Defining the protein complexome of translation termination factor eRF1: Identification of four novel eRF1‐containing complexes that range from 20S to 57S in size

The eukaryotic eRF1 translation termination factor plays an important role in recognizing stop codons and initiating the end to translation. However, which exact complexes contain eRF1 and at what abundance is not clear. We have used analytical ultracentrifugation with fluorescent detection system to identify the protein complexome of eRF1 in the yeast Saccharomyces cerevisiae. In addition to eRF1 presence in translating polysomes, we found that eRF1 associated with five other macromolecular complexes: 77S, 57S, 39S, 28S, and 20S in size. Generally equal abundances of each of these complexes were found. The 77S complex primarily contained the free 80S ribosome consistent with in vitro studies and did not appear to contain significant levels of the monosomal translating complex that co‐migrates with the free 80S ribosome. The 57S and 39S complexes represented, respectively, free 60S and 40S ribosomal subunits bound to eRF1, associations not previously reported. The novel 28S and 20S complexes (containing minimal masses of 830 KDa and 500 KDa, respectively) lacked significant RNA components and appeared to be oligomeric, as eRF1 has a mass of 49 KDa. The majority of polysomal complexes containing eRF1 were both substantially deadenylated and lacking in closed‐loop factors eIF4E and eIF4G. The thirteen percent of such translating polysomes that contained poly(A) tails had equivalent levels of eIF4E and eIF4G, suggesting these complexes were in a closed‐loop structure. The identification of eRF1 in these unique and previously unrecognized complexes suggests a variety of new roles for eRF1 in the regulation of cellular processes.     

EFFECTS OF AVIAN SEED DISPERSAL ON THE GENETIC STRUCTURE OF WHITEBARK PINE POPULATIONS

We used allozyme analysis to examine family structure, the spatial patterning of related individuals, in two populations of whitebark pine (Pinus albicaulis), a subalpine conifer that commonly displays a multistem form. The individual stems within clumps are genetically distinct individuals, having arisen from separate seeds. Individuals within a clump are genetically more similar than individuals in different clumps, but individuals in neighboring clumps do not appear to be more similar than individuals in distant clumps. This family structure appears to be a direct result of the seed-caching behavior of Clark's nutcrackers (Nucifraga columbiana), the primary dispersal agent for whitebark pine seeds.     

Female axillary secretions influence women''s menstrual cycles: A critique

Preti, Cutler, Garcia, Huggins, and Lawley report (1986, Horm. Behav. 20, 474-482) that women's menstrual cycles can be modulated with applications of female-derived secretions. An experimental sample of 10 women who reported that they had 29.5 +/- 3 day menstrual cycles was treated on a 22- to 25-day cycle with an extract of axillary secretions from a group of female donors. After two menstrual cycles, the mean absolute difference between the women's menses onsets and the treatment applications decreased significantly. A control sample of 9 women similarly treated with blank/ethanol showed no significant change. Reanalysis of the data indicates that four subjects in the extract sample synchronized with the extract cycles because of "errors" in the extract applications and another four synchronized as a result of experimental design, mathematical properties of cocycling menses onsets, and chance variations. After these factors are accounted for, no evidence suggests that the cycles of the subjects in the extract sample were modulated by the female-derived axillary secretion.     

Micro quantitative Edman manual sequencing

A manual Edman technique is described which allows sequential quantitative determination of from 3 to 10 amino terminal residues on quantities of peptides or proteins down to one nanomole. This is achieved by a fast, efficient method of obtaining the anilinothiazolinone or phenylthiohydantoin amino acid, and quantitating by either back hydrolysis and amino acid analysis or by a new, rapid, high resolution, quasi-isocratic, high-pressure liquid chromatographic procedure. The overall method has been extensively tested successfully on both peptides and proteins of known and unknown amino-terminal sequence and the results included here. In addition, a wide variety of applications relevant to primary structure analysis such as sequencing blocked polypeptides, use of denaturing agents as coupling buffers, reduction of protein or peptide losses on consecutive sequencing and peptide mixture analysis are all incorporated in the methodology outlined.     

Measurement of protein synthesis in rat lungs perfused in situ

Compartmentalization of amino acid was investigated to define conditions required for accurate measurements of rates of protein synthesis in rat lungs perfused in situ. Lungs were perfused with Krebs–Henseleit bicarbonate buffer containing 4.5% (w/v) bovine serum albumin, 5.6mm-glucose, normal plasma concentrations of 19 amino acids, and 8.6–690μm-[U-¹⁴C]phenylalanine. The perfusate was equilibrated with the same humidified gas mixture used to ventilate the lungs [O₂/CO₂ (19:1) or O₂/N₂/CO₂ (4:15:1)]. [U-¹⁴C]Phenylalanine was shown to be a suitable precursor for studies of protein synthesis in perfused lungs: it entered the tissue rapidly (t_½, 81s) and was not converted to other compounds. As perfusate phenylalanine was decreased below 5 times the normal plasma concentration, the specific radioactivity of the pool of phenylalanine serving as precursor for protein synthesis, and thus [¹⁴C]phenylalanine incorporation into protein, declined. In contrast, incorporation of [¹⁴C]histidine into lung protein was unaffected. At low perfusate phenylalanine concentrations, rates of protein synthesis that were based on the specific radioactivity of phenylalanyl-tRNA were between rates calculated from the specific radioactivity of phenylalanine in the extracellular or intracellular pools. Rates based on the specific radioactivities of these three pools of phenylalanine were the same when extracellular phenylalanine was increased. These observations suggested that: (1) phenylalanine was compartmentalized in lung tissue; (2) neither the extracellular nor the total intracellular pool of phenylalanine served as the sole source of precursor for protein; (3) at low extracellular phenylalanine concentrations, rates of protein synthesis were in error if calculated from the specific radioactivity of the free amino acid; (4) at high extracellular phenylalanine concentrations, the effects of compartmentalization were negligible and protein synthesis could be calculated accurately from the specific radioactivity of the free or tRNA-bound phenylalanine pool.     

