Terminal Regions Of Chromosomes Research Articles

Analysis and location of a rice BAC clone containing telomeric DNA sequences

BAC2, a rice BAC clone containing (TTTAGGG)(n) homologous sequences, was analyzed by Southern hybridization and DNA sequencing of its subclones. It was disclosed that there were many tandem repeated satellite DNA sequences, called TA352, as well as simple tandem repeats consisting of TTTAGGG or its variant within the BAC2 insert. A 0. 8 kb (TTTAGGG)(n)-containing fragment in BAC2 was mapped in the telomere regions of at least 5 pairs of rice chromosomes by using fluorescencein situ hybridization (FISH). By RFLP analysis of low copy sequences the BAC2 clone was localized in one terminal region of chromosome 6. All the results strongly suggest that the telomeric DNA sequences of rice are TTTAGGG or its variant, and the linked satellite DNA TA352 sequences belong to telomere-associated sequences.

Chromosome studies in first polar bodies from hamster and human oocytes.

Most studies on preconception diagnosis published so far have used polymerase chain reaction (PCR) analysis to identify single gene defects. Although fluorescent DNA probes have been used to obtain a partial cytogenetic diagnosis of aneuploidies in first polar bodies without defined chromosome structures, the analysis of structural chromosome anomalies in the interphase nucleus is not adequate. We describe a procedure to obtain first polar body chromosome complements from hamster and human oocytes. In 63.6% (105 of 165) of hamster first polar bodies the chromosome complement showed a defined chromosome morphology and in 94.1% (16 of 17) of human oocytes fixed after follicular puncture it was possible to obtain high quality, well spread chromosome complements. First polar body chromosomes are fuzzy and shorter than oocyte chromosomes, but fluorescent in-situ hybridization results obtained in human first polar bodies clearly show that it is possible to detect whole chromosomes, centromeres and unique sequences, including the terminal regions of small chromosomes. This suggests that in fresh oocytes, DNA loss resulting from apoptotic chromosome fragmentation has not yet occurred. Using the procedure described, first polar bodies could be used to analyse the meiotic segregation of maternal structural abnormalities and to detect numerical chromosome anomalies in humans.

Induction of small-segment-translocation between wheat and rye chromosomes

A new approach to produce wheat-rye translocation, based on the genetic instability caused by monosomic addition of rye chromosome in wheat, is described. 1 283 plants from the selfed progenies of monosomic addition lines with single chromosome of inbred rye line R12 and complete chromosome complement of wheat cultivar Mianyang 11 were cytologically analyzed on a plant-by-plant basis by the improved C-banding technique. 63 of the plants, with 2n = 42, were found containing wheat-rye translocation or substitution, with a frequency of 4.91%. Compared with the wheat parent, other 32 plants with 2n = 42 exhibited obvious phenotypic variation, but their component of rye chromosome could not be detected using the C-banding technique.In situ hybridization with a biotin-la-beled DNA probe was used to detect rye chromatin and to determine the insertion sites of rye segments in the wheat chromosomes. In 20 out of the 32 variant wheat plants, small segments of rye chromosomes were found being inserted into different wheat chromosomes and form small-segment-translocation (SS translocation). The physical mapping of the translocated small segments of rye chromosomes indicated that alien insertion could occur in both the terminal and intermediate regions of wheat chromosomes. The technique described appeared to be an effective means to induce SS translocation. The wide application of SS translocation in the study of molecular cytogenetics and plant breeding is also discussed.

