The karyotype of Nodipecten nodosus (Bivalvia: Pectinidae)

Earlier karyotypical work on Nodipecten nodosus embryos indicated that this species has a diploid number of 38, with six pairs respectively of metacentric and submetacentric chromosomes and seven pairs of subtelocentric chromosomes, although there were some difficulties in obtaining complete metaphases. The present work provides additional results on specific regions of the chromosomes in N. nodosus and, by meiotic studies, confirms the chromosome number with more reliability. Active nucleolar organizer regions (NOR), detected in mitotic metaphases from embryos, can be characterized in N. nodosus by a high level of heteromorphism of NOR-sites, indicating that these regions are not appropriate as chromosomal markers in this species. The procedure for detecting constitutive heterochromatin of chromosomes allowed us to observe most of the heterochromatic blocks at a pericentromeric position and some at telomeric and interstitial positions. The analysis of meiotic chromosomes from gonad tissue revealed the presence of 19 bivalents during metaphase I, all homomorphic and isopicnotic, confirming the previously described diploid chromosomal number of 38 for N. nodosus. From these results, some evolutionary aspects of the Pectinidae are briefly discussed.     

Population differences in the expression of nucleolus organizer regions in the grasshopperEyprepocnemis plorans

Summary Fluorescence in situ hybridization revealed the presence of ribosomal RNA genes in paracentromeric regions of all A chromosomes and in the distal half of B chromosomes in embryonic cells from Moroccan specimens of the grasshopperEyprepocnemis plorans. The expression of these genes was monitored by the presence of nucleoli attached to each chromosome bivalent in diplotene cells from males collected from two different Moroccan populations and was compared to previous data of Spanish populations. Whereas only the nucleolus organizer regions (NORs) on S₉–S₁₁ and X chromosomes were active in the Spanish specimens. Moroccan individuals showed NOR activity in all chromosomes. The rRNA genes on the B chromosome were inactive in both populations. The S₉ and S₁₀ NORs were less active in Moroccan specimens than in Spanish specimen, which might be partly explained by the negative interdependence for expression of the S₁₀ NOR with respect to those on L₂ and X chromosomes. On the other hand, the X NOR was more active in Moroccan specimens than in Spanish specimens, and this might be partly due to the positive effect that the presence of B chromosomes has on the expression of this NOR. The implications of these observations on current models of NOR activity regulation are discussed.Abbreviation NOR nucleolus organizer region     

Satellited Y chromosomes: Structure,origin, and clinical significance

Summary Three cases of inherited satellited Y chromosomes (Yqs) were analysed using several cytogenetic techniques. The cytogenetic data of the 14 cases of Yqs chromosomes described to date were reviewed. All Yqs chromosomes carry an active nucleolus organizer region (NOR) in their long arm and must have developed from translocations involving the short arms of the acrocentric autosomes. The structure of the heterochromatic satellite region in the Yqs chromosomes shows conspicuous inter-familial differences; this permits the reconstruction of the translocations from which the various Yqs were derived. Some causal factors leading to the development of Yqs chromosomes are considered: the specific localization of the four satellite DNAs and highly methylated DNA sequences in the karyotype, and some new experimental data on the spatial arrangement of heterochromatic regions in interphase nuclei. These provide distinct evidence for a preferential involvement of the autosomes 15 and 22 in the translocations with the Y heterochromatin. All clinical reports documenting Yqs males born with malformations were reviewed. It appears that the presence of an extra NOR and NOR-associated heterochromatin in the Yqs chromosomes does not cause any phenotypic abnormalities (as long as the Y euchromatin is intact). The possibility that a Yqs chromosome predisposes to non-disjunction and/or to translocations of other chromosomes is discussed.     

The origin of mitotic sex-chromosome association in the brush-tailed possum, Trichosurus vulpecula (marsupalia:phalangeridae)

Nonrandom associations between the sex chromosomes of the brush-tailed possum, Trichosurus vulpecula, were found to be due to association of nucleolar organizer regions (NOR's) on the X and Y chromosomes. NOR association was also observed between an autosome and the X chromosome. These findings, based on silver staining, are in contrast to the report of MURRAY (1977), who observed sex-chromosome association in this animal and indicated that these nonrandom associations may reflect an association between heterochromatic regions during interphase. We observed only two pairs of NOR's per cell in this animal, one autosomal and one on the sex chromosomes, rather than the several such regions observed by MURRAY, who used an N-banding technique. We discuss the problem of nonhomologous chromosome association in mammalian cells as influenced by heterochromatin and NOR's and find little support for nonhomologous chromosome associations at mitotic metaphase due to heterochromatin association.     

