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The unusually high error rate of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) suggests that polymerization errors by this enzyme contribute to the genetic variability of the AIDS virus. We have analyzed the mechanism for HIV-1 RT infidelity by studying two distinct steps that might lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Our results indicate that the capacity of HIV-1 RT to polymerize nucleotides onto mispaired termini is a major factor in the production of mutations by this enzyme. When a noncomplementary dAMP was inserted opposite a template adenine by HIV-1 RT, the nascent 3'-terminal A.A mispair was readily extended by subsequent incorporation of the next complementary nucleotide. The frequencies of nucleotide addition onto 3'-terminal A-A, A-C, and A-G mispairs were determined by quantitating the amount of extended primers with a gel electrophoresis assay and by measuring mutagenesis after hybridization of mismatched primers opposite an amber mutation in bacteriophage phi X174 DNA. The mispair extension frequencies are approximately 50-fold higher by HIV-1 RT than by the mammalian replicative enzyme DNA polymerase alpha.

Addition of the 3'----5' proofreading exonuclease, epsilon subunit of Escherichia coli DNA polymerase III, to DNA polymerase alpha from calf thymus has been studied. Alone, calf thymus DNA polymerase alpha terminates in vitro DNA synthesis upon insertion of noncomplementary nucleotides. Upon addition of the epsilon subunit, DNA polymerase alpha elongates the newly synthesized DNA as a result of hydrolysis of the 3'-terminal mispair. The fidelity of DNA polymerase alpha in vitro is increased 7-fold by addition of the exonuclease. The functional interaction between DNA polymerase alpha and the epsilon subunit is independent of any detectable physical association. This suggests that a mechanism for proofreading could exist in mammalian cells involving sequential catalysis by DNA polymerase alpha excision of errors by a separate 3'----5' exonuclease, and further elongation onto correctly base-paired 3' termini by DNA polymerase alpha.

The DNA polymerase-primase from Drosophila lacks 3'----5' exonuclease activity. However, a potent exonuclease can be detected after separating the 182-kDa polymerase subunit from the other three subunits of the enzyme (73, 60, and 50 kDa) by glycerol gradient sedimentation in the presence of 50% ethylene glycol. The exonuclease activity cosediments with the polymerase subunit, suggesting that the two activities reside in the same polypeptide. The 3'----5' exonuclease excises mismatched bases at the 3' termini of primed synthetic and natural DNA templates. Excision of a mispaired base at the 3' terminus occurs at a 10-fold greater rate than excision of the correctly paired base. When replication fidelity is measured by the bacteriophage phi X174 am3 reversion assay, the isolated polymerase subunit is at least 100-fold more accurate than either the intact polymerase-primase or a complex of the 182- and 73-kDa subunits. These results suggest that the 3'----5' exonuclease functions as a proofreading enzyme during Drosophila DNA replication in vitro and very likely in vivo.

In order to better understand the role of the -35 sequence motif in transcription initiation by Escherichia coli sigma 70 RNA polymerase holoenzyme, -35 promoter elements of limited nucleotide composition have been selected from random DNA sequences. Functional promoter elements have been identified that are composed of just two nucleotide species (A,T; G,C; T,C; and T,G) and one nucleotide species (poly(dT)). From this study of 81 promoter mutations, two conclusions can be made. First, given a population of random DNA sequences, there exist sequences that form stronger -35 promoter elements than the consensus sequence. These sequences lack some features of the consensus sequence and reveal unexpected homology in the ordinarily nonconserved spacer sequence. Second, the sequence requirements at the -35 site may suggest a possible role for DNA secondary structure in the recognition of promoters by RNA polymerase.

The effect of several divalent cations on the accuracy of DNA replication in vitro has been examined. Only Be2+ altered the accuracy of DNA synthesis using purified DNA polymerase (DNA nucleotidyltransferase; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase; EC 2.7.7.7) from avian myeloblastosis virus. The Be2+-induced base substitutions occurred with all templates and with all nucleotides tested. Analysis of the product by equilibrium density centrifugation and processive hydrolysis with snake venom phosphodiesterase suggested that the noncomplementary nucleotides were present in phosphodiester linkage. Nearest neighbor studies indicated that many of the Be2+-induced errors were present as single base substitutions. The enhancement of error frequency could be duplicated by the pretreatment of the enzyme, but not the template, with Be2+. Glycerol gradient centrifugation dissociated the Be2+-DNA polymerase complex and restored the initial error frequency of the polymerase. Thus, the weak binding of a metal cation to a DNA polymerase could alter the accuracy with which that polymerase copied DNA. Beryllium is a known carcinogen. The potential use of this system as a screening technique to detect chemical mutagens and carcinogens is considered.

