Analysis of chromosome replication by a BrdU antibody technique

Chromosome replication was studied without synchronization in human lymphocyte and amniotic cell cultures visualizing very short 5-bromodeoxyuridine (BrdU) pulses by an immunologic technique (BAT). The findings agree in general with those facts known from earlier BrdU staining techniques. The very high sensitivity of BAT was shown to allow the detection of replication in a band where 1 in 200 nucleotides is replaced by BrdU. The main observations are: though the replication patterns after BAT appear strange the bands correspond to those described by the Paris Conference (1971). At the beginning of the S-phase a stepwise onset of replication in only a subset of R-bands is confirmed. There is a considerable difference in the sensitivity between early and late S (S_E and S_L) for the detection of BrdU pulses. This difference probably reflects a different spatial arrangement of chromatin in R-bands as compared with G-bands below the level of cytogenetic analysis. The use of short pulses did not reveal any additional subdivision of S_E or S_L. The correspondence between chromosomal bands and replicon clusters is discussed briefly with respect to the different time they need for replication.     

BrdU pulse/reverse staining protocols for investigating chromosome replication

By using a reverse Giemsa staining procedure (TT chromatin pale, TB chromatin dark) it is possible to detect replication in metaphase chromosomes with short (~10 min) 5-bromodeoxyuridine (BrdU) pulses. A pulse protocol allows us to consider the question “What is replicating at this point in time?” and we have investigated replication patterns during cycle transit in stimulated human female lymphocytes. A clear-cut demarcation between R-zone early and G-zone late was not found. Instead, whilst replication commences (with a very staggered start) in R-zones, activity soon appears to transgress band boundaries and gives rise to cells with unclassifiable patterns where chromosomes take on a mottled or reticulate appearance. Replication in R-zones dies out leaving a clear G-zone pattern persisting for the remainder of S which terminates with a very staggered finish. When pulse duration is increased (~1 h) the frequency of unclassifiable cells falls and occasional “mixed-pattern” cells appear which have, within the same cell, typical R- and G-zone regions. The existence of such cells indicates that if a mid-S replication pause exists (and the absence of any mid-S wave of pale stained cells suggests that it does not) it does not make exclusive separation between dark R- and G-band zones.     

A replication technique for scanning electron microscopy: Applications for anthropologists

Scanning electron microscopy has become an increasingly useful tool for anthropologists, particularly because of the development of improved methods of replicating specimens. One of the best replication techniques involves silicone-based dental impression materials to make negative impressions, in conjunction with epoxy resins, which are used to make positives or casts. The technique outlined here is particularly useful for anthropologists. Using this technique allows the examination of bone surfaces, teeth, and fossils for taphonomic, microwear, and experimental studies. Reproduction of detail is faithful at magnifications of × 1,500 to × 2,000, routinely giving resolutions of .1 to .25 μm.     

Demonstration of immune complexes on erythrocytes by scanning electron microscopy

If anti-sera are combined with native whole blood by an in-vitro technique, immunocomplexes are formed. They are fixed to erythrocytes and can be made visible by a scanning electron microscope on blood smears especially pretreated.     

Frequency and distribution of chiasmata in Syrian hamster spermatocytes studied by the BrdU antibody technique

The frequency and distribution of chiasmata and the nature of terminal associations was re-examined in Syrian hamster spermatocytes using the 5-bromo-2-deoxyuridine (BrdU) antibody technique (BAT) for differential chromatid labelling. Differential chromatid substitution was achieved by BrdU incorporation at the penultimate pre-meiotic S-phase followed by one of three different staining protocols: (i) fluorescence plus Giemsa (FPG), (ii) acridine orange staining or (iii) BAT. For analysis of chiasmata frequency and localization in the diplotene/diakinesis stages the resolution of FPG and acridine orange staining was comparable to that of BAT. In metaphase II chromosomes BAT was more informative than FPG and acridine orange staining and revealed small, terminal crossover exchanges. This finding proves that many terminal associations of meiotic chromosomes actually represent chiasmata at the end of the first meiotic division. Some crossover exchanges were localized in the constitutive heterochromatin of autosomes. Using BAT we also detected crossover exchanges in close vicinity to each other. This observation is reminiscent of the fact that crossing over interference means a reduction in frequency and does not imply total inhibition.     

Human chromosome banding specific for electron microscopy

Electron microscopy (EM) provides much higher resolution than that obtained with light microscopy (LM). Until now, however, no chromosome banding procedure specifically adapted for EM was available. To produce an easy and reproducible banding method that would allow accurate chromosome identification, we investigated the applicability of an immunochemical method. BrdU-substituted chromosomal regions can be accurately visualized by applying a monoclonal antibody against BrdU, followed by a gold-tagged secondary antibody. Since BrdU is incorporated during the last part of the S-phase, regions of darkly stained G- and C-bands are substituted. A characteristic C-banding pattern is revealed, and the G-banding obtained is sharp and allows discrimination between subbands. Its similarity with the classical G-banding observed by LM makes it easy to interpret and facilitates karyotyping.     

