Evidence that all messenger RNA molecules (except histone messenger RNA) contain Poly (A) sequences and that the Poly(A) has a nuclear function

The appearance of newly formed messenger RNA in polyribosomes of HeLa cells Is inhibited by over 85% by 3′deoxyadenosine (Penman, Rosbash &; Penman, 1970) probably due to the failure of normal attachment of poly(A) to heterogeneous nuclear RNA in the presence of this drug (Darnell, Philipson, Wall &; Adesnik, 1971). Results presented here show that the labeled RNA which does reach polysomes in the presence of 3′deoxyadenosine can be characterized as messenger RNA which contains smaller poly(A) segments than normal messenger RNA. The results of the present experiments suggest that all, or almost all, HeLa cell messenger RNA molecules (except for histone messenger RNA) are derived from nuclear RNA molecules which contain poly (A).     

Human submandibular gland statherin and basic histidine-rich peptide are encoded by highly abundant mRNA's derived from a common ancestral sequence

The molecular cloning of sequences encoding human submandibular gland (SMG) statherin and a basic histidine-rich peptide is described. The corresponding mRNA's were highly abundant in the human and the Rhesus monkey (Macaca mulatta) SMG, but no homologous message was detectable in the murine SMG. Sequence analysis revealed strong homology between the statherin and basic histidine-rich mRNA's, suggesting their evolution from a common ancestral sequence.     

Elf Electromagnetic Fields Affect Gene Expression of Hegenerating Rat Liver Following Partial Hepatectomy

Pulsed extremely-low-frequency magnetic fields (ELF-PEMFs) influence the expression of oncogenes c-myc and c-ras and of ornithine decarboxylase (ODC) in the regenerating rat liver following partial hepatectomy. In fact, while the mRNA's encoding both oncogenes are present in very low amounts in the normal liver, their concentration is dramatically increased during regeneration. Ornithine decarboxylase and c-myc mRNA's reach a maximum during the early phases of regeneration (3 hours after surgery) and decrease thereafter. c-ras mRNA reaches a maximum 40 hours after the operation. Treatment with ELF-PEMFs delivered to the animals immediately after the operation and every 12 hours thereafter increases The concentration of both oncogenes and of ornithine decarboxylase mRNA' s at 3 hours (c-myc and ODC) and at 40 hours (c-ras) respective-ty.     

Re-examination of histone changes during development of newt embryos

Embryos of Triturus pyrrhogaster (BOIE) were labeled with Na₂¹⁴CO₃ and the incorporation of radioactivity into histone fractions was determined by the electrophoresis of the acid-soluble protein from isolated nuclei on a polyacrylamide gel with or without Triton X-100. The results supported the previous observation that the content of H1 histone might be low in blastulas and increased during development but they did not confirm the displacement of blastula H1 by other H1 molecular species in later embryos. The rate of H2b or H2a histone synthesis did not change much during development which contrasted sharply with the case of histone synthesis in sea urchin embryos. By changing the label duration or by culturing various durations after the label it was suggested that the histone fractions were synthesized or degraded as a set and any particular fraction that had a markedly long or short life could not be detected. The results were discussed in relation to the possible functions of H1 histone and to the histone synthesis in sea urchin embryos.     

The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Cell cycle-regulated histone mRNAs end in a conserved 26-nt sequence that can form a stem-loop with a six-base stem and a four-base loop. The 3' end of histone mRNA has distinct functions in the nucleus and in the cytoplasm. In the nucleus it functions in pre-mRNA processing and transport, whereas in the cytoplasm it functions in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), present in both nuclei and polyribosomes, is likely the trans-acting factor that binds to the 3' end of mature histone mRNA and mediates its function. A nuclear extract that efficiently processes histone pre-mRNA was prepared from mouse myeloma cells. The factor(s) that bind to the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide containing the conserved stem-loop sequence. Using this depleted extract which is deficient in histone pre-mRNA processing, we show that SLBP found in polyribosomes can restore processing, suggesting that SLBP associates with histone pre-mRNA in the nucleus, participates in processing, and then accompanies the mature mRNA to the cytoplasm.     

