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.     

Characterization of the canine karyotype by counterstain-enhanced chromosome banding

The application of the counterstain-contrasted fluorescent banding technique to canine chromosomes provided an improved capability to highlight specific heterochromatic regions and to produce well defined banding patterns both on mitotic and meiotic chromosomes. Triple staining with chromomycin A3 - distamycin A - DAPI revealed the occurrence of DA - DAPI positive heterochromatin in chromosomes 33, 36, 37, and 38. Pachytene nuclei present more favourable material for the detection of very small amounts of DA - DAPI material than mitotic division stages. Counterstain-enhanced chromomycin R-banding greatly facilitated chromosome identification. A standard R-band karyotype of Canis familiaris is proposed and described in some detail. DAPI - actinomycin D staining produced a QFH-type banding pattern and enhanced differentiation of some polymorphic regions.     

Expression of heterochromatin by restriction endonuclease treatment and distamycin A/DAPI staining of Indian muntjac (Muntiacus muntjak) chromosomes

The constitutive heterochromatin of the Indian muntjac (Muntiacus muntjak) was examined following digestion with various restriction endonucleases (AluI, HaeIII, HinfI, and MboI), as well as by selective fluorescence staining with distamycin A plus 4'-6-diamidino-2-phenylindole. Distinct areas within the C-bands were found to have characteristic staining patterns which were more conspicuous in the sex chromosomes. Two dot-like structures resistant to AluI were found in the X and Y1 chromosomes in the same position as the nucleolus organizer regions.     

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.     

Correlation of pachytene chromomeres and metaphase bands of human chromosomes, and distinctive properties of telomeric regions

By means of double staining with DAPI and chromomycin A3, we show that the chromomeres of human pachytene chromosomes are generally DAPI positive and chromomycin negative, like the G- or Q-bands of mitotic chromosomes. Thus we have demonstrated, using an objective technique not based on morphological comparisons, that chromomeres and G-bands are equivalent. However, terminal chromomeres and the ends of mitotic chromosomes, as well as a few other sites, are chromomycin positive and DAPI negative. The ends of human chromosomes appear, therefore, to contain a distinctive class of GC-rich DNA.     

Characterization of heterochromatin by sequential counterstain-enhanced fluorescence in three domestic bird species: Meleagris gallopavo, Columba livia domestica, and Anser anser L

The karyotypes of three avian species--Meleagris gallopavo, Anser anser L., and Columba livia domestica--were investigated by means of counterstain-enhanced fluorescence techniques (chromomycin A3/distamycin A/DAPI followed by DAPI/actinomycin D staining). A heterochromatin characterization of macro- and microchromosomes was performed. CMA3-positive (GC-rich) regions in the turkey included the telomeres of chromosomes 1, 3, 4, and Z. In the goose, the chromosome 2 was also CMA3-bright at the telomeres. The W chromosome possessed large amounts of CMA3-bright material on the short arm in both the turkey and the goose. Two types of centromeric heterochromatin were distinguished on acro- to telocentric chromosomes 6 to 14 in the pigeon. The microchromosomal heterochromation of the turkey and goose was GC-rich but had a high degree of variation. In the pigeon, several microchromosomes possessed predominantly AT-rich heterochromatin.     

Ribosomal RNA expression in a mammalian hybrid,the hinny

The expression of nucleolus organizer region (NOR) activity in diploid cells was investigated in a model mammalian hybrid system, the hinny (female ass x male horse), by sequential Ag-NOR and chromomycin A3/distamycin A/DAPI (CDD) staining in lectin-stimulated peripheral blood lymphocytes. In the majority of cases we found non-expression of the horse-derived NOR chromosomes in the hinny. However, in one case there was strong NOR expression on horse-derived chromosome no. 1.     

Characterization of chromosomes and localization of the rDNA locus in the aye-aye (Daubentonia madagascariensis).

The karyotype of a prosimian primate, the aye-aye (Daubentonia madagascariensis), is described. Results from a variety of staining methods (Q-, R-, G-, and C-banding, distamycin A/DAPI and methyl-green/DAPI) are reported. Sites of methylation were visualized using antibodies against 5-methylcytosine. Digestion of aye-aye fixed metaphase chromosomes with the restriction endonuclease HaeIII produced G-banding. No other restriction enzymes tested produced clear G- or C-banding patterns. Ag-staining of the nucleolar organizer regions (NORs) revealed the location of rDNA sites on the short arms of the smallest pairs of acrocentric chromosomes, 13p and 14p.     

