Biochemical characterization of the RNA-hydrolytic activity of a pumpkin 2S albumin

A pumpkin 2S albumin with ribonuclease (RNase) activity was purified from pumpkin seeds (Cucurbita sp.) by liquid chromatographic techniques. It manifested potent RNase activity toward baker’s yeast RNA and calf liver RNA, and some polyhomoribonucleotides, including poly(A), poly(U) and poly(C) but not poly(G). Moreover, it was able to hydrolyze total RNA of both animal and plant origins. Ions such as Na⁺, Mg²⁺, Ca²⁺, and Zn²⁺ inhibited its RNase activity. Since RNase activity has not been previously reported in 2S albumins, this work may shed further light on the biological importance of this group of proteins.     

The Effect of Red Irradiation on Plastid Ribosomal RNA Synthesis in Dark-grown Pea Seedlings

Dark-grown pea seedlings (Pisum sativum L.) were irradiated for a short period each day with low intensity red light (662 nm), red light immediately followed by far red light (730 nm), or far red light alone. Other plants were transferred to a white light regime (14 hours light/10 hours dark). There was no change in the amount of RNA in the tissue on a fresh weight basis after the various treatments. However, compared with dark-grown seedlings, those plants irradiated with red light showed an increase in the net RNA content per stem apex. In addition there was a two- to three-fold increase in ribosomal RNA of the etioplasts relative to the total ribosomal RNA. These increases were comparable to those found in plants grown in the white light regime. The changes were much smaller if the dark-grown plants were irradiated either with red light followed by far red light, or with far red light alone. Thus continuous light is not essential for the production of ribosomal RNA in plastids, and the levels of ribosomal RNA found in chloroplasts can also be attained in etioplasts of pea leaves in the dark provided the leaf phytochrome is maintained in its active form.     

The detection of single-stranded RNA in an isometric virus-like particle from Shiitake mushroom [Lentinus edodes (Berk.) Sing.]

Isometric virus-like particles (VLPs) with diameter of approximately 34 nm containing ss-RNA were purified from abnormal mycelium of Shiitake mushroom, Lentinus edodes (Berk.) Sing. SDS-polyacrylamide gel elec-trophoresis demonstrated that the virions contain a single capsid polypeptide with molecular weight of about 22 000 daltons. The nucleic acid extract from purified VLPs preparations showed only one band with a size of approximately 7.3 kilobases. The susceptibility to RNase 1 and S1 digestions, resistance to DNase and thermal denaturation behaviour of the viral genome indicated that it is a single-stranded RNA. To our knowledge, isometric single-stranded RNA VLPs isolated from Shiitake mushroom mycelia have not been reported before.     

Enhanced expression of a distinct plastid DNA region in mustard seedlings by continuous far-red light

Fragments of chloroplast DNA from mustard (Sinapis alba L.) seedlings, generated by the restriction endonuclease Eco RI, were used to assess the frequency of complementary sequences in mustard RNA by DNA/RNA hybridization. A pronounced increase in hybridization to a single DNA fragment was found with RNA from seedlings irradiated with continuous far-red light, compared to RNA from dark-grown seedlings.     

PATTERNS OF GROWTH AND CELL COMPOSITION OF FRESHWATER ALGAE IN LIGHT-LIMITED CONTINUOUS CULTURES1

Selenastrum minutum (Näg.) Collins and three species of Scenedesmus were grown in light-limited continuous cultures. Comparative growth patterns were explained in terms of differences in the rates of light absorption and utilization. For all species, increasing light intensity or lowering temperature caused chl a/C to decrease and RNA/C to increase. Similar shifts in pigment composition have been widely reported. However, the RNA:light interaction has only been reported infrequently, and the RNA:temperature interaction is believed to be the first reported observation of its kind in algae. Distinct differences in RNA/C among the test algae may be size-dependent. Similar environmentally induced shifts in chl a/C and RNA/C among all test algae point to the existence of a common control mechanism which maximizes the specific growth rate through adjustments to cell composition.     

