Tritium labelling and cytochemistry of extra DNA in Acheta

Females of Acheta domesticus were injected with H³-thymidine and H³-uridine at various stages of development in order to study DNA and RNA synthesis in the DNA body present in the oocytes. Staining with alkaline fast green, azure B and the Feulgen reaction were employed as cytochemical tests. The following main results were obtained.

1. The DNA body appears in the oogonia at interphase as a Feulgen positive spherical structure 2 microns in diameter and is seen in subsequent mitotic divisions as a slightly smaller structure of variable shape. H³-thymidine autoradiography discloses that the DNA present in this body is synthesised at a different time from the chromosomal DNA.
2. At interphase and during the early prophase of meiosis the DNA body increases in size becoming a large Feulgen positive sphere 6 microns in diameter. Small nucleoli are present within this body. The DNA of the body is complexed with histone as revealed by alkaline fast green staining. H³-thymidine labelling discloses that it is at these stages that the bulk of the DNA synthesis takes place in the body.
3. Every oocyte contains a DNA body, and no body of comparable size or shape seems to be present in the male meiotic prophase.
4. At pachytene and diplotene the DNA body acquires the appearance of a “puff”. Two zones can be distinguished inside the DNA body: (1) an inner core of DNA and an outer shell of RNA. The inner core is Feulgen positive and stains light green with azure B, the outer shell is Feulgen negative and stains purple-violet with azure B, as does the cytoplasm. From the inner DNA core many Feulgen positive fibrils radiate into the outer RNA shell. These fibrils appear unstained or slightly greenish with Azure B, forming a transparent network in a purple-violet background. This gives the body the typical appearance of a “puff”. H³-uridine incorporation reveals that the RNA synthesis occurs in the outer RNA shell of the body and in the chromosomes. RNase treatment removes the H³-uridine incorporated into these regions.
5. At the end of diplotene the DNA body starts to disintegrate. The DNA core breaks up into minor components and the outer RNA zone also begins to disintegrate. By late diplotene the whole body has vanished, releasing DNA, histone and RNA into the nucleus. Subsequently the nuclear envelope disintegrates as it regularly does at the end of prophase of meiosis.
6. The simplest interpretation of the above results is that the DNA body represents hundreds of copies of the genes of the nucleolar organizing region.

     

Conservation of nucleolar structure in polytene tissues of Ceratitis capitata (Diptera: Tephritidae)

Nucleolar structure was studied in mitotic and three polytene tissues of the Mediterranean fruit fly, Ceratitis capitata using in situ hybridization with a tritium-labelled rDNA probe and silver staining. In mitotic metaphase chromosomes nucleolar organiser regions were localised in the short arms of both sex chromosomes. In polytene nuclei of trichogen cells, salivary glands and fat body rDNA was detected within nucleoli. Nucleoli in these tissues have a similar structure with rDNA labelling concentrated in a central core. Silver staining resulted in very heavy staining of polytene nucleoli and interphase nucleoli in diploid cells. Silver staining of nucleolar organisers in metaphase chromosomes is weak or absent although the X chromosome has numerous dark silver bands in other locations. The results suggest that nucleolar structure is conserved in polytene tissues contrasting with the variability of autosomal banding patterns and sex chromosome structure. They also indicate that silver staining is not necessarily specific for nucleolar regions.     

Silver staining of the lampbrush chromosomes ofTriturus cristatus carnifex

Lampbrush chromosomes fromTriturus cristatus carnifex were stained using the ammoniacal silver staining (AgAS) technique. Many of the recognized marker structures proved to be silver positive, plus between six and fifteen lateral loop pairs. None of the stained loop pairs corresponded to known sites of the nucleolus organizers, although the extrachromosomal nucleoli were silver positive. The ammoniacal silver staining technique does not demonstrate the specificity for active ribosomal cistrons in lampbrush chromosomes that it does in a wide variety of mammalian mitotic chromosomes.     

