A case of trisomy 22 in Pongo pygmaeus.

A behaviorally and clinically abnormal female orangutan was analyzed cytologically using general banding techniques and by an alkaline silver method for staining nucleolus organizer regions. The karyotype had 49 chromosomes, including an extra chromosome 22 (49,XX + 22). No variant chromosome types or heterozygous structural rearrangements were found. Nine of the 14 large acrocentric chromosomes, Nos. 11--17, and three of the five presumptive human G-group equivalents, i.e., two of three chromosomes 22, and one chromosome from pair 23, exhibited positive silver staining of the nucleolus organizer region (NOR).     

Nucleolar organizing regions of human chromosomes

Summary Silver-Stained cells from 49 parents with a history of several abortions were compared with cells from 35 parents with normal liveborn children. The modal and mean number of silver-stained NORs (Ag-NORs) observed on D- or G-group chromosomes was similar in both groups and between males and females. Ag-NORs were randomly distributed on all five acrocentric pairs. The distribution and size of Ag-NORs within an individual was not random and was fairly consistent from cell to cell.The mean number of associations per cell was similar in both males and females of the abortion group and was less than the number of associations in controls. The probability of D- or G-group chromosomes being associated was near the expected probability of 0.6 for D-association and 0.4 for G-association. The frequency of association of any chromosome combination did not differ statistically from the expected values, though the number of associations, 15/22, was higher than expected.     

Cytologic demonstration of differential activity of rRNA gene clusters in different human tissues

Summary rRNA gene activity was evaluated by cytologic methods in cultured human cells from two different tissues grown under controlled experimental conditions. The modal and average numbers of silver positive nucleolus organizers (NOs) per cell as well as the distribution of cells with different numbers of silver positive NOs and different combinations of D-plus G-group silver stained chromosomes, were evaluated. Statistically significant differences in the average number of silver positive NOs per cell between leukocytes and fibroblasts grown under standard experimental conditions have been demonstrated. The observed differences became sharper in cells cultured under more restrictive conditions. Also, differences in the frequency of silver positivity of specific chromosomal NOs located on individually indentified chromosomes were observed in cells from the same tissue. Furthermore, differences in the frequency of activation of rDNA clusters located on the same chromosome were also observed between cells from the two tissues. The possible biologic meanings of these findings are discussed.This paper is dedicated to Professor G. Montalenti on the occasion of his 80th birthday     

Nucleolus organizer and N-band distribution in morphologic and fluorescence variants of human chromosomes

Summary Three cases of morphologic variants of human D- or G-group chromosomes have been studied by N- and Ag-AS banding techniques. The results confirm our previous findings about the localization of nucleolus organizers on the secondary constrictions of acrocentric chromosomes. Preliminary results on the distribution and number of N bands in D- and G-group chromosomes with morphologic and fluorescence variants are reported     

Silver staining of the lampbrush chromosomes ofTriturus cristatus carnifex

Lampbrush chromosomes fromTriturus cristatus carnifex were stained using the ammoniacal silver staining (AgAS) technique. Many of the recognized marker structures proved to be silver positive, plus between six and fifteen lateral loop pairs. None of the stained loop pairs corresponded to known sites of the nucleolus organizers, although the extrachromosomal nucleoli were silver positive. The ammoniacal silver staining technique does not demonstrate the specificity for active ribosomal cistrons in lampbrush chromosomes that it does in a wide variety of mammalian mitotic chromosomes.     

Suppression of human nucleolus organizer activity in mouse-human somatic hybrid cells

Cells from four different mouse-human somatic cell hybrids were stained with quinacrine to identify each metaphase chromosome and with ammoniacal silver by the Ag-AS method to locate nucleolus organizer regions. Each of the hybrids contained human acrocentric chromosomes. None of these human acrocentric chromosomes was stained with silver in any hybrid cell. Diploid cells were available from the human parent of one of the hybrids. In these cells both copies of nos. 13 and 15 stained with silver; the same chromosomes in the hybrid cell were not stained. These results support earlier reports that the expression of human ribosomal RNA (rRNA) genes is suppressed in mouse-human hybrid cells. Further, they suggest that silver staining by the Ag-AS method reflects activity of rRNA genes rather than just the presence of these genes.     

