Messenger Discriminating Species of Initiation Factor F<Subscript>3</Subscript>

Species of the polypeptide chain initiation factor F₃ with high selectivity toward either MS2 or T4 phage messenger RNAs have been isolated from normal E. coli cells.     

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.     

A population-based statistical approach identifies parameters characteristic of human microRNA-mRNA interactions

Background  

MicroRNAs are ~17–24 nt. noncoding RNAs found in all eukaryotes that degrade messenger RNAs via RNA interference (if they bind in a perfect or near-perfect complementarity to the target mRNA), or arrest translation (if the binding is imperfect). Several microRNA targets have been identified in lower organisms, but only one mammalian microRNA target has yet been validated experimentally.     

Stage-specific initiation factors for protein synthesis during insect development

In insects, as in bacteria, the smaller (40 S) ribosomal subunit binds messenger RNA during initiation of protein synthesis. An 80 S ribosomal unit is formed by association of free 40 S and 60 S subunits. Formation of the complete initiation complex requires GTP, aminoacyl-tRNA, protein initiation factors and messenger RNA. The complex sediments as an 80 S band on sucrose gradient. Protein initiation factors are extracted from unwashed ribosomes and appear to be able to discriminate between messenger RNAs obtained from different stages of development. They promote formation of the 80 S complex only when messenger RNA is extracted from the same stage of development, providing a mechanism for control of protein synthesis by which ribosomes can select the messenger RNA to be translated. Two possibilities have been proposed to explain this phenomenon: (1) that a group of messenger RNAs from a given stage of development may have a specific sequence of nucleotides preceding the AUG codon. This sequence is recognized by a stage-specific element of the initiation machinery; (2) and or, the secondary structure of messenger RNA from a given stage of development may be specific and therefore recognized by a unique initiation factor.     

Quantitation and characterisation of poly(A)-containing messenger RNAs from mouse neuroblastoma cells

Comparison of several isolation procedures for neuroblastoma poly(A)-containing mRNAs shows that the highest percentage recovery of undegraded and biologically active messenger RNAs is obtained using proteinase K prior to phenol extraction. The messenger RNAs thus isolated comprise approximately 1.5% of the total ribosomal RNAs and have negligible contamination with 18 and 28 S RNAs. On denaturing polyacrylamide gels they have an average molecular weight of 6.5-10(5) with a range from 2.2-10(5) to 1.53-10(6). The messenger RNAs have an average poly(A) content of 154 nucleotides. They are highly active in wheat germ in vitro protein synthesizing systems, giving as much as 4.3 pmol [35S]methionine incorporation into total protein per mol of mRNA. This is almost as active as a control globin mRNA preparation.     

Genetic localization of two polycistronic messenger RNAs from bacteriophage lambda as deduced from their base compositions and annealing properties

Summary We have analyzed the base composition of two heavy messenger RNAs synthesized by bacteriophage lambda early after induction:l ₃ and r ₂. The GC content of these two RNAs is very similar to the GC content of corresponding parts of DNA. This result confirms the approximate genetic location of l ₃ and r ₂ as determined by DNA-RNA hybridization experiments.     

Two different species of messenger RNAs specify synthesis of M1 and M2 pyruvate kinase subunits

A study was performed to determine whether M₁ and M₂ pyruvate kinases were synthesized under the direction of one or two messenger RNAs. We compared M₁ and M₂ pyruvate kinases purified from fresh tissues with those neosynthesized under the direction of messenger RNAs from tissues synthesizing either M₁ or M₂. RNA was isolated from rat muscle, lung, spleen and kidney by ethanol precipitation in 7 M guanidium chloride, translated in rabbit reticulocyte system and newly-synthesized pyruvate kinase subunits were purified by microimmunoaffinity chromatography. Pyruvate kinase from fresh muscle and spleen was purified in one step by a similar process. Muscle and spleen RNA directed the synthesis of M subunits with molecular weights of approx. 61000 and 62000, respectively, the same as those of the corresponding fresh tissue monomers. In addition, peptide maps obtained by partial digestion of neosynthesized M₁ and M₂ with V₈ protease from Staphylococcus aureus confirmed that these polypeptides were clearly different.     

Cyclic AMP and NH3/NH4+ both regulate cell-type-specific mRNA accumulation in the cellular slime mold, Dictyostelium discoideum

Dictyostelium discoideum cells plated for development until aggregation stage, and then dissociated into media containing glucose, albumin, and cAMP will form into clumps and undergo prespore and prestalk differentiation. Differentiation in this in vitro system is dependent on three components: cAMP, multicellularity, and the acquisition of "differentiation competence" which the cells acquire in a period between interphase and aggregation stage when plated on Millipore filters. We have used this system to explore aspects of the multicellular environment which are involved in regulation the accumulation of the different prespore- and prestalk-specific messenger RNAs. Two classes of prespore messenger RNA, as well as a prestalk-specific messenger RNA, all require the acquisition of differentiation competence in order to be expressed in response to cAMP. Additionally, all of these messenger RNAs require agglomerate formation for maximal expression. The addition of 33 mM ammonium sulfate (NH4)2SO4, however, can entirely replace the requirement for agglomerate formation for expression of the prestalk-specific messenger RNA, and can partially substitute for agglomerate formation in inducing the expression of both classes of prespore-specific messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs. In this system, cAMP is essential for the initial induction of expression of all three classes of messenger RNAs while agglomerate formation or elevated NH3/NH+4 is essential only for the maintenance of the elevated levels of the messenger RNAs.     

Structural analysis of a 1.7-kilobase mouse mammary tumor virus-specific RNA

We have detected a mouse mammary tumor virus (MMTV)-specific 1.7-kilobase (kb) polyadenylated RNA in mammary glands of several mouse strains. In BALB/c mice, it is the only MMTV-specific RNA species present. C3H and GR mammary glands and tumors contain, in addition, 3.8- and 7.8-kb MMTV RNAs. Nuclease S1 analysis was performed to map 1.7-kb polyadenylated RNA. It contains predominantly long terminal repeat (LTR) sequences. The 5' end maps approximately 134 nucleotides upstream from the 3' end of the LTR. Colinearity with complete proviral DNA continues to a site about 153 nucleotides downstream from the left (5') LTR. No sequences from the middle part of proviral DNA were found. Colinearity with proviral DNA is resumed 72 nucleotides upstream from the right (3') LTR. The nucleotide sequence in this area is TTCCAGT, which is a splice acceptor consensus sequence. The anatomy of 1.7-kb RNA indicates that it may serve as a messenger for the 36,700-dalton protein encoded by the LTRs of MMTV.