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.     

Differential characterization of holocentric chromosomes in triatomines (Heteroptera, Triatominae) using different staining techniques and fluorescent in situ hybridization

A comparative study of holocentric chromosomes in the triatomine species Panstrongylus megistus, Rhodnius pallescens and Triatoma infestans was carried out in order to characterize heterochromatin, rDNA active sites and nucleolar proteins. Cytological preparations of seminiferous tubules were stained by silver impregnation, C banding, fluorochromes cma3/da and dapi/da, and fluorescent in situ hybridization (FISH) with Drosophila melanogaster 28S rDNA probe. Our results showed interesting aspects of the organization of chromatin and chromosomes in the meiotic cells of these insects. In R. pallescens, sex chromosomes (X, Y) were distinct from autosomes, when submitted to silver impregnation, C banding, CMA3 staining, and FISH, confirming that these chromosomes bear nucleolar organizer regions (NORs). In P. megistus, two of the three sex chromosomes were CMA3/DAPI-; at early meiotic prophase and at diakinesis, silver impregnation corresponded with FISH signals, indicating that in this species, two chromosomes (probably a sex chromosome and an autosome) bear NORs. In T. infestans, silver nitrate and FISH also stained corresponding areas on meiotic chromosomes. Our data suggest that in triatomines, in general, the number and location of NORs are species-specific. These regions may be considered important chromosome markers for comparative studies to improve the understanding of evolutionary mechanisms in these hematophagous insects.     

Comparative cytogenetics of three species of Dichotomius (Coleoptera, Scarabaeidae)

Meiotic and mitotic chromosomes of Dichotomius nisus, D. semisquamosus and D. sericeus were analyzed after conventional staining, C-banding and silver nitrate staining. In addition, Dichotomius nisus and D. semisquamosus chromosomes were also analyzed after fluorescent in situ hybridization (FISH) with an rDNA probe. The species analyzed had an asymmetrical karyotype with 2n = 18 and meta-submetacentric chromosomes. The sex determination mechanism was of the Xy(p) type in D. nisus and D. semisquamosus and of the Xy (r) type in D. sericeus. C-banding revealed the presence of pericentromeric blocks of constitutive heterochromatin (CH) in all the chromosomes of the three species. After silver staining, the nucleolar organizer regions (NORs) were located in autosomes of D. semisquamosus and D. sericeus and in the sexual bivalent of D. nisus. FISH with an rDNA probe confirmed NORs location in D. semisquamosus and in D. nisus. Our results suggest that chromosome inversions and fusions occurred during the evolution of the group.     

Molecular cytogenetics and characterization of a ZZ/ZW sex chromosome system in Triportheus nematurus (Characiformes, Characidae)

Chromosomes of Triportheus nematurus, a fish species from family Characidae, were analyzed in order to establish the conventional karyotype, location of C-band positive heterochromatin, Ag-NORs, GC- and AT-rich sites, and mapping of 18S and 5S rDNA with fluorescence in situ hybridization (FISH). The diploid number found was 2n = 52 chromosomes in both males and females. However, the females presented a pair of differentiated heteromorphic chromosomes, characterizing a ZZ/ZW sex chromosome system. The Z chromosome was metacentric and the largest one in the karyotype, bearing C-positive heterochromatin at pericentromeric and telomeric regions. The W chromosome was middle-sized submetacentric, appearing mostly heterochromatic after C-banding and presenting heterogeneous heterochromatin composed of GC- and AT-rich regions revealed by fluorochrome staining. Ag-NORs were also GC-rich and surrounded by heterochromatic regions, being located at the secondary constriction on the short arms of the second chromosome pair, in agreement with 18S rDNA sites detected with FISH. The 18S and 5S rDNA were aligned in tandem, representing an uncommon situation in fishes. The results obtained reinforce the basal condition of the ZZ/ZW sex system in the genus Triportheus, probably arisen prior to speciation in the group.     