The effect of Escherichia coli single-strand binding protein on the accuracy of in vitro DNA synthesis has been determined by using two independent methods. By using the synthetic polynucleotide poly[d(A-T)] and measuring dGTP misincorporation or by using phi X174 DNA and measuring nucleotide substitutions, we found that binding protein increases the fidelity of DNA synthesis by as much as 10-fold. This increase is observed with DNA polymerases of divergent sources and is progressive with increasing concentration of binding protein. The increased accuracy observed with DNA polymerases lacking a 3' leads to 5' exonuclease points to a mechanism other than augmented proofreading. In accord with the properties of single-strand binding proteins, it is suggested that increased fidelity is a result of enhanced base selection by the DNA polymerase, resulting from increased rigidity of the template due to its interaction with binding protein.

The fidelity with which wild type T4 DNA polymerase copies phi X174 amber 3 plus strand DNA at position 587 in vitro has been measured. Synthesis is initiated by hybridizing to the template a HaeIII restriction fragment whose 3'-OH terminus is 83 nucleotides from the amber 3 site. Based on gel electrophoresis of product DNA molecules and genetic marker rescue data, T4 DNA polymerase copies significantly beyond the mutant site. Transfection analysis shows that the A X T leads to G X C mutation at position 587 occurs 10- to 100-fold less frequently with T4 DNA polymerase than with E. coli DNA polymerase I. The aberrant incorporation of cytosine opposite adenine at position 587 by the T4 polymerase alone is occurring at a frequency not greater than about 10(-7) which, for this particular locus, may be similar to the fidelity exhibited by the T4 accessory proteins plus the polymerase comprising the replication complex. A comparison of the accuracy of mutator L56 and antimutator L141 T4 DNA polymerases relative to wild type shows at most a 2- to 4-fold decrease and increase, respectively, in fidelity. When compared to 10- to 1000-fold effects on mutation frequencies that these same mutant alleles have in vivo, these results suggest that the wide range in expression of mutator and antimutator phenotypes in vivo may be dependent on an abnormal interaction of the aberrant DNA polymerases with other protein components of the replication complex.

The fidelity of DNA synthesis with purified DNA polymerase alpha and beta from human placenta has been studied. With poly[d(A-T)] as the template-primer and Mg2+ as the metal activator, DNA polymerase alpha incorporates 1 mol of dGMP for every 6,000 to 12,000 mol of complementary nucleotides polymerized. Under the same conditions, DNA polymerase beta is more accurate, the error rate being 1/20,000 to 1/60,000. This greater accuracy of DNA polymerase beta is observed with a variety of homopolymer templates. With both enzymes, substitution of Mg2+ with activating concentrations of Mn2+ or Co2+ enhances the frequency of misincorporation. At greater than activating concentrations of Mn2+ and Co2+, there is an inhibition of complementary nucleotide incorporation, further increasing the frequency of misincorporation. Nearest neighbor analysis of the products synthesized with both enzymes indicates that the noncomplementary nucleotides are incorporated predominantly as single base substitutions. The greater accuracy of DNA polymerase beta over DNA polymerase alpha should be considered in relationship to their possible roles in DNA replication and repair.

We have measured the efficiencies of utilization of 8-oxo-dGTP and 8-NH2-dGTP by human immunodeficiency virus type 1 and murine leukemia virus reverse transcriptases and compared them to those of DNA polymerases alpha and beta. Initially, we carried out primer extension reactions in the presence of dGTP or a dGTP analog and the remaining three dNTPs using synthetic DNA and RNA templates. These assays revealed that, in general, 8-NH2-dGTP is incorporated and extended more efficiently than 8-oxo-dGTP by all enzymes tested. Second, we determined rate constants for the incorporation of each analog opposite a template cytidine residue using steady state single nucleotide extension kinetics. Our results demonstrated the following. 1) Both reverse transcriptases incorporate the nucleotide analogs; discrimination against their incorporation is a function primarily of Km or Vmax depending on the analog and the enzyme. 2) Discrimination against the analogs is more stringent with the DNA template than with a homologous RNA template. 3) Polymerase alpha exhibits a mixed kinetic phenotype, with a large discrimination against 8-oxo-dGTP but a comparatively higher preference for 8-NH2-dGTP. 4) Polymerase beta incorporates both analogs efficiently; there is no discrimination with respect to Km and a significantly lower discrimination with respect to Vmax when compared with the other polymerases.

