Chromosome banding in Amphibia

The structure of the nucleolus organizer regions (NORs) in mitotic chromosomes, diploid nuclei and spermatogenesis was studied in 260 individual animals from 23 genera of the Anura. The analyses were performed with conventional cytogenetic methods as well as with Ag-staining, GC- and AT-specific fluorochromes (mithramycin, chromomycin A₃, quinacrine) and C-banding. Most of the species have only one pair of NORs in their karyotypes. The majority of individuals of all species exhibited considerable differences in the sizes of their homologous NORs. Most of these heteromorphisms are due to tandem duplications or triplications in one of the two NORs. However, duplicated or triplicated NORs never occur in a homozygous form, but are instead always in combination with a normal-structured NOR in the homologous chromosome. In three animals, a complete deletion of one NOR and its closely associated constitutive heterochromatin was determined. The cytochemistry of the specific NOR-stainings are discussed. The size differences of the Ag-, mithramycin- and chromomycin A₃-stained NORs can be traced to differences in the rDNA content in these NORs.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Chromosome banding in Amphibia

The karyotypes of 14 species of Anura from 9 genera of the suborders Amphicoela, Aglossa, Opisthocoela and Anomocoela were analysed with various banding techniques and conventional cytogenetic methods. The 18S + 28S and 5S ribosomal RNA genes were localized by means of in situ hybridization. No Q-, R- and G-banding patterns in the euchromatic segments of the metaphase chromosomes could be demonstrated in any of the species; this does not seem to be caused by a higher degree of spiralization of the amphibian chromosomes, but by the special DNA organization in these organisms. In most karyotypes, constitutive heterochromatin is present at centromeres, telomeres and nucleolus organizer regions (NORs), but rarely in interstitial positions. The heterochromatic regions are either quinacrine positive and mithramycin negative or vice versa. All species examined possess only one homologous pair of NORs; these display the brightest mithramycin fluorescence in the karyotypes. Many specimens exhibited unequal labelling of the two NORs both after silver and mithramycin staining as well as after in situ hybridization with ³H-18S + 28S rRNA. In four species, between one and six chromosome pairs with homologous 5S rRNA sites could be identified. The 5S rRNA genes and the 18S + 28S rRNA genes are closely linked in two species. In the male meiosis of the Amphicoela and Opisthocoela, there are intersitial, subterminal and terminal chiasmata in the bivalents, whereas only terminal chiasmata are observed in the bivalents of the Aglossa and Anomocoela. No heteromorphic sex-specific chromosomes could be demonstrated in any of the species. The differential staining techniques revealed that the chromosomal structure in these four suborders is largely the same as in the highly evolved anuran suborders Procoela and Diplasiocoela.     

Nucleolus organizer regions and heterochromatin in the zebu (Bos indicus L.)

Summary Ag-NOR staining and a counterstain enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI-staining = CDD-method) and G-banding, respectively, have been applied to the zebu (Bos indicus L.) chromosomes. The nucleolus organizer regions (NORs) were found in the telomeric regions of chromosomes nos. 2, 3, 4, 11, and 28. CDD staining led to a well-defined R-banding pattern along the chromosome arms and to the visualization of centric heterochromatic bands of variable sizes.     

Chromosome banding in Amphibia

The distribution and quantity of constitutive heterochromatin and of the nucleolus organizer regions (NORs) on the chromosomes of 22 species of bufonids and hylids (Amphibia, Anura) was investigated. Three different kinds of constitutive heterochromatin were found and the frequency of brightly fluorescing heterochromatic regions was remarkably high. On almost all chromosomes there is centric and telomeric heterochromatin. Quantitative estimates of heterochromatin demonstrate that large DNA differences among closely related species can not be attributed to differing quantities of constitutive heterochromatin. In all species investigated, only one homologous pair of NORs was found, which lies preferentially in the proximal and interstitial segments of the long chromosome arms. The NORs are always associated with constitutive heterochromatin on both sides. The size variability between homologous NORs is very high. In the euchromatic regions of the metaphase chromosomes, neither Q- nor G-bands can be demonstrated; this can be attributed to an extremely strong contraction of the anuran chromosomes. On the basis of these results various mechanism of the chromosomal evolution in Anura are discussed.     