A positive regulatory gene is required for accumulation of the functional messenger RNA for the glucose-repressible alcohol dehydrogenase from Saccharomyces cerevisiae

The amount of glucose-repressible alcohol dehydrogenase is regulated by the amount of its functional messenger RNA. ADHII² protein was detected by a radioimmune assay and differentiated from ADHI, the classical ADH isozyme, by limited proteolysis with Staphylococcus aureus protease. When yeast containing the wild-type alleles for ADR2 (the ADH II structural locus) and for ADR1 (its positive regulatory gene) were pulse-labeled with [³⁵S]methionine during derepression, radioactive label accumulated in the antibody-precipitated ADHII coterminously with the appearance of ADHII activity. The kinetics of functional ADHII mRNA appearance during derepression in this strain were shown to be the same as those for ADHII protein synthesis in vivo when RNA, extracted from derepressed cells, was translated in a wheat germ cell-free translation system.The role of the positive regulatory gene, ADR1, in ADHII expression was analyzed using two strains mutated at that locus. Yeast containing the adr1-1 allele are incapable of derepressing ADHII activity. When this strain was pulselabeled with [³⁵S]methionine during derepression, approximately one-tenth to one-twentieth the level of ADHII protein synthesis was detected as in the wild-type strain. When RNA was extracted during derepression from cells containing the udr1-1 allele and translated in a wheat germ cell-free system, little functional ADHII mRNA was found to be present.The role of the ADR1 gene was further analyzed using a strain containing the ADR1-5^c allele, which allows constitutive synthesis of ADHII activity. In this strain during glucose repression. ADHII protein synthesis and amount of functional mRNA were at levels comparable to those found for the wild-type strain after complete derepression. Similar kinetics of ADHII protein synthesis and of mRNA accumulation during derepression were observed in the strain carrying the ADR1-5^c allele when compared to that carrying the ADR1 allele, but the absolute amounts were greater by three- to fourfold in cells containing the ADR1-5^c allele. These results indicate that the ADR1 gene acts to increase the level of functional ADHII mRNA during derepression.     

Specific Endonuclease R Fragments of Bacteriophage φX174 Deoxyribonucleic Acid

The restriction enzyme from Hemophilus influenzae, endonuclease R, cleaves phiX174 replicative-form deoxyribonucleic acid (DNA) into at least 13 specific limit fragments. The molecular weights of 12 of the fragments have been estimated by gel electrophoresis and electron microscopy. Using the genetic assay for small fragments of phiX DNA, we have shown that we can salvage markers from the endonuclease R phiX-RF fragments.     

BOOK REVIEWS

Book reviewed in this article:
NORTH AND SOUTH AMERICA: The Agricultural and Hunting Methods of the Navaho Indians . W. W. H ill .
NORTH AND SOUTH AMERICA: A Brief History of Navajo Silver smithing . A rthur W oodward .
NORTH AND SOUTH AMERICA: Navaho Life of Yesterday and Today . K atharine L uomala .     

Encapsulation of coagulase-negative staphylococci of bovine origin.

Capsule expression was assessed in six coagulase-negative staphylococcal strains in serum-soft agar and by india ink and electron microscopy. Classification of strains as encapsulated by serum-soft agar and india ink methods differed. Staphylococcus chromogenes, Staph. hyicus, and Staph. simulans grew as diffuse colonies in serum-soft agar and unstained halos were detected in india ink preparations. Staphylococcus hominis and Staph. simulans grew as diffuse colonies in serum-soft agar but no unstained halo was seen in india ink preparations. Staphylococcus hyicus was the only strain that gave negative results with serum-soft agar and india ink assays. Conventional electron microscopy revealed the presence of capsular polysaccharides on the cell surface of Staph. chromogenes, Staph. hominis and Staph. hyicus. Conventional electron microscopic technique used to examine the surface of cells was detrimental to capsule structure. During dehydration the capsule collapsed and appeared as electron dense aggregates at the surface of cells. To confirm results of conventional electron microscopy and to visualize clearly the cell surface, encapsulated Staph. hyicus and unencapsulated Staph. simulans were observed after freeze-fracture and etching by scanning electron microscopy. The fibrous nature of capsular polysaccharides surrounding cells of Staph. hyicus were distinct and confirmed observation by conventional electron microscopy. A rapid transmission electron microscopic technique is described also for observation of capsule. Results of the rapid TEM method agreed with conventional TEM and SEM. The finding that coagulase-negative staphylococci isolated from bovine milk are capable of capsule production may be important when investigating pathogenicity of these micro-organisms.