Chromosoma

Substituting one of the (sub)metacentric chromosomes of a simple interchange complex by the two corresponding telocentrics marks one of the unchanged arms, one of the interchange segments and one of the interstitial segments. When the two telocentrics can be distinguished and when only infrequently MI configurations are formed for which more than a single origin is possible, rather exact estimates of the frequency of occurrence of chiasmata in the six segments can be obtained. When the two telocentrics can not be distinguished and in addition the frequency of MI configurations with more than one possible origin is not negligible, the analysis is more complex. With some previous knowledge of the interchange and some simplification an acceptable simple solution remains possible. — Of both cases an example for rye, Secale cereale, is given, together with an analysis of interference between these segments, and a conversion of probabilities of being bound at MI to genetic length.

Monoclonal antibodies A33/22 and La11G7 have been used to study the distribution of the corre-sponding antigens, PwA33 and La, on the lampbrush chromosome loops and nucleoplasmic structures of P. waltl oocytes, using immunofluorescence, confocal laser scanning microscopy and immunogold labeling. The results obtained with these antibodies have been compared with those obtained with the Sm-antigen-specific monoclonal antibody Y12. All these monoclonal antibodies (mAbs) labeled the matrices of the majority of normal loops along their whole length. Nucleoplasmic RNP granules showed a strong staining with the mAbs La11G7 and Y12 throughout their mass, but with the mAb A33/22, they showed only a weak peripheral labeling in the form of patches on their surface. This patchy labeling was confirmed by confocal laser scanning microscopy. Electron microscopy revealed that this patchy labeling might be due to a hitherto undescribed type of submicroscopic granular structure, around 100 nm in either dimension, formed by 10-nm particles. Such granules were observed either attached to the RNP granules or free in the nucleoplasm, but rarely in relation with the normal loop matrices. These 100-nm granules may have a role in the movement of proteins and snRNPs inside the oocyte nuclei for storage, recycling, and/or degradation. Our results also suggest that all the microscopically visible free RNP granules of the nucleoplasm of P. waltl oocytes correspond to B snurposomes. The granules forming the B (globular) loops showed a labeling pattern similar to that of B snurposomes; their possible relationship is discussed.

The spatial distribution of the three centromere-associated proteins α-tubulin, CENH3, and phosphorylated histone H2A (at threonine 120, H2AThr120ph) was analysed by indirect immunodetection at monocentric cereal chromosomes and at the holocentric chromosomes of Luzula elegans by super-resolution light microscopy and scanning electron microscopy (SEM). Using structured illumination microscopy (SIM) as the super-resolution technique on squashed specimens and SEM on uncoated isolated specimens, the three-dimensional (3D) distribution of the proteins was visualized at the centromeres. Technical aspects of 3D SEM are explained in detail. We show that CENH3 forms curved “pads” mainly around the lateral centromeric region in the primary constriction of metacentric chromosomes. H2AThr120ph is present in both the primary constriction and in the pericentromere. α-tubulin-labeled microtubule bundles attach to CENH3-containing chromatin structures, either in single bundles with a V-shaped attachment to the centromere or in split bundles to bordering pericentromeric flanks. In holocentric L. elegans chromosomes, H2AThr120ph is located predominantly in the centromeric groove of each chromatid as proven by subsequent FIB/FESEM ablation and 3D reconstruction. α-tubulin localizes to the edges of the groove. In both holocentric and monocentric chromosomes, no additional intermediate structures between microtubules and the centromere were observed. We established models of the distribution of CENH3, H2AThr120ph and the attachment sites of microtubules for metacentric and holocentric plant chromosomes.

In spermatocytes of Helix aspersa, the structure of the synaptonemal complexes undergoes changes in the course of the pachytene, the lateral elements being transformed into wide bands of lesser density than the chromatin. By using the uranyl-EDTA-lead sequence, which preferentially stains RNA, the lateral elements can be made to appear positive in the early pachytene while the corresponding areas, which become wider and more diffuse, are positive during late pachytene. — Apparently, the lateral elements do not persist in the diplotene and remnants of the central element can occasionally be observed. Using the uranyl-EDTA-lead method reveals some positively stained material surrounding the chromatin, mostly granular in appearance, which is observed in late pachytene and attains its maximum amount during diplotene. Several aspects of these observations are here discussed.

