Restriction enzyme and fluorochrome banding analysis of the constitutive heterochromatin of Saguinus species (Callitrichidae, Primates)

Metaphases of Saguinas fuscicollis fuscicollis and Saguinas mystax were subjected to restriction enzyme banding (Alu I, Hae III, Hin fI, Rsa I, Dde I, Mbo I and Msp I) and sequenced C-banding, together with fluorochrome staining (CMA3 and DAPI). Both species showed large C-bands in the pericentromeric regions. S. f. fuscicollis also manifested distal C-bands in both arms of pair 5 and in the short arms of pairs 8-15. In each species the heterochromatin revealed different reactions to the restriction enzymes and fluorochromes. This was related to its location in the genome (centromeric, pericentromeric, distal), making possible the identification of distinct categories of constitutive heterochromatin. In S. f. fuscicollis there were at least five types, namely centromeric in bi-armed chromosomes, centromeric in acrocentrics, pericentromeric, distal, and cryptic bands, detected only with the Alu I. There were three types in S. mystax, viz centromeric in bi-armed chromosomes, centromeric in acrocentric, and pericentromeric chromosomes. Several aspects of their constitution and origin are discussed.     

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.     

Equilocality of heterochromatin distribution and heterochromatin heterogeneity in acridid grasshoppers

Comparative fluorescence studies on the chromosome of ten species of acridid grasshoppers, with varying amounts and locations of C-band positive heterochromatin, indicate that the only regions to fluoresce differentially are those that C-band. Within a given species there is a marked tendency for groups of chromosomes to accumulate heterochromatin with similar fluorescence behaviour at similar sites. This applies to all three major categories of heterochromatin — centric, interstitial and telomeric. Different sites within the same complement, however, tend to have different fluorescence properties. In particular, centric C-bands within a given species are regularly distinguishable in their behaviour from telomeric C-bands. Different species, on the other hand, may show distinct forms of differential fluorescence at equilocal sites. These varying patterns of heterochromatin heterogeneity, both within and between species, indicate that whatever determines the differential response to fluorochromes has tended to operate both on an equilocal basis and in a concerted fashion. This is reinforced by the fact that structural rearrangements that lead to the relocation of centric C-bands, either within or between species, may also be accompanied by a change in fluorescence behaviour.We dedicate this paper, with affection, to Professor Hans Bauer on the occasion of his 80th birthday, and in appreciation of his singular contribution to the study of chromosomes     

Heterochromatin and interstitial telomeric DNA homology

The purpose of this investigation was twofold. The first objective was to demonstrate that, in most of ten mammalian species commonly used in biomedical research, not all constitutive heterochromatin (C-bands) represents telomeric DNA. For example, the C-bands in human chromosomes, the long arm of the X and the entire Y chromosome of Chinese hamster, and most of the short arms of Peromyscus and Syrian hamster chromosomes are not telomeric DNA. In addition to the usual terminal telomeric DNA in the chromosomes of these mammalian species, the pericentromeric regions of seven or eight Syrian hamster chromosomes and all Chinese hamster chromosomes except pair one have pericentromeric regions that hybridize with telomeric DNA, some in C-bands and some not. The second objective was to describe a simple fluorescence in situ hybridization (FISH) reverse-printing procedure to produce black-and-white microphotographs of metaphase and interphase cells showing locations of telomeric DNA with no loss of resolution. Thus, at least three different types of heterochromatin (telomeric heterochromatin, nontelomeric heterochromatin and a combination of both) are present in these mammalian species, and this simple black-and-white reverse printing of telomeric FISH preparations can depict them economically without sacrificing clarity.     

Counterstained enhancement of TaqI resistant sites after distamycin A/diamidinophenylindole treatment

Numerous selective and differential staining techniques have been used to investigate the hierarchical organisation of the human genome. This investigation demonstrates the unique characteristics that are produced on fixed human chromosomes when sequential procedures involving restriction endonuclease TaqI, distamycin A (DA) and 4,6-diamidino-2-phenylindole (DAPI) are employed. TaqI produces extensive gaps in the heterochromatic regions associated with satellite II and III DNAs of human chromosomes 1, 9, 15, 16 and Y. DA/DAPI selectively highlights, as brightly fluorescent C-bands, the heterochromatin associated with the alpha, beta, satellite II and III DNAs of these chromosomes. When DA and DAPI are used on chromosomes before TaqI digestion, and then stained with Giemsa, the centromeric regions appear to be more resistant, producing a distinct C-banding pattern and gaps in the heterochromatin regions. Sequential use of the DA/DAPI technique after TaqI treatment produces a bright fluorescence on the remaining pericentromeric regions of chromosomes 1, 9, 16 and Y, which also displayed a cytochemically unique banding pattern. This approach has produced specific enhanced chromosomal bands, which may serve as tools to characterize genomic heterochromatin at a fundamental level.     

