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The O protein of bacteriophage lambda localizes the initiation of DNA replication to a unique site on the lambda genome, ori lambda. By means of electron microscopy, we infer that the binding of O to ori lambda initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages of this prepriming reaction, the first two of which we have characterized previously. First, dimeric O protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, lambda P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A + T-rich region adjacent to the O binding sites. Third, the addition of the DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two O binding sites and the adjacent A + T-rich region to the right of the binding sites. We conclude that the lambda O protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.

We have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZ alpha-complementation gene, which encodes the alpha region (specifying the amino-terminal portion) of beta-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase alpha-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes.

Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.

The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR. The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate. If butyryl-CoA is involved in the condensation, caproate is the ultimate product. ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product. In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway. The rapid initial production of acetate indicates equally rapid production of ATP. Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate. The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium. The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds. 13C-13C coupling in the butyrate and caproate produced from [1-13C]ethanol and [2-13C]acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated. The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources.

We have determined the fidelity of DNA replication by human cell extracts in reactions containing excess dGTP. Replication errors were scored using two M13 DNA substrates having the replication origin on opposite sides of the lacZ alpha-complementation gene. The data suggest that the average rates for replication errors resulting from G(template), T.dGTP, and A.dGTP mispairs are 25 x 10(-6), 12 x 10(-6), and 3 x 10(-6), respectively. The data also suggest that error rates for both the (+) and (-) strands differ by less than 2-fold when they are replicated either as the leading or lagging strand. This is in contrast to the 33- and 8-fold differences observed earlier for G.dTTP and C.dTTP mispairs on the (+) strand when replicated by the leading or lagging strand complex (Roberts, J. D., Izuta, S., Thomas, D. C., and Kunkel, T. A. (1994) J. Biol. Chem. 269, 1711-1717). Thus, the relative fidelity of the leading and lagging strand replication proteins varies with the mispair and sequence considered. Misincorporation of dGTP preferentially occurs at template positions where dGTP is the next correct nucleotide to be incorporated. This "next nucleotide" effect is characteristic of reduced exonucleolytic proofreading and suggests that these replication errors are normally proofread efficiently. Fidelity measurements performed in the absence or presence of dGMP, an inhibitor of proofreading exonuclease activity, suggest that the leading strand replication complex proofreads some mispairs more efficiently than does the lagging strand replication complex.

A general repair process for DNA heteroduplexes has been detected in HeLa cell extracts. Using a variety of M13mp2 DNA substrates containing single-base mismatches and extra nucleotides, extensive repair is observed after incubation with HeLa cell cytoplasmic extracts and subsequent transfection of bacterial cells with the treated DNA. Most, but not all, mispairs as well as two frameshift heteroduplexes are repaired efficiently. Parallel measurements of repair in HeLa extracts and in Escherichia coli suggest that repair specificities are similar for the two systems. The presence of a nick in the molecule is required for efficient repair in HeLa cell extracts, and the strand containing the nick is the predominantly repaired strand. Mismatch-dependent DNA synthesis is observed when radiolabeled restriction fragments, produced by reaction of the extract with heteroduplex and homoduplex molecules, are compared. Specific labeling of fragments, representing a region of approximately 1,000 base pairs and containing the nick and the mismatch, is detected for the heteroduplex substrate but not the homoduplex. The repair reaction is complete after 20 min and requires added Mg2+, ATP, and an ATP-regenerating system, but not dNTPs, which are present at sufficient levels in the extract. An inhibitor of DNA polymerase beta, dideoxythimidine 5'-triphosphate, does not inhibit mismatch-specific DNA synthesis. Aphidicolin, an inhibitor of DNA polymerases alpha, delta, and epsilom, inhibits both semiconservative replication and repair synthesis in the extract. Butylphenyl-dGTP also inhibits both replicative and repair synthesis but at a concentration known to inhibit DNA polymerase alpha preferentially rather than delta or epsilon. This suggests that DNA polymerase alpha may function in mismatch repair.

Pathways of ammonia assimilation into glutamic acid in Bacillus macerans were investigated by measurements of the specific activities of glutamate dehydrogenase (GDH), glutamine synthetase, and glutamate synthase. In ammonia-rich medium, GDH was the predominant pathway of ammonia assimilation. In nitrogen-fixing cells in which the intracellular NH4+ concentration was 1.4 +/- 0.5 mM, the activity of GDH with a Km of 2.2 mM for NH4+ was found to be severalfold higher than that of glutamate synthase. The result suggests that GDH plays a significant role in the assimilation of NH4+ in N2-fixing B. macerans.

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha, gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.

