Scanning electron microscopy of giemsa-stained chromosomes and surface-spread chromosomes

Giemsa-stained chromosomes as prepared for light microscopy, and including G-banded, C-banded, and FPG-stained chromosomes, were examined by scanning electron microscopy. Although suitable for light microscopy, these chromosomes were too flat for a close examination of their fine structure by scanning electron microscopy. The surface of Giemsa-positive regions was rough and bright, whereas that of unstained or poorly stained regions was smoother and less bright. Giemsa-staining, therefore, seems to produce the bulkiness of the chromosomes. On topographical examination by scanning electron microscopy, the transparent chromosomes as observed with the light microscope proved to be footprints. Stereographical examinations of surface-spread chromosomes showed that minimally stretched chromosomes were composed of a mass of nodular and twisted looping fibers with an average diameter of about 300 Å. The substructure of these chromosome fibers was not determined. The kinetochore region was discernible as a constriction in the mass of the chromosome fibers, and was distinguishable from gaps by the presence of several chromosome fibers parallel to the axis of the chromatid. The organization of the chromosome fibers, however, was disordered rather than regular.     

Ultrastructural characterization of the sex chromosomes during spermatogenesis of spiders having holocentric chromosomes and a long diffuse stage

An ultrastructural study has been made of spermatogenesis in two species of primitive spiders having holocentric chromosomes (Dysdera crocata, XO and Segestria florentia X₁X₂O). Analysis of the meiotic prophase shows a scarcity or absence of typical leptotene to pachytene stages. Only in D. crocata have synaptonemal complex (SC) remnants been seen, and these occurred in nuclei with an extreme chromatin decondensation. In both species typical early prophase stages have been replaced by nuclei lacking SC and with their chromatin almost completely decondensed, constituting a long and well-defined diffuse stage. Only nucleoli and the condensed sex chromosomes can be identified. — In S. florentina paired non-homologous sex chromosomes lack a junction lamina and thus clearly differ from the sex chromosomes of more evolved spiders with an X₁X₂O male sex determination mechanism. In the same species, sex chromosomes can be recognized during metaphase I due to their special structural details, while in D. crocata the X chromosome is not distinguishable from the autosomes at this stage. — The diffuse stage and particularly the structural characteristics of the sex chromosomes during meiotic prophase are reviewed and discussed in relation to the meiotic process in other arachnid groups.     

The genetic conditions in heterozygous and homozygous populations ofDrosophila I. The fate of alien chromosomes

In experimental populations ofDrosophila melanogaster lethal chromosomes with dominant markers and inversions were introduced and the frequency changes of the markers studied during a period of several generations. The base populations of the various experiments differed from each other with respect to their degree of heterozygosity. Monochromosomal populations were isogenic for a quasinormal + chromosome, dichromosomal populations contained the genetic material of two different + chromosomes, trichromosomal of three, tetrachromosomal of four, hexachromosomal of six and polychromosomal populations of many normal chromosomes. Marker chromosomes with the dominant genesLCy, Cy, Pm orD respectively were added to the populations with an initial frequency of 16,6 per cent. The fate of the dominant markers was different in different populations. In some the marker chromosome reached equilibrium frequencies, in others they were eliminated with variable speed. As a rule the lethal marker chromosomes were accepted by monochromosomal populations; however, they were eliminated from populations with a higher degree of heterozygosity. Since in all populations one genotype, namely the homozygote for the marker chromosome, was lethal, the adaptive values c of the +/LCy, +/Cy, +/Pm or +/D heterozygotes could easily be calculated from the experimental data. This c value can be used as a measure for the combining ability of the marker chromosomes. It could be shown that c depends on the degree of heterozygosity of a population or in other words that the average degree of heterozygosity of the marker free individuals determines the selection processes. An equation can be arrived at which fits the experimental results very well if superiority of heterozygous +/+ individuals over +/+ homozygotes is assumed. From that it was concluded that heterosis is the determining variable in our experiments. An attempt was undertaken in order to decide if in our case the observed heterosis was due to dominance or to overdominance. It was postulated that in di-, tri-, tetra- or hexachromosomal populations the adaptive values of the marker free normals should progressively increase if recessive detrimental genes are the cause of heterosis but not if heterozygosity on many loci leads to overdominance. The a values of the +/+ individuals were ealeulated from the frequency changes of the marker chromosomes for each subsequent two-generation period. Unfortunately only two different dichromosomal populations were available. These showed increasing adaptive values for the normals. The tri-, tetra-, and hexachromosomal populations, however, gave different results, some with increasing, some with fluctuating adaptive values. From that it was concluded that heterosis can be due in one case to dominance and in the other to overdominance. In either case, the recessive genetic load may be rather important as a determinating factor in the dynamics of populations.Dedicated to Prof. Th. Dobzhansky on the occasion of his seventieth birthday in deepest gratitude.     