High Resolution Mapping of the/ndica-Derived Rice Blast Resistance Genes. I.Pi-b

Rice is a good material for studying positional cloning of disease resistance genes because its ratio of physical to genetic distance is small (100 to 300 kb/cM), and abundant genetic information is available. As a first step in cloning, the rice blast resistance gene Pi-b was finely mapped near the terminal region of chromosome 2 using restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) markers. Various near-isogenic lines (NILs) developed by the introgression of the indica-derived Pi-b gene into japonica recurrent parents facilitated its rapid and accurate localization and also the F2 analyses. Although Pi-b was introgressed independently from four cultivars, all the donors showed the same RFLP profile for all the nearby probes used, whereas there was some polymorphism among japonica recurrent parents, suggesting a single origin of the resistance gene. Pi-b cosegregated with RAPD (b-1) and RFLP (RZ123) markers, and it was bracketed between flanking RFLP markers at 0.5 cM on the centromeric side, and 1.9 cM on the telomeric side. The centromeric and telomeric limits of the indica regions of NILs BL-1 and BL-7 were closer to Pi-b than the flanking nucleic markers on each side, respectively, providing better (< 0.5 and < 1.9 cM) delineating markers. A long-range Southern blot with rare-cutter restriction enzymes revealed an 85-kb common band between the two 0-cM markers. These markers provide an excellent environment for the positional cloning of Pi-b.

Rat 5S rDNA spacer sequences and chromosomal assignment of the genes to the extreme terminal region of chromosome 19

Fragments of the spacer region of genes for rat 5S ribosomal RNA (rDNA), which are tandemly repeated, were amplified by PCR with primers specific to the two ends of the coding region for 5S rRNA. Two amplified fragments of approximately 1.6 kb were subcloned and sequenced. The spacer sequences showed a high degree of sequence identity to each other (99.2%) but substantial divergence from those of analogous mouse clones. The homologous regions in the mouse clones were interrupted by the duplication or deletion of small segments of DNA. A 12-mer, 5'-GGCTCTTGGGGC-3', thought to be responsible for efficient transcription, was located from position -33 to position -22 in the rat -5S clones. The genes were mapped by fluorescence in situ hybridization with cloned fragments of rat 5S rDNA as probe. The genes were localized exclusively in a single telomeric region of chromosome 19.

Genes from Lycopersicon chmielewskii affecting tomato quality during fruit ripening

Three chromosomal segments from the wild tomato, L. chmielewskii, introgressed into the L. esculentum genome have been previously mapped to the middle and terminal regions of chromosome 7 (7M, 7T respectively), and to the terminal region of chromosome 10 (10T). The present study was designed to investigate the physiological mechanisms controlled by the 7M and 7T segments on tomato soluble solids (SS) and pH, and their genetic regulation during fruit development. The effects of 7M and 7T were studied in 64 BC2F5 backcross inbred lines (BILs) developed from a cross between LA 1501 (an L. esculentum line containing the 7M and 7T fragments from L. chmielewskii), and VF145B-7879 (a processing cultivar). BILs were classified into four homozygous genotypes with respect to the introgressed segments based on RFLP analysis, and evaluated for fruit chemical characteristics at different harvest stages. Gene(s) in the 7M fragment reduce fruit water uptake during ripening increasing pH, sugars, and SS concentration. Gene(s) in the 7T fragment were found to be associated with higher mature green fruit starch concentration and red ripe fruit weight. Comparisons between tomatoes ripened on or off the vine suggest that the physiological mechanisms influenced by the L. chmielewskii alleles are dependent on the translocation of photosynthates and water during fruit ripening.

Study on Diffused Centromeric Nature of Drosera Chromosomes.

Drosera dichrosepala was exposed to different doses of Gamma radiation. Fragmented and fused chromosomes were observed as a consequences. Diffused centromeres in every fragment chromosome was detected by centromeric banding (Cd-banding) and was supported by its typical disjunction and totally lack of lagging chromosomes or micronuclei from anaphase to telophase. C-banding revealed that the fragment chromosomes were highly heterochromatic and fragmentation might be occurred at the terminal regions of chromosomes. Fluorescent banding suggested that most of the fragment chromosomes were rich in GC base composition in the species. Alteration of karyotype due to Gamma irradiation also indicated that spontaneous fragmentation or fusion of chromosomes might be a possible factor for promoting the bimodal karyotype in this genus.

A genetically engineered V79 cell line SD1 expressing rat CYP2B1 exhibits chromosomal instability at the integration site of the transfected DNA.

