Assignment of the gene coding for human cytochrome c oxidase subunit VIb to chromosome 19, band q13.1, by fluorescence in situ hybridisation

Summary A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-11-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit VIb gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.     

Localization of chromosome regions in potoroo nuclei (<Emphasis Type="Italic"> Potorous tridactylus </Emphasis>Marsupialia: Potoroinae)

Chromosome paints of the rat kangaroo ( Aepyprymnus rufuscens, 2n =32) were used to define chromosome regions in the long nosed potoroo ( Potorous tridactylus, 2n =12 female, 13 male) karyotype and localize these regions in three-dimensionally preserved nuclei of the potoroo to test the hypothesis that marsupial chromosomes have a radial distribution. In human nuclei chromosomes are distributed in a proposed radial fashion. Gene-rich chromosomes in the human interphase nucleus are preferentially located in the central area while gene-poor chromosomes are found more at the periphery of the nucleus; this feature is conserved in primates and chicken. Chromosome ordering in nuclei of P. tridactylus is related to their size and centromere position. Its relationship with replication patterns in interphase nuclei and metaphase was studied. In addition it was observed that the nucleus was not a smooth entity but had projections occupied by specific chromosome regions. Edited by: R. Allshire     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

SUPERNUMERARY CHROMOSOMES IN SAXIFRAGA VIRGINIENSIS (SAXIFRAGACEAE)

Acetocarmine squashes of root tips have demonstrated that 2n = 20 and 38 in Saxifraga virginiensis. These contrast with the earlier reported count of 2n = 28 for this species. In several populations supernumerary chromosomes were detected. Both intrapopulational and interpopulational variation in supernumerary chromosome number were detected, with the largest number of supernumerary chromosomes observed being six. Because these supernumerary chromosomes are equal in size to many of the smaller A chromosomes during mitotic metaphase, the presence of supernumerary chromosomes in this species could not be ascertained by analysis of mitotic metaphase preparations alone. During mitotic prophase, however, the supernumerary chromosomes of S. virginiensis are highly heterochromatic, appearing more densely coiled and darkly stained than the A chromosomes. This characteristic facilitated the recognition of supernumerary chromosomes in this species. The similarity in size of A and supernumerary chromosomes during mitotic metaphase and the observation of six supernumerary chromosomes in one population suggest that the count of 2n = 28 reported earlier for S. virginiensis may actually be a misinterpretation of 2n = 20 plus 8 supernumerary chromosomes. Furthermore, these findings and the observation of this same supernumerary chromosome phenomenon in other species of Saxifraga raise the possibility that some of the many disparate chromosome counts attributed to aneuploidy in the large genus Saxifraga may also be the result of misinterpretations of supernumerary chromosomes as A chromosomes.     

The ultrastructure of G- and C-banded chromosomes

A method of visualizing chromosome bands by electron microscopy has been used to investigate the fine structural organization of G- and C-banded chromosomes. The following information has been obtained:

1. 1. G-bands, produced by trypsinization, were electron dense regions of highly packed chromatin fibres separated by regions in which the chromatin fibres were much less densely packed (interbands).

2. 2. Several degrees of chromatin dispersion were apparent in trypsinized chromosomes. Such dispersion was not a prerequisite for the initial visualization of G-bands, however the progressive pattern of dispersion indicated that the bands were relatively more resistant to dispersion than the interbands.

3. 3. After fixation and trypsinization, individual chromatin fibres measured 250 Å in diameter and appeared morphologically similar to control chromatin fibres seen by whole mount electron microscopy.

4. 4. In trypsinized chromosome complements, the chromosomes often appeared to be interconnected to one another by chromatin fibres. The evidence indicates that these interchromosomal fibres are artefacts produced by the overlapping of dispersed chromatin fibres.

5. 5. When the same metaphase chromosome was observed by both light and electron microscopy, some of the light microscopic G-bands were represented by two or more ultrastructural bands. The number of bands seen in metaphase chromosomes by electron microscopy appears to approach the increased number of bands generally seen in prometaphase chromosomes by light microscopy.

