Epitope mapping by photobleaching fluorescence resonance energy transfer measurements using a laser scanning microscope system.

The donor photobleaching method (T. M. Jovin and D. J. Arndt-Jovin. 1989. Annu. Rev. Biophys. Biophys. Chem. 18:271-308.) has been adapted to an ACAS 570 (laser scanning microscope) system to measure fluorescence resonance energy transfer (FRET) on individual human peripheral blood T cells. Photobleaching was completed in approximately 100 ms in our case and it followed double-exponential kinetics. The energy transfer efficiency (E) was approximately 20% between the CD4 epitopes OKT4-FITC and Leu-3a-PE as well as between OKT4E-FITC and OKT4-PE. E was approximately 8% between OKT4-FITC and Leu-4-PE (alpha CD3) and barely detectable (approximately 4%) from OKT4-FITC to Leu-5b-PE (alpha CD2). The E values obtained by the photobleaching method were highly reproducible both in repeated measurement of identical samples and in experiments with different batches of cells and were in agreement with the flow cytometric donor quenching measurements. As expected, E measured between primary and secondary layers of antibodies increased (from approximately 14% to approximately 28%) when F(ab')2 fragments were substituted for whole antibody molecules as the donor. On a T cell line we mapped the distance between the idiotypic determinant of the T cell receptor (TcR) and the Leu-4 epitope of CD3 as proximal as E = 28%, as compared to E = 4% between a framework TcR epitope and Leu-4. In the latter case, however, approximately 40% less Leu-4 was bound suggesting that the antigen binding site of TcR is in close proximity with one of the two CD3 epsilon chains, which hence are not equivalent.     

Spatial relations of human chromosomes identified by quinacrine fluorescence at metaphase

Summary Data on centromere locations from 168 fully identified normal human complements were subjected to special analyses of variance by computer. Aggregation specifically attributable to homologue associations seemed definitely to be absent. Chromosomes 1, 9 and 16, which contain large heterochromatic blocks were distributed as typical non-acrocentrics. X chromosomes in female cells behaved much like other chromosomes of their size, but may have an atypically large homologue distance. Acrocentrics aggregate as a group without specific homologue associations; smaller chromosomes in the group are often nearer the center of the metaphase plate. The data do not suggest stronger acrocentric association in females than males.

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Zusammenfassung Daten über die Zentromerlokalisation von 168 vollständig identifizierten normalen menschlichen Metaphaseplatten wurden einer speziellen Varianzanalyse unterworfen. Irgendeine Aggregation, die etwa auf eine Assoziation homologer Chromosomen hätte zurückgeführt werden können, fehlte eindeutig. Die Chromosomen 1, 9 und 16, die große heterosomen bei Frauen verhielten sich wie andere Chromosomen der gleichen Größenklasse; die Homologen sind aber etwas weiter voneinander entfernt. Akrozentrische liegen als Gruppe zusammen ohne spezifische Assoziation zwischen Homologen. Kleinere Chromosomen der Gruppe finden sich oft näher dem Zentrum der Metaphaseplatte. Es finden sich keine Hinweise für eine stärkere Assoziation der Akrozentrischen bei Frauen im Vergleich zu Männern.

     

Distribution of human chromosomes on the metaphase plate using banding techniques

Summary This study deals with the problem of distribution of the 46 centromeres on the human metaphase plate after treatment with colchicine and hypotonic shock.The location of the centromeres was recorded for 400 metaphases in which the chromosomes were identified by bands.A quantitative analysis of chromosome distribution ascertains the absence of proximities between homologous chromosomes. The already known ones between acrocentric chromosomes are found at a very high level. The nature and intensity of these proximities vary from one sample to another as is the case between heterologues.A three-dimensional graphic technique is proposed to summarize all of the interchromosome proximities.     

Banding and spiralization of human metaphase chromosomes

Summary A new technique is described which produces spiralization of human metaphase chromosomes. The important feature is heat followed by trypsin treatment. By varying conditions, it is possible to produce bands, spirals and intermediate stages. This provides a new approach to the understanding of banding and chromosome structure.     

Comparison of areas of quinacrine mustard fluorescence and modified Giemsa staining in human metaphase chromosomes

The methods of quinacrine mustard fluorescence and modified Giemsa staining were compared in view of the structural details revealed in human mitotic chromosomes derived from the peripheral blood of normal healthy humans. Over the chromatids both techniques produced a crossbanding pattern where larger segments of heavy staining in the latter technique and the fluorescing bands in the former occurred at similar locations. The centromeric heterochromatin, intensely stained with Giemsa was, however, negative in fluorescence, except for chromosome no. 3 and less often no. 6. The regularly occurring secondary constrictions in chromosomes 1, 9, and 16 behaved generally like areas of centromeric heterochromatin. The area of secondary constriction in the Y chromosome as also that of chromosome 9 in the ASG modification of the Giemsa technique was both non-fluorescent and non-staining.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The association pattern was studied in 2715 mitoses of 90 meningiomas with different numbers of acrocentric chromosomes. In cells with monosomy 22, a significant increase of mitoses with associations was observed in comparison to cells with a normal karyotype. The number of associating acrocentric chromosomes was highly significantly increased. This surplus was not only caused by a highly significant increase of associating G chromosomes but also of D chromosomes. The loss of further acrocentric chromosomes had no significant influence on the number of mitoses with associations or the number of associating chromosomes. Based on the well-known correlations between the nucleolus organization and the association pattern, the results seem to indicate a compensation mechanism among the nucleoles organizing regions (NOR's) which keeps the supply of nucleolar material constant and simultaneously causes a higher association tendency between the remaining acrocentric chromosomes. The increase of associations in the 22 monosomic cells was interpreted as a overcompensation after the loss of only one NOR.     

