Regulation of the in vitro synthesis of E. coli ribosomal protein L12.

The $\text{in}\text{vitro}$ DNA dependent synthesis of ribosomal protein L12 and the β subunit of RNA polymerase has been investigated using DNA from a plasmid which contains the genetic information for ribosomal protein L12 and the β subunit of RNA polymerase. This DNA, however, lacks the promoter region and the genetic information for the first 26 amino acids of ribosomal protein L10. It was found that L12 and the β subunit of RNA polymerase are efficiently synthesized $\text{in}\text{vitro}$ from this DNA. These results suggest that L12 and the β subunit of RNA polymerase can be synthesized from a promoter situated within the L10 gene.     

Conformational changes at the peptidyl transferase center of antibiotic resistant mutants of Saccharomyces cerevisiae

Ribosomes from $\text{Saccharomyces}\text{cerevisiae}$ wild type (Y166) and trichodermin (TR1) and narciclasine (NAR2b) resistant strains were affinity labeled with p-nitrophenylcarbamyl-(³H)Phe-tRNA. Resistant ribosomes were able to bind covalently two to five times more aminoacyl-tRNA derivative than control. The labeling pattern of the individual ribosomal proteins was also altered in the two mutants. These results evidence a change in the conformation of the peptidyl transferase center that brings the 3′ end of the aminoacyl-tRNA closer to several ribosomal proteins.     

Concanavalin A-induced suppressor cell activity for T-cell proliferative responses: Autologous and allogeneic suppression in aging humans

Peripheral blood mononuclear cells from 48 healthy subjects of ages varying from 20 to 94 years were evaluated for the ability to generate suppressor cell activity following in vitro incubation with concanavalin A. The suppression of proliferative responses by autologous and young, allogeneic lymphocytes to phytohemagglutinin was assessed using suppressor/ responder cell ratios ($\text{S}\text{R}$) of 2:1 and 1:1 and by using a summation index. Inducible suppressor cell activity for autologous responder cells was comparable between 24 aged (76.0 ± 10.9 years) and 20 young (26.8 ± 4.6 years) subjects. However, aged subjects exhibited a significant decrease in suppressor cell activity ($\text{S}\text{R}\text{= 1}$) when allogeneic responder cells were utilized. Our results indicate that autologous inducible suppressor cell activity is preserved in the aged population, whereas allogeneic activity is impaired.     

Induction kinetics of human suppressor cells in the mixed lymphocyte reaction and influence of prednisolone on their genesis

The induction kinetics of human suppressor cells in mixed lymphocyte cultures (MLC) and the influence of prednisolone on the genesis of these suppressor cells is reported. We induced over 1 to 6 days suppressor cells in one-way MLC (MLC-1), the inhibitory activity of which was tested on a secondary MLC (MLC-2), and on responder cells alone, where lymphocytes were obtained from the same lymphocyte donors as for the MLC-1. In four experiments the degree of inhibition ($\text{x}\text{?}\text{±}\text{SE}$) when suppressor cells were induced for 2, 4, or 6 days was 38.5 ± 11.8, 79.5 ± 7, and 85 ± 6%, respectively, compared to 50.5 ± 9.4, 83.3 ± 7.8, and 85.3 ± 9.8% when 500 ng/ml prednisolone was added to the MLC-1. A similar inhibition pattern was observed when the generated suppressor cells were incubated with responder cells only. The inhibitory activity of these MLC-induced suppressor cells was abrogated by irradiation with 3000 R. Suppressor cells apparently are generated in MLCs between Days 1 and 4; furthermore, their genesis is not affected by usual therapeutic concentrations of prednisolone.     

Dephosphorylation of 40S ribosomal subunits by phosphoprotein phosphatase activity from rabbit reticulocytes.