Cytogenetic characterization of Rhamdia quelen (Siluriformes, Heptapteridae) from the Bodoquena Plateau, Mato Grosso do Sul, Brazil

We made a cytogenetic study of the fish Rhamdia quelen collected from the Bodoquena Plateau, an isolated national park region in Mato Grosso do Sul State, Brazil. The diploid number was 2n = 58, with 36 metacentric + 16 submetacentric + 6 subtelocentric chromosomes. We found one to three B chromosomes, which were metacentric and submetacentric and of medium size, showing both intra- and interindividual variation. The nucleolus organizer region (NOR) was located in the terminal region of the short arm of submetacentric pair 20. Staining with CMA3 fluorochrome revealed the NOR location, while there was no evidence of fluorescent staining with DAPI. C banding revealed heterochromatin mainly in the terminal regions of the chromosome arms, including the NOR pair. In addition, metacentric pair 2 showed three heterochromatic blocks in the terminal portions and in the pericentromeric region. The B chromosomes appeared euchromatic. The CB + CMA3 staining combination demonstrated only one chromosome pair with fluorescence, probably the NOR-bearing one, while CB + DAPI gave various fluorescent signals, including metacentric pair 2, indicating that these heterochromatic regions are AT-rich in this population of R. quelen. The R. quelen population in this isolated region of Brazil is chromosomally distinct from that of other populations that have been studied.     

Longitudinal differentiation of metaphase chromosomes of Indian muntjac as studied by restriction enzyme digestion,in situ hybridization with cloned DNA probes and distamycin A plus DAPI fluorescence staining

The longitudinal differentiation of metaphase chromosomes of the Indian muntjac was studied by digestion with restriction enzymes, in situ hybridization with cloned DNA probes and distamycin A plus DAPI (4-6-diamidino-2-phenylindole) fluorescence staining. The centromeric regions of chromosomes 3 and 3 + X of a male Indian muntjac cell line were distinct from each other and different from those of other chromosomes. Digestion with a combination of EcoRI^* and Sau3A revealed a pattern corresponding to that of C-banding. Digestion with AluI, EcoRII or RsaI yielded a band specific to the centromeric region only in chromosomes 3 and 3 + X. Furthermore, HinfI digestion yielded only a band at the centromeric region of chromosome 3, whereas DA-DAPI staining revealed a single band limited to the extreme end of the C-band heterochromatin of the short arm of 3 + X. These results suggest that centromeres of Indian muntjac chromosomes contain at least four different types of repetitive DNA. Such diversity in heterochromatin was also confirmed by in situ hybridization using specific DNA probes isolated and cloned from highly repetitive DNA families. Heterozygosity between chromosome homologs was revealed by restriction enzyme banding. Evidence is presented for the presence of nucleolus organizer regions (NORs) on the long arm of chromosome 1 as well as on the secondary constrictions of 3 and 3 + X.Abbreviations DA distamycin A - DAPI 4-6-diamidino-2-phenylindole - NOR(s) nucleolus organizer region(s) - PBS phosphate-buffered saline - PI propidium iodide     

Nature and distribution of constitutive heterochromatin and NOR location in the grasshopper Phaeoparia megacephala (Romaleidae: Orthoptera)

The constitutive heterochromatin (CH) of Phaeoparia megacephala was studied using C-banding and fluorochrome staining (CMA3, DAPI and acridine orange). The nucleolar organizer regions (NOR) were identified with silver staining. The chromosome complement of this species was 2n = 23, XO in males, and 2n = 24, XX in females. The CH was pericentromeric in all chromosomes. L1, L2, L3 and X chromosomes showed large blocks of CH, while the medium and small chromosomes had small blocks. The staining procedure with acridine orange revealed the same pattern. All the pericentromeric regions showed small blocks of CMA3-positive constitutive heterochromatin (GC-rich regions), while only part of the large C-band positive chromosome segments (L1, L2, L3 and X) were CMA3 positive. This character demonstrates an uncommon heterogeneity of constitutive heterochromatin in P. megacephala. The fluorochrome DAPI did not reveal DAPI-positive regions (AT-rich regions). Silver staining revealed only one pair of medium chromosomes with NOR.     

