Sequence homology to the Drosophila per locus in higher plant nuclear DNA and in Acetabularia chloroplast DNA.

In plant cells a DNA sequence was found which is homologous to the Drosophila per locus. In rape and spinach the homologous sequence occurs in the nuclear but not in the chloroplast genome while in Acetabularia it is found in the chloroplast but not in the nuclear genome. A 1.175 kb EcoRi-SalI fragment of the chloroplast genome of Acetabularia containing the homologous sequence was subcloned into pUC12 and sequenced. The core of the 1.175 kb fragment is a repetitive tandemly arranged sequence of 43 units of the hexamer GGA ACT coding for glycine and threonine.

Individual selection, culture and manipulation of higher plant cells.

Due to the heterogeneity in morphology, physiological and morphogenetical capabilities of higher plant cells in mass culture, the development of methods for individually culturing defined cells seemed to be useful and necessary. Individual cell culture represents a powerful tool for studies on the physiology of different cell types, the analysis of differentiation programs, the genetic manipulation of plant cells and cell-cell interactions. An improved microculture system based on a computer-controlled set-up for the efficient selection, transfer and individual culture of defined higher plant cells until regeneration of whole plants is described. Related experimental approaches for individually manipulating higher plant cells under controlled conditions, such as electrofusion of defined pairs of protoplasts and subprotoplasts, cell reconstruction and intranuclear microinjection of protoplasts and karyoplasts - mainly performed with cells of the crop plant Brassica napus L. - are presented.

Evidence for a circaseptan and a circasemiseptan growth response to light/dark cycle shifts in nucleated and enucleated Acetabularia cells, respectively.

Nucleated as well as enucleated Acetabularia mediterranea cells were subjected to 14 different patterns of shifts in a regimen of 12 hr of light alternating with 12 hr of darkness in four 30-day long experiments. With one exception, which might be due to a circannual modulation, these experiments showed that nucleated cells had maximal growth rates when a shift was performed every 7th or 15th day. In enucleated cells, maxima were observed on shift schedules that were about 3-4 days rather than about 7 days apart. The results indicate that in the unicellular green alga Acetabularia a rhythm of about 7 days (circaseptan) exists and that removal of the nucleus results in a circaseptan frequency multiplication.

High yield and stable transformation of the unicellular green alga Acetabularia by microinjection of SV40 DNA and pSV2neo.

SV40 DNA and pSV2neo were microinjected into isolated nuclei of Acetabularia mediterranea. The injected nuclei were implanted into anucleate cell fragments of the same species. Such combinations not only survived but also formed progeny. The F(1), F(2) and F(3) generations of these combinations were analyzed. In the case of SV40-treated cells T-antigen was expressed and accumulated in the nuclei of all three generations studied as shown by indirect immunofluorescence. Nuclear exchange experiments revealed expression of the T-antigen only if a transformed nucleus but not if only a transformed cytoplasm was involved. Transformation by pSV2neo, a chimeric gene with a selectable marker was demonstrated by the induction of G-418 resistance as well as immunofluorescence. Genomic DNA was isolated from gametes, originating in cysts from the F(1), F(2) and F(3) generations of injected cells, and subjected to Southern analysis. These experiments demonstrated that both types of DNA are integrated into the host genome.

Increased sensitivity in antigen detection during immunoblot analysis resulting from antigen enrichment via immunoprecipitation.

The sensitivity in antigen detection during immunoblot analysis is greatly increased if the antigen is first immunoprecipitated from the crude extract before electrophoresis and transfer to nitrocellulose. Not only does the method allow detection of antigens which are minor components of crude mixtures or antigens which cannot be radiolabeled, but the method also resolves problems, such as high background, which are often associated with immunoprecipitation. Also, by modifying the method, whether or not monoclonal antibodies recognize the same or different antigens and/or epitopes can be easily determined.

Perinuclear dense bodies: characterization as DNA-containing structures using enzyme-linked gold granules.

Cytoplasmic structures of the Acetabularia cell that appear close to the nucleus during the vegetative phase of the life cycle have been characterized cytochemically. The results obtained by using RNase and DNase linked to gold granules indicate that RNA and also DNA occur in these cytoplasmic structures.

Synchronization of protoplasts from Glycine max (L.) Merr. and Brassica napus (L.).

Cells of Glycine max originating in a suspension culture and cells of Brassica napus prepared from hypocotyls were synchronized. Synchronization was achieved by preparing protoplasts in the usual way and subsequently letting the protoplasts regenerate into cells by removing the cell-wall-digesting enzymes. More than 70% of the cells had divided synchronously at the end of the first cycle as determined by the mitotic index. The high frequency of mitosis critically depended on the osmolality of the medium. The duration of the S-phase was estimated by measuring the activity of thymidylate kinase as well as incorporation of [(3)H]deoxythymidine into acid-insoluble material. The data indicate that synchronization is induced by resetting the cell cycle.