The structure of the chromosomes in human primordial oocytes

Primordial oocytes (oocytes in primordial follicles) from human ovaries aged 51/2 months post conception to 11 3/4 years post partum were examined in: (a) squash preparations of fresh and fixed tissue; (b) histological preparations; and (c) thin sections by electron microscopy, in order to study the structure of the chromosomes. — The light microscope shows that the chromosome consists of a thread bearing numerous fine lateral appendages. Cytochemical tests indicate that the thread contains DNA, and is surrounded by material containing RNA and protein. — The electron microscope shows that there are three main structural components in the chromosome: (i) an axis or core containing at least two longitudinal strands about 200 Å thick; (ii) a surrounding sheath composed of coiled fibrils which form symmetrically arranged columns and loops, and (iii) clusters of large granules which are associated with the outer parts of the sheath. Small nucleoli and other granular bodies are also present. — These observations indicate the presence of lampbrush chromosomes in the human oocyte. The significance of this type of chromosome in mammals is discussed in relation to the differential radiosensitivity of the oocytes, and to the form of chromosomes at the dictyate stage in rodents.     

The behavior of allocyclic chromosomes in Bloom's syndrome

The behavior of individual allocyclic chromosomes has been analyzed in lymphocytes of a sister and a brother with Bloom's syndrome. Of 4,633 diploid cells, 115 showed allocyclic chromosomes, and 74 of these had 44, 45 or 46 normal metaphase chromosomes accompanied by one or two allocyclic chromosomes. Of 56 tetraploid cells, 9 contained such chromosomes. The allocyclic chromosomes appeared pulverized or extended corresponding to S or G₂ PCC. We have proposed the hypothesis that individual allocyclic chromosomes do not, as a rule, come from micronuclei, as has often been assumed, but have been left behind in their cycle. This would be caused by a mutation or deletion of a hypothetical coiling center situated near the centromere of each chromosome arm. The following observations agree with our explanation but less well or not at all with the idea of micronuclei: (1) In only 9.6% of the cells does the allocyclic chromosome lie at the edge of the metaphase plate. (2) In 24 cells a part of a chromosome is pulverized while the rest is in metaphase. (3) Both a pulverized and an extended chromosome were present in the same cell. (4) A pulverized acrocentric is often nose-to-nose with a normal D or G chromosome. (5) No allocyclic chromosomes corresponding to G₁ PCC have been found in our material. (6) When a ring is replaced by an allocyclic chromosome, it is usually a member of a 46-chromosome complement. Furthermore, the occurrence of allocyclic chromosomes is correlated with that of other chromosome anomalies which do not follow a Poisson distribution. Allocyclic chromosomes are also more frequent (16%) in tetraploid than in diploid cells (2%).     

Structure of isolated protein-depleted chromosomes of plants

Chromosomes from poppy (Papaver somniferum L.) and wheat (Triticum monococcum L.) were obtained from cell suspension cultures using a mass isolation procedure. Protein-depleted isolated chromosomes were produced using different modes of extraction (e.g., sodium chloride, dextran sulphate-heparin) and examined by protein electrophoresis as well as light and electron microscopy. The results are discussed as they relate to the reported structure of protein-depleted animal chromosomes. With respect to the scaffold model of mitotic chromosomes we conclude that i) nonhistone proteins seem to play a fundamental role in plant chromosome architecture; ii) DNA is a structural component of protein-depleted chromosomes; iii) centromeric regions may be of structural importance for the higher order organization of chromosomes; iv) the existence of a 2M NaCl resistant scaffold appears not to be a common feature to both plant and animal chromosomes; v) despite the absence of a typical scaffold in plant chromosomes our results suggest that the higher order organization of plant and animal chromosomes is similar if not the same.     

