Characterization of kinetochores in multicentric chromosomes

Long-term cultures of certain rat and mouse cell lines carry several dicentric and some multicentric chromosomes. Using antikinetochore antibodies obtainable from serum of scleroderma (var. CREST) patients we studied the number of kinetochores formed along the length of these chromosomes. The rat cells displayed as many kinetochores as there were centromeres. However, mouse cells showed the synthesis of only one kinetochore in dicentric and multicentric chromosomes which had been in the culture for a period of 1 year or more. When translocations were induced by bleomycin in mouse L cells, the newly formed dicentric chromosomes showed the formation of two kinetochores. It is not known when the accessory centromeres lose their capacity to assemble kinetochore proteins. Possibly, in the rat the latent kinetochores lack a specific component which renders them ineffective for microtubule binding. The reason for the formation of only one kinetochore in mouse multicentric chromosomes is not clear. It may be due to the accumulation of mutations, modification of the kinetochore protein so that it lacks the antibody binding component, or a more effective regulatory gene than in the rat.     

Sequence of centromere separation: differential replication of pericentric heterochromatin in multicentric chromosomes

The dicentric and multicentric chromosomes in L cells and a brain tumor cell line of mouse display only one site of kinetochore formation associated with the active centromere. The accessory or inactive centromeres show premature separation. These cell lines were treated with 10^–6 M 5-bromodeoxyuridine (BrdUrd) followed by anti-BrdUrd antibody to study the pattern of replication of pericentric heterochromatin flanking the active vs inactive centromeres. Regardless of its quantity, heterochromatin around the inactive centromere replicates earlier than that associated with the active centromere. There appears to be a relationship between the timing of separation of a centromere and the timing of replication of pericentric heterochromatin. The premature replication of heterochromatin associated with an inactive centromere may be responsible for its premature separation and, hence, inactivity.     

The kinetochores of Caenorhabditis elegans

Light microscopy of the mitotic chromosomes of Caenorhabditis elegans suggests that non-localized kinetochores are present, since the chromosomes appear as stiff rods 1 to 2 m in length and lack any visible constriction. The holokinetic structure was confirmed by reconstructions of electron micrographs of dividing nuclei in serially sectioned embryos. In prophase the kinetochore appears as an amorphous projection approximately 0.18–0.2 m in diameter in cross section and in longitudinal section it appears to be continuous along the chromatin. At prometaphase and metaphase the kinetochore is a convex plaque covering the poleward face of the chromosome and extending the length of the chromosome. In longitudinal section the kinetochore is a trilaminar structure with electron dense inner and outer layers of 0.02 m, and an electron lucent middle layer of 0.03 m. The inner layer is adjacent to a more electron dense region of chromatin. The kinetochore was also seen as a band extending the length of the chromosome in whole mount preparations of chromosomes stained with ethanolic phosphotungstic acid. Most gamma ray induced chromosome fragments segregate normally in embryonic mitoses, but some fragments display aberrant behavior. Similar behavior was seen in embryos carrying a genetically characterized free duplication. It is suggested that mitotic segregation of small fragments may be inefficient because the probability of attachment of microtubules to the kinetochore is proportional to kinetochore length.     

Formation of spindle fibers,kinetochore orientation,and behavior of the nuclear envelope during mitosis in endosperm

The formation of kinetochore (chromosomal) and continuous fibers, and the behavior of the nuclear envelope (NE) was described in studies combining light and electron microscopy. Microtubules (MTs) push and pull the NE which becomes progressively weaker before breaking. It breaks to a certain extent due to mechanical pressure. Clear zone MTs penetrate into the nuclear area as dense bundles and form continuous fibers. These MTs also attach to some kinetochores during this process. Some kinetochore fibers seem to be formed by the kinetochores themselves which are also responsible for further development and changes of kinetochore fibers. Formation of kinetochore fibers is asynchronous for different chromosomes and even for two sister kinetochores. Often temporary faulty connections between different kinetochores or the polar regions are formed which usually break in later stages. This results in movements of chromosomes toward the poles and across the spindle during prometaphase. The NE, whose fine structure has been described, breaks into small pieces which often persist to the next mitosis. Old pieces of NE are utilized in the formation of new NE at telophase. Several problems concerning the mechanism of chromosome movements, visibility of the NE, etc., have also been discussed.     

