A Method for Combined C-Banding and Silver Staining

A reliable technique for combined C-banding and silver staining of metaphase chromosomes which uses trypsinization is described. Slides are first immersed in dilute HCl to remove residual cytoplasm from around the chromosomes. They are then treated with saturated barium hydroxide and incubated overnight in saline sodium citrate (0.30 M NaCl, 0.03 M sodium citrate, adjusted to pH 7.0 with HCl). Following the C-banding pretreatment, a two-step method of silver staining which employs a protective colloidal developer is used to stain the nucleolar organizer regions (NORs) of the chromosomes. Silver staining is followed by trypsinization to remove extraneous silver precipitate from the chromosome arms which permits the C-bands to be stained with Giemsa. The method works equally well with fresh and aged mitotic chromosome preparations and gives consistent staining of both heterochromatin and active NORs in metaphases across the slide.     

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.     

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.     

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.     

Cytogenetic analysis of Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) with Xyp sex determination system using standard staining, C-bands, NOR and synaptonemal complex microspreading techniques

The mitotic and meiotic chromosomes of the beetles Epicauta atomaria (Meloidae) and Palembus dermestoides (Tenebrionidae) were analysed using standard staining, C-banding and silver impregnation techniques. We determine the diploid and haploid chromosome numbers, the sex determination system and describe the chromosomal morphology, the C-banding pattern and the chromosome(s) bearing NORs (nucleolar organizer regions). Both species shown 2n = 20 chromosomes, the chromosomal meioformula 9 + Xyp, and regular chromosome segregation during anaphases I and II. The chromosomes of E. atomaria are basically metacentric or submetacentric and P. dermestoides chromosomes are submetacentric or subtelocentric. In both beetles the constitutive heterochromatin is located in the pericentromeric region in all autosomes and in the Xp chromosome; additional C-bands were observed in telomeric region of the short arm in some autosomes in P. dermestoides. The yp chromosome did not show typical C-bands in these species. As for the synaptonemal complex, the nucleolar material is associated to the 7th bivalent in E. atomaria and 3rd and 7th bivalents in P. dermestoides. Strong silver impregnated material was observed in association with Xyp in light and electron microscopy preparations in these species and this material was interpreted to be related to nucleolar material.     

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.     

A new approach to the auchenorrhyncha (Hemiptera, Insecta) cytogenetics: chromosomes of the meadow spittlebug Philaenus spumarius (L.) examined using various chromosome banding techniques

The karyotype of the meadow spittlebug Philaenus spumarius (L.) was studied using conventional chromosome staining, C- and AgNOR- banding, and fluorescent CMA3- and DAPI- techniques. This is the first report on differential staining of the holocentric chromosomes of Auchenorrhyncha. The karyotype of Ph. spumarius includes 2n = 22 + XX/X0. The autosomal pair 1 is large and carries a gap in every homologue. After silver staining, NORs were revealed in both this chromosome pair and a middle-sized pair, most likely 6 or 7. In spermatocyte meiosis, the majority of bivalents formed one chiasma each. The bivalent 1 showed from 1 to 4 chiasmata, the value of 1 or 2 being prevalent. Two further bivalents also showed two chiasmata in some cells. After C-banding, terminal and interstitial dot-type C-heterochromatic blocks were revealed in the chromosomes. In 4 of 11 studied males, the autosomal pair 1 was polymorphic for an extra segment attached to one of the homologues. The segment consisted of both heterochromatic and euchromatic portions. No defined signals were observed in any chromosome treated with DAPI. After CMA3- staining, bright fluorescent signals were obtained in the NOR-bearing chromosomes, suggesting GC-rich DNA bound to the NORs.     