The genetically engineered cell line SD1 was constructed by co-transfection of V79 Chinese hamster cells with two plasmids: one containing a full-length cDNA encoding rat CYP2B1 and the second incorporating a selective marker gene. This cell line has been used in gene mutation tests and in cytokinesis-block micronucleus assays to identify procarcinogens which are metabolized by CYP2B1 to reactive metabolites. An elevated frequency of spontaneous micronuclei was recorded in SD1 cells compared to parental V79 cultures. Karyotypic analysis revealed a chromosomal instability which was manifested by amplification of the p-arms of a chromosome designated 'n' (derived from chromosome 8). This chromosome was variable in length and sometimes exhibited a telomeric fusion which led to the formation of a dicentric chromosome. Fluorescence in situ hybridization with digoxigenin-labelled plasmid DNA showed the presence of pSV450 plasmid DNA coamplified with genomic DNA sequences located in the terminal region of chromosome 'n'.

Cytogenetical Studies in Rhoeo spathacea (Commelinaceae) II. Characterization of an acrotrisomic plant.

The objective of this study in Rhoeo spathacea was the cytological identification of a trisomic plant which includes an acrocentric chromosome. The homology of the extra chromosome was not identified with certainty by karyotype analysis because of its lack of similarity with any other chromosome. Somatic metaphase analysis indicates that interchanges take place in terminal regions of Rhoeo chromosomes. The presence of the acrocentric also supports this idea. On the basis of behavior at MI, this extra chromosome seems to be homologous with one of the largest chromosome. MI was as expected for ring forming Rhoeo (Chains of 13, 45.7%; 12+1I, 15.7%; or 11+2I, 15%) and not as expected for a desynaptic. The extra chromosome was either associated by its long arm with its supposed homologue or free; when associated, its short arm was free. At AI the segregation was mostly 6:7, with microspores with n=5 to n=26, n=7, 34.4% and also producing SDR with n=13 (22.4%). When all the progeny is included in the observations, a possible apomictic mechanism of origin was postulated.

In situ hybridization to metaphase chromosomes in six species ofPhaseolus andVigna using ribosomal DNA as the probe

In situ hybridization with a biotin-labeled rice ribosomal DNA (rDNA) probe to the somatic metaphase chromosomes of six species ofPhaseolus andVigna (P. angularis, P. calcaratus, P. coccineus, P. vulgaris, V. sesquipedalis andV. sinensis) was done to determine the sites of rDNA. Hybridization signals were present in the terminal and subterminal chromosome regions of each of the six species. The number of rDNA sites was two inP. angularis andP. calcaratus, four inP. coccineus andP. vulgaris, and six inV. sesquipedalis andV. sinensis.

The arrangement and transcription of telomere DNA sequences at the ends of lampbrush chromosomes of birds

The arrangement of loops and chromomeres at the ends of lampbrush chromosomes in four species of bird is described with reference to chromomeres, loops and transcription units. Unlike the situation described in lampbrush chromosomes of amphibians, the lampbrush chromosomes of birds end in a terminal chromosome with conspicuous loops emerging from it. The fine-scale morphology of the ribonuclear protein matrix of these terminal loops is different from that of the majority of loops elsewhere on the chromosomes. In many cases the loops associated with the terminal chromomere are open ended, emerging from the chromomere but not returning to it at the other end. The distal ends of terminal open-ended loops therefore represent the true ends of the chromatids that make up a lampbrush half-bivalent. The pattern of binding of three telomeric DNA sequence probes to the terminal regions of bird lampbrush chromosomes, under conditions of DNA/DNA and DNA/RNA transcript in situ hybridization has been investigated by fluorescence in situ hybridization. All three probes gave the same results. With DNA/DNA and DNA/RNA transcript hybridization, three classes of structure were labelled: the terminal chromomere, a small number of interstitial chromomeres and the terminal transcription unit on telomere loops. Labelling of telomere loops, but not of terminal or interstitial chromomeres, was eliminated by ribonuclease treatment before in situ hybridization. The labelled regions of telomere loops were spaced away from the labelled terminal chromomere by an unlabelled sub telomeric transcription unit. After DNA/DNA in situ hybridization, no labelled loops were seen. DNA/RNA transcript in situ hybridization with single-stranded hexamers of each strand of telomeric DNA showed that the terminal transcription unit on telomere loops represents transcription exclusively from the C-rich strand of the repeat outwards towards the end of the chromosome. It is concluded that transcription specifically of the C-rich strand of strictly terminal clusters of telomere repeats is an obligatory event on the lampbrush chromosomes of birds and is unlikely to represent indiscriminate readthrough from proximally located gene elements.

