NOR Sites Detected by Ag-DAPI Staining of an Unusual Autosome Chromosome of Bradysia Hygida (Diptera:Sciaridae) Colocalize with C-banded Heterochromatic Region

The study of chromosomes in insects is a good tool in mitotic process analysis, zoographic localization and evolution investigation. Among them, the Sciaridae offers a karyotype with a small number of chromosomes, where the heterochromatin and nucleolar organizer region, NOR, are easily analyzed in metaphase chromosomes obtained from cerebral ganglia squashes. In this work, the heterochromatic regions on Bradysia hygida mitotic chromosomes, revealed by C-banding, were identified as centromeric blocks on A and C chromosomes and as dark interstitial region in B and X chromosomes. By Ag-DAPI staining, active nucleolus organizer region, NOR, was revealed associated to the constitutive heterochromatin in the end of the C autosome chromosome. The C-band regions and the unusual ribosomal site localization are discussed.     

Morphometric analysis of human chromosome satellites and NOR asymmetries by transmission electron microscopy

Human chromosome satellites appear as roughly spherical telomeric structures of 0.215 +/- 0.013 microns mean diameter by electron microscopy. Morphometric evaluations showed that in the short arms of D and G chromosomes lacking secondary constrictions, the chromatin which constituted the satellites appeared virtually integrated within the short arms. Asymmetry was detected in sister satellites from almost identical dimensions to the near absence of one of them. When Ag-NOR staining was employed to locate active nucleolar organizer regions (NORs) these appeared associated with satellite short arms and active NORs were never found in non-satellite chromosomes. Asymmetry was also evident between sister NORs.     

Human nucleolus organizers: the satellites or the stalks?

A silver-staining technique specific for demonstrating nucleolus organizer regions (NOR) showed that the achromatic stalks of the 10 acrocentric autosomes of the human complement represent the NORs. Some variability in number of stained stalks is observed from cell-to-cell and from individual-to-individual. The silver-stained masses may extend beyond the stalks and cover the satellites, especially in chromosomes with short stalks or minute satellites.     

‘Nucleolar dominance’ as observed in barley translocation lines with specifically reconstructed SAT chromosomes

Summary Diploid homo- and heterokaryotypes of barley translocation lines with only one satellite chromosome pair containing two nucleolus organizer regions (NORs) in opposite arms were found to show repressed nucleolus formation by the transposed NOR as evident from the formation of only micronucleoli. The same was true for auto-tetraploid homokaryotypes and for translocation lines with all NORs tandemly arranged into the same chromosome arm. When NORs were transposed to chromosomes without NOR in the standard karyotype, the normal pattern of nucleolus formation remained unaffected. The modified mode of nucleolus formation after the combination of all NORs in one chromosome pair is interpreted to be due to intrachromosomal nucleolar dominance analogous to interchromosomal nucleolar dominance observed in certain interspecific hybrids.     

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.     

Silver staining of nucleolar organizer regions (NOR) in some species of Hymenoptera (bees and parasitic wasp) and Coleoptera (lady-beetle)

Adaptations of the nucleolar organizer regions (NOR) banding technique using precipitation of silver salts significantly improved the NOR characterization of some species of hymenopterans and one coleopteran. The bee Melipona marginata (2n = 18) showed one metacentric pair of chromosomes with a NOR in the pericentromeric position. The parasitic wasp Mellitobia australica (2n = 12) also showed one metacentric pair with a strongly Ag-positive NOR. The male lady-beetle Cycloneda sanguinea (2n = 18 + Xy(p)) displayed a NOR on a pair of acrocentric autosomes. In the male Euglossa sp. (a haplodiploid species) (n = 21) the NOR were multiple, and occurred in five chromosomes. In the bee Plebeia sp. 1 (2n = 34) the NOR seemed restricted to one of the homologues of a metacentric pair. The systematic advances brought out by using this technique in the context of current theories of karyotypic evolution of these taxa are described and discussed.     

