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Human allospecific TLCs generated against HLA antigens associated with DR1 through DRw8. III. Family segregation analyses.

We have studied the complexity and fine specificity of the HLA-D region using a panel of T lymphocyte clones generated against alloantigens associated with HLA-DR1 through DRw8. After extensive testing in population studies, 89 clones were tested in proliferation assays with 14 families. Segregation patterns were analyzed for haplotype associations by calculating sequential lod scores to test the likelihood that genes encoding epitopes detected by TLCs were linked to HLA genes. Four general categories were identified: (1) clonal responses that segregated with the same HLA-D region haplotype in all informative pedigrees; (2) clonal responses that segregated with HLA in all pedigrees but not always with the same haplotype; (3) clonal responses that segregated with HLA in some families but failed to segregate in others or produced equivocal results; (4) clonal responses that did not segregate with HLA haplotypes.

Cell Line↗

Transgressive segregation of erucic acid content in Brassica carinata A. Braun.

Two Ethiopian mustard ( Brassica carinata A. Braun) lines with low (about 10%) and zero erucic acid (C22:1) have been obtained. The low C22:1 mutant line L-2890 was isolated after a chemical-mutagen treatment of C-101 seeds (about 40% C22:1). The zero C22:1 line L-25X-1 was obtained by interspecific crossing. Our objective was to determine the genetic control of low and zero C22:1 contents in these lines and the relationship between the loci controlling these traits. Reciprocal crosses between L-2890, L-25X-1 and high C22:1 lines, and between L-2890 and L-25X-1, were made. The F(1), F(2) and BC(1) F(1) generations were obtained. No maternal or cytoplasmic effects for C22:1 content were observed in any of the crosses. The analysis of the fatty acid composition in the segregating populations from the crosses of L-2890 with the high C22:1 lines C-101 and L-1630 indicated that the segregation patterns fitted a model of two alleles at two loci, M1 and M2, with partial (near complete) dominance for high concentration. The segregation patterns in the cross of the zero C22:1 line L-25X-1 with the high C22:1 line L-1630, were explained on the basis of two genes, E1 and E2, with additive gene action. The F(1) and segregating generations of the crosses L-2890 x L-25X-1 showed a strong transgressive segregation with C22:1 values of up to 50.0%, four-fold higher than those of L-2890. The analyses of the F(2), BC(1)F(1) and F(3) generations indicated that the combination of alleles at four loci, M(1) and M(2) in L-2890 and E(1) and E(2) in L-25X-1, controlled the transgressive segregation for C22:1. The proposed genotypes (C22:1 content) for each parent were as follows: L-2890 (10% C22:1) = m(1) m(1) m(2) m(2) E(1) E(1) E(2) E(2); L-25X-1 (0% C22:1) = M(1) M(1) M(2) M(2) e(1) e(1) e(2) e(2); and C-101 (45% C22:1) = M(1) M(1) M(2) M(2) E(1) E(1) E(2) E(2).

Brassica↗

Cellular autophagic capacity changes during azaserine-induced tumour progression in the rat pancreas. Up-regulation in all premalignant stages and down-regulation with loss of cycloheximide sensitivity of segregation along with malignant transformation.

The knowledge of alterations in regulation of autophagy during tumorigenesis may also help our understanding of its normal control. We established an experimental system and reported recently that autophagic capacity, measured as the cell's capability of increasing segregation (formation of autophagosomes) and subsequent degradation of cytoplasmic quanta were highly increased in premalignant nodule cells 6 months after initiation by azaserine in the rat pancreas in vivo. In the present study, we followed changes of these autophagic functions throughout the tumour progression. We carried out electron-microscopic morphometrical analysis of the expansion of autophagic vacuole compartment and subcompartments induced by vinblastine (an in vivo segregation enhancer), as well as their regression upon segregation-inhibitor cycloheximide post-treatment. Premalignant tumour samples were taken at month 5, month 8 (nodules), month 10 and month 15 (adenomas) after initiation. In all these stages, a highly increased and varying autophagic capacity was found compared with the host tissue. The basal (non-stimulated) autophagic compartment was measurable only at month 5 and month 15, and its regression upon cycloheximide was consistent with increased basal autophagic activity. Compared with the host tissue, autophagic capacity profoundly decreased in the differentiated and anaplastic adenocarcinomas at month 20, when, surprisingly, cycloheximide was unable to inhibit segregation. Our conclusion is that down-regulation of the cycloheximide sensitive segregation and a partly compensatory up-regulation of an alternative pathway of segregation might occur along with malignant transformation.