Cytogenetic characterization of the bay scallop, Argopecten irradians irradians, by multiple staining techniques and fluorescence in situ hybridization

The chromosomes of Argopecten irradians irradians were studied by various cytogenetic approaches. Conventional chromosome characterization built on C-banding, DAPI-staining, and silver staining was complemented by the physical mapping of ribosomal DNA and telomeric sequence (TTAGGG)n by FISH. Results showed that the constitutive heterochromatin revealed by C-banding was mainly distributed at telomeric and centromeric regions. However, interstitial C-bands were also observed. The pattern of DAPI banding was almost consistent with that of C-banding. Silver staining revealed that NORs were located on the short arms of chromosome 3 and 10, and this was further confirmed by FISH using 18S-28S rDNA. 5S rDNA was mapped as two distinguishable loci on the long arm of chromosome 11. 18S-28S and 5S rDNA were located on different chromosomes by sequential FISH. FISH also showed that the vertebrate telomeric sequence (TTAGGG)n was located on both ends of each chromosome and no interstitial signals were detected. Sequential 18S-28S rDNA and (TTAGGG)n FISH demonstrated that repeated units of the two multicopy families were closely associated on the same chromosome pair.     

Diversification of a ZZ/ZW sex chromosome system in Characidium fish (Crenuchidae, Characiformes)

Karyotype and other chromosomal markers of Characidium cf. gomesi were analyzed using conventional (Giemsa-staining, Ag-NOR and C-banding) and molecular (Fluorescent in situ hybridization (FISH) with 18S and 5S rDNA biotinylated probes) techniques. Both sexes had invariably diploid chromosome number 2n = 50 while karyotypes of males and females differed. That of male consisted of 32 metacentric + 18 submetacentric chromosomes and that of female consisted 31 metacentric + 18 submetacentric + 1 subtelocentric chromosomes. The Z chromosome was medium-sized metacentric, while W was highly heterochromatinized subtelocentric element. NORs as revealed by Ag-staining were situated at 2–7 telomeric regions while FISH with 18S probes showed consistently 10 signals at telomeric regions. FISH with 5S rDNA probe showed constantly signals at one metacentric pair. Distribution of centromeric heterochromatin was mostly in all chromosome pairs, besides some telomeric sites. The common origin of the sex chromosome system of ZZ/ZW type in the karyotypes of other representatives of the genus analyzed so far might be hypothesized based on biogeography and partial phylogeny of the group.     

Cytogenetic analysis of the neotropical spider Nephilengys cruentata (Araneomorphae, Tetragnathidae): standard staining nors, C-bands and base-specific fluorochromes.

The aim of this work is to characterize Nephilengys cruentata in relation to the diploid number, chromosome morphology, type of sex determination chromosome system, chromosomes bearing the Nucleolar Organizer Regions (NORs), C-banding pattern, and AT or GC repetitive sequences. The chromosome preparations were submitted to standard staining (Giemsa), NOR silver impregnation, C-banding technique, and base-specific fluorochrome staining. The analysis of the cells showed 2n = 24 and 2n = 26 chromosomes in the embryos, and 2n = 26 in the ovarian cells, being all the chromosomes acrocentric. The long arm of the pairs 1, 2 and 3 showed an extensive negative heteropycnotic area when the mitotic metaphases were stained with Giemsa. The sexual chromosomes did not show differential characteristics that allowed to distinguish them from the other chromosomes of the complement. Considering the diploid numbers found in N. cruentata and the prevalence of X1X2 sex determination chromosome system in Tetragnathidae, N. cruentata seems to possess 2n = 24 = 22 + X1X2 in the males, and 2n = 26 = 22 + X1X1X2X2 in the females. The pairs 1, 2 and 3 showed NORs which are coincident with the negative heteropycnotic patterns. Using the C-banding technique, the pericentromeric region of the chromosomes revealed small quantity or even absence of constitutive heterochromatin, differing of the C-banding pattern described in other species of spiders. In N. cruentata the fluorochromes DAPI/DA, DAPI/MM and CMA3/DA revealed that the constitutive heterochromatin is rich in AT bases and the NORs possess repetitive sequences of GC bases.     

Constitutive heterochromatin, 5S and 18S rDNA genes in Apareiodon sp. (Characiformes, Parodontidae) with a ZZ/ZW sex chromosome system