Purification of 5S RNA from Plant Leaves

A simple and rapid method for large scale preparation of 5S RNA from plant leaves is described. To begin, all nucleic acids were extracted from the leaves with a mixture of phenol-chloroform-n-butyl alcohol and extracting buffer. After precipitation of the high-molecular-weight nucleic acids from the crude extract with 2 M LiCl, the low, molecular-weight RNA in the supernatant (containing about 6% 5S RNA) could be separated by eleetrophoresis on denatured polyaerylamide gels. The band of 5S RNA was excised from the preparatory slab gel under UV light and then purified by eleetrophoretie elution. In our experiments, several mg pure 5S RNA was obtained from 100 g leaves in a single run which takes about 4 days. The purified final produet was pure as showing a single hand on denatured polyaerylamide gel and a typical UV absorption peak of nucleic acid. The sedimentation coefficient (S20w) of the product was 4.6 as determined by ultracentrifugation.     

Effect of light on cell elongation,nucleic acid and protein synthesis in hypocotyls of Lupinus angustifolius

Summary The synthesis of DNA, RNA, protein and dry matter was followed in the elongating cells of hypocotyl of Lupinus (Lupinus angustifolius L.) germinating in total darkness or in continuous light. Light strongly inhibits the synthesis of DNA, RNA and protein. It was shown by histofluorometric DNA determinations that a reduced synthesis of DNA in continuous light is accompanied by a lower level of endomitosis in the cortex cells of the hypocotyl. In the dark cortex nuclei become 8 C while in the light they become only 4 C. The more pronounced differences between dark and light germinated hypocotyls of lupinus in comparison with pea epicotyls is explained to a great extent by mitotic growth.     

Effect of Bacteriophage R17 Infection on Hostdirected Synthesis of Ribosomal Ribonucleates

Studies were performed on the synthesis of ribosomal ribonucleates in cells of Escherichia coli K-12 infected by the ribonucleic acid (RNA) bacteriophage R17. Host-specific RNA was measured in the presence of phage RNA by in vitro hybridization of the purified ribonucleates with E. coli deoxyribonucleic acid. The results showed that, although the overall rate of RNA synthesis was only slightly affected by phage infection, the level of host RNA synthesis was decreased by 70 to 80%. Fractionation of the purified ribonucleates by sucrose gradient sedimentation, followed by hybridization of fractions sedimenting in the 23S and 16S regions, revealed that the level of ribosomal RNA synthesis was also decreased by 70 to 80%, and that this inhibition occurred during the first 15 to 20 min after infection. These findings are discussed in light of what is known about the inhibition of host RNA synthesis by other virus systems.     

Capacity for RNA synthesis in 70S ribosome-deficient plastids of heat-bleached rye leaves

In the leaves of rye seedlings (Secale cereale L.) grown at an elevated temperature of 32°C the formation of plastidic 70S ribosomes is specifically prevented. The resulting plastid ribosome-deficient leaves, which are chlorotic in light, represent a system for the identification of translation products of the 80S ribosomes among the chloroplastic proteins. Searching for the primary heat-sensitive event causing the 70S ribosome-deficiency, the thermostability of the chloroplastic capacity for RNA synthesis was investigated. The RNA polymerase activity of isolated normal chloroplasts from 22°-grown rye leaves was not inactivated in vitro at temperatures between 30° and 40°C. The ribosome-deficient plastids purified from bleached 32°-grown leaf parts contained significant RNA polymerase activity which was, however, lower than in functional chloroplasts. After application of [³H]uridine to intact leaf tissues [³H]uridine incorporation was found in ribosome-deficient plastids of 32°C-grown leaves. The amount of incorporation was similar to that in the control chloroplasts from 22°C-grown leaves. According to these results, it is unlikely that the non-permissive temperature (32°C) causes a general inactivation of the chloroplastic RNA synthesis in rye leaves.     

Purification of immunuglobulin light chain messenger RNA by immunoprecipitation from mouse myeloma tumor, MOPC-31C.

Polysomes producing IgGl(kappa) myeloma protein were specifically selected by an immunoprecipitation method, and immunoglobulin light chain mRNA was purified from the precipitated polysomes. The purified mRNA migrated predominantly as a single band and the molecular weight of this mRNA was calculated to be 410.000 by polyacrylamide gel electrophoresis in 98% formamide. A protein possessing a molecular weight of 25,000, which is the size of the light chain precursor, was synthesized as a major product of translation in a wheat germ cell-free system. DNA complementary to the mRNA (cDNA) was prepared with avian myeloblastosis virus RNA-dependent DNA polymerase. This cDNA had an average size of 8.3S as determined by sedimentation through an alkaline sucrose gradient. Using this cDNA, Crt 1/2 values of template RNA and RNA from various preparations were calculated from the results of molecular hybridization. The relative content of the mRNA increased 4,4-fold during the immunoprecipitation of polysomes.     

