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%).     

Two phases of DNA replication in human cells

The course of DNA synthesis in the chromosomes was studied in synchronized human lymphocyte cultures, by means of the BrdU-Hoechst-Giemsa method. In comparing replication patterns and G-banding it was found that with regard to banding the process of DNA replication can be divided into two separate phases, an early replication period which is characterized by DNA synthesis in R bands of the autosomes and active X chromosome, and a late replication period which concerns the G-positive regions of the autosomes and all the bands of the heterochromatic X and Y chromosomes. No overlapping was found between the two phases mentioned. The possible role of regulatory mechanisms was discussed.     

NADH dehydrogenase: a new molecular marker for homoeology group 4 in Triticeae. A map of the 4RS chromosome arm in rye

Summary Structural gene loci encoding the monomeric isozymes nicotin adenin dinucleotide dehydrogenase (NADH dehydrogenase or NDH) have been located on the 4AL, 4B, and 4DS chromosome arms of Triticum aestivum cv Chinese Spring, on the 4RS chromosome arm of Secale cereale cultivars Imperial, King II, Dakold, and Ailes, on the 4S¹ S/7S¹ chromosome of Aegilops longissima, the 4E of Elytrigia elongata, and the CSU-A of Aegilops umbellulata. All the results support the homoeologous relationships among these chromosomes in the five species studied. In addition, a map of the 4RS chromosome arm in cv Ailes has been realized, linking loci Pgm-1 (located on the 4RS chromosome arm) and Ndh-1 (17.91 cM), with an estimated distance between both loci and the centromere of 20.00 cM and 32.12 cM, respectively.     

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (diptera,sciaridae)

The so-called controlling element (CE), which normally programs the curious behavior of the sex chromosome in this genus, has been localized in the short right arm of the polytene X in S. coprophila. The localization was accomplished by use of five X-autosome translocations whose break points define three blocks of heterochromatin (heterochromomeres) extending from the X centromere to the very end (right) of the chromosome. The behavior of the translocation chromosomes at the crucial second spermatocyte division was examined and the precocious chromosome identified in all five cases. Then, knowing the heterochromomere make-up of each chromosome, the position of the CE could be mapped; it is located in heterochromomere H2, the same block of heterochromatin that contains 50% of the ribosomal RNA cistrons. — The question of whether the CE can manipulate any centromere in the nucleus has been only partially answered. It can manipulate translocation chromosomes which possess the centromere of the metacentric autosome (salivary chromosome IV) or that of the shorter rod (salivary chromosome II); but the longer rod (salivary chromosome III) whose proximal end, as seen in the polytene nucleus, is heavily laden with heterochromatin of its own, has not been brought under CE control. — In one of the translocations, T23, the precocious chromosome is a very large metacentric chromosome which resembles the peculiar V-shaped X of S. pauciseta. This peculiarity is not observed in the J-shaped precocious chromosome of T29. These points are discussed.Dedicated to Professor Hans Bauer on the occasion of his 75th birthday.     

C-banding and non-homologous associations

A chromosome complement formed by 16 autosomes and an Xy_p sex chromosome system was found in Epilachna paenulata Germar (Coleoptera: Coccinellidae). All autosomes were metacentric except pair 1 which was submetacentric. The X and the Y chromosomes were also submetacentric but the Y was minute. The whole chromosome set carried large paracentric heterochromatic C-segments representing about 15% of the haploid complement length. Heterochromatic segments associated progressively during early meiotic stages forming a large single chromocenter. After C-banding, chromocenters revealed an inner networklike filamentous structure. Starlike chromosome configurations resulted from the attachment of bivalents to the chromocenters. These associations were followed until early diakinesis. Thin remnant filaments were also observed connecting metaphase I chromosomes. Evidence is presented that, in this species, the Xy_p bivalent resulted from an end-to-end association of the long arms of the sex chromosomes. The parachute Xy_p bivalent appeared to be composed of three distinct segments: two intensely heterochromatic C-banded corpuscles formed the canopy and a V-shaped euchromatic filament connecting them represented the parachutist component. The triple constitution of the sex bivalent was interpreted as follows: each heterochromatic corpuscle corresponded to the paracentric C-segment of the X and Y chromosomes; the euchromatic filament represented mainly the long arm of the X chromosome terminally associated with the long arm of the Y chromosome. The complete sequence of the formation of the Xy_p bivalent starting from nonassociated sex chromosomes in early meiotic stages, and progressing through pairing of heterochromatic segments, coiling of the euchromatic filament, and movement of the heterochromatic corpuscles to opposite poles is described. These findings suggest that in E. paenulata the Xy_p sex bivalent formation is different than in other coleopteran species and that constitutive heterochromatic segments play an important role not only in chromosome associations but also in the Xy_p formation.     