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.     

Chromosome heteromorphisms in the Japanese

Summary The nucleolus organizer regions (NORs) of variant D- and G-group chromosomes characterized by enlargements of the short arms including secondary constrictions and satellites, were examined using the silver-staining method. Of a total of nine variants examined, four were found to have double Ag-stained NORs in the enlarged short arm, two were found to involve chromosome 22, one was a 13, and one, a 14. Four of the other variants had only one Ag-stained NOR. From the positions of the NORs, three of them were judged to have enlarged satellites (two chromosomes 15 and one 22) and the other an enlarged short arm (a 15). In the remaining variant (a 14), no Agstained material was noted in the short arm, so it could not be determined whether this variant chromosome was derived from the enlargement of the short arm or from satellites. Based on the position of the Ag-stained NORs and staining intensity of the Q and C methods in the short arms, mechanisms of producing the enlarged short arms of D- and G-group chromosomes are discussed.     

Identification of a silver binding protein associated with the cytological silver staining of actively transcribing nucleolar regions.

Nucleoli isolated from Novikoff hepatoma cells were stained with AgNO3 to demonstrate the typical staining of active ribosomal cistrons. Pre-treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 2.0 M NaCl did not interfere with silver staining. Treatment of the nucleoli with 80 mM Tris-HCl (pH 7.5) -- 0.15 M NaCl did, however, eliminate silver binding. Serial extraction of nucleoli with 2.0 M NaCl buffer followed by 0.15 M NaCl buffer also abolished silver staining. Analysis of the supernatant fraction of these extracts by polyacrylamide gel electrophoresis indicates that, although more than one nucleolar protein can bind silver, only one protein is associated with the staining of active ribosomal cistrons.     

Cytological research on the argentophilic nucleolus-organizer regions of the metaphase chromosomes in parthenogenetic mouse embryos]

Silver staining technique visualizing argentophilic nucleolus organizer regions (Ag-NORs) was used for studying parthenogenetic mouse embryos produced by artificial activation of oocytes in Ca(2+)-Mg(2+)-free medium. Ag-NOR-containing chromosomes were detected in metaphases of parthenogenetic embryos during six successive cleavage divisions starting with the two-cell stage. The frequency of metaphases with varying AG-NOR number in diploid parthenogenones was similar to that in the control (fertilized) embryos. Average number of metaphase Ag-NOR chromosomes (calculated per diploid chromosome set) in haploid parthenogenones exceeded that in the control; in some cases all NORs were stained by silver. This is evidence that latent ribosomal cistrons in some chromosomes can be activated.     

Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.     

Patterns of silver staining on NORs of prematurely condensed muntjac chromosomes following RNA inhibition

Prematurely condensed chromosomes of muntjac G0 lymphocytes as well as contact-inhibited and Actinomycin D (actD)-treated fibroblasts have been stained with silver nitrate to estimate the correlation between RNA suppression and the NOR staining. The results demonstrate that actD treatment for up to 36 h does not significantly affect the staining. Only partial suppression occurs in contact-inhibited cells, whereas complete abolition is obtained in long quiescent lymphocytes. We conclude that the reduction of the staining occurs only gradually from the NORs over a number of days or even weeks. We assume that the silver staining proteins may be associated with rDNA having a regulatory or structural role to play in rDNA activity.     