Adenocarcinoma↗

Polarization of ooplasmic segregation and dorsal-ventral axis determination in ascidian embryos.

During ooplasmic segregation in ascidians, the myoplasm moves from its original location in the periphery of the unfertilized egg to the vegetal pole of the zygote. The vegetal cap of myoplasm marks the future site of gastrulation and the dorsal side of the embryo. The purpose of this investigation was to determine the mechanism for polarizing the myoplasm during ooplasmic segregation. To test the possibility that the myoplasm moves toward the sperm and that the vegetal pole is the exclusive site of sperm entry, we examined fertilization in egg fragments of the ascidians Styela plicata and Ciona savignyi. Similar frequencies of fertilization were exhibited by various egg fragments, including animal and vegetal fragments or multiple fragments prepared from the same egg. These results indicate that sperm do not enter the egg exclusively at the vegetal pole. Experiments with egg fragments and constricted eggs, combined with chalk marking of the animal pole, demonstrated that after fertilization the myoplasm segregates parallel to the animal-vegetal axis, usually toward the vegetal end of the cell. Activation of primary oocytes with the Ca2+ ionophore A23187 caused the myoplasm to segregate independently of the animal-vegetal axis. This confirms previous experiments in which eggs aligned along a glass fiber coated with A23187 segregated their myoplasm toward the fiber (W.R. Jeffery, 1982, Science 216, 545-547) and suggests that the intrinsic cue for polarization is a release of sequestered Ca2+ at fertilization. Therefore, it appears that ooplasmic segregation and the dorsal-ventral axis are polarized by maternal factors distributed in a concentration gradient along the animal-vegetal axis of the ascidian egg.

Animals↗

Perceived texture segregation in chromatic element-arrangement patterns: high intensity interference.

An element-arrangement pattern is composed of two types of elements that differ in the ways in which they are arranged in different regions of the pattern. We report experiments on the perceived segregation of chromatic element-arrangement patterns composed of equal-size red and blue squares as the luminances of the surround, the interspaces and the background (surround plus interspaces) are varied. Perceived segregation was markedly reduced by increasing the luminance of the interspaces. Perceived segregation was approximately constant for constant ratios of interspace luminance to square luminance and increased with the contrast ratio of the squares. Unlike achromatic element-arrangement patterns composed of squares differing in lightness [Beck et al (1991). Vision Research, 32, 719-743] perceived segregation did not decrease when the luminance of the interspaces was below that of the squares. Similar results were obtained for red and yellow, red and green, green and yellow, green and blue, and blue and yellow squares. Perceived segregation based on edge alignment was not interfered with by high intensity interspaces. Stereoscopic cues that caused the squares composing the element-arrangement pattern to be seen in front of the interspaces did not greatly improve perceived segregation. One explanation of the results is in terms of inhibitory interactions among achromatic and chromatic cortical cells tuned to spatial frequency and orientation. Alternately, the results may be explained in terms of how the luminance of the interspaces affects the grouping of the squares for encoding surface representations. Neither explanation accounts fully for the data and both mechanisms may be involved.

Color Perception↗

Segregation of photosystems in thylakoid membranes as a critical phenomenon.

The distribution of the two photosystems, PSI and PSII, in grana and stroma lamellae of the chloroplast membranes is not uniform. PSII are mainly concentrated in grana and PSI in stroma thylakoids. The dynamics and factors controlling the spatial segregation of PSI and PSII are generally not well understood, and here we address the segregation of photosystems in thylakoid membranes by means of a molecular dynamics method. The lateral segregation of photosystems was studied assuming a model comprising a two-dimensional (in-plane), two-component, many-body system with periodic boundary conditions and competing interactions between the photosystems in the thylakoid membrane. PSI and PSII are represented by particles with different values of negative charge. The pair interactions between particles include a screened Coulomb repulsive part and an exponentially decaying attractive part. The modeling results suggest a complicated phase behavior of the system, including quasi-crystalline phase of randomly distributed complexes of PSII and PSI at low ionic screening, well defined clustered state of segregated complexes at high screening, and in addition, an intermediate agglomerate phase where the photosystems tend to aggregate together without segregation. The calculations demonstrated that the ordering of photosystems within the membrane was the result of interplay between electrostatic and lipid-mediated interactions. At some values of the model parameters the segregation can be represented visually as well as by analyzing the correlation functions of the configuration.