DNA replication and inactivation patterns in structural abnormality of sex chromosomes

Summary High resolution chromosome analysis and bromodeoxyuridine (BrdUrd) incorporation have been applied to study patterns of chromosomal replication (inactivation) in two cases of unbalanced X-autosome translocations, seven cases of X and Y chromosome rings or fragments, and five cases of dicentric isochromosomes (Xq). Our results indicate the following: (1) In (X-A) translocations, detailed replicational analysis of the translocated autosomal segment is informative. Absence of spreading effect and partial-incomplete spreading effect are the most common observations. (2) Sex chromosome derived fragments and rings can be differentiated based on their replicational features. (3) Dicentric isochromosomes (Xq) can be classified based on intercentromeric distances, replicational asynchrony, and centromere inactivation. (4) A correlation between intercentromeric distance and degree of 45,X mosaicism was observed in dicentric i(Xq) chromosomes.Evidence for spreading effect based on our results and on the review of the literature has been critically analyzed and general rules in evaluating spreading effects (SE) proposed. The cytologic detection of active regions on the late replicating X chromosome and the inactivation capacity of the juxtacentromeric region of Xp is evaluated. It is proposed that centromere suppression and underreplication are related phenomena. Finally, the analysis of informative replicational stages is emphasized and the application of their analysis in basic and clinical cytogenetics demonstrated.     

Population cytogenetics of the genus Caledia (Orthoptera: Acridinae)

The acridine grasshopper, Caledia captiva exists as two chromosomal races in south-east Queensland. One of these, the Moreton race inhabits the coastal region to the east of the Great Dividing Range. All chromosomes of the complement (2n=11II+XO/XX) have been involved in centromeric rearrangement, which transforms the acro- and telocentric chromosomes into submeta- and metacentric elements. The second, or Torresian race is widely distributed through southern Papua, Arnhem Land, Cape York Peninsula and down the east coast of Australia as far south as Brisbane. This race, which is characterised by a completely acro- and telocentric chromosome complement, approaches the Moreton race in south-east Queensland where the two races are separated by less than 1 km, along a front of at least 150 km. Evidence is presented to show that chromosome introgression is occurring across the contact zone and this takes place in one direction only, namely the Torresian chromosomes are infiltrating into the Moreton race but not reciprocally. Furthermore, the introgression of chromosomes across the zone is limited to certain members of the Torresian complement and even then these successful chromosomes show highly variable degrees of penetrance into the Moreton race. It is proposed that a tension zone exists between these two races which is maintained by the interaction of (a) ecological tolerance differences on either side of the zone and (b) by partial competitive exclusion due to the interracial differences in phenology. This case of parapatric association with limited hybridisation is unique in its clarity due to the marked differences in the appearance of the chromosome complements of these races which permits direct assessment of the behaviour of most members of the genome in hybrids and their derivatives.     

Fractionation of wheat gliadin and glutenin subunits by two-dimensional electrophoresis and the role of group 6 and group 2 chromosomes in gliadin synthesis

