Size of poly (A) +-mRNA in Saccharomyces cerevisiae polysomes.

The size of poly (A) +-mRNA in different classes of yeast polysomes is estimated. The average molecular weight of long-term labelled polysomal poly (A) +-mRNA is about 0,65 x 10(6) daltons. Approximately 60% of the poly (A) +-mRNA polynucleotide chains located at the 5' end, are unprotected by ribosomes and degraded by nucleases upon incubation of cell lysates, to yield a population of poly (A) +-mRNA with an average molecular weight of 0,25 x 10(6) daltons.     

Comparison of the crystallin mRNA populations from rat, calf and duck lens. Evidence for a longer alpha A2-mRNA and two distinct alpha B2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding alpha-, beta-, gamma- and delta-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two alpha A2-mRNAs of approximately 1250 and 1350 nucleotides and the alpha AIns-mRNA with a size similar to that of the largest alpha A2-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of alpha A2-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with alpha A sequences. The duck lens alpha A2-mRNA appears to be 400-450 bases longer than the rat and calf lens alpha A2-mRNAs. Furthermore, in contrast to the single alpha B2-mRNA in rat and calf lens, two alpha B2-mRNAs have been identified in duck lens, one, the major species, similar in size to the alpha B2-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the alpha A2- and alpha B2-mRNAs most likely reside in their 3'-untranslated sequences.     

The effect of tryptrophan on nucleocytoplasmic translocation of RNA in rat liver.

The effect of the administration of tryptophan on the transport of nuclear poly (A)-containing mRNA to the cytoplasm in rat liver was investigated. Administration of tryptophan to fasted rats pretreated with cordycepin and actinomycin D led to decreased levels of nuclear poly (A)-mRNA and a concomitant increase in the levels of polyribosomal poly (A)-mRNA in the cytoplasm as determined by measuring in vivo incorporation of labeled precursors into hepatic RNA. Using isolated hepatic nuclei of rats prelabeled in vivo with [14C]orotic acid, there was greater release of labeled poly(A)-mRNA into the incubation medium from nuclei of tryptophan-treated rats than from nuclei of control animals. The increased release of RNA from hepatic nuclei of tryptophan-treated animals was not related to the cell sap present in the media since cell saps from livers of control and experimental rats gave similar results. These results support earlier findings which suggest that in the rat tryptophan increases the rate of translocation of hepatic poly(A)-mRNA from nucleus to cytoplasm.     

Target organ regulation of substance P in sympathetic neurons in culture

The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.     

Studies on a rat liver subcellular poly A-rich RNA fraction with glutamate dehydrogenase template activity

A heterogeneous poly A-mRNA fraction was isolated from rat liver microsomes by phenol:chloroform extraction, millipore filtration, and poly U-agarose affinity chromatography. The fractions were characterized by their secondary structures and poly A contents. From translational studies, the isolated fraction was found to have high glutamate dehydrogenase template activity in cell-free systems containing microsomes or polysomes. A spectrophotometric procedure for following enzyme biosynthesis was also developed.     

Accumulation of polyadenylated mRNA during liver regeneration.

Cytoplasmic and polysomal polyadenylated mRNA [poly(A)+-mRNA] increased by 120% prior to the onset of DNA synthesis during the regeneration of rat liver following partial hepatectomy. Despite this large change in cytoplasmic mRNA and an approximately 50% increase in total nuclear RNA, the amount of polyadenylated nuclear RNA increased by only 15--20% during this time. Neither the average size of nuclear or of cytoplasmic polyadenylated mRNA nor the length of their poly(adenylic acid) [poly(A)] tracts changed during liver regeneration. Polysomal poly-(A)+-mRNA increased proportionately more and at a faster rate than rRNA during the first day following partial hepatectomy. Normal livers contained a substantial proportion of cytoplasmic poly(A)+-mRNA not associated with polysomes but this proportion was not altered in 3-h regenerating liver. Thus, in regenerating liver, most preexisting cytoplasmic mRNA does not appear to be recruited into polysomes prior to the substantial increase in the amount of cytoplasmic poly(A)+-mRNA.     

Comparison of the crystallin mRNA populations from rat,calf and duck lens. Evidence for a longer αA2-mRNA and two distinct αB2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding α-, β-, γ- and δ-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two αA₂-mRNAs of approximately 1250 and 1350 nucleotides and the αA^Ins-mRNA with a size similar to that of the largest αA₂-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of αA₂-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with αA sequences. The duck lens αA₂-mRNA appears to be 400–450 bases longer than the rat and calf lens αA₂-mRNAs. Furthermore, in contrast to the single αB₂-mRNA in rat and calf lens, two αB₂-mRNAs have been identified in duck lens, one, the major species, similar in size to the αB₂-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the αA₂- and αB₂-mRNAs most likely reside in their 3′-untranslated sequences.     

Subcellular distribution of total poly (A) -containing RNA and ferritin-mRNA in the cytoplasm of rat liver.

Poly(A)-containing RNA was isolated from rat liver microsomes and from the post-microsomal supernatant fraction. Approximately 15% of total rat liver poly(A)-containing RNA was found to be present in the post-microsomal supernatant. The relative capacity for apoferritin synthesis of each poly(A)-containing RNA preparation was measured in a cell-free system derived from wheat germ. The post-microsomal supernatant fraction was found to be highly enriched with ferritin mRNA and accounted for 40–50% of the total ferritin-mRNA present in the cytoplasm of rat liver.     

The subcellular distribution of poly-A-degrading activity in mouse kidney.

