Gibberellinsäure-stimulierte Genaktivität im Endosperm von Phaseolus

Summary Gibberellic acid, injected into maturing ovules of Phaseolus vulgaris, induces 3.2-fold enhancement of the number of additional nucleoli within the endopolyploid endosperm nuclei. The additional nucleoli originate at different sites of polytene chromosome-like chromosome bundles. They contain RNA and are sensitive to actinomycin D. Similar nucleolar bodies are extruded by the main nucleolus. It is assumed that gibberellic acid stimulates the chromosomal and nucleolar RNA synthesis, i.e., the gene activity, in the endosperm nuclei.RNA synthesis was tested by ³H-thymidine it could be seen that gibberellin-treated and gibberellin-treated endosperms were labeled, but those of actinomycin-treated endosperms were not. Using ³H-thymidine it could be seen that gibberellin-treated endosperm nuclei continue to replicate their DNA for a longer period than untreated nuclei. Hence it follows that the treated nuclei become more highly endopolyploid and are capable of functioning longer than the untreated ones.

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Mit Unterstützung durch den Fonds zur Förderung der wissenschaftlichen Forschung der Republik Österreich.     

Alcuni Aspetti Citologici Dell'Endosperma di Phaseolus Coccineus L.

Abstract

Some cytological aspects of Phaseolus coccineus L. endosperm. — Cytological observations were made on the endosperm of Phaseolus coccineus, in a limited stage of development of the seed, using different dyes and labelling with ³H-thymidine and ³H-uridine. The results seem to indicate that, contrary to suspensor cells, in the endosperm of Phaseolus, at least in the considered stage, extra DNA synthesis is not present. Chromosomes in these nuclei, however, undergo several endoreduplication cycles. Nucleoli may be either one or more in a cell, and show a characteristic structure. They are often eccentric in the nucleus and extrusions of nucleolar content in the nucleoplasm or cytoplasm are seen. An apparent alternate activity of RNA synthesis in the nucleoli of the suspensor and in those of endosperm cells is discussed.     

Localization and characterization of chromatin within the interphase nucleus of Amoeba proteus by means of in situ centrifugation and radioautography

Centrifugation of living Amoeba proteus labeled with ³H-thymidine permits the identification by electron microscopic radioautography of chromatin in the interphase nucleus by segregating (through centrifugation-induced stratification) the relatively dilute chromatin from the remainder of the nuclear contents. This procedure reveals that the bulk of the chromatin is in the form of a network of 800 to 900 Å fibrils that are moved by centrifugation to a region just centripetal to the rapidly sedimenting nucleoli. — There is a surprising absence of ³H-thymidine labeling associated with the numerous A. proteus nucleoli, raising the possibility that in this organism the genes specifying ribosomal RNA are non-nucleolar. ³H-thymidine label also is absent from nuclear helixes, membranes, and all other recognizable nuclear regions.     

OBSERVATIONS ON SYNAPTONEMAL COMPLEXES IN PREMEIOTIC INTERPHASE OF WHEAT

Synaptonemal complexes (SCs) were found in stage 3, premeiotic (S phase) pollen mother cell (PMC) nuclei of wheat which were labeled with ³H-thymidine. Three nucleoli are present in PMC nuclei at the beginning of stage 3, premeiotic interphase (S3). During S3, nucleoli move toward the nuclear envelope and fuse to form one nucleolus near the end of the stage. PMC nuclei labeled with ³H-thymidine were serially sectioned to show that more than one nucleolus was present and that SCs were also present in these DNA synthetic nuclei. Entire S3 PMC nuclei were serially sectioned to show the presence of SCs and all three nucleoli. Entire leptotene nuclei were also serially sectioned and segments of SCs were found. It is concluded that the association of homologous chromosomes in S3 of wheat is an early step in SC formation which proceeds through leptotene and is completed in zygotene and pachytene. Thus there is evidence that the continuum of chromosome pairing in wheat starts much earlier than was once thought.     

