Cytogenetic analyses of a Salvelinus hybrid reveal an evolutionary relationship between the parental species

Mitotic analyses of the brook trout (Salvelinus fontinalis) x arctic char (S. alpinus) hybrids (sparctic trout) revealed a mode of 2n = 82 with 18 metacentric and 64 acrocentric chromosomes. The brook trout had 2n = 84 with 16 metacentric chromosomes and the arctic char had 2n = 80 with 20 metacentric chromosomes; both species are derivatives of a single tetraploid event. Variable multivalent-like configurations that may be centromeric associations of bivalents were observed in C-banded pachytene figures of female sparctic trout. Metaphase I analyses of sparctic trout males indicated that two fusions of nonhomologous acrocentric chromosomes representing two duplicated chromosome sets must have occurred in the arctic char after its evolutionary divergence from the brook trout. A mode of seven tetravalent rods per cell suggests that preferential multivalent pairing occurs in the sparctic hybrid; metaphase I analyses of S. alpinus males revealed a mode of only five tetravalent rods per cell. The presence of multivalents implies that the arctic char, like the brook trout, is still undergoing diploidization. Cytochemical detection of the nucleolar organizer region (NOR) revealed intra- and interspecific as well as intraindividual variability in the numbers and types of chromosomes (metacentric or acrocentric) on which NORs appeared in arctic char and sparctic trout. Brook trout only had NORs on acrocentric chromosomes. This may indicate that different chromosomal fusions occurred in the evolution of brook trout from arctic char.     

Supernumerary chromosome system,C-banding pattern characterization and multiple nucleolus organizer regions in Moenkhausia sanctaefilomenae (Pisces,Characidae)

Chromosomes of Moenkhausia sanctaefilomenae (10 males and 20 females) collected from a tributary of the Tietê River (Botucatu, S.P. Brazil) were examined using kidney and testicular cells. Both males and females presented 2n=50 chromosomes and 1 to 8 small supernumerary microchromosomes. C-banding demonstrated positively stained heterochromatic blocks in almost every chromosome and a pattern of interstitial bands located in the same position in relation to the centromere on the long arm of a large number of chromosomes. Two large NORs were detected in pair 19 of all the silver-stained metaphases; zero to 6 additional small NORs were detected in other chromosomes. A general survey of the occurrence of supernumerary chromosomes in fishes is presented.     

Distribution of silver-stained nucleolus-organizing regions in the chromosomes of the Equidae

The distribution of silver-stained nucleolus-organizing regions (NORs) in fibroblast chromosomes from all seven extant species of Equidae are described. There are variations in the number and locations of silver-stained NORs in different species but most of the cells in an individual of any speies had only 2 to 4 silver-stained NORs. In Equus przewalskii and E. caballus NORs were detected on up to three different chromosomes. In E. asinus 11 different chromosomes were observed to possess NOR sites. E. hemionus kulan and E. hemionus onager had NORs on 2 large metacentric pairs and a small acrocentric pair. In E. grevyi and E. burchelli NORs were located on 3 to 4 different pairs. E. zebra hartmannae had silver-staining over the telomere regions of the short arms of the chromosomes 1, 3 and 4. A comparison of G-banded chromosomes and silver-stained NORs has revealed one autosome with conserved G-band patterns and possessing a silver-staining NOR in all the species except E. asinus. Variations in the number and multichromosomal locations of NORs in various species could have evolved by pairing and exchanges between non-homologous chromosomal heterochromatin having similar satellite DNA sequences.     