Structure, chromosomal localization, and expression of 12 genes of the MAGE family.

We reported previously that human gene MAGE-1 directs the expression of a tumor antigen recognized on a melanoma by autologous cytolytic T lymphocytes. Probing cosmid libraries with a MAGE-1 sequence, we identified 11 closely related genes. The analysis of hamster-human somatic cell hybrids indicated that the 12 MAGE genes are located in the q terminal region of chromosome X. Like MAGE-1, the 11 additional MAGE genes have their entire coding sequence located in the last exon, which shows 64%-85% identity with that of MAGE-1. The coding sequences of the MAGE genes predict the same main structural features for all MAGE proteins. In contrast, the promoters and first exons of the 12 MAGE genes show considerable variability, suggesting that the existence of this gene family enables the same function to be expressed under different transcriptional controls. The expression of each MAGE gene was evaluated by reverse transcription and polymerase chain reaction amplification. Six genes of the MAGE family including MAGE-1 were found to be expressed at a high level in a number of tumors of various histological types. None was expressed in a large panel of healthy tissues, with the exception of testis and placenta.

Characterization of the effect of introgressed segments of chromosome 7 and 10 from Lycopersion chmielewskii on tomato soluble solids, pH, and yield

Three chromosomal segments from the wild tomato L. chmielewskii have been introgressed into the L. esculentum genome. Using molecular markers they have been mapped to the middle and terminal regions of chromosome 7 (7M, 7T respectively), and to the terminal region of chromosome 10 (10T). This study was conducted to further clarify the physiological influence of the introgressed segments of chromosome 7 and 10 on tomato soluble solids (SS), and other fruit and yield parameters. The effect of the 10T segment was evaluated using five lines that differ for the presence of this segment. As previously reported this segment increased fruit pH with no significant effect on SS. Sixty-four BC2F5 backcross inbred lines (BILs) were developed from a cross using LA1501 (an L. esculentum line that contains the 7M and 7T fragments from L. chmielewskii) as the donor parent, and VF145B-7879 (a processing cultivar) as the recurrent parent. BILs were classified in four groups (+ +, inbreds without either of the L. chmielewskii segments; 7M +, lines with only the 7M segment; + 7T, inbreds with only the 7T segment, and 7M7T, inbreds with both segments) based on RFLP information, and then compared to each other for all the parameters under study. Inbreds homoyzgous for the 7M fragment displayed greater SS (26%) and higher pH (0.10) than the control group (+ +). The 7L fragment did not influence either SS or pH, but was observed to significantly increase fruit yield by 12% when compared to the recurrent parent. A gene or genes that increase yield without affecting SS or pH may have potential in the development of commerical cultivars.

