The maps of the lampbrush chromosomes of Triturus (Amphibia urodela)

The lampbrush chromosomes of Triturus vulgaris meridionalis were isolated from the germinal vesicle of medium and large-sized oocytes and studied with phase-contrast microscope. The maps were constructed on the basis of the lengths and major morphological features of the chromosomes. The length of each map is equal to the mean of the relative lengths of the corresponding chromosome from different oocytes (the relative length of each chromosome is represented by the ratio between its absolute length and that of chromosome XII from the same complement, conventionally considered as 100 units long). The maps arranged in decreasing length order, were oriented according to the most frequent position of chiasmata, as centromeres were not always evident. — Chromosomes VI and XI bear a sphere in subterminal position. Landmarks typical for T. vulgaris meridionalis are the loops inserted on chromosomes VIII (47 units), X (23 units), XI (34 units) and XII (34 units) frequently presenting themselves under the form of double loop bridges of considerable extension. On chromosomes I (4 units), VI (13 units), X (4 units) and XI (36 units) giant bodies were found that are sometimes comparable to dense-matrix loops. Chromosome XI includes a nucleolus-organizing region, sometimes identifiable by the presence of an inserted nucleolus. Normal and granular loops (much extended at times), axial granules, globules, and loopless bars supplement the morphology of the lampbrush chromosomes of this species.     

Localization of antigens PwA33 and La on lampbrush chromosomes and on nucleoplasmic structures in the oocyte of the urodele Pleurodeles waltl: Light and electron microscopic immunocytochemical studies

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 lampbrush chromosomes of Salamandra salamandra (L.) (Amphibia Urodela)

Lampbrush chromosomes isolated from the germinal vesicle of medium sized oocytes can be individually identified by differences in two characters: (1) chromosome regions rich in well developed loops, and (2) number and position of spheres. Actually the lateral loops are not all equally extended, but those which are inserted in a certain region of the axis of some chromosomes are more developed and sometimes are loaded with dense and copious matrix; chiasmata do not occur inside these regions. One or more spheres are present on eight chromosomes in the complement (chromosomes I–VI, VIII and X): the total number of spheres inserted on S. salamandra lampbrush chromosomes is the highest among the salamandrid species studied so far. These landmarks as well as the maximally developed normal loops are schematically drawn on the maps of the single lampbrush chromosomes. The length of the maps corresponds to the mean value of the lengths of each chromosome relative to that of chromosome XII, taken as 100 units long.Also bivalents from first metaphase spermatocytes have been analysed: they are generally ring-shaped with two terminal or subterminal chiasmata.     

Observations on the cytology of Bipes (Amphisbaenia) with special reference to its lampbrush chromosomes

The positions and general anatomical and histological characteristics of the gonads of Bipes biporus and B. canaliculatus are described. The amounts of DNA per haploid chromosome set have been measured in both species, the values being 1.83 and 2.0 pg for biporus and canaliculatus respectively. The karyotypes of both species are described on the basis of data from mitotic and meiotic metaphase chromosome sets and from lampbrush chromosomes. B. biporus has 10 macrochromosomes and 11 microchromosomes. B. canaliculatus has 11 macrochromosomes and 11 microchromosomes. The karyotypes of the two species differ distinctly with regard to the shapes of 3 of the macrochromosomes. Chiasma distribution is described for male meiosis in B. biporus. Studies of the lampbrush chromosomes of both species show the chiasma distribution in the female to be generally similar to that found in the male biporus. In B. canaliculatus, lampbrush chromosomes with maximally extended lateral loops are found in oocytes that are oblate spheroids measuring 0.7×1.0 mm along their short and long axes respectively, these being well before the start of the major phase of vitellogenesis. Smaller oocytes have more distinct chromomeres and shorter loops. Microchromosomes take the form of typical small lampbrush chromosomes in oocytes. There are at the most 1,000 chromomeres per haploid set of lampbrush chromosomes in B. canaliculatus. Chiasmata are described from lampbrush preparations in which the two half-bivalents are firmly attached to one another without evident association of their axes, indicating the possibility of chiasmate association between the DNA axes of lateral loops. There are remarkably few extrachromosomal nucleoli in Bipes oocytes, and its is suggested that this may indicate a level of ribosomal gene amplification that is much lower than that found in fish and Amphibia. The observations are particularly discussed in relation to current ideas concerning the structure and function of lampbrush chromosomes.     