Phosphoprotein phosphatase activities which remove phosphoryl groups from ribosomal protein have been partially purified from rabbit reticulocytes by chromatography on DEAE-cellulose. Two major peaks of phosphoprotein phosphatase activity were observed when 40S ribosomal subunits, phosphorylated $\text{in vitro}$ with cyclic AMP-regulated protein kinases and (γ-³²P)ATP, were used as substrate. The phosphatase activity eluting at 0.14 M KCl was characterized further using ribosomal subunits phosphorylated $\text{in situ}$ by incubation of intact reticulocytes with radioactive inorganic phosphate. Phosphate covalently bound to 40S ribosomal subunits and 80S ribosomes was removed by the phosphatase activity. The enzyme was not active with phosphorylated proteins associated with 60S ribosomal subunits.     

Molecular cloning of part of the mitochondrial DNA of Drosophila melanogaster

We report the construction of recombinant plasmids containing part of the mitochondrial DNA of $\text{Drosophila}\text{melanogaster}$. Of the four fragments of this DNA generated by the restriction endonuclease HindIII, two were successfully cloned into the HindIII site of the plasmid pCM2. Unexpectedly the other two fragments could not be isolated by cloning into the HindIII site of either pCM2 or pBR322. Part of a third fragment, containing the gene for the large ribosomal RNA, was incorporated into the PstI site of pBR322. We show that this recombinant plasmid contains sequences complementary to an abundant RNA species which is present in $\text{Drosophila}$ embryos and which binds to oligo-dT-cellulose.     

Synthesis and characterization of a DNA probe complementary to rat liver 28S ribosomal RNA.

Conditions for the production of a complementary DNA sequence for use in studies of ribosomal RNA are described. $\text{E}$. $\text{coli}$ DNA polymerase I is used to transcribe highly purified 28S ribosomal RNA from rat liver. The reaction is sensitive to the tertiary structure of the rRNA template-primer. The complementary DNA hybridizes to its rRNA template with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.02. The hybrid formed between 28S ribosomal RNA and complementary DNA has a T_m of 73°C. The probe reacts with total rat nuclear RNA with a $\text{R}{}_{\mathrm{o}}\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 1.0.     

An Escherichia coli K12 mutant carrying altered ribosomal protein (S10).

An $\text{Escherichia coli}$ mutant (JE14373) carrying decreased stability of stable RNA species was found to have altered electrophoretic mobility of a 30S ribosomal protein (S10). Recombinants covering $\text{str}$ gene (76 min on $\text{E. coli}$ linkage map by Bachmann, Low and Taylor, 1976 (ref. 1)) obtained from a cross of CSH64 × JE14373, restored normal S10 protein. The size analysis of RNAs labeled for 15 min with [³H]uridine showed 50 to 60 % decrease of 16S RNA in this mutant strain, but almost no decrease of 23S RNA at 10 or 40 min after addition of rifampicin. On the other hand, no change was observed in the stability of both rRNA pieces in its parental PA3092 strain even at 40 min after addition of rifampicin.     

Structural polypeptides of primate derived type C RNA tumor viruses

Proteins of gibbon ape lymphosarcoma virus (GaLV) and woolly monkey sarcoma virus, type 1, together with its associated virus ($\text{SSV-1}\text{SSAV-1}$) were analyzed by guanidine-agarose chromatography and the separation patterns were compared with those of mouse and feline type C viruses. GaLV contained five major proteins, including two glycoproteins, whereas lower mammalian viruses contained six major proteins, including two glycoproteins. The molecular weights of the five GaLV proteins closely resembled the molecular weights of the five equivalent lower mammalian viral proteins. $\text{SSV-1}\text{SSAV-1}$ showed a separation pattern similar to GaLV except it contained a low but detectable amount of an additional glycoprotein. Both GaLV and $\text{SSV-1}\text{SSAV-1}$ were deficient in a protein of molecular weight about 15,000 daltons which is found in all known type C viruses of avian, reptilian and lower mammalian species.     

