G. J. P. A. Anders

AGE-DEPENDENT VARIABILITY OF RIBOSOMAL RNA-GENE ACTIVITY IN MAN AS DETERMINED FROM FREQUENCIES OF SILVER STAINING NUCLEOLUS ORGANIZING REGIONS ON METAPHASE CHROMOSOMES OF LYMPHOCYTES AND FIBROBLASTS

Frequencies of silver staining nucleolus organizing regions (NORs) have been determined in lymphocytes and fibroblasts from very young and from aged persons. Since silver staining of NORs is associated with activity of ribosomal RNA-genes, we used this approach to investigate a possible inactivation of these genes during aging. Our lymphocyte data are based on a study per age-group of 220 metaphases from 10 subjects. Although in both age-groups modal numbers of silver staining chromosomes per metaphase had similar ranges over the subjects, the frequency of metaphases containing the maximal number of staining chromosomes was in the old age-group (80--89 years) significantly lower than in the young age-group (less than 1 year old). In fibroblasts, of which 75 metaphases from 4 subjects were included per age-group, differences were more pronounced. Modal numbers of silver staining chromosomes were for the aged persons (69--83 years) lower than for the young children (less than 1 year old). Highly significant differences were observed between both groups in frequency of metaphases containing the maximal number of positively reacting acrocentric chromosomes and, more in general, in frequencies of silver staining D- and G-group chromosomes, the lower frequencies being found in the old age-group. We propose the term NOR-junctions as distinct from satellite associations for arrangements of acrocentric chromosomes which after silver staining are visibly connected at their NORs. The number of acrocentric chromosomes involved in lymphocyte NOR-junctions of aged people was significantly higher than the number of joined acrocentrics in young children. The frequency of these NOR-junctions themselves, irrespective of the number of chromsomes involved, was higher for aged persons than for young children, although this difference appeared to be statistically not significantly higher than in fibroblasts. Also based on qualitative observations from our study we discuss tcehnical and biological problems of our approach to study cell aging in vivo by means of silver staining of NORs. We conclude that in man, reflected by the difference in frequencies of silver staining NORs between young and aged persons, a rather extensive loss of ribosomal RNA-gene activity may occur during aging.

Unilateral agenesis of the diaphragm

in th is jou rna l Passarge et al. (I968) descr ibed a b ro the r and sis ter wi th unil a te ra l agenesis of the d i aphragm, and col lected four comparab le repor ts of famil ia l occurrence f rom the l i te ra ture . They a d v a n c e d the hypothes i s t h a t th is defect is e t iological ly different f rom o ther congeni ta l lesions of the d i aphragm, and pos tu l a t ed a rare au tosomal recessive gene as the cause of the a bno rma l i t y . W e repor t here ano the r ease of a large, famil ia l occurring, congeni ta l d i a p h r a g m a t i c defect .

Paternal transmission of a B/D translocation, t(4p-; 14p+ or 15p+), resulting in a partial 4p trisomy

SummaryIn a mentally retarded boy without gross malformations, karyotype analysis showed a partial 4p trisomy. His phenotypical normal father is a carrier of the balanced translocation t(4p-; 14p+ or 15p+).ZusammenfassungBei einem Jungen, der in seiner geistigen Entwicklung zurückgeblieben war, jedoch keine schweren Mißbildungen aufwies, wurde eine partielle 4p-Trisomie gefunden. Der phänotypisch normale Vater ist Träger einer balancierten Translokation t(4p-; 14p+ oder 15p+).

A case of 18q- in a family with a translocation t(6p+;18q-), identified by the Giemsa-banding technique

SummaryIn a male infant with multiple congenital malformations a deletion of the long arm of chromosome No. 18 was identified by means of the A.S.G. technique. His mother and sister were balanced heterozygotes with the karyotypes 46,XX,t(6p+;18q-).ZusammenfassungBei einem männlichen Neugeborenen mit multiplen Mißbildungen surde mit der ASG-Technik eine Deletion des langen Armes eines Chromosomes 18 identifiziert. Mutter und Schwester des Patienten waren balancierte Heterozygoten mit dem Karyotyp 46,XX,t(6p+;18q-).

