Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains. The 19 control strains included 10 strains trisomic for autosomes other than 2, and 9 euploid strains. Our estimate of the mean AchE activity in the control strains did not differ significantly from zero and might, in any case, have originated from a minute amount of activity contributed to the cells by an esterase in our culture medium. Despite the striking elevation of AchE activity in fibroblasts trisomic for chromosome 2, extracts of these cells had only about 1.5% of the specific AchE activity (per microgram DNA) present in extracts of human cerebral cortex. None of the 22 strains studied had detectable activity for two other enzymes which, like AchE, have a restricted distribution among human tissues: xanthine oxidase and choline acetyltransferase.     

Dosage effects for superoxide dismutase-1 in nucleated cells aneuploid for chromosome 21.

Assays of the activity of chromosome 21 determined superoxide dismutase-1 (SOD-1) in lymphocytes and polymorphonuclear granulocytes have demonstrated 38% and 40% increases, respectively, in cells from individuals with trisomy 21. Similarly, SOD-1 activity in trisomic fibroblasts is increased by 81%, while cells monosomic for chromosome 21 have only 60% of normal activity. Taken together with the data on SOD-1 activities in trisomic erythrocytes and platelets, the present results firmly confirm the existence of a true dosage effect for this enzyme in cells aneuploid for chromosome 21. However, the results of assays of the activity of glutathione peroxidase in trisomic fibroblasts did not confirm the possibility previously reported of a chromosome 21 related dosage effect for this enzyme.     

A neural crest deficit in Down syndrome mice is associated with deficient mitotic response to Sonic hedgehog

Trisomy 21 results in phenotypes collectively referred to as Down syndrome (DS) including characteristic facial dysmorphology. Ts65Dn mice are trisomic for orthologs of about half of the genes found on human chromosome 21 and exhibit DS-like craniofacial abnormalities, including a small dysmorphic mandible. Quantitative analysis of neural crest (NC) progenitors of the mandible revealed a paucity of NC and a smaller first pharyngeal arch (PA1) in Ts65Dn as compared to euploid embryos. Similar effects in PA2 suggest that trisomy causes a neurocristopathy in Ts65Dn mice (and by extension, DS). Further analyses demonstrated deficits in delamination, migration, and mitosis of trisomic NC. Addition of Sonic hedgehog (Shh) growth factor to trisomic cells from PA1 increased cell number to the same level as untreated control cells. Combined with previous demonstrations of a deficit in mitogenic response to Shh by trisomic cerebellar granule cell precursors, these results implicate common cellular and molecular bases of multiple DS phenotypes.     

Effect of a partial trisomy in the mouse upon cellular and nuclear RNA

In a previous paper (Rake and Edwards, 1987) it was shown that the majority of the chromatin from trisomic mouse cells has nucleosomes with a smaller repeat length of DNA than the nucleosome repeat length of normal cells. Here it is shown that the RNA content of the total cell and of the nuclei is the same in all tissues studied, in both normal and trisomic cells. However, the amount per unit time or rate of RNA synthesis is depressed in the trisomic liver and brain nuclei. The depression of RNA synthesis could not be specified to the small trisomic section of the chromatin but instead must reflect the overall nuclear activity. These results, along with those of Devlinet al. (1988), indicate that the trisomic condition alters a substantial part of nuclear organization and activity, not just the small trisomic part.     

A complex study of strains of human cells with karyotype anomalies. III. An immunoelectrophoretic analysis of water-soluble antigens]

Antisera to diploid, trisomic and triploid embryonic fibroblast-like cells were obtained after hyperimmunization of rabbits. Immunoelectrophoretic analysis with these antisera revealed up to 9 water-soluble antigens in embryonic cells, which were present in skin fibroblasts from adult donors as well. Trisomic and triploid strains did not differ from the diploid ones by the spectrum of water-soluble antigens. The content of the number of antigens (especially of cathode fractions) in trisomic cells was significantly low as compared with those in control diploid cells, whereas in triploid ones it differed slightly. All the strains were characterized by the presence of proteins immunologically identical to alpha-globulin of human serum.     