ULTRASTRUCTURE AND CYTOCHEMISTRY OF METABOLIC DNA IN TIPULA

A DNA body is present in the females of the fly Tipula oleracea and is formed in contact with the sex chromosomes in the oogonial interphases. At each oogonial mitosis, the DNA body follows the chromosomes to one anaphase group and is included in one of the telophase nuclei. The body increases appreciably in size during the interphase of meiosis. All oocytes have the body, but only a few nurse cells possess it. The DNA body synthesizes its DNA at a different time than the chromosomes, as is shown by incorporation of tritiated thymidine, and contains 59% of the DNA of the nucleus, as is disclosed by spectrophotometric measurements. At late diplotene the DNA body disintegrates, releasing its DNA into either the nucleus or the cytoplasm. When studied in the electron microscope, the DNA body appears composed of a tight mass of intertwined fibrils. Demonstration that the main mass of the body is composed of DNA is obtained from cytochemical tests which reveal that the DNA body is Feulgen positive, stains green with azure B, incorporates H³-thymidine, and after digestion with DNase is Feulgen negative. The DNA of the body is complexed with histone, like the DNA of the chromosomes, as is revealed by an intense alkaline fast green staining. Electron microscope examination of oocytes reveals that one side of the DNA body is in close contact with the nuclear envelope and that the other side possesses an outer shell composed mainly of particles 150 to 250 A in diameter. Between the outer shell and the chromosomes there is a band of low electron opacity, 4000 to 7000 A thick. In the light microscope, this light band together with the outer shell is Feulgen negative and stains violet with azure B; this is confirmation of the presence of RNA. In the oocytes the nucleoli are found inside the DNA body. These nucleoli have a nucleolonema composed mainly of particles 150 to 250 A. The nucleoli are Feulgen negative, alkaline fast green negative, stain violet with azure B, and do not stain with azure B after RNase digestion, thus confirming their RNA content. The presence of the nucleoli inside the DNA body and of a band of RNA between the body and the chromosomes is indicative of a high RNA synthetic activity. Since the DNA of the body is complexed with histone, as in the chromosomes, and the nucleoli are located inside the body, the simplest interpretation of the DNA body is that it represents hundreds of copies of the operons of the nucleolar organizing region or neighboring regions. The situation found in Tipula has several basic features in common with the polytene chromosomes of other Diptera and with the hundreds of nucleoli present in Triturus oocytes. In all three cases, genes seem to be copied hundreds of times but are kept in different types of packages. A DNA body like the one in Tipula oleracea is found in other species of Diptera and in the Coleoptera. There is no indication, from the present investigation, that the DNA body is in any way associated with a virus.     

A Method for Preparation of Synaptonemal Complexes of Meiotic Chromosomes from Basidial Protoplasts of the White Button Mushroom Agaricus bisporus (Lange) Imbach.

For the first time, preparations of synaptonemal complexes (SCs) were made from meiotic chromosomes of white button mushroom (Agaricus bisporus) basidia. It is the first experience of obtaining SC preparations of filamentous fungi from isolated meiosporangium protoplasts. Previously, only yeast SC preparations were obtained following this approach. The method includes four major stages: isolation of basidium protoplasts by treatment of basidia with lytic enzymes, spreading of protoplast nuclei on a filmy support by osmotic shock, staining the preparations with silver nitrate, and examination under light and electron microscopes. The structures of spread premeiotic nuclei, axial elements of chromosomes, SCs, chromatin, and nucleoli were studied at the leptotene–diplotene stage of meiotic prophase I.     

Localization and development of kinetochores and a chromatid core during meiosis in grasshoppers

Positive staining of kinetochores and a chromatid core has been achieved using a simplified silver staining method in squash preparations from meiotic chromosomes of grasshoppers. This technique permits the exact localization of kinetochores on the chromosomes whether metacentric, acrocentric or ‘telocentric’. The sister kinetochores can be observed from mid-diplotene stages but they are not differentiated during first meiotic metaphase. However they can be observed again at the onset of anaphase 1. The existence of a positively stained chromatid core in meiotic divisions is also reported. This core appears well defined inside each chromatid from diplotene to the end of the second meiotic division. The visualization of these cores in first meiotic metaphase clearly shows the points at which the chiasmata took place.     

Cytochemical study of pseudoisocyanine stained human chromosomes

Summary Human meiotic and mitotic chromosomes were studied with N-N diethyl pseudoisocyanine stain. Following methylation and oxydation, the staining allowed microscopic observation of slides with both monochromatic light and fluorescence. In addition, stained preparations can be permanently conserved.Preceeded by diverse methods of chromosome denaturation or 5-BUDR incorporation, PIC lends itself to a large number of banding techniques.Cytochemical study of stained chromosomes demonstrated a certain PIC affinity for DNA although tests performed do not exclude the possibility of PIC reaction with certain proteins.This investigation was supported by INSERM (ATP no. 8-74-29).     