Differences in amino acid sequence of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from two species of Dasycladaceae.

In contrast to other plants the plastid genome of Acetabularia is larger in size and shows a high degree of variability. This study on the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase demonstrates that strongly conserved areas also exist in the plastid genome of the Dasycladaceae. Searching for differences in the amino acid sequence of the large subunit from Acetabularia mediterranea and Acicularia schenckii, proteolytic peptides which differ in their elution behaviour in reverse-phase high-performance liquid chromatography were sequenced. Only six amino acids were found to be exchanged in the large subunit from these two species. Since these two species diverged approx. 150 million years ago, these results imply that 0.84 amino-acid exchanges per 100 amino acids have occurred in 10(8) years, underlining the strong conservatism of the large subunit.

Cytosolic protein kinase activity associated with the maturation of the malaria parasite Plasmodium berghei.

Seven cytosolic phosphoproteins with relative molecular masses of 110, 58, 52, 46, 38, 36 and 34kDa and isoelectric points between 4.2 and 5.0 are identified from the rodent malaria parasite Plasmodium berghei. Similar patterns of phosphorylated proteins are obtained from parasite cytosol after incubation of intact infected erythrocytes with [32P]orthophosphate, or from parasite cytosol incubated with [gamma-32P]ATP. The characteristics of the phosphorylation reaction are similar to the previously described Plasmodium protein kinase [Wiser, M.F., Eaton, J.W. and Sheppard, J.R. (1983) J. Cell. Biochem. 21, 305-314], suggesting that the same protein kinase is involved. More protein phosphorylation activity is associated with the mature parasites than the immature forms, suggesting that these phosphoproteins may play some role in the parasite's erythrocytic stage cycle.

Identification of a high molecular weight polypeptide that may be part of the circadian clockwork in Acetabularia.

In the chloroplast fraction of the unicellular and uninucleate green alga Acetabularia, we have detected a M(r) approximately 230,000 protein (p230) whose synthesis exhibits a pronounced endogenous diurnal rhythm. As judged by scanning densitometry of fluorographs of NaDodSO(4)/polyacrylamide gels, the synthesis of other proteins in the same fraction was independent of the time in the cycle. The incorporation of [(35)S]methionine into p230 was completely inhibited by cycloheximide, whereas chloramphenicol had no effect. This strongly suggests that p230 is translated on 80S ribosomes. Eighthour periods of exposure to cycloheximide produced a shift in the phase of the oscillation of p230 synthesis. The results are consistent with the hypothesis that p230 is essential for expression of circadian rhythms in Acetabularia.

Tandemly repeated nonribosomal DNA sequences in the chloroplast genome of an Acetabularia mediterranea strain.

A purified chloroplast fraction was prepared from caps of the giant unicellular green alga Acetabularia mediterranea (strain 17). High molecular weight DNA obtained from these chloroplasts contains at least five copies of a 10-kilobase-pair (kbp) sequence tandemly arranged. This unique sequence is present in DNA from chloroplasts of all stages of the life cycle examined. A chloroplast rDNA clone from mustard hybridized with some restriction fragments from Acetabularia chloroplast DNA but not with the repeated sequence. An 8-kbp EcoRI-Pst I fragment of the repeated sequence was cloned into pBR322 and used as a hybridization probe. No homology was found between the cloned 8-kbp sequence and chloroplast DNA from related species Acetabularia crenulata or chloroplast DNA from spinach.

Regulation of a ribonucleoside reductase during the early generative phase in Acetabularia.

The activity of a ribonucleoside reductase was estimated during the life cycle of Acetabularia. During the early generative phase the enzyme activity was dramatically increased. Regulation of the ribonucleoside reductase was observed even in the absence of the nucleus. The increase in activity was inhibited by chloramphenicol but not by cycloheximide. These results indicate that the enzyme is translated on 70 S ribosomes.

Enzymic ribonucleoside reduction at the non-phosphorylated level in Acetabularia.

The occurrence of an enzyme that catalyses the conversion of cytidine into deoxycytidine was demonstrated in homogenates of Acetabularia. Cytidine was identified as the substrate by comparing cytidine, cytidine 5'-monophosphate, cytidine 5'-diphosphate and cytidine 5'-triphosphate as potential substrates. Experiments with ATP analogues whose inhibitory effect on kinase reactions is well established, supplied evidence that the nucleoside is reduced without a phosphorylation step before the reduction. Further evidence in this line came from incubations with cytidine in the presence of phosphatase and from trap-type experiments in which the effects of excess non-labelled cytidine 5'-phosphate and deoxycytidine, respectively, on the formation of deoxycytidine phosphates from cytidine were studied.

Methotrexate-resistant Somatic Cells of Glycine max (L.) Merr.: Selection and Characterization.