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.     

Polymorphism of heterochromatic regions of flax chromosomes

The C-banding technique was used to study flax chromosomes (Linum usitatissimum L., 2n = 30). Heterochromatin was located mainly in pericentromeric regions of chromosomes. In spite of small size (1.5-3.5 microm), all 15 pairs of homologous chromosomes were identified on the basis of the C-banding pattern and morphology. An idiogram of C-banded chromosomes of L usitatissimum L. is presented. Polymorphism of chromosomal heterochromatic regions was studied in karyotypes of three flax samples: L usitatissimum L., accession K-603 (L usitatissimum var. usitatissimum), and accession K-594 (L. usitatissimum var. humile (Mill.)). A common C-banding pattern was observed in all forms studied, although there were some distinctions in the individual band size. The fibre flax (accession K-603) karyotype had the C-banding pattern similar to that of L usitatissimum L., but some intercalary and telomeric C-bands were somewhat larger, and a satellite (NOR) was observed in the short arm of chromosome I. In crown flax, (K-594) chromosomal C-banding pattern exhibited smaller pericentromeric and larger intercalary bands; telomeric bands were present on almost all chromosomes. Thus, the intraspecies polymorphism revealed in the chromosomal C-banding pattern makes possible the use of C-bands as chromosome markers in the studies of genetic and genomic polymorphism of this species.     

Specific arrangement of chromosomes in the spermiogenesis of Gallus domesticus

On the chromosomes of the rooster the constitutive heterochromatin (C-bands) is to be found for the most part at the centromeres. The position of the centric heterochromatin in spermatids and sperm is not randomly distributed. In early, round spermatids one heterochromatic block lies at this exact position on the membrane that develops into the tip of the sperm nucleus (acrosomal chromocenter). During the elongation of the spermatid nucleus another heterochromatic block comes to lie on the basal nuclear membrane. The other centromeres arrange themselves tandem-wise between the acrosomal and the basal chromocenters. Comparisons have been made between this specific arrangement and the location of chromosomes in the sperm of amphibians and mammalians.     

Location of ribosomal genes in the chromosomes of Anopheles darlingi and Anopheles nuneztovari (Diptera,Culicidae) from the Brazilian Amazon

Fluorescence in situ hybridization of Anopheles darlingi and A. nuneztovari demonstrated nucleolar organizer region activity at the end of the fourth larval instar, when the nucleolar organizer regions underwent gradual condensation. The heteromorphic sex chromosomes showed intraindividual size variation in the rDNA blocks located in the pericentromeric region and this coincided with the location of constitutive heterochromatin (C-banding).     

Constitutive heterochromatin DNA polymorphisms in diploid Medicago sativa ssp. falcata.

A Giemsa C-banding technique was used to study the amount and location of constitutive heterochromatin in diploid (2n = 2x = 16) Medicago sativa ssp. falcata (L.) Arcangeli. Most accessions had the standard C-banding pattern with centromeric bands on all the chromosomes and a prominent heterochromatic band at the nucleolar organizer regions (NOR) of the satellited (SAT) chromosomes. However, we observed in various accessions that constitutive heterochromatic C-bands can exist at the telomeric ends of all the chromosomes. Interstitial bands occurred on the short arms of all chromosomes except for chromosome 3 and on the long arms of chromosomes 1, 2, 3, and 6, only. Rearranged chromosomes such as isochromosomes have been observed for the short arms of chromosomes 2 and 6. This is the first report on the existence of C-banding polymorphisms and the detection of putative isochromsomes in the chromosomes of diploid ssp. falcata which could have contributed to the variation observed in cultivated alfalfa.     