15N NMR spectra were taken of 15N-enriched tRNA extracted from bakers yeast; ammonium sulfate was used as a nitrogen source. The increase in the degree of denaturation of tRNA, which occurs with increase in temperature from 30 degrees C to 70 degrees C, resulted in no large changes in 15N chemical shifts at acidic and neutral pH but quite pronounced changes in proton-15N nuclear Overhauser effects.

We have developed a soluble enzyme system that replicates exogenously added plasmid DNA (lambda dv) bearing the replication origin of the bacteriophage lambda chromosome. The system contains pure phage lambda O and P replication proteins and a partially purified mixture of Escherichia coli replication proteins [the enzyme system of Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374). The features of lambda dv replication in this system closely resemble the known characteristics of phage lambda DNA replication in vivo. The system (i) depends completely on exogenously supplied DNA, (ii) specifically replicates supercoiled plasmid DNA that contains a lambda replication origin, (iii) depends on both the lambda O protein and the lambda P protein, (iv) depends on RNA polymerase, (v) depends on host replication proteins (e.g., primase, dnaB protein, and several others that function in the priming of DNA synthesis in E. coli) as judged by antibody inhibitions, and (vi) replicates as much as 32% of added lambda dv plasmid DNA through a single complete round to generate catenated daughter molecules. Furthermore, replication of lambda dv DNA in vitro requires DNA gyrase and an ATP-regenerating system. It is notable that addition of lambda O and P proteins to the mixture of E. coli replication proteins inhibits replication of plasmids bearing the origin of the E. coli chromosome. Exploitation of this enzyme system should allow a detailed investigation of the biochemical mechanisms involved in bacteriophage lambda DNA replication and its regulation.

We determined the fidelity of avian myeloblastosis virus and Moloney murine leukemia virus reverse transcriptases (RTs) during DNA synthesis in vitro using the M13mp2 lacZ alpha gene as a mutational target. Both RTs commit an error approximately once for every 30,000 nucleotides polymerized. DNA sequence analysis of mutants generated in a forward mutation assay capable of detecting many types of errors demonstrated that avian myeloblastosis virus RT produced a variety of different mutations. The majority (58%) were single-base substitutions; all of which resulted from the misincorporation of either dAMP or dGMP. Minus-one frameshifts were also common, composing about 30% of the mutations. In addition to single-base events, eight mutants contained sequence changes involving from 2 to 59 bases. The frequency of these mutants suggests that, at least during DNA synthesis in vitro, RTs also commit errors by mechanisms other than classical base miscoding and misalignment. We examined the ability of RTs to synthesize DNA from a mismatched primer terminus at a sequence where the mismatched base was complementary to the next base in the template. Unlike cellular DNA polymerases which polymerize from the mismatched template-primer, RTs preferred to polymerize from a rearranged template-primer containing a matched terminal base pair and an unpaired base in the template strand. The unusual preference for this substrate suggests that the interactions between RTs and the template-primer are different from those of cellular DNA polymerases. The overall error rate of RT in vitro is sufficient to account for the estimated mutation rate of these viruses.

Approximately one year ago, Dr. J. D. Roberts submitted for microscopic study an unusual polypoid mass removed by him from the nose of a patient residing in eastern Texas. The sections prepared from this mass revealed large endosporulating organisms in various stages of their development and obviously different from any organisms previously encountered in the study of surgical or of autopsy material. No great difficulty, however, was experienced in finding adequate descriptions of these organisms and of the lesions which they produce. About the same time a paper on the pathology of rhinosporidiosis by Karunaratne1 of the Ceylon Medical College appeared, covering the main features of the disease and its causative agent. Rhinosporidiosis, as the name implies, involves most frequently nasal structures, especially the nasal septum. It is a comparatively rare disease of world-wide distribution, produced by a readily demonstrable endo-sporulating vegetable parasite known as Rhinosporidium seeberi. The disease has been recognized most frequently in India and on the neighboring island of

The conformational equilibria of 2-carboxy-1,4-butanedioic acid and its mono-, di-, and trianions were estimated by NMR couplings in dimethyl sulfoxide (DMSO). Intramolecular hydrogen bonding was inferred for the mono- and dianions, but not for the triacid. For the di- and trianions, the ³JHH couplings were consistent with the negative carboxylate groups being much closer together than might be expected from electrostatic repulsion considerations. The successive triacid pKa values were estimated as 7.0, 13.4, and ~20(?) on the Bordwell scale.

Vicinal proton−proton NMR couplings have been employed to estimate the differences in conformational equilibria for 1,4-butanedioic-2,3-¹³C2 acid in the progression of its diprotic to monoprotic to di-ionized forms as a function of solvent in water and in a series of alcohols ranging from methanol to tert-butyl alcohol. Except for water, the percentage of gauche increases from diacid to monoanion and then decreases from monoanion to dianion. The substantial gauche preference for the monoanion species in the less-polar alcohols is clearly the result of intramolecular hydrogen bonding.