Chromosome location of the ribosomal RNA genes in Triturus vulgaris meridionalis (Amphibia,Urodela)

Ribosomal genes have been localized on mitotic and lampbrush chromosomes of 20 specimens of Triturus vulgaris meridionalis by in situ hybridization with ³H 18S+28S rRNA. The results may be summarized as follows: 1) each individual shows positive in situ hybridization at the nucleolus organizing region (NOR) on chromosome XI; 2) in addition, many specimens exhibit a positive reaction in chromosomal sites other than the NOR (additional ribosomal sites); 3) the chromosomal distribution of the additional sites appears to be identical in different tissues from the same specimen and to follow a specific individual pattern; 4) the additional ribosomal sites are preferentially found at the telomeric, centromeric or C-band regions of the chromosomes involved.Abbreviations rRNA ribosomal RNA - NOR nucleolus organizer region - rDNA the DNA sequences coding for 18S+28S rRNA plus the intervening spacer sequences - SSC 0.15 M sodium chloride, 0.015 sodium citrate, pH 7     

Cytogenetic analyses of two Curimatidae species (Pisces; Characiformes) from the Paranapanema and Tietê Rivers.

Cytogenetic analyses were performed in two Curimatidae species (Steindachnerina insculpta and Cyphocharax modesta) from the Paranapanema and Tietê Rivers (S?o Paulo State, Brazil), showing a karyotype composed of 54 meta-submetacentric chromosomes in both species. Silver- and chromomycyn-staining and fluorescent in situ hybridization (FISH) using a 18S rDNA probe indicated that the nucleolar organizer regions (NORs) of both species are localized in the terminal region of the long arm of two metacentric chromosomes. Although a single NOR system was evidenced in both analyzed species, S. insculpta and C. modesta presented the nucleolar organizer regions in distinct chromosome pairs, indicating that these cistrons can be considered cytogenetic markers. Variation on the amount and distribution of the constitutive heterochromatin (C-bands) could also be detected between the two species - while S. insculpta presented few heterochromatic blocks, intensely stained C-bands were evidenced in C. modesta specially in the terminal region of the long arm of the NOR-bearing chromosomes. Although most Curimatidae species have been characterized by homogeneous karyotypes, isolated populations could be established under different environmental conditions leading to karyotype micro-structure variations specially related to the NORs localization and C-banding distribution. The obtained data were useful for the cytogenetic characterization and differentiation of S. insculpta and C. modesta and could be used in evolutionary inferences in the Curimatidae group.     

Cytogenetic analysis of Astylus antis (Perty, 1830) (Coleoptera, Melyridae): Karyotype, heterochromatin and location of ribosomal genes

Cytogenetic analysis of Astylus antis using mitotic and meiotic cells was performed to characterize the haploid and diploid numbers, sex determination system, chromosome morphology, constitutive heterochromatin distribution pattern and chromosomes carrying nucleolus organizer regions (NORs). Analysis of spermatogonial metaphase cells revealed the diploid number 2n = 18, with mostly metacentric chromosomes. Metaphase I cells exhibited 2n = 8II+Xyp and a parachute configuration of the sex chromosomes. Spermatogonial metaphase cells submitted to C-banding showed the presence of small dots of constitutive heterochromatin in the centromeric regions of nearly all the autosomes and on the short arm of the X chromosome (Xp), as well as an additional band on one of the arms of pair 1. Mitotic cells submitted to double staining with base-specific fluorochromes (DAPI-CMA(3) ) revealed no regions rich in A+T or G+C sequences. Analysis of spermatogonial mitotic cells after sequential Giemsa/AgNO (3) staining did not reveal any specific mark on the chromosomes. Meiotic metaphase I cells stained with silver nitrate revealed a strong impregnation associated to the sex chromosomes, and in situ hybridization with an 18S rDNA probe showed ribosomal cistrons in an autosomal bivalent.     

