Behaviour of the X chromosomes during growth and maturation of parthenogenetic eggs of Amphorophora tuberculata (Homoptera,Aphididae), in relation to sex determination

Prophase chromosomes of growing oocytes from thelytokous, viviparous females of Amphorophora tuberculata Brown and Blackman (n=2) were studied using a modified propionic acid squash technique with Feulgen staining. In early prophase, prior to the growth phase of the oocyte, the X chromosomes are partially condensed and looped together so that all four ends appear to be associated. Later in prophase the X chromosomes separate in oocytes destined to be female, but remain associated in presumptive male oocytes. The autosomes condense gradually throughout prophase. The nucleus of the presumptive male oocyte is further characterised by the formation of a spherical Feulgen-positive body, which attains a large size (7 m diameter) in late prophase. At this stage, the X chromosomes are no longer visible as separate entities, and are apparently included in the spherical body. At metaphase this disappears, leaving the X chromosomes still united as a condensed bivalent. The spherical body seems to have nucleolar as well as chromatin constituents; nucleolar organisers are present at the ends of the X chromosomes where it first arises. It may function in maintaining the cohesion between the X chromosomes through prophase, and could also facilitate correct orientation of the X bivalent on the spindle of the maturation division. As sex determination in aphids is controlled by juvenile hormone concentration, it appears that the hormone may interact with the X chromosomes during prophase, bringing about their separation in female oocytes, perhaps by inhibiting the formation of the spherical body.     

NUCLEAR DIVISION IN ALTERNARIA TENUIS

A study of the hyphal and conidial nuclei of Alternaria tenuis with the Feulgen nuclear reaction indicates that division is mitotic. Five chromosomes and a spindle apparatus are formed during nuclear division.     

Nuclear Division in the Ameboflagellate Adelphamoeba galeacystis

Nuclear division in synchronized cultures of the ameboflagellate Adelphamoeba galeacystis has been described. Division in this organism is typically promitotic. It occurs within an intact nuclear membrane and is characterized by the persistence of the nucleolus and its transformation into 2 polar masses. The nucleolus is stained with pyronin-Y by the methyl green pyronin-Y technic, and with Heidenhain's hematoxylin, but is unstained by the Feulgen reaction. The reaction with these stains is removed after digestion of the nucleolus by ribonuclease. During mitosis the nucleolus undergoes an orderly series of vacuolizations before forming the polar masses. The chromatin is Feulgen positive, stains with methyl green by the methyl green pyronin-Y technic and undergoes a series of characteristic changes during the division process. Synchronization of amebae grown on coverglasses was accomplished by transfer of cells from 30 to 38.5 C for a period of 100 min. A temporal sequence of nucleolar and chromatin participation in the nuclear division of this organism is suggested.     

Chromosome Banding and DNA Methylation Patterns, Chromatin Organisation and Nuclear DNA Content in Zingeria biebersteiniana

Chromosome structure and chromatin organisation of a two-chromosome model cereal Zingeria biebersteiniana (Claus) P. Smirnov were studied: nuclear DNA content was determined by microdensitometric analysis after Feulgen staining; Feulgen absorption at different thresholds of absorbance in interphase nuclei also provided evidence on the organisation of chromatin, allowing quantitative estimation of condensed chromatin within interphasic nucleus. The DNA methylation pattern of Z. biebersteiniana metaphase chromosomes was examined with a specific monoclonal antibody. 5-methyl-cytosine residues are present in several chromosome sites and differences may be present between corresponding regions of homologues. Chromosome banding pattern reveals large bands in the centromeric regions of each chromosome, showing constitutive heterochromatin; by fluorochromes staining pericentromeric blocks are evidenced. After the cold and 9-aminoacridine pre-treatments and after aceto-carmine and aceto-orceine staining, respectively, the metaphase chromosomes were analysed by image analysis system revealing a segmentation of the chromosome body that resembles Giemsa/Reverse banding in animal chromosomes. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Feulgen banded karyotype of the mouse: analysis of the mechanisms of banding