Rapid identification of chromosomes carrying silver-stained nucleolus-organizing regions

SummaryThe use of a combination of transmitted light and epiluminescence after silver and fluorescent staining of chromosome preparations makes it possible to achieve simultaneous visualization of silver-stained NORs and fluorescent chromosomes. This technique permits exact localization of silver precipitates on normal and BrdU-substituted chromosomes. After previous silver impregnation, fluorescent staining by actinomycin-daunomycin-DAPI was used to induce a banding pattern that enables identification of specific chromosomes while observing silver-stained NORs at the same time. Application of this method to Downs syndrome patient revealed a 21/21 Robertsonian translocation with NORs eliminated.

SHORT-TERM MICROCULTURES OF LYMPHOCYTES FROM CHINESE-HAMSTER PERIPHERAL-BLOOD

A microtechnique for the culture of Chinese Hamster lymphocytes is described using Cooke microtiter plates with 100,000 leucocytes in a culture volume of 0.1 ml and a culture time of three days. The culture media used were RPMI 1640 and Trowell T8 supplemented with 20% foetal calf serum (FCS) and 2 mu PHA. The cells were harvested with a Skatron cell culture harvester using glass fibre filters. Various technical aspects of the lymphocyte cultures from the Chinese Hamster are described. The relevance of changes in culture conditions to the variability of culture results was analysed for PHA and FCS concentrations, different culture media, cell concentration, vessel shape and culture duration.

A comparison of constitutive heterochromatin staining methods in two cases of familial heterochromatin deficiencies.

SummaryUsing DAPI staining after pretreatment with distamycin A we detected a familial deficiency of chromosome 16 heterochromatin. A distinct positively staining band, however, was seen after C-banding. Thus, by using these different heterochromatin staining methods, heterogeneity of the constitutive heterochromatin in the centromeric region of human chromosome 16 was indicated. The same C-banding procedure was also applied to a previously described familial deficiency of chromosome 9 heterochromatin evidenced using distamycin A/DAPI staining and G 11 staining (Buys et al., 1979). In this case a C-band appeared to be virtually absent on the relevant chromosome. These staining methods may be valuable tools in the study of chromosome polymorphisms.

The influence of the serum/PHA ratio, microplate well shape and 2-mercaptoethanol on the stimulation of different numbers of cells in lymphocyte cultures from the chinese hamster.

Conditions for microculture of Chinese hamster lymphocytes are described which allow measurement of thymidine uptake with 6000 to 1000 lymphocytes per culture. The relationship between degree of cell stimulation, PHA concentration culture surface and cell concentration is described, as well as the influence of addition of 2-mercaptoethanol to the cultures.

Chinese hamster lymphocyte cultures. Relationship between lymphocyte proliferation, cell concentration, culture time and culture area.

Different numbers of Chinese hamster lymphocytes were cultured in microtiter plates with flat-, round- and V-bottomed wells for different culture times. The smallest number of cells could be stimulated in plates with V-bottomed wells. At least 25,000-50,000 cells and a longer culture time than for round- or V-bottomed plates were required for maximal stimulation in flat-bottomed plates. For a given well conformation the optimal day of culture is earlier with higher and later with lower cell concentrations. The optimal culture time for a given number of cells is shortest in V- and longest in flat-bottomed plates. The amount of PHA producing the highest thymidine incorporation for a given number of cells depends upon the well conformation. It decreases with lower cell concentration and increases with longer culture time.

TIME AND CELL SYSTEMS AS VARIABLES IN FUSION EXPERIMENTS WITH POLYETHYLENE-GLYCOL

SummaryCell hybridization was done between a monolayer of B14-150 Chinese hamster cells and a suspension of either mouse leukemia cells or normal human lymphocytes. Cell contact was obtained by centrifugation of the suspension cells onto the monolayer cells in a culture plate. Cell fusion was done by means of polyethylene glycol (PEG). The optimum time for PEG exposure as well as the yield of hybrid cells differed markedly with the different combinations.