Simultaneous activity of nucleolus organizer regions of human and Chinese hamster chromosomes in somatic cell hybrids

In an interspecific human-Chinese hamster hybrid that retains 13 and 85.6% of the chromosomes of each parental complement, activity of nucleolus-organizing regions (NOR) of both type chromosomes is observed in 18.9% of the cells. Interspecific chromosomal associations are also noted. Unlike the parental lines of Chinese hamster cells, the hybrids show the associations of the NOR of Chinese hamster chromosomes. In hybrid cells, there occurs partial suppression of NOR activity in human and Chinese hamster chromosomes, while the NOR of the 3d chromosome of the Chinese hamster is completely suppressed.     

Genetic determination of NOR activity in human lymphocytes from twins

Summary Activity of nucleolar organizer regions (NORs) was studied in cultured blood lymphocytes from 20 monozygotic (MZ) and 20 dizygotic (DZ) twin pairs. The number of Agstained NORs, the degree of staining, and the frequency of acrocentric associations were used as criteria of the NOR activity, the acrocentric chromosomes being identified by G-banding. Analysis of intrapair concordance as well as of intrapair variance showed the number of Ag+NORs and the size of Ag-deposits to be highly heritable traits. Intrapair differences in acrocentric association frequency were not significantly higher in DZ compared with MZ twins.     

Cytogenetic investigations in families with ataxia-telangiectasia.

Chromosomal studies were performed on peripheral blood lymphocytes and cultured skin fibroblasts from five Israeli-Moroccan families with ataxia-telangiectasia. A total of 24 individuals, including seven propositi, was investigated. Among the probands, significantly elevated rates of chromosome damage were observed in both blood and skin. Skin fibroblasts of affected individuals showed several orders of magnitude more chromosome breakage than lymphocytes. Increased rates of chromosome damage were also observed in the fibroblasts of some phenotypically normal family members (obligate heterozygotes and sibs) when compared to normal controls. An apparent abnormal clone of cells, possessing a large acrocentric marker chromosome (14q+), was observed in varying proportions among cells of all the propositi (2-5% of lymphocytes; 1-9% of fibroblasts).     

Frequency of Ag-stained nucleolus organizer regions in the acrocentric chromosomes of man

Summary The Ag-stainability of the nucleolus organizer region (NOR) was studied in the acrocentric chromosomes identified by Q-banding of cultured lymphocytes in 51 karyotypically normal persons (31 males and 20 females). A consistent pattern of Ag-positive NORs was found in each individual. Ninety percent of the individuals have a modal number of 8–10 Ag-positive NORs per cell. The frequency of Ag-positive NORs is similar in all five acrocentrics. A statistically nonsignificant lower frequency is found in chromosome 22. Ag-negative NORs on both homologues were found in four cases. The observed frequency distribution of individuals with homozygous NOR-positive, heterozygous, and homozygous negative acrocentric chromosomes was in accordance with the Hardy-Weinberg law in all five pairs of the acrocentric chromosomes as well as in total. No sex difference was observed in our material.A.-V. Mikelsaar is visiting exchange scientist of the Österreichische Bundesministerium für Wissenschaft und Forschung     

Tissue-specific heterogeneity in DNA replication patterns of human X chromosomes

Regional DNA replication kinetics in human X chromosomes have been analysed using BrdU-33258 Hoechst-Giemsa techniques in five cell types from human females: amniotic fluid cells, fetal and adult skin fibroblasts, and fetal and adult peripheral lymphocytes. In all cell types, the late-replicating X chromosome can be distinguished from its active, earlyreplicating homologue, and both the early and late X exhibit temporally and regionally characteristic internal sequences of DNA replication. The replication pattern of the early X in amniotic fluid cells and skin fibroblasts is similar to that of the early X in lymphocytes, although certain discrete regions are later-replicating in these monolayer tissue culture cells than are the corresponding regions in lymphocytes. However, DNA replication kinetics in late X chromosomes from amniotic fluid cells and skin fibroblasts are strikingly different from those observed in lymphocytes with respect both to the initiation and termination of DNA synthesis. The predominant late X pattern observed in 80–95% of lymphocytes, in which replication terminates in the long arm in bands Xq21 and Xq23, was never seen in amniotic fluid cells or skin fibroblasts. Instead, in these cell types, bands Xq25 and Xq27 are the last to complete DNA synthesis, while bands Xq21 and Xq23 are earlier-replicating; this pattern is similar to the alternative replication sequence observed in 5–20% of lymphocyte late X chromosomes. This replication sequence heterogeneity is consistent with the existence of tissue-specific influences on the control of DNA replication in human X chromosomes.     