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     

Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.

The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.     

Quantitative studies on the arrangment of human metaphase chromosomes

Summary The pattern of association of acrocentric chromosomes was examined in ten and five carriers of a 15/21 and a 13/14 Robertsonian translocation, respectively, and was compared with that of the same numbers of relatives with normal karyotypes. In the carriers of 15/21 translocation, the number of large associations (involving more than two acrocentrics) and the association frequencies for individual acrocentric chromosomes, were significantly higher than in the control group. The mean number of associations of the single homologs of the translocation chromosomes was much higher than that of the other acrocentrics. In the carriers of 13/14 translocations, only the association frequency for chromosome 13 was higher than in the normal relatives. The uninvolved chromosomes homologous to those involved in translocations showed an insignificant increase in associations in comparison with the other acrocentrics. These results suggest that some mechanism within the cells compensates for the effect of missing acrocentrics or of acrocentrics lacking NORs on the number of associations. The possible relations of this phenomenon to the activity of the nucleolus organizing regions are discussed.     

Quantitative studies on the arrangement of human metaphase chromosomes

Summary The spatial relationships between the homologous pairs of chromosomes in the normal human colcemid-treated metaphase plate were tested by two different mathematical approaches: (a) determination of the distances between the centromeres of the homologous chromosomes compared to the mean distance of all centromeres of the mitosis in question; (b) measuring the distances of the different chromosomes from the center of the mitosis.The following results were obtained: (1) The arrangement of human metaphase chromosomes does not follow a normal distribution; the distribution is narrower and taller, probably due to an impairment of free chromosome spreading by the cell membrane. We believe that only in membraneless mitotic cells should the chromosome-spread correspond to a normal distribution under the same preparation conditions. (2) There is a positive correlation between decreasing chromosome size and decreasing mean distance between homologous chromosomes. (3) A close positive correlation exists between increasing chromosome size and increasing distance to the barycenter of the mitosis. (4) There is also a close positive correlation between the distance of homologous chromosome pairs and their distance from the center of the mitosis, i.e., with increasing distance from the center of the mitosis, the distance between the homologous partners increases. (5) The following statistically significant deviations from these rules could be established: (a) The large acrocentric chromosomes are closer associated, as one would expect from their size, probably due to their participation in the nucleolus organization; (b) in the female cell one of the two X chromosomes has an extremely peripheral localization; the X chromosomes are furthest apart of all pairs of homologous chromosomes; (c) the chromosome pairs 6 and 8 are relatively close together in spite of their peripheral position, suggesting a truc close association of the homologus partners; (d) the chromosome pair 18 has a more peripheral position than expected, and a relatively large mean distance between the homologous partners; (e) the chromosome pair 1 has a much more central position and a closer association than is expected from its size.     

3-D cell-level chlorophyll fluorescence imaging of ozone-injured sunflower leaves using a new passive light microscope system

A passive light microscope system has been developed, capable of reconstructing an extended-focus 3-D cell-level image of chlorophyll fluorescence and Phi(PSII) of intact attached leaves using a limited number of focal plane images of chlorophyll fluorescence. Using this system, the relationships between the depth of the mesophyll cells in spongy tissue and the intensity of the chlorophyll fluorescence and the Phi(PSII) were investigated in sunflower leaves exposed to 300 ppb ozone for 12 h at a PPFD of 300 micromol m(-2) s(-1) actinic light. After ozone exposure, fluorescence intensity (F) largely decreased in the cells just under the epidermal cells (within approximately 20 microm of the epidermal cells), but the sites where fluorescence intensity decreased had no relationship to the position of the stomata. By contrast, the distribution of Phi(PSII) showed no change after the ozone exposure. These findings suggest that ozone-induced inhibition occurs in the cells just under the epidermal cells by reducing the light absorption of the chloroplasts, while the operating quantum efficiency of PSII photochemistry is maintained.     

Chromatin organization and restriction endonuclease activity on human metaphase chromosomes

Human metaphase chromosomes were treated with HaeIII, HindIII, EcoRI, and AluI restriction endonucleases and subsequently stained with either Giemsa or ethidium bromide. The results obtained seemed to suggest that the structural organization of specific chromosome regions can play a primary role in determining the cytological effect after digestion with specific restriction endonucleases.     

Preparation of extended metaphase chromosomes from human cultured cells using a topoisomerase II inhibitor, ICRF-193.

Effects of ICRF-193, a topoisomerase II inhibitor, on metaphase chromosome preparations were examined. A short-time exposure of this drug to human HL60 cells in a suspension culture before harvest resulted in obtaining more extended metaphase chromosomes. The length of chromosome 6 identified by fluorescence in situ hybridization was twice as long with this drug treatment. Together with effectiveness for adherent HepG2 cells, these results suggest that treatments with ICRF-193 provide a simple and reliable method for extended metaphase chromosome preparations from cultured cells.     

Effect of isoniazid on structure of human metaphase chromosomes]

Isoniazid gives rise to chromomerization of human bone marrow metaphase chromosomes in vitro or in vivo. The discontinuous appearance of the chromatids seems to be related to their progressive spiralization during the mitotic division. The medicinal substance tested would have the property of revealing the distribution of heterogenous chromatine by showing down its condensation procedure.