We describe catalytically active mutants of HIV RT (human immunodeficiency virus reverse transcriptase) generated by random sequence mutagenesis and selected in Escherichia coli for ability to complement the temperature-sensitive phenotype of a DNA polymerase I (Pol Its) mutant. We targeted amino acids Asp-67 through Arg-78 in HIV RT, which form part of the beta3-beta4 flexible loop and harbor many of the currently known mutations that confer resistance to nucleoside analogs. DNA sequencing of 109 selected mutants that complement the Pol Its phenotype revealed substitutions at all 12 residues targeted, indicating that none of the wild-type amino acids is essential. However, single mutations were not observed at Trp-71, Arg-72, and Arg-78, consistent with evolutionary conservation of these residues among viral RTs and lack of variation at these positions among isolates from patients. The mutations we recovered included most of those associated with drug resistance as well as previously unidentified mutations. Purification and assay of 14 mutant proteins revealed correlation between their DNA-dependent DNA polymerize activity in vitro and ability to complement the Pol Its phenotype. Activity of several mutants was resistant to 3'-azidothymidine triphosphate. We conclude that random sequence mutagenesis coupled with positive genetic selection in E. coli yields large numbers of functional HIV RT mutants. Among these are less active variants which are unlikely to be isolated from HIV-infected individuals and which will be informative of the roles of individual amino acids in the catalytic functions of the enzyme.

Oxygen free radicals are highly reactive species generated by many cellular oxidation-reduction processes. These radicals damage cellular constituents and have been causally implicated in the pathogenesis of many human diseases. We report here that oxygen free radicals generated by Fe2+ in aqueous solution are mutagenic. Aerobic incubation of luminal diameter X174 am3 (amber 3 mutation) DNA with Fe2+ results in decreased phage survival when the treated DNA is transfected into Escherichia coli spheroplasts. Transfection of the treated DNA into SOS-induced spheroplasts results in an increase in mutagenesis as great as 50-fold. Both killing and mutagenesis can be prevented by binding of Fe2+ with deferoxamine or by the addition of catalase or mannitol. These results suggest that DNA damage and mutagenesis brought about by Fe2+ are likely to occur by a Fenton-type mechanism that involves the generation of (i) hydrogen peroxide by the autoxidation of iron and (ii) hydroxyl radicals by the interaction of the hydrogen peroxide with Fe2+. DNA sequence analysis of the Fe2+-induced mutants indicates that reversion of the phage phenotype to wild type occurs largely by a transversion type of mutation involving substitution of deoxyadenosine for thymidine opposite a template deoxyadenosine. Mutagenesis is not abolished by incubation of Fe2+-treated luminal diameter X174 am3 DNA with an apurinic endonuclease and only partially abolished by incubation with alkali, suggesting that a large fraction of the mutagenesis by oxygen free radicals is not caused by formation of apurinic sites but instead involves an as-yet-to-be-defined alteration in deoxyadenosine. These findings raise the possibility that free iron localized in cellular DNA may cause mutations by the generation of oxygen free radicals.

A series of colon carcinomas, adenomas, and adjacent tissues were analyzed for ploidy alterations and mutations in KRAS2. To increase the sensitivity for identifying mutations, we used histological enrichment, cell sorting, and DNA amplification by the polymerase-catalyzed chain reaction followed by direct DNA sequence analysis. Of the 40 carcinomas analyzed, 27 contained aneuploid cells and 26 contained mutations at the first position of codon 12 of KRAS2. Of the 12 adenomas studied, 4 contained aneuploid cells and 9 contained the same mutation at codon 12. In both adenomas and carcinomas, mutations were identified in both diploid and aneuploid cells. In some cases, regions of histologically benign mucosa adjacent to the carcinoma contained mutations. These combined results suggest that mutations in KRAS2 occur early in the development of human colon carcinoma, before change in ploidy, and that these mutations exist in diploid cells from which an aneuploid subpopulation arises. Furthermore, mutations may exist in histologically normal mucosa in regions adjacent to carcinoma, suggesting that a field of genetically abnormal mucosa may surround these tumors.