小熊猫染色体异染色质的显示

以培养的小熊猫外周淋巴细胞为实验材料,结合Ｃ－显带技术及ＣＭＡ３／ＤＡ／ＤＡＰＩ三竽荧光杂色的方法,对小熊猫的染色体组型、Ｃ－带带型及ＣＭＡ３／ＤＡ／ＤＡＰＩ荧光带带型进行了研究,发现：（１）经Ｃ－显带技术处理,可在小熊猫染色体上呈现出一种极为独特的Ｃ－带带型。在多数染色体上可见到丰富的插入Ｃ－带及端粒Ｃ－带。而着丝区仅显示弱阳性Ｃ－带;（２）除着丝粒区外,ＣＭＡ３诱导的大多数强荧光带纹与Ｃ－阳性     

Localisation of NORs and counterstain-enhanced fluorescence studies in Salmo gairdneri and Salmo trutta (Pisces,Salmonidae)

Summary The karyotypes of the rainbow trout (Salmo gairdneri R.) and the brown trout (Salmo trutta L.) were analyzed by means of silver staining and the chromomycin A₃/distamycin A/DAPI fluorescence banding technique. The nucleolus organizer regions (NORs) were localized at the secondary constrictions of chromosome no. 14 in S. gairdneri and of chromosome no. 10 in S. trutta. Additional silver positive dots were observed at or close to several centromeres in S. gairdneri. Brilliant chromomycin A₃ (CMA₃) fluorescence heterochromatin blocks were localized on both sides of the nucleolar constrictions in S. gairdneri. A polymorphic CMA₃ positive band was detected close to the NORs of S. trutta. No distamycin A/DAPI intense heterochromatin blocks were detected in the genomes of the two Salmo species investigated.     

Chromosome banding and FISH with rDNA probe in the diploid and tetraploid loach Misgurnus anguillicaudatus

The chromosomes of the diploid and tetraploid loach Misgurnus anguillicaudatus were analyzed by staining with Ag, chromomycin A₃ (CMA₃)/distamycin A (DA), and DA/4′,6-diamidino-2-phenylindole (DAPI), and using fluorescence in situ hybridization (FISH) with 5.8S + 28S rDNA as a probe. Nucleolus organizer regions (NORs) were mapped to the telomeric region of the short arms of the largest (first) metacentric chromosome pair in the diploid loach with 2n = 50 and the homologous quartet in the tetraploid loach with 4n = 100. The NORs were positive at the same region of the first metacentric chromosome for Ag and CMA₃/DA stainings, but negative for DA/DAPI staining. Four signals at the homologs within the same quartet suggest the duplication of the entire genome from diploid to tetraploid status. However, a size difference was detected between the rDNA signals by FISH and CMA₃ banding.     

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.     

Polymorphism of 5-methylcytosine-rich DNA in human acrocentric chromosomes

Summary The acrocentric chromosomes of 18 unrelated individuals were analyzed by sequential staining by the chromomycin A₃/methyl green R-banding technique to identify the chromosomes, followed by an indirect immunoperoxidase technique to detect 5-methylcytosine (5MeC)-rich DNA. The short arms of both chromosomes 15 usually (92% of the chromosomes) had a large collection of 5MeC-rich DNA, which was always rich in AT base pairs. Much less commonly (11% of the possible occasions), a collection of 5MeC-rich DNA was seen on the short arm of a chromosome 13, 14, 21 or 22, and this DNA was always rich in GC base pairs. Sequential distamycin A/DAPI (DA/DAPI) and R-banding studies were carried out in 13 of these 18 individuals. There was bright DA/DAPI fluorescence of the 5MeC-rich region on the short arm of chromosome 15 but not on that of any other acrocentric chromosome. One implication of these findings is that bisatellited or other abnormal chromosomes that are DA/DAPI negative and 5MeC positive cannot be derived from number 15. In the case of a de novo chromosome of this type, the specific origin from any other acrocentric chromosome could be demonstrated by examining 5MeC-binding of the parental chromosomes.     

Further resolution of the quail karyotype and characterization of microchromosomes by counterstain-enhanced fluorescence

Sequential staining with a counterstain-contrasted fluorescent R-banding technique (chromomycin A3/distamycin A-DAPI) followed by DAPI-actinomycin D-induced quinacrine-fluorescence-Hoechst 33258 (QFH)-type banding allowed the identification of quail chromosomes up to chromosome 19. The chromomycin A3-positive staining behavior of the W chromosome and of the heterochromatic areas of most microchromosomes indicated their GC-rich nature.     

Cytochemical heterogeneity of the C-band in human chromosome 1

Summary The heterogeneity of the C-band of human chromosome 1 has been evaluated using several selective staining methods: C-banding (CBG), distamycin A plus 4-6-diamidino-2-phenylindole (DA/DAPI) and Giemsa G-11 pattern following the treatment with the restriction endonucleases AluI and HaeIII. The bands produced by each method are characteristic but not identical. The total C-band is resistant to AluI treatment. The bands induced by HaeIII and the one stained by DA/DAPI are markedly similar but smaller than the C-band. The G-11 technique stains yet smaller regions than those of HaeIII and DA/DAPI. Depending on the expression of staining properties, the C-band of chromosome 1 usually consists of three subdivisions: the proximal, intermediate and distal regions, suggesting an extremely heterogeneous nature. The staining variations between different regions are further substantiated by studies of a reciprocal translocation where the proximal region and the remaining C-band of chromosome 1 are separate.     

