Site and timing of synthesis of tubulin and other proteins during oogenesis in Drosophila melanogaster

Protein synthetic patterns during oogenesis in Drosophila melanogaster were examined; in particular the site, time, and rate of tubulin synthesis and accumulation during oogenesis were determined. Ovarian proteins were labeled with [³⁵S]methionine in vivo or in organ culure in vitro, and the proteins synthesized in egg chambers of specific developmental stages displayed by two-dimensional gel electrophoresis. A dissection technique was devised to examine proteins synthesized in each of the three cell types present in stage 10B egg chambers. The majority of proteins which were resolved by two-dimensional gel electrophoresis, including tubulin and actin, were synthesized throughout oogenesis and, at least to some extent, in each of the stage 10B cell types. Protein synthesis specific to developmental stage and/or cell type was also observed; for example, two nonchorion proteins were synthesized only in follicle cells and primarily at stage 10. A sensitive and specific radioimmune assay was developed in order to quantitate tubulin accumulation. Synthesis of several α-tubulin subunits and one β-tubulin subunit was observed. The tubulin content per egg chamber increased from 3 ng in stage 9 to 17 ng in stage 14, a period of about 13 hr. An accumulation rate of 1 ng/hr suggests that tubulin mRNA can account for about 4% of the total, nonmitochondrial, poly(A)⁺ RNA of the egg. Analysis of separated cell types at stage 10B revealed that both the follicle and nurse cells synthesize and accumulate appreciable amounts of tubulin. The stage 10B oocyte contains relatively little tubulin but actively synthesizes it. These two complementary analyses demonstrate that the tubulin present in the egg is synthesized within the oocyte-nurse cell syncytium, first in the nurse cells and later in the oocyte.     

Modulation of a neural antigen during metamorphosis in Drosophila melanogaster

Transplantation of the acousticolateral placode to the evacuated eye position in embryos of the frog Rana pipiens has been used to force axons of the VIIIth cranial nerve to penetrate the diencephalon. These ectopic axons establish a growth trajectory that is strikingly similar to their normal growth trajectory within the medulla oblongata despite the fact that no other axons within the diencephalon normally follow this route. The result is discussed in terms of the "blueprint" and substrates pathway hypotheses which have been advanced to explain the initial development of axon tracts within the central nervous system.     

Changing rates of DNA and RNA synthesis in Drosophila embryos

Rates of DNA and RNA synthesis during Drosophila embryogenesis were measured by labeling octane-treated embryos with [¹⁴C]thymidine and [³H]uridine. Radioactivity incorporated per hour was converted to rates of synthesis using measurements of the pool-specific activity during the labeling periods. The rate of DNA synthesis during early embryogenesis increases to a maximum at 6 hr after oviposition and then decreases sharply. Measured rates of DNA synthesis were used to calculate that the total amount of DNA per embryo doubles every 18 min at blastoderm, every 70–80 min during gastrulation, and less than once every 7 hr at later stages. The rate of RNA accumulation per embryo increases continuously during the first 14 hr of embryogenesis. The rate of nuclear RNA synthesis per diploid amount of DNA, however, decreases fivefold between blastoderm and primary organogenesis. The cytoplasmic poly(A)⁺ RNA synthesized by blastoderm embryos associates rapidly with polysomes. The relatively high rate of synthesis of polysomal poly(A)⁺ RNA per nucleus at blastoderm allows the small number of nuclei present at blastoderm to make a significant quantitative contribution to the informational RNA active in the early embryo. At the end of blastoderm, approximately 14% of the mRNA being translated in the embryo has been synthesized after fertilization.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