Chromosome studies of Saguinus midas niger (Callithricidae,Primates) from Tucurui,Para, Brazil: Comparison with the karyotype of Callithrix jacchus

The karotype of Saguinus midas niger was studied by G-, C-, and nuclear organizer region (NOR)-banding techniques. Variations in C-banding patterns were observed in some chromosomes. The banding patterns obtained were compared with those previously described for Callithrix jacchus. The two species differ by a reciprocal translocation involving pairs 9 and 16; by a paracentric inversion in chromosomes 1, 13, 14, 18, and 22; and by a pericentric inversion in at least four subtelocentric pairs (chromosomes 19, 20, 21, and 22), dislocating the nucleolar organizer region from the small short arm in C. jacchus to the proximal segment of the long arm in S. m. niger (or vice versa). The amount of constitutive heterochromatin is greater in S. m. niger than in C. jacchus, especially in chromosomes 4, 7, and 14. The Y chromosome is smaller in C. jacchus than in S. m. niger.     

Regional differences in immunostainability of isolated metaphase chromosomes of Indian muntjac with anti-Z-DNA antibody

The distribution of Z-form DNA along the length of metaphase chromosomes of Indian muntjac was studied by indirect immunofluorescence procedures using an antibody specific to the Z-DNA conformation. Several fixation conditions were compared for reproducible detection of Z-DNA in isolated metaphase chromosomes. Fixation of chromosomes with 45% acetic acid alone gave reproducible reactivity with the antibody. When fixation was done either with Carnoy's solution (3:1 methanol:acetic acid) or with 75% alcohol alone, the antibody binding was at background level. Acetic acid-fixed chromosomes exhibited intense fluorescence both at C-band heterochromatin and at nucleolus organizer regions (NORs). The euchromatic regions had weakly, but clearly, stained bands, which were quite similar to the chromomycin A₃ R-bands. After treatment with topoisomerase I, the immunofluorescence at NORs and R-bands disappeared, but only a slight decrease in immunofluorescence intensity was observed at C-band regions. We suggest that this difference in the immunoreactivity of NORs and R-bands from C-bands reflects a difference in gene activity among these regions. Possible molecular mechanisms involved in Z-DNA immunoreactivity are discussed, based on SDS-polyacrylamide gel electrophoretic analysis of chromosomal proteins after extraction of metaphase chromosomes with different fixative solutions.Abbreviations PI propidium iodide - NOR(s) nucleolus organizer region(s) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis Deceased, April 23, 1988     

Alpha and beta heterochromatin in polytene chromosome 2 ofDrosophila melanogaster

The formation of alpha and beta heterochromatin in chromosomes of Drosophila melanogaster was studied in salivary glands (SGs) and pseudonurse cells (PNCs). In SGs of X0, XY, XYY, XX and XXY individuals the amounts of alpha heterochromatin were similar, suggesting that the Y chromosome does not substantially contribute to alpha heterochromatin formation. Pericentric heterochromatin developed a linear sequence of blocks in PNCs, showing morphology of both alpha and beta heterochromatin. In situ hybridization with Rsp sequences (H _o clone) revealed that the most proximal heterochromatic segment of the mitotic map (region h39) formed a polytenized block in PNCs. Dot analysis showed that the clone had a hybridization rate with PNC-DNA very close to that with DNA from mainly diploid head cells, whereas the homologous SG-DNA was dramatically underrepresented. A similar increase of DNA representation in PNC was found for AAGAC satellite DNA. The mitotic region h44 was found not to polytenize in the SG chromosome, whereas in PNC chromosome 2 this region was partly polytenized and presented as an array of several blocks of alpha and beta heterochromatin. The mapping of deficiencies with proximal breakpoints in the most distal heterochromatin segments h35 in arm 2L and h46 in 2R showed that the mitotic eu-heterochromatin transitions were located in SG chromosomes distally to the polytene 40E and 41C regions, respectively. Thus, the transition zones between mitotic hetero- and euchromatin are located in banded polytene euchromatin. A scheme for dynamic organization of pericentric heterochromatin in nuclei with polytene chromosomes is proposed. Received: 17 November 1995; in revised form: 10 April 1996 / Accepted: 18 September 1996     

Unusual primitive heteromorphic ZZ/ZW sex chromosomes in Proceratophrys boiei (Anura, Cycloramphidae, Alsodinae), with description of C-Band interpopulational polymorphism