Biophysical Phenomena↗

Feature-specific electrophysiological correlates of texture segregation.

Discrimination between a figure and its surround is an important first step of pattern recognition. This discrimination usually relies, as a first step, on the detection of borders between a figure and its surround, for example based on spatial gradients in luminance, colour, or texture. There is evidence that neurones in the visual cortex are specifically activated by segregation between textures, but the relation between segregation based on different types of features such as colour, luminance, and motion is unclear. Evoked EEG potentials specific to texture segregation were investigated in 17 observers in two separate experiments and by means of functional magnetic resonance imaging in a separate study (Fahle et al., in preparation). Differences in either luminance, colour, line orientation, motion, or stereoscopic depth defined a checkerboard pattern. Patterns defined by each of these features elicited segregation-specific potentials. In contrast to earlier reports (Vision Research 37 (1997) 1409), however, we find pronounced differences between the segregation-specific potentials evoked through different features, especially regarding their peak latencies. The topographical distribution of the activity evoked reveals different polarities and partly specific locations for different stimulus features, indicating the existence of different processors for texture segregation based on different features.

Adult↗

Subnanometer three-dimensional atom-probe investigation of segregation at MgO/Cu ceramic/metal heterophase interfaces.

Three-dimensional atom-probe (3DAP) microscopy has been applied to the study of segregation at ceramic/metal (C/M) interfaces. In this article, results on the MgO/Cu(X) (where X = Ag or Sb) systems are summarized. Nanometer-size MgO precipitates with atomically clean and atomically sharp interfaces were prepared in these systems by internal oxidation. Segregation of the ternary component (Ag or Sb) at the MgO/Cu heterophase interface was enhanced by extended low-temperature anneals. Magnesia precipitates in the 3DAP reconstructions were delineated as isoconcentration surfaces, and segregation of each ternary component at the C/M interfaces was analyzed with the proximity histogram method developed at Northwestern University. This method allows the direct extraction of the Gibbsian interfacial excess of solute at the C/M interfaces from the experimental data. A value of (3.2+/-2.0) x 10(17)m(-2) at 500 degrees C is obtained for the segregation of Ag at a MgO/Cu(Ag) interface, while a value of (2.9+/-0.9) x 10(18) m(-2) at 500 degrees C is obtained for the segregation of Sb at a MgO/Cu(Sb) interface. The larger Gibbsian excess for Sb segregation at this ceramic/metal heterophase interface is most likely due to the so-called pdeltaV effect.

Journal Article↗

Parental nucleosomes segregated to newly replicated chromatin are underacetylated relative to those assembled de novo.

Antibodies specific for acetylated histone H4 were used to examine the acetylation state of parental histones that segregate to newly replicated DNA. To generate newly replicated chromatin containing only segregated parental nucleosomes, isolated nuclei were labeled with [3H]TTP in vitro; alternatively, whole cells were labeled with [3H]thymidine in the presence of cycloheximide. Soluble chromatin was prepared by micrococcal nuclease digestion, and subjected to immunoprecipitation with "penta" antibodies (Lin et al., 1989). In sharp contrast to nucleosomes containing newly synthesized, diacetylated H4 (Perry et al., 1993), chromatin replicated in vitro was only marginally susceptible to immunoprecipitation. Control experiments established that bona fide acetylated chromatin was selectively immunoprecipitated by the same techniques and that segregated nucleosomes were not disassembled during treatment with "penta" antibodies. When replication was coupled to an in vitro histone acetylation system, the enrichment for segregated nucleosomes in the immunopellet increased approximately 3-fold, demonstrating that changes in the acetylation state of segregated histones can be detected immunologically and that parental histones on new DNA are accessible to acetyltransferases during, or immediately after, DNA replication. In vivo pulse-chase experiments, performed in the presence of cycloheximide, confirmed these results. Uptake experiments further established that concurrent histone acetylation did not alter the rate of DNA synthesis in vitro. Our results provide evidence that replication-competent chromatin is not obligatorily acetylated, and indicate that the acetylation status of segregated histones may be maintained during chromatin replication. The possible significance of this, with respect to the regulation of chromatin higher order structures during DNA replication, and the propagation of transcriptionally active vs inactive chromatin structures, is discussed.