Regulation of DNA synthesis in cytochalasin B (CB)-induced binucleate HeLa cells

The effects of timing and duration of cytochalasin B (CB) treatment on the kinetics of the initiation of DNA synthesis in mono- and binucleate HeLa cells, synchronized in the G1 phase of the cell cycle by the reversal of a mitotic block (N₂O at 80 PSI), were studied. In the control, bi-, tri- and tetranucleate cells entered S phase slightly earlier than the mononucleate cells at a rate proportional to the number of their nuclei. The difference between any two adjacent sub-populations was less than 0.5 h. However, the binucleate cells produced by a 90 min CB treatment immediately after the reversal of the mitotic block exhibited a considerably shorter G1 period as compared to mononucleate cells (a difference of 1.5 h). This exaggerated difference in the duration of G1 period between mono- and binucleate cells disappeared when the CB treatment was delayed by 75 or 90 min indicating that it was an experimental artifact. From this study, we conclude that there is naturally some degree of nuclear cooperation in the multinucleate systems, particularly with regard to the initiation of DNA synthesis, which is not influenced by CB treatment.     

Identification of vitelline membrane proteins in Drosophila melanogaster

In Drosophila melanogaster, proteins involved in vitelline membrane production are secreted by ovarian follicle cells during stages 9 and 10 of oogenesis. We have used SDS-PAGE and two-dimensional electrophoresis to identify six major size classes of radiolabeled components in purified vitelline membrane preparations. Analyses of in vivo labeled proteins from egg chambers of different developmental stages and stage 10 follicle cells show that components of five of these size classes are synthesized by follicle cells during the period of vitelline membrane deposition. Immunological analysis of eggshell antigens utilizing complex antisera raised to purified eggshell fragments has confirmed the identity of components of three size classes.     

Chromatin proteins of sea urchin embryos: Dual origin from an oogenetic reservoir and new synthesis

The chromatin proteins of different embryonic stages, ranging from 16 cell to gastrula, of the sea urchin Strongylocentrotus purpuratus were labeled, in vivo, with ¹⁴C and were labeled, in vitro, with ³H. The proteins thus labeled were separated by high resolution two-dimensional electrophoresis. The extent of possible cytoplasmic contamination has been examined with reconstruction experiments. Gastrula chromatin contains over 200 separable nonhistone proteins, and about 90% of them are also detected at the 60-cell stage; cleavage stages have over all protein gel patterns displaying numerous differences with the pattern shown by chromatin from later stages. Differences in the proportion of histone to nonhistone proteins that are synthesized are observable at the different embryonic stages, with histones predominating in midcleavage. About half of the nonhistone proteins of the developing embryo that can be labeled with ³H, in vitro, are not labeled with ¹⁴C, in vivo, and hence, must originate from a reservoir of nonhistone proteins assembled during oogenesis.     

Determination of the rates of synthesis and degradation of vitamin D-dependent chick intestinal and renal calcium-binding proteins

Vitamin D₃ and its biologically active metabolite 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] are shown to induce in the chick intestine and kidney the biosynthesis of a calcium binding protein (CaBP). In vitamin D₃-replete chickens raised under adequate dietary calcium (Ca) and phosphorus (P) conditions, the steady-state level of intestinal CaBP (30–50 g/mg protein) is 5- to 20-fold greater than that of renal CaBP. Whereas dietary phosphorus restriction is known to elevate both intestinal and renal CaBP levels, dietary calcium restriction elevates only intestinal CaBP. The present study reports the rates of biosynthesis in vivo and in vitro, and of biodegradation in vivo, of both intestinal and renal CaBP after administration of vitamin D₃ or 1,25(OH)₂D₃ to rachitic chicks. The apparent rate constant of degradation for intestinal CaBP was 0.024 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 29}\text{h}$) and that for renal CaBP was 0.019 h^?1 ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 36}\text{h}$) while total cellular soluble protein in the intestine and kidney had half-lives of 43 and 70 h, respectively. The time course of induction of the synthesis of CaBP was determined in intestine and kidney after administration of a physiological dose of 1,25(OH)₂D₃ to rachitic chicks. Intestinal CaBP synthesis was detectable by 3 hours, reached a maximal rate by 10 hours, and sharply decayed by 16–20 hours. The time course of induction of renal CaBP synthesis was very similar, although the rate of renal CaBP synthesis was readily detectable at the initial time of administration of 1,25(OH)₂D₃. The relative rates of synthesis of CaBP in the intestine and kidney under a variety of dietary Ca and P conditions in the vitamin D₃-replete chick exactly paralleled the steady-state level of CaBP in these two tissues. These results are consistent with a model in which the steady-state levels of intestinal and renal CaBP are solely determined by their respective rates of biosynthesis; the CaBP biosynthetic capability, in turn, is regulated by the availability of 1,25(OH)₂D₃ to each target organ.     

