Cytoplasmic RNP Research Articles

Instability of the anteroposterior axis in spontaneous double abdomen (sda), a genetic variant of Chironomus samoensis (Diptera, Chironomidae)

ABSTRACT We have isolated a laboratory strain of Chironomus samoensis in which determination of the anteroposterior egg polarity is disturbed. Most conspicuous is the spontaneous formation of ‘double abdomen’ embryos where head and thorax are replaced by a mirror image of the abdomen. Such double abdomens are found in about half of the egg clusters in this strain, which we call the spontaneous double abdomen (sda) strain as opposed to the normal (N) strain. Also observed in the sda strain, although less frequently, are ‘double cephalon’ embryos showing a mirrorimage duplication of cephalic segments in the absence of thorax and abdomen. Moreover, embryos from the sda strain tend to form cells at the anterior pole resembling the pole cells at the posterior pole. Reciprocal crossings between the sda and the N strain indicate that the sda trait is inherited maternally. Spontaneous double abdomen formation is correlated with signs of disturbed egg architecture, including extruded yolk and detached cells. Double cephalons can also be generated by centrifuging embryos from the N strain, whereas centrifugation of sda embryos produces mostly double abdomens. Double abdomen formation can be induced experimentally by anterior u.v. irradiation of embryos from either strain. The sda trait and u.v. irradiation act in a synergistic fashion. The data suggest that the sda trait may be caused by one or more genomic mutations interfering indirectly with the activity of anterior determinants, i.e. cytoplasmic RNP particles necessary for the development of anterior segments. The sda defects may be ascribed to alterations in cytoskeletal components involved in anchoring anterior determinants and segregating them into anterior blastoderm cells.

Prosomes are involved in the repression of viral mRNA.

Prosomes are small cytoplasmic RNP complexes. We present evidence that their RNA is a potential and selective inhibitor of viral mRNA translation while translation of normal cellular mRNA e.g. rabbit globin mRNA or HeLa cell mRNA is not affected.

Distribution and utilization of 5 S-RNA-binding proteins during the development of Xenopus oocytes.

At early stages of oogenesis in Xenopus laevis most of the ribosomal 5S RNA is complexed with three proteins to form two types of cytoplasmic RNP storage particle. A particle sedimenting at 42S contains 5S RNA and tRNA together with two proteins of Mr 48000 (P48) and Mr 43000 (P43) and a second particle sedimenting at 7S contains 5S RNA plus a protein of Mr 40000 (P40, also known as the transcription factor, TFIIIA). In this report we use antibodies monospecific for each protein to follow the movement of 5S RNA from nucleus to cytoplasm to nucleolus to cytoplasm and to determine the fate of each of the proteins that associate with 5S RNA during these transitions. Both P48 and P43 have roles additional to the formation of the 42S RNP storage particle; P48 is detected in the nucleus during early oogenesis and is cleaved to yield an Mr-33000 fragment that remains associated with 5S RNA that is excess to ribosome requirement during late oogenesis; P43 appears to be cleaved to yield fragments of Mr 28000 and 17000, the latter being present in ribosomal fractions. Apparently, there is no function for P40 in addition to those already described in transcription of 5S RNA genes and in storage of 5S RNA as a 7S RNP particle.

Association of cap binding protein-related polypeptides with cytoplasmic RNP particles of chick embryonic muscle

Cap binding protein (CBP)-related polypeptides were identified in different cytoplasmic RNP particles of embryonic chick muscles using monoclonal antibody to purified CBP. A single immunoreactive peptide ( M r 78000) was present in preparations of both free mRNP particles and a novel 10 S translation inhibitory RNP particle. In contrast, proteins isolated from these particles showed two new low- M r immunoreactive peptides ( M r 43000 and M r 29000). No CBP related protein could be detected in polysomal mRNP, although an immunoreactive M r 43000 CBP-related protein was present in polysomes. The relevance of the association of different CBP-related polypeptides with cytoplasmic RNP particles and polysomes are discussed.

Analysis of reconstruction of an RNP particle which stores 5S RNA and tRNA in amphibian oocytes.