Characterization of heterochromatin in different species of the Scilla siberica group (Liliaceae) by in situ hybridization of satellite DNAs and fluorochrome banding

Satellite DNAs have been isolated from the monocotyledonous plants Scilla siberica, S. amoena, S. ingridae (all are highly GC-rich), and S. mischtschenkoana by using the Ag⁺ –Cs₂SO₄ density centrifugation technique. Hybridization in situ has been performed with ₃H-cRNA to these satellite DNAs in all four species. In each species, the endogenous satellite DNA is located mainly in intercalary and major heterochromatin bands associated with terminal regions and nucleolar organizer regions (NORs) but not in centromeric regions. Patterns observed after cross-species hybridization show a high degree of satellite DNA homology between S. siberica, S. amoena, and S. ingridae. By contrast, satellite DNA of S. mischtschenkoana consists largely of different, non homologous DNA sequences, with two exceptions: (i) the NORs of all four species contain similar satellite sequences, and (ii) a strong homology exists between the satellite DNA of S. mischtschenkoana and centromeric DNA of S. siberica but not with those of S. amoena and S. ingridae. — Heterochromatin has also been characterized by the AT-specific fluorochromes quinacrine (Q) and DAPI and the GC-specific agent chromomycin A₃ (CMA₃), in combination with two counterstaining techniques. While CMA₃-fluorescence is largely in agreement with data on base composition and location of the specific satellite DNAs, the results with Q and DAPI are conflicting. Prolonged fixation has been found to change the fluorescence character in certain instances, indicating that other factors than the base sequence of the DNA also play a role in fluorochrome staining of chromosomes. The results are discussed in relation to the taxonomy and phylogeny of the four species.     

Reverse fluorescent chromosome banding with chromomycin and DAPI

Two DNA binding guanine-specific antibiotics, chromomycin A₃ (CMA) and the closely related mithramycin (MM), were used as chromosome fluorescent dyes. Root-tip metaphase chromosomes of three plant species and human metaphase chromosomes were sequentially stained with CMA or MM and the DNA binding AT-specific fluorochrome 4-6-diamidino-2-phenylindole (DAPI). In some cases a non-fluorescent counterstain was used as contrasting agent: methyl green in conjunction with CMA, and actinomycin D (AMD) in combination with DAPI. — In all three plant species, Vicia faba, Scilla siberica, and Ornithogalum caudatum, the nucleolus organiser regions and/or associated heterochromatin displayed very bright fluorescence with CMA and MM and, in general, heterochromatic segments (C-bands) which were bright with CMA and MM were pale with DAPI whereas segments which were dim with CMA and MM displayed very bright fluorescence with DAPI. — Human metaphase chromosomes showed a small longitudinal differentiation in CMA fluorescence, which was essentially the reverse of the banding pattern obtained with AMD/DAPI double-staining, but of lower contrast. The CMA-banding pattern appears to be similar to the pattern found by R-banding procedures.     

An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae,Cyprinodontiformes)

The mitotic and meiotic chromosomes of the American cyprinodont fish Poecilia sphenops var. melanistica were analysed. All 46 chromosomes are telocentric. By specific staining of the constitutive heterochromatin with C-banding and various AT-specific fluorochromes, the homomorphic chromosome pair 1 could be identified as sex chromosomes of the ZW/ZZ type. All female animals exhibit a W chromosome with a large region of telomeric heterochromatin that is not present in the Z chromosome. These sex chromosomes cannot be distinguished by conventional staining; they represent the first demonstration of sex chromosomes in fishes in an early stage of morphological differentiation. The W heterochromatin and the telomeric heterochromatin in the two autosomes 18 show a very bright fluorescence when stained with AT-specific fluorochromes. This allows the direct identification of the chromosomal sex by examining the interphase nuclei: females exhibit three, males only two brightly fluorescent heterochromatic chromocenters in their nuclei. The significance of these ZW/ ZZ sex chromosomes and their specific DNA sequences, the dose compensation of the Z-linked genes, and the experimental possibilities using sex-reversed ZW males are discussed.     