Karyotype, sex chromosome system and cytogenetics characteristics of an unidentified species of the genus Apareiodon originating from Piquiri River (Paraná State, Brazil) were investigated using differential staining techniques (C-banding and Ag-staining) and fluorescent in situ hybridization (FISH) with 5S and 18S rDNA probes. The diploid chromosome number was 2n = 54 with 25 pairs of meta- (m) to submetacentric (sm) and 2 pairs of subtelocentric (st) chromosomes. The major ribosomal rDNA sites as revealed by Ag-staining and FISH with 18S rDNA probe were found in distal region of longer arm of st chromosome pair 26, while minor 5S sites were observed in the interstitial sites on chromosome pairs 2 (smaller cluster) and 7 (larger one). The C-positive heterochromatin had pericentromeric and telomeric distribution. The heteromorphic sex chromosome system consisted of male ZZ (pair 21) and female middle-sized m/st Z/W chromosomes. The pericentric inversion of heterochromatinized short arm of ancestral Z followed by multiplication of heterochromatin segments is hypothesized for origin of W chromosome. The observed karyotype and chromosomal markers corresponded to those found in other species of the genus.     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

Cytogenetic variability in genus odontocheila (Coleoptera,Cicindelidae): karyotypes,C-banding,NORs and localisation of ribosomal genes of O. confusa and O. nodicornis

Two species of Odontocheila, O. confusa and O. nodicornis, from the Neotropical Region were studied regarding their karyotypes, localisation and activity of ribosomal genes and C-banding. The species, although belonging to the same genus, have quite distinct karyotypes. O. confusa has 10 pairs of autosomes and a single sex chromosome mechanism of the XY/XX type, thus a diploid value of 2n = 22 in males and females. One aneuploid male with a diploid number of 2n = 20 and one male with three B chromosomes were found in a total of eight males studied. O. nodicornis has 17 autosomal pairs and also a single chromosome system but of the X0/XX type, thus a diploid value of 2n = 35 in males and 2n = 36 in females. Fluorescence in situ hybridisation (FISH) revealed the presence of rDNA clusters in two autosomes in both species in mitotic and meiotic figures. Silver staining of male interphase nuclei confirmed the FISH results and showed that all rDNA genes were active. C-banding analysis revealed the presence of constitutive heterochromatin in the centromeres of all chromosomes in the two species plus two pairs in O. nodicornis with terminal positive C-bands. These results are discussed from the cytogenetic and evolutionary point of view.     

Molecular cytogenetics of <Emphasis Type="Italic">Oligosarcus hepsetus</Emphasis> (Teleostei,Characiformes) from two Brazilian locations

Karyotype and cytogenetic markers of Oligosarcus hepsetus from two Brazilian locations in the Paraíba do Sul River Basin (Brazil) were investigated using differential staining techniques (C-banding, silver (Ag)- and chromomycin A₃ (CMA₃)-staining) and fluorescent in situ hybridization (FISH) using 18 S rDNA and 5 S rDNA probes. The diploid chromosome number was invariably 2n = 50 with 3 pairs of metacentric, 5 pairs of submetacentric, 8 pairs of subtelocentric and 9 pairs of acrocentric chromosomes. No heteromorphic sex chromosomes were observed. The nucleolar organizer regions (NORs) were detected in the short arms of the largest acrocentric pair using Ag-, CMA₃- stainings and FISH with 18 S rDNA probe, the latter showing also positive labeling in the short arms of a small acrocentric pair, not visualized by the former methods. FISH with 5 S rDNA probe showed positive labeling in the two chromosome pairs. While the CMA₃-staining exhibited GC-rich heterochromatin segments in two pairs of chromosomes, including those coincided with Ag-NORs, the DAPI staining did not reveal any signal, indicating the absence of AT-rich heterochromatin. FISH with an As-51 satellite DNA probe derived from the closely related Astyanax scabripinnis did not reveal any positive signal, demonstrating the absence of this class of DNA in the genome of the specimens under study.     

Comparative cytogenetics and chromosomal characteristics of ribosomal DNA in the fish genus Vimba (Cyprinidae)

Karyotypic and cytogenetic characteristics of Vimba vimba and V. elongata were investigated using differential staining techniques (sequential C-banding, Ag- and CMA₃-staining) and fluorescent in situ hybridization (FISH) with 28S rDNA probe. The diploid chromosome number in both species was 2n = 50 with 8 pairs of metacentrics, 14 pairs of submetacentrics to subtelocentrics and 3 pairs of subtelo- to acrocentrics. The largest chromosome pair of the complements was characteristically subtelo- to acrocentric. The nucleolar organizer regions (NORs) in both species were detected in the telomeres of a single, middle-sized subtelocentric chromosome pair, a pattern common in a number of other Leuciscinae. FISH with rDNA probe produced consistently positive hybridization signals detected in the same regions indicated by Ag-staining and CMA₃-fluorescence. The distribution of C-positive heterochromatin was identical in both species, including a conspicuous size polymorphism of heterochromatic blocks in the largest metacentric and subtelo- to acrocentric chromosomal pairs. No heteromorphic sex chromosomes were detected. A single analyzed individual of V. melanops possessed the same karyotype and NOR phenotype as V. vimba and V. elongata. The apparent karyotype homogeneity and chromosomal characteristics of ribosomal DNA in all three species of the genus Vimba is consistent to that found in most other representatives of the European leuciscine cyprinid fishes.     