Rejoining between 9q+ and Philadelphia chromosomes results in normal-looking chromosomes 9 and 22 in Ph1-negative chronic myelocytic leukemia

Summary Rearrangement of the breakpoint cluster region (bcr) and the chromosomal location of c-abl and 3-bcr were studied in two patients with Philadelphia chromosome (Ph¹)-negative chronic myelocytic leukemia (CML). One patient (patient 1) had a normal karyotype and the other (patient 2), 46,XY,inv(3)(q21q26). Both patients showed the bcr rearrangement by Southern blot analysis with a 1.2 kb 3-bcr probe. In situ hybridization studies demonstrated the location of the homologous sequences of bcr on chromosome 22 in patient 1, and on chromosomes 9 and 22 in patient 2. These findings indicate that the morphologically normal-looking chromosomes 9 and 22 in patient 2 are the result of a retranslocation between chromosomes 9q+ and 22q-, abnormalities which were first formed by a standard Ph¹ translocation.     

Stability of a multiple X chromosome system and associated B chromosomes in birch aphids (Euceraphis spp.; Homoptera: Aphididae)

Autosomal dissociations are a common feature of aphid karyotype evolution, but multiple X chromosome systems are rare. Birch-feeding aphids of the genus Euceraphis, however, have X₁X₂O males as a general rule, X₁ being always much larger than X₂. Only one species has XO males, and this condition appears to be secondary. Most Euceraphis karyotypes also have one or more, usually heterochromatic, elements that occur in the same numbers in both males and females, yet behave like X chromosomes at male and female meiosis I. They appear to be supernumerary, non-functional X chromosomes, although showing greater within-species stability in size and number than typical B chromosomes. Euceraphis gillettei forms a separate group within the genus and feeds on alders (Alnus species), yet has a similar system, and the two most closely related genera, Symydobius and Clethrobius, also have additional chromosomal elements possibly representing non-functional X chromosomes. Thus the multiple X chromosome system in these aphids seems to be a primitive condition.     

Ultrastructure and division behaviour of dinoflagellate chromosomes

Chromosomes of Prorocentrum triestinum and P. micans have similar substructural and morphometrical values as revealed by electron microscopy of thin sections. However, differences were found between the species in mean length, volume and numerical density of chromosomes, and the volume of the chromosome complement, the nuclear volume and the chromosome number. When examined by a whole-mount procedure both Prorocentrum species have left-handed screw-like chromosomes which end in differentiated telomeres. The chromosomes divide sequentially from one telomere towards the other, presenting a Y and finally a V configuration. At the region where each chromosome divides nascent sister chromatids are connected by two bridges. Sister chromatids have similar quantitative values when compared with each other and with the still undivided chromosome, which suggests that both replication and division take place as coupled events.Supported by CAICYT, grant 2409/83     

Satellite DNA and evolution of sex chromosomes

The satellite DNA (satellite III) which is mainly represented in the female of Elaphe radiata (Ophidia, Colubridae) has been isolated and its buoyant density has been determined (=1.700 g cm^–3). In situ hybridisation of radioactive complementary RNA of this satellite DNA with the chromosomes of different species has revealed that it is mainly concentrated on the W sex chromosome and its sequences are conserved throughout the sub-order Ophidia. From hybridisation studies these sequences are absent from the primitive family Boidae which represents a primitive state of differentiation of sex chromosomes. Chromosome analysis and C-banding have also revealed the absence of heteromorphism and of an entirely heterochromatic chromosome in the species belonging to the primitive family and their presence in the species of highly evolved families. It is suggested that the origin of satellite DNA (satellite III) in the W chromosome is the first step in differentiation of W from the Z in snakes by generating asynchrony in the DNA replication pattern of Z and W chromosomes and thus conceivably reducing the frequency of crossing-over between them which is the prerequisite of differentiation of sex chromosomes. Presence of similar sex chromosome associated satellite DNA in domestic chicken suggests its existence in a wider range of vertebrates than just the snakes.     