Evolutionary diversity of reverse (R) fluorescent chromosome bands in vertebrates

Mitotic chromosomes, interphase cell nuclei, and male meiosis of 41 species representing all vertebrate classes were analyzed with distamycin A/mithramycin counterstaining. The purpose of the study was to recognize differences and common characteristics in the reverse (R) fluorescent banding patterns in the chromosomes of vertebrate species at various stages of evolution. In contrast to the warm-blooded mammals and birds, the euchromatic segments in the chromosomes of most reptiles, amphibians, and fishes contain no multiple fluorescent R-bands. This is thought to be due to the absence of the long homogeneous regions (isochores) in the DNA of the cold-blooded vertebrates. Distamycin A/mithramycin banding specifically reveals the GC-rich constitutive heterochromatin in all vertebrates. In most of the vertebrate chromosomes examined, the heterochromatic regions have opposite staining properties with mithramycin and quinacrine. Mithramycin labels the nucleolus organizer regions very brightly in the karyotypes of fishes, amphibians, reptiles and birds, but not of mammals. The lack of mithramycin fluorescence at the nucleolus organizer regions of mammals is attributed to the relatively low level of redundancy of the GC-rich ribosomal DNA in their genomes. Studies on the various meiotic stages of the cold-blooded vertebrates show that the mithramycin labeling of the nucleolus organizers is independent of their state of activity. This can be confirmed by mithramycin fluorescence at the nucleoli of actinomycintreated cells.Dedicated to the memory of Professor Dr. Hans Bauer     

Nucleolus-organizer regions and heterochromatin in three species of Bovidae

Ag-NOR staining and a counterstain-enhanced fluorescence technique (chromomycin A₃/distamycin A/DAPI staining = CDD-method) have been applied to ibex (Capra ibex L.), chamois (Rupicapra rupicapra L.) and bison (Bison bison L.) chromosomes.Chromomycin A₃ visualization led to a well defined R-banding pattern along the chromosome arms and to a clear demonstration of centric heterochromatic bands of variable size. The nucleolus organizer regions (NORs) were found in the telomeric regions of the chromosomes 2, 3, 4, 5 and 28 of the ibex, of the chromosomes 1/3 (short arm), 2, 4, 5 and 28 of the chamois and of the chromosomes 2, 3, 4, 11 and 28 of the bison.     

Heterochromatins and band karyotypes in three species of salmonids

Summary The heterochromatins of rainbow trout (Salmo gairdneri R.), brown trout (Salmo trutta fario L.) and brook trout (Salvelinus fontinalis M.) were characterized by sequential chromomycin A₃/distamycin A/DAPI (CDD) and DAPI/actinomycin D (DAPI/AmD) fluorescence. On most biarmed chromosomes, an equilocal localization of prominent DAPI/AmD positive, chromomycin A₃ negative, AT-rich blocks at the centromeres were observed in all three species. Band karyotypes of the three species were established. In rainbow trout, several DAPI/AmD positive heterochromatin blocks behaved positive in a silver-staining method. Mitotic and interphase studies proved the presence of inter-individual NOR variation in brown trout. The NORs of brook trout were localized on chromosomes 5, 10, 14, 15 and 29.     

NORs and counterstain-enhanced fluorescence studies in Cyprinidae of different ploidy level

The ribosomal RNA gene expression in the genomes of evolutionary diploid (Scardinius erythrophthalmus, Leucaspius delineatus, Tinca tinca) and polyploid species (Cyprinus carpio, Carassius carassius, Carassius auratus gibelio, Carassius auratus auratus) of Cyprinidae has been investigated by means of a silver nitrate technique. The diploid species investigated exhibited only one pair of chromosomes with nucleolus organizers (NOR). Higher numbers of rRNA-expressing chromosomal sites in several evolutionary polyploid species (Carassins) gave evidence against a complete functional diploidization, at least with regard to the NOR bearing chromosomes in these species. The NORs displayed a heterochromatic brilliant chromomycin A₃ fluorescence. No distamycin-A/DAPI-bright heterochromatic blocks were detected in the genomes of the Cyprinidae.     

