Ribsome and messenger specificity in protein synthesis by bacteria

Ribosomes from Escherichia coli and Pseudomonas fluorescens, two Gram-negative bacteria, translated messenger RNA preparations from all bacteria tested (3 Gram-negative species, and 6 Gram-positive species) as well as f2 RNA and T4 early messenger RNA. Ribosomes from Clostridium pasteurianum, Streptococcus faecalis, and Bacillus subtilis, three Gram-positive organisms, did not translate messenger RNA preparations derived from any of the Gram-negative organisms, the f2 RNA, or the T4 early messenger RNA but did translate messenger preparations from all 6 Gram-positive bacterial species tested.     

A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

The translation of rabbit hemoglobin messenger RNA in a cell-free extract from chick embryo brain

The translation of rabbit hemoglobin messenger RNA in an unfractionated cytoplasmic extract from chick embryo brain was studied. This translation was not dependent upon reticulocyte-specific factors. An analysis of the product synthesized in vitro with the embryo brain cell-free extract and rabbit hemoglobin messenger RNA by carboxymethyl cellulose chromatography showed that the system was capable of synthesizing both the α and β globin chains. Analysis of the tryptic peptides of the in vitro synthesized α chain by ion-exchange chromatography showed that the embryo brain extract with rabbit hemoglobin messenger RNA was capable of synthesizing the complete α chain of rabbit hemoglobin. The results suggest that no stringent tissue-specific controls exist for the translation of globin messenger RNA and were discussed in this context.     

In vitro synthesis of phase-specific flagellin of Salmonella

Chromatography of Salmonella flagellin at pH 8 on DEAE-cellulose separated at least four serologically distinct kinds of flagellin, a, enx, i and 1,2, eluting in that order with increasing concentration of sodium chloride. By this chromatographic technique, the preincubated cell-free extract of Escherichia coli given saltprecipitable RNA of Salmonella was shown to synthesize flagellin characteristic of the flagellar antigen type of the cells from which the RNA was derived. Two of the in vitro synthesized flagellins specifically reacted with their corresponding antiserum.When RNA was extracted from the cells of the diphasic strain propagated from a single colony, expressing either phase 1 or phase 2, the in vitro synthesized flagellin was predominantly the same as that produced by the original colony. Translation of messenger RNA specific for phase 1 flagellin was not inhibited by the presence of messenger RNA specific for phase 2. RNA extracted from the cells of a diphasic strain without any selection directed synthesis of both phase 1 and phase 2 flagellins in the ratio expected if the culture was at equilibrium with respect to phase variation. Experimental evidence is presented to support the hypothesis that phase variation is due to the alternative synthesis of phase-specific messenger RNA.     

Auxin- and ethylene-induced changes in the population of translatable messenger RNA in Basal sections and intact soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated basal sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. Within one hour of 2,4-dichlorophenoxyacetic acid treatment, the translatable messenger RNAs for at least twelve in vitro translation products are modulated upward. In vitro translation products of polyadenylated RNA from untreated, auxin-treated and Ethephon-treated intact soybean hypocotyl were also analyzed. Within two hours of treatment with either 2,4-dichlorophenoxyacetic acid or Ethephon, the translatable messenger RNAs for a group of high molecular weight in vitro translation products are modulated upward. There is a particular set of translatable messenger RNA, encoding in vitro translation products in the 24,000 to 32,000 molecular weight range, that is specifically modulated upward by auxin treatment in intact soybean hypocotyl and in hypocotyl sections.     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

Comparative Study of Ribosomal Ribonucleic Acid Cistrons in Enterobacteria and Myxobacteria

Deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybrids are formed by Escherichia coli 16S or 23S ribosomal RNA or pulse-labeled RNA with the DNA of various species of the Enterobacteriaceae. The relative extent of hybrid formation is always greater for ribosomal RNA. These DNA-RNA hybrids have been further characterized by their stability to increasing temperature, and, in every case, the stability of pulse-labeled RNA hybrids was lower than that of the corresponding ribosomal RNA hybrids, although 16S and 23S ribosomal RNA hybrids had very similar stabilities. Therefore, ribosomal RNA showed a greater degree of apparent conservation in base sequence than pulse-labeled or messenger RNA both in the extent of cross-reaction and in the stability of hybrid structures. Similar results were obtained with Myxococcus xanthus RNA. Since in this case the base composition of the pulse-labeled or messenger RNA is richer in guanine plus cytosine than ribosomal RNA, the higher cross-reaction of ribosomal RNA is more readily attributable to conservation of base sequence in these cistrons than to its base composition. Thus, the base sequence of ribosomal RNA cistrons of bacilli, enteric bacteria, and myxobacteria is conserved relative to those of the rest of the genomes. This conservation is, however, not absolute since the stability of heterologous ribosomal RNA hybrids is always lower than that of homologous hybrids.     