Meiosis in trisomic female mice with Robertsonian translocations. II. Chromosome behaviour at first and second meiotic metaphases

First and second meiotic metaphases (MI and MII, respectively) from female mice of Robertsonian translocation (Rb) stock, trisomic for chromosome 16 (Ts16) or 19 (Ts19), were studied. The mature trisomic oocytes were derived from explanted fetal ovaries that had been cultured and then transplanted so as to mature heterotopically. Multivalent configurations involving the Rb chromosomes and the additional trisomic acrocentric were analysed. Pentavalent configurations occurred in 74.5% of 98 Ts16 MI and 44.2% of 249 Ts19 MI oocytes; quadrivalents (with a univalent acrocentric) were found in 9.2% of Ts16 MI and 10.8% of Ts19 MI oocytes. In 1% of Ts16 MI and 4% of Ts19 MI oocytes, there were two Rb bivalents and a univalent trisomic acrocentric. Rb trivalents and Rb bivalents occurred together in 14.3% of Ts16 MI and 39.4% of Ts19 MI oocytes. Chiasma frequencies were similar in trisomic and chromosomally balanced MI. Chiasma position, distribution, and localization were nearly identical, whether they were found in Rb multivalents or acrocentric bivalents, but one control group (from chromosomally balanced Ts19 littermates) had significantly more terminal chiasmata. Within the triple homologous region of 8% of Rb pentavalents, two chiasmata were observed in the same relative position in the two sister chromatids of one of the three homologs, suggesting a lapse in chiasma position interference. Assortment at MI anaphase was influenced by secondary nondisjunction of the Rb. The ratio of balanced to unbalanced MII oocytes was 1:4 in both trisomies.     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.     

Trisomics from triploid-diploid crosses in self-incompatible Lycopersicum peruvianum

Summary The transmission rate of trisomy was determined for two primary trisomic types, triplo-1 and triplo-3, of the self-incompatible species Lycopersicum peruvianum. Chromosome counts in somatic metaphases of root-tip squashes from 112 progeny plants showed that 8 individuals (7.2 %) were trisomic and 104 (92.8%) were diploid. The average frequency of transmission approximated 2.6% in triplo-1 and 8.6% in triplo-3. Data are presented on the karyotype and the morphological features of the 8 trisomics detected in the progenies of triplo-1 and triplo-3 and the various factors affecting the transmission rate of trisomy are discussed.The transmission rate of trisomy was also determined for the trisomic plant 269 which displayed a complete deletion of the satellited part of chromosome 2 and was characterized by ovate fruits. Out of 18 progeny plants analysed, 8 (44.4%) were trisomic and 10 (55.6%) were diploid. Cytological and morphological analyses of the 8 trisomic individuals revealed that only two of them (11.1 %) resembled the parental trisomic. A number of diploid and trisomic progenies exhibited a partial or a complete deletion of the satellited segment of chromosome 2.This work has been supported by a contract between the European Communities and the CNEN. This publication is contribution n ° 484 from The Division Applicazioni delle Radiazioni del CNEN and contribution n ° 1482 from the Biology Radioprotection Medical Research programme of the Directorate General XII of the European communities     

Uniparental disomy for chromosome 16 in humans.

