In situ hybridization of nuclear and cytoplasmic RNA to locus 2-48BC in Drosophila hydei

The maximum grain density over the heat-shock locus 2-48BC of Drosophila hydei polytene chromosomes obtained after in situ hybridization of nuclear RNA extracted from tissue culture cells labelled during incubation at 37° C is five times higher than that obtainable by using polysomal RNA isolated from the same cells. Furthermore, the addition of a large excess of unlabelled polysomal RNA reduced the amount of in situ hybridization of nuclear RNA by only 20% showing that nuclear 2-48BC RNA contains sequences not present in polysomal 2-48BC RNA. — The polysomal 2-48BC RNA is polyadenylated, as are the RNA sequences present in the polysomes complementary to the other two major heat shock loci 2-32A and 2-36A. Polyadenylated RNA, with an apparent size of 15S, complementary to locus 2-48BC is also found in the cytoplasm of D. hydei salivary glands.     

A reliable method for extraction of RNA from various conifer tissues

Summary A simple and efficient procedure suitable for extraction of high-quality RNA from cultured conifer tissues, somatic embryos, zygotic embryos, needles, stem and root tissues was developed. It produced from 100 g up to 700 g total RNA per gram tissue dependent on the types of tissues used. RNA quality was estimated by spectrophotometry, agarose gel electrophoresis, in vitro translation of mRNA, cDNA synthesis and Northern blot analysis. The method also worked well with Arabidopsis thaliana and tobacco tissues.Abbreviations CTAB cetyltrimethylammonium bromide - DEPC diethylpyrocarbonate - PVP polyvinylpyrrolidone - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

The relationship between RNA catalytic processes

Proposals that an RNA-based genetic system preceeded DNA, stem from the ability of RNA to store genetic information and to promote simple catalysis. However, to be a valid basis for the RNA world, RNA catalysis must demonstrate or be related to intrinsic chemical properties which could have existed in primordial times. We analyze this question by first classifying RNA catalysis and related processes according to their mechanism. We define: (A) thedisjunct nucleophile class which leads to 5-phosphates. These include Group I and II intron splicing, nuclear mRNA splicing and RNase P reactions. Although Group I introns and its excision mechanism is likely to have existed in primordial times, present-day examples have arisen independently in different phyla much more recently. Comparative methodology indicates that RNase P catalysis originated before the divergence of the major kingdoms. In addition, alldisjunct nucleophile reactions can be interrelated by a proposed mechanism involving a distant 2-OH nucleophile. (B) theconjunct nucleophile class leading to 3-phosphates. This class is composed of self-cleaving RNAs found in plant viruses and the newt. We propose that tRNA splicing is related to this mechanism rather than the previous one. The presence of introns in tRNA genes of eukaryotes and archaebacteria supports the idea that tRNA splicing predates the divergence of these cell types.     

In vitro transcription by isolated nuclei of Rhynchosciara americana salivary glands

A method for the isolation of polytene nuclei from salivary glands cells of the Diptera Rhynchosciara americana is described. The stage-specific morphological pattern of the chromosome is maintained during the isolation. The isolated nuclei show two distinct RNA polymerase activities, namely I and II, characterized on the basis of ionic requirements and -amanitin sensitivity. Studies of the product under the incubation conditions show that the system allows the synthesis of high-molecular weight RNA, beside a low molecular weight peak which may comprise pre-4S and 5S RNAs.-Autoradiographic studies carried out in the presence or absence of the toxin -amanitin showed that micronucleoli contain products of RNA polymerase type I activity (ribosomal RNA) and that the DNA puffs are engaged in -amanitin sensitive RNA synthesis and thus are sites of polymerase type II activity.     

Active transcription of the pseudogene for subunit 7 of the NADH dehydrogenase in Marchantia polymorpha mitochondria

A pseudogene, nad7, which has significant sequence similarity (66.7% amino acid identity) with the bovine nuclear gene for a 49 kDa subunit of the NADH dehydrogenase (NADH:ubiquinone oxidoreductase, EC 1.6.99.3), has been identified on the mitochondrial genome of the liverwort Marchantia polymorpha. The predicted coding region, which includes six termination codons, is actively transcribed into RNA molecules of 16 and 9.6 kb in length, but RNA splicing products were not detected in the liverwort mitochondria. Genomic DNA blot analysis and RNA blot analysis using poly(A)⁺ RNA suggest that a structurally related nuclear gene encodes the mitochondrial ND7 polypeptide. These results imply that this nad7 is a relic of a gene transfer event from the mitochondrial genome into the nuclear genome during mitochondrial evolution in M. polymorpha.Communicated by R. G. Herrmann     