Chromatin Bridges between Sister Chromatids Induced in Late G2 Mitosis in CHO Cells by Trimethylpsoralen + UVA

A hypothesis is proposed that sister chromatid pairing is due, at least in part, to the pairing between DNA strands belonging to each of the two sister chromatids (conservative pairing). To test this hypothesis interstrand DNA cross-links were induced in late G2-mitosis in CHO cells in order to bind covalently paired DNA strands eventually coming from both sister chromatids and detect the consequent chromatin bridges between sister chromatids (SCCBs). Therefore cells were treated with trimethylpsoralen (TMP) + UVA (365 or 405 nm). Chromatin bridges in ana-telophase were induced by an UVA irradiation at 365 nm, which gives rise to both monoadducts and cross-links, but not by a 405-nm irradiation, which gives rise only to monoadducts. An analysis of colchicine-induced c-anaphases demonstrated that such chromatin bridges were really SCCBs and that terminal regions of chromosomes were particularly involved. The evolution of SCCBs was studied to rule out that they were masked isochromatid exchanges. So TMP + UVA-treated cells were induced to polyploidize with colchieine and labeled with 5-bromodeoxyuridine. Cells treated with TMP + UVA in G2-mitosis appeared as M1 tetraploid c-metaphases; in such cell populations there was not an increase in isodicentric chromosomes, which are derived from isochromatid exchanges. The present data, as a whole, support the hypothesis that a "conservative pairing" between DNA strands of sister chromatids can be present in mitosis.

Repetitive sequences in the genome of Anemone blanda: identification of tandem arrays and of dispersed repeats.

A tandemly repetitive sequence family (AbS1) and a repetitive sequence (Hd) forming part of a larger dispersed element (dorf-1) of Anemone blanda were characterised. The AbS1 satellite sequence family is located in all 4',6-diamidino-2-phenylindole (DAPI) positive intercalary heterochromatic bands and in the DAPI positive heterochromatic terminal region of chromosome 3, while the dispersed Hd homologous sequences are preferentially associated with euchromatic chromosome regions. The major component of the AbS1 satellite is AbS1-H1 with a basic repeat unit of 1640 bp; a minor fraction (AbS1-H5) consists of 320 bp units. A subsection of the AbS1-H1 repeat unit exhibits homologies to the 25S rRNA gene of flowering plants suggesting that the 1.64 kb satellite was generated by amplification of a precursor satellite and/or single copy sequence together with an rDNA fragment. The rDNA homologous region is considered to evolve at a rate similar to pseudogenes and thus the age of this satellite DNA fraction can be roughly estimated as about 27 million years. The dispersed repeated sequence Hd (about 1300 bp) is associated with the 8 kb element dorf-1. A. blanda dorf-1 constitutes about 0.2% of the genome (3 x 10(4) copies), is bounded by identical long terminal repeats, and exhibits partial homology to the Lilium gypsy-type element del1, but has yet to be confirmed as a retrotransposon. In contrast to the AbS1 satellite sequence family, Hd homologous sequences were found not only in A. apennina, the closest relative of A. blanda, but also in A. nemorosa and A. ranunculoides indicating that a progenitor sequence of dorf-1 was present in a common ancestor before speciation occurred.

Clustering of hypervariable minisatellites in the proterminal regions of human autosomes

Six of the human minisatellites detected by DNA fingerprint probes have been localized by in situ hybridization to human metaphase chromosomes. These hypervariable loci are not dispersed at random in the human genome, but show preferential, though not exclusive, localization to terminal G-bands of human autosomes. Two of the proterminal minisatellites are very closely linked to other variable loci. Sequence analysis of one of these additional minisatellites suggests that the two linked minisatellites arose by independent amplification of different repeat units. The proterminal regions of human autosomes may therefore be rich in minisatellites, analogous to the pseudoautosomal terminal pairing region of human sex chromosomes that is similarly abundant in hypervariable minisatellites.