Morphological observations on cultured lampbrush-stage Rana pipiens oocytes.

Early lampbrush-stage oocytes are characterized by small lampbrush chromosome loops, a small amount of ribonucleoprotein (RNP) matrix on the loops, small nucleoli, few RNP particles in the nucleoplasm, and a smooth germinal vesicle contour. In vitro culture of these oocytes in serum-free culture medium for 24 hr at 18°C promotes a number of morphological changes in the oocytes: The lampbrush loops increase in diameter and acquire extensive RNP matrix, the nucleoli increase in size and complexity, the nucleoplasm accumulates numerous polymorphic RNP particles, and the germinal vesicle envelope acquires a sacculated contour. These characteristics are typical of the in vivo maximum lampbrush stage, and their appearance is due to an apparent in vitro acceleration of the lampbrush phase. Two possible interpretations of these observations are discussed.     

Identification of the lampbrush chromosome loops which transcribe 5S ribosomal RNA in Notophthalmus (Triturus) viridescens

The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. — The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.     

DNA Topoisomerase II Is Not Detectable on Lampbrush Chromosomes but Enriched in the Amplified Nucleoli of Xenopus Oocytes

In somatic cells DNA topoisomerase II (topo II) is thought to be involved in the domain organization of the genome by anchoring the basis of chromatin loops to a chromosomal scaffold. Lampbrush chromosomes of amphibian oocytes directly display this radial loop organization in cytological preparations. In order to find out whether topo II may play a role in the organization of these meiotic chromosomes, we performed immunofluorescence studies using antibodies against Xenopus topo II. Our results indicate that topo II is apparently absent from lampbrush chromosomes and is hence unlikely to act as a "fastener" of the numerous lateral chromosomal loops. Topo II was, however, enriched in the amplified nucleoli of Xenopus oocytes.     

Characterization of the lampbrush chromosomes of the marbled newt Triturus marmoratus (Latreille, 1800)

The maps of the lampbrush chromosomes of Triturus marmoratus oocytes were constructed on the basis of their lengths and major morphological characters such as giant fusing loops, dense matrix loops, lumpy objects, axial granules, lateral globules and reflected fusions; a nucleolus organizing region occurs subterminally on the right side of chromosome X. — Bivalent I appears morphologically asymmetrical, its two partners being of different lengths and bearing heteromorphic loops and other heterozygous structures: this heteromorphism may indicate that the two partners of bivalent I represent the ZW heterochromosomes of the species. Finally, an autoradiographic study has been performed in order to ascertain the pattern of ³H-uridine incorporation shown by the most typical landmarks and nucleoli.This work was financially supported by C.N.R., Roma.     

Cytogenetic Maps of Lampbrush Chromosomes of Newts of the Genus Pleurodeles: An Algorithm of Lampbrush Chromosome Identification inPleurodeles waltl by Immunocytochemical Staining of Landmark Loops with Polyclonal Anti-Ro52 Antisera

Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodelesnewts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoproteins of landmark lateral loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain lampbrush chromosome preparations in newts of the genes Pleurodeles. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker landmark loops along the chromosome length were constructed.     

Intranuclear structures containing RNA maturation factors in early vitellogenic oocytes of Rana temporaria

We have examined the distribution of RNA processing factors in the germinal vesicle (GV) of the common frog Rana temporaria during early vitellogenesis by immunostaining on light- and electronmicroscopic levels and by in situ nucleic acid hybridization. Small nuclear RNPs (snRNP) and factor SC35 involved in pre-mRNA splicing occur in lampbrush chromosome loops and numerous granules 1-3 microns in size. These granules are identical to B snurposomes of Xenopus laevis and Notophtalmus viridescens described earlier (Wu et al., 1991). Some of B snurposomes are attached to homologous loops of lampbrush chromosomes. Immunofluorescent study of Cajal bodies/coiled bodies (CB) showed that sometimes CB have B snurposomes attached to their surface. In this case splicing factor SC35 is found in B snurposomes and B-like inclusions in CB matrix. In CB without attached B snurposomes splicing factor SC35 localizes throughout the whole organelle. Staining of GV spreads with antibodies against nucleolar protein NO38 revealed this protein in CB, nucleoli and micronucleoli. Using in situ nucleic acid hybridization and immunofluorescent staining we have found that on GV spreads from hibernating frogs B snurposomes contact nucleoli. Nucleoli contain snRNP. These data suggest that nucleoli may be storage sites of snRNPs during natural inactivation of RNA synthesis. During winter season in Rana temporaria GV nucleoli become compacted and a number of micronucleoli (less than 2 microns) dramatically increases. Analysis of micronucleoli showed that they contain rRNA, protein NO38, trace amount of U3 small nucleolar RNA and do not contain fibrillarin, involved as U3 in pre-rRNA processing. We suggest that decrease of rRNA synthesis during frog hibernation results in transformation of part of nucleoli in micronucleoli.     

