characterized by Ag staining, C-banding, CMA3 staining, and ribosomal fluorescent in situ hybridization (rDNA FISH). Extensive intraspecific variation in NOR number and distribution were observed in O. costlowi, O. sp. macrovifera, O. notoglandulata, O.l. labronica, O. l. pacifica (2n = 6), O. p. puerilis, O. diadema (2n = 8), O. hartmanni, O. gracilis (2n = 10). In O. sp. robusta (2n = 10), Ag-NORs were always located on a single chromosome pair. CMA3 staining suggests a possible trend toward a GC-rich rDNA compartmentalization. In O.l. labronica, O. p. puerilis, O. diadema, and O. sp. robusta rDNA FISH shows that Ag and FISH signals coincide. Results from C-banding seem to indicate that the increased genome size (GS) observed in O. sp. macrovifera (0.8 pg) and O. hartmanni (1.16 pg) compared to the base GS value of the genus (0.4 pg) cannot be attributed to variation in the heterochromatin content.

Chromosomal analysis of repeated DNAs in the rainbow wrasse Coris julis (Pisces, Labridae).

Despite the interest of several authors, the karyotype of the labrid C. julis is still debated and in particular the presence of sex-chromosomes is still contradictory. In order to analyze the karyotype organization of C. julis we have performed an analysis with classical and molecular cytogenetic techniques. Our results after silver-, CMA3- and DAPI-staining, C-banding and fluorescent in situ hybridization (FISH) using 28S rDNA, 5S rDNA, and telomeric repeat (TTAGGG)n, as probes allowed us to characterize the chromosomal location of several repetitive DNAs of C. julis. Finally, regardless of the technique used, no difference in the chromosome complement was found between males and females.

18S-28S rDNA is interspersed throughout heterochromatin in the millipede Enologus oxypygum (Diplopoda).

In the present study, somatic metaphase chromosomes of the millipede Enologus oxypygum (Diplopoda: Julida) were hybridized in situ with a sea urchin (Echinodermata) ribosomal probe (prR14) in order to map major RNA genes (rDNA). Chromosomal preparations were also silver stained (Ag-NOR) to evaluate the rDNA transcriptional activity. Our results indicate that RNA genes are throughout heterochromatin in eight chromosomes involving 1/4 of the total heterochromatin which, in this species, is about 67% of the total DNA. Ag-NOR and FISH patterns were not coincident.

Chromosomal location of ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n telomeric repeats in the neogastropod Fasciolaria lignaria (Mollusca: Prosobranchia).

This paper reports on a successful application of fluorescent in situ hybridization (FISH) with three repetitive DNA probes (ribosomal DNA (rDNA), (GATA)n and (TTAGGG)n) in the chromosomes of Fasciolaria lignaria (Mollusca: Prosobranchia: Neogastropoda). rDNA FISH consistently identified four chromosome pairs per spread in the three examined specimens. The telomeric sequence (TTAGGG)n hybridized with termini of all chromosomes. GATA FISH revealed abundant, dispersed minisatellite regions which were not associated to the XY sex-determining mechanism as indicated by the absence of a Y specific pattern of labelling.

Chromosome analysis and rDNA FISH in the stag beetle Dorcus parallelipipedus L. (Coleoptera: Scarabaeoidea: Lucanidae).

In the present work the chromosome complement (2n = 18; 8AA + XY) of the stag beetle Dorcus parallelipipedus L. (Scarabaeoidea: Lucanidae) is analyzed using conventional Giemsa staining, banding techniques and ribosomal fluorescent in situ hybridization (rDNA FISH). rDNA FISH remains the unique tool for providing a clear-cut identification of Nucleolar Organizer Regions (NORs) when conventional banding methods such as silver- and CMA3-staining proved to be inadequate. The dull, homogeneous CMA3 fluorescence of all chromosomes indicates the absence of markedly GC rich compartmentalized regions in D. parallelipipedus genome. Silver impregnation inadequacy in detecting NOR regions is to be sought in the unusual extensive silver stainability of heterochromatic material which, on the contrary of what stated for vertebrates, seems to be a common feature in Scarabaeoidea species.

Ribosomal DNA location in the scarab beetle Thorectes intermedius (Costa) (Coleoptera: Geotrupidae) using banding and fluorescent in-situ hybridization.

Mitotic metaphase chromosomes of the scarab beetle Thorectes intermedius (Costa) (Coleoptera Scarabaeoidea: Geotrupidae) were analyzed using various banding methods and fluorescent in-situ hybridization (FISH) with a ribosomal probe. The results obtained indicate that silver and CMA3 staining are unable to localize the chromosome sites of nucleolar organizer regions (NORs). Such an inadequacy is a consequence of the extensive silver and CMA3 stainability of both constitutive heterochromatin and heterochromatin associated to the NORs.

Cytogenetic studies in some species of Scorpaeniformes (Teleostei: Percomorpha).

Cytogenetic studies were performed in four species of two scorpaeniform teleost families: Scorpaenidae and Triglidae. The karyotypes of Trigla lucerna, Trigloporus lastoviza (Triglidae), Scorpaena porcus and S. notata (Scorpaenidae) were analysed using various banding methods and in situ hybridization with a telomeric probe. In the two Scorpaena species, modest morphological divergence corresponded to considerable karyotype reorganization, while in the two Triglidae substantial phenotypical divergence corresponded to limited chromosomal changes. These data stress the need for a taxonomical re-evaluation of these teleosts based on characters independent of morphology.

High heterochromatin content in somatic chromosomes of two unrelated species of Diplopoda (Myriapoda).

For the first time, a conventional analysis of C-banded karyotypes was carried out in two distantly related diplopod species; this revealed an impressive percentage of heterochromatin in both genomes. In Acanthopetalum sicanum (Order Callipodida) (2n = 12), heterochromatin constitutes about 60% of the total DNA in females and 56% in males, whereas in Enologus oxypygum (Order Julida) (2n = 22) it is about 67% in both sexes. Heterochromatin of the two species was found to be similar in base composition (AT rich) and heterochromatin distribution, indicating that it has accumulated in a species-specific manner. Sex-determining mechanisms of the XY type were detected in both A. sicanum and E. oxypygum. In A. sicanum, the Y presented the lowest heterochromatic content of all chromosomes in the karyotype, whereas the X presented the highest.

Spermatocyte chromosome analysis of Helicella virgata (Pulmonata: Helicidae): silver-stained and C-banded chromosomes.

Chromosome numbers of the snail Helicella virgata from the fields of Castellammare del Golfo (Sicily) are n = 26 and 2n = 52. Silver-staining analyses of testicular cells suggest that both mitotic and meiotic chromosomes are involved in nucleolus organization. A within-individual variability in NOR-banding pattern is present in each of the 20 specimens analyzed.

Karyological studies in Coris julis (Pisces, Labridae).

In the present investigation the diploid number 2n = 48 (NF = 58) has been determined for females, primary males, and secondary males of Coris julis from the Gulf of Palermo. Differentiated sex chromosomes have not been observed in the population under study.