Metaphase association of mammalian chromosomes

The association behavior of chromosomes bearing nucleolar organizer region (NOR) and (or) C-heterochromatin in metaphase plates was analyzed. Different species with an informative chromosomal localization of NOR and C-heterochromatin were evaluated. Several examples indicate that the well-known metaphase association is not due to NORs or NOR activity per se. Other mechanisms such as ectopic pairing are responsible for the association. These types of pairing seem to be enhanced by the chromatin-decondensing effect of nearby NOR activity.     

Banding patterns of the chromosomes of Presbytis cristatus pyrrhus and P. obscurus

The G- and Q-bands and the location of the nucleolar organizer regions (NOR) in the chromosomes of Presbytis obscurus and the Q- and C-bands of P. cristatus pyrrhus are described. Their chromosomes are compared to those of Macaca mulatta and to other Cercopithecidae and Hylobatidae. The origin of the two different banding patterns of pair no. 1 in our specimen of P. cristatus pyrrhus is discussed.     

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.     

New insights into the effects of extra nucleolus organizer regions

Summary Chromosome studies were carried out in four members of a sibship with a 15p+chromosome. Two carriers had normal offspring, one was unmarried, and the index case had three abortions and no live children. By means of different banding techniques, up to four satellites and four stalks could be observed on the abnormal chromosome. The Ag-I method showed from zero to four silver precipitates on the 15p+marker. Mean Ag-staining for 15p+and the ten acrocentric chromosomes were obtained in the carriers. Statistically significant differences between sibs were found. These results suggest the existence of: (a) An interindividual and intercellular variation of nucleolus organizer region (NOR) activity in man. (b) An optimal threshold of NOR activity, so that disturbances at the meiotic level could occur when it is exceeded. Our conclusions lead us to advise analysis of NOR activity in individuals with extra nucleolus organizer 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.     

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.     

Expression of ribosomal genes under the action of 5-azacytidine in the African green monkey RAMT cell line

The use of cytosine analogue--5-Azacytidine(5AzaC), derepression of ribosomal genes has been studied in one of organising chromosomes in the African green monkey RAMT cell line in which the nucleolar organizer region (NOR) in parental cells was active. The effect of 5AzaC on the functional state of NOR was assessed by the length of the secondary constriction in this chromosome and by the intensity of Ag-staining of NOR. 5AzaC was added to the cell culture at concentrations 2-16 M, either immediately after the cell passage or at the 24th h from the beginning of cell cultivation for the following 17-34 hours. As a control the cells cultivated in the absence of 5AzaC were used. Comparison of control cells with those treated with 5AzaC showed: 1) increase of the length of the second constriction in the chromosome with the initial inactive NOR in the 5AzaC--treated cells; 2) a marked increase of the intensity of NOR's Ag-staining in the same chromosome after incorporation of 5-AzaC into DNA. The conclusion about the methylation of cytosine bases in the DNA of ribosomal genes in one NOR organising chromosomes in RAMT cell line was made.     

Genetic determination of NOR activity in human lymphocytes from twins

Summary Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Agstained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag+NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acrocentric association frequency were not significantly higher in DZ compared with MZ twins.     

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.     

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.     

Protective effect of wheat germ agglutinin on the course of mitosis in the roots of <Emphasis Type="Italic">Triticum aestivum</Emphasis> seedlings exposed to cadmium

Effect of pretreatment with 28 nM wheat germ agglutinin (WGA) on cell divisions in the root apical meristem of 4-day-old seedlings of wheat (Triticum aestivum L.), distribution of cells among mitotic phases, cadmium-induced disruptions of normal progression through mitosis, and activity of nucleolar organizer regions (NOR) of the chromosomes was studied after 7-h-long exposure to 1 mM cadmium acetate. Pretreatment with WGA has a pronounced protective effect on divisions of root meristem cells exposed to cadmium. Progression of the cells through mitotic phases was normalized, abnormal mitoses became much less numerous, and the share of binuclear cells decreased. Activity of NOR remained at the control level that much depended on the ability of WGA to prevent reduction in cytokinin content under cadmium stress.