Recombinant DNA techniques have been used to quantitate the amount of nucleotide sequence divergence in the mitochondrial DNA population of individual normal humans. Mitochondrial DNA was isolated from the peripheral blood lymphocytes of five normal humans and cloned in M13 mp11; 49 kilobases of nucleotide sequence information was obtained from 248 independently isolated clones from the five normal donors. Both between- and within-individual differences were identified. Between-individual differences were identified in approximately 1/200 nucleotides. In contrast, only one within-individual difference was identified in 49 kilobases of nucleotide sequence information. This high degree of mitochondrial nucleotide sequence homogeneity in human somatic cells is in marked contrast to the rapid evolutionary divergence of human mitochondrial DNA and suggests the existence of mechanisms for the concerted preservation of mammalian mitochondrial DNA sequences in single organisms.

The Chinese hamster V79 cell mutant aphr-4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase alpha (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase alpha has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the alpha-type polymerase: sensitivity to 0.2 M KCl, resistance to heat denaturation (45 degrees C for 15 min), an apparent Km of 5 microM for dATP, and an ability to copy poly(dT)X(rA)10 but not poly(rA)X(dT)12. The crude extracts and purified DNA polymerase alpha from the mutant cells are not inhibited by aphidicolin (greater than 0.6 microM). The apparent Km for dCTP with DNA polymerase alpha is 1.0 +/- 0.4 microM (mean +/- SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent Km for dCTP of 10 +/- 4 microM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.6.1.8) locus, is higher for mutant cells (42-73 x 10(-8)) than for parental cells (3-16 x 10(-8)). These data suggest a mechanism for aphidicolin resistance of the mutant--i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase alpha may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.

We have selected a group of Escherichia coli promoters from random DNA sequences by replacing 19 base pairs at the -35 promoter region of the tetracycline resistance gene tetr of the plasmid pBR322. Substitution of 19 base pairs with chemically synthesized random sequences results in a maximum of 4(19) (about 3 X 10(11)) possible replacement sequences. From a population of about 1000 bacteria harboring plasmids with these random substitutions, tetracycline selection has revealed several functional -35 promoter sequences. These promoters have retained only partial homology to the -35 promoter consensus sequence. In three of these promoters, the consensus alignment shifts 10 nucleotides downstream, allowing the RNA polymerase to recognize another Pribnow box from within the original pBR322 sequence. Two of the sequences promote transcription more strongly than the native promoter. This technique may have application for the selection of additional DNA sequences with varied biological activity.

Two families of platelet phospholipase A2 activity, were chromatographically resolved by anion exchange chromatography and were functionally distinguishable by their differential phospholipid subclass substrate specificity and calcium ion requirements. The major phospholipase A2 activity was present in the cytosolic compartment, eluted from DEAE-cellulose at 230 mM NaCl (hereafter referred to as phospholipase A2(beta)), and demonstrated a 100-fold selectivity in catalyzing the hydrolysis of 1-(O)-(Z)-hexadecenyl-2-oleoyl-sn-glycero-3-phosphocholine (plasmenylcholine) in comparisons with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (phosphatidylcholine). Phospholipase A2(beta) was purified to homogeneity by sequential gel filtration and Mono Q column chromatographies. Phospholipase A2(beta) eluted with an apparent molecular mass of 58 kDa during gel filtration chromatography and migrated as a single band with an apparent molecular mass of 30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that its native quaternary structure is dimeric. Fast protein liquid chromatography demonstrated that the polypeptides catalyzing this activity were comprised of multiple isoforms which possessed different specific activities. Each isoform required Ca2+ ion for activity and was completely activated over the range through which Ca2+ ion concentration is augmented in stimulated platelets (i.e. 300-800 nM).

The effect of pyrophosphate on the fidelity of in vitro DNA synthesis has been examined. Pyrophosphate enhances misincorporation by Escherichia coli DNA polymerase I in copying phi X174 DNA. The increased misincorporation is directly proportional to the extent of inhibition of the rate of polymerization. In contrast, pyrophosphate is not detectably mutagenic with avian myeloblastosis virus DNA polymerase or DNA polymerases alpha and beta from animal cells, which lack associated proofreading activities. This suggests that increased misincorporation by pyrophosphate is not due to an increase in misinsertions by DNA polymerase, but rather due to inhibition of proofreading by pyrophosphate. However, the pyrophosphate-induced infidelity has a different specificity from, and is not competitive with, two experimental markers of 3'----5' exonuclease proofreading; i.e. the effects of the next nucleotide or the addition of deoxynucleoside monophosphates. These distinctive features suggest a second mode of proofreading susceptible to inhibition by pyrophosphate. This concept is discussed in relation to models for proofreading described in the literature.