Molecular cloning and sequencing analysis of a β-tubulin gene from Lupinus albus

Genomic -Dash library constructed from Lupinus albus nuclear DNA was screened using a fragment of the -tubulin cDNA ( 8–31) clone of Chlamydomonas reinhardtii as probe. One of the positive recombinant phages was isolated, subcloned and analysed by sequencing. We present here nucleotide and derived amino acid sequences of the -tubulin gene, designated as L1 and identified by similarity with other -tubulins. The L1-encoded protein reveals a very high degree of similarity with other plant tubulins and contains consensus sequences for binding guanine base, phosphate and Mg²⁺. Northern analysis of total RNA isolated from roots, leaves, flowers and pools revealed that Lupinus albus -tubulin genes are constitutively expressed in all studied plant tissues.     

Relative positions of homologous chromosomes or groups in male and female metaphase figures

Summary This study investigates statistically, with computer assistance, the square distances between chromosome centromeres in homologous pairs or groups after circularizing transformation. By the method described here, it is possible to eleminate all subjective measurments and just use the coordinates x _i and y _i of the centromeres. The values obtained for homologous chromosomes are shown to be specific but not always small. Low square distance values occur in greater number for the chromosomes that are most frequently involved in aneuploïdies. This is true for acrocentric chromosomes which, moreover, tend to lie close together significantly more often in female than in male mitoses; it is also true for group 17–18 especially in males, and of XX in female mitoses. Furthermore, we find significantly low square distances in chromosome pair 1.     

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).     

Quantitative utilization of selected grasses by fall armyworm larvae

Food utilization by larvae of the fall armyworm (Spodoptera frugiperda [J. E. Smith]) (Lepidoptera: Noctuidae), showed greater consumption of corn (Zea mays L.) than pinto bean diet, Tifton 10, or Coastal bermudagrass (Cynodon dactylon [L.] Pers.). Transfer of larvae from diet to susceptible grasses such as corn, Tifton 10 or Coastal produced differences in growth rates as a result of food consumption rates. Transfer of larvae from diet to resistant grasses such as common centipedegrass (Eremochola ophiuroides [Munro] Hack) Tifton 292 bermudagrass, and zoysiagrass (Zoysia sp.) reduced larval growth as a result of low consumption rates and/or greater metabolic expenditures. Larvae initially fed Tifton 10, Coastal, or centipedegrass before feeding on corn grew significantly faster than when they fed continuously on corn.

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Résumé Des chenilles de S. frugiperda ont consommé plus de maïs (Zea mays), que d'un régime à base de Phaseolus vulgaris de la variété Pinto, ou de Tifton 10, ou de Cynodon dactylon de la variété Coastal. Des chenilles, transférées du régime artificiel sur des plantes sensibles comme le maïs, le Tifton 10 ou le Coastal, ont présenté des taux de croissance différents provenant de modifications de leur consommation. Le transfert du régime à des plantes résistantes, telles que Eremochola ophiuroides, Tifton 292, C. dactylon et Zoysia sp., a entraîné une diminution de la croissance larvaire par suite d'une plus faible consommation et/ou de dépenses métaboliques plus élevées. Des chenilles ayant consommé du Tifton 10, du Coastal ou de l'E. ophiuroides avant de consommer du maïs, se sont développées significativement plus vite que celles qui s'étaient alimentées continuellement sur maïs.