Nucleoprotein methylation in salivary gland chromosomes of Sciara coprophila: Correlation with DNA synthesis

Methylation and ethylation of chromosomal nucleoproteins have been demonstrated in the salivary glands of Sciara coprophila larvae. A difference in grain distribution pattern was observed between chromosomes incubated in ³H-methyl-methionine and ³H-ethyl-ethionine. Like DNA synthesis, methylation could only be demonstrated at certain developmental stages, and during some of these a close correlation was observed between labelling patterns following ³H-methyl-methionine and ³H-thymidine in separated gland pairs. No such correlation was observed with RNA synthesis. Ethylation of chromosomal non-histone protein could not be correlated with nucleic acid biosynthesis. Feeding ethionine during larval development produced similar nucleolar abnormalities to those previously described in rat liver nucleoli.     

Chromosome constitution and nucleolar behaviour in the endosperm of Cucurbita pepo L

At early stages of development the endosperm of Cucurbita pepo L. was found to be triploid with 3n=60. Higher levels of ploidy (6n, 12n, 24n, 48n) were encountered at later stages. The euploid increase in chromosome number is the result of endopolyploidy. The volume of the nucleolus increased with the increase in nuclear volume. Cells with varying numbers of nucleoli of different sizes and shapes were observed in the endosperm at all stages. Fragmentation of the nucleolus is believed to be responsible for the increase in nucleolar number. Signs of nuclear disorganisation in the endosperm were visible from the 17th day onwards. As the endosperm tissue gets absorbed by the developing embryo, the mature seed is non-endospermous.     

Nuclear DNA content in differentiated tissues of sunflower (Helianthus annuus L.)

Summary Scanning cytophotometry following Feulgen-staining was used to determine nuclear DNA content in many differentiated tissues of nine cultivars, hybrids or selfed lines ofHelianthus annuus. Apart from such ephemeral tissues as endosperm and anther tapetum, it was found that tissue differentiation in sunflower occurs in the diploid condition, cells being arrested in the DNA presynthetic phase (G₁). In certain cases, however, the nuclear DNA content of differentiated G₁ cells does not exactly match the 2C DNA content found in meristematic cells, but may be either higher or lower. In endosperm and anther tapetum cells, nuclear DNA content may be as high as 24 C and 32 C, respectively. Cytological and autoradiographic analyses after³H-thymidine incorporation reveal that polyploidy in the tapetal cells is due to chromosome endoreduplication. No detectable difference between male-fertile and male-sterile plants exists as far as occurrence and level of cell polyploidy are concerned. The results are discussed in the context of previous investigations on the nuclear condition of differentiatedHelianthus annuus tissue.     

SOME NUCLEAR CHANGES ACCOMPANYING DEVELOPMENT IN THE ENDOSPERM OF GLEDITSIA AND PODOPHYLLUM

The development of the endosperm in Gleditsia is marked by the growth of the average nucleus from 141 μ³ on June 12 to 978 μ³ on July 14. At the time of formation of a cellular endosperm the average nuclear volume commences dropping and by July 20 reaches an average of 224 μ³ throughout the endosperm. In Podophyllum at the time of cell formation the average nuclear volume begins to drop from a value of about 2850 μ³ and falls to a value of from 500 μ³ to 770 μ³, the latter estimates depending upon the mode of fixation of the material. The volume decrease in the Podophyllum endosperm nucleus is accompanied by a fall in mean relative DNA content of the average nucleus, as determined by microspectrophotometry with the two wavelength method. The possibility of degeneration of some of the higher “ploid” types is considered, as well as depolyploidization.     

Rapporti Embrione-Endosperma in Triticum Durum: Velocità Di Utilizzazione,Da Parte Dell'Embrione,Di 3H-Tdr. Proveniente Dall'Endosperma

Abstract

Embryo-endosperm relationship in «Triticum durum» seeds: embryo utilization speed of ³H-Tdr from the endosperm. — The transferring speed of DNA labeled precursor (³H-Tdr) from endosperm to embryo, in Triticum, has been detected by embryo transplantation technique. The results show that the first hour (after transplant) some cells (3%) in root meristem incorporated ³H-Tdr. Labeled cells frequency is increasing between the first and the third hour of experiment, up to 25%; thereafter the percentage keeps constant. The histological location of cells and/or an insufficient availability of ³H-Tdr (from the endosperm) might explain the low labeling index detected in the above experiment.     