Summary Subunits of wheat endosperm proteins have been fractionated by two-dimensional electrophoresis. To determine which subunits in the two-dimensional electrophoretic pattern belong to gliadin or glutenin the endosperm proteins have also been fractionated by a modified Osborne procedure and by gel filtration on Sephadex G-100 and Sepharose CL-4B prior to separation by two-dimensional electrophoresis.The control of production of five major grain protein subunits is shown to be determined by chromosomes 6A, 6B and 6D by comparing two-dimensional electrophoretic protein subunit patterns of aneuploid lines of the variety Chinese Spring. From these and previous studies it is concluded that some , and gliadins (molecular weights by SDS-PAGE 30,000 to 40,000) are specified by genes on the short arms of homoeologous Group 6 chromosomes, the gliadins (molecular weights by SDS-PAGE 50,000 to 70,000) are specified by genes on the short arms of homoeologous Group 1 chromosomes and the glutenin subunits (molecular weights by SDS-PAGE > 85,000) are specified by genes on the long arms of homoeologous Group 1 chromosomes.No major gliadins or glutenin subunits were absent when any of the chromosomes in homoeologous Groups 2, 3, 4, 5 or 7 were deleted. However two gliadins whose presumed structural genes are on chromosome 6D were absent in aneuploid stocks of Chinese Spring carrying two additional doses of chromosome 2A. Two out of thirty-three intervarietal or interspecific chromosome substitution lines examined, involving homoeologous Group 2 chromosomes, lacked the same two gliadins. All the subunits in the other thirty-one chromosome substitution lines were indistinguishable from those in Chinese Spring. It is therefore concluded that the major variation affecting gliadin and glutenins in wheat is concentrated on the chromosomes of homoeologous Groups 1 and 6 but Group 2 chromosomes are candidates for further study.An endosperm protein controlled by chromosome 4D in Chinese Spring is shown to be a high molecular weight globulin.     

Normal meiosis in two 47,XYY men

Summary Details of testicular histology and meiosis are given for two 47,XYY men, one an oligospermic childless individual, the other a fertile man with near-normal spermatogenic activity in his testes. Examination of the chromosomes at meiosis, with Q and C staining, gave no evidence for the occurrence of the second Y chromosome in the germ line of either individual.     

Quantitative comparisons between the karyotypes of Australian marsupials from three different superfamilies

Relative DNA values and percent lengths of chromosome arms have been studied for six species of the super-family Dasyuroidea, five species of Phalangeroidea and four species of Perameloidea. By multiplying by relative DNA values all percent lengths have been expressed in the same units. Two species are said to share a chromosome if neither of the arms differs at the 5% level of probability. It is argued that if species share at least two two-armed chromosomes, this can be taken as evidence of relationship. A standard dasyurid karyotype has been defined and individual species of Dasyuridae show only small deviations from it. The nature and significance of the bi-modal distribution of chromosome numbers in marsupials is discussed.     

Complete characterization of a large marker chromosome by reverse and forward chromosome painting

Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the degenerate oligonucleotide primed — polymerase chain reaction, we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads (reverse chromosome painting) and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome (forward chromosome painting) and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Silver staining the chromosome scaffold

Cytological silver-staining procedures reveal the presence of a core running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this core and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic core staining was unaffected. Control experiments suggested that the core does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific core staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.     

Chromosome size variation during pollen grain development in Scilla sibirica

Summary The first pollen grain mitosis in Scilla sibirica takes place within three weeks after the completion of meiosis. Within one anther the duration of the first pollen grain mitotic cycle varies substantially. The duration of the mitotic cycle affects the length of chromosomes at metaphase of the first pollen grain mitosis. In grains which divide early the chromosomes at metaphase are longer, up to twice the length, of the chromosomes in grains dividing late. The diminution in length with increase in the mitotic cycle is due to more intensive coiling which, in turn, is explained by a lengthening of G2 and of prophase. The relationship between the duration of the mitotic cycle and chromosome length at metaphase would account, at least largely, for the variation in chromosome length between different tissues within organisms. It explains also why the chromosome at metaphase of mitosis are shorter in polyploids than in their diploid ancestors.     

Multiple sex chromosomes in Drosophila miranda: A system to study the degeneration of a chromosome

Drosophila miranda possesses an intriguing sex chromosome constitution. While female metaphase plates have 10 chromosomes (diploid set), in males only 9 chromosomes can be identified. The missing homologue has been translocated to the Y, forming a neo-Y chromosome which is polytenized in the salivary gland cells. This report presents a detailed characterization of DNA, isolated from D. miranda flies. In situ hybridizations, using cRNA transcribed from unfractionated D. miranda DNA, reveal hybridization to the neo-Y with label distributed over the entire chromosome. The original partner of the translocated chromosome, X², is essentially unlabelled. These results suggest that repetitive DNA sequences invade the translocated chromosome. This result is discussed with reference to the hypothesis of degeneration of the Y chromosome, formulated by Muller (1918, 1932a).     