Previtellogenic oocytes of Triturus cristatus accumulate a free cytoplasmic RNP which sediments at 40S and contain 5S RNA and tRNA in association with two proteins of MW 45,000 and 39,000 daltons (P45 and P39). The 40S particle has a buoyant density of 1.53 g . cm-3 in CsCl and consists of four identical RNP subunits. Each monomeric subunit contains one molecule of 5S RNA, three molecules of tRNA, two molecules of P45 and one molecule of P39. The 40S particle can be completely dissociated by SDS treatment into its individual components, and the subunits, and even the complete 40S particle, can be reformed by removal of SDS in the presence of 0.2 M NaCl. RNA/protein binding experiments with isolated components, and analysis of reformed RNP complexes in CsCl gradients, demonstrate that the stable interactions are: 5S RNA/P45, 3(tRNA)/P45, 5S RNA/P39 and 5S RNA/P45/P39. Immunological studies show that P45 has also a nuclear location and may bind to the 5S RNA transcript in the chromatin, whereas P39 is predominantly cytoplasmic and is possibly related to proteins associated with 5S RNA in the ribosomal 60S subunit. It is suggested that the 40S RNP particle not only stores 5S RNA and tRNA but also provides a means for the exchange of the 5S RNA transcript binding protein (P45) for the 5S RNA ribosome associated protein (P39).

Mobile dispersed genetic element MDGI of Drosophila melanogaster: transcription pattern

Virtually the whole region of the mobile dispersed genetic element mdg1 including the direct repeats that frame mdg1 /1/ is transcribed. The end of the transcriptional unit is located within the direct repeat. Although the transcription of mdg1 is symmetric, one direction is transcribed preferentially and is a full-length transcript represented by approximately 29S poly(A)+RNA. Besides it, approximately 15S poly(A)+RNA is present in the cytoplasm of culture cells which corresponds to the 3'-part of 29S RNA and possibly to a small region near the 5'-end of the latter. The major fraction of mdg1 transcripts is present in the fraction of free cytoplasmic RNP particles while only 10-20% are recovered in polysomes. In this respect, mdg1 is similar to mdg3 and quite different from two single-copy genes transcripts of which are almost completely recovered in polysomes. The relationship between mobile dispersed genetic elements and endogeneous viral genomes is discussed.

Studies on informational RNAs and ribonucleoproteins of normal rat liver, Zajdela hepatoma and liver of hepatoma-bearing rats

DNA-like RNA (D-RNA) species, both as constituents of RNP particles and protein-free molecules, isolated from normal rat liver, Zajdela ascites hepatoma and liver of tumor-bearing animals were compared with respect to their sedimentation and chromatographic behaviour. A selective labelling of D-RNA was carried out under actinomycin D-induced block of rRNA synthesis. No detectable differences in nuclear D-RNAs of the tissues compared could be observed whereas cytoplasmic D-RNAs of hepatoma and liver of tumor-bearing rats appear to be much more heterogeneous as compared to those of normal liver. The kinetics of the labelled D-RNAs appearing in the cytoplasm in case of malignant cells also differs from that characteristic of normal liver cells. The comparative analysis of cytoplasmic RNP complexes in the sucrose density and CsCl isopycnic band centrifugation revealed a shift in the ratio of polyribosome-bound and free complexes in favour of the latter in the tumor and liver of tumor-bearing rat as compared with normal liver. The data presented are interpreted to indicate a certain impairment in the selective nucleus-to-cytoplasm transport of RNA in tumor cells, the cells of tumor-bearing animal occupying in this respect an intermediate position between normal and tumor tissues.