6. 6. C-banding methods (NaOH treatment or overtrypsinization) resulted in the extraction of variable amounts of chromatin from the non C-band regions of the chromosomes, however the constitutive heterochromatin remained highly condensed and resistant to extraction. This result supports the hypothesis that the mechanism of C-banding involves the selective loss of non C-band chromatin.

     

The lampbrush chromosomes of Salamandra salamandra (L.) (Amphibia Urodela)

Lampbrush chromosomes isolated from the germinal vesicle of medium sized oocytes can be individually identified by differences in two characters: (1) chromosome regions rich in well developed loops, and (2) number and position of spheres. Actually the lateral loops are not all equally extended, but those which are inserted in a certain region of the axis of some chromosomes are more developed and sometimes are loaded with dense and copious matrix; chiasmata do not occur inside these regions. One or more spheres are present on eight chromosomes in the complement (chromosomes I–VI, VIII and X): the total number of spheres inserted on S. salamandra lampbrush chromosomes is the highest among the salamandrid species studied so far. These landmarks as well as the maximally developed normal loops are schematically drawn on the maps of the single lampbrush chromosomes. The length of the maps corresponds to the mean value of the lengths of each chromosome relative to that of chromosome XII, taken as 100 units long.Also bivalents from first metaphase spermatocytes have been analysed: they are generally ring-shaped with two terminal or subterminal chiasmata.     

<Emphasis Type="Italic">In vitro</Emphasis> root induction in weedy <Emphasis Type="Italic">amaranthus</Emphasis> species to obtain mitotic chromosomes

Summary Mitotic chromosome analysis has proven to be an important tool in monitoring the potential for genetic exchange among related plant species. One major obstacle to using mitotic chromosome analysis in any species is obtaining large numbers of clear, well-spread metaphase chromosomes necessary to perform cytological techniques such as chromosome banding and fluorescent in situ hybridization. The ability to obtain good chromosome spreads is in part determined by the number and morphology of the roots, which contain the metaphase tissue. Many Amaranthus species produce very thin, delicate roots. The technique used in the process described herein provides for much more substantial roots, allowing for higher probability of obtaining well-spread metaphase chromosomes. Seeds were planted in a soilless mixture, and then cuttings and leaves were taken from the plants. The cuttings were sterilized and placed in Murashige and Skoog (MS) media, while leaf tissue was analyzed by flow cytometry, both pre-and post-propagation, to obtain DNA contents. No changes in DNA content were observed. The in vitro procedure produced significantly larger roots than were produced in soilless mix. Furthermore, all of the in vitro roots observed had 32 chromosomes of normal morphology. In vitro root propagation allowed large numbers of roots to be obtained from a single plant, thereby resulting in increased probability of obtaining cells with metaphase chromosomes that reflected the original plants' chromosome numbers and therefore may be used for molecular cytogenetic analysis.     

Molecular cytogenetic analysis of Brassica rapa-Brassica oleracea var. alboglabra monosomic addition lines

Interspecific alien chromosome addition lines can be very useful for gene mapping and studying chromosome homoeology between closely related species. In this study we demonstrate a simple but robust manner of identifying individual C-genome chromosomes (C5, C8 and C9) in the A-genome background through the simultaneous use of 5S and 25S ribosomal probes on mitotic and meiotic chromosomes of three different Brassica rapa-B. oleracea var. alboglabra monosomic addition lines. Sequential silver staining and fluorescence in situ hybridisation indicated that 18S-5.8S-25S rRNA genes on the additional chromosome C9 are expressed in the A-genome background. Meiotic behaviour of the additional chromosomes was studied in pollen mother cells at diakinesis and metaphase I. In all of the addition lines the alien chromosome was most frequently observed as a univalent. The alien chromosome C5, which carries an intercalary 5S rDNA locus, occasionally formed trivalents that involved either rDNA- or non rDNA-carrying chromosomes from the A genome. In the case of chromosomes C8 and C9, the most frequently observed intergenomic associations involved the regions occupied by 18S-5.8S-25S ribosomal RNA genes. It is possible that not all such associations represent true pairing but are remnants of nucleolar associations from the preceding interphase. Variations in the numbers and distribution of 5S and 25S rDNA sites between cultivars of B. oleracea, B. oleracea var. alboglabra and B. rapa are discussed.This revised version was published online in April 2005 with corrections to Fig. 2.     