We performed cytogenetic analyses on specimens from three population samples of Proceratophrys boiei from southeastern and northeastern Brazil. We stained chromosomes of mitotic and meiotic cells with Giemsa, C-banding and Ag-NOR methods. All specimens of P. boiei presented a karyotype with a full chromosome complement of 2n=22, metacentric and submetacentric. We observed the secondary constriction within the short arm of pair 8, which was in the same position of the nucleolus organizer region (NOR). NOR heteromorphism was observed within two specimens from the municipality of Mata de S?o Jo?o (northeastern Bahia State). The C-banding evidenced an unusual heterochromatic pattern in the genome of P. boiei. In the southern most population samples (S?o Paulo State), we observed large blocks of heterochromatin in the centromeric regions of all chromosomes, whereas the northernmost samples (Bahia State) presented a small amount of constitutive heterochromatin. We suppose that this geographic variation in heterochromatin quantities could be due to heterochromatinization of some chromosome regions in the genome of the S?o Paulo samples. Furthermore, females from S?o Paulo presented, within chromosome pair 1 from C-banded karyotypes, one homologous chromosome almost heterochromatic, whereas males had heterochromatin restricted to the centromeric region. This unusual heterochromatic arrangement led us to assume that P. boiei owns a ZZ/ZW type of sexual determination system. This finding is very important, as this is the first record of ZZ/ZW sex chromosomes within Cycloramphidae. We believe that the cytogenetic differences found between southeastern and northeastern Brazilian population samples of P. boiei strongly supports the existence of a species complex under the name P. boiei, and the requirement of taxonomic and systematic reviews by morphological, bioacoustical, molecular, and cytogenetic data could define this taxonomic issue in the future.     

Complex sex chromosome system in Eigenmannia sp. (Pisces,Gymnotiformes)

Chromosomes of Eigenmannia sp. (7 males and 15 females) collected from the Tietê River in Botucatu (SP, Brazil) were examined from gill, kidney and testicular cells. The diploid chromosome number in males was 2n=31 and in females, 2n=32. In both sexes the number of chromosomal arms was 40. The difference in diploid number was due to the fusion of two acrocentrics. Mitotic and meiotic studies suggested that one of the fused acrocentrics was the Y chromosome. The sex-determining mechanism in Eigenmannia sp. could therefore be XX, AA in the female and X, \-YA A in the males. One of the males presented 2n=30 chromosomes due to the occurrence of another fusion of acrocentrics. C-banding analysis of the mitotic chromosomes revealed constitutive heterochromatin in the centromeric regions of all acrocentrics. However, small metacentrics were C-band negative. The YA chromosome is C-band negative except for a small amount of heterochromatin in the centromeric region. The nucleolar organizer region as identified by Ag-staining is present in the interstitial region of chromosome pair No. 10.     

Levels of conservation and variation of heterochromatin and nucleolus organizers in the Bovidae

Chromomycin A3 banding of the mitotic sets of 10 species of Bovidae (cattle, wisent, yak, banteng, gaur, red buffalo, swamp buffalo, sheep, mufflon, and goat) serves to demarcate both centromeric constitutive heterochromatin and R-banding patterns capable of identifying all the chromosomes within a given complement. In all species significant amounts of chromomycin-bright heterochromatin are present at the centromeres of all autosomes, though there was a high degree of intra- and inter-individual variation in the size of the heterochromatic blocks. Marked interspecies differences in the centromeric patterns were evident. The X chromosomes contained appreciable amounts of centromeric heterochromatin only in the two buffaloes. All the animals studied lacked distamycin A - diamidinophenylindole type heterochromatin. AgNO3 staining was applied sequentially to detect the location of active nucleolus organizer regions (NORs). The distribution of NORs was reasonably conservative in most of the species. An exceptional situation was found in the two buffaloes, where only one NOR pair matched with the standard karyotype of the Bovidae.     