Most (95%) of the poly-A-degrading activity of the mouse kidney was found in the cytoplasmic fraction and only 5% was found in the nuclear fraction; 43% of the poly-A-degrading activity of the cytoplasm was found in the mitochondria, 22% in the microsomes, and 30% in the soluble fraction. Differences in activity and specificity indicate that poly A is degraded in the nucleus by enzymes that are separate and distinct from the enzymes in the cytoplasm that degrade poly A. The nuclear poly-A-degrading activity can be separated into an endonuclease with a general specificity and exonuclease, similar to one found in Ehrlich ascites tumor cells, which shows some specificity for poly A.     

Complex population of nonpolyadenylated messenger RNA in mouse brain

The complexity of nonadenylated mRNA [poly(A)-mRNA] has been determined by hybridization with single-copy DNA (scDNA) and cDNA. Our results show that poly(A)- and poly(A)+ mRNA are essentially nonoverlapping (nonhomologous) sequence populations of similar complexity. The sum of the complexities of poly(A)+ mRNA and poly(A)- mRNA is equal to that of total polysomal RNA or total mRNA, or the equivalent of approximately 1.7 x 10(5) different sequences 1.5 kb in length. Poly(A)- mRNA, isolated from polysomal RNA by benzoylated cellulose chromatography, hybridized with 3.6% of the scDNA, corresponding to a complexity of 7.8 x 10(4) different 1.5 kb sequences. The equivalent of only one adenosine tract of approximately 20 nucleotides per 100 poly(A)- mRNA molecules 1.5 kb in size was observed by hybridization with poly(U). cDNA was transcribed from poly(A)- mRNA using random oligonucleotides as primers. Only 1-2% of the single-copy fraction of this cDNA was hybridized using poly(A)+ mRNA as a driver. These results show that poly(A)- mRNA shares few sequences with poly(A)+ mRNA and thus constitutes a separate, complex class of messenger RNA. These measurements preclude the presence of a complex class of bimorphic mRNAs [that is, species present in both poly(A)+ and poly(A)- forms] in brain polysomes.     

Increased synthesis of ribonucleic acids in mouse liver after treatment with a nonionic detergent.

A single injection of Tween 40 (polyoxyethylene(20)sorbitan monohexanoate) in the dose range of 600--800 mg/kg body weight induced an increased short-term labeling especially of poly(A)-containing mRNAs in mouse liver (using either [3H]orotic acid or [32P]orthophosphate as RNA precursors), and apparently increased the turnover rates of both rRNAs and mRNAs in this organ over a period of 24 h. In the early period (4 h) after the injection of Tween 40, there was also a significant increase in the content of 32P-labeled adenylic acid in microsomal poly(A)-mRNA fraction. The activity of DNA-dependent RNA polymerases in the nuclei of treated animals was stimulated up to 60% over that in control nuclei in the same period after detergent injection.     

Identification of messenger RNA for glutamate dehydrogenase using a spectrophotometric probe

Specific messenger RNA for glutamate dehydrogenase was partially purified from a calf liver polysomal poly(A)-mRNA fraction by sucrose density gradient centrifugation. Enzyme activity in the translational incubation mixture was detected by measuring NADH oxidation in the presence of -ketoglutarate and ammonia as a decrease in absorbancy 340–442 nm in a dual wavelength Aminco DW-2 spectrophotometer.     

RNA metabolism and poly(A) distribution in mouse liver following administration of dimethylnitrosamine

Dimethylnitrosamine (DMNA) strongly inhibited RNA synthesis in mouse liver under conditions when the nucleotide pattern, rate of nucleotide synthesis and phosphorylation ratio were unaffected. (An unidentified, probably non-nucleotide, component in the acid-soluble liver fraction was selectively reduced.) The inhibition of RNA synthesis was associated with a decrease in the RNA polymerase activity of isolated liver nuclei, well established already 45 min after DMNA administration. The reduced activity included both Mg2+- and Mn2+/(NH4)2SO4-stimulated polymerase functions. The inhibition in vivo involved the whole complement of RNA, including poly (A)-containing RNA and isolated poly(A) sequences. The transfer of labelled RNA from the nucleus to the cytoplasm was not impaired. There was no detachment of poly(A)-containing RNA from the microsomes, and the proportion of tightly membrane-bound microsomal RNA and poly(A) sequences was not reduced as determined by use of a flotation technique. No breakage or shortening of the poly(A) chains was indicated by sedimentation analysis.     

Photosynthesis as a prototype of solar energetics of new type. I. Chlorophyll apparatus of photosynthesis

The hybridization of double-stranded regions of pre-mRNA from mouse Ehrlich ascites carcinoma cells, rabbit bone marrow cells and primary culture of rabbit kidney cells with an excess of total poly(A)+-mRNA of mouse or rabbit globin mRNA respectively was studied. The hybrids were detected as RNAase-stable acid precipitable material or by adsorbtion of the hybrid complexes of poly(U)-sepharose. The sizes of the hybrid complementary sequences and their thermal stability were estimated.     

Structure of nuclear pre-mRNA. IX. Hybridization of double-stranded portions of pre-mRNA with excess mRNA

The hybridization of double-stranded regions of pre-mRNA from mouse Ehrlich ascites carcinoma cells, rabbit bone marrow cells and primary culture of rabbit kidney cells with an excess of total poly(A)+-mRNA of mouse or rabbit globin mRNA respectively was studied. The hybrids were detected as RNAase-stable acid precipitable material or by adsorbtion of the hybrid complexes of poly(U)-sepharose. The sizes of the hybrid complementary sequences and their thermal stability were estimated.