Evolutionary changes in non-histone chromosomal proteins within the Drosophila melanogaster group revealed by monoclonal antibodies

Monoclonal antibodies directed against nonhistone chromosomal proteins of D. melanogaster were tested for crossreactivity with the homologous antigens of various Drosophila species. — By indirect immunofluorescence it could be shown that three antibodies react only with polytene chromosomes of species of the D. melanogaster subgroup, and only much less with chromosomes of other species of Drosophila. — With chromosomes of various other species of the Sophophora or Drosophila radiations only a reaction at background level could be observed. — The results suggest that the three antibodies react with different antigenic determinants of a single protein whose conformation changed rather fast during evolution of the Drosophilidae.     

A monoclonal antibody that recognizes a phosphorylated epitope stains lampbrush chromosome loops and small granules in the amphibian germinal vesicle

An mAb library was produced against proteins from the germinal vesicle (GV) of the frog Xenopus laevis; mAb 104 was selected from this library on the basis of its immunofluorescent staining of lampbrush chromosome loops. Chromosomes from several species of frogs and salamanders stained equally well. The antibody also stained the surface of numerous small granules in the GV nucleoplasm. The interior of the same granules was stained by antibodies against small nuclear ribonucleoproteins (snRNPs). mAb 104 also stained somatic nuclei from many vertebrate and invertebrate species, usually in a finely punctate pattern similar to that described for anti-snRNP and other antinuclear antibodies. The staining of somatic nuclei was much stronger during the mitotic stages than during interphase. Immunoblot analysis showed that mAb 104 recognizes a phosphorylated epitope.     

Light and electron microscopic immunolocalization of two nuclear antigens in the liver of Pleurodeles waltl using monoclonal antibodies

The distribution of 2 nuclear antigens in the interphase nuclei of liver of Pleurodeles waltl was determined with the help of monoclonal antibodies, using immunofluorescence for light microscopy and indirect immunoperoxidase and immunogold labeling procedures for electron microscopic localization. The antibodies C36/1 and A33/22 label antigens with relative molecular masses of 270 kDa and 80 kDa, and isoelectric points of 7.0 and 6.4, respectively. The liver of urodels is characterized by the presence of a peripheral layer of hematopoietic cells around the parenchymatous tissue formed by typical hepatocytes. The antibody C36/1 labels the nuclei of both types of cells, whereas the antibody A33/22 labels the nuclei of hepatocytes but not those of the peripheral hematopoietic cells. With both these antibodies, labeling, whenever observed, is restricted to fibrillar structures in the interchromatin space, i.e., to peri- and inter-chromatin fibrils; condensed chromatin, nucleoli, and nuclear envelope are not labeled.     

Cytogenetic maps of lampbrush chromosomes of newts of the genus Pleurodeles: an algorithm of lampbrush chromosome identification in Pleurodeles waltl by immunocytochemical marker-loop staining with polyclonal anti-Ro52 antisera

Our work was aimed at developing a simple and effective method of identification of most or all chromosomes of Pleurodeles newts. To this end, we used DAPI staining of the chromomeres of newt lampbrush chromosomes and immunochemical reactions between the ribonucleoprotein (RNP) marker loops and polyclonal antibodies against human zinc-finger protein Ro52 (52-kDa Ro/SS-A). A method has been developed to obtain newt lampbrush chromosome preparations. Cytological maps of P. waltl chromosomes (Spanish population/subspecies) showing distributions of chromomeres and marker loops along the chromosome length were constructed.