Acetylation↗

The yeast kinetochore protein Slk19 is required to prevent aberrant chromosome segregation in meiosis and mitosis.

BACKGROUND: Slk19 is a coiled-coil protein, which locates to the kinetochores of S. cerevisiae. Most cells lacking Slk19 undergo incomplete meiosis and form dyads during sporulation. Endogenous chromosomes appeared to be predominantly divided in an equational manner during single-division meiosis of slk19 null mutants. RESULTS: We have monitored the segregation of artificial chromosomes (YACs) in slk19 null mutants during both single-division meiosis and complete meiosis. In contrast to the results obtained with endogenous chromosomes, YACs only rarely undergo equational segregation during single division meiosis, although high rates of aberrant segregation were detected. This accounts for the high frequency of lethal spores among dyads of slk19 delta null mutants. The fraction of slk19 delta cells that were able to form tetrads solely exhibited YAC segregation defects in meiosis II, whereas the segregation of YACs in meiosis I was normal in these cells. This result might indicate that correct chromosome division in meiosis I is a prerequisite for tetrad formation. slk19 null mutants also showed YAC instability in mitosis and reduced survival after the induction of mitotic spindle damage. CONCLUSION: Slk19 is required to avoid aberrant segregation of chromosomes in meiosis I and II and in mitosis. We suggest that the absence of Slk19 leads to uncoupling of chromosome movement from completion of microtubule attachment and resolution of chromosome cohesion.

Base Sequence↗

Segregation analysis of prostate cancer in France: evidence for autosomal dominant inheritance and residual brother-brother dependence.

Four segregation analyses concerning prostate cancer (CaP), three conducted in the United States and one in Northern Europe, have shown evidence for a dominant major gene but with different parameter estimates. A recent segregation analysis of Australian pedigrees has found a better fit of a two-locus model than single-locus models. This model included a dominantly inherited increased risk that was greater at younger ages and a recessively inherited or X-linked increased risk that was greater at older ages. Recent linkage analyses have led to the detection of at least 8 CaP predisposing genes, suggesting a complex inheritance and genetic heterogeneity. To assess the nature of familial aggregation of prostate cancer in France, segregation analysis was conducted in 691 families ascertained through 691 CaP patients, recruited from three French hospitals and unselected with respect to age at diagnosis, clinical stage or family history. This mode of family inclusion, without any particular selection of the probands, is unique, as probands from all previous analyses were selected according to various criteria. Segregation analysis was carried out using the logistic hazard regressive model, as incorporated in the REGRESS program, which can accommodate a major gene effect, residual familial dependences of any origin (genetic and/or environmental), and covariates, while including survival analysis concepts. Segregation analysis showed evidence for the segregation of an autosomal dominant gene (allele frequency of 0.03%) with an additional brother-brother dependence. The estimated cumulative risks of prostate cancer by age 85 years, among subjects with the at-risk genotype, were 86% in the fathers' generation and 99% in the probands' generation. This study supports the model of Mendelian transmission of a rare autosomal dominant gene with high penetrance, and demonstrates that additional genetic and/or common sibling environmental factors are involved to account for the familial clustering of CaP.

Age of Onset↗

Nonrandom segregation of centromeres following mitotic recombination in Drosophila melanogaster.

Mitotic recombination is widely used in Drosophila as a technique to study genetic and developmental problems. It has been generally assumed that, following mitotic exchange between homologous chromatids during the G2 stage, the centromeres attached to the chromatids involved in the exchange segregate randomly. As a result, two equally frequent types of segregation, yielding genetically different products, are produced. However, when epidermal or enzymatic cell-marker mutants are used, only one type of segregation gives rise to marked cells. In the present work we test this assumption of random segregation using cytological markers. With cytological markers, larval neuroblast cells resulting from mitotic recombination exhibit recognizably all possible products from mitotic recombination. We find that one type of segregation is favored, in that, after mitotic recombination, the centromeres attached to the chromatids involved in the mitotic exchange preferentially migrate to opposite poles during anaphase. This favored segregation could be the result of exchange between previously oriented chromatids or could be due to the effect of the exchange upon subsequent orientation of homologous chromosomes. In either case, frequencies of mitotic recombination have been overestimated in the past.