The association between chromosomal mosaicism observed on chorionic villus sampling (CVS) and poor pregnancy outcome has been well documented. CVS mosaicism usually represents abnormal cell lines confined to the placenta and often involves chromosomal trisomy. Such confined placental mosaicism (CPM) may occur when there is complete dichotomy between a trisomic karyotype in the placenta and a normal diploid fetus or when both diploid and trisomic components are present within the placenta. Gestations involving pure or significant trisomy in placental lineages associated with a diploid fetal karyotype probably result from a trisomic zygote which has lost one copy of the trisomic chromosome in the embryonic progenitor cells during cleavage. Uniparental disomy would be expected to occur in one-third of such cases. Trisomy of chromosome 7, 9, 15, or 16 is most common among the gestations with these dichotomic CPMs. Nine pregnancies with trisomy 16 confined to the placenta were prenatally diagnosed. Pregnancy outcome, levels of trisomic cells in term placentas, and fetal uniparental disomy were studied. Intrauterine growth retardation (IUGR), low birthweight, or fetal death was observed in six of these pregnancies and correlated with high levels of trisomic cells in the term placentas. Four of the five cases of IUGR or fetal death showed fetal uniparental disomy for chromosome 16. One of the infants with maternal uniparental disomy 16 had a significant malformation (imperforate anus). All infants with normal intrauterine growth showed term placentas with low levels of trisomic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural studies of the nucleoli in diploid and trisomic chickens

Chickens having a trisomic complement for the nucleolar organizer region (NOR) serve as a model for investigations on gene expression, molecular regulation, and physiological alterations resulting from extra genetic material. Of fundamental importance is the question of whether the extra set of ribosomal cistrons in trisomic chickens generates a morphologically and biochemically complete nucleolus. Ultrastructural and cytochemical studies of diploid and trisomic embryonic and adult cells were undertaken. All structures identified in trisomic cells as nucleoli by gross morphology were shown to be RNA rich by acridine orange fluorescence. At the ultrastructural level, the expected granular and fibrillar components were identified in all nucleoli of trisomic cells. Fibrillar centers, indicative of NOR's, were seen in all cases. These and other results from silver staining suggest full gene activity for each of the three chromosomes in the trisomic cell at all stages of development.     

Involvement of glutathione and glutathione-related enzymes in the protection of normal and trisomic human fibroblasts against daunorubicin

We measured the glutathione content, and the activity of glutathione-related enzymes and DT-diaphorase in cultured normal (cell line: S-126) and trisomic (cell lines: S-158, S-240) human fibroblasts exposed to daunorubicin (DNR). Determination of reduced and total glutathione levels, and measurement of the activity of glutathione peroxidase, glutathione reductase, glutathione-S-transferase and DT-diaphorase were performed spectrophotometrically. Human fibroblasts were exposed to 4 microm DNR for 2 h, and the cells placed in drug-free medium for 6, 12, 24, 48, and 72 h. Cellular levels of GSH and total glutathione decreased following exposure to DNR. However, the ratio of GSH to total glutathione returned to control levels only in trisomic cells. These changes were concomitant with increasing glutathione-S-transferase and glutathione reductase activities. DNR also significantly increased the activity of Se-independent peroxidase and DT-diaphorase in trisomic fibroblasts. Marked increases in the activity of Se-dependent peroxidase and DT-diaphorase alone were seen in normal cells. The results provide the first evidence that DNR can induce alterations in the level of glutathione and glutathione-dependent enzymes in trisomic fibroblasts as compared to normal cells, which may provide additional protection against daunorubicin-induced oxidative stress in trisomic fibroblasts.     

Characteristics of intracellular metabolism of procollagen and other proteins in human embryo fibroblasts in trisomy for chromosome 7

A comparative study of the relative rates of intracellular total protein metabolism in diploid and aneuploid (with trisomy for chromosome 7) human embryo fibroblasts in the logarithmic and stationary growth phases was carried out. Using double labeling with [14C]proline (24 hrs) and [3H]proline (3 hrs), it was found that: the rates of intracellular protein metabolism during transition to the stationary phase of growth are increased in diploid cells and decreased in cells with trisomy for chromosome 7; the relative rate of protein metabolism in the logarithmic phase is higher in trisomic cells than in diploid ones. The intracellular degradation of procollagen in trisomic cells is increased approximately by 17% as compared to normal fibroblasts. Treatment of cell lysates with bacterial collagenase revealed the presence of procollagen incomplete degradation products in anomalous fibroblasts. The observed differences in the rates and mode of protein metabolism during transition of diploid and trisomic fibroblasts to the stationary phase of growth suggest that the odd autosome interferes with the normal coordinated activity of genes in chromosomes.     