THE REACTION OF A GROUP OF ROTHSCHILD'S GIRAFFE TO A NEW ENVIRONMENT

The behaviour of a translocated group of 11 Rothschild's giraffe (Giraffa camelopardalis rothschildi Lydekker) was studied for 9 weeks in 1968. A comparison was made between their new and former habitats, the food plants eaten in both places, and behaviour. For 3 ¹/₂ months after release, they appeared to have settled in the area, but they suddenly started extending their range. Possible reasons are suggested for their failure to stay in the release area and their eventual disappearance.     

SLBP is associated with histone mRNA on polyribosomes as a component of the histone mRNP

The stem–loop binding protein (SLBP) binds the 3′ end of histone mRNA and is present both in nucleus, and in the cytoplasm on the polyribosomes. SLBP participates in the processing of the histone pre-mRNA and in translation of the mature message. Histone mRNAs are rapidly degraded when cells are treated with inhibitors of DNA replication and are stabilized by inhibitors of translation, resulting in an increase in histone mRNA levels. Here, we show that SLBP is a component of the histone messenger ribonucleoprotein particle (mRNP). Histone mRNA from polyribosomes is immunoprecipitated with anti-SLBP. Most of the SLBP in cycloheximide-treated cells is present on polyribosomes as a result of continued synthesis and transport of the histone mRNP to the cytoplasm. When cells are treated with inhibitors of DNA replication, histone mRNAs are rapidly degraded but SLBP levels remain constant and SLBP is relocalized to the nucleus. SLBP remains active both in RNA binding and histone pre-mRNA processing when DNA replication is inhibited.     

In maize,glutelin-2 and low molecular weight zeins are synthesized by membrane-bound polyribosomes and translocated into microsomal membranes

Summary Experiments to establish the site of biosynthesis and the possible translocation into microsomes of glutelins-2 (28 kD G2) and low molecular weight zeins (10, 14, 15 kD Z2) have been carried out. Free and membrane-bound polyribosomes as well as microsomal membranes were isolated from immature endosperms of W64A Zea mays L. In vitro translation studies were carried out in the presence and in the absence of membranes using [³⁵S]-methionine or [³⁵S]-cysteine as precursors. Cell-free translation products were characterized by electrophoretic mobility, solubility and antigenic properties. The results obtained indicate that 28 kD G2 and low molecular weight zeins are primarily synthesized on membrane-bound polysomes. From experiments using proteinase K as a probe, we also conclude that these proteins are translocated into microsomes where they accumulate. The translocated and pre-28 kD G2 proteins do not present changes in the apparent molecular weight. However we show that there are differences in their isoelectric points, a fact that indicates the existence of 28 kD G2 processing.     

The virus-specific RNA species in free and membrane-bound polyribosomes of transformed cells replicating murine sarcoma-leukemia viruses

Ribonucleic acids extracted from polyribosomes of cells replicating murine sarcoma-leukemia viruses (M-MSV(MLV)) were resolved by electrophoresis on 2.5% polyacrylamide gels. Virus-specific RNA was detected by hybridization of RNA in the gel fractions with the ³H-DNA product of the viral RNA-directed DNA polymerase. The postmicrosomal supernatant and the free polyribosomes contained one peak of virus-specific RNA with a molecular weight of about 2.9 × 10⁶ (35S). In contrast, the microsomes and the membrane-bound polyribosomes contained two peaks of virus-specific RNA in approximately equal amounts with molecular weights of 2.9 × 10⁶ (35S) and 1.5 × 10⁶ (approximately 20S). The high molecular weight viral RNA species might serve as polycistronic mRNA for the synthesis of large polypeptides that are cleaved to form the smaller viral proteins.     