Animals↗

Zebrafish vasa RNA but not its protein is a component of the germ plasm and segregates asymmetrically before germline specification.

Work in different organisms revealed that the vasa gene product is essential for germline specification. Here, we describe the asymmetric segregation of zebrafish vasa RNA, which distinguishes germ cell precursors from somatic cells in cleavage stage embryos. At the late blastula (sphere) stage, vasa mRNA segregation changes from asymmetric to symmetric, a process that precedes primordial germ cell proliferation and perinuclear localization of Vasa protein. Analysis of hybrid fish between Danio rerio and Danio feegradei demonstrates that zygotic vasa transcription is initiated shortly after the loss of unequal vasa mRNA segregation. Blocking DNA replication indicates that the change in vasa RNA segregation is dependent on a maternal program. Asymmetric segregation is impaired in embryos mutant for the maternal effect gene nebel. Furthermore, ultrastructural analysis of vasa RNA particles reveals that vasa RNA, but not Vasa protein, localizes to a subcellular structure that resembles nuage, a germ plasm organelle. The structure is initially associated with the actin cortex, and subsequent aggregation is inhibited by actin depolymerization. Later, the structure is found in close proximity of microtubules. We previously showed that its translocation to the distal furrows is microtubule dependent. We propose that vasa RNA but not Vasa protein is a component of the zebrafish germ plasm. Triggered by maternal signals, the pattern of germ plasm segregation changes, which results in the expression of primordial germ cell-specific genes such as vasa and, consequently, in germline fate commitment.

Actins↗

Centromeric regions control autonomous segregation tendencies in single-division meiosis of Saccharomyces cerevisiae.

We have previously shown that yeast cdc5 or cdc14 homozygotes can be led through a single-division meiosis in which some of the chromosomes segregate reductionally whereas others, within the same cell, segregate equationally. Chromosomes XI tend to segregate reductionally, whereas chromosomes IV tend to segregate equationally. In this report we present experiments with cdc5 homozygous strains, in which the centromeres of one or both chromosomes XI was replaced by the centromeric region from chromosome IV. Analysis of the products of single-division meioses in these strains demonstrates that the choice between reductional or equational segregation is directed by sequences in the vicinity of the centromeres. Although the choice is made separately for each individual chromosome, the analysis also reveals the existence of a system responsible for coordinated segregation of the two chromosomes of a given pair.

Cell Division↗

Enhancement of telomere-plasmid segregation by the X-telomere associated sequence in Saccharomyces cerevisiae involves SIR2, SIR3, SIR4 and ABF1.

We have previously shown that circular replicating plasmids that carry yeast telomere repeat sequence (TG1-3) tracts segregate efficiently relative to analogous plasmids lacking the TG1-3 tract and this efficient segregation is dependent upon RAP1. While a long TG1-3 tract is sufficient to improve plasmid segregation, the segregation efficiency of telomere plasmids (TEL-plasmids) is enhanced when the X-Telomere Associated Sequence (X-TAS) is also included on the plasmids. We now demonstrate that the enhancement of TEL-plasmid segregation by the X-TAS depends on SIR2, SIR3, SIR4 and ABF1 in trans and requires the Abf1p-binding site within the X-TAS. Mutation of the Abf1p-binding site within the X-TAS results in TEL-plasmids that are no longer affected by mutations in SIR2, SIR3 or SIR4, despite the fact that other Abf1p-binding sites are present on the plasmid. Mutation of the ARS consensus sequence within the X-TAS converts the X-TAS from an enhancer element to a negative element that interferes with TEL-plasmid segregation in a SIR-dependent manner. Thus, telomere associated sequences interact with TG1-3 tracts on the plasmid, suggesting that the TASs have an active role in modulating telomere function.

Base Sequence↗

Mitochondrial genotype segregation in a mouse heteroplasmic lineage produced by embryonic karyoplast transplantation.