Identification of Haynaldia villosa chromosomes added to wheat using a sequential C-banding and genomic in situ hybridization technique

Genomic in situ hybridization (GISH) offers a convenient and effective method for cytological detection, but can not determine the identity of the chromosomes involved. We integrated C-banding with GISH to identify Haynaldia villosa chromosomes in a wheat background. All chromosomes of H. villosa showed C-bands, either in telomeric regions or in both telomeric and centromeric regions, which allowed unequivocal identification of each H. villosa chromosome. The seven pairs of H. villosa chromosomes were differentiated as 1–7 according to their characteristic C-bands. Using a sequential C-banding and GISH technique, we have analyzed somatic cells of F₃ plants from the amphiploid Triticum aestivum-H. villosa x Yangmai 158 hybrids. Three plants (94009/5-4,94009/5-8 and 94009/5-9) were shown to contain H. villosa chromosome(s). 94009/5-4 (2n = 45) had three H. villosa chromosomes (2, 3 and 4); 94009/5-8 (2n = 45) possessed one chromosome 4 and a pair of chromosome 5, and 94009/5-9 (2n = 43) was found to have one chromosome 6 of H. villosa. The combination of GISH with C-banding described here provides a direct comparison of the cytological and molecular landmarks. Such a technique is particularly useful for identifying and localizing alien chromatin and DNA sequences in plants.     

Genetic regulation of the replication pattern of polytene chromosomes in interspecific hybrids of Drosophila

The sixth chromosome (microchromosome) of D. littoralis changed its replication pattern in nuclei of the salivary gland cells in reciprocal F₁ hybrids between D. virilis and D. littoralis. — To locate the factor (or factors) in the D. virilis chromosomes, which may influence the replication pattern of the sixth chromsome of D. littoralis, we produced hybrid stocks with synthetic karyotypes characterized by different combinations of D. littoralis homologous chromosomes and hybrid chromosomes. Based on autoradiographical studies of DNA synthesis in synthetic karyotypes, it may be concluded that the dominant factor (or factors), which influences the replication of the sixth chromosome of D. littoralis, is located on the homeologous microchromosomes of D. virilis. The possible interrelation between the changed replication pattern of D. littoralis sixth chromosome and its atypical behaviour at early embryogenesis in (D. virilis x D. littoralis) F₁ hybrids is discussed.     

Linkage relationships between prolamin genes on chromosomes 1A and 1B of durum wheat

Gliadin and glutenin electrophoresis of F₂ progeny from four crosses of durum wheat was used to analyse the linkage relationships between prolamin genes on chromosomes 1A and 1B. The results showed that these genes are located at the homoeoallelic lociGlu-1,Gli-3,Glu-3 andGli-1. The genetic distances between these loci were calculated more precisely than had been done previously for chromosome 1B, and the genetic distances betweenGli-A3,Glu-A3 andGli-A1 on chromosome 1A were also determined. Genes atGli-B3 were found to control some-gliadins and one B-LMW glutenin, indicating that it could be a complex locus.     

The B chromosome system in the varying lemming <Emphasis Type="Italic">Dicrostonyx torquatus</Emphasis> Pall., 1779 from natural and laboratory populations

In varying lemmings from seven natural populations (from Bolshezemelskaya tundra to Chukotka Peninsula), the number of B chromosomes ranged from 0 to 15. In populations surveyed for several years, B chromosome frequencies were stable. Two laboratory colonies (founded by Dicrostonyx torquatus torquatus from the Polar Urals and D. t. chionopaes from Yakutia) produced more than 3000 animals, of which 1699 were karyotyped. A small excess of B chromosomes in the progeny over that in their parents was observed in each generation. Coefficients of transmission k ₁ of additional chromosomes were on average 0.519 in D. t. torquatus and 0.511 in D. t. chionopaes. In oocytes I and II, an accumulation of B chromosomes was observed as compared to somatic cells ( k₂ = 0.66). The reproductive output of animals from both laboratory colonies did not depend on size of the B genome. The reduction of body and scull sizes observed in animals carrying numerous B chromosomes may confer negative selective value in the conditions of the Extreme North. In general, the system of B chromosomes in D. Torquatus is well balanced and very stable, being apparently under strong genetic control.Translated from Genetika, Vol. 40, No. 12, 2004, pp. 1686–1694.Original Russian Text Copyright © 2004 by Gileva.     