BIOSYNTHESIS OF COLLAGEN : Biochemical and Physicochemical Characterization of Collagen-Synthesizing Polyribosomes

Synthesis of collagen on polyribosomes has been demonstrated in vitro in chick embryo corium by radioisotope incorporation, zone centrifugation through sucrose gradients, and analytical ultracentrifugation. Collagen synthesis was associated with polyribosomes ranging in size, as reflected by their sedimentation constants, from about 180S to approximately 1600S. Most of the newly formed collagen, hydroxyproline, was present on the largest polyribosome aggregates (~ 350–1600S), but small polyribosomes (~180–200S) also contained collagen. On the basis of the proline-¹⁴C/hydroxyproline-¹⁴C ratios and the disrupting effect of collagenase, the proposal is made that the 350–1600S polyribosomes from this tissue are involved predominantly in collagen synthesis. The large polyribosomes are disrupted extensively by collagenase but only partially by ribonuclease and trypsin. Therefore, it appears that they are stabilized by the interaction of newly forming collagen chains. Evidence is presented consistent with the hypothesis that these large polyribosomes are formed by the aggregation of small polyribosomes (180–200S) through the interaction of collagen polypeptides. It is suggested that these small polyribosomes might be involved in the synthesis of subunits of the collagen alpha chain.     

Characterization of Late Polyoma mRNA

Polyoma-infected mouse kidney cell cultures were labeled with [³H]uridine for 3 h late in the lytic cycle (26 to 29 h after infection) and RNA was extracted from cytoplasm and nuclei and from isolated polyribosomes. Sedimentation velocity analysis in sucrose gradients showed that polyoma-specific “giant” and 26S RNAs are present exclusively in the nucleus. RNA associated with cytoplasmic polyribosomes was analyzed by sedimentation in aqueous sucrose density gradients and dimethylsulfoxide sucrose gradients, as well as by polyacrylamide gel electrophoresis. Polyoma-specific RNA in polyribosomes consists of at least two classes, with sedimentation coefficients of 16 (major fraction) and 19S (minor fraction) in aqueous sucrose gradients and 15 and 17S, respectively, in dimethylsulfoxide gradients. Estimates based on dimethylsulfoxide gradient and analysis suggest a molecular weight of approximately 500,000 for 16S RNA and 700,000 for 19S RNA. These polyoma RNAs seem to undergo reversible conformational changes under the different conditions of analysis. We cannot exclude the possibility that they contain more than one molecular species.     

Three mRNA populations differing in turnover and processing in mouse liver

Using kinetics of approach to steady state labeling, we have found that liver poly(A)⁺ mRNAs fall into three populations, differing in stability and probably in processing, as reflected in their dissimilar delays in reaching polyribosomes and turnover times. There are mRNA-1 (delay 10 min, half-life 1 hr); mRNA-2 (delay 3 hr, half-life 2 hr); and mRNA-3 (delay 40 min, half-life 2.6 hr). The first two species function on free polyribosomes while the third one is operating on bound polyribosomes. The populations listed contribute 10, 20 and 70%, respectively, to the total steady state labeled poly(A)⁺ mRNAs.Abbreviations Poly(A)⁺ and poly(A)^- mRNAs mRNAs with and without poly(A) segments on the 3 end - HnRNA heterogeneous nuclear RNA     

The in vitro reconstitution of a functional rough membrane active in protein synthesis

Rough endoplasmic reticulum was reconstituted from free polyribosomes and rough membrane stripped from its ribosomes by KCl and puromycin. The reconstituted rough membrane resembled the native rough membrane in the following aspects: RNA/protein ratio, buoyant density in a continuous sucrose gradient, amino acid incorporation capacity and sensitivity towards protein synthesis inhibitors. When the reconstitution was done with double labelled polyribosomes ([³²P] polyribosomes, [³H] leucine labelling of nascent peptide chain before or after the attachment of the polyribosomes to the membrane) both labels banded together with the reconstituted rough membrane band. Hybrid rough membrane could be formed from rat liver stripped rough membrane and wheat germ ribosomes. This hybrid membrane could translate globin mRNA.