Cytogenetic study in Corydoras britskii (Siluriformes: Callichthyidae), using different chromosomal banding and fluorescence hybridization in situ with rDNA probes

Cytogenetic analyses were performed on Corydoras britskii from the Miranda River basin, an important river located in the Pantanal, Mato Grosso do Sul State, Brazil. The karyotype of this species comprises 90 chromosomes and a karyotype formula of 4m + 10 sm + 22 st + 54a. The nucleolus organizer regions were detected by impregnation with silver nitrate and FISH with an 18S rDNA probe on the short arm of three acrocentric chromosomes. The constitutive heterochromatin is distributed in pericentromeric and interstitial positions, and also associated with the NORs. The HinfI restriction endonuclease was used and showed homology with practically all types of heterochromatin observed in C. britskii, except for two interstitial heterochromatic blocks present in a subtelocentric pair. The fluorochrome staining evidenced six chromosomes with chromomycin-positive signals indicating that both the heterochromatin interspersed with NORs and some heterochromatic blocks were rich in GC base pairs. FISH using a 5S rDNA probe revealed the presence of these regions in only one subtelocentric pair in the interstitial position. The obtained data substantiate the karyotype diversity of the genus Corydoras and provide novel information about the composition of heterochromatin and location of 5S and 18S rDNA sites.     

NORs inheritance analysis in crossings including individuals from two stocks of rainbow trout (Oncorhynchus mykiss)

Silver nitrate staining of rainbow trouts (Oncorhynchus mykiss) chromosomes, for the identification of the nucleolar organizing regions (NORs), revealed that in individuals from Núcleo Experimental de Salmonicultura de Campos do Jord?o (Brazil) NORs were located in the long arms of submetacentric pair while in specimens from Mount Shasta (USA) NORs were located in the short arms of a submetacentric pair. Cytogenetic analysis of the offspring, obtained through artificial crosses including individuals from both stocks, allowed the identification of NORs in two submetacentric chromosomes, one in the short arms and the other in the long arms, confirming the effectiveness of the hybridization process. Complementary results obtained using the FISH technique with 18S and 5S rDNA probes showed that NOR-bearing chromosomes exhibited a cluster of 5S genes located in tandem with the 18S gene cluster in both stocks. The results allow us to suggest that the difference in NOR-bearing chromosomes found between the two stocks is likely to be due to pericentric inversion involving the chromosome segment where 18S and 5S rDNA genes are located. The presence of ribosomal genes in the long arms of a submetacentric chromosome is apparently a particular characteristic of the rainbow trout stock of Campos do Jord?o and might be used as a chromosome marker in studies of controlled crosses in this species.     

Sex chromosomes and associated rDNA form a heterochromatic network in the polytene nuclei of Bactrocera oleae (Diptera: Tephritidae)

The olive fruit fly, Bactrocera oleae, has a diploid set of 2n?=?12 chromosomes including a pair of sex chromosomes, XX in females and XY in males, but polytene nuclei show only five polytene chromosomes, obviously formed by five autosome pairs. Here we examined the fate of the sex chromosomes in the polytene complements of this species using fluorescence in situ hybridization (FISH) with the X and Y chromosome-derived probes, prepared by laser microdissection of the respective chromosomes from mitotic metaphases. Specificity of the probes was verified by FISH in preparations of mitotic chromosomes. In polytene nuclei, both probes hybridized strongly to a granular heterochromatic network, indicating thus underreplication of the sex chromosomes. The X chromosome probe (in both female and male nuclei) highlighted most of the granular mass, whereas the Y chromosome probe (in male nuclei) identified a small compact body of this heterochromatic network. Additional hybridization signals of the X probe were observed in the centromeric region of polytene chromosome II and in the telomeres of six polytene arms. We also examined distribution of the major ribosomal DNA (rDNA) using FISH with an 18S rDNA probe in both mitotic and polytene chromosome complements of B. oleae. In mitotic metaphases, the probe hybridized exclusively to the sex chromosomes. The probe signals localized a discrete rDNA site at the end of the short arm of the X chromosome, whereas they appeared dispersed over the entire dot-like Y chromosome. In polytene nuclei, the rDNA was found associated with the heterochromatic network representing the sex chromosomes. Only in nuclei with preserved nucleolar structure, the probe signals were scattered in the restricted area of the nucleolus. Thus, our study clearly shows that the granular heterochromatic network of polytene nuclei in B. oleae is formed by the underreplicated sex chromosomes and associated rDNA.     