1) The distribution pattern of heterochromatin characterized by Giemsa-banding, Quinacrine-banding and DNA-late replication has been studied in a reconstructed karyotype of Vicia faba with all chromosome pairs interdistinguishable. 2) By means of two Giemsa-banding methods both an interstitial and a centromeric Giemsa-banding pattern are described. The former one comprehends 14 “marker” and 18 “additional” bands of lower but characteristic visualization frequencies. The centromeric Giemsa-banding pattern consists of 7 bands, located in the centromeric and in the secondary constrictions of the metaphase chromosomes. Chromosomes with banding patterns intermediate between the interstitial and the centromeric Giemsa-banding have also been observed. 3) Quinacrine-banding revealed 10–12 brightly fluorescent bands and 1–2 regions of dim fluorescence. Most Q-bands occupy chromosomal positions also characterized by interstitial Giemsa bands. 4) The DNA-late replication pattern, analyzed both by autoradiography and by FPG-technique, revealed 9 late replicating chromosome regions; all of these correspond positionally to the sites of interstitial Giemsa bands. 5) The results are discussed with respect to (a) the relationships between the banding- and the DNA-late replication pattern; (b) banding and heterochromatin characteristics; (c) the correlations between the distribution of chromatid aberrations and special types of heterochromatin. — The patterns of heterochromatin distribution found are in basic conformity with the corresponding patterns reported for the standard karyotype of Vicia faba. The heterochromatin type characterized by both Giemsabanding and late replication is characteristic of all those chromosome regions which after mutagen treatments show up as aberration hot spots. Positional correlations between interstitial Giemsa marker bands and chemically induced isochromatid breaks are indicative of preferential aberration clustering in heterochromatin/euchromatin junctions.

A discrete fraction of heterogeneous nuclear RNA, in addition to the previously described 75 S RNA, has been identified in salivary gland cells of Chironomus tentans. On the basis of its electrophoretic mobility in 1% agarose gel, it is tentatively designated as 35 S RNA. The 35 S RNA possesses distinct and characteristic labelling kinetics. After a lag of 20–45 min it emerges on the chromosomes and in the nuclear sap, but significant quantities of labelled 35 S RNA in the cytoplasm can be found only after 45–90 min of incorporation. The origin of 35 S RNA from genes manufacturing heterogeneous nuclear RNA has been established on the basis of its sensitivity against the nucleoside analogue, 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole, DRB. RNA extract derived from chromosome IV, carrying the Balbiani rings, lacks 35 S RNA. Furthermore, an essentially normal appearance of 35 S RNA was observed in glands having very small or no Balbiani rings and no detectable 75 S RNA synthesis. Both these findings are consonant with the interpretation that 35 S RNA is not generated by genes harboured in the Balbiani rings. When the continuous synthesis of chromosomal RNA is interrupted by actinomycin D, most of the 35 S RNA accumulated in the nuclear sap disappears, whereas a corresponding RNA fraction arrives in the cytoplasm. Kinetic studies as well as the chase experiment with actinomycin D suggest the existence of a kinetic relationship between nuclear sap 35 S RNA and its cytoplasmic counterpart. In addition, studies with the initiation inhibitor DRB strongly indicate that 35 S RNA derives from chromosomal RNA molecules of higher S values than 35 S, probably from RNA in the 75–100 S range, by post-transcriptional cleavages.

Applying a Giemsa staining technique to the meiotic chromosomes of Anemone blanda demonstrates that Giemsa bands similar to those seen in the mitotic chromosomes are discernible at all the principal stages of meiosis. The bands are not a product of the Giemsa procedure since they can be seen in unstained preparations using phase-contrast optics as chromocentres in interphase nuclei and as condensed regions in prophase chromosomes. That the bands seem to be permanent features of the nucleus, whether it is dividing or otherwise is an important consideration for understanding their nature and function. Bands and chiasmata do not coincide indicating on the one hand that chiasmata are not responsible for differences in banding patterns and on the other hand that the conservation of bands is an indication that they are either inert regions or specialised regions with considerable adaptive significance. These alternatives can only be resolved by genetical studies of the banding phenomena.