Genetic localization of a follicle cell protein locus in Drosophila melanogaster

Summary Three variant forms of a novel set of follicle cell proteins (Fc) were found when screening geographic wild-type strains of Drosophila melanogaster by SDS-polyacrylamide gel electrophoresis of ³⁵S-methionine labelled ovaries. These variant forms were used to establish X chromosomal linkage and for further genetic localization by both recombinant analysis and by cytogenetical mapping. A locus involved in the synthesis of Fc proteins was localized to the 7C1-9 region, i.e. very close to the singed locus (21.0 cM). The number of Fc proteins, their variation and possible function is discussed.     

Expression of storage-protein genes during soybean seed development

Mature seeds of Glycine max (L.) Merr. contain two major storage proteins, a glycosylated 7S protein (conglycinin) and a non-glycosylated 11S protein (glycinin). Accumulation of these proteins and their mRNAs during seed development in cv. Provar was studied by SDS polyacrylamide gel electrophoresis and by Northern (DNA-RNA) hybridization. The 11S acidic and basic subunits and the 7S and subunits began to accumulate 18–20 d after pollination, shortly after the termination of cell division in developing cotyledons, whereas the 7S and 11S A-4 subunits were not detected until one to two weeks later, during the maturation phase of development. Messenger RNAs for 7S and 11S proteins were first detected 14–18 d after pollination, several days before the accumulation of storage proteins. Extracts from embryonic axes contained reduced levels of the 7S subunit, very little 11S protein, no detectable 7S or 11S A-4 subunits, and an additional 7S subunit not found in cotyledons. Soybean axes and cotyledons therefore differ in their synthesis of seed storage proteins.Abbreviations cDNA complimentary DNA - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Determination of the backbone torsion angle ɛ in nucleic acids

Summary The multiplet structure of cross peaks in double-quantum-filtered COSY NMR spectra is analysed for those resonances that include passive heteronuclear couplings. Interestingly, the cross peak involving the sugar-ring protons H2 and H3 in nucleic acids display an E.COSY-type appearance exclusively when the backbone torsion angle (C4-C3-O3-P) adopts a gauche(-) conformation. This observation allows an unambiguous analysis of the conformation around , without the knowledge of ³J_cp coupling constants.     

Oligomerization of deoxyguanosine 5′-phosphoro-2-methylimidazolide on a polycytidylate template

The oligomerization of deoxyguanosine 5-phosphoro-2-methylimidazolide on a polycytidylate template is much less efficient than the oligomerization of the corresponding activated ribonucleotide. Nonetheless oligomers containing up to eight nucleotide residues are detected. The products are 3–5-linked oligodeoxyribonucleotides capped at the 5-terminus with a pyrophosphate-linked monomer.     

Repair and plasmid R46 mediated mutation requires inducible functions in Proteus mirabilis

Summary In Proteus mirabilis nalidixic acid or a predose of UV induce Rec protein formation, a portion of post-UV replication repair and post-UV replication enhancement. These inducible functions are not significantly affected by the plasmid R46, which renders P. mirabilis efficiently UV-mutable. The R46-mediated UV induction of rif ^r mutations requires additional inducible functions, as existing after malidixic acid treatment in rec ⁺ strains. After a nalidixic acid pretreatment UV efficient induction of rif ^r mutations occurs without an otherwise obligatory period of post-UV incubation prior to plating on rifampicin agar. The inducible character of this qualification of plasmid R46-mediated UV mutagenesis in P. mirabilis is evident from the inhibitory effects of chloramphenicol and starvation. Constitutive high-level synthesis of Rec protein in cells harboring the recombinant (multi-copy) rec ⁺ plasmid pPM1 reduced plasmid R46-mediated UV mutagenesis, probably by preventing (inducible?) functions required by the plasmid R46 repair-mutator.     

Interphase development and beginning of mitosis in the different nuclei of polynucleate homokaryotic cells

The degree of synchrony in the course of the interphase periods G₁, S and G₂ and in the initiation of mitosis in the several nuclei of each cell of a polynucleate population induced by treatment with 0.1% caffeine, in root meristems of Allium cepa, through inhibition of cytokinesis in two successive cell divisions is analysed by means of labelling with ³H-thymidine.—The S period is initiated simultaneously in all the nuclei of each polynucleate cell, which supports the hypothesis of a factor present in the cytoplasm that is responsible for inducing DNA synthesis.—However, all the nuclei in a polynucleate cell do not pass from the S period to the G₂ period simultaneously, those surrounded by the greatest amount of cytoplasm, generally the outer nuclei, being the first to complete the S period (early nuclei) and beginning the prophase before their fellow-nuclei in the same cell (late nuclei).—From the metaphase onwards, however, all the nuclei in a polynucleate cell continue to develop synchronously. The synchronizing mechanism has a twofold aspect: the shortening of the G₂ period in the late nuclei and the lengthening of it in the early ones and, on the other hand, an arrest of prophase in the early nuclei until the late ones have caught up, which suggests the existence of an inhibiting factor produced by the late nuclei capable of acting upon the early ones through the cytoplasm.     