The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5′-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5′-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5′-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.

The mutagenic potential of apurinic sites in vivo has been studied by transfection of depurinated phi X174 DNA containing amber mutations into SOS-induced Escherichia coli spheroplasts. Mutagenicity is abolished by treatment of the depurinated DNA with an apurinic endonuclease from Hela cells, establishing the apurinic site as the mutagenic lesion. The frequency of copying apurinic sites in vitro was analyzed by measuring the extent of DNA synthesis using E. coli DNA polymerase I and avian myeloblastosis DNA polymerase. The inhibition of DNA synthesis by apurinic sites was less with avian myeloblastosis DNA polymerase, suggesting that this error-prone enzyme copies apurinic sites with greater frequency. Consistent with this conclusion is the observation that, upon transfection into (normal) spheroplasts, the reversion frequency of depurinated phi X174 am3 DNA copied with avian myeloblastosis virus DNA polymerase is much greater than that of the same DNA copied with E. coli DNA polymerase I. Sequence analysis of the DNA of 33 revertant phage produced by depurination indicates a preference for incorporation of deoxyadenosine opposite putative apurinic sites. The combined results indicate that mutagenesis resulting from apurinic sites is associated with bypass of these noncoding lesions during DNA synthesis.

The error frequency of in vitro DNA synthesis using a natural DNA template has been measured with a biological assay for nucleotide substitutions. phiX174 DNA containing an amber mutation was copied in vitro by Escherichia coli DNA polymerase I, and the reversion frequency of the progeny DNA was determined by transfection of E. coli spheroplasts. E. coli polymerase I makes less than 1 mistake at the am3 locus for every 7700 nucleotides incorporated under standard reaction conditions. Substitution of Mn2+ for Mg2+ and unequal concentrations of deoxynucleoside triphosphate substrates raises this mutation frequency to greater than 1 in 1000. Thus, E. coli DNA polymerase I can copy natural DNA templates with high fidelity and its accuracy can be affected by alterations in reaction conditions.

To assess the involvement of terminal transferase in generating immunoglobulin diversity, the mutagenic potential of this enzyme has been measured. The frequency of single base substitutions during copying of phi X174 DNA by DNA polymerase beta is increased by, at most, 3-fold upon the addition of terminal transferase. However, terminal transferase is highly mutagenic, either alone or with DNA polymerase beta, in a forward mutation assay using M13mp2 DNA. The frequency of complex mutants, as determined by DNA sequence, is increased by greater than 100-fold. These mutants involve the deletion of a variable number of bases initially present in the template sequence and the addition of a sequence of nucleotides rich in guanine residues. Analysis of these mutants suggests an antibody diversity model implicating terminal transferase in the imprecise linkage of variable, joining, and diversity segments during the formation of functional immunoglobulin genes.

O4-Alkylthymine-DNA adducts have been implicated as causative lesions in chemical mutagenesis and carcinogenesis. To directly assess the mutagenic potential of these adducts in vivo, we have designed an enzymatic technique for introducing nucleotide analogues at predetermined sites of biologically active DNA. Escherichia coli DNA polymerase I was used in vitro to incorporate a single O4-methylthymine residue at the 3' terminus of an oligonucleotide primer opposite the adenine residue of the amber codon in bacteriophage phi X174 am3 DNA. After further extension of the primer with unmodified nucleotides, the partial-duplex product was transfected into E. coli spheroplasts. Replication of the site-specifically methylated DNA in E. coli deficient in O4-methylthymine-DNA methyltransferase (ada-) yielded 10-fold more mutant progeny phage than replication of nonmethylated DNA; no increase in mutation frequency was observed after replication in repair-proficient (ada+) E. coli. The DNA from 20 independently isolated mutant plaques all contained A.T----G.C transitions at the original site of O4-methylthymine incorporation. These data demonstrate that O4-methylthymine induces base-substitution mutations in E. coli and suggest that this adduct may be involved in mutagenesis by N-nitroso methylating agents. This enzymatic technique for site-specific mutagenesis provides an alternative to the chemical synthesis of oligonucleotides containing altered bases.

