Translational specificity of Bacillus stearothermophilus ribosomes.

The translational specificity of Bacillus stearothermophilus ribosomes was studied by determining the effectiveness of various synthetic RNAs as templates at 37 degrees and at higher temperatures. The effectiveness of poly(G,U) was maximal at a G:U ratio of 1:3; it declined with lower G content because of reduced ribosomal affinity for the RNA and, with higher G, because of interference by secondary structure. The effectiveness of poly(A,C,G,U) also declined when secondary structure was increased by increasing (G+C) content. Escherichia coli ribosomes exhibited a similar specificity for poly-(G,U), but had a lower sensitivity to interference by RNA secondary structure. In both bacterial species, sensitivity to secondary structure was determined by the 30S ribosomal subunit.     

Lack of ribosomal protein S1 in Bacillus stearothermophilus.

The 30S ribosomal subunit of Bacillus stearothermophilus migrated as a single band when electrophoresed on agarose-acrylamide composite gels. The addition of the ribosomal protein S1 purified from Escherichia coli resulted in the appearance of an additional band migrating more slowly; 14C-labeled S1 of E. coli was shown to be associated only with this form. Antibody against E. coli protein S1 did not crossreact with either the total 30S ribosomal proteins or the postribosomal supernatant from B. stearothermophilus. These results indicate that B. stearothermophilus lacks a protein equivalent to E. coli S1 and may explain our previous finding [Eur. J. Biochem. 56, 15-22 (1975) that E. coli S1 greatly stimulated the translation by B. stearothermophilus ribosomes of f2 phage RNA.     

Dictyostelium discoideum contains a gene encoding a myosin I heavy chain.

We have cloned and completely sequenced a gene encoding the heavy chain of Dictyostelium myosin I. Like the myosin I molecules from Acanthamoeba, the Dictyostelium myosin I heavy chain is composed of a globular head domain fused to a 45-kDa glycine-, proline-, and alanine-rich carboxyl-terminal domain, rather than the coiled-coil rod domain of conventional myosins. Comparisons of the Dictyostelium myosin I heavy-chain amino acid sequence with those of the Acanthamoeba myosins I reveal that they are highly similar throughout, including the unconventional carboxyl-terminal domains. The Dictyostelium myosin I gene is expressed in growing cells as a 3600-nucleotide mRNA. Measurements of the steady-state level of this mRNA at different times during starvation-induced aggregation and development are consistent with a role for myosin I in chemotaxis and aggregation. Generation of Dictyostelium cells lacking myosin I by gene disruption and/or antisense RNA production should provide a way to test directly the role of this nonfilamentous myosin in cell motility. These experiments will be simplified by the fact that Southern blot analyses of Dictyostelium genomic DNA are consistent with there being a single myosin I heavy-chain gene.     

Association Factor of Ribosomal Subunits from Bacillus stearothermophilus

The isolation of a new factor, which can cause the in vitro association of 30S and 50S ribosomal subunits at low Mg⁺⁺ concentration, is described. The association factor is eluted together with the dissociation protein when ribosomes of Bacillus stearothermophilus are washed with salt solutions of high concentration. The association activity is heat-stable, whereas dissociation factor is inactivated after 10 min at 80°C. This treatment allows the separation of both factors. Several properties rule out the possibility that uncharged, amino-acyl-, or peptidyl-tRNA are responsible for the association process described in this report. Digestion with trypsin shows that the association factor contains at least two components, one of which is a protein.     

Degradation of penicillin G to phenylacetylglycine by D-alanine carboxypeptidase from Bacillus stearothermophilus.

D-Alanine carboxypeptidase from Bacillus stearothermophilus is a membrane-bound enzyme which is inhibited by covalent interaction with penicillin G. The penicilloyl enzyme spontaneously reactivates and simultaneously releases a penicillin G degradation product; 0.2 mumol of the latter was isolated after incubation of 4.2 mumol of [8-14C]penicillin G with 10 g of membrane protein. It was identified as phenylacetylglycine by chromatographic techniques, infrared spectroscopy, and mass spectrometry. A mechanism for the degradation is proposed in which the remaining part of penicillin G would be released as 5,5-dimethyl-delta2-thiazoline-4-carboxylic acid. The implications of this finding are discussed.     

Chicken lysozyme gene contains several intervening sequences.

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.     

Eliminated chromatin of Ascaris contains a gene that encodes a putative ribosomal protein.