Genetic variation in the subunits of globulin-1 storage protein of French bean

Summary Charge and molecular weight heterogeneity of globulin-1 (G1) polypeptides of the bean, Phaseolus vulgaris L., were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Different bean cultivars were classified into three groups: Tendergreen, Sanilac, and Contender on the basis of their protein subunit composition. Nine distinct major bands: 51,49, 48.5,48^T, 48^S, 47, 45.5, 45^S, and 45^C, and two minor bands: 46^T and 46^S were found to account for the three profiles seen on one-dimensional SDS-PAGE. Two-dimensional analysis revealed these eleven protein bands to be composed of a minimum of fourteen distinct protein subunits. The Tendergreen and Sanilac types differ in their G1 polypeptide composition. The protein patterns of the Contender types are intermediate, containing many protein subunits found in the patterns of the Tendergreen and Sanilac types suggesting a genetic and evolutionary relationship.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

Characterization of recurrent somatic embryogenesis of alfalfa on auxin-free medium

Callus cultures from 300 genotypes of alfalfa (Medicago sativa L.) were initiated from leaf, petiole, and internode explants placed on Blaydes medium containing 10.74 M -naphthaleneacetic acid, 11.42 M indole-3-acetic acid, and 9.29 M kinetin. Five genotypes produced somatic embryos. Upon transfer of these embryos to growth regulator-free Murashige and Skoog medium with B₅ vitamins, new somatic embryos repeatedly formed directly on older somatic embryos without an intervening callus phase in a cycle lasting about 30 days. These cultures have been maintained for two years, during which time their embryogenic capacity has remained stable. New embryogenic cultures could be started repeatedly from these genotypes. The elimination of sugars from the medium could stop recurrent embryogenesis. Glucose, maltose, and fructose stimulated recurrent embryogenesis more effectively than sucrose. Sucrose was superior to lactose, while sorbitol and mannitol did not stimulate recurrent somatic embryogenesis. The absence of nicotinic acid in the medium, as long as sucrose was present, was lethal to embryos of three of the five tested genotypes. The ability of this system to propagate embryos exponentially offers potential for development of new gene transfer systems and application to artificial seed technology.Abbreviations NAA -naphthaleneacetic acid - RSE recurrent somatic embryogenesis     

In situ study of chorion gene amplification in ovarian follicle cells ofDrosophila nasuta

The temporal and spatial pattern of replication of chorion gene clusters in follicle cells during oogenesis inDrosophila melanogaster andDrosophila nasuta was examined by [^3H thymidine autoradiography and byin situ hybridization with chorion gene probes. When pulse labelled with [³H] thymidine, the follicle cells from stage 10–12 ovarian follicles of bothDrosophila melanogaster and,Drosophila nasuta often showed intense labelling at only one or two sites per nucleus.In situ hybridization of chorion gene probes derived fromDrosophila melanogaster with follicle cell nuclei ofDrosophila melanogaster andDrosophila nasuta revealed these discrete [³H] thymidine labelled sites to correspond to the two amplifying chorion gene clusters. It appears, therefore, that in spite of evolutionary divergence, the organization and programme of selective amplification of chorion genes in ovarian follicle cells have remained generally similar in these two species. The endoreplicated and amplified copies of each chorion gene cluster remain closely associated but the two clusters occupy separate sites in follicle cell nucleus.     