Satellite association of the nucleolar chromosomes in a plant

Summary A method for preparing chromosomes that included enzyme maceration and subsequent flame-drying allowed us to easily detect satellite association in the mitotic cells ofNothoscordum fragrans (2 n=19), which has six acrocentric nucleolar chromosomes in its chromosome complement. Of 593 metaphase plates examined, approximately 60% had satellite association. The number of chromosomes involved in the association varied from two to six, and the incidence decreased as the number of chromosomes involved in the association increased. Comparison of the same chromosomes stained with Giemsa and subsequently with silver demonstrated that the nucleolar organizing regions (NORs) that responded almost negatively to Giemsa and positively to silver was responsible for satellite association. The nucleoli may strongly correlate with satellite association since persistent nucleoli associated with a few metaphase chromosomes were sometimes found and the nucleoli had a strong tendency to fuse with each other at interphase. Four types of acrocentric chromosomes could be discriminated on the basis of the bands negatively staining with Hoechst. All four types were involved in satellite association and there were significant deviations from the expectation for random participation in the association.     

Multiple synaptinemal complexes at the region of gene amplification in Acheta

Ovaries of Acheta domesticus (house cricket) were fixed for electron microscopy at two stages of development: (1) ovaries containing mainly oocytes at interphase and early prophase of meiosis, and (2) ovaries with oocytes mainly at pachytene and diplotene. The E.M. study was accompanied by three types of light microscopy controls consisting mainly of cytochemical tests. Every oocyte contains a DNA body which at pachytene and diplotene acquires the appearance of a puff. In the light microscope two zones can be distinguished inside the body: (1) an inner core of DNA and (2) and outer shell of RNA. In the E. M. the inner core consists of a fibrillar material and the outer shell is composed of areas of high electron opacity consisting mainly of tightly packed particles and fibrils. At these stages synaptinemal complexes are seldom seen associated with the DNA body but are present throughout the nucleus as part of the paired chromosomes. The complexes are present as single units. — In the oocytes at interphase and early prophase of meiosis, where the DNA body is active in DNA replication, the body appears in the light microscope as a large Feulgen positive sphere containing Feulgen negative areas. In the E. M. at these stages the DNA body consists of: (a) the two components found at pachytene, (b) a third electron dense component which is more homogeneous than the other two, and (c) of large assemblies of synaptinemal complexes originating from several centers. The most significant features of the axial complexes are: (1) the circular packing of the complexes, (2) their occurrence in packages of 300 to 400 units and (3) the fact that not all of the DNA body forms complexes but only a part of it.Biochemical experiments (Lima-de-Faria, Birnstiel and Jaworska, 1969) have demonstrated the amplification of ribosomal cistrons in the DNA body of Acheta. The simplest explanation is that the multiple complexes are formed either between the extra gene copies of the two homologues, or between the extra copies of each chromosome as well. There seems to be a correlation between the presence of multiple axial complex formation and gene amplification in Acheta but the exact relation between the two phenomena demands further study.Dedicated to Dr. Sally Hughes-Schrader on the occasion of the seventyfifth birthday on the twentyfifth of January 1970.     

Relationship between secondary constrictions and nucleolus organizing regions inAllium sativum chromosomes

Summary Allium sativum L. (2 n=16) had three types of clones with regard to the number of chromosomes carrying well-defined secondary constrictions: the first type had two secondary constricted chromosomes (type I), the second had three (type II) and the third had four (type III). Silver staining was applied to these three types of cells to determine the number of nucleolus organizing regions (NORs) per cell and to study the relationship between the morphological appearance of the secondary constrictions and the ability of the chromosomes to form nucleoli. Ag-positive regions appeared on two chromosomes in type I, on three in type II and on four in type III. The comparison of Giemsa and Feulgen stained chromosomes with the silver stained ones clearly indicated that the positive reaction with silver occurred exclusively on the secondary constricted regions that responded negatively to both Giemsa and Feulgen staining, indicating that the size of the achromatic secondary constrictions directly reflects the volume of the Ag-positive materials. However, all three types of clones had a maximum of four nucleoli at interphase. Of the four nucleoli, either two or one was extremely small (less than 1 m in diameter) in types I and II respectively. The size variations of the other nucleoli seemed to be positively correlated with those of the Ag-positive regions. This and the observation that the maximum number of nucleoli per cell did not coincide with the number of Ag-positive regions on the metaphase chromosome complement suggest strongly that the NORs responsible for the minute nucleoli cannot be detected on the metaphase chromosomes. The present observations indicate that not all NORs are indicated by the morphological appearance of secondary constrictions.     