The chromosomes of the mouse have been identified by specific banding patterns revealed by the Feulgen stain. Comparison of the patterns of the Feulgen-stained karyotype with those of acetic-saline-Giemsa stain and quinacrinemustard-fluorescence demonstrates a high order of similarity among the three, with the localization of Feulgen dense bands and regions closely paralleling that of Giemsa dark and fluorescence bright bands. Since the stained substrate of the Feulgen reaction is known to be DNA, it is suggested that all three banding methods reveal the distribution of DNA or of some moiety that closely follows DNA distribution in metaphase chromosomes. The preparative procedure of the Feulgen banding method consists of a 15 to 20 minute exposure to PO₄ buffer at pH 10 and a prolonged (60–72 hrs) exposure to 12xSSC. Omission or curtailment of either step results in preparations with chromosome sets that are not karyotypable, although some stain differentiation is produced. HCl extraction prior to the preparative treatment blocks banding, but acid extraction following the preparative treatment, either that of the HCl hydrolysis of the Feulgen reaction of that of an almost fourfold extension of the standard hydrolysis time, does not obliterate bands already formed. By extrapolation from biochemical studies of chromatin, it is postulated that the localization of Feulgen dark and light stain, representing relative DNA densities, reflects the regional protein association of the DNA; the Feulgen dense regions may result from aggregation of a specific class of histones by the alkaline buffer with consequent condensation of the DNA bound to those histones; the Feulgen pale or negative regions may represent those in which non-aggregated proteins, histone and non-histone, have been solubilized in the saline incubation, rendering the DNA of those regions subject to diffusion or vulnerable to fragmentation in the Feulgen hydrolysis.     

Chromatin diminution and a chromosomal mechanism of sexual differentiation in Strongyloides papillosus

Eggs obtained from feces of rabbits infected with Strongyloides papillosus were squashed and the karyotypes were determined. They contained cells with either two long and two medium sized chromosomes (2L2M), or one long, three medium and one short chromosome (L3MS). Two types of parasitic female gonad could be distinguished on the basis of oocyte chromosome morphology at prometaphase of the maturation division. All the oocytes in a gonad contained either two upaired long chromosomes and two unpaired medium sized chromosomes, or two unpaired medium sized chromosomes and two unpaired chromosomes segmented into beads in one region. At the maturation division in mitotic parthenogenesis the beads appear to be lost from one of the chromosomes. This generates a medium sized and a shorter chromosome, which together with the undiminished chromosomes make up the L3MS karyotype. Animals with beaded oocyte chromosomes lay eggs that develop into males. It is suggested that males are heteromorphic for the long homologue due to chromatin diminution, that occurs in the maturation division of mitotic parthenogenesis.     

STUDIES ON NUCLEI USING CORRELATED CYTOCHEMICAL, LIGHT, AND ELECTRON MICROSCOPE TECHNIQUES

In this paper, a procedure for correlating electron microscope and light microscope cytochemical studies using immediately adjacent serial thin and thick sections has been described and discussed. This technique, combined with the Feulgen reaction for DNA, has been of particular value in framing and answering both general and specific questions about the nucleus. The results may be summarized as follows:— Apparent nuclear homogeneity in the electron microscope is not due to loss of DNA as evidenced by positive Feulgen reactions in such nuclei. Arrangement of Feulgen-positive material in chromosomes, heterochromatin, perinuclear and perinucleolar chromatin, etc., is similar to that customarily observed in the light microscope but this is not necessarily reflected in a cursory survey of the electron image. Careful comparison of light and electron images shows that fine differences in structure are associated with chromatin localization. Primary spermatocyte prophase chromosomes of crayfish have been positively identified by their Feulgen-positive nature. Core-like axial structures in such chromosomes have been observed (9) and are described further. A remarkable feature of spermiogenesis in the crayfish is an elaboration of the nuclear envelope of the spermatid accompanying the formation of what becomes a mass of convoluted membranes in the sperm. In the spermatid, perinuclear chromatin follows outpocketings of the nuclear envelope into the cytoplasm. In the early sperm, on the other hand, although the nuclear envelope is continuous with the system of convoluted membranes, the chromatin is distinct from it and is retained in the nucleus proper by some mechanism independent of the nuclear envelope. None of the above observations was apparent from the electron microscope images alone; they were possible only by virtue of the correlated cytochemical and electron microscope study of adjacent sections. The successful use of other cytochemical tests, such as the PAS reaction for certain carbohydrates, in such correlated studies is also described.     