X-Y-autosome translocation, chromosome compaction, NOR expression and heterochromatin insulation in the Scarabaeid beetle Dynastes hercules hercules

The karyotype of the giant beetle Dynastes hercules hercules is composed of only 16 autosomes and large sex chromosomes. Meiotic studies in the males showed that a large part of the sex chromosomes undergo synapsis at pachynema similarly to autosomes, demonstrating that both derived from an autosome-gonosome translocation. Therefore, karyotype formula is 18,neoXY. The heterochromatisation of the neoX short arm at pachynema indicates that it corresponds to the ancestral X. It carries the nucleolar organizer region (NOR) in its proximal part, which is undercondensed, especially in male mitotic and meiotic cells. In female mitotic cells, both NOR staining and undercondensation were more difficult to observe in the neoX short arms. In somatic interphase nuclei, NOR expression strongly varies with the sex. Two separated compact groups of silver dots were observed in female nuclei, while a single dispersed and large group of silver deposit exists in the males. Both the lower condensation and the higher NOR expression of the single neoX of the males, compared to each of the two neoXs of the females, is interpreted to be a consequence of dosage compensation, a mechanism not yet described in Coleoptera. In mammals as well as in Coleoptera, the carriers of gonosome-autosome translocations not exhibiting deleterious phenotypes show constitutive heterochromatin at the autosome-gonosome junction. Thus, heterochromatin may play an important universal role by clearly separating chromosome segments with different regulations of gene expression, such as inactivation or dosage compensation of the X chromosome on the one side and a conventional autosomal structure on the other side.     

Localization of chromosomal DNA sequences homologous to ribosomal gene type I insertion DNA in Drosophila melanogaster

Summary Chromosomal sites which have DNA homology to the 1 kb (kilobase pair) BamHI restrictable fragment of the 5 kb type I insertion present in many ribosomal genes in Drosophila melanogaster, were identified by using in situ hybridization and autoradiography. XX and XY complements of polytene chromosomes showed the nucleolus and chromocenter to be heavily labeled. Of the light label over euchromatic regions, the 102C band of chromosome 4 labeled particularly intensely. In mitotic XX and XY complements, the NORs (nucleolus organizer regions) of both sex chromosomes labeled as did the centromeric heterochromatin of autosomes. Label also appeared less frequently over telomeric and euchromatic regions.     

Heterochromatin markers: A search for heterochromatin specific middle repetitive sequences in Drosophila

We sought for cloned sequences of middle repetitive (MR) complexity that mark obligatory heterochromatic regions. Total genome probes were employed in a differential screening procedure to recover X-specific, Y-specific and autosomal specific heterochromatic sequences. X- and Y-linked sequences were recovered in the same experiment. (Y-linked clones will be described elsewhere). All nine independent, non-identical X-specific clones were found to be partially homologous to one another and to type I rDNA insertion. No other X-specific Bam HI or HindIII clones were found. In situ hybridization to normal and inverted chromosomes revealed extensive homology in the heterochromatin spanning the nucleolus organizer (NOR) and the eu-heterochromatin junction. Eleven clones which are underrepresented in polytene chromosomes were selected in another differential screening. None was autosome-specific. Five were of nucleolar origin. Among them a presumptive type II 28SrDNA insertion sequence was clearly localized within the X-chromosome proximal heterochromatin in addition to the known localization of the X and Y nucleolar organizers. We mapped three clones to major sites on the Y chromosome and to secondary autosomal sites. The results are discussed with regard to the complexity of heterochromatin organization.     

Karyology of the Antarctic scallop Adamussium colbecki, with some comments on the karyological evolution of pectinids

Karyotype, location of the nucleolar organiser region (NOR) and heterochromatin presence and composition were studied in the Antarctic scallop Adamussium colbecki Smith, 1902. The karyotype exhibits 2n = 38 chromosomes with 11 pairs of metacentrics, 5 of submetacentrics, one subtelocentric and two telocentrics. Ag–NOR, CMA₃, DA/MM and NOR–FISH evidenced paracentromeric NORs on the short arm of 2nd pair chromosomes. Digestion with three restriction endonucleases followed by sequential staining with Giemsa, CMA₃ and DAPI evidenced on all chromosomes centromeric heterochromatin positive for both DAPI and CMA₃. In situ hybridisation analysis showed the presence of an AT-rich satellite DNA in the centromeric heterochromatin of several chromosomes. A mosaicism was detected in the germinal cell lines of one specimen, as in six of the 20 plates examined the set had 37 chromosomes with a missing pair of telocentrics and an unpaired metacentric. Comparison of the chromosome sets of all the pectinids studied to date and comparison with a phyletic tree obtained from molecular mitochondrial genes studies yielded good agreement between karyotype morphology and taxonomic classification.