Nucleolus,nucleolar chromosomes,and nucleolus-associated chromatin from early diplotene to dictyotene in the human oocyte

Summary The shape, relationships, relative DNA content, and nucleolar activity of the short arm of acrocentric bivalents were studied in human oocytes from early diplotene to dictyotene. At the beginning of diplotene, the short arms of the previously paired chromosomes were again separated and displayed the same morphological features as in mitotic prophase chromosomes. They were connected only with the nucleolus. In situ hybridization and silver staining showed that the nucleolar organizer regions (NORs) were located in the peripheral region of the nucleolus. Tritiated-uridine incorporation was active. At birth, the relationships of the acrocentric short arms showed increasing complexity. The chromosomes ended in nucleolus-associated chromatin blocks of irregular shape, containing large quantities of DNA as demonstrated by intense binding of³H-actinomycin D. The number of chromosomes converging on these chromatin blocks exceeded the number of acrocentrics, suggesting that heterochromatic regions of other chromosomes were associated with the short arm of acrocentrics. In the electron microscope, the NORs were represented by fibrillar centers located on the periphery of the nucleolus and consistently connected with the blocks of dense chromatin. These relationships remained unchanged in the primordial oocyte in the adult ovary. Persistence of³H-uridine uptake showed that the oocyte was not at a resting stage. The possible cytogenetic consequences of these observations are discussed.     

Ovarian nests in cultured females of the Siberian sturgeon Acipenser baerii (Chondrostei,Acipenseriformes)

Ovaries of Acipenser baerii are of an alimentary type and probably are meroistic. They contain ovarian nests, individual follicles, inner germinal ovarian epithelium, and fat tissue. Nests comprise cystoblasts, germline cysts, numerous early previtellogenic oocytes, and somatic cells. Cysts are composed of cystocytes, which are connected by intercellular bridges and are in the pachytene stage of the first meiotic prophase. They contain bivalents, finely granular, medium electron dense material, and nucleoli in the nucleoplasm. Many cystocytes degenerate. Oocytes differ in size and structure. Most oocytes are in the pachytene and early diplotene stages and are referred to as the PACH oocytes. Oocytes in more advanced diplotene stage are referred to as the DIP oocytes. Nuclei in the PACH oocytes contain bivalents and irregularly shaped accumulation of DNA (DNA‐body), most probably corresponding to the rDNA‐body. The DNA‐body is composed of loose, fine granular material, and comprises multiple nucleoli. At peripheries, it is fragmented into blocks that remain in contact with the inner nuclear membrane. In the ooplasm, there is the rough endoplasmic reticulum, Golgi complexes, free ribosomes, complexes of mitochondria with cement, fine fibrillar material containing granules, and lipid droplets. The organelles and material of nuclear origin form a distinct accumulation (a granular ooplasm) in the vicinity of the nucleus. Some of the PACH oocytes are surrounded by flat somatic cells. There are lampbrush chromosomes and multiple nucleoli present (early diplotene stage) in the nucleoplasm. These PACH oocytes and neighboring somatic cells have initiated the formation of ovarian follicles. The remaining PACH oocytes transform to the DIP oocytes. The DIP oocytes contain lampbrush chromosomes and a DNA‐body is absent in nuclei. Multiple nucleoli are numerous in the nucleoplasm and granular ooplasm is present at the vegetal region of the oocyte.     

An early stage of ZW/ZZ sex chromosome differentiation in Poecilia sphenops var. melanistica (Poeciliidae,Cyprinodontiformes)

The mitotic and meiotic chromosomes of the American cyprinodont fish Poecilia sphenops var. melanistica were analysed. All 46 chromosomes are telocentric. By specific staining of the constitutive heterochromatin with C-banding and various AT-specific fluorochromes, the homomorphic chromosome pair 1 could be identified as sex chromosomes of the ZW/ZZ type. All female animals exhibit a W chromosome with a large region of telomeric heterochromatin that is not present in the Z chromosome. These sex chromosomes cannot be distinguished by conventional staining; they represent the first demonstration of sex chromosomes in fishes in an early stage of morphological differentiation. The W heterochromatin and the telomeric heterochromatin in the two autosomes 18 show a very bright fluorescence when stained with AT-specific fluorochromes. This allows the direct identification of the chromosomal sex by examining the interphase nuclei: females exhibit three, males only two brightly fluorescent heterochromatic chromocenters in their nuclei. The significance of these ZW/ ZZ sex chromosomes and their specific DNA sequences, the dose compensation of the Z-linked genes, and the experimental possibilities using sex-reversed ZW males are discussed.     