High-resolution analysis of Allium cepa chromosome replication by an FPG technique

In Allium cepa meristematic cells, BrdU incorporation into late-replicating DNA and its detection by FPG technique has shown to allow the visualization of characteristic late replicated segments (LRS) in metaphase chromosomes not detected till now. Since these LRS have very similar characteristics with the 'dot-like' exchanges reported previously in this system, they seem to represent a new support for the idea of a regular organization of chromatin in metaphase.     

Nucleopolyhedrovirus: scanning electron microscopy technique

A simplified methodology was developed to study the geometric form of multiple Bombyx mori Nucleopolyhedrovirus by scanning electron microscopy. The virus belongs to Baculoviridae family and was isolated from the silkworm Bombyx mori (L.) (Lepidoptera: Bombycidae). The polyhedra of Nucleopolyhedrovirus were obtained from the filtrate, inoculum and hemolymph of the silkworm experimentally infected with nuclear polyhedra. This material was placed on stubs, where a copper tape was previously adhered. After dry at room temperature the virus was covered with carbon and gold. Scanning electron microscopy analysis revealed a well defined morphology for the polyhedra of multiple Bombyx mori Nucleopolyhedrovirus, making possible the mathematical study that identified it as a truncated octahedron. The form of the polyhedron can present taxonomic value, once it is specific for each viral lineage.     

Membrane turnover in crab photoreceptors studied by high-resolution scanning electron microscopy and by a new technique of thick-section transmission electron microscopy

Summary Crab photoreceptors were examined after treatment by the osmium-DMSO-osmium method for high-resolution scanning electron microscopy. This technique of specimen preparation was also adapted for transmission electron microscopy, enabling sections up to 1 urn thick to be viewed in a conventional microscope at 75 kV. With appropriate pretreatment, some cytoskeletal elements can be visualised by both techniques. The methods were then used to investigate some of the daily changes known to occur in photoreceptor cell structure. Striking differences were found in the structure of Golgi bodies present in retinula cells during the synthesis and breakdown phases of the daily cycle of photoreceptor membrane turnover. Cyclic changes were also noticed in the mitochondria of retinula cells, and additional evidence was found for a previously proposed model of rhabdomeral microvillus formation.     

Morphometric analysis of human chromosome satellites and NOR asymmetries by transmission electron microscopy

Human chromosome satellites appear as roughly spherical telomeric structures of 0.215 +/- 0.013 microns mean diameter by electron microscopy. Morphometric evaluations showed that in the short arms of D and G chromosomes lacking secondary constrictions, the chromatin which constituted the satellites appeared virtually integrated within the short arms. Asymmetry was detected in sister satellites from almost identical dimensions to the near absence of one of them. When Ag-NOR staining was employed to locate active nucleolar organizer regions (NORs) these appeared associated with satellite short arms and active NORs were never found in non-satellite chromosomes. Asymmetry was also evident between sister NORs.     

Demonstration of replication patterns in the last premeiotic S-phase of male Chinese hamsters after BrdU pulse labeling

Chromosome replication in the last premeiotic S-phase of male mammals has been previously studied by [³H]thymidine autoradiography and by a 5-bromodeoxyuridine (BrdU)/Giemsa technique. We used a recently developed BrdU-antibody technique (BAT) in this study. The following conclusions were drawn: (1) The replication patterns observed are similar to that of somatic cells. (2) The heterochromatin starts replication in early S-phase. (3) The euchromatic part of the X chromosome of the male Chinese hamster replicates together with the autosomes and therefore behaves isocyclicly and not allocyclicly as hitherto assumed. Hence, genetic inactivity of the X chromosome may be brought about by a mechanism different from that in somatic cells.by P.B. Moens     

Demonstration of PAS-positive material in electron microscopy with lead staining

Summary 1. Two types of lead staining for electron microscopy, with different staining mechanisms, are described.2. The first type of staining, leading to an increase of the contrast already available, is referred to as intensifying staining.3. The second type of staining leads to the appearance of lead precipitates at several sites where PAS-positive material can be expected. This type of staining is therefore referred to as PAS-like staining.4. Preliminary hypotheses for the mechanisms of these different stainings are given.With 10 Figures in the Text     

Demonstration by light and electron microscopy of capsules on gonococci recently grown in vivo.

A study by light microscopy, using Leishman's stain alone or Leishman's stain followed by nigrosin, showed the presence of capsules on gonococci of two strains subcultured from subcutaneous chambers in guinea pigs. With the Alcian blue method of preparation for electron microscopy, gonococci of both strains recently grown in vivo showed densely stained capsules on some cells, while others in the same preparation showed only irregular masses of dense material on their surfaces with strands connecting adjacent bacteria. Treatment with antiserum, complement and conglutinin revealed irregular masses and strands of extracellular material with fixatives that did not contain Alcian blue.