Cytogenetic characterization of <Emphasis Type="Italic">Eurysternus caribaeus</Emphasis> (Coleoptera: Scarabaeidae): evidence of sex-autosome fusion and diploid number reduction prior to species dispersion

Mitotic and meiotic chromosomes of several populations of Eurysternus caribaeus (Coleoptera: Scarabaeidae) were analysed through conventional staining, C-banding, base-specific fluorochromes, silver nitrate staining and fluorescent in situ hybridization (FISH). All specimens showed 2n = 8 in their karyotypes, with a neo-XY sex system (Y is a submetacentric and X a metacentric) and three pairs of submetacentric autosomes. The analysis of constitutive heterochromatin (CH) revealed small blocks located in the centromeric region of all chromosomes which do not present positive staining under the fluorochromes CMA3 and DAPI. Silver nitrate staining revealed that the nucleolar organizer region (NORs) is associated with the sex chromosomes. The FISH technique revealed that rDNA sites in the X and Y are different in size. Data from different populations indicate that the diploid number reduction (2n = 8) observed in E. caribaeus is established and presumably has preceded the dispersion of this species. Moreover, this reduction occasioned the translocation of rDNA sites to the sex chromosomes, X and Y, an uncommon pattern in Scarabaeidae that was observed for the first time by the FISH in this work.     

Chromosomal location of 18S and 5S rDNA sites in Triportheus fish species (Characiformes, Characidae)

The location of 18S and 5S rDNA sites was determined in eight species and populations of the fish genus Triportheus by using fluorescent in situ hybridization (FISH). The males and females of all species had 2n = 52 chromosomes and a ZZ/ZW sex chromosome system. A single 18S rDNA site that was roughly equivalent to an Ag-NOR was detected on the short arms of a submetacentric pair in nearly all species, and up to two additional sites were also observed in some species. In addition, another 18S rDNA cluster was identified in a distal region on the long arms of the W chromosome; this finding corroborated previous evidence that this cluster would be a shared feature amongst Triportheus species. In T. angulatus, a heterozygotic paracentric inversion involving the short arms of one homolog of a metacentric pair was associated with NORs. The 5S rDNA sites were located on the short arms of a single submetacentric chromosomal pair, close to the centromeres, except in T. auritus, which had up to ten 5S rDNA sites. The 18S and 5S rDNA sites were co-localized and adjacent on the short arms of a chromosomal pair in two populations of T. nematurus. Although all Triportheus species have a similar karyotypic macrostructure, the results of this work show that in some species ribosomal genes may serve as species-specific markers when used in conjunction with other putatively synapomorphic features.     

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.     

Chromosomal Mapping and Molecular Characterization of Ribosomal RNA Genes in Lebias fasciata (Teleostei, Cyprinodontidae)

Chromosome location of major (18S, 5.8S and 28S) and 5S ribosomal RNA genes (rDNAs) was examined in Lebias fasciata collected from different Italian blackish-waters, using silver (Ag)- and chromomycin A3 (CMA3)-staining and/or fluorescence in situ hybridization (FISH). Both 18S and 5S rDNA probes for FISH were obtained with polymerase chain reaction-directed cloning from genomic DNA of the examined species. Nucleolar organizer regions (NORs) containing the major rDNAs showed intraspecific polymorphism in number as detected by Ag-and CMA3-staining and FISH with the 18S rDNA probe. On the other hand, 5S rDNA loci constantly occurred on one chromosome pair and co-localized with a pair of the major rDNA loci as evidenced by two-color FISH using the 5S and 18S rDNA probes. Sequential CMA3- and Ag-NOR staining and FISH revealed apparent inactivation of some NORs. The cloned 5S rDNA was found to contain some TATA-like sequences that might play an important role in the regulation of gene expression.