Extrachromosomal rDNA amplification in the oocytes of Polystoechotes punctatus (Fabricius) (Insecta-Neuroptera-Polystoechotidae)

The ovary of Polystoechotes punctatus consists of several ovarioles of meroistic-polytrophic type. Histological, histochemical and ultrastructural studies revealed that the extrachromosomal amplification of rDNA takes place in the oocyte nucleus. Prior to previtellogenic growth the oocyte nucleus contains the chromosomes of meiotic prophase and a condensed extra DNA body. Initial split of extrachromosomal DNA material into several fragments coincides with the appearance of a spherical, fine granular body (referred to as primary nucleolus). Its gradual fragmentation accompanied by further dispersion of amplified DNA results in the formation of a growing number of multiple nucleoli. Until mid previtellogenesis each multiple nucleolus contains detectable amount of rDNA. In the advanced stages of previtellogenesis rDNA can hardly be visualized within the multiple nucleoli, while chromosomes form a few dense aggregates randomly disposed in the karyoplasm. At the onset of vitellogenesis the chromosomes assemble to form a karyosome. In its close vicinity DNA-positive material reaggregates. Multiple nucleoli are either found on the periphery of this aggregation or merge within it. At the final stages of vitellogenesis the number of multiple nucleoli significantly decreases.     

Gene amplification in oocytes with 8 germinal vesicles from the tailed frog Ascaphus truei Stejneger

In the last 3 oogonial mitoses in Ascaphus truei all daughter nuclei remain in the same cell. The oocyte is 8-nucleate at the start of meiotic prophase and remains so until late in oogenesis when 7 of the nuclei disappear. All 8 nuclei in a single oocyte resemble one another with respect to size and chromatin distribution at all stages of meiotic prophase. Much of the Feulgen-positive material in pachytene nuclei is concentrated into one region of the nucleus. — All of the 8 germinal vesicles of yolky oocytes have a full set of lampbrush diplotene bivalents. Germinal vesicles from oocytes of up to 0.8 mm diameter have less than 100 nucleoli, some of which are multiple nucleoli in the sense that they have more than one core region. Each of the 8 nuclei in oocytes from one animal had about the same volume of nucleolar material. — Two values have been obtained for the amount of DNA in a diploid nucleus from Ascaphus. A biochemical estimate utilizing erythrocyte nuclei and the diphenylamine reaction yielded a value of 7.1 pg per nucleus. Microphotometry of erythrocyte nuclei stained with Feulgen's reagent gave a value of 8.2 pg per nucleus. — Microphotometric measurements of Feulgen-stained nuclei at various stages of meiotic prophase up to diplotene indicate that each nucleus synthesizes up to 5 pg of extrachromosomal DNA during and immediately after pachytene. This DNA is considered to be nucleolar. Autoradiography of nuclei from oocytes which had been incubated for 6h in ³H thymidine showed silver grains over pachytene and early diplotene nuclei only. In pachytene nuclei the silver grains overlaid that part of the nucleus where Feulgen-positive material was most concentrated. Most of the chromosomal material was unlabelled. — The significance of the 8-nucleate condition in Ascaphus oocytes is discussed, and the amount of nucleolar DNA synthesized at pachytene and of nucleolar material present in germinal vesicles is compared with corresponding situations in other amphibians.     

Effects of Potassium Cyanide on Silver Stainability of Specific Cell Structures

Treatment with potassium cyanide prevents the occurrence of diffuse silver precipitates on chromosome preparations. It therefore allows greatly increased times of incubation with silver solutions on both mitotic and meiotic preparations. By this method selective silver staining of active nucleolar organizers, centromeric regions and kinetochores of mitotic chromosomes and of specifically staining structures of meiotic chromosomes has been obtained.     

Ribosomal gene amplification in multinucleate oocytes of the egg brooding hylid frog Flectonotus pygmaeus

The multinucleate oocytes of Flectonotus pygmaeus begin as cysts containing 2,000 or more meiotic nuclei. Each nucleus amplifies its ribosomal DNA early in oogenesis. The level of amplification is widely different from one nucleus to another, and ranges from less than 0.1 × C to more than 8 × C. The C value for this species is 1.7 × 10^–12g of DNA. In oocytes of about 0.5 mm diameter the nuclei sort themselves out into an outer shell of several hundred nuclei that swell up to become much larger than the nuclei that remain in the inner compact mass. Later the outer nuclei continue to swell and decrease in number while the inner nuclei disintegrate and disappear. By the time the oocyte reaches 1.2mm diameter there are only a few large nuclei left and each has many large nucleoli and a full set of lampbrush chromosomes. Eventually, only one germinal vesicle nucleus is left, and this has all the characteristics of the single germinal vesicles that are typical of oocytes from other amphibians. It is suggested that the sorting out of nuclei into the inner mass and the outer shell of larger nuclei in middle sized oocytes is a consequence of the positions the nuclei happen to be in at the time, but that the contest for the role of germinal vesicle may be won by the nucleus of the outer shell that has the highest ribosomal DNA content.