Visualization of nucleolar organizer regions in mammalian chromosomes using silver staining

A simple ammoniacal silver staining procedure, designated Ag-AS, differentially stains the chromosomal locations of ribosomal DNA in certain mammalian species. This was critically demonstrated by Ag-AS staining of the nucleolus organizer regions in karyotypes of the same species and cell lines used for locating the ribosomal cistrons by DNA/RNA in situ hybridization. With Ag-AS, silver stained NORs (Ag-NORs) are visualized as black spherical bodies on yellow-brown chromosome arms. Ag-NORs were visualized throughout mitosis at the secondary constrictions in the rat kangaroo, Seba's fruit bat, Indian muntjac, and Rhesus monkey. The Chinese hamster and cattle have telomeric Ag-NORs, the mouse subcentromeric Ag-NORs, and the field vole Ag-NORs as minute short arms or choromosomal satellites. Ag-NORs occur at both secondary constrictions and at telomeres in the cotton rat. Variability in Ag-NOR pattern included differences in the number of Ag-NORs per cell within a cell population, size of Ag-NORs among chromosomes of a complement, and presence of Ag-NOR on particular chromosomes in two cell lines of the Chinese hamster. The available cytochemical data suggest that the Ag-AS reaction stains chromosomal proteins at the NOR rather than the rDNA itself.     

Analysis of nucleolus organizer regions (NORs) in mitotic and polytene chromosomes ofPhaseolus coccineus by silver staining and Giemsa C-banding

Chromosomes with active nucleolus organizer regions (NORs) were visualized in root tip metaphases ofPhaseolus coccineus using the silver staining technique. A mean number of 5.5 Ag-NORs per cell was observed in 54 cells from eight plants. In the endopolyploid nuclei of the suspensor the silver technique did not demonstrate the reported specificity for nucleolus organizer activity, because there was usually pale staining of nucleoli and preferential staining of heterochromatic regions in the polytene chromosomes including pericentromeric material, telomeres and NORs. The mean number of NORs per nucleolus as detected by this method was 5.8 (28 nucleoli analysed). Using a modified preparation technique, giant chromosomes stained pale, but nucleoli of suspensor cells displayed darkly silver staining internal domains, each of which originating from a nucleolus organizer.—Giemsa C-banding of endopolyploid suspensor nuclei revealed C-positive nucleolus organizers with darkly staining intranucleolar fibrils. The latter were frequently involved in inter-NOR associations. In 34 nucleoli analysed, the mean number of Giemsa C-positive NORs per nucleolus was 6.0.Dedicated to Professor Dr.Lothar Geitler on the occasion of his 80th birthday.     

红翅皱膝蝗减数分裂染色体的螺旋与轴结构

用低渗处理和苯酚品红染色,在经过卡诺液(甲醇3∶冰醋酸1)固定和未经固定的红翅皱膝蝗减数分裂染色体上都看到了螺旋结构。观察和测量结果表明,每条染色单体都是由430nm左右的染色线螺旋形成的。由染色线到染色体的压缩率为4∶1。低渗处理后固定的材料经过银染,则显示了染色体轴结构。同样,未经低渗处理直接固定的材料银染时也出现了轴结构。银染的轴结构位于每个染色单体的中央,并贯穿整个染色单体。在光镜下,这个轴并不是直径均一的棒状结构,而似乎是由许多大小相近的颗粒相连而成。本文对染色体结构的有关模型、骨架和轴结构的真实性以及轴和螺旋的关系等问题进行了讨论。     

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.     

Detection of nucleolus organizer regions in chromosomes of human,chimpanzee, gorilla,orangutan and gibbon

Nucleolus organizer regions were detected by the Ag-AS silver method in fixed metaphase chromosomes from human and primates. In the human, silver was deposited in the secondary constriction of a maximum of five pairs of acrocentric chromosomes: 13, 14, 15, 21 and 22. The chimpanzee also had five pairs of acrocentric chromosomes stained, corresponding to human numbers 13, 14, 18, 21 and 22. A gibbon had a single pair of chromosomes with a secondary constriction, which corresponded to the nucleolus organizer region. In each case the Ag-AS method detected the sites which have been shown by in situ hybridization to contain the ribosomal RNA genes. An orangutan had eight pairs of acrocentric chromosomes stained with Ag-AS, probably corresponding to human numbers 13, 14, 15, 18, 21 and 22, plus two others. Two gorillas had silver stain over two pairs of small acrocentric chromosomes and at the telomere of one chromosome 1. The larger gorilla acrocentric chromosomes had no silver stain although they all had secondary constrictions and entered into satellite associations.