Yolk synthesis in ovarian follicles ofDrosophila

Summary The autonomous synthesis of yolk proteins in ovarian follicles ofDrosophila melanogaster was analyzed. Vitellogenic follicles were labelled with³⁵S-methionine in vitro and the newly synthesized yolk proteins were separated by SDS-polyacrylamide gel electrophoresis. Possible contamination of the follicle preparations caused by adhering fat body cells could be excluded by culturing follicles in males prior to labelling in vitro. When labelled follicles were cut at the nurse cell/oocyte border the three yolk proteins (YP1, YP2, YP3) were found only in posterior fragments containing ooplasm and follicle cells, whereas two radioactive protein bands (A and B) were detected in nurse cells (anterior fragments). The yolk proteins of these five bands were characterized by peptide mapping. Band A protein, migrating a little more slowly than YP2, is closely related to both YP1 and YP2 while band B contains a yolk protein which is very similar to YP3. Hence, the nurse cells have been identified as a site of vitellogenin synthesis within the ovary ofDrosophila.Supported by the Deutsche Forschungsgemeinschaft, SFB 46     

Mobilization of the transposable element mdg4 by hybrid dysgenesis generates a family of unstable cut mutations in Drosophila melanogaster

Summary A family of unstable mutations at the cut locus in Drosophila melanogaster was obtained under the conditions of hybrid dysgenesis (Gerasimova 1981, 1982). The in situ hybridization experiments have shown that, in the original unstable ct ^MR2 mutation, the 7B region of the X chromosome (where cut is located) contains a mobile dispersed genetic element, mdg4. All other unstable ct mutations derived from ct ^MR2 including visible and lethal alleles and unstable ct ⁺ reversions, also contain mdg4 in the 7B region. The X chromosomes of the parent strain (wild type) do not contain mdg4 at all. All stable revertants derived from ct ^MR2, from other unstable ct mutations, or from ct lethals lost mdg4 from the 7B region. The ct ^MR2 X chromosome does not contain P-elements, although a few copies are present in the autosomes. The instability of the ct ^MR2./ct ^MR2 strain remained at a high level for 50 generations (1.5 years) and then rapidly decreased. A new cross with an MRh12/Cy strain (originally used for dysgenesis induction and containing a number of P-elements) increased the instability to a level exceeding the original one. The data strongly suggest that unstable ct mutations in our system are induced by transpositions of mdg4, possibly activated by P-elements.     

An EM autoradiographic study on ovarian follicle cells of Drosophila melanogaster with special reference to the formation of egg coverings

Summary Ovarian follicle cells of Drosophila melanogaster have been studied by ultrastructural and autoradiographic analyses.During their migration through the germarium, follicle cells undergo several structural changes and, of these, the most conspicuous one occurs at the level of the nucleolus. By the time the first ovarian chamber is formed, follicle cells have formed a layer of uniform thickness all around a cluster or nurse cells and the oocyte. Following the initiation of vitellogenesis, the follicle cells overlying the oocyte become columnar while those over the nurse cells become very thin. During stages 9–10, the columnar follicle cells are involved in the formation of the vitelline membrane, while from stages 11 to 13 these cells produce the endochorion.An EM autoradiographic analysis has shown that the rate of ³H-uridine incroporation in follicle cell nuclei is low in previtellogenic chambers, while it becomes very high in nuclei of stage 9–10 chambers. After short exposure to uridine, silver grains are located predominantly over nucleoli.Evidence from incorporation studies with ³H-lysine indicates that the columnar follicle cells and the region of the various egg coverings are highly labelled within an hour of incubation in the tracer.The observations confirm that columnar follicle cells are the only cells in the chamber involved in the formation of materials which make up the egg coverings.This work was partly supported by C.N.R. (Italy)I am indebted to Dr. J. Jacob from the Institute of Animal Genetics (Edinburgh) for introducing me to the use of EM autoradiography     

Comparison between crude Interleukin-2 and recombinant Interleukin-2 in maintaining killing activity of cultured lymphocytes

Peripheral blood lymphocytes, regional lymph node lymphocytes or malignant effusion lymphocytes from cancer patients were incubated with crude IL-2 (cIL-2) for 13 days. These effectors, which frequently expressed IL-2 receptor (IL-2R), proliferated well and possessed augmented killing activity against fresh autologous tumor cells and K562. However, when recombinant IL-2 (rIL-2) was added for the last 4 days of culture instead of cIL-2, IL-2R expression and killing activity against fresh autologous tumor cells decreased significantly (P<0.05). Phenotypic analysis indicated that cIL-2 significantly promoted the expansion of the cytotoxic population (CD8⁺ .11b^–)(P<0.05). The decreases in killing activity and IL-2R expression were restored by 0.004% PHA plus rIL-2, but not in the presence of rIFN-, rIL-1, rIL-l, rIL-4 or rIL-6. PHA-free cIL-2 maintained killing activity, but not IL-2R expression.We conclude that some factors in cIL-2 and a low dose of PHA-P are necessary for the maintenance of killing activity and IL-2R expression of cultured lymphocytes in the late phase of culture.     