Evidence for a compensatory mechanism regulating Ag-NOR activity in families with de novo 21;21 translocation Down syndrome

Nucleolus organizing region (NOR) activity in seven probands with Down syndrome due to a de novo (21;21) translocation and their parents was analyzed on the basis of total Ag-NOR size per cell, mean Ag-NOR size per cell (Xc), mean Ag-NOR size per acrocentic (Xa), and the characteristic Ag-NOR number of each subject. The results showed intercellular variations in total Ag-NOR size per cell in all subjects, as well as interindividual variations in mean Ag-NOR size per cell. When the subjects were grouped according to their characteristic Ag-NOR number and the mean Ag-NOR size per cell for each group (GXc) and the mean Ag-NOR size per acrocentric for each group (GXa) were calculated, a number of interesting and significant correlations were found: (1) GXc correlated perfectly with the group's characteristic Ag-NOR number, (2) GXa varied inversely with the group's characteristic Ag-NOR number, and (3) GXc and GXa varied inversely with each other. These results suggest that if the Ag-NOR number of a cell decreases, the total NOR activity of the cell also decreases, but the NOR activity of its chromosomes increases. This finding supports the existence of a compensatory mechanism that regulates NOR activity on the cellular level.     

Immune responses to Herpesvirus sylvilagus infection in cottontail rabbits

Immunologic changes produced by Herpesvirus sylvilagus infection of cottontail rabbits were investigated to evaluate this virus infection system as an animal model for EBV infection in humans. H. sylvilagus neutralizing antibodies appeared as early as 7 days after infection, peaked 2 to 4 wk postinfection and decreased to low levels by 8 to 10 wk postinfection. Complement-dependent antibodies mediating the protection of in vitro infection of monocytes and Con A-stimulated lymphoblasts with H. sylvilagus were observed as were complement-dependent cytotoxic antibodies against H. sylvilagus-infected cells. No cytolytic activity was present in sera taken either before or 3 days after infection; cytolysis was first observed 7 days after infection. The development of cytolytic antibodies appeared to be biphasic during an infection course of 12 to 16 wk. In vivo induction of a primary cytotoxic lymphocyte response to H. sylvilagus was also investigated. Splenic lymphocytes from infected animals lysed H. sylvilagus-infected skin fibroblasts; however, similar activity was not observed when PBMC or mesenteric lymph node lymphocytes were used as effector cells. H. sylvilagus-infected autologous skin fibroblasts were preferentially lysed as compared to heterologous skin fibroblasts. This virus-specific cytotoxic activity appeared 5 days postinfection and peaked 7 days postinfection. By 28 days postinfection, only low levels of cytotoxic activity were detected in spleen cells. Herpesvirus sylvilagus infection of cottontail rabbits provides an animal model for the study of lymphoproliferative disorders induced by herpesviruses.     

Study of the activity of the nucleolus organizer segments in the chromosomes of normal, leukemic and neoplastic human cells by silver nitrate staining

The application of a modified Ag-1 method for staining nucleolar organizer regions (NOR) in chromosomes has shown that the total number of AgNO3-stained NOR varies from 6 to 8 in cells of normal individuals as well as in the phytohemagglutinin (PHA) stimulated blood cells of patients suffering from acute leukemia (AL) and chronic myelocytic leukemia (CML). No AgNO3-stained NOR were detected either in non-PHA stimulated blood cells of AL or CML patients, apart from one case of a 48 hours blood culture without PHA from blast crisis CML, where 100 per cent mitotic cells displayed 3-6 AgNO3-stained chromosomes. In a Hela cell line with the modal number equal to 50 and the average number od acrocentrics being 9.3 per cell, the AgNO3-stained NOR numbered constantly 5. In tumor cells from pleural fluid of metastatic ovary tumor patient, with the modal number of cells varying from 50 to 160, the total number of AgNO3-stained NOR increased from 13 to 26 per metaphase. A hypothesis is forward to explain the negative Nor staining property in leukemia, according to which the activities of ribosomal cistrons (rDNA) in the miotic cells of relatively mature granulocytic and erythrocytic cells are either very much reduced or totally arrested.     

Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.     

Genomic compatibility between two phyllotine rodent species evaluated through their hybrids