Morphological and cytochemical analysis of an unusual nucleus-pyrenoid association in a unicellular red alga

Summary Protrusions of the nucleus that extend into the pyrenoid of the unicellular red algaRhodella maculata have been characterised morphologically and cytochemically. Serial reconstructions indicate that cells frequently have two tubular nuclear protrusions that are up to 1.2 m long. Cytochemical analyses were undertaken to investigate the nucleic acid content of the protrusions. DNA was not detected in the nuclear protrusions, though clear labelling was seen in all other DNA-containing zones (viz. the main nuclear compartment, the chloroplast and the mitochondria). High concentrations of RNA were observed in the nuclear protrusion. In situ hybridization experiments indicate that ribosomal RNAs are not a major component of the protrusion RNA. Possible roles of the protrusion and its RNA content are discussed.Abbreviations DNA deoxyribonucleic acid - RNA ribonucleic acid - rRNA ribosomal RNA     

Extreme heterogeneity of polyadenylation sites in mRNAs encoding chloroplast RNA-binding proteins in Nicotiana plumbaginifolia

We have previously characterized nuclear cDNA clones encoding two RNA binding proteins, CP-RBP30 and CP-RBP-31, which are targeted to chloroplasts in Nicotiana plumbaginifolia. In this report we describe the analysis of the 3-untranslated regions (3-UTRs) in 22 CP-RBP30 and 8 CP-RBP31 clones which reveals that mRNAs encoding both proteins have a very complex polyadenylation pattern. Fourteen distinct poly(A) sites were identified among CP-RBP30 clones and four sites among the CP-RBP31 clones. The authenticity of the sites was confirmed by RNase A/T1 mapping of N. plumbaginifolia RNA. CP-RBP30 provides an extreme example of the heterogeneity known to be a feature of mRNA polyadenylation in higher plants. Using PCR we have demonstrated that CP-RBP genes in N. plumbaginifolia and N. sylvestris, in addition to the previously described introns interrupting the coding region, contain an intron located in the 3 non-coding part of the gene. In the case of the CP-RBP31, we have identified one polyadenylation event ocurring in this intron.     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

Balbiani ring RNA content and half-life in nucleus and cytoplasm of Chironomus tentans salivary gland cells

The content of RNA with an origin in the Balbiani rings 1 and 2 (BR 1+2) has been determined in chromosomes, nuclear sap and cytoplasm of Chironomus tentans salivary gland cells. Together with information on rate and completeness of export this permits an estimation of half-life of this RNA in cytoplasm and its residence time in the nucleus. The quantities in the BR, nuclear sap, and cytoplasm are roughly related as 110200. The 75 S RNA in the nuclear sap with an origin in the BR 1+2 must to a high extent be a precursor to the cytoplasmic 75 S RNA in vivo. The half-life of the cytoplasmic component is about 20 h and the half-life (residence time) for BR 1+2 RNA in the nuclear sap around one hour. The presence of a large pool of BR RNA in the sap explains the previously observed delay in its cytoplasmic appearance in vivo.     

The yeast,Saccharomyces cerevisiae,RNase P/MRP ribonucleoprotein endoribonuclease family

Ribonuclease P (RNase P) is a ribonucleoprotein responsible for the endonucleolytic cleavage of the 5-termini of tRNAs. Ribonuclease MRP (RNase MRP) is a ribonucleoprotein that has the ability to cleave both mitochondrial RNA primers presumed to be involved in mitochondrial DNA replication and rRNA precursors for the production of mature rRNAs. Several lines of evidence suggest that these two ribonucleoproteins are related to each other, both functionally and evolutionarily. Both of these enzymes have activity in the nucleus and mitochondria. Each cleave their RNA substrates in a divalent cation dependent manner to generate 5-phosphate and 3-OH termini. In addition, the RNA subunits of both complexes can be folded into a similar secondary structure. Each can be immunoprecipitated from mammalian cells with Th antibodies. In yeast, both have been found to share at least one common protein. This review will discuss some of the recent advances in our understanding of the structure, function and evolutionary relationship of these two enzymes in the yeast,Saccharomyces cerevisiae.Abbreviations LRI long range interaction - mt mitochondrial - MRP mitochondrial RNA processing - NME nuclear mitochondrial endonuclease - POP processing of precursor - RNase ribonuclease - SNM suppressor of NME - RNP ribonucleoprotein     