The fidelity of DNA polymerase-α-primase from calf thymus has been analyzed by measuring mutagenesis in vitroand by site-specific nucleotide misinsertion and mispair extension. Using the ϕX174 am3 DNA reversion assay errors are detected at the amber3 site only when both dATP and dCTP are significantly biased during in vitrocopying reactions. Analysis of these products on DNA sequencing gels reveals pause sites due to the slow extension of mispaired 3′ termini. Measurements of misinsertion rates opposite template A show that the rates of dAMP or dCMP misinsertion are similar and occur 40–50 times more rapidly than dGMP misinsertion. The rate of extension from an A:C mispair is 100- and 400-fold greater than from an A:A mispair and an A:G mispair, respectively. Nucleotide misinsertions to generate all 12 possible mispairs have been measured kinetically on ϕX174 DNA templates that contain either A, C, G, or T at position 587. Misinsertion frequencies range from 1/4000 to 1/106depending on the mispairs generated. Extension from all 12 different mispairs was examined by starting with oligonucleotide primers that contain different 3′-terminal mispairs. Rates of extension from mispairs are 103to 106times slower than from correctly paired bases. Extension frequencies were purine:pyrimidine > pyrimidine:pyrimidine > purine:purine. Lack of extension of misincorporated bases suggests the involvement of exonucleolytic proofreading to enable continued DNA synthesis and to guarantee the high fidelity of eucaryotic DNA replication.

The relative mutagenicities of O-alkylthymine-DNA adducts were analyzed in vivo by site-specific mutagenesis. Purified DNA polymerases were used to incorporate O4-methyl (Me)-, O4-ethyl (Et)-, O4-isopropyl (iPr)-, or O2-Me-dTTP onto the 3' terminus of a synthetic oligonucleotide (15-mer) hybridized to phi X174 am3 DNA. The product oligonucleotides were further extended in the presence of unmodified dNTPs to yield 21-mers containing single O-alkylthymine adducts opposite the adenine residue of the bacteriophage amber codon. Polyacrylamide gel electrophoresis and nearest-neighbor analyses confirmed the identities and nucleotide positions of the adducts. Transfection and replication of the site-specifically alkylated DNAs in ada- Escherichia coli (defective in the alkyltransferase capable of repairing O4-alkylthymine-DNA adducts) yielded mutant progeny phage with reversion frequencies of: O4-Me-dThd (19.5 × 10(-6)) greater than O4-Et-dThd (7.5 × 10(-6)) greater than O4-iPr-dThd (3.0 × 10(-6)) greater than or equal to O2-Me-dThd (1.0 × 10(-6)) approximately equal to dThd (2.0 × 10(-6)). None of the adducts produced mutations above background following replication in ada+ E. coli. DNA sequence analyses of 40 independently isolated mutant phage derived from the O4-Me- or O4-Et-dThd-containing DNAs showed that all mutants contained guanine residues opposite the original site of the alkylthymines. These data are consistent with a mechanism of mutagenesis involving the formation of O4-alkyl-T.G base pairs during DNA replication in E. coli and suggest that the formation of A.T—G.C transition mutations is characteristic of mutagenesis by O4-Me- and O4-Et-dThds in vivo.

Error rates for conventionally purified DNA polymerase-alpha from calf thymus, chicken, and human sources have been reported to be one in 10,000 to one in 40,000 nucleotides incorporated. Isolation of polymerase-alpha by immunoaffinity chromatography yields a multiprotein high molecular weight replication complex that contains an associated DNA primase (Wong, S. W., Paborsky, L. R., Fisher, P. A., Wang, T. S-F., and Korn, D. (1986) J. Biol. Chem. 261, 7958-7968). We have isolated DNA polymerase-primase complexes from calf thymus, from a human lymphoblast cell line (TK-6), and from Chinese hamster lung cells (V-79) using two different methods of immunoaffinity chromatography. These enzyme complexes are 12- to 20-fold more accurate than conventionally purified calf thymus DNA polymerase-alpha when assayed using the phi X174am3 fidelity assay; estimated error rates are one in 460,000 to one in 830,000 nucleotides incorporated when the enzyme complex is freshly isolated. The polymerase-primase complex from calf thymus exhibited no detectable 3'—5' exonuclease activity using a heteroduplex substrate containing a single 3'-terminal mismatched nucleotide. Upon prolonged storage at −70 degrees C, the error rate of the immunoaffinity-purified calf thymus DNA polymerase-primase complex increases to about one in 50,000 nucleotides incorporated, an error rate similar to that exhibited by conventional isolates of DNA polymerase-alpha.