Cohesive single-stranded ends of Streptomyces temperate bacteriophage R4.

Summary The cohesive single-stranded termini of temperate Streptomyces phage R4 were found to be complementary 11 base single-stranded 3-extended DNAs with the sequence: 5-CGCCGTGTCTT-3 3-GCGGCACAGAA-5     

Nuclear changes induced by the nematodeXiphinema diversicaudatum in root-tips of strawberry

Summary Feeding by the nematodeX. diversicaudatum caused a progressive increase in the DNA content and size of strawberry nuclei. After four days feeding, nuclei had DNA values intermediate between 8C and 16C and had increased in size from a mean of 17 m² for control root tips to 49 m². Multinucleate cells were present after two and four days feeding. There were no ultrastructural differences in the composition of nuclei from control and parasitized root tips, but strawberry nuclei consisted mainly of dispersed chromatin whereas ryegrass nuclei contained a large proportion of condensed chromatin.     

Follicle cell development is partly independent of germ-line cell differentiation in Drosophila oogenesis

Summary The developmental potential of the cells of the somatic follicular epithelium (follicle cells) was studied in mutants in which the differentiation of the germ-line cells is blocked at different stages of oogenesis. In two mutants, sn ^36a and kelch, nurse cell regression does not occur, yet the follicle cells around the small oocyte continue their normal developmental program and produce an egg shell with micropylar cone and often deformed operculum and respiratory appendages. Neither the influx of nurse cell cytoplasm into the oocyte nor the few follicle cells covering the nurse cells are apparently required for the formation of the egg shell. In the tumor mutant benign gonial cell neoplasm (bgcn) the follicle cells can also differentiate to some extent although the germ-line cells remain morphologically undifferentiated. Vitelline membrane material was synthesized by the follicle cells in some bgcn chambers and in rare cases a columnar epithelium, which resembled morphologically that of wild-type stage-9 follicles, formed around the follicle's posterior end. The normal polarity of the follicular epithelium that is characteristic for mid-vitellogenic stages may, therefore, be established in the absence of morphologically differentiating germ-line cells. However, the tumorous germ-line cells do not constitute a homogeneous cell population since in about 30% of the analyzed follicles a cell cluster at or near the posterior pole can be identified by virtue of its high number of concanavalin A binding sites. This molecular marker reveals an anteroposterior polarity of the tumorous chambers. In follicles mutant for both bgcn and the polarity gene dicephalic the cluster of concanavalin A-stained germ-line cells shifts to more anterior positions in the follicle.     

Untersuchungen am Ovar von Bruchidius obtectus Say. (Coleoptera-Polyphaga) zur Klärung des Oocytenwachstums in der Prävitellogenese

Zusammenfassung Im ersten Teil der Arbeit werden die Ovariolen adulter Imagines von Bruchidius obtectus licht- und elektronenmikroskopisch untersucht. Durch den Nachweis von Nährsträngen, die die Oocyten mit den Nährzellen der Endkammer verbinden, konnte erstmals gezeigt werden, daß Bruchidius telotroph-meroistische Ovariolen besitzt. Die Nährzellen, deren Kerne kettenförmige Nukleolen aufweisen, bilden bei den Imagines ein Syncytium, das von einem räumlichen Maschennetz aus interstitiellen Zellen stabilisiert wird. In den Oocytenkernen entsteht während der Prävitellogenese eine Karyosphäre, von der aus Nukleolarkörper, Binnenkörper und segmentierte Längsstrukturen gebildet werden. Die Nukleolarkörper und die kettenförmigen Nährzellnukleolen werden als multiple Nukleolen diskutiert.Der zweite Teil der Arbeit stellt eine ontogenetische Untersuchung des Ovariolengewebes dar. Danach entsteht das Nährzellsyncytium in der Phase der Imaginalhäutung aus einem zellulär-fusomalen Verband. Die morphologische Abgrenzung der Ei- und Nährzellen voneinander sowie die Ausbildung von Nährsträngen finden ebenfalls in dieser Entwicklungsphase statt. Die präsumptiven Ei- und Nährzellen durchlaufen auf dem Puppenstadium das Pachytän der Prophase der Meiose. Damit weisen sich die Nährzellen als Keimbahnabkömmlinge aus.Im dritten Teil der Untersuchungen erfolgt eine Analyse der DNA- und RNA-Synthese sowie des RNA-Transports in dem Ovariolengewebe adulter Imagines. DNA Markierungen mit ³H-Thymidin lassen auf einen, wenn auch geringen, Polyploidiegrad der Nährzellkerne schließen. Markierungen mit ³H-Uridin belegen eine hohe RNA-Syntheserate der Nährzellkerne. Mit nahezu gleicher Intensität wie die Nährzellkerne synthetisieren auch die Oocytenkerne RNA, obwohl sie eine Karyosphäre bilden. Mit Hilfe von Markierungsgradienten im Ooplasma sowie von Nährstrangmarkierungen gelang der Nachweis eines RNA-Transportes von Nährzellsyncytium über die Nährstränge in die Oocyten.Abschließend wird das telotrophe Ovar von Bruchidius (Coleoptera-Polyphaga) dem telotrophen Ovar der Heteropteren gegenübergestellt. Der Vergleich legt eine konvergente Entwicklung dieses Ovartyps bei Insekten nahe.