Non-reciprocal gonadal dysgenesis in hybrids of the chironomid midge Chironomus thummi II. Gonadal-dysgenesis inducing chromosomes

Females of Chironomus thummi thummi (stock H1) were backcrossed with hybrid males of the cross Ch. thummi thummi (H1) x Ch. thummi piger (E) in order to test which of the four piger chromosomes determine the temperature sensitive non-reciprocal gonadal dysgenesis originally observed in the thummi x piger hybrids. In the backcross rudimentary gonads as well as normal gonads were found irrespective of the chromosome constitution of the individuals. The highest frequency of abnormal gonads (63%) occurred in backcross larvae which received piger chromosomes I and III. Statistical evaluation of the data showed that piger chromosome I is able to induce 44% dysgenic gonads. The gonadal-dysgenesis inducing ability of piger chromosome III is only recognizable if also piger chromosome I is present in the genome. The piger chromosomes II and IV do not have a statistically significant influence on the frequency of abnormal gonads.Arguments are presented for the assumption that the factors determining rudimentary development of the gonads are located distally to those pericentric chromosome regions of the piger chromosomes I and III that in polytene chromosomes show differences in the size of bands in comparison with the homologous thummi regions. The question is discussed whether the rudimentary development of the gonads is caused by gene pairs with an epistatic form of interaction or by the activity of transposable elements.     

Decreased DNA content of birch (Betula) chromosomes at high ploidy as determined by cytophotometry

Relative amounts of DNA were determined on telophase nuclei by Feulgen cytophotometry for euploid taxa of birch (Betula) with somatic chromosome numbers of 28, 42, 56, 70, and 84. A direct correlation was found between observed DNA absorbance and chromosome number except for plants of B. papyrifera with 84 somatic chromosomes. The DNA density value for nuclei of the 84-chromosome plants fitted a 12.25 ratio instead of the expected 13.0 ratio. The DNA density value for these plants was calculated to be approximately equivalent to plants which would possess 63 somatic chromosomes. The average DNA value per chromosome was 2.73 for the 84-chromosome plants in contrast to 3.50 per chromosome in each of the lower euploids. Nuclear diameters of the 84-chromosome plants were directly related to chromosome number and not to DNA density value. The genomic number of Betula was considered to be x=7, rather than x=14, since a DNA value equivalent to 63 chromosomes is a multiple of 7 and not 14. Diploid birch species (2n=2x=28), therefore, would actually be tetraploids (2n=4x=28). The reduction in DNA content may be an adaptation for the establishment of higher ploidy in birches.     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

     

Modulating crossover positioning by introducing large structural changes in chromosomes

Background

Crossing over assures the correct segregation of the homologous chromosomes to both poles of the dividing meiocyte. This exchange of DNA creates new allelic combinations thus increasing the genetic variation present in offspring. Crossovers are not uniformly distributed along chromosomes; rather there are preferred locations where they may take place. The positioning of crossovers is known to be influenced by both exogenous and endogenous factors as well as structural features inherent to the chromosome itself. We have introduced large structural changes into Arabidopsis chromosomes and report their effects on crossover positioning.

Results

The introduction of large deletions and putative inversions silenced recombination over the length of the structural change. In the majority of cases analyzed, the total recombination frequency over the chromosomes was unchanged. The loss of crossovers at the sites of structural change was compensated for by increases in recombination frequencies elsewhere on the chromosomes, mostly in single intervals of one to three megabases in size. Interestingly, two independent cases of induced structural changes in the same chromosomal interval were found on both chromosomes 1 and 2. In both cases, compensatory increases in recombination frequencies were of similar strength and took place in the same chromosome region. In contrast, deletions in chromosome arms carrying the nucleolar organizing region did not change recombination frequencies in the remainder of those chromosomes.

Conclusions

When taken together, these observations show that changes in the physical structure of the chromosome can have large effects on the positioning of COs within that chromosome. Moreover, different reactions to induced structural changes are observed between and within chromosomes. However, the similarity in reaction observed when looking at chromosomes carrying similar changes suggests a direct causal relation between induced change and observed reaction.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1276-z) contains supplementary material, which is available to authorized users.