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.

Two distinct mechanisms of action for intercalating agents have been delineated: one leading to the production of frameshift misincorporations and the other leading to the production of single-base substitutions. Addition misincorporations are competitive with respect to DNA template (a measure of classical intercalation) but are not competitive with respect to deoxynucleotide substrates. Single-base substitutions are not competitive with template, polymerase, or deoxynucleotide as tested individually, but are proportional to the absolute drug concentration, indicating a ternary complex involving intercalator, polymerase, and template. Increased frequencies of single-base substitutions have not been considered as a general property of intercalators. Using a mutant phi X174 DNA, we demonstrate that intercalators also induce single-base substitutions with natural DNA templates. Reversion of am3 phi X174 DNA occurs only by single-base substitutions at position 587; this is increased 8-fold when the DNA is copied in vitro in the presence of intercalators.

The effects of intercalating agents on the fidelity of DNA synthesis in vitro have been investigated. The accuracy of DNA synthesis with Escherichia coli DNA polymerase I with both the poly[d(A-T)] and poly[d(G-C)] templates is decreased in the presence of the intercalating agents proflavin, ethidium bromide, acridine orange, ICR-170, and ICR-191. Nearest neighbor analyses of the product of the reaction indicate that two different types of misincorporations occur in the presence of intercalating agents, frameshifts, and single-base substitutions. With alternating polynucleotide templates, frameshifts involving pyrimidines are the most frequent change in sequence observed. Overall, frameshift misincorporations occur with frequencies of one complementary pyrimidine for each intercalated site and one noncomplementary pyrimidine for each 150 sites. From analysis of nearest neighbor frequencies in the product, it is inferred that the intercalating agents interact specifically with pyrimidine (3' leads to 5') purine sequences. An analysis of ratios of correct nucleotide incorporations as a function of intercalator concentration indicates that frameshifts are predominantly additions; however, one cannot rule out infrequent deletions. Base substitutions in the presence of intercalators occur less frequently than frameshifts. From the results of reaction kinetics and nearest neighbor frequencies, it is concluded that the noncomplementary nucleotides are incorporated in phosphodiester linkage and are present as single-base substitutions. Taken together, the results of these studies suggest at least two different modes of action for intercalating agents on the accuracy of DNA synthesis: one leading to frameshift misincorporations and the other leading to single-base substitutions.

According to Fraenkel,1 emphysema is found in more than 5 per cent of all autopsies. It is rarely found in children, but increases in frequency with each decade. It is often present in persons about 40 years of age, and frequently occurs during old age. Generally it is described as occurring in three types (excluding interstitial emphysema, which has no relation to the disease under discussion) namely: compensatory, atrophic and hypertrophic or vesicular emphysema. TYPES OF EMPHYSEMA COMPENSATORY. —Compensatory emphysema is a condition in which one portion of the lung increases in size and function, when another portion is destroyed or temporarily useless. It occurs, for instance, in association with pneumonias, pleural effusions and pneumothorax. Anatomically, there is found an enlargement of the normal lung; there are no variations from the normal structure; the unaffected lung, as a result of distention, has an increased vital capacity and is able to

(From the Pathological Laboratory of Rush Medical College,)In numerous journals at short intervals beginning in April, 1898, the Lyonese observers, Arloing and Courmont, have announced the discovery of a means for the diagnosis of early tuberculosis more certain and less harmful than any hitherto known. This naturally attracted great attention at once, although on account of the many difficulties in experimentation and control of results relatively few repeated the original measures. Many means and ways for the diagnosis of incipient tuberculosis have been suggested of late years, but all of them are either harmful or insufficient. By "incipient" tuberculosis is meant that period of the disease in which Koch's bacillus can not be clinically detected in the lesion it produces or in the products thereof.A few of the methods devised, with the objections to them, are the following: 1. The tuberculin test and its modifications by Koch and

A study has been made of the parameters affecting the retention of liquid water by cotton fabric during centrifugal extraction. In addition to the obvious effect of centrifugal field strength, data are given on the effects of initial water content of the fabric load, surface tension and temperature of the water being expelled, thickness of the fabric bed, and others. The results are discussed in relation to a water retention theory based on the equilibrium between surface forces and gravitational forces which was proposed by J. M. Preston and co-workers.