Heterochromatin, synaptonemal complex, and NOR activity in the somatic and germ cells of a male domestic dog, Canis familiaris (Mammalia, Canidae)

C-banding and silver staining of the somatic and germ cells of the male domestic dog. Canis familiaris, have shown that: (1) the amount of C-banding is small compared to most other mammalian species, (2) three pairs of autosomes have nucleolus organizer regions (NORs) at the terminal ends of their long arms, whereas the Y chromosome has an NOR on the terminal end of the short arm, (3) the organization of the synaptonemal complex (SC) is similar to that of other mammalian species, (4) a distinct SC is formed between the long arm of the Y chromosome and probably the short arm of the X chromosome, and (5) the differential axes of both sex chromosomes do not demonstrate fusiform thickenings nor do they stain darkly with silver as do the XY bivalents in many other mammalian species.     

经甫树蛙的染色体组型、C带和Ag-NORs的研究

本文分别用骨髓细胞染色体标本制作法、BSG技术和一种快速、简便的Ag-NORs显带技术,首次研究了经甫树蛙的染色体组型、C带和Ag-NORs。结果表明,经甫树蛙2n=26,有5对大型和8对小型染色体,次缢痕在No.11染色体长臂末端,为C带负染;银染表明,此次缢痕处即是经甫树蛙的“标准NORs”经甫树娃的C带结构异染色质主要是着丝点型和插入型的。文章初步讨论了树蛙属的细胞分类、经甫树蛙次缢痕、Ag-NORs和C带的关系。     

Nucleolar organizer activity in wheat,rye and derivatives analyzed by a silver-staining procedure

Nucleolar activity was analyzed in wheat (Triticum sp.), rye (Secale cereale) and several types of wheat-rye derivatives using a modified, highly reproducible, silver staining procedure (Lacadena et al. 1984). A comparative analysis of the nucleolar organizer regions (NORs) of somatic metaphase chromosomes was made by phase contrast, C-banding, and silver staining. The frequency distribution of the number of nucleoli visualized at interphase by silver staining was also used to infer the activity of NORs. The results agree quite well with data from in situ hybridization reported by other authors. The behavior of euploid, ditelosomic and nulli-tetrasomic plants of common wheat showed the relative nucleolar activity of the four organizer chromosomes to be: 6B > 1B > 5D > 1A. — Several types of wheat-rye derivatives were analyzed: interspecific hybrid, triticale, addition and substitution lines, and plants with the genome constitutions, AABBDR, ABDR + 5D, ABRR, and ABRRR. In all cases the nucleolar organizer chromosome 1R of rye was suppressed by the presence of wheat chromosomes.     

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.     

Are NORs easily translocated to deleted chromosomes?

Summary The karyotype of two human meningiomas are reported in which, besides other aberrations, a deleted chromosome 1 and 6 could be observed. In these chromosomes most of the short arm is missing. After silver staining for the detection of NORs, not only the satellite regions of most of the acrocentric chromosomes were stained but also the deleted ends of chromosomes 1 and 6. Besides other explanations for this unusual NOR-staining, the possibility is discussed that NORs of acrocentric chromosomes are easily translocated to deleted chromosomes.Parts of this work are included in the doctoral thesis (M.D.) of H.H.     

Karyotype similarities among Pimelodidae (Pisces, Siluriformes) from the Brazilian amazon region.

Three neotropical species of freshwater fish (Pseudoplatystoma fasciatum, P. tigrinum and Sorubim lima) of the Amazon region (Brazil) were cytogenetically analysed by means of conventional, silver staining and C-banding techniques. All showed 2n = 56, with a predominance of biarmed chromosomes. The NORs were located in the terminal position on the short arms, and only two chromosomes bearing NORs were detected. Heterochromatin was found to be equilocally distributed in some chromosomes of the three species, which may share homologous chromosomes. The present data indicate a somewhat conservative karyotypic evolution in this fish group.     