Rescue of blocked cells by reinnervation in denervated forelimb stumps of larval Ambystoma

Cells of amputated, denervated larval Ambystoma forelimbs dedifferentiate and enter the cell cycle but do not subsequently proliferate sufficiently to form a blastema. The denervated limb stump resorbs slowly until reinnervation stimulates regeneration. We used this system to investigate the fate of cells in denervated limbs which undergo early but limited cycling in response to amputation. In Experiment 1, cells were labeled with [3H]thymidine (3H-T) on Day 4 postamputation (PA)/Day 3 postdenervation (PD). Labeled cells were still present on Day 7 PA, but were less frequently observed on Day 13 PA when the limbs were reinnervated and beginning to regenerate. In Experiment 2 we denervated 1 day preamputation to obtain earlier reinnervation and prevent loss of Day 4 PA labeled cells. Cells labeled with 3H-T on Day 4 PA/Day 5 PD were present throughout the denervation period and most were still present on Day 13 PA. Little or no mitotic activity was found among the labeled cells after the initial round of cycling. The apparent cell cycle block was released upon reinnervation on Days 12 and 13 PA when cycling resumed. Labeled mitotic figures were present on Day 13 PA, and the mitotic index of the labeled population increased as a result of reinnervation. These results demonstrate that blocked cells are rescued by nerves, re-enter the cell cycle, and thus contribute to the reinnervation blastema.     

A library of monoclonal antibodies to nuclear proteins from Drosophila melanogaster embryos. Characterization by a cultured cell assay

To study the structure and function of the cell nucleus, a library of 170 monoclonal antibodies was produced to nuclear antigens from 3-6 h old Drosophila embryos. In preparation for immunization, nuclei were separated, at neutral pH and in the presence of polyamines, into two fractions containing either urea-soluble non-histone nuclear proteins or histones plus small quantities of non-histone proteins complexed to DNA. The antibodies were characterized in a rapid, indirect immunofluorescent assay employing cultured Drosophila cells (Schneider's line 2). Low backgrounds and high specific fluorescence were achieved in this assay by purifying the rhodamine-labelled second antibody on a polystyrene resin and washing the cells with optimal concentrations of detergents. The assay categorized antigens according to their cellular locations: in nuclei, in nuclei plus cytoplasm, or primarily in cytoplasm. A subset of nuclear antigens reacted specifically with the nuclear envelope. In addition, some antibodies were characterized by their reactions with polytene chromosomes. The cultured cell assay provides a new, efficient method for expanding this antibody library. The monoclonal antibodies in the library now provide highly specific tools for investigating structural nuclear proteins and proteins that may be regulatory during embryonic development.     

Rapid appearance of multivesicular bodies in the cortex of Drosophila eggs at ovulation

Following activation of Drosophila oocytes, acid phosphatase-positive multivesicular bodies (MVB) rapidly form in the cortex. From observations of eggs during the first few minutes of activation, MVBs are thought to form from invaginations of the oolemma. Since there is no evidence for turnover of vitellogenin receptors at earlier times, it is postulated that these receptors are synchronously removed at activation.     