Cytochemical study of the elimination chromatin in silkworm meiosis]

The elimination chromatin separating from chromosomes during the first maturation division of female sex cells of lepidoptera insects was studied cytochemically on paraffin sections of eggs of Bombyx mori L. Ocytes at the metaphase-telophase stage of the first meiotic division were stained for RNA by metyl green-pyronin and gallocyanin with negative results. These data differ from earlier positive results for Solenobia triquetrella reported by Ris and Kleinfeld, 1952.     

CYTOLOGICAL AND CYTOCHEMICAL STUDIES OF GREEN MONKEY KIDNEY CELLS INFECTED IN VITRO WITH SIMIAN VIRUS 40

Cytological and cytochemical studies of green monkey kidney cells infected with SV40 virus indicated that the type of lesion produced was influenced by the multiplicity of infection and that the lesions appeared later and progressed more slowly when the inoculum was diluted. The earliest change consisted of enlargement of ribonucleoprotein-containing spherules in the nucleolus (nucleolini). This was followed by rarefaction, with or without condensation, of the chromatin and the appearance of one or more homogeneous masses of inclusion material containing DNA, RNA, and non-histone protein which eventually filled the nucleus. In some instances the chromatin appeared to be directly transformed into inclusion material. In the later stages of infection, the ribonucleoprotein of the nucleolini was no longer stainable and material resembling the nucleoprotein of the intranuclear inclusions was found in the nucleolar vacuoles and in the cytoplasm. The nucleic acids in the inclusions were stained by toluidine blue, toluidine blue-molybdate, the Feulgen stain, and by methyl green. The stainable material was extractable by nuclease digestion or by hot trichloroacetic acid. Green or yellowish green staining by acridine orange was apparently due to binding of dye by protein and not by nucleic acids since the staining reaction was not reduced by extraction of nucleic acids by hot trichloroacetic acid. Extraction with pepsin in combination with ribonuclease or deoxyribonuclease removed practically all the inclusions from the cells; consequently they could not be stained with acridine orange. The cytochemical studies suggest that the use of pepsin together with nuclease is not a meaningful technique.     

Exposure and removal of stainable groups during Feulgen acid hydrolysis of fixed chromatin at different temperatures

Summary Hyperdiploid Ehrlioh's ascites tumour cells grown in male mice (strain NMRI) were labeled with radioactive nucleotides. The nucleic acids were extracted from fixed, air-dried smears by fractionated hydrolysis and their radioactivity measured by liquid scintillation. The experiments showed that the exposure of aldehydes through removal of purine bases and the elimination of these aldehydes through depolymerisation of DNA were the two main processes responsible for the Feulgen hydrolysis curve. They were shown to be independent and overlapping. The depurination can be described as a simple hydrolytic reaction, while the extraction of DNA depends on a number of different factors. This entails that, in the Feulgen acid hydrolysis procedure, the part of DNA measured is dependent upon the stability of the chromatin. It was found that it is possible accurately to determine the depolymerisation process and thereby roughly correct the measured amount of Feulgen DNA.     

Vegetative nuclei and differentiation of chromosomes inTrichophyton vanbreuseghemii

The stages of nuclear division were observed inTrichophyton vanbreuseghemii microcultures stained with Giemsa and by the Feulgen reaction. The course of karyokinesis is discussed. It takes the form of mitosis, modified by narrowing of the lumen of the hyphal cell and the movement of the cytoplasm. No spindle or centrioles were found. In vegetative hyphae (1.2–1.5 μm in diameter), the metaphase chromosomes were arranged lineally in a row following the direction of the long axis of the hyphal cell. The distribution of anaphase chromosomes occurs perpendicularly or obliquely to the cell walls. The chromosomes were spherical, with a diameter of about 0.3 μm. A haploid number of chromosomes (five) was found.     