Appearance of a kidney-specific ribosomal protein during mouse embryonic development

Ribosomes were prepared from adult mouse liver and kidney and the protein components examined by SDS polyacrylamide gel electrophoresis. A kidney specific protein was identified and was found to be associated with the large ribosomal subunit. $\text{In}$$\text{vitro}$ labelling of 11- and 14-day embryonic kidneys and subsequent analysis of the ribosomal proteins indicated a change in the ribosomal population during development. The kidney specific protein was synthesized during the first four days of kidney organogenesis.     

Release of the "ammonia effect" on three catabolic enzymes by NADP-specific glutamate dehydrogenaseless mutations in Saccharomyces cerevisiae

Mutations which inactivate the NADP-glutamate dehydrogenase (anabolic GDHase) pleiotropically release the ammonia inhibition (NH₄⁺ effect) on a number of distinct catabolic activities. In addition to releasing inhibition on several permeability functions (1), these mutations suppress the NH₄⁺ effect on the synthesis of arginase, urea amidolyase and allantoinase. They do not affect the NH₄⁺ effect on the NAD-glutamate dehydrogenase.Two mechanisms of action of these mutations have to be considered, namely a modification of the process of $\text{induction}$ (such as removal of inducer exclusion) and a suppression of $\text{nitrogen catabolite repression}$.     

DNA-dependent in vitro synthesis of Escherichia coli ribosomal protein L10 and the formation of an L10L12 complex

The $\text{in vitro}$ synthesis of ribosomal protein L10 has been demonstrated using λrif^d18 DNA as template. The L10 synthesized $\text{in vitro}$ forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum.     

Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation.

The binding of [${}^3\text{H}$]kanamycin to $\text{E. coli}$ ribosomes and ribosomal subunits was studied by equilibrium dialysis and Millipore filter methods. The 70S ribosome bound ca. two molecules up to the antibiotic concentration of 10 uM, and more at higher concentrations. Each ribosomal subunit was observed to possess one major binding site, and the affinity of the small ribosomal subunit was greater than that of the large subunit. The binding of [${}^3\text{H}$]kanamycin to ribosomes and ribosomal subunits was reversed by neomycin or gentamicin, but not by streptomycin and chloramphenicol. Kanamycin, neomycin and gentamicin interfered with the binding of [${}^{14}\text{C}$] tuberactinomycin O. Translocation of N-Ac-Phe-tRNA was markedly inhibited by kanamycin, neomycin or gentamicin, but not by streptomycin.     

An amber mutation in the gene rpsA for ribosomal protein S1 in Escherichia coli

Summary An amber mutation has been induced in the gene rpsA (which codes fo ribosomal protein S1) of Escherichia coli K-12 strain in the presence of an amber suppressor (supD) and mutations sueA, sueB and sueC that additively enhance the efficiency of suppression. That the amber mutation has occurred in the gene rpsA was confirmed by complementation with a plasmid which carried the wild-type allele of rpsA. The mutation is lethal in the absence of an amber suppressor, indicating that ribosomal protein S1 is indispensable to E. coli.     

U.V. induced covalent crosslinking of E.coli ribosomal RNA to specific proteins

$\text{E.}\text{coli}$ 70S ribosomes uniformly labeled $\text{in}\text{vivo}$ with ³²PO₄ were subjected to varying doses of u.v. radiation and then to the combined action of the RNases A and T₁. Following these treatments the ribosomal proteins were separated by trichloroacetic acid precipitation from the noncovalently attached RNA degradation fragments. Subsequent two-dimensional gel electrophoresis and autoradiography of these proteins revealed that significant ³²PO₄ was associated with unique ribosomal proteins, L2 was among these.     