The ultrastructure of the nuclear envelope of amphibian oocytes

Summary In order to investigate the chemical composition of the nuclear pore complexes isolated nuclei from matureXenopus laevis oocytes were manually fractioned into nucleoplasmic aggregates and the nuclear envelopes. The whole isolation procedure takes no more than 60–90 sec, and the pore complexes of the isolated envelopes are well preserved as demonstrated by electron microscopy. Minor nucleoplasmic and cytoplasmic contaminations associated with the isolated nuclear envelopes were determined with electron microscopic morphometry and were found to be quantitatively negligible as far as their mass and nucleic acid content is concerned. The RNA content of the fractions was determined by direct phosphorus analysis after differential alkaline hydrolysis. Approximately 9% of the total nuclear RNA of the matureXenopus egg was found to be attached to the nuclear envelope. The nonmembranous elements of one pore complex contain 0.41×10^–16 g RNA. This value agrees well with the content estimated from morphometric data. The RNA package density in the pore complexes (270×10^–15 g/³) is compared with the nucleolar, nucleoplasmic and cytoplasmic RNA concentration and is discussed in context with the importance of the pore complexes for the nucleo-cytoplasmic transport of RNA-containing macromolecules.Additionally, the results of the chemical analyses as well as of the³H-actinomycin D autoradiography and of the nucleoprotein staining method of Bernhard (1969) speak against the occurence of considerable amounts of DNA in the nuclear pore complex structures.The author thanks Miss Ulrika Lempert, Miss Marianne Winter, and Miss Sigrid Krien for skilful technical help as well as Dr. W. W. Franke for many helpful discussions. The work has been supported by a Deutsche Forschungsgemeinschaft grant given to Dr. W. W. Franke (SFB Molgrudent, 46).     

Ribonuclease P from plant nuclei and photosynthetic organelles

RNase P consists of both protein and RNA subunits in all organisms and organelles investigated so far, with the exception of chloroplasts and plant nuclei where no enzyme-associated RNA has been detected to date. Studies on substrate specificity revealed that cleavage by plant nuclear RNase P is critically dependent on a complete and intact structure of the substrate. No clearcut answer is yet possible regarding the order of processing events at the 5 or 3 end of tRNAs in the case of nuclear or chloroplast processing enzymes. RNase P from a phylogenetically ancient photosynthetic organelle will be discussed in greater detail: The enzyme from theCyanophora paradoxa cyanelle is the first RNase P from a photosynthetic organelle which has been shown to contain an essential RNA subunit. This RNA is strikingly similar to its counterpart from cyanobacteria, yet it lacks catalytic activity. Properties of the holoenzyme suggest an intermediate position in RNA enzyme evolution, with an eukaryotic-type, inactive RNA and a prokaryotic-type small protein subunit. The possible presence of an RNA component in RNase P from plant nuclei and modern chloroplasts will be discussed, including a critical evaluation of some criteria that have been frequently applied to elucidate the subunit composition of RNase P from different organisms.Abbreviations RNase P Ribonuclease P - (pre-)tRNA transfer ribonucleic acid (precursor) - tRNA ^Ser (- ^Tyr , - ^Phe ) transfer ribonucleic acid specific for serine (tyrosine, phenylalanine) - CyRP RNA RNA component of cyanelle RNase P     

Nuclear buoyant density determination and the purification and characterization of wild-type neurospora nuclei using percoll density gradients

A procedure has been developed using Percoll density gradients for the isolation and purification of nuclei from germinated conidia of wild-type Neurospora crassa St. Lawrence strain 74A. Crude nuclei were purified isopycnically in gradients of Percoll, which is silica coated with polyvinylpyrrolidone. A DNA:RNA:protein ratio of 1:3.5:6.5 was found in purified nuclei. Cytoplasmic contamination was found to be negligible in the nuclear preparations, as determined by electron microscopy and by following a radioactively-labeled ribosome tag during the isolation procedure. A small amount of endogenous ribonuclease activity was detected in the crude nuclear preparations, but not in suspensions of nuclei purified in the Percoll gradients. Ribosomal RNA was extracted from the nuclei in good yields, and electrophoretic analysis indicated the presence of precursor rRNA molecules, as well as the mature 17S and 25S rRNA species. Using the Percoll gradient system, the buoyant density of purified Neurospora nuclei was determined to be 1.08 grams per milliliter based on refractive index measurements.     