Structural Characteristics of the Chromocenter in Ovary Cells ofc(3)Gand nodMutants of Drosophila melanogaster

Homolog pairing, chromosome morphology, and chromosome disjunction in the first meiotic division were studied in the oocytes of c(3)G/c(3)Gfemale Drosophila melanogasterat developmental stages 3–4 and 14. It was found that homologs were completely or partly paired in some cells (about 20% in either case). The lengths of chromosomes in +/+, +/c(3)G, and c(3)G/c(3)Gcells were at a ratio of 1.0 : 1.6 : 2.2. The chromocenters of homozygous cells had an abnormal structure. There was no meiotic block in metaphase 1, and chromosomes only segregated equally in about 80% of anaphases of the first meiotic division. The data obtained correspond to the abnormal variants of the formation of the chromocenter in c(3)G/c(3)Gfemales that could be predicted based on the two-ring structure of the chromocenter. The mechanism of the effect of the homo- and heterozygosity for the hypomorphic mutation c(3)Gon the formation of the synaptonemal complex (SC) and crossing over frequency was suggested. In nod/nodhomozygous females, asynapsis of pericentromeric regions of homologs was observed in the chromocenter. It was assumed that NOD kinezin is necessary at the last stages of pairing of the pericentromeric regions of homologs and formation of the coordinating bonds between them.     

Chromosome banding in the genusPinus

Somatic chromosomes (2n=24) ofPinus luchuensis Mayr at metaphase were observed by fluorescent banding methods with chromomycin A₃ (CMA) and DAPI. CMA-bands appeared at the interstitial and/or proximal regions of nearly all chromosomes. DAPI-bands appeared at the interstitial and/or centromeric regions of nearly all chromosomes, and pairs of DAPI-dots appeared at the centromeric regions. Each homologous pair of chromosomes in the chromosome complement was identified by the CMA and DAPI fluorescent banding patterns. The interstitial CMA-bands were mostly localized at the secondary constrictions of the Feulgen-stained chromosomes. The fluorescent banding pattern ofP. luchuensis was very similar to that ofP. thunbergii, but was different from that ofP. densiflora.     

Electron microscopic identification of the interphase chromosomes of Amoeba proteus and Amoeba discoides using autoradiography; with some notes on helices and other nuclear components

Data obtained from light and electron microscope autoradiographs of cells of Amoeba proteus and Amoeba discoides previously incubated in medium supplemented with H³ thymidine, indicate that fibrous material, the basic unit of which is about 150 Å in diameter, represents the interphase chromosomes of these amoebae. The helices of interphase nuclei do not appear to incorporate H³ thymidine, which is in opposition to the hypothesis of Taylor (1963) that they are G₂ chromosomes, and makes it unlikely that they represent any form of the DNA-containing component of the amoeba's interphase nucleus. Stereo-electron microscopy reveals that the direction of spiralization of helices may be either left or right handed and that the direction of spiralization of a single helix can reverse. The specific location of helices and of 850 Å–1150 Å electron dense bodies suggests that they are either primary chromosome products which subdivide before entering the cytoplasm, or units for the intranuclear transportation of primary chromosome products. In each nuclear membrane pore complex one central and eight peripheral regions of dense material are found. At each of the nine points, the dense material appears to traverse the nuclear membrane.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Studies on the mitotic chromosome scaffold of Allium sativum

An argentophilic structure is present in the metaphase chromosomes of garlic(Allium sativum),Cytochemical studies indicate that the main component of the structure is non-histone proteins(NHPs).The results of light and electron microscopic observations reveal that the chromosme NHP scaffold is a network which is composed of fibres and granules and distributed throughout the chromosomes.In the NHP network,there are many condensed regions that are connected by redlatively looser regions.The distribution of the condensed regions varies in individual chromosomes.In some of the chromosomes the condensed regions are lognitudinally situsted in the central part of a chromatid while in others these regions appear as coillike transverse bands.At early metaphase.scaffolds of the sister chromatids of a chromosome are linked to each other in the centromeric region,meanwhile,they are connected by scafold materials along the whole length of the chromosome.At late metaphase,however,the connective scaffold materials between the two sister chromatids disappear gradually and the chromatids begin to separate from one another at their ends.but the chromatids are linked together in the centromeric region until anaphase.This connection seems to be related to the special structure of the NHP scaffold formed in the centromeric region.The morphological features and dynamic changes of the chromosome scaffold are discussed.     