Variation in S phase in synchronous human cell lines

Summary Growth parameters of diploid and trisomic human fibroblasts were determined. The rate of growth of both classes of cells was examined in asynchronous cultures, and diploid and trisomic cells had similar growth rates. Synchronous cultures were developed using simple mitotic selection. The patterns and length of the DNA synthetic period (S phase) were found to be altered in trisomy 21 cells when compared to diploid human or to heteroploid HeLa cells. Early S-phase synthesis was absent or reduced and the overall length of the S phase was extended. However, the trisomic cells have apparently normal rates of DNA chain elongation and normal replicon sizes. This work was supported by the United States Department of Energy and the National Institutes of Health.     

Variation in S phase in synchronous human cell lines.

Growth parameters of diploid and trisomic human fibroblasts were determined. The rate of growth of both classes of cells was examined in asynchronous cultures, and diploid and trisomic cells had similar growth rates. Synchronous cultures were developed using simple mitotic selection. The patterns and length of the DNA synthetic period (S phase) were found to be altered in trisomy 21 cells when compared to diploid human or to heteroploid HeLa cells. Early S-phase synthesis was absent or reduced and the overall length of the S phase was extended. However, the trisomic cells have apparently normal rates of DNA chain elongation and normal replicon sizes.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 1182 mitoses of 21 patients with trisomy 13 and in a control group. In addition, 173 trisomic mitoses were compared with the same number of diploid mitoses in a case of mosaicism.The number of mitoses with associations was no higher in the trisomic cells than in cells with normal karyotypes. Some differences were observed in the frequency of associations per cell and of the types of associations in the patient group and in the trisomic cells of the mosaic case. The number of associations in which more than two acrocentric chromosomes were involved was unexpectedly low in the cells with a supernumerary chromosome 13.The result are interpreted as suggesting the existence of a compensatory mechanism activated by the additional acrocentric chromosome.Parts of this work are included in the doctoral (MD) thesis of DM     

Altered chromatin structure associated with a partial trisomy of chromosome 7 in the mouse

The chromatin of a mouse that is trisomic for part of chromosome 7 was investigated. Chromatin from trisomic tissue has a smaller average nucleosome DNA repeat length than chromatin from tissue taken from normal diploid littermates. DNA of the nucleosome cores is the same size in both normal and trisomic tissues. Not all of the nucleosome monomers have different repeat lengths. Normal and trisomic mouse kidney cells in tissue culture maintained their nucleosome repeat-length differences.     

Cytogenetics of an unstable trisomie in barley (Hordeum vulgare).

The origin, identification, meiotic chromosome behavior, and breeding behavior of an unstable trisomic barley were studied. The extra chromosome originated by breakage and fusion of an acrocentric chromosome 3 in a plant from an F2 population of a cross between acrotrisomic 3L3S (2n = 14 + 1 acro3L3S) and a balanced lethal stock, xc. (xantha) ac (albino). The F2 population segregated only for the albino trait. The genotypic constitution of the trisomic plant was ac ac (for both normal chromosome 3) and Ac (for the unstable metacentric chromosome). The unstable extra metacentric chromosome was designated as metacentric 3B (abbreviated as meta3B). Meiotic chromosome behavior in plants with 2n = 14 + 1 meta3B differed from plant to plant and within spikes. Some plants showed only trisomic cells with a chromosome configuration of 1 III + 6 II and 7 II + 1 I at metaphase I, whereas other plants showed both trisomie and disomic cells (7 II) that resulted from the elimination of the extra meta3B. The frequency of ring trivalents was low (6.8%). An average transmission rate of unstable meta3B ranged from 4.3 to 12.9%. The elimination of meta3B, and hence loss of the dominant Ac allele, resulted in albino seedlings as well as white stripes on plants, leaves, and spikes. Chromosome numbers of albino seedlings in the progeny of 2n = 14 + 1 meta3B were all diploid (2n = 14), while green seedlings contained 2n = 14 + 1 meta3B. However, progenies of some spikes of one trisomic plant showed a low frequency of green diploids and metatrisomics (2n = 14 + 1 meta3B), which was attributed to crossing-over.     