Variant Ph translocations in chronic myeloid leukemia

Variant translocations were found in eight of 142 consecutive patients with Ph-positive, chronic myeloid leukemia encountered in our laboratory during the last decade. Two patients had simple, two-way variant translocations: t(17;22)(p13;q11) and t(16;22)(q24;q11). Both of these patients had an additional translocation involving chromosome #9: t(7;9) (q22;q34) and t(9;17)(q34;q21), respectively. Complex variant translocations were found in four cases: t(2;9;22)(p23q12;q34;q11), t(3;9;22)(p21;q34;q11), t(9;12;22)(q34;p13;q11q13), and t(13;17;22)(p11;p11q21;q11). In two cases, the only discernable cytogenetic aberration was del(22)(q11). A review of the chromosomal breakpoints involved in this series and in 185 cases of variant Ph translocations previously reported in the literature reveals that a disproportionately large number of breakpoints are located in light-staining regions of G-banded chromosomes. Furthermore, the breakpoints in simple variant translocations are more often located in terminal chromosomal regions, whereas, the breakpoints in complex translocations typically affect nonterminal bands. No obvious correlation was detected between variant Ph translocation breakpoints and either fragile sites, oncogene locations, or consistent chromosome breakpoints in other malignancies.

Use of nalidixic acid for constructing the replication map of the Mycobacterium phlei chromosome.

Nalidixic acid was used for describing more accurately the terminal replication region of the Mycobacterium phlei chromosome. Cell division in synchronized cultures was not sensitive to this acid any more between 185-190 min, i.e. about 10 min after replication of the ser gene, the last of 24 genes of the replication map described so far. The replication of the chromosome was controlled by determining the position of the bac gene. Microscopic studies in phase contrast of the cells that were subjected for long time periods to nalidixic acid treatment at a bactericidal concentration showed elongated cells. The electronmicroscopic observation showed that a portion of the population influenced by nalidixic acid lyses, whereas other cells remain intact and resemble control cells.

MEIOTIC ASSOCIATIONS IN BARLEY LINES WITH EIGHT PAIRS OF CHROMOSOMES AND THEIR HAPLOIDS

Meiosis was studied in pollen mother cells and embryo sac mother cells of seven lines of eight-paired barley (Hordeum vulgare L.) in diploids (2n = 16) and their haploids (2n = 8). Translocation 5-7a, whose breakpoints are distal on chromosome 5 and in the satellite of chromosome 7, was found in all lines. The extra fragment chromosome (8) is a normal or translocation form of chromosome 5 deficient for terminal regions. In diploids, the extra chromosome behaves regularly at meiosis except for occasional univalent-formation and can form a symmetric ring bivalent, but it never forms chiasmata with other chromosomes. In haploids, however, it forms a low frequency of chiasmata with chromosome 5 but this frequency is reduced when the terminal region of chromosome 5 pairs homologously with the satellite region of 7. Based on the hypothesis used to explain chromosome pairing behavior, it should be possible to derive and identify additional eight-paired lines that would be useful for breeding.

A specific chromosome element, the telomere of Drosophila polytene chromosomes.

The submicroscopic organization of terminal chromosome regions of Drosophila hydei polytene chromosomes is described. A compact region composed of tightly packed fibrils of 100 to 125 A diameter embedded in an amorphous material is located at each of the chromosome ends of the 5 long chromosome arms. From this compact region, sometimes containing cavities, fibrils extend onto the nearest normal band region. The diameter of the extending fibrils is 100–125 A, 200–250 A or 400 A. Pronase digestion of fixed and squashed chromosomes reduced the electron density of the amorphous matrix in the compact regions but failed to affect the diameter of the fibrils. The extending fibrils, however, showed a decrease in diameter after pronase digestion. The most frequently observed diameter values were 100–125 A. — The volume of the terminal structures, including the compact region as well as the extending fibrils, is characteristically different for the various elements of the karyotype. Chromosome 2 displays the largest terminal structure, whereas chromosome 4 only occasionally shows the presence of compact regions. — End to end association of the long chromosome arms involves the fusion of the compact terminal structures. The non-random distribution of end to end association seems to be correlated with the volume of the terminal structures. Chromosome 2 which contains the largest compact terminal region is more frequently involved in end to end associations than any other chromosome arm. — The terminal regions show replication of DNA. They belong to the group of regions which display a discontinuous labeling pattern along the chromosomes, representing a late phase of the replication cycle. — The unique structural organization of the terminal chromosome regions, which is never observed at any other location of the genome supports the idea that they are morphological manifestations of the postulated telomeres.