Nukleolarvakuolen in den Zellen des Embryosackes vonJasione montana L.

Summary The transparent integument ofJasione montana ovules enables the observation of the nucleolar vacuolar pulsation in egg cell, in secondary nucleus and after the fertilization in zygote, in primary endosperm nucleus and in cells of embryo and endosperm too. The rate of vacuolar pulsation depends on their volume, on the total cell activity and with the endosperm cells on the length of their mitotic cycle. Vacuolar activity can be observed continuously during the developmental cycle of the endosperm cell nucleoli. It starts immediately after the nucleolus formation in telophase, it continues during entire interphase and it ends immediately before the nucleolus disappearence in the late prophase. The pulsation is not interrupted during the nucleoli fusion in prophase.

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Das Material für die Arbeit erhielten wir beim Drehen des wissenschaftlichen Filmes E 1751 am IWF in Göttingen. Für fachliche RatschlÄge, Durchführung der Filmaufnahmen und technische Mitarbeit danke ich Herrn Dr. H. K.Galle, Herrn H. H.Heunert und FrÄulein B.Milthaler. Die finanziellen Mittel stellte in dankenswerter Weise die DFG zur Verfügung.     

Arabidopsis replication factor C4 is critical for DNA replication during the mitotic cell cycle

Replication factor C (RFC) is a conserved eukaryotic complex consisting of RFC1/2/3/4/5. It plays important roles in DNA replication and the cell cycle in yeast and fruit fly. However, it is not very clear how RFC subunits function in higher plants, except for the Arabidopsis (At) subunits AtRFC1 and AtRFC3. In this study, we investigated the functions of AtRFC4 and found that loss of function of AtRFC4 led to an early sporophyte lethality that initiated as early as the elongated zygote stage, all defective embryos arrested at the two‐ to four‐cell embryo proper stage, and the endosperm possessed six to eight free nuclei. Complementation of rfc4‐1/+ with AtRFC4 expression driven through the embryo‐specific DD45pro and ABI3pro or the endosperm‐specific FIS2pro could not completely restore the defective embryo or endosperm, whereas a combination of these three promoters in rfc4‐1/+ enabled the aborted ovules to develop into viable seeds. This suggests that AtRFC4 functions simultaneously in endosperm and embryo and that the proliferation of endosperm is critical for embryo maturation. Assays of DNA content in rfc4‐1/+ verified that DNA replication was disrupted in endosperm and embryo, resulting in blocked mitosis. Moreover, we observed a decreased proportion of late S‐phase and M‐phase cells in the rfc4‐1/–^{FIS2;DD45;ABI3pro::AtRFC4} seedlings, suggesting that incomplete DNA replication triggered cell cycle arrest in cells of the root apical meristem. Therefore, we conclude that AtRFC4 is a crucial gene for DNA replication.     

An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules

Excised, unfertilized cotton (Gossypium hirsutum L.) ovules were cultured for 1–5 days postanthesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2–3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The morphology of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the amount of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the plasmalemma and the membranes of the plasmalemma and the membranes of the RER. The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac.     

STUDIES ON THE GERMINATION OF SEEDS OF THE ROOT PARASITES,ALECTRA VOGELII AND STRIGA GESNERIOIDES. II. DNA SYNTHESIS AND DEVELOPMENT OF THE QUIESCENT CENTER IN THE RADICLE

DNA synthetic activity in the radicle meristem of embryos of germinating seeds of the obligate root parasites, Alectra vogelii and Striga gesnerioides was followed by autoradiography of ³H-thymidine incorporation. Incorporation of ³H-thymidine occurred in the nuclei of cells destined to form the vascular tissues, ground meristem and epidermis. An analysis of the distribution of labeled nuclei demonstrated the presence of a quiescent center of 2-4 cells in the radicle at the beginning of seed germination, becoming more prominent at later stages of germination. During continued growth of the radicle which resulted in a reduction in size of the meristem, cells of the original quiescent center were activated to undergo DNA synthesis.     