Frequency of Ag-stained nucleolus organizer regions in the acrocentric chromosomes of man

Summary The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding of cultured lymphocytes in 51 karyotypically normal persons (31 males and 20 females). A consistent pattern of Ag-positive NORs was found in each individual. Ninety percent of the individuals have a modal number of 8–10 Ag-positive NORs per cell. The frequency of Ag-positive NORs is similar in all five acrocentrics. A statistically nonsignificant lower frequency is found in chromosome 22. Ag-negative NORs on both homologues were found in four cases. The observed frequency distribution of individuals with homozygous NOR-positive, heterozygous, and homozygous negative acrocentric chromosomes was in accordance with the Hardy-Weinberg law in all five pairs of the acrocentric chromosomes as well as in total. No sex difference was observed in our material.A.-V. Mikelsaar is visiting exchange scientist of the Österreichische Bundesministerium für Wissenschaft und Forschung     

Karyotype, Ag-NOR, CMA3 and SEM studies in a fish (Mystus tengara, Bagridae) with indication of female heterogamety

Somatic karyotypes in M. tengara contained 54 chromosomes, comprising 26 homomorphic pairs in both sexes and one pair of heteromorphic nature in female (one big submetacentric and one small subtelocentric chromosomes), while in males this pair was homomorphic (with two big sub-metacentric chromosomes). The Nucleolus Organizer Regions (NORs) were located at one arm of the suspected sex elements in both sexes, while another pair of metacentric chromosomes (No.7) also showed Ag-positive arm. The CMA3 technique revealed relatively bright fluorescing zones in the regions of chromosomes that showed Ag-positive staining, revealing thereby the preponderance of GC-rich active sites of rRNA genes in NORs. SEM studies revealed clear heteromorphism to exist in the elements suspected as sex chromosomes in females.     

Evidence for male XO sex-chromosome system in Pentodon bidens punctatum (Coleoptera Scarabaeoidea: Scarabaeidae) with X-linked 18S-28S rDNA clusters

In scarab beetle species of the genus Pentodon, the lack of analysis of sex chromosomes in females along with the poor characterization of sex chromosomes in the males, prevented all previous investigations from conclusively stating sex determination system. In this study, somatic chromosomes from females and spermatogonial chromosomes from males of Pentodon bidens punctatum (Coleoptera: Scarabaeoidea: Scarabaeidae) from Sicily have been analyzed using non-differential Giemsa staining. Two modal numbers of chromosomes were obtained: 2n = 20 and 19 in females and males, respectively. This finding along with other karyological characteristics such as the occurrence of one unpaired, heterotypic chromosome at metaphase-I and two types of metaphase-II spreads in spermatocytes demonstrate that a XO male/XX female sex determining mechanism - quite unusual among Scarabaeoidea - operates in the species investigated here. Spermatocyte chromosomes have also been examined after a number of banding techniques and fluorescent in situ hybridization with ribosomal sequences as a probe (rDNA FISH). The results obtained showed that silver and CMA(3) staining were inadequate to localize the chromosome sites of nucleolus organizer regions (NORs) due to the over-all stainability of both constitutive heterochromatin and heterochromatin associated to the NORs. This suggests that heterochromatic DNA of P. b. punctatum is peculiar as compared with other types of heterochromatin studied so far in other invertebrate taxa. By rDNA FISH major ribosomal genes were mapped on the X chromosome.     

Behavior of the nucleolus organizer regions of the chromosomes in polykaryocytes consisting of micronuclei

The nucleolar organizer regions (NORs) of Chinese hamster chromosomes (clone 237, cell line BIId-ii-FAF28) were studied in mononuclear cells and polykaryocytes induced with colcemid. The chromosomes with NORs were marked as 1, 2, 3, 4. The activity of NORs in mononuclear cells was higher in chromosomes 1, 2, 3. The associations of NORs were observed between chromosomes I and 2 (3% of all metaphases). In polykaryocytes the chromosomal pairs 1, 2, 3 showed different NOR activity in different metaphases. The associations of NORs in pairs of chromosome I were found in 51.3% of cases. The associations of NORs in pairs of chromosome 2 were observed in 7.5% of cases. This method may be used for the estimation of association potency of NORs in chromosomes.     