Nucleotide Composition of the Cold-Sensitive Heterochromatic Regions in Paris hainanensis Merrill

Cold-induced decondensation of heterochromatic regions (CSR-bands) in Paris hainanensis(=Daiswa hainanensisMerrill Takht.) (2n= 10; 10 + b) was studied. The comparison of CSR-banding patterns with those obtained by nucleotide-specific staining with fluorochromes DAPI and chromomycin A₃demonstrated that low temperatures induced decondensation only of large AT-rich heterochromatic regions. It is suggested that this is characteristic of all plant species.     

Localisation of NORs and counterstain-enhanced fluorescence studies inPerca fluviatilis (Pisces,Percidae)

The karyotype of the perch (Percafluviatilis L.) was analysed by means of silver-staining, the chromomyc in A/3 Distamycin A/DAPI and Actinomycin D fluorescence technique in order to locate active NORs and to selectively highlight certain heterochromatic regions. The ribosomal RNA genes are localized at the secondary constrictions of the short arms of chromosomes no. 16. Additionally, bright chromomycin fluorescence was observed in the same regions. No DAPI/ Distamycin A bright heterochromatic block was detected in the perch karyotype.     

Karyotype characterization of the lake sturgeon, Acipenser fulvescens (Rafinesque 1817) by chromosome banding and fluorescent in situ hybridization.

A karyotype analysis using several staining techniques was carried out on the North American lake sturgeon, Acipenser fulvescens. The chromosome number was found to be 2n = 262 +/- 6. A representative karyotype of 264 chromosomes was composed of 134 meta- and submetacentrics, 70 telo- and acrocentrics, and 60 microchromosomes. The constitutive heterochromatin, revealed by C banding, was localized in various positions on several chromosomes, including microchromosomes. The signals of fluorescent in situ hybridization (FISH) with a HindIII satellite DNA probe were visible as centromeric heterochromatin blocks on 48 chromosomes. The telomeric repeat (TTAGGG)n detected by FISH was localized at both ends of all chromosomes and two chromosomes were entirely marked. Fluorescent staining with GC-specific chromomycin A3 showed recognizable fluorescent regions, whereas a more uniform base composition was revealed by the AT-specific 4',6-diamidino-2-phenylindole (DAPI). After silver staining, the active nucleolar organizer regions (NORs) were detected on 12 chromosomes. FISH with the 5S probe showed four signals on four small chromosomes. Our data suggest that A. fulvescens is a tetraploid species.     

Heterochromatin and nucleolus-organizer-region behaviour at male pachytene of Sus scrofa domestica

In the domestic pig (2n=38) two types of constitutive heterochromatin can be differentiated by fluorescence counterstaining techniques. All 24 biarmed autosomes and the X chromosome have chromomycin A₃-positive centromeric C-bands, whereas all 12 acrocentric chromosomes exhibit DA-DAPI-positive centromeric heterochromatin. Fluorescence analysis of male pachytene nuclei revealed that the DA-DAPI-positive C-bands form one or two large chromocentres per cell, while the chromomycin A₃-bright C-material is well scattered. Hence, the bivalents formed by the acrocentric chromosome pairs are centromerically associated, whilst the submetacentric bivalents are not. —Counce-Meyer spreading techniques were used to study the structure of synaptonemal complexes (SCs) both by light and electron microscopy. In general, the SCs of the domestic pig resemble those described for other mammals. The SC formed by the X and the Y may include up to 94.5% of the Y chromosome. In silver-stained microspreads each of the bivalents (nos. 8 and 10) bearing the nucleolus-organizer-regions (NORs) is connected to a pair of nucleoli, indicating that all four NORs are active during early meiotic stages. By contrast, in the majority of mitotic metaphases of phytohaemagglutinin-stimulated lymphocytes only one pair (no. 10) exhibited Ag-NOR staining. — The significance of the chromosome disposition in the pachytene nucleus is discussed with regard to heterochromatin composition and karyotype evolution.This paper is dedicated to Prof. Hans Bauer on the occasion of his 80th birthday     