Phytochrome-mediated light regulation of nitrate reductase expression in squash cotyledons

In etiolated squash (Cucurbita maxima L.) cotyledons, nitrate-inducible NADH:nitrate reductase activity and protein were increased in darkness by red light pulses with red/far-red photoreversibility. Continuous far-red light also led to increased levels of nitrate reductase activity and protein. Poly(A)+RNA, which hybridizes to squash nitrate reductase cDNA, was also increased by light treatments. Thus, we found that after nitrate triggering, nitrate reductase expression appears to be regulated by light via phytochrome.     

Transient down-regulation of phytochrome mRNA abundance in etiolated cucumber cotyledons in response to continuous white light

Phytochrome mRNA abundance decreased to 20% of the initial level in etiolated cucumber (Cucumis sativus L.) cotyledons exposed to continuous white light. Unexpectedly, by 12 hours of continuous white light, phytochrome mRNA had reaccumulated to 60% of the control level. High stringency RNA blot analyses suggest that it is the mRNA encoding type I phytochrome that reaccumulates.     

Transcription of chromatin from mouse fibroblasts

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended «light and a compact «heavy component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II).Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.     

Electrophoretically homogeneous myeloma light chain mRNA and its translation in vitro

mRNA coding for the light chain of a myeloma protein has been purified to give one band in acrylamide gel electrophoresis. This pure RNA (S～13.5) could be translated $\text{in}\text{vitro}$ into the light chain in a heterologous cell-free translation system. The light chain synthesized $\text{in vitro}$ is apparently slightly larger than the light chain secreted by the tumor.     

Transcription of chromatin from mouse fibroblasts.

Chromatin purified from mouse fibroblasts can be fractionated after shearing by sedimentation in a sucrose gradient into an extended "light" and a compact "heavy" component. Further purification of these classically described components can be achieved by a second cycle of centrifugation of the light and heavy components through an equilibrium density gradient of metrizamide. The light component purified from sucrose gradient sediments faster (peak I) on metrizamide than its heavy counterpart (peak II). Template activity for DNA directed RNA synthesis in the presence of E. coli RNA polymerase is negligible in peak II but very pronounced in the peak I fraction. The difference in template activity appears to be connected with differences in propagation rather than initiation rates. Comparison of gel electrophoresis patterns of proteins indicate that the active subcomponent includes high molecular weight components not present in the inactive one, but that its histone content is somewhat lower. Using a very highly sensitive automatic recording apparatus for the measurement of melting profiles, no clear cut difference has been observed in the behaviour of active and inactive chromatin subcomponents nor in that of their total DNA.     

Activity of multiple light chain genes in murine myeloma cells producing a single, functional light chain

Two cloned lambda 1-producing myelomas (HOPC-1, MOPC-104E) contain rearranged kappa genes and levels of mature-sized kappa RNA comparable to those found in kappa-producing myeloma cells. Another lambda 1-producing myeloma tumor line (HOPC-2020) and a lambda 1-containing B cell leukemia line (BCL1) also contain significant levels of kappa RNA. One lambda 11-producing line (MOPC-315) contains no detectable kappa RNA, but it also has no kappa genes in the embryonic configuration. kappa-related proteins are not detectable in the lambda 1-producing lines by standard procedures, but by sensitive methods at least two lines contain kappa protein fragments. The MOPC-104E line produces both a 14.5K kappa fragment that is not readily detectable because of its low rate of synthesis and short half-life (T 1/2 less than 5 min), and a major 16.5K protein that lacks kappa cross reactivity but is demonstrable by translation of purified MOPC-104E kappa RNA. The HOPC-1 kappa RNA also encodes a short-lived 14K kappa fragment. The MPC-11 line, which produces a mature kappa RNA and protein as well as an 800 base kappa fragment RNA and kappa protein fragment, has both kappa alleles rearranged, one apparently aberrantly between J and C kappa. Two different kappa RNA species, one the same size as the MPC-11 kappa fragment RNA, frequently are present in kappa RNA-containing Abelson murine leukemia virus-transformed lymphoid cells as well as in 18 and 19 day murine fetal liver. For light chains, neither allelic nor isotype exclusion is generally evident in myeloma and lymphoma cells; rather both produce only a single functional light chain. Models of light chain activation must explain restriction by considering the functional properties of the light chain rather than light chain gene expression.