     

Regioselective deacetylation of peracetylated monosaccharide derivatives by polyethylene glycol-modified lipase for the oligosaccharide synthesis

Lipase modified with polyethylene glycol became soluble and active in organic solvents, and catalyzed regioselective deacetylation of peracetylated monosaccharide derivatives in 1,1,1-trichloroethane. The deacetylation occurred only at the positions of C-4 and C-6 of the glycopyranoside ring. Especially, peracetylated methyl -D-xylopyranoside and peracetylated L-serine--D-xylopyranoside were hydrolyzed only at the position of C-4. Subsequently, one of the resulting products, that is L-serine-2,3-di-O-acetyl--D-xylopyranoside, was coupled with galactose residue to obtain L-serine-4-O-(-D-galactopyranosyl)--D-xylopyranoside, a model compound of the carbohydrate-protein linkage region of proteoglycans.     

Pac-Man does not resolve the enduring problem of anaphase chromosome movement

Summary The Pac-Man hypothesis suggests that poleward movement of chromosomes during anaphase A is brought about by: disassembly of kinetochore microtubules (MTs) at the kinetochore; generation of the poleward force exclusively at or very close to the kinetochore; and the required energy coming from coupled disassembly of these MTs. This model has become widely accepted and cited as the sole or major mechanism of anaphase A. Rarely acknowledged are several significant phenomena that refute some or all of these postulates. We summarise these anomalies as follows: poleward movement of chromosomes occurring without insertion of any MTs at the kinetochore; anaphase shortening of kinetochore fibres in spindles entirely devoid of chromosomes and, presumably, kinetochores; continued movement of chromosomes while their severed kinetochore stub elongated poleward after treatment with UV microbeams; and fluxing of tubulin subunits through kinetochore MTs during anaphase A, indicating that during anaphase, kinetochore MTs disassemble partly or solely at the poles.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

In vitro plant regeneration via somatic embryogenesis from root culture of some rhizomatous irises

A method for plant regeneration of Iris via somatic embryogenesis is described. Root and leaf pieces from in vitro-grown plants of several genotypes of rhizomatous Iris sp. were cultured in vitro. Callus induction occurred only on root cultures incubated under low light intensity (35 mol m^-2 s^-1) on two induction media containing 2,4-D (4.5 or 22.5 M), NAA (5.4 M) and kinetin (0.5 M). Somatic embryos developed after transfer of callus onto four regeneration media containing 9 or 22 M BA, or 5 M kinetin and 2 M TIBA or 9 M BA and 4 M TIBA. Plantlets could be obtained from these somatic embryos. Genotypic differences were found both in callus induction and somatic embryo formation, with I. pseudacorus responding better than I. versicolor or I. setosa. Cytological analysis performed on root tips of 80 regenerated plants revealed that two of the I. pseudacorus regenerants were tetraploid.Abbreviations 2,4-D dichlorophenoxy acetic acid - NAA naphthaleneacetic acid - BA 6-benzyladenine - TIBA 2,3,5-triiodobenzoic acid - IBA indolebutyric acid     

A Cytogenetic Study of the Leaf Beetle Genus Cyrtonus (Coleoptera, Chrysomelidae)

The chromosomes of ten species of Cyrtonus and the genome sizes of six are surveyed. Among the total of 15 chromosomally studied species, 11 have 2n=28 chromosomes and a 13+Xy_p male meioformula, three have 2n=40 and 19+Xy_p and one 2n=46 and 22+Xy_p. All but one species with 28 chromosomes show only metacentric or submetacentric chromosomes, whereas the species with 40 and 46 chromosomes display some telocentrics or subtelocentrics, that are probably derived from the former by centric fissions. However, since the number of major chromosome arms is strikingly higher in these latter species (NF=70 and 78) than in the 28-chromosome species (mostly NF=56), other chromosomal rearrangements such as pericentric inversions or heterochromatin accretions could also be involved. The genome sizes display a narrow range, from 1C=0.6–1.22pg, and they are not significantly correlated with the chromosome numbers. Some possible factors implied in the rough chromosomal evolution of Cyrtonus are discussed in relation to a few other genera of the subfamily Chrysomelinae.     