鱼类染色体的荧光显带研究

应用GC碱基特异性荧光染料色霉素A,辅以AT减基特异性荧光染料Hoechst33258,DAPI或喹吖因对鲤鱼,鲫鱼,大鳞副泥鳅和的有丝分裂染色体及黄鳝的有丝分裂和减数分裂染色体进行了荧光显带研究,结果发现,色霉素A3可以特异性地显示鱼类有丝分裂及减数分裂各个时期核仁组织区NORS的存在,Hoechst33258,DAPI或喹吖因则使这些区域（NORs)淡染,大鳞副泥鳞的染色体NORs 分布位置具有性别,根据实验结果,对有关鱼类染色体的荧光染色研究及其应用进行了讨论。     

Band karyotypes and specific types of heterochromatins in several species of European percid fishes (Percidea,Pisces)

The chromosomes of the European Percidae (Lucioperca lucioperca L., Gymnocephalus cernuus L., Gymnocephalus schraetser L. and Perca fluviatilis L.) were analyzed by means of silver staining chromomycin A₃/distamycin A/DAPI and DAPI/actinomycin D fluorescence banding techniques. The nucleolus organizer regions (NORs) were localized at the satellite stalks of chromosome no. 16 in Lucioperca lucioperca and Perca fluviatilis, and of chromosome no. 18 in both Gymnocephalus species. Bright chromomycin A₃ fluorescence clusters were associated with them.Bright distamycin A-DAPI and DAPI/actinomycin D heterochromatic blocks were detected in Lucioperca lucioperca and the Gymnocephalus species.     

NOR polymorphism in the Iberian species Chondrostoma lusitanicum (Pisces: Cyprinidae)

Chromosomal polymorphism regarding number of NOR sites in the cyprinid fish Chondrostoma lusitanicum was examined using C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ (CMA₃). The analysis of heterochromatic regions allowed a more precise identification of the centromeric regions and the proposal of a revised haploid chromosome formula (7M: 15S: 3A). We describe variability in the number of NOR regions per genome, number of active NOR sites per cell, and relative size of individual NORs. Individuals expressed two or four NOR-bearing chromosomes. Polymorphism was detected in all the populations studied and sex-related differences were not found. The observed chromosomal NOR phenotypes suggest the occurrence of structural rearrangements during the evolutionary process of this diploid leuciscine cyprinid.     

Banded karyotype of spined loach Cobitis taenia and triploid Cobitis from Poland

The present work provides new data on the banding pattern of diploid Cobitis taenia and its triploid hybrid females, which belong to the diploid–polyploid complex in the Vistula River tributary. C-banding, silver-staining (Ag), and fluorescent staining with chromomycin A₃ techniques were used to describe the diploid and triploid karyotype. The karyotype of Cobitis taenia of 2n=48 was characterised by one pair of NOR-bearing subtelocentric chromosomes and at least four chromosomes with CMA₃-positive sites. The C-positive heterochromatin was present in the centromeres of almost all chromosomes and the pericentromeric regions of several metacentric and submetacentric chromosomes. The triploid females of 3n=74 had two pairs of chromosomes with active NORs. The NORs-sites were located terminally on two biarmed and two uniarmed chromosomes. The CMA₃-staining revealed at least six A₃-positive sites. The C-banded and A₃-stained triploid karyotype was composed of haploid set of Cobitis taenia and diploid set of unidentified species, so heterochromatin pattern confirmed the possibility of their hybrid origin. The characteristics of banded diploid and triploid karyotype, and the hypothetical karyotype of an unknown species of 2n=50 is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.