Double Staining for Comparative Measurements in Squash Preparations

Comparative measurements of nuclei or chromosomes following different treatments are seldom made on squash preparations, since variations which arise during preparation of the slides may easily mask genuine treatment differences. This drawback may be overcome by making use of dyes which, when substituted for basic fuchsin in Schiff's reagent, will give a Feulgen-type reaction with chromatin. By selecting dyes of contrasting colours, it is possible to intermingle cells from different treatments in the same squash preparation, and to perform comparative measurements on adjacent cells.

Suitable dyes which contrast well with basic fuchsin are toluidine blue, or azure A (which stain chromatin blue) and chrysoidin yellow (which stains chromatin yellow). These dyes are made up and used in the same manner as ordinary Feulgen reagent.

Samples of cells from the two treatments to be compared are fixed, washed and hydrolysed in 1 N HCl at 60 C. One sample is stained in regular Feulgen reagent, the other in the contrast dye, then both are macerated and thoroughly mixed on the same slide in a single drop of 45% acetic acid. A coverslip is added, and the preparation flattened to the required amount and made permanent after dry-ice removal of the cover. This technique may also be utilised for comparative grain counts in autoradiography, provided that the contrast dye does not cause chemical fogging of the film.     

Die Cytologie der Parthenogenese bei dem Tardigraden Hypsibius dujardini

Hypsibius dujardini Doy. (Articulata, Tardigrada) shows obligatory parthenogenesis under given cultivating conditions. Males were never found. The first meiotic division reduces the number of chromosomes; the (2n=10) chromosomes are divided between a small polar body and the egg nucleus. Prior to the second division the dyads divide, thus restoring the diploid number. A diploid polar body is formed subsequent to the second division. After the egg nucleus has moved toward the center of the egg, the cleavage divisions begin. — During meiosis II and the first cleavage divisions the chromosomes can develop into “large chromosomes” which presumably consist mostly of RNA. No “large chromosomes” are found after the seventh cleavage division. Sometimes a plate of coloured material (“elimination chromatin”) can be observed between the anaphase daughter plates of the first cleavage divisions. In this case the chromosomes are always small.     

Peculiarities of cytochemical properties of cancer cells as revealed by study of deoxyribonucleoprotein susceptibility to Feulgen hydrolysis.

Chromatin of human squamous carcinoma cells reacts more intensively to short (1-2 min) acid hydrolysis in the Feulgen reaction and is, after such treatment, more intensively stained by Schiff reagent than chromatin of normal cells of the same origin. To reveal this difference in chromatin properties the use of a fluorescence variant of the Feulgen reaction is necessary because nuclei-binding of Schiff reagent after such short hydrolysis is so weak that the amount of the stain bound by means of absorption technique is hardly possible. The use of increased sensitivity of cancer cells chromatin to acid hydrolysis for cancer cytodiagnosis is suggested, especially for the diagnosis of so called diploid cancers for which detection on the basis of deoxyribonucleic acid content determination is impossible.     

Nuclear division in the ameboflagellate Adelphamoeba galeacystis.

Nuclear division is synchronized cultures of the ameoboflagellate Adelphamoeba galecystis has been described. Division in this organism is typically promitotic. It occurs within an intact nuclear membrane and is characterized by the persistence of the nucleolus and its transformation into 2 polar masses. The nucleolus is stained with pyronin-Y by the methyl green pyronin-Y technic, and with Heidenhain's hematoxylin, but is unstained by the Feulgen reaction. The reaction with these stsins is removed after digestion of the nucleolus by ribonuclease. During mitosis the nucleolus undergoes an orderly series of vacuolizations before forming the polar masses. The chromatin is Feulgen positive, stains with methyl green by the methyl green pyronin-Y technic and undergoes a series of characteristic changes during the division process. Synchronizationof amebae grown on coverglasses was accomplished by transfer of cells from 30 to 38.5 C for a period of 100 min. A temporal sequence of nucleolar and chromatin participation in the nuclear division of this organism is suggested.     