δ-Aminolaevulinic acid and amino acid neurotransmitters

Summary The effects of the porphyrin precursor -aminolaevulinic acid (ALA) on -aminobutyric acid (GABA) and L-glutamate transmitter systems was investigated in rat brain. It was found that ALA inhibited GABA and glutamate uptake and stimulated basal efflux of the amino acids in purified nerve endings. These effects were evident only at relatively high concentrations of ALA (at least 100 M). Such concentrations probably do not occur in the nervous systems of patients suffering from acute porphyria. In addition, it was found that ALA inhibited the stimulated release of GABA from nerve endings probably by acting as an agonist at GABA autoreceptors. This effect was found at very low concentrations of ALA (1 M). It is therefore likely that the neuropsychiatric manifestations of the acute porphyric attack are attributable, to some extent, to reduced GABA release at central synapses.     

The fine structure of chickpea (Cicer arietinum L.) chromosomes as revealed by pachytene analysis

A standard pachytene karyotype of chickpea (Cicer arietinum L.) is presented for the first time. Individual pachytene chromosomes were identified and described in detail. An idiogram was prepared on the basis of chromosome length, arm ratio, and distribution of heterochromatin and euchromatin. Chickpea pachytene chromosomes belong to the differentiated type with darker staining heterochromatin proximal to and lighter staining euchromatin distal to the centromeres. Chromosomes were numbered from 1 to 8 following a descending order of length. The total length of the chromosome complement at pachytene was 335.33 , and chromosome size ranged from 58.05 to 30.53 .     

Population cytogenetics of the genus Caledia (Orthoptera: Acridinae)

The genus Caledia contains two species. C. species nova 1 is restricted to the Oriomo Plateau of S.W. Papua and has a complement of twelve telocentric chromosomes. The second species C. captiva has a much wider distribution pattern—from S.W. Papua in the North, down the entire Eastern seaboard of Australia to Southern Victoria. It is also found in the Northern Territory. Although the chromosome number is the same as C. species nova 1, four major and distinct chromosomal races can be distinguished in C. captiva. — The basic ancestral race is found in Tropical North Queensland at the base of the Cape York Peninsula. All twelve chromosomes are telocentric and the karyotypic organization is similar to that found in C. species nova 1 and in other Acridines. A second, general purpose karyotypic race has a wide distribution between S.W. Papua, Arnhem Land and the East Australian coast as far South as Brisbane. It is considered a derivative form of the ancestral type and is fixed for small pericentric inversions on seven pairs of chromosomes. In the South-Eastern Queensland region there exists a further race which carries large pericentric inversions on all the autosomes and the X chromosome. The situation here is confounded since the basic chromosomes can be represented as either acro or telocentrics. Various levels of polymorphism for the inversions exist between different chromosomes in different populations indicating considerable differentiation within this zone. This race is almost completely surrounded by the general purpose karyotype where the races are contiguous in certain parts of the range. — The South-Eastern corner of Australia is characterised by a chromosome race quite different from those found further North. Here a complex pericentric inversion system exists involving a series of seven small inversions and larger inversions on chromosomes 1, 2, 4 and 10. Chromosomes 2 and 4, in particular, are highly polymorphic. — The presence and persistence of these 4 chromosomal races can be accounted for in terms of the known climatic changes which have occurred in this region in the recent past.     

Further histochemical properties of rabbit skeletal muscle fibres

Summary The histochemical activities of succinic dehydrogenase (SDH), creatine kinase (CK), sarcoplasmic reticular ATPase (SR-ATPase) and myosin ATPase were studied in serial sections of rabbit adductor muscle. Three fibre types were distinguished depending upon the distribution of the enzyme activities. The type II white fibres posessing minimal SDH showed high myosin ATPase, SR-ATPase and ATPase dependent CK activities. Red oxidative fibres showing high SDH fell into two distinct groups: One category had mainly a peripheral localization of SDH and showed an enzymatic profile identical to that of type II white fibres. The second category of red fibres displayed both a homogeneous distribution of small diformazan granules throughout the fibre as well as a sub-sarcolemmal collection when tested for SDH activity but possessed very low amounts of reaction product of the various enzymes of the energetic metabolism studied. Since it is well established that the myosin ATPase of a fibre correlates with its contraction time, the present histochemical investigation provides further support for this concept by demonstrating the presence of high SR-ATPase and ATPase dependent CK activities in all white and red fibres rich in myosin ATPase.     