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The growth of oocytes during previtellogenesis in the ovary of Bruchidius obtectus say. (Colepotera-Polyphaga)

Summary In the first part of the investigation the ovarioles of adult imagines are analyzed by light and electron microscopy. It is shown that nutritive cords connect the oocytes with the apical trophic tissue, demonstrating that Bruchidius has telotroph-meroistic ovarioles. The trophic tissue, in which the nurse cell nuclei contain chain-like nucleoli, is a syncytium stabilized by a three-dimensional network of interstitial cells. During previtellogenesis, a karyosphere is formed in oocyte nuclei in which nucleolar bodies, endobodies, and filament bodies originate. The nucleolar bodies and the chain-like nucleoli of nurse cells are considered to be multiple nucleoli.In the second part, the development of the ovariole tissue during ontogenesis is studied. The syncytial trophic tissue derives from a cellular-fusomal organization during the phase of molting. During the same period, the morphological distinction between nurse cells and oocytes as well as the development of nutritive cords take place. Nurse cells are derived from the germ-line, since, during pupal stages, both the prospective oocytes and the prospective nurse cells undergo the prophase of meiosis up to pachytene.The third part is an investigation of DNA- and RNA-synthesis and RNA-transport in the ovariole tissue of adult imagines. DNA labelling with tritiated thymidine shows a small degree of polyploidisation in nurse cell nuclei. By labelling with tritiated uridine, a high rate of RNA-synthesis could be demonstrated in nurse cell nuclei. A similar amount of RNA-synthesis exists in oocyte nuclei, even if they form a karyosphere. The transport of RNA from the apical trophic tissue via the nutritive cords into the oocytes is demonstrated by silver grain gradients over the ooplasm and by the labelling of nutritive cords.Finally, the telotrophic ovary of Bruchidius (Coleoptera-Polyphaga) is compared with the telotrophic ovary of Heteroptera, suggesting a convergent development of telotroph-meroistic ovaries in insects.

Meinem verstorbenen Lehrer Herrn Prof. Dr. Karlheinz Bier bin ich für die Überlassung des Themas und für die rege Anteilnahme am Fortgang der Arbeit zu großem Dank verpflichtet. Ebenso gilt mein Dank Herrn Prof. Dr. Klaus Heckmann, der mir die Fortsetzung der Arbeit ermöglichte. Für zahlreiche Diskussionsbeiträge und kritische Anmerkungen danke ich Herrn Prof. Dr. Oswald Hess, Herrn Dr. Udo Mays, Herrn Prof. Dr. Karl Müller und Herrn Dr. Fritz Weber. Frau Marianne Unger danke ich für die technische Hilfe bei der Anfertigung des Schemas.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, Progress No. 9 and Green Arrow, and two tall cultivars, Alaska and Alderman, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering Alderman cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

The cytology of the vitellogenic stages of oogenesis in Drosophila melanogaster. I. General staging characteristics

The cytology of the vitellogenic stages in the development of the oocyte of Drosophila melanogaster has been studied using whole mounts and sections of plastic-embedded ovaries and single egg chambers for light microscopy and cytochemistry. The migrations, changes in morphology, and synthetic products of the follicle cells are described as a function of developmental stage. The follicle cells synthesize the egg coverings, the vitelline and chorionic membranes, and elaborate the micropyle and dorsal chorionic appendages. The changing structure of the nurse cell nucleus and changes in organelle composition of its cytoplasm are described. The nurse cells synthesize ribosomes, lipid droplets, and mitochondria. These components pass through the ring canal system into the oocyte, which increases in volume some 200,000 times during its 78 hours of development.