The relationship between patterns of DNA replication and of Quinacrine fluorescence in the human chromosome complement

Cultured human peripheral blood lymphocytes were labelled with ³H-thymidine in the early or late S phase prior to mitosis. Quinacrine fluorescence patterns in metaphase chromosomes were then recorded photographically and the slides reprocessed for autoradiography so that the same metaphase cells were examined with the two techniques. The intensity and distribution of ³H-thymidine labelling was compared with the intensity and distribution of Q fluorescence with particular reference to chromosomes 1, 13, 14, 15, 17, 18, 19, 20, 21 and 22. It was found that chromosome regions showing bright fluorescence were also late replicating and that, in general, patterns of late replications reflected the patterns of fluorescence. Exceptions to this generalisation included the late labelling X chromosome in cells of female origin and areas near the centromeres on chromosomes 1, 9, 16 and 22. These centromeric regions show a dull fluorescence but, with exception of chromosome 9, are strongly Giemsa-positive in the ASG staining technique. On the basis of staining reaction, late replicating heterochromatic regions fall into five categories, the relationships and functional significance of these categories is discussed.     

The Location of the nucleolus organizer regions in Drosophila hydei

The positions of the nucleolus organizer regions in metaphase chromosomes of Drosophila hydei were detected by in situ hybridization experiments. In agreement with earlier conclusions the nucleolus of the X chromosome was found to originate in a terminal region of the heterochromatic arm. The Y chromosome contains two nucleolus organizers, one in a terminal position of the long arm, and the other in the short arm. The implications with respect to the evolution of the Y chromosome are discussed.     

Chromosome alterations in tissue culture cells ofAllium fistulosum

Heterochromatin behaviour and structural alterations in chromosomes of cells derived from callus culture ofAllium fistulosum have been studied.The diploid chromosome complement ofAllium fistulosum consists of 16 chromosomes with significant amount of heterochromatin mainly of telomeric nature. In eight collections of callus cells analysed, a high rate of numerical and structural chromosome abnormalities was observed. After 12 months in culture about 20% of metaphase chromosomes possessed distinct signs of mutational events.C-banded preparations revealed that many structural alterations involved regions of heterochromatin. Interchromosomal connections and chromatid fusions occurred at telomeric heterochromatin segments. Also formation of the end-to-end associations and polycentric chromosomes often took place without visible loss of telomeric heterochromatin.     

ELECTRON MICROSCOPIC OBSERVATIONS ON THE SUBMICROSCOPIC MORPHOLOGY OF THE MEIOTIC NUCLEUS AND CHROMOSOMES

Thin sections of the testicular follicles of the grasshopper Laplatacris dispar were studied under the electron microscope. In the primary spermatocytes, during meiotic prophase, three main regions can be recognized within the nucleus: (1) the nucleolus and associated nucleolar material; (2) the interchromosomal regions with the dense particles; and (3) the chromosomes. The nucleolus is generally compact and is surrounded by nucleolar bodies that comprise aggregations of dense round particles 100 to 250 A in diameter. A continuous transition can be observed between these particles and those found isolated or in short chains in the interchromosomal spaces. Particles of similar size (mean diameter of 160 A) can be found associated with the nuclear membrane and in the cytoplasm. The chromosomes show different degrees of condensation in different stages of meiotic prophase. The bulk of the chromosome appears to be made of very fine and irregularly coiled filaments of macromolecular dimensions. Their length cannot be determined because of the thinness of the section but some of them can be followed without interruption for about 1000 to 2000 A. The thickness of the chromosome filaments seems to vary with different stages of prophase and in metaphase. In early prophase, filaments vary between 28 ± 7 A and 84 ± 7 A with a mean of 47 A, in late prophase the mean is about 70 A. In metaphase the filaments vary between 60 and 170 A with a mean of about 100 A. Neither the prophase nor the metaphase chromosomes have a membrane or other inhomogeneities. The finding of a macromolecular filamentous component of chromosomes is discussed in relation to the physicochemical literature on nucleoproteins and nucleic acids and as a result it is suggested that the thinnest chromosome filaments (28 ± 7 A) probably represent single deoxyribonucleoprotein molecules.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