Chromatin diminution in the nematodes Parascaris equorum and Ascaris lumbricoides leads to the formation of somatic cells that contain less DNA than the germ-line cells. We present molecular evidence for the coding potential of germ-line-specific DNA. We report on a cDNA clone that codes for a putative ribosomal protein (ALEP-1, for A. lumbricoides eliminated protein 1). That the corresponding gene is located in the eliminated portion of the genome indicates a difference in germ-line and somatic ribosomes of A. lumbricoides and P. equorum. Elimination of the ALEP-1 gene from all somatic cells in its fully active state may represent an alternative way to gene regulation.     

Yeast signal peptidase contains a glycoprotein and the Sec11 gene product.

Partially purified yeast microsomal signal peptidase appears to be a complex of four polypeptides of 13, 18, 20, and 25 kDa. The 18-kDa chain is the product of the Sec11 gene, which is necessary for signal peptidase activity. The 25-kDa subunit is a glycoprotein that binds Con A. Two related methods for purification of the enzyme are presented; the first includes removal of peripheral membrane proteins from microsomes by alkali extraction, solubilization of the enzyme by nonionic detergent and high salt, and four different chromatographic procedures. An alternative method was developed based on lectin-affinity chromatography.     

Correlation between Thermal Death and Membrane Fluidity in Bacillus stearothermophilus

Paramagnetic resonance spectra of spin labels partitioned into spheroplast membranes of Bacillus stearothermophilus indicate lateral lipid phase separations. Cells adjust their lipid composition in response to temperature changes so that the same change of state in membrane phospholipids is achieved at the respective growth temperature. A temperature-sensitive mutant that fails to change its lipid composition above a certain temperature can survive only up to the higher temperature boundary for lateral phase separation. These data are interpreted to indicate that the maximal and minimal growth temperatures of thermophiles are regulated by the onset and conclusion of phase separations of the particular lipid composition they synthesize. It is suggested that isolated lipid domains are required for functional membrane assembly.     

The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase.

Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences. In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene. The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function. However, the presence of the enzyme is not a conserved property of herpesviruses. We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups. The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.     

Reevaluation of the accepted allosteric mechanism of phosphofructokinase from Bacillus stearothermophilus

The binding of phosphoenolpyruvate (PEP) to the single allosteric site on phosphofructokinase (EC ) from Bacillus stearothermophilus (BsPFK) diminishes the ability of the enzyme to bind the substrate fructose 6-phosphate (Fru-6-P). Comparisons of crystal structures with either Fru-6-P or phosphoglycolate, an analog of PEP, bound have shown that Arg-162 interacts with the negatively charged Fru-6-P. Upon the binding of phosphoglycolate, Arg-162 is virtually replaced by Glu-161, which introduces a potential coulombic repulsion between enzyme and substrate [Schirmer, T. & Evans, P. R. (1990) Nature (London) 343, 140-145]. It has previously been proposed that this structural transition explains the allosteric inhibition in BsPFK, and this explanation has appeared in textbooks to illustrate how an allosteric ligand can influence substrate binding at a distance. Site-directed mutagenesis has been employed to create three mutants of BsPFK that substitute an alanine residue for Glu-161, Arg-162, or both. The E161A mutation does not affect the inhibition of BsPFK by PEP at 25 degrees C, and while the R162A mutation decreases BsPFK's affinity for Fru-6-P by approximately 30-fold, R162A diminishes the effectiveness of PEP inhibition by only 1/3. Combining E161A and R162A produces behavior comparable to R162A alone. These and other data suggest that the movement of Glu-161 and Arg-162 does not play the central role in producing the allosteric inhibition by PEP as originally envisioned in the Schirmer and Evans mechanism.     

A lectin gene encodes the alpha-amylase inhibitor of the common bean.

An alpha-amylase inhibitor that inhibits insect and mammalian alpha-amylases but not plant alpha-amylases, is present in seeds of the common bean (Phaseolus vulgaris). We have purified the alpha-amylase inhibitor by using a selective heat treatment in acidic medium and affinity chromatography with porcine pancreas alpha-amylase coupled to agarose. Under sodium dodecyl sulfate gel electrophoresis, the purified inhibitor gave rise to five bands with mobilities corresponding to molecular masses ranging from 14 to 19 kDa. N-terminal sequencing (up to 15 amino acids) of the polypeptides obtained from these bands resulted in only two different sequences matching two stretches of the amino acid sequence deduced from an already described lectin gene [Hoffman, L. M. (1984) J. Mol. Appl. Gen. 2,447-453]. This gene is different from but closely related to the genes that code for phytohemagglutinin, the major lectin of bean. Further evidence based on amino acid composition, identification of a precursor, and recognition of the product of the gene (expressed in Escherichia coli) by an anti-alpha-amylase inhibitor serum confirms that the inhibitor is encoded by this or a closely related lectin gene. This finding assigns a biological function, which has been described at the molecular level, to a plant lectin gene product and supports the defense role postulated for seed lectins. The lack of homology with other families of enzyme inhibitors suggests that this may be the first member of a new family of plant enzyme inhibitors.     