RNA synthesis and coding capacity of polyadenylated and nonpolyadenylated mRNA from cultures of differentiating Drosophila melanogaster myoblasts

We have investigated the synthesis and coding capacity of RNA isolated from cultures of differentiating Drosophila embryonic muscle cells. We find that following muscle cell fusion, the sedimentation profile of newly synthesized polyadenylated RNA becomes somewhat lighter. In vitro translation products analyzed by two-dimensional gel electrophoresis indicate that the coding capacity of translatable myogenic mRNA changes during differentiation. A group of several muscle-specific proteins (including the contractile proteins) is translated only from mRNA isolated after the initiation of fusion. This pattern coincides with proteins synthesized in vivo during differentiation. Additionally, we find that polyadenylated and nonpolyadenylated myogenic mRNA from a given developmental stage in culture have extremely similar coding potentials.     

A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster

Individual segments of the chromosomal DNA in D. melanogaster were isolated, and the sequences they contain were analyzed for repetition and mapped within the polytene chromosomes. Isolation was achieved by first constructing circular hybrid DNA molecules consisting of single chromosomal segments linked by poly(dA):poly(dT) joints to single molecules of the tetracycline resistance plasmid, pSC101. Tetracycline-sensitive E. coli were transformed to resistance by this heterogeneous population of hybrid molecules and homogeneous populations of different hybrids were isolated from the clones of transformants. Three hybrid plasmids (pDm 1, 2, and 4) were studied in detail. Each exhibits the structure expected from the method of construction and none exhibits internal sequence repetition detectable by reassociation kinetics. The D. melanogaster sequences in pDm2 and 4 belong to a single class defined by little or no repetition within the genome and localization to a single chromomeric region in the polytene chromosomes. The characteristics of this class, which also includes 4 of a second set of 6 hybrids, are not compatible with tandem repetition models for the chromomere. The sequences in pDm1 are repeated 90 times and are located in 15 different chromomeric regions and within the chromocentric β-heterochromatin. This distribution is of the kind predicted by certain regulatory models, for example, that of Britten and Davidson (1969).     

Drosophila: the genetics of two major larval proteins.

A series of irradiation-induced deficiencies covering 62 polytene chromosome bands in chromosome arm 3L of Drosophila melanogaster includes the loci of two abundant developmentally regulated larval proteins. The structural gene for larval serum protein 2 (LSP 2) lies at 68E3 or 4, and that for salivary glue secretion protein 3 between 68A8 and 68C11, coincident with a major intermoult puff active in the salivary gland at the time of glue synthesis. The structural genes for esterase 6 and four visible recessive loci lie within the same region.     

Temperature sensitivity of muscle and neuron differentiation in embryonic cell cultures from the Drosophila mutant, shibire.

The sex-linked temperature-sensitive mutation, shibire^ts1, which causes, at the restrictive temperature, adult paralysis and pleiotropic morphological defects in embryonic, larval, and pupal development, has been shown to exhibit temperature-sensitive inhibition of differentiation in embryonic cultures in vitro. When shi cultures were incubated at 30°C for 24 hr, both muscle and neuron differentiation were inhibited more than 90% compared to control shi cultures incubated at 20°C. Heat shift experiments showed that the temperature-sensitive periods for neuron and muscle differentiation occurred at 11 to 18 and 14 to 16 hr, respectively, where zero time was the initiation of gastrulation in donor embryos. Short heat pulses (4 and 8 hr) which extended into the temperature-sensitive period resulted in moderate inhibition of differentiation; greater inhibition occurred as the duration of the pulses increased. In contrast, heating wild-type Oregon-R cultures at 30°C for 24 hr did not inhibit muscle cell differentiation and inhibited neuron differentiation relatively little. The temperature-sensitive period in shibire for muscle differentiation occurred well after myoblast division, during the period of myocyte elongation, aggregation, and fusion, whereas that for neuron differentiation took place during a period of enzyme synthesis (acetylcholinesterase and choline acetyltransferase) and axon elongation. Thus, the shi temperature-sensitive gene product affects at least two different cell types, in vitro, at different times during differentiation.