Initiation factor-independent translation of mRNA derived from bacillus phage 429 DNA occurs with translation systems derived from Bacilus subtilis or Escherichia coli. This is in sharp contrast to the strict dependence on ribosome salt wash fraction ofE. coli ribosomes for the translation ofT7 and other mRNAs derived from Gram-negative organisms.Although the classification of bacteria as Gram-negative or Gram-positive is based on differences in cell wall architecture (1), additional distinctions between these two groups ofbacteria are known. Evolutionary trees based on 16S and 5S RNA sequences (2, 3) indicate that the Gram-positive bacteria represent some of the most primitive species, such as the clostridia, that are characterized by fermentative metabolism and the ability to form endospores in a hostile environment. The Grampositive bacilli appeared somewhat later with perhaps the first use of aerobic respiratory metabolism, but they retain sporeforming ability. The Gram-negative bacteria evolved even later. They are often capable offacultative respiratory metabolism but have lost the ability to sporulate. Instead, the Gram-negative bacteria, such as Escherichia coli, are equipped to adapt to a changing environment.One manifestation of the increased adaptability of E. coli is its ability to express genes from a wide variety of bacteria including both Gram-positive and Gram-negative species. In contrast, many E. coli genes carried on hybrid plasmids are not expressed in the Gram-positive species Bacillus subtilis (4-6). These in vivo restrictions of heterospecific gene expression in B. subtilis could result from limitations in transcription, translation, or posttranslational processing of genetic information.There is increasing evidence that the translational machinery isolated from E. coli differs significantly from that of its Grampositive predecessor, B. subtilis. Studies from several laboratories indicate that ribosomes isolated from Gram-positive bacteria are extremely inefficient in translating mRNAs derived from Gram-negative bacteria (7-13). Ribosomes from E. coli, on the other hand, show comparable translational efficiency in response to mRNAs from Gram-positive or Gram-negative bacteria.Reconstitution ofhybrid 30S ribosomal subunits has revealed differences in the protein components which affect the efficiencies with which Gram-negative mRNAs are translated (11,14,15). For example, Isono and Isono (16) reported that translation of f2 RNA by Bacillus stearothermophilus ribosomes is stimulated by the addition of E. coli ribosomal protein S1. We have demonstrated here that E. coli ribosomal protein S1 has very little effect on the efficiency with which f2 RNA and T7 mRNA are translated by the B. subtilis translation system and concluded that S1 is not the sole determinant ofspecies-specific initiator recognition.Another approach to understanding the basis for selective mRNA recognition by B. subtilis ribosomes involves characterization of their interaction with mRNAs from Gram-positive organism...

To investigate the function of base 792 of 16S rRNA in 30S ribosomes of Escherichia coli, the wild-type (adenine) residue was changed to guanine, cytosine, or uracil by oligonucleotide-directed mutagenesis. Each base change conferred a unique phenotype on the cells. Cells containing plasmid pKK3535 with G792 or T792 showed no difference in generation time in LB broth containing ampicillin, whereas cells with C792 exhibited a 20% increase in generation time in this medium. To study the effect on cell growth of a homogeneous population of mutant ribosomes, the mutations were cloned into the 16S rRNA gene on pKK3535 carrying a spectinomycin-resistance marker (thymine at position 1192), and the cells were grown with spectinomycin. Cells containing G792 or C792 showed 16% and 56% increases in generation time, respectively, and a concomitant decrease in 3S assimilation into proteins. Cells with T792 did not grow in spectinomycin-containing medium. Maxicell analyses indicated decreasing ability to form 70S ribosomes from 30S subunits containing guanine, cytosine, or uracil at position 792 in 16S rRNA. It appeared that C792-containing 30S ribosomes had lost the ability to bind initiation factor 3. MATERIALS AND METHODSBacteria, Plasmids, and Bacteriophage. Escherichia coli HB101 (10), XL1 Blue (11), and BL21(DE3) (12) have been described. E. coli BL21(DE3) contains a chromosomal gene for phage T7 RNA polymerase under control of the UV5 lac promoter. Plasmid pAR3056 contains the rrnB operon under control of the T7 promoter (13). Plasmid pKK3535 contains the rrnB operon (14, 15). Plasmid pKK3535 with a thymine at position 1192 (T1192) of the 16S rRNA gene, which confers spectinomycin resistance on host cells (16, 17), pAR3056 with the Sma 1-6 deletion, and pKK3535 with the mutation at position 791 were gifts from A. E. Dahlberg (Brown University, Providence, RI). Bacterial strains carrying plasmids were grown in LB broth with either ampicillin (200 ,ug/ml) 3700The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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The spot corresponding to hypoxanthine phosphoribosyltransferase (HPRT; IMP:pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) has been identified in twodimensional polyacrylamide gels of HeLa cell extracts. This spot is absent in gels of 24 HPRT deficient mutants. A missense mutant displays a new HPRT spot at the same molecular weight but different isoelectric focusing position. Five (3)(4)(5)(6). In the present work, we demonstrate that HPRT mutants and revertants can be analyzed by two-dimensional polyacrylamide gel electrophoresis of crude cell extracts. In this procedure, proteins are separated in one dimension by isoelectric focusing, and then in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (7).We have identified the spot corresponding to HPRT on two dimensional gels of HeLa cell extracts even though the enzyme represents only 0.02% of the soluble protein. We have analyzed 24 HPRT deficient mutants, and for every one, the spot corresponding to the wild-type enzyme disappears. A missense mutant labeled H23 displays a new spot at the same molecular-weight location, but at a different isoelectric focusing position. Unexpectedly, five independently isolated revertants of H23 display spots corresponding to both the wild-type and Abbreviations: HPRT, hypoxanthine phosphoribosyltransferase; DME medium, Dulbecco's modified Eagle's medium; TG medium, DME medium containing 6-thioguanine; MTH medium, DME medium containing methotrexate, thymidine, and hypoxanthine; TH medium, DME medium containing thymidine and hypoxanthine. * Address reprint requests to this author. Mutagen Treatment. One million wild-type HeLa cells growing exponentially are placed in plates containing 10 ml of fresh DME medium. The medium is removed after 12-24 hr and replaced with DME medium containing mutagen. The mutagens used are ethyl methanesulfonate at 400, 500, and 600 ,ug/ml and N-methyl, N'-nitro, N-nitrosoguanidine at 4, 5, and 6 ,ug/ml. After 24 hr, the medium containing mutagen is removed, the cells are rinsed with 5 ml of phosphate-buffered saline, and 10 ml of fresh DME medium is added to the plates. The cells are grown in DME medium for 5-10 days prior to the addition of selective medium to allow time for the residual HPRT concentration in mutants to decrease. During this time, the cells are transferred so that they do not exhaust the medium. The three levels of ethyl methanesulfonate and nitrosoguanidine kill approximately 60%, 90%, and 98% of the cells, respectively.Mutant Selections. Ten milliliters of selective "TG medium" containing 0.1 mM 6-thioguanine in DME-medium is added to plates containing approximately 4 to 8 X 106 mutagentreated cells. After 3-7 days, the medium and cell debris are removed and fresh TG medium is added. Colonies are observed 14-21 days after addition of TG medium. Colonies are removed with a pasteur pipet and grown in TG medium. Each mutant cell line is recloned by plating the cells at a low density and isolating individual colonies. To avoid duplication o...