A total of 70 methotrexate-resistant cell lines were isolated from UV-irradiated fast growing suspensions of Glycine max(L.) Merr. The resistant cells grew in 4 X 10(-4) M MTX without significant reduction in growth rate, whereas 100 % of wild type cells died in 2.2 x 10 (-7) M within 24 h. The frequency of resistant cell clones was 2 x 10(-8) per cell generation. Two different selection strategies, a single-step and a multiple-step protocol yielded phenotypically identical resistant lines. All resistant lines have retained their resistance over more than 500 cell generations in the absence of selection. Resistant cells plated on MTX could be selected from an excess of wild type cells. Ten percent of the resistant lines internalized MTX at a reduced rate. DHFR activity in wild type and resistant cells was equally sensitive to inhibition by MTX. Resistant cells did not display an increased activity of DHFR. Likewise MTX binding to cell extracts was the same for all cell lines. In the wild type MTX-blockage of cell division could be partially released by 2 x 10(-4) M thymidine. In the presence of MTX thymidine had no influence on the growth of resistant cells.

Possible translocation of a gene for thymidylate kinase from the chloroplast to the nuclear genome during evolution.

On the basis of the fact that in Acetabularia three out of four enzymes that are involved in deoxyribonucleotide metabolism are translated on 70 S ribosomes, while a fourth one is synthesized on 80 S ribosomes, the suggestion was made that translation on 80 S ribosomes might reflect an evolutionarily more recent situation while translation on 70 S ribosomes might be of more ancient origin. This hypothesis prompted experiments aimed at defining the site of translation of thymidylate kinase in a species closely related to Acetabularia but long ago separated from it: Batophora oerstedii. It was demonstrated that in this species, in contrast to a number of Acetabulariae, thymidylate kinase is translated on 70 S ribosomes. A possible, appealing interpretation of this finding is that during evolution the coding site for thymidylate kinase has been translocated from the chloroplast to the nuclear genome.

Enhancer-controlled expression of the simian virus 40 T-antigen in the green alga Acetabularia.

Nuclei from Acetabularia mediterranea were isolated, microinjected with simian virus 40 (SV40) DNA and fused with cytoplasts from the same species. Various times after fusion of the injected nuclei the fusion products were screened for expression of the T-antigen by indirect immunofluorescence. One and two days after injection a bright fluorescence could be observed in the nuclei of Acetabularia. On the basis of this immunofluorescence we conclude that in Acetabularia cells the T-antigen is expressed and accumulated in the nucleus. Moreover, evidence is presented that the Acetabularia cell recognizes the SV40 enhancer sequence. The expression product of the SV40 DNA appears significantly earlier than the expression products of other foreign genes in Acetabularia. The results suggest that the well characterized SV40 can be used as a vector system for the introduction and expression of foreign genes in Acetabularia.

Circadian temperature rhythm and circadian-circaseptan (about 7-day) aspects of murine death from malaria.

About-7-day (circaseptan) and circadian rhythms were sought and found in host-parasite relations of mice infected with Plasmodium berghei. Five inbred male DBA mice, about 18 weeks of age, were implanted with transsensors for temperature telemetry. Core temperature, monitored every 10 min for 3 days before the intravenous or intraperitoneal inoculation of 10(5) infected erythrocytes and thereafter until death, was analyzed by cosinor. A statistically highly significant circadian rhythm exhibited similarly synchronized acrophases. Core temperatures on the days immediately after malarial infection were mostly within the range of temperatures observed before injection. A mesor-hypothermic stage preceded death by several days. In a second study, 24 male BALB/c and 42 male DBA mice, 12 weeks of age, housed in three rooms on different regimens of light and darkness (alternating at 12-hr intervals), staggered by 8 hr, were inoculated ip with 10(4) infected erythrocytes, one-half at noon, the other half at midnight, within 0.5 hr of blood withdrawal. Thus, one endeavored to cover six circadian host stages (02, 06, 10, 14, 18, and 22 hr after light-on). At 54 and 51% overall mortality (irrespective of inoculation time), a circadian rhythm in susceptibility to malaria was demonstrated in these mice by the single cosinor fit of a 24-hr period (P less than 0.003 and less than 0.020, respectively). The single cosinor fit of a 7-day period further demonstrated a circaseptan rhythm (P = 0.014) in the mortality of both strains following the inoculation of P. berghei. The acrophase (360 degrees = 168 hr) was at -325 degrees from the inoculation time with 95% confidence limits extending from -276 to -378 degrees. Such predictable time relations of P. berghei to its murine host await the exploration of mechanisms underlying the circadian and infradian (7-day) rhythmicities here demonstrated and quantified with their uncertainties. Irrespective of mechanisms, information on such periodicities may also guide attempts to optimize treatment by timing according to the interactions of plasmodial virulence and host resistance that remain to be quantified separately.