Heterochromatin composition and nucleolus organizer activity in four canid species

Sequential staining with a counterstain-contrasted fluorescent banding technique (chromomycin A3-distamycin A-DAPI) revealed the occurrence of distamycin A-4,6-diamidino-2-phenylindole (DA-DAPI) staining heterochromatin in the centromeric regions of chromosomes 33, 36, 37, and 38 in the wolf (Canis lupus pallipes) and of chromosomes 13, 16, and 23 in the blue fox (Alopex lagopus). The red fox (Vulpes vulpes) lacked such regions. Staining with DAPI--actinomycin D produced a QFH-type banding pattern with clearcut differences in the staining behaviour of DA-DAPI positive regions between these three canid species. Staining with the fluorochrome D 287/170 did not preferentially highlight any of the DA-DAPI positive regions in any of them. Counterstain-enhanced chromomycin A3 R-banding and studies of nucleolus organizer region location and activity confirmed a close relationship between the karyotype of the wolf and the domestic dog. Few heterochromatic marker bands were encountered in these two species, but heterochromatin polymorphism was evident in the blue fox.     

Modification of DAPI banding on human chromosomes by prestaining with a DNA-binding oligopeptide antibiotic, distamycin A

A DNA-binding AT-specific oligopeptide antibiotic, distamycin A, was used as non-fluorescent counterstain in conjunction with the DNA-binding AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) to investigate the effect of the antibiotic on DAPI fluorescent banding of human chromosomes. Distamycin A-pretreated metaphases and interphase nuclei exhibited a significantly lower overall fluorescence intensity than DAPI controls. Chromosome arms were pale and intercalary DAPI bands (Q bands) were obliterated, but some specific regions of constitutive heterochromatin remained brightly fluorescent. These were mainly the constrictions of chromosomes 1, 9 and 16, the short arm of chromosome 15, and the distal part of the Y. The distamycin A/DAPI banding pattern appears to be comparable to that reported for anti-5-methylcytosine binding [11]. The observations are discussed as they relate to the roles of chromosomal DNAs and proteins in chromosome banding.     

Prediction of mammalian meiotic synaptic and recombinational behavior of inversion heterozygotes based on mitotic breakpoint data and the possible evolutionary consequences

The karyotype of moose (2n=68) is characterized by very large C-bands close to the centromeres of most chromosomes. The C-banded material represents 40% of the genome. For further characterization of the heterochromatin chromosome spreads were treated with restriction endonucleases and the restriction enzyme (Re) banding pattern was analysed. HaeIII, AluI, MboI, RsaI and HinfI produced informative Re-bands. DdeI induced an even digestion with no banding. Staining with chromomycin A3 produced bright fluorescence in regions corresponding to C-bands. Labeling with BrdUrd during late S phase differentiates four regions in the C banded area. The sequence of these regions from centromere to telomere are: late, early, late and early replicating.The authors propose the existence of five satellite DNA families with distinctive characteristics of G-C and A-T richness and different replication timing, and point out the different clusters for the endonucleases detailed above and their varying location in the chromosomes examined.     

Cytological characterization of sex chromosomes and ribosomal DNA location in Anastrepha species (Diptera, Tephritidae)

This paper reports a comparative analysis of heterochromatin organization in the sex chromosomes of the fruit fly Anastrepha. Mitotic chromosomes of eight Anastrepha species from different taxonomic groups were stained with DAPI and chromomycin A3 fluorochromes followed by C-banding. A specific sex-chromosome banding pattern was obtained for each of the analyzed species. Fluorescence in situ hybridization (FISH) was performed to investigate the chromosomal location of rDNA loci. In all cases the rDNA sequences were found to localize exclusively to the sex chromosomes. The results further extend the chromosomal knowledge of Anastrepha and allow a precise species identification.     

Chromosome banding study of European catfish,Silurus glanis (Pisces,Siluridae)

The karyotype of European catfish (Silurus glanis L.) was analyzed sequentially by means of silver staining and the chromomycin A₃ (CMA₃)/distamycin A (DA)/DAPI fluorescence technique and by C-banding, respectively. The nucleolus organizer regions (NORs) were localized on the submetacentric pair No. 14. Brilliant CMA₃ fluorescent heterochromatin blocks corresponded to the NORs visualized by silver staining. No DA/DAPI-bright positive fluorescent patterns were detected while C-banding led to the detection of specific banding patterns on several chromosome pairs.—Using these banding data, the karyotype of S. glanis was redescribed.