Alteration of ringer pH and its effects on precursor incorporation into Drosophila salivary gland nuclei

Drosophila salivary glands were explanted and incubated with ³H-uridine (or ³H-thymidine) in Ringer's solution (Ephrussi-Beadle modified saline) adjusted to pH values in the integral range, 4 to 10. The results of autoradiographic investigations indicate a differential effect of altering Ringer pH on ³H-uridine as opposed to ³H-thymidine incorporation: a) Relatively uniform levels of chromosomal incorporation of ³H-thymidine occurred over the range of test pH. Some decrease of incorporation was noted at pH 5 and some increase at pH 9. b) Chromosomal incorporation of ³H-uridine was severely depressed at pH 4 and 7 relative to the high incorporation levels observed at other pH values. Controlling pH of Ephrussi-Beadle Ringer's solution in such experiments seems a necessity. This appears especially important for studies involving ³H-uridine incorporation.     

Modification of culture medium for Xenopus embryonic cells : I. Effects of albumin and other proteins

Stearns medium that had originally been developed for culture of Rana pipiens embryonic cells was modified and used for Xenopus laevis embryonic cells. Modification was done either by reducing a concentration of albumin or by replacing it with other proteins, such as α-globulin, β,γ-globulin, horse serum and protamin. Comparisons were made between the original and the modified medium with regard to their ability to support not only cellular uptake of ³H-uridine, ³H-thymidine and ¹⁴C-protein hydrolysate, but also aggregate formation. From the results obtained it has been shown that the original Stearns medium containing 0.5% albumin and the modified medium, which includes 0.1% globulin in place of albumin, seem to be most suited also for Xenopus embryonic cells. A concentration of albumin can be reduced to 0.1% without lowering the rate of ³H-uridine incorporation or RNA synthesis, but with an apparent decrease in the ³H-thymidine and ¹⁴C-protein hydrolysate incorporation. In a medium containing α-globulin the cells are more active to incorporate ³H-thymidine and ¹⁴C-protein hydrolysate than in media containing β,γ-globulin and horse serum as well as albumin. No remarkable difference is found among the media tested in their ability to support aggregate formation and in the appearance of aggregates formed. In a medium with protamin there occurs neither incorporation of the precursors nor formation of aggregates.     

Gibberellins and pea seed development

The gibberellin (GA)-biosynthesis mutations, lh ⁱ , ls and Ie ⁵⁸³⁹ have been used to investigate the role(s) of the GAs in seed development of the garden pea (Pisum sativum L.). Seeds homozygous for lh ⁱ possess reduced GA levels, are more likely to abort during development, and weigh less at harvest, compared with wild-type seeds due to expression of the lh ⁱ mutation in the embryo and/ or endosperm. Compared with wild-type seeds, the lh ⁱ mutation reduces endogenous GA₁ and gibberellic acid (GA₃) levels in the embryo/endosperm a few days after anthesis and fertilizing lh ⁱ plants with wild-type pollen dramatically increases GA₁ and GA₃ levels in the embryo/ endosperm and restores normal seed development. By contrast, the ls and le ⁵⁸³⁹ mutations do not appear to reduce GA levels in the embryo/endosperm of seeds a few days after anthesis, and do not affect embryo or endosperm development. However, both the ls and lh ⁱ mutations substantially reduce endogenous GA levels in embryos at contact point (the first day the liquid endosperm disappears). Levels of GAs in seeds from crosses involving the ls and lh ⁱ mutations suggest that GAs are synthesised in both the embryo/endosperm and testa and that the expression of ls depends on the tissue and developmental stage examined. These results suggest that GAs (possibly GA₁ and/or GA₃) play an important role early in pea seed development by regulating the development of the embryo and/or endosperm. By contrast, the high GA levels found in wild-type seeds at contact point (and beyond) do not appear to have a physiological role in seed development.Abbreviations GA_n gibberellin A_n - DAA days after anthesis - WT wild-type We thank Noel Davies, Katherine McPherson and Peter Bobbi for technical assistance, Professor L. Mander (ANU, Canberra) for dideuterated GA standards, and the Australian Research Council and Frontier Research Program, The Institute of Physical and Chemical Research (RIKEN, Japan), for financial support.     