An X1X1X2X2/X1X2Y mechanism of sex determination in a South American rodent, Deltamys kempi (Rodentia, Cricetidae)

Chromosome studies on 14 specimens of Deltamys kempi disclosed six males with 2n = 37, NF = 38, six females with 2n = 38, NF = 38, and two females with 2n = 37, NF = 38. G- and C-band analyses revealed a Y-autosome translocation in the males leading to a multiple chromosome system of sex determination of the type X1X1X2X2/X1X2Y, this being the second case of such a mechanism described in rodents. At meiosis the males presented a trivalent in which C-banding studies showed an alternate orientation of the sex chromosomes due to end-to-end association of the X1 and Y chromosomes, the Y and the X2 being held together by interstitial chiasmata. At metaphase II both n = 17 + Y and n = 18 + X1 are regularly observed. The two females with 2n = 37, NF = 38, are heterozygous for an autosomal centric fusion involving chromosomes 1 and 13. The product of the Y-autosome translocation constitutes the largest element of the karyotype (9.4% of the haploid set); the X1 chromosome amounts to 7.8% of this set, including a large heterochromatic block. When only its euchromatic region is considered, this percentage decreases to 4.6%. From two to seven NORs were observed at the telomeres, with a mean of 4.4 +/- 1.1 per cell.     

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.     

Rapid identification of chromosomes carrying silver-stained nucleolus-organizing regions

Summary The use of a combination of transmitted light and epiluminescence after silver and fluorescent staining of chromosome preparations makes it possible to achieve simultaneous visualization of silver-stained NORs and fluorescent chromosomes. This technique permits exact localization of silver precipitates on normal and BrdU-substituted chromosomes. After previous silver impregnation, fluorescent staining by actinomycin-daunomycin-DAPI was used to induce a banding pattern that enables identification of specific chromosomes while observing silver-stained NORs at the same time. Application of this method to Down's syndrome patient revealed a 21/21 Robertsonian translocation with NORs eliminated.     

Effect of increase in ploidy on the activation of nucleolar organizer regions in Chinese hamster ovary (CHO) cells

The effect of increased ploidy on the activation of specific nucleolar organizer regions (NORs) was examined by comparing the distribution and frequency of active NORs in pseudodiploid Chinese hamster ovary (CHO) cells with a quasi-tetraploid hybrid line. Active NORs were identified on both unrearranged chromosomes and isochromosomes of the Z group by silver staining. The increase in cell ploidy in the hybrid did not result in the complete inactivation of specific NORs or the activation of a previously silent NOR. However, for several chromosome pairs identified as carrying NORs, apparent translocations and deletions which produced the karyotype of the pseudodiploid cells deleted or inactivated the NOR of one member of a homologous pair. When two copies of such chromosomes were present in the quasi-tetraploid hybrid line, the activity of their NORs showed apparent coordination. Furthermore, the frequency of activity of individual NORs in two CHO lines and in a quasi-tetraploid hybrid line suggests that active NORs are not inherited directly.     

A rapid technique for producing silver-stained nucleolus organizer regions and trypsin-Giemsa bands on human chromosomes

Summary A simple and rapid technique is described whereby the nucleolus organizer regions (NORs) of human chromosomes can be differentially stained with silver. This staining is followed by trypsin-Giemsa banding on the same metaphase chromosomes. The metaphases simultaneously exhibit silverstained NORs and G bands, allowing for the unequivocal identification of all chromosomes and greatly facilitating studies involving the NOR-bearing acrocentrics.     

The role of cytologic NOR variants in the etiology of trisomy 21.

Silver-stained chromosomes from 29 couples with a trisomy 21 offspring and from 25 control couples were studied to determine whether there was an association of nucleolar-organizing-region variants in parents of children with trisomy 21. A reproducible scoring system for the analysis of silver-stained chromosomes was developed, and this was applied to the analysis of study participants in a blinded fashion. Seven of the 58 parents of children with trisomy 21 and seven of the 50 control parents were found to have variant NORs on silver staining. Therefore, we do not find a demonstrable risk for nondisjunction of chromosome 21 in individuals with silver-staining variants.