Nuclear division in the cyst,and white spot nuclei preceding cyst formation,inAcetabularia wettsteinii

Summary Electron microscopy of nuclear division in young cysts ofAcetabularia wettsteinii shows that the dividing nucleus hat two additional cisternae of endoplasmic reticulum immediately outside the nuclear envelope. These additional cisternae are attached to, and apparently formed from a membrane body which develops outside the nucleus in early prophase. The interphase nucleus does not have the additional cisternae. The nucleoli are extruded from the nucleus at anaphase, the nucleolar bodies remaining in the peri-nuclear cytoplasm. The chromosomes have localized centromeres; the stratified ultrastructure characteristic of some chlorophycean and animal kinetochores has not been found inAcetabularia, although the kinetochore appears distinct, projecting from the chromatid, and has attached microtubules. The condensed bodies of the white spot nucleus are discussed.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Karyotype of North American shortnose sturgeon Acipenser brevirostrum with the highest chromosome number in the Acipenseriformes

The shortnose sturgeon Acipenser brevirostrum was revealed to have a larger number of chromosomes than previously reported for other sturgeon species. Its chromosome number ranged from 362 to 372 (of ten specimens examined), showing intraindividual variation. The karyotype of metaphase with the highest chromosome number (372) consisted of 89 pairs of macrochromosomes and 97 pairs of microchromosomes (fundamental number; NF=550). Although the microchromosomes were relatively shorter than the macrochromosomes, most of them had discernible arms and centromeres. Silver-stained nucleolar organizer regions (Ag-NORs) were localized on the telomeric regions of 5 pairs of chromosomes (Ag-NORs=10): 4 were made up of small meta/submetacentrics and 1 of acrocentrics. Polyploidy of A. brevirostrum should be hexaploid based on the karyotype, numerous chromosomes, Ag-NORs, and previously reported large genome size (ca. 13pg DNA/cell).Supplementary material to this paper is available in electronic format at http://dx.doi.org/10.1007/s10228-004-0257-z     

The genomic sequence for Prader-Willi/Angelman syndromes' loci of human is apparently conserved in the great apes

Chromosomal changes through pericentric inversions play an important role in the origin of species. Certain pericentric inversions are too minute to be detected cytogenetically, thus hindering the complete reconstruction of hominoid phylogeny. The advent of the fluorescence in situ hybridization (FISH) technique has facilitated the identification of many chromosomal segments, even at the single gene level. Therefore the cosmid probe for Prader-Willi (PWS)/Angelman syndrome to the loci on human chromosome 15 [ql 1-12] is being used as a marker to highlight the complementary sequence in higher primates. We hybridized metaphase chromosomes of chimpanzee (PTR), gorilla (GGO), and orangutan (PPY) with this probe (Oncor) to characterize the chromosomal segments because the nature of these pericentric inversions remains relatively unknown. Our observations suggest that a pericentric inversion has occurred in chimpanzee chromosome (PTR 16) which corresponds to human chromosome 15 at PTR 16 band pl 112, while in gorilla (GGO 15) and orangutan (PPY 16) the bands q11-12 complemented to human chromosome 15 band q11-12. This approach has proven to be a better avenue to characterize the pericentric inversions which have apparently occurred during human evolution. Genetic divergence in the speciation process which occurs through chromosomal rearrangement needs to be reevaluated and further explored using newer techniques.Correspondence to: R.S. Verma