DNA replication of a polytene chromosome section in Drosophila prior to and during puffing

Polytene chromosome sections 63E1-6 of 3L in Drosophila melanogaster were studied by ³H-uridine and ³H-thymidine autoradiography in late third instar larvae and prepupae. In late third instar larvae 63E does not incorporate ³H-uridine. In prepupae, however, a large puff is formed in 63E which is most active in RNA synthesis. — ³H-thymidine labeling patterns and frequencies of regions 61A-64C were analysed and the non-puffed and puffed 63E sections were compared with reference sections. Both in late third instar larvae and in prepupae 63E shows late replication behavior. It is concluded that the decondensation of chromosome bands does not necessarily entail earlier and/or faster DNA replication.     

Stimulation of uptake of tritiated thymidine into mouse marrow cells in culture by a factor from L-cell conditioned medium

Uptake of ³H-thymidine into suspension cultures of mouse marrow cells was stimulated by the addition of serum-free conditioned medium harvested from cultures of mouse L-cells. Characterization of the “conditioning factor activity” (CFA) by gel filtration, ion exchange chromatography, velocity sedimentation, polyacrylamide gel electrophoresis and susceptibility to trypsin digestion indicated that the CFA detected by stimulation of ³H-thymidine uptake is the same as the CFA detected previously by its ability to stimulate colony formation by marrow cells in vitro. The ³H-thymidine uptake assay was used to investigate the kinetics of disappearance of CFA as a function of time in the presence of mouse marrow cells. The CFA recoverable from the cultures decreased rapidly during the first day, and approached background levels by the fifth day. There was no evidence of inhibitory substances in the depleted media. Even if the cultures continued to receive fresh conditioned medium at daily intervals, ³H-thymidine uptake decreased sharply after the fifth day, indicating that the marrow cells had lost their capacity to respond to CFA.     

INCORPORATION OF TRITIATED THYMIDINE DURING VARIOUS STAGES OF LEAF DEVELOPMENT OF XANTHIUM PENNSYLVANICUM

³H-thymidine was incorporated into leaf tissue of Xanthium pennsylvanicum during the stage of active cell division, during cellular differentiation, and into mature cells. Incorporation into nuclear DNA was high in the early stages of development. No nuclear incorporation was found after cessation of cell division. However, significant incorporation could be demonstrated in cytoplasm of differentiating and mature cells. Depending upon the time of growth in the radioisotope and the time of growth after treatment, ³H-thymidine, or its metabolized fraction, was incorporated into the secondary wall depositions of epidermal cells, mesophyll parenchyma cells, xylem cells, and chloroplasts. Autoradiographic technique and liquid scintillation spectrometry were used in these studies. The significance of ³H-thymidine incorporation into various organelles is discussed in relation to cell metabolism and its regulation during leaf development.     

Altered dihydrofolate reductase in fol regulatroy mutants of Escherichia coli K12.

Summary Culture of the thymine-dependent mutant of Rhizobium trifolii T37 was synchronized with phenethanol. During bacterial synchronous growth with synchronized replication of DNA, cells were differentially labeled using subsequently ³H-thymidine of low and high specific activity. The grain tracks produced in autoradiographs of chromosomes were denser on both ends than in the middle. In control experiments with bacteria labeled only with ³H-thymidine of low specific activity, the grain density was uniform throughout the grain track. The results constitute clear evidence of bidirectional replication of R. trifolli chromosome.