Involvement of synaptonemal complex proteins in sex chromosome segregation during marsupial male meiosis

Marsupial sex chromosomes break the rule that recombination during first meiotic prophase is necessary to ensure reductional segregation during first meiotic division. It is widely accepted that in marsupials X and Y chromosomes do not share homologous regions, and during male first meiotic prophase the synaptonemal complex is absent between them. Although these sex chromosomes do not recombine, they segregate reductionally in anaphase I. We have investigated the nature of sex chromosome association in spermatocytes of the marsupial Thylamys elegans, in order to discern the mechanisms involved in ensuring their proper segregation. We focused on the localization of the axial/lateral element protein SCP3 and the cohesin subunit STAG3. Our results show that X and Y chromosomes never appear as univalents in metaphase I, but they remain associated until they orientate and segregate to opposite poles. However, they must not be tied by a chiasma since their separation precedes the release of the sister chromatid cohesion. Instead, we show they are associated by the dense plate, a SCP3-rich structure that is organized during the first meiotic prophase and that is still present at metaphase I. Surprisingly, the dense plate incorporates SCP1, the main protein of the central element of the synaptonemal complex, from diplotene until telophase I. Once sex chromosomes are under spindle tension, they move to opposite poles losing contact with the dense plate and undergoing early segregation. Thus, the segregation of the achiasmatic T. elegans sex chromosomes seems to be ensured by the presence in metaphase I of a synaptonemal complex-derived structure. This feature, unique among vertebrates, indicates that synaptonemal complex elements may play a role in chromosome segregation.     

Heteropycnosis of an underreplicating chromosome

Drosophila nasutoides has an extraordinary genome since 62% of its DNA resides in chromosome4. This element mainly consists of constitutive heterochromatin which does not polytenize. Earlier studies of heterochromatin attributed little attention to the fact that condensed chromosomes often vary in condensation. This paper reports that chromosomes of the same complement display different degrees and kinetics of condensation. InD. nasutoides, even sex specific differences can be observed. The results of a comparative microphotometric study on neuroblast metaphases in both sexes revealed the following picture. The process of chromosome condensation is not restricted to mitotic prophase but continues into the metaphase. The mean condensation is not equal for all chromosomes. In the metaphase of the female, Feulgen density increases from theX chromosome, via3 and2, to chromosome4. In the male, the order isX, 2, 3, Y, and4. During the metaphase of the male, chromosomes condense with similar kinetics. In contrast, chromosomes of the female display asynchrony as monitored by area and length determinations. TheX chromosomes of the female probably have enhanced shortening during prophase. This would explain the metaphase of the female where theX chromosomes shorten less than the autosomes, and why each of theX chromosomes is 15% shorter than theX chromosome in the metaphase of the male. Further differences were observed in the longitudinal and lateral compaction of the chromosomes in males and females. The sex chromosomes and chromosome3 condense by shortening, while chromosomes2 and4 preferentially reduce their diameter. The large amount of DNA engaged in heteropycnosis and the isochromosome nature allow the identification of chromosome4 during interphase. At this stage, a new category of extreme DNA packaging was detected. The interphase density of chromosome4 can exceed that of metaphase by a factor of up to 8. Two events account for this high degree of condensation:(1) the homologues are particularly associated due to somatic pairing and (2) the arms are further tightened as a result of pericentric folding. The features of the isochromosome suggest that the interaction of chromatids during interphase is essentially caused by specific DNA sequences. The data confirm that heteropycnosis not only interferes with gene expression but also strongly inhibits DNA synthesis in endocycles.