Differential staining of tick chromosomes: techniques for C-banding and silver-staining and karyology of Rhipicephalus sanguineus (Latreille)

A new karyotypic procedure is described for tick chromosomes along with modifications of accepted techniques for C-banding and silver-staining. The staining procedures produce patterns with staining limited to the centromeric regions and the nucleolar organizer regions (NORs). The possible reasons for the concordant patterns are discussed based on the staining characteristics of the 2 techniques. The karyotype of Rhipicephalus sanguineus possesses 20 acrocentric autosomes with a single pair of NORs located proximal to the centromere on the largest autosomal pair. The sex-determining system is XX:XO with the X chromosome being the largest chromosome in the complement and subtelocentric.     

Cytogenetic examination of the NOR activity in a proband with 13/13 translocation and in her relatives

Summary NOR activity in a proband with 13/13 translocation and in her relatives was examined by NOR silver impregnation and by determination of the association frequencies. In the proband, besides the fused chromosomes 13, also a chromosome 14 and a 15 showed no NOR staining. Therefore the possiblity could be ruled out that the loss of NORs was compensated by the activation of inactive NORs. However, in the proband, one chromosome 22 seemed to be more intensively stained by silver nitrate than in her parents. As in the proband, the association frequency remained constant because of an increased association tendency of chromosomes 22. The possibility is discussed that the loss of NORs was compensated by a higher NOR activity of one chromosome 22.Parts of this work are included in the doctoral thesis (M. D.) of S. H.     

小山蛙和中国雨蛙的核型及其C—带和银染的研究

采用骨骼细胞直接制备染色体标本法和BSG及Ag-NORs显带技术,研究了小山蛙(Rana minimus)和中国雨蛙(Hyla chinensis)的常规核型、C-带和Ag-NORs。小山蛙2n=26,有5对大型和8对小型染色体。第6对染色体短臂近着丝点处具次缢痕,并为C-带染色阳性,银带显示此次缢痕处,即是标准NORs。C-带显现于几乎所有染色体的着丝点区,插入型C-带少且弱,无端位带。中国雨蛙2n=24,有6对大型和6对小型染色体。次缢痕在第10对染色体长臂上,亦为C-带染色阳性,并与银染显示的标准NORs相一致。C-带分析表明,亦以着丝点C-带为主,插入型C-带少,无端位带。本文初步讨论了蛙科和雨蛙科的核学特征以及它们间的演化关系。     

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.     

Karyotype analysis of Lilium longiflorum and Lilium rubellum by chromosome banding and fluorescence in situ hybridisation.

Detailed karyotypes of Lilium longiflorum and L. rubellum were constructed on the basis of chromosome arm lengths, C-banding, AgNO3 staining, and PI-DAPI banding, together with fluorescence in situ hybridisation (FISH) with the 5S and 45S rDNA sequences as probes. The C-banding patterns that were obtained with the standard BSG technique revealed only few minor bands on heterologous positions of the L. longiflorum and L. rubellum chromosomes. FISH of the 5S and 45S rDNA probes on L. longiflorum metaphase complements showed overlapping signals at proximal positions of the short arms of chromosomes 4 and 7, a single 5S rDNA signal on the secondary constriction of chromosome 3, and one 45S rDNA signal adjacent to the 5S rDNA signal on the subdistal part of the long arm of chromosome 3. In L. rubellum, we observed co-localisation of the 5S and 45S rDNA sequences on the short arm of chromosomes 2 and 4 and on the long arms of chromosomes 2 and 3, and two adjacent bands on chromosome 12. Silver staining (Ag-NOR) of the nucleoli and NORs in L. longiflorum and L. rubellum yielded a highly variable number of signals in interphase nuclei and only a few faint silver deposits on the NORs of mitotic metaphase chromosomes. In preparations stained with PI and DAPI, we observed both red- and blue-fluorescing bands at different positions on the L. longiflorum and L. rubellum chromosomes. The red-fluorescing or so-called reverse PI-DAPI bands always coincided with rDNA sites, whereas the blue-fluorescing DAPI bands corresponded to C-bands. Based on these techniques, we could identify most of chromosomes of the L. longiflorum and L. rubellum karyotypes.