RNA Polymerase B levels during the cell cycle ofPhysarum polycephalum

Summary Tritiated -amanitin has been used as a specific and sensitive probe to estimate the number of RNA polymerase B molecules in isolated nuclei, chromatin and nucleoids, obtained from macroplasmodia ofPhysarum polycephalum. During mitosis (metaphase±10 min) there is at least 10-fold less RNA polymerase B than at all phases of the cell cycle, even if DNA replication has been blocked in vivo. It is concluded that many of the RNA polymerase B molecules leave the chromatin during decondensation of the chromosomes in telophase of the synchronous nuclear division ofPhysarum.     

Protoplast culture and female plant regeneration in the dioecious plant Melandrium album

Conditions for protoplast isolation and culture in the dioecious species Melandrium album (Silene alba) are described and differences between female and male plants subjected to in vitro culture identified. Plant regeneration was achieved in the case of female plants only. Selection for high regeneration capacity lines was initiated aiming at creating responsive experimental material for further studies. The regeneration behaviour in this dioecious plant is discussed in the context of hormonal models on sex determination.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - 2iP N⁶-[2-isopentenyl]adenine - NAA -naphthalene-acetic acid - PVP polyvinylpyrrolidone     

Two related,low-temperature-induced genes from Brassica napus are homologous to the human tumour bbc1 (breast basic conserved) gene

In order to identify genes involved in cold acclimation, we have constructed a cDNA library from Brassica napus (cv. Samouraï) cold-acclimated etiolated seedlings. By differential screening, a cDNA clone named pBnC24 (Brassica napus Cold), corresponding to a new cold-inducible plant gene, was isolated. Northern blot hybridizations using total RNA from acclimated and unacclimated seedlings confirmed that BnC24 represents a cold-regulated gene. In contrast with a number of cold-inducible plant genes, BnC24 does not seem to be responsive to abscisic acid (ABA). In addition, further screening of the cold-acclimated cDNA library using pBnC24 cDNA as a probe, allowed the isolation of a second type of homologous cDNA. Sequence analysis showed that the two BnC24 genes encode basic 24 kDa proteins, which are highly hydrophilic and rich in alanine, lysine and arginine. The nucleotide and deduced amino acid sequences of these clones do not show any homology with other previously described cold-induced plants genes. However they have strong homology with a recently discovered human tumour gene, bbc1 (breast basic conserved), which seems to be highly conserved in eukaryotes.     

Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 M 6-benzyladenine, 4.5 M 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid monohydrate - PVP polyvinylpyrrolidone - PDS potassium dextran sulfate - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate     

The expanding realm of ballistosporous yeasts

The recent progress in the systematics of ballistosporous yeasts is discussed. The extensive isolation studies carried out in the last decade resulted in a marked increase in the number of ballistosporous yeast species which now number nearly fifty. The increased number of species has expanded the complexity of taxonomic properties in the following ways: expansion of the range of mol% G+C of nuclear DNA to 39–68.5, thus overlapping that of all basidiomycetous yeasts; increased complexity of conidiogenesis with the finding of the generaBallistosporomyces andKockovaella. Based on partial sequencing of 18S ribosomal RNA (positions 1451–1618 inSaccharomyces cerevisiae), it is suggested that mode of conidiogenesis has little value for defining genera. Consequently, ballistosporous yeasts merely represent the ballistosporous stage of various taxa which cover the whole evolutionary spectrum of basidiomycetous yeasts. The importance of continuing isolation of ballistosporous yeasts is stressed, which together with molecular studies, will aid further progress in the systematics of basidiomycetous yeasts.     

The separation of solids from the liquefied mash of cassava tuber and continuous saccharification by immobilized glucoamylase

Summary The solid material in liquefied mash of cassava tuber was very efficiently separated by a mixture of Trichoderma cellulase and Aspergillus niger pectinase. The solid content of the residue after the treatment and centrifugation decreased from 29.5% to 7.0%. The transparent digested solution from cassava tuber after centrifugation was continuously saccharified by glucoamylase immobilized in a gel which was prepared using polyvinylpyrrolidone and -ray irradiation. The addition of 50 ppm of sulfite ion completely prevented microbial contamination during the 18 days of operation. The final DE (dextrose equivalent), glucose content and disaccharide content in the hydrolyzate were 98, 94.4 and 3.3%, respectively.