Secretion of Escherichia coli beta-lactamase from Bacillus subtilis by the aid of alpha-amylase signal sequence.

We describe a secretion vector system for introducing foreign genes into Bacillus subtilis. We constructed secretion vectors from the plasmid pUB110 and the promoter and signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens. Foreign structural genes can be inserted into the various vectors after the signal sequence region of the alpha-amylase gene. Demonstrating secretion of a foreign gene product from Bacillus, we here report that the Escherichia coli beta-lactamase gene, devoid of its own signal sequence coding region, can be expressed in B. subtilis by the aid of the secretion vectors so that greater than 95% of the enzyme activity is secreted to the growth medium. Efficient secretion of beta-lactamase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is observed if the complete signal sequence coding region of the alpha-amylase gene precedes the beta-lactamase structural gene. However, an incomplete alpha-amylase signal peptide lacking the six carboxy-terminal amino acid residues does not promote secretion of the fused beta-lactamase, which remains unprocessed and cell-associated.     

Nuclear matrix DNA from chicken erythrocytes contains beta-globin gene sequences.

Nuclear matrices containing residual DNA were isolated from chicken erythrocytes after extraction of purified nuclei with buffered 2 M NaCl. After further purification of this residual DNA, it was found to contain high concentrations of beta-globin gene sequences as assayed by dot hybridization with 32P-labeled nick-translated pHB1001. Electron microscopy of a random sample of this residual DNA fraction shows the DNA to be intimately associated with protein at various intervals. A hypothesis for enrichment of active genes in residual DNA from purified chromatin or in nuclear matrix DNA is also discussed.     

Bacillus licheniformis penicillinase synthesized in Escherichia coli contains covalently linked fatty acid and glyceride.

DNA sequence analysis of the structural gene for Bacillus licheniformis penicillinase has revealed a tetrapeptide sequence of Leu-Ala-Gly-Cys within the NH2-terminal part of the precursor form of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6). The same tetrapeptide occurs in the signal sequence of the prolipoprotein of Escherichia coli, and the cysteine residue in the tetrapeptide of prolipoprotein is modified to form glyceride-cysteine which becomes the NH2 terminus of Braun's lipoprotein. On the basis of labeling, with [2-3H]glycerol, [3H]palmitate, [35S]methionine, and [35S]sulfuric acid, of an E. coli strain lysogenic for a lambda vector containing the penicillinase gene from B. licheniformis and of immunoprecipitation with rabbit antisera against purified B. licheniformis penicillinase, we conclude that B. licheniformis penicillinase synthesized in E. coli contains covalently linked glyceride and fatty acid. These results strongly suggest the operation of a modification system in E. coli, and presumably in other Gram-negative bacteria, which results in the formation of a glyceride-cysteine residue if the proper peptide sequence is present in the signal sequence of membrane proteins.     

Scrapie agent contains a hydrophobic protein.

The scrapie agent causes a degenerative nervous system disorder of sheep and goats. Considerable evidence indicates that the scrapie agent contains a protein that is necessary for infectivity [Prusiner, S. B., Groth, D. F., Cochran, S. P., Masiarz, F. R., McKinley, M. P. & Martinez, H. M. (1980) Biochemistry 19, 4883-4891], but direct demonstration of a protein moiety has been hampered by lack of sufficiently purified preparations. Employing preparations of the scrapie agent enriched 100- to 1000-fold with respect to protein, we found that digestion by proteinase K destroyed more than 99.9% of the infectivity. Diethylpyrocarbonate, which chemically modifies amino acid residues in proteins with high efficiency, also inactivated the scrapie agent in these purified preparations. Reductions of infectivity by proteinase K and diethylpyrocarbonate were not observed with less purified preparations. The agent bound to phenyl-Sepharose could not be eluted with 8.5 M ethylene glycol; however, a combination of ethylene glycol and detergents did release the agent. These observations provide good evidence for a protein and for hydrophobic domains within the scrapie agent. Whether the protein required for infectivity is the same protein responsible for the hydrophobic properties of the scrapie agent remains to be established.