In order to investigate the genomic compatibility between allopatric rodent species, Phyllotis darwini and Phyllotis magister, we have studied several cytogenetic and reproductive features of their laboratory hybrids. Of thirty-one pairings between species, only five were successful, producing eleven newborns. Like parents, hybrids had 38 metacentric chromosomes, except for the subtelocentric Y chromosome inherited from P. magister. There was almost total C and G band correspondence between homeologous autosomes. However, parental sex chromosomes had different morphology, C and G bands. Ag-NOR bands appeared as small telomeric Ag+ regions, distributed in four chromosomal pairs of darwini, three of magister and four homeologous chromosomes of the hybrids. The three forms had similar indexes of NOR activity per cell, in spite of the variability in NOR expression which was always detected. Usually, only one member of parental homologous chromosomes showed AgNOR+; nevertheless, both homeologous chromosomes were active in many hybrid cells. The frequencies of cells that expressed their ribosomal genes in the two homologous or homeologous NOR chromosomes were similar in parental and hybrid cells. These results strongly suggest that ribosomal genes of both parental genomes would function codominantly in the hybrids. The gonad histological and morphometric analyses showed that hybrids conformed to Haldane's rule, since females were fertile and males were infertile. Our results indicate that P. darwini and P. magister genomes can function in relative harmony and compatibility when they are placed together in their laboratory generated hybrids, suggesting that these species have few genetic differences, probably because they have recently diverged.     

SCE variability in lymphocytes and fibroblasts

Summary To determine whether the sister chromatid exchange (SCE) distributions obtained in lymphocytes and fibroblasts from different individuals are comparable, a controlled study was set up. Peripheral blood and skin biopsies were taken on the same day from five individuals living for years under the same environmental conditions. All samples were treated in the same fashion, and the SCEs were scored in 50 metaphases of peripheral blood lymphocytes and of skin fibroblasts in an early and in a late passage. A repeat blood sample was taken from the same five indivuduals 1 year later. Based on the results obtained in this first part of the study, five randomly chosen healthy blood donors were sampled at different times and studied in the same fashion. Each chromosome was identified, and the SCE scores were tabulated per chromosome over 50 metaphases. The statistical analysis consisted of fitting log linear models to these scores and examining the best fit by determining the exceedance probabilities (observed significance level). For lymphocytes, the results indicated that the SCE distributions depended only on the chromosome examined, and not on BrdU-exposure time, individuals, or time of sampling. Treatment with ethyl methane sulfonate (EMS) increased the number of SCEs proportionally on all chromosomes. Analysis of the SCE scores on lymphocytes and fibroblasts of the five individuals and on their low and high passage fibroblast cultures revealed the necessity of including higher order interactions in order to fit a suitable model to the data. Therefore comparison of the SCE scores of lymphocytes with those of fibroblasts or comparison of scores on fibroblasts from different individuals could not be done. In practice, to compare samples or individuals, it suffices to score the SCE on a limited number of chromosomes (e.G., the A group) of 50 metaphases.     

Increased SCE inducibility by low doses of methylcholanthrene in lymphocytes obtained from patients with Down's disease

The inducibility of sister-chromatid exchanges (SCEs) following 3-methylcholanthrene (MC) treatment of normal human peripheral blood lymphocytes and of in vitro cultured normal human embryo fibroblasts, as well as peripheral blood lymphocytes of patients with Down's disease and of fibroblasts of an embryo with trisomy 21 has been investigated. 10(-6) M MC treatment increased the frequency of SCEs by 2.5 in the case of Down lymphocytes when compared to the healthy control. The fibroblasts with trisomy 21, however, did not show an increased sensitivity to MC treatment when compared with normal fibroblasts, expressed in the number of SCEs per nucleus found in the cells.     

An immunochemical method for the detection of the expression of human gene loci in human-rodent somatic cell hybrids with special reference to the GPI locus

Species-specific antibodies, prepared against unpurified human and Chinese hamster fibroblast extracts, were used to identify the parental origins of enzymes in human-hamster somatic cell hybrids. Results of the detection of the expression of the human glucosephosphate isomerase gene locus (GPI) by electrophoretic and immunochemical techniques were concordant in 17 instances. The human GPI synthesized by fibroblasts derived from skin explants and by somatic cell hybrids retaining the human GPI locus, regardless of whether the human parental cells were lymphocytes or fibroblasts, appeared to be antigenically identical.This work was supported by the Medical Research Council of Canada (Grant MA-4061). Personnel and operating support were provided by The Children's Hospital of Winnipeg Research Foundation, Inc.     

Adhesion and transcellular migration of neutrophils and B lymphocytes on fibroblasts

During tissue inflammation, infiltrated leukocytes may have physical contacts with fibroblasts. We observed that neutrophils and B lymphocytes adhered in a larger proportion than T cells on cultured fibroblasts. Microscopy showed that adhesion was also characterized by leukocyte engulfment by the fibroblasts. In migration assays, only neutrophils and B lymphocytes were selectively able to migrate through a fibroblast barrier. Adhesion and migration were increased by stimulation with tumor necrosis factor-α (TNF-α) and phorbol-12-myristate-13-acetate (PMA). Antibodies against ICAM-1/β2 integrin blocked the interaction of neutrophils to fibroblasts. For B lymphocytes the couple VCAM-1/α4 integrin was also involved in this interaction. Human skin fibroblasts presented similar adhesion characteristics as rat cardiac fibroblasts. By measuring the distance between the border of migration holes and cadherin-positive adherens junctions, more than 65% of the holes correspond to the transcellular route over the paracellular route. Furthermore, vimentin staining revealed that the migration holes were highly nested by intermediate filaments in accordance with the transcellular route. Our results demonstrated that engulfment of neutrophils and B lymphocytes by fibroblasts resulted in selective passage by a transcellular route.     