Membrane associated poly (A)-containing ribonucleoproteins in Artemia Salina gastrula embryos

The embryonic development of Artemia salina is often interrupted by a period of dormancy at the gastrula stage. Although no endogeneous protein synthesis could be detected in these encysted embryos [ 11, the presence of stored mRNA has been demonstrated [2-4]. Messenger activity is associated with both poly(A)+-RNA and poly(A)--RNA extracted from the membrane fraction but only with the poly(A)-RNA of the free cytoplasmic ribonucleoproteins [3]. Poly(A)*-RNA of the cytoplasmic RNP is complexed with a translational inhibitor RNA, and messenger activity is restored if the inhibitor RNA is dissociated from the mRNA by the use of EDTA during the fractionation procedure [ 51. We have shown by sucrose gradient centrifugation that free cytoplasmic poly(A)+RNPs exhibited a heterogeneous distribution consisting of several discrete peaks ranging from 20-120 S [4]. Approximately 50% of the RNPs sedimented faster than 50 S and were complexed with ribosomes or ribosomal subunits. The presence of EDTA converted all these poly(A)*-RNPs into free 20-40 S particles. We demonstrate here that the poly(A)‘-RNPs of the membrane fraction exhibit a different size distribution on sucrose gradients as compared with the

Antibodies to cytoplasmic antigens in lupus erythematosus

Seven patients with classic cutaneous lupus erythematosus are described. Three of these patients had features satisfying four of the American Rheumatism Association (ARA) preliminary criteria for the diagnosis of systemic lupus erythematosus (SLE). Their sera, however, lacked antinuclear antibodies but demonstrated precipitating antibodies reactive against cytoplasmic RNP (La) and non-nucleic acid (Ro) antigens. Four additional ANA-negative patients lacking significant skin disease but having a lupus-like multisystem disease were found to have antibodies to soluble cytoplasmic antigens. Thirty-three of 130 ANA-positive SLE patients, but none of 16 discoid lupus patients, possessed these anticytoplasmic antibodies. These findings suggest that antibodies to Ro and La may be a marker for systemic disease in ANA-negative patients with 1) cutaneous lupus and 2) a distinct subpopulation of patients with a lupus-like syndrome without skin disease.

Long-Term Persistent Vesicular Stomatitis Virus and Rabies Virus Infection of Cells In Vitro

BHK 21 carrier cells persistently infected with VSV Indiana for over 2 years have been shedding generally very low levels of mature infectious virus or mature T particles (averaging less than one-hundredth p.f.u./cell/day) yet most cells are producing virus antigens and are resistant to homologous superinfection. However, large amounts of biologically active T particle RNP can be recovered from cytoplasmic extracts of these carrier cells even at times when they are shedding no detectable infectious virus. This recovered cytoplasmic RNP replicates (with helper B virions) to produce mature T particles, interferes strongly after DEAE dextran-facilitated uptake and, together with B virions, allows the establishment of a persistent carrier state in exposed cells. No 'provirus' DNA copies of the VSV RNA genome are detectable (less than 1/40 copy/cell or I copy per 40 cells) in carrier cells after more than 2 years of persistent infection, and all transfection attempts have failed using DNA from these VSV carriers or DNA from carrier cells persistently infected with some other negative strand RNA viruses (measles, mumps, LCM, influenza, rabies). Infectious viruses shed after more than I year from carrier cells originally infected with wild-type B virions are small plaque mutants showing a slight temperature sensitivity. Cured cell populations can be obtained from the long term VSV carrier culture by cloning in the presence or absence of antiviral antibody.

Differences in the fractional composition of cytoplasmic RNP particles in Zajdela ascites hepatoma cells and the liver cells of animals with tumors

The relative content of poly(A)-RNA in the cytoplasm is higher in cells of Zajdela's ascites hepatoma and of the liver of rats with tumors than in normal rat liver cells. Another distinguishing feature of the tumor cells (and also, to a lesser degree, of the liver cells of animals with tumors) is a change in the ordinary ratio between polyribosomes and monoribosomes (and, correspondingly, between mRNP particles and informosomes) for normal liver cells in favor of the latter, indicating the occurrence of definite changes in their protein-synthesizing apparatus. According to some of the indices investigated, cells of tumor-bearing animals occupy an intermediate position between normal and tumor cells.

RNase sensitivity of an anterior morphogenetic determinant in an insect egg (Smittia sp., Chironomidae, Diptera).