Identification and characterization of a new methylated amino acid in ribosomal protein L33 of Escherichiacoli

Methylated amino acids from ribosomal protein L33 of various $\text{Escherichia}\text{coli}$ strains (Q13, B and MRE600) were analyzed. It was found that while protein L33 from $\text{E.}\text{coli}$ Q13 contains two methylated neutral amino acids (peaks I and II), only one methylated neutral amino acid (peak I) was found in protein L33 derived from both $\text{E.}\text{coli}$ strains B and MRE600. The methylated amino acid present in peak I was identified as N-monomethylalanine by ion-exchange column chromatography, high-voltage paper electrophoresis and descending paper chromatography using different solvent systems. This marks the first time that N-monomethylalanine was found in any ribosomal protein.     

RNA-protein interactions in the ribosome. I. Characterization and ribonuclease digestion of 16 S RNA-ribosomal protein complexes

Proteins from the 30 S ribosomal subunit of Escherichia coli were fractionated by column chromatography and individually incubated with 16 S ribosomal RNA. Stable and specific complexes were formed between proteins S4, S7, S8, S15 and S20, and the 16 S RNA. Protein S13 and one or both proteins of the $\text{S}\text{16}\text{S}\text{17}$ mixture bound more weakly to the RNA, although these interactions too were apparently specific. The binding of $\text{S}\text{16}\text{S}\text{17}$ was found to be markedly stimulated by proteins S4, S8, S15 and S20. Limited digestion of the RNA-protein complexes with T₁ or pancreatic ribonucleases yielded a variety of partially overlapping RNA fragments, which retained one or more of the proteins. Since similar fragments were recovered when 16 S RNA alone was digested under the same conditions, their stability could not be accounted for by the presence of bound protein. The integrity of the fragments was, however, strongly influenced by the magnesium ion concentration at which ribonuclease digestion was carried out. Each of the RNA fragments was characterized by fingerprinting and positioned within the sequence of the 1600-nucleotide 16 S RNA molecule. The location of ribosomal protein binding sites was delimited by the pattern of fragments to which a given protein bound. The binding sites for proteins S4, S8, S15, S20 and, possibly, S13 and $\text{S}\text{16}\text{S}\text{17}$ as well, lie within the 5′-terminal half of the 16 S RNA molecule. In particular, the S4 binding site was localized to the first 500 nucleotides of this sequence while that for S15 lies within a 140-nucleotide sequence starting about 600 nucleotides from the 5′-terminus. The binding site for the protein S7 lies between 900 and 1500 nucleotides from the 5′-terminus of the ribosomal RNA.     

Suppression of carboxy-terminal truncations of the yeast mitochondrial mRNA-specific translational activator PET122 by mutations in two new genes, MRP17 and PET127

Summary The PET122 protein is one of three Saccharomyces cerevisiae nuclear gene products required specifically to activate translation of the mitochondrially coded COX3 mRNA. We have previously observed that mutations which remove the carboxy-terminal region of PET122 block translation of the COX3 mRNA but can be suppressed by unlinked nuclear mutations in several genes, two of which have been shown to code for proteins of the small subunit of mitochondrial ribosomes. Here we describe and map two more new genes identified as allele-specific suppressors that compensate for carboxy-terminal truncation of PET122. One of these genes, MRP17, is essential for the expression of all mitochondrial genes and encodes a protein of M_r 17343. The MRP17 protein is a component of the small ribosomal subunit in mitochondria, as demonstrated by the fact that a missense mutation, mrp17-1, predicted to cause a charge change indeed alters the charge of a mitochondrial ribosomal protein of the expected size. In addition, mrp17-1, in combination with some mutations affecting another mitochondrial ribosomal protein, caused a synthetic defective phenotype. These findings are consistent with a model in which PET122 functionally interacts with the ribosomal small subunit. The second new suppressor gene described here, PET127, encodes a protein too large (M_r 95900) to be a ribosomal protein and appears to operate by a different mechanism. PET127 is not absolutely required for mitochondrial gene expression and allele-specific suppression of pet122 mutations results from the loss of PET127 function: a pet127 deletion exhibited the same recessive suppressor activity as the original suppressor mutation. These findings suggest the possibility that PET127 could be a novel component of the mitochondrial translation system with a role in promoting accuracy of translational initiation.