Comparative analysis of chromosomal nucleolar organizing region activity in spontaneous and medical abortuses]

Comparative analysis of functional activity of silver stained nucleolar-organizing regions (NOR) activity was carried out in human fibroblasts of 70 spontaneous and 50 medical abortuses. The NOR activity was significantly higher in spontaneous abortuses compared with medical ones. This phenomenon which was observed in female, but not in male abortuses, did not depend upon the tissue origin of fibroblasts (embryonic or extraembryonic) and was due to increase of the NOR activity of the all acrocentric chromosomes to the level at which individual differences in the NOR activities among some acrocentrics were smoothed. It has been suggested that the ribosomal genes activity in different sex is likely to be differently involved in polygenic systems determining vitally important features of organism that may lead to different selection intensity in different sex resulted in deviation of the ribosomal gene copy number from the optimum. The possible role of changes in DNA methylation at the genome level in activation of ribosomal genes in spontaneous abortuses is discussed.     

Regulation of collagen gene expression in the Tsk2 mouse

The tight skin 2 (Tsk2) mutation is an ENU induced dominant mutation localized on mouse chromosome 1. While the molecular defect is unknown, Tsk2/+ mice display cutaneous thickening associated with excessive matrix production and are used as a model of scleroderma. The purpose of this study was to examine the cellular mechanisms associated with the excessive synthesis of matrix macromolecules using a collagen promoter GFP reporter transgene (pOBCol3.6GFP) as a marker of Col1a1 expression. This analysis of pOBCol3.6GFP expression in Tsk2/+ skin showed an increase in transgene activity compared to wild-type (+/+) samples. In addition, an increased area of "high" GFP fluorescence in Tsk2/+ dermis in both 1- and 4-month-old mice was observed that was also associated with an increased number of dermal fibroblasts per unit area of dermis. These data collectively suggest an important mechanism of Tsk2/+ skin fibrosis; an increased number of collagen expressing cells as well as elevated collagen expression on a per cell basis. During this study it was noted that Tsk2/+ mice appeared consistently smaller than wild-type (+/+) siblings and measurements of body length revealed a decrease (5-10%) in 1- and 2-month-old Tsk2/+ mice as well as a decrease in body weight in both age groups as compared to wild-type (+/+) control mice. Femur length was also decreased (2-9%) in Tsk2/+ mice. Finally, in contrast to Tsk/+ mice that display an emphysema-like lung pathology, histological sections of lungs from Tsk2/+ mice were normal and indistinguishable from wild-type (+/+) controls.     

Polymorphisms of Ag-stained nucleolar organizer regions in man

Summary Polymorphisms of the NORs as tested by Ag-staining of metaphase G-banded chromosomes were investigated in cultured blood lymphocytes of karyotypically normal individuals from the Moscow population.The study of cell-to-cell variability in the number of Ag-stained NORs carried out on 14 monozygotic twin pairs showed the phenomenon to have some features of real intercellular variation.In 40 unrelated individuals the individual acrocentric chromosomes were compared by the number of Ag-stained NORs, their degree of staining, and their participation in acrocentric association. Chromosome 21 was found to be significantly more active than four others by all the criteria, and chromosome 15 was less active compared with the others by the size of the Ag deposits and the frequency of participation in NOR associations. The frequency distribution of homozygotes and heterozygotes for Ag-stained NORs in the same group of 40 individuals was in accordance with the Hardy-Weinberg law.     

Telomerase expression restores dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model

The lifespan of human fibroblasts and other primary cell strains can be extended by expression of the telomerase catalytic subunit (hTERT). Since replicative senescence is accompanied by substantial alterations in gene expression, we evaluated characteristics of in vitro-aged dermal fibroblast populations before and after immortalization with telomerase. The biological behavior of these populations was assessed by incorporation into reconstituted human skin. Reminiscent of skin in the elderly, we observed increased fragility and subepidermal blistering with increased passage number of dermal fibroblasts, but the expression of telomerase in late passage populations restored the normal nonblistering phenotype. DNA microarray analysis showed that senescent fibroblasts express reduced levels of collagen I and III, as well as increased levels of a series of markers associated with the destruction of dermal matrix and inflammatory processes, and that the expression of telomerase results in mRNA expression patterns that are substantially similar to early passage cells. Thus, telomerase activity not only confers replicative immortality to skin fibroblasts, but can also prevent or reverse the loss of biological function seen in senescent cell populations.