In chironomid midges, the development of head and thorax in the embryo requires the function of cytoplasmic determinants localized near the anterior pole of the egg. Experimental inactivation of these determinants causes a dramatic switch in the developmental program of the embryo. Instead of the normal segment pattern, the aberrant pattern "double abdomen" is formed. Head, thorax, and anterior abdominal segments are then replaced by an additional set of posterior abdominal segments joined in mirror image symmetry to the original abdomen. Such double abdomens have been produced, with a maximum yield of 29%, by application of RNase (ribonuclease I, ribonucleate 3'-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) to the anterior pole region of the egg. This was achieved by microinjection or puncturing the eggs during submersion in RNase. Control experiments with inactive RNase S fragments reliably proved that double abdomen formation resulted from RNase activity. Neither application of other enzymes to the anterior pole region nor application of RNase to other egg regions produced double abdomens in significant yields. The effects of RNase concentration and stage of development were determined. The data from these and earlier experiments are compatible with the idea that stored cytoplasmic RNP particles act as anterior determinants. Similarities to genetically caused switches in developmental pathways (homeotic mutations) are discussed.

Nuclear RNA in sea urchin embryos: I. Some characteristics of ribonucleoprotein complexes

Using cesium chloride gradient analysis, we have examined the kinetics of labeling and some physical properties of nuclear ribonucleoprotein complexes in sea urchins. Our results strongly indicate that these complexes are not artifacts of procedure but are definite members of chromatin that are readily distinguishable from cytoplasmic RNP complexes. The nuclear complexes can be separated into two major classes: (1) A class in which the protein to RNA ratio is at least 4:1; (2) a second class in which the protein to RNA ratio is much less. Using [ 3H]uridine, long-term labeling and short pulses of [ 32P], we have studied the labeling kinetics of RNA in the two classes. The nuclear RNA which is associated to a high degree with protein turns over very rapidly; over 70% of the nuclear RNA labeled in a short pulse appears in this fraction, whereas the nuclear RNA synthesized in long-term pulses is present to a greater extent in complexes containing a much smaller proportion of protein. The difference in the rate of turnover of RNA in these two classes may be significant in the regulation of RNA processing in the nucleus.

Leucoplast of the cryptomonad Chilomonas paramecium: Evidence for presence of a true plastid in a colorless flagellate

Chilomonas paramecium is a colorless cryptomonad flagellate which contains a leucoplast. The structural features of the leucoplast parallel those shown in the cryptomonad chloroplast, exemplified by Cryptomonas ovata. An electron microscopic study of this colorless organelle shows it to be divided into two compartments: the outer compartment contains starch granules and 220 Å ribosomes similar to cytoplasmic RNP particles. The inner compartment often contains carotenoid-like masses, 160 Å ribosomes significantly smaller than those in the outer compartment, and DNA-like filaments. Cesium-chloride density gradients of the whole-cell DNAs show three DNase-sensitive satellites in addition to the major fraction. One of them is presumed to be associated with the leucoplast. Evidence for the existence of true plastids in bleached and colorless flagellates is reviewed.

Changes in hepatic ribonucleoproteins in kwashiorkor.

Using the toluidine blue-ammonium molybdate staining method, different types of RNPs were demonstrated in the hepatocytes of patients with kwashiorkor. DCR-I and DCR-III were found to be reduced. Nucleolar chromatin and nucleolar parachromatin showed an apparent increase. Chromosomal, perichromosomal and nuclear parachromatin remained unaffected. Granular cytoplasmic RNP was nonreactive in all the cases.

Potassium permanganate staining of ultrathin sections for electron microscopy

Ultrathin sections of Araldite- and Maraglas-embedded tissues may be cleanly stained by submersion for 30 minutes in a freshly prepared 1.0% aqueous solution of KMnO4. Results resemble those achieved with lead stains. By submersion of the sections throughout the staining and washing procedure, contamination of the specimen is avoided. Capricious precipitation of the stain, granularity of the section, tissue damage, and fragility of the section in the electron beam (reported disadvantages of KMnO4 as an electron stain) have not been encountered. Destructive effects associated with KMnO4 staining may be due to excessive or indiscriminate use of agents, e.g., citric acid, to remove precipitated stain. Use of such bleaching agents is unnecessary after properly executed staining procedures. The only disadvantage encountered is failure of cytoplasmic RNP particles to stain completely after OsO4 fixation.