The spatial localization of homologous chromosomes in human fibroblasts at mitosis

Chromosomes from ten human male fibroblast metaphases were completely reconstructed from electron micrographs of serially sectioned material. Chromosome centromere positions were determined by finding the three-dimensional coordinates of the centromere midpoint. The data set showed the identity of nine chromosome types (chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and the Y chromosome) preserved as they are positioned in vivo. The results indicate that there is (1) no significant association of the homologous chromosomes examined, (2) a significant tendency for a central location of the Y chromosome and of chromosome 18, (3) a significant tendency for a peripheral location of chromosome 6, (4) no significant tendency for homologous chromosomes to reorganize as metaphase advances and (5) no significant differential condensation across the metaphase plate. Therefore, the only organization pattern observed for the centromeres of the homologous chromosomes studied is some sorting by size across the metaphase plate. These results may be typical of dividing cell types. Different chromosome arrangements are found in some non-dividing cell types (e.g. mammalian brain cells). The different distributions of chromosomes in different cell types can be considered as forms of nuclear differentiation. It is postulated that nuclear differentiation may be related to cell differentiation.     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

Position-specific effects in the mutagenic action of mitomycin C on the chromosomes of Hordeum vulgare L.

Summary The mutagenic action of mitomycin C (MMC) on the chromosomes of two reconstructed karyotypes of barley was studied. MMC-induced chromatid aberrations were found to be distributed non-randomly along the chromosomes. The regions situated next to the secondary constrictions of chromosomes 6 and 7 appeared to be clearly pronounced aberration hot spots. In these segments, intercalary deletions and duplication-deletions were the most frequently induced aberration types. The comparative analysis of the frequency and localization of MMC-induced aberrations in the chromosomes of the two karyotype variants, which differ from each other by the position of the hot spot segments, provided new evidence about the influence of the segment transposition on the hot spot expressivity. The most remarkable finding obtained in the study is that the size of the segment involved in both intercalary deletions and duplication-deletions proved to be strongly affected by the structural peculiarity of the reconstructed chromosome. The possible reasons underlying this finding are discussed.     

Cytotaxonomy of the triatominae (Reduviidae: Hemiptera)

The chromosome number and meiotic cycle of 20 species of Triatominae have been investigated. In the male, there are five types of chromosome complement: 20+XY, 20+X₁X₂Y, 20+X₁X₂X₃Y, 18+XY and 22+XY.The cytological data suggest that the type number for the subfamily is 22 (20+XY). In the hybrids: Triatoma barberi () and T. protracta (), anomalous behavior of certain chromosomes has been observed. Phylogenetic relationships based on chromosome evidence in the subfamily have been discussed. It is suggested that fragmentation is the major factor for chromosome evolution in the group.     

Selective staining of Y chromosomal loops in Drosophila hydei,D. neohydei,and D. eohydei

Modified Giemsa procedures have been developed which elicit differential and highly selective staining of individual Y chromosomal lamp-brush loops in spermatocyte nuclei of Drosophila hydei, D. neohydei, and D. eohydei. In all three species the Y loop pair known as the clubs stains a brilliant dark red with Giemsa at pH 10. With the same treatment other loop pairs either remain unstained, e.g. the threads, or show a differentiation between light blue and pink staining matrical material, e.g. pseudonucleolus and cones in D. hydei and D. eohydei. With eosin at pH 2.8 the threads in D. hydei can be stained intensely, as well as one matrical component of the pseudonucleolus. Pretreatment with RNase or TCA removes all stainability from the Y loops with Giemsa at pH 10. TCA treatment enhances eosin staining at pH 2.8. These and other variations of Giemsa may be utilized to establish homologies between Y loops in different species. The molecular basis of the staining reactions remains to be elucidated.