Mitochondrial genotypes have been shown to segregate both rapidly and slowly when transmitted to consecutive generations in mammals. Our objective was to develop an animal model to analyze the patterns of mammalian mitochondrial DNA (mtDNA) segregation and transmission in an intraspecific heteroplasmic maternal lineage to investigate the mechanisms controlling these phenomena. Heteroplasmic progeny were obtained from reconstructed blastocysts derived by transplantation of pronuclearstage karyoplasts to enucleated zygotes with different mtDNA. Although the reconstructed zygotes contained on average 19% mtDNA of karyoplast origin, most progeny contained fewer mtDNA of karyoplast origin and produced exclusively homoplasmic first generation progeny. However, one founder heteroplasmic adult female had elevated tissue heteroplasmy levels, varying from 6% (lung) to 69% (heart), indicating that stringent replicative segregation had occurred during mitotic divisions. First generation progeny from the above female were all heteroplasmic, indicating that, despite a meiotic segregation, they were derived from heteroplasmic founder oocytes. Some second and third generation progeny contained exclusively New Zealand Black/BINJ mtDNA, suggesting but not confirming, an origin from an homoplasmic oocyte. Moreover, several third to fifth generation individuals maintained mtDNA from both mouse strains, indicating a slow or persistent segregation pattern characterized by diminished tissue and litter variability beyond second generation progeny. Therefore, although some initial lineages appear to segregate rapidly to homoplasmy, within two generations other lineages transmit stable amounts of both mtDNA molecules, supporting a mechanism where mitochondria of different origin may fuse, leading to persistent intraorganellar heteroplasmy.

Animals↗

Fixation, segregation and linkage of allozyme loci in inbred families of the Pacific oyster Crassostrea gigas (Thunberg): implications for the causes of inbreeding depression.

The effect that inbreeding has on the fixation and segregation of genes has rarely been confirmed by direct observation. Here, fixation, segregation, and linkage of allozymes is investigated in the progeny of self-fertilized hermaphrodites of the normally outcrossing Pacific oyster Crassostrea gigas. The estimate of fixation pooled over loci, individuals, and families, F = 0.462, is significantly lower than the expected value of 0.5. Log-likelihood ratios reveal significant heterogeneity in fixation among individuals, among families, and among loci. In addition, the grand pooled segregation ratio, 127:243:54, deviates significantly from 1:2:1, with a bias against homozygotes for alleles of lesser frequency in the natural population. Segregation ratios for 11 of 14 loci are significantly heterogeneous among families, and exact tests for segregation within families reveal 16 significant results out of 51 tests. Thus, fixation and segregation of allozyme markers in inbred oyster families deviates from the expectations of neutral inbreeding theory. Di-genic disequilibria are significant for four of 74 di-locus pairs revealing two linkage groups. Strong viability selection is apparently conditional on the genotype of the hermaphrodite-founders and is largely focused on these two linkage groups. These genetic effects are explained by interaction between cis-linked factors and polymorphic regulatory backgrounds.

Animals↗

Molecular mapping of segregation distortion loci in Aegilops tauschii.

Distorted segregation ratios of genetic markers are often observed in progeny of inter- and intraspecific hybrids and may result from competition among gametes or from abortion of the gamete or zygote. In this study, 194 markers mapped in an Aegilops tauschii F2 population were surveyed for distorted segregation ratios. Region(s) with skewed segregation ratios were detected on chromosomes 1D, 3D, 4D, and 7D. These distorter loci are designated as QSd.ksu-1D, QSd. ksu-3D, QSd.ksu-4D, and QSd.ksu-7D. Three regions of segregation distortion identified on chromosome 5D were analyzed in two sets of reciprocal backcross populations to analyze the effect of sex and cytoplasm on segregation distortion. Extreme distortion of marker segregation ratios was observed in populations in which the F1 was used as the male parent, and ratios were skewed in favor of TA1691 alleles. There was some evidence of differential transmission caused by nucleo-cytoplasmic interactions. Our results agree with other studies stating that loci affecting gametophyte competition in male gametes are located on 5DL. The distorter loci on 5DL are designated as QSd.ksu-5D.1, QSd.ksu-5D.2, and QSd.ksu-5D.3.

Chromosome Mapping↗