Isolation and sequence analysis of sea urchin (Lytechinus pictus) histone H4 messenger RNA

Histone messenger RNAs isolated from early blastula stage Lytechinus pictus sea urchin embryos have been separated into discrete RNA bands on polyacrylamide gels. The most rapidly migrating of these molecules, the putative histone H4 mRNA, has been digested with T₁ ribonuclease to generate oligonucleotides for nucleotide sequence analysis. Many of these sequences are colinear with the highly conserved amino acid sequence of histone H4 protein as determined for both cows and peas.Histone H4 messenger RNA hybridizes in conditions of DNA excess to sea urchin DNA which is repeated approximately 470-fold. Despite this level of repetition the nucleotide sequence of the H4 messenger RNA reflects little evolutionary divergence within the H4 genes of L. pictus as judged by the stoichiometric yield of T₁ oligonucleotides and the hybridization and thermal stability of histone H4 mRNA-DNA hybrids.     

Alteration of Coding Properties of Polysome-Associated Messenger RNA in Potato Tuber Slices during Aging

Kinetics of polysome formation, translational capacity, and coding properties of polysome-associated messenger RNA were investigated in potato tuber tissue discs during aging. Polysome content rapidly increased immediately after slicing from 14% of total ribosomes in freshly sliced discs to 55% within 12 hours of aging. The amount of polysomal RNA also increased 5-fold during this period. Translational capacity of polysome-associated messenger RNA increased in parallel with the increase in content of polysomal RNA of the tissue discs when measured in a wheat germ cell-free system. Analysis of the in vitro translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the majority of polypeptides coded by the messenger RNA did not vary greatly during the period of rapid polysome formation. Three types of messenger RNA were found to change in amount during that period: those which appeared only after aging, those which disappeared during aging, and those which disappeared early but reappeared later in the aging period.     

Auxin-induced changes in the population of translatable messenger RNA in elongating sections of soybean hypocotyl

In vitro translation products of polyadenylated RNA from untreated and auxin-treated elongating sections of soybean (Glycine max var. Wayne) hypocotyl were analyzed by two-dimensional polyacrylamide gel electrophoresis. The levels of translatable messenger RNA for at least ten in vitro translation products are increased by auxin treatment. The induction by auxin occurs rapidly (within 15 minutes), and the amounts of the induced in vitro translation products increase with time of auxin treatment. Indoleacetic acid has the same effect on the population of translatable messenger RNA as 2,4-dichlorophenoxyacetic acid. The auxin-induced in vitro translation products disappear rapidly when Actinomycin D is present during the last two hours of a three-hour auxin treatment.     

Hybridization maps of early and late messenger RNA sequences on the adenovirus type 2 genome.

Specific fragments of adenovirus type 2 DNA, generated by cleavage with restriction endonucleases endoR.EcoRI, endoR.HpaI and endoR.HindIII were used in hybridization-mapping experiments. The complementary strands of individual cleavage fragments were separated by the method of Tibbetts &; Pettersson (1974). Liquid hybridizations were performed with ³²P-labeled separated strands of cleavage fragments and messenger RNA extracted from cells early and late after adenovirus infection. The fraction of each fragment strand which was represented in “early” and “late” messenger RNA was determined by chromatography on hydroxylapatite. Early messenger RNA was found to be derived from four widely separated regions, two on the 1- and two on the h-strand (h- and l- refer to the strand with heavy and light buoyant density in CsCl when complexed with poly(U, G)). Messenger RNA, present exclusively late after infection, is derived from several locations, predominantly from the l-strand with a major block of continuous sequences extending between positions 0.25 and 0.65 on the unit map of the adenovirus type 2 genome.     

De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin.     

Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[¹⁴C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.     

Further studies on the synthesis of RNA in vitro by enzyme--template complexes isolated from induced lambda lysogens

RNA synthesized in vitro by enzyme-template complexes isolated from λ lysogens at early or late times following induction has been shown by competition-hybridization procedures to resemble messenger RNA transcribed in vivo at the same stage of viral development, and to differ from RNA made in vitro by purified Escherichia coli RNA polymerase. It is demonstrated here that RNA synthesis by such complexes involves elongation of chains which have been started in vivo, rather than initiation of new RNA chains in vitro.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.