Pairing Competition between Identical and Homologous Chromosomes in Rye and Grasshoppers

Meiotic pairing preferences between identical and homologous but not identical chromosomes were analyzed in spontaneous tetraploid/diploid chimeras of three male grasshoppers (Eyprepocnemis plorans) whose chromosome pair 11 were heterozygous for C-banding pattern and in four induced tetraploid/diploid chimaeral rye plants (Secale cereale) heterozygous for telomeric heterochromatin C-bands in chromosomes 1R and 2R. In the grasshoppers, a preference for identical over homologous pairing was observed, whereas in rye both a preference for homologous rather than identical pairing and random pairing between the four chromosomes of the set was found. From the results in rye, it can be deduced that pairing preferences do not depend exclusively on the similarities between chromosomes involved. It is suggested that genotypic or cryptic structural differences between the homologous chromosomes of each pair analyzed might be responsible for the pairing preferences found. This hypothesis can also explain the results obtained in grasshoppers, although the possibility of premeiotic association cannot be excluded in this material.     

Integrative study on chromosome evolution of mammals, ants and wasps based on the minimum interaction theory

There is well-known evidence that in many eukaryotes, different species have different karyotypes (e.g. n=1-47 in ants and n=3-51 in mammals). Alternative (fusion and fission) hypotheses have been proposed to interpret this chromosomal diversity. Although the former has long been accepted, accumulating molecular genetics evidence seems to support the latter. We investigated this problem from a stochastic viewpoint using the Monte Carlo simulation method under the minimum interaction theory. We found that the results of simulations consistently interpreted the chromosomal diversity observed in mammals, ants and wasps, and concluded that chromosome evolution tends to evolve as a whole toward increasing chromosome numbers by centric fission. Accordingly, our results support the fission hypothesis. We discussed the process of chromosome evolution based on the latest theory of the molecular structure of chromosomes, and reconfirmed that the fission burst is the prime motive force in long-term chromosome evolution, and is effective in minimizing the genetic risks due to deleterious reciprocal translocations and in increasing the potential of genetic divergence. Centric fusion plays a biological role in eliminating heterochromatin (C-bands), but is only a local reverse flow in contrast to the previously held views.     

Homologous chromosomes characteristics by sequential banding procedures in rainbow trout Oncorhynchus mykiss

The development of high resolution methods of chromosome banding helped the finding of homologous chromosomes, detecting chromosomal abnormalities, and assigning the gene loci to particular chromosomes in mammals. Unfortunately, small and numerous fish chromosomes do not show GC rich and GC poor compartments, this preventing the establishment of G banding pattern. The combination of techniques enabling the identification of constitutive heterochromatin (C-banding), heterochromatin resistant to restriction endonucleas, NOR bearing chromosomes (AgNO3 banding), or AT rich regions on chromosomes (DAPI banding) in sequential staining provides a better characteristic of fish chromosomes. In this work sequentially DAPI, DdeI, AgNO3 stained chromosomes of rainbow trout resulted in the characteristic banding pattern of some homologous chromosomes. Procedure of FISH with telomere probe and DAPI as a counterstaining fluorochrome visualized simultaneous hybridization signals and DAPI banding. Possibility of detection both FISH and DAPI signals can help in procedures of gene mapping on chromosomes.     

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     

Comparison of the Giemsa C-banded karyotypes of Hystrix coreana and H. patula (Poaceae, Triticeae)

The karyotypes of Hystrix coreana from eastern USSR and H. patula from USA were investigated by Giemsa C-banding. Both species are outbreeders and have 2n = 4x = 28. The karyotype of two plants of H. coreana has 10 metacentric, 6 submetacentric, 8 heterobrachial and 4 SAT chromosomes; two plants differed by having 12 metacentric, 4 submetacentric, 8 heterobrachial and 4 SAT-chromosomes, and 10 metacentric, 4 submetacentric, 9 heterobrachial and 5 SAT-chromosomes, respectively. The C-banding pattern had no or few inconspicuous intercalary bands, but conspicuous telomeric C-bands in one or both arms giving a high content of heterochromatin (16.3–18.2%). The chromosome complement of one plant of H. patula had 8 metacentric, 6 submetacentric, 8 heterobrachial and 6 SAT-chromosomes. The C-banding pattern had between 1 and 4 intercalary or centromeric bands and conspicuous telomeric bands on one or both arms giving a high content of constitutive heterochromatin (16.4%).     