Meiotic disjunction and embryonic lethality in trisomies derived from an X-linked translocation in the onion fly,Hylemya antiqua (Meigen)

Translocation- and tertiary trisomies (for the X-chromosomes) were obtained after testcrossing translocation heterozygous females of an X-linked “simple” translocation stock. Meiotic disjunction as judged from segregations at M II (males) and in young eggs of testcrosses (males and females) in translocation trisomics was studied. No progeny of tertiary trisomic males and females was found, but male M II could be studied. Six different orientation types appeared in translocation trisomie (2n + 1) males and these were present in equal frequencies. No adjacent II configurations were found. The small X- and Y-chromosomes and the large translocated X-chromosome of the translocation complex disjoin at random (n and n + 1 gametes) in both translocation- and tertiary trisomic males. In translocation trisomic females four different orientation types appeared. From the high frequency of two of these (together, 94.5%) it is concluded that the two normal X-chromosomes show preferential pairing and disjunction, while the translocated X-chromosome moves to either one of the two poles at random. Primary trisomic (for the X-chromosome) males (XXY) and females (XXX) were obtained from testerossed translocation trisomics. Cytological analysis of adult male progeny of testerossed XXY males showed that no random orientation for the X-, X- and Y-chromosomes occurred because half of the sons was disomic (XY) and half of them trisomic (XXY). A possible mechanism is discussed. Analysis of young eggs of testerossed XXX females indicated a segregation of 2X∶1X=1∶1. The level of “semi”-sterility as scored from testcrosses of translocation trisomies appeared to be as in translocation heterozygotes. Here again a close relation exists between “semi”-sterility and deficiencies in eggs for a large chromosomal segment. The possible use of this translocation for genetic control of insect pests is discussed.     

Skewed X-chromosome inactivation is associated with trisomy in women ascertained on the basis of recurrent spontaneous abortion or chromosomally abnormal pregnancies

An increase in extremely skewed X-chromosome inactivation (XCI) (> or = 90%) among women who experienced recurrent spontaneous abortion (RSA) has been previously reported. To further delineate the etiology of this association, we have evaluated XCI status in 207 women who experience RSA. A significant excess of trisomic losses was observed among the women who had RSA with skewed XCI versus those without skewed XCI (P=.02). There was also a significant excess of boys among live births in this group (P=.04), which is contrary to expectations if the cause of skewed XCI was only that these women carried X-linked lethal mutations. To confirm the association between skewed XCI and the risk of trisomy, an independent group of 53 women, ascertained on the basis of a prenatal diagnosis of trisomy mosaicism, were investigated. Only cases for which the trisomy was shown to be of maternal meiotic origin were included. The results show a significantly higher level of extreme skewing (> or = 90%) in women whose pregnancies involved placental trisomy mosaicism (17%) than in either of two separate control populations (n=102 and 99) (P=.02 compared with total control subjects). An additional 11 cases were ascertained on the basis of one or more trisomic-pregnancy losses. When all women in the present study with a trisomic pregnancy (n=103) were considered together, skewed XCI was identified in 18%, as compared with 7% in all controls (n=201) (P=.005). This difference was more pronounced when a cutoff of extreme skewing of 95% was used (10% vs. 1.5% skewed; P=.002). Maternal age was not associated with skewing in either the patient or control populations and therefore cannot account for the association with trisomy. Previous studies have shown that a reduced ovarian reserve is associated with increased risk of trisomic pregnancies. We hypothesize that the association between skewed XCI and trisomic pregnancies is produced by a common mechanism that underlies both and that involves a reduction of the size of the follicular pool.