Feulgen-DNA absorption curves of polytene chromosome regions ofRhynchosciara americana

Summary Feulgen absorption spectra of polytene chromosome regions, differing in types of DNA composition and of chromatin compactness, were determined microspectrophotometrically in squashed salivary glands ofRhynchosciara americana. The absorption curves exhibited a secondary maximum at their ascending branch after the chromatins were maximally depurinated. The degree of shoulder prominence could not be specifically correlated with the packing state or even with richness in satellite repetitive DNA of the chromatins. It is assumed that other factors may exist which play a part in the phenomenon that gives rise to the Feulgen absorption shoulder characteristics. Differences in the extraction rate of the apurinic acid fragments due to their binding to specific non-histone proteins are suggested as contributing to the above-mentioned absorption patterns when comparing the various chromatin types.     

Some Evidence for the Specificity of the Feulgen Reaction

Carr has attacked the specificity of the Feulgen reaction on three grounds: that the chromosomes are adsorbents capable of regenerating the color of the Schiff reagent; that selectivity for the nucleus depends on destruction of cytoplasm by acid hydrolysis preceding staining; and that the reaction is not blocked by SO₂ water, as he says it should be if staining occurs by a chemical reaction. The first point was tested by staining chromosomes treated with nuclease. They were Feulgen negative, but their protein basis remained intact. The second point was tested by hydrolyzing fixed tissues, washing off solutes, drying, and comparing weight loss with controls. As differences were negligible, the fixed cytoplasm must not have been made soluble by hydrolysis. Carr's third point was not tested experimentally. It is concluded that these objections to specificity of the Feulgen reaction are not valid.     

The localisation of an Mr 74,000 major chromatin antigen on native salivary chromosomes of Drosophila melanogaster

An antigen making a major contribution to the immune response to Drosophila melanogaster chromatin resides primarily on a nonhistone charge-class family of proteins of Mr 74,000. Immunofluorescence detects this antigen at interbands, puffs and diffuse bands of D. melanogaster salivary chromosomes isolated without exposure to acid fixatives, and on nucleoplasmic ribonucleoprotein droplets. In the electron microscope, gold labelling reveals the binding of monoclonal antibodies specific for the antigen at chromosomal loci generally bearing putative ribonucleoprotein (RNP) particles. However, the locus 3C 11–12 is remarkable in that it bears putative RNP particles but is virtually unlabelled, suggesting protein specificity at different active loci.     

Feulgen-DNA response and chromatin condensation in Malpighian tubules of Melipona rufiventris and Melipona quadrifasciata (Hymenoptera, Apoidea)

Melipona quadrifasciata and Melipona rufiventris are stingless bee species which present low and high heterochromatin content, respectively, on their mitotic chromosomes as assessed visually after a C-banding assay. However, these species do not show differences in the C-banding responses of their Malpighian tubule interphase nuclei. In the present study, the Feulgen-DNA response, which could inform on differences in DNA depurination due to differences in chromatin condensation, was compared in the cell nuclei of the Malpighian tubules of these species. It was hypothesized that differences in acid hydrolysis kinetics patterns, as assessed by Feulgen reaction and studied microspectrophotometrically, could discriminate M. quadrifasciata and M. rufiventris interphase nuclei not distinguishable with the C-banding method. Feulgen-DNA values corresponding to more than one ploidy class were found in both species; these values at the hydrolysis time corresponding to the maximal DNA depurination for each ploidy degree were higher in M. quadrifasciata, reflecting a higher DNA content in the Malpighian tubule cell nuclei of this species compared to those of M. rufiventris at the same larval instar. The maximal Feulgen-DNA values of M. quadrifasciata after short (50 min) and long (90 min) hydrolysis times were found to be closer to each other, while those of M. rufiventris occurred sharply at the long hydrolysis time, indicating that DNA depurination in M. quadrifasciata occurred faster. This result is probably related to the involvement of differences in chromatin condensation; it agrees with the idea that M. rufiventris contains more heterochromatin than M. quadrifasciata, which is supported by the analysis of results obtained with the image analysis parameter average absorption ratio. The depurination kinetics studied here with the Feulgen reaction were revealed to be more pertinent than the C-banding technique in establishing differences in levels of chromatin condensation for these cell nuclei.