Cytogenetic study of Anopheles albitarsis (Diptera: Culicidae) by C-banding and in situ hybridization

The C-banding pattern and the size and location of the nucleolar organizer regions (NORs) are described for the first time in Brazilian populations of Anopheles (Nyssorhynchus) albitarsis sensu lato. C-banding revealed variation in the size of the centromeric heterochromatic blocks in autosomal chromosomes and in the acrocentric (X) and puntiform (Y) sex chromosomes. Fluorescence in situ hybridization showed that the NORs were located in the pericentromeric region of the sex (XX/XY) chromosomes and that this coincided with the number and location of centromeric constitutive heterochromatin blocks previously revealed by C-banding. The NORs varied in size among the homologues of the three populations. These findings of the populations studied support the hypothesis that the stability of NORs in the A. albitarsis complex is characterized by the presence of clustered and conserved sites in a unique pair of chromosomes.     

Chromosome banding in amphibia

In the chromosomes of 12 frog species of the suborder Diplasiocoela (Amphibia, Anura), the constitutive heterochromatin and the nucleolus organizer regions (NORs) have been specifically stained. On most of the chromosomes, aside from the centric heterochromatin, telomeric and interstitial C-bands were also found. The various C-bands display a very variable reaction to alkaline pretreatment; this indicates heterogeneity in the constitutive heterochromatin. Sex chromosomes could not be identified in any of the species studied. The number and chromosomal positions of the NORs vary quite strongly between species and between families. In 4 species of the genus Rana, there were, aside from the standard-NORs in chromosome pair 10, between 4 and 14 extra, small NORs detectable in the smaller chromosome pairs. As possible causal mechanism of these additional small NORs the reintegration of amplified rDNA during amphibian oogenesis is suggested. Q- or G-bands could only be recognized in mitotic prophase chromosomes. The strong spiralization of metaphase chromosomes prevents the differential demonstration of Q- or G-bands in the euchromatic regions.     

Heterochromatic banding patterns on chromosomes of twelve weevil species (Insecta,Coleoptera, Curculionoidea: Apionidae,Curculionidae)

The C-banding patterns of twelve weevil species are presented. The obtained results confirm the existence of two groups of species: with a small or large amount of heterochromatin in the karyotype. The first group comprises seven species (Apionidae: Holotrichapion pisi; Curculionidae: Phyllobius urticae, Ph. pyri, Ph. maculicornis, Tanymecus palliatus, Larinodontes turbinatus, Cionus tuberculosus). In weevils with a small amount of heterochromatin, tiny grains on the nucleus in interphase are visible, afterwards in mitotic and meiotic prophase appearing as dark dots. The absence of C-bands does not indicate a lack of heterochromatin but heterochromatic regions are sometimes so small that the condensation is not visible during the cell cycle. The second group comprises five species (Otiorhynchus niger, O. morio, Polydrusus corruscus, Barypeithes chevrolati, Nedyus quadrimaculatus) which possess much larger heteropicnotic parts of chromosomes visible during all nuclear divisions. The species examined have paracentromeric C-bands on autosomes and the sex chromosome X, except for Otiorhynchus niger, which also has an intercalary bands on one pair of autososomes. All the species examined differ in the size of segments of constitutive heterochromatin. The y heterochromosome is dot-like and wholly euchromatic in all the studied species.     

黄牡丹八个居群的Giemsa C-带比较研究

应用ＢＳＧ方法对黄牡丹（Ｐａｅｏｎｉａ ｄｅｌａｖａｙｉ ｖａｒ．ｌｕｔｅａ）８个居群的Ｇｉｅｍｓａ Ｃ－带进行了比较研究。８个居群的所有染色体都在着丝点附近显示出了Ｃ－带,所有染色体的长臂上都没有显示Ｃ－带,而短臂上的Ｃ－带数量和位置在居群之间表现出了一定的差异。花甸凡居群手第二、第三、第四和第五对染色体显示端带;卓干山居群的第一、第三、第四和第五对染色体的短臂上没有显示Ｃ－带。在所研究的８个居