Site-directed mutagenesis in single cells: transitions produced by DNA carrying a single O6-alkylguanine residue.

Using a single burst assay based on a Poisson Distribution, I have determined the mutant virus frequency in single spheroplasts transfected with phi X174 form I' DNA carrying an O6-methyl-, ethyl-, n-propyl- or n-butylguanine residue at position 2401 of the minus strand. One set of experiments was performed with spheroplasts derived from Escherichia coli AB1157, which has normal DNA-repair systems. Of the cells examined after transfection with DNA carrying a methylguanine moiety, 30% produced mutant virus and 12% contained only mutants; with ethylguanine, 55% of the cells had mutants and 41% produced only mutants; with butylguanine, 6% of the cells had mutants and 3% contained only mutants; with propylguanine no mutants were detected in the 33 cells examined. In similar experiments carried out with spheroplasts defective in excision repair (E. coli AB1157 uvrA6) the percentage of cells producing mutant phage after transfection with DNA carrying an O6-butylguanine residue increased from 6 to 21%, and the percentage of cells producing only mutants increased from 3 to 8%; with DNA carrying an O6-methylguanine moiety, the percentage of cells producing mutants decreased from 30 to 6% and the percentage of cells producing only mutants fell from 12% to 0. In order for an individual uvrA cell to produce exclusively mutant phage from a single O6-alkylguanine residue some form of selection must occur during replication because one strand of the transfecting DNA is wild-type, and excision repair, which could lead to a homoduplex with the transition in both strands, is defective in these cells. This selection must also occur in cells with normal DNA repair. The first event in the selection process is critical; if replication of the alkyl-DNA occurs first and if a mutation is produced, then there is a significant probability that the cell will produce only mutant virus regardless of whether or not repair occurs in subsequent events, but the frequency one observes is influenced strongly by the status of the repair systems in the cell.

Site-specific mutagenesis in cells with normal DNA repair systems: transitions produced from DNA carrying a single O6-alkylguanine.

This paper describes a systematic study of transition frequencies produced in vivo when a homologous series of O6-alkylguanine residues located at a preselected position in gene G of phi X174 form I' DNA (double-stranded, circular, covalently-closed, relaxed) is transfected into spheroplasts from two strains of Escherichia coli having normal DNA repair systems. Mutant frequencies were measured as percent of total phage produced by single bursts. The results are: (A) Synthetic DNA without any alkyl group gave a transition frequency of 0.02%. (B) In E. coli AB1157, the frequencies fall into two groups depending on the alkyl group: methyl and ethyl, 8-11%; n-propyl and n-butyl approximately 0.9%. (C) The average transition frequencies were higher in AB1157 than in C600. These data demonstrate that a single O6-alkylguanine residue can produce a specific transition at significant frequencies in cells with normal repair systems and that the mutant frequency depends upon the nature of the alkyl group and the cell type.

Solid-phase synthesis and side reactions of oligonucleotides containing O-alkylthymine residues.

As part of our studies on the molecular mechanism of mutation [Chambers, R. W. (1982) in Molecular and Cellular Mechanisms of Mutagenesis (Lemontt, J. F., & Generoso, W. M., Eds.) pp 121-145, Plenum, New York and London], we wanted to prepare specific oligonucleotides carrying O2- or O4-alkylthymidine residues. Since O-alkylthymine moieties are known to be alkali labile, side reactions were expected during the deprotection procedures used for synthesis of oligonucleotides on a solid support by the classical phosphoramidite method. We have studied these side reactions in detail. Kinetic data show the deprotection procedures displace most O-alkyl groups at rates that make these procedures inappropriate for synthesis of most oligonucleotides carrying O-alkylthymine moieties. We describe alternative deprotection procedures, using readily accessible reagents, that we have used successfully to synthesize a series of oligonucleotides carrying several different O-alkylthymine moieties. The oligonucleotides synthesized are d(A-A-A-A-G-T-alkT-T-A-A-A-A-C-A-T), where alk = O2-methyl, O2-isopropyl, O4-methyl, O4-isopropyl, and O4-n-butyl. This work extends the previously described procedure for the chemical synthesis of oligonucleotides carrying an O4-methylthymine moiety [Li, B. F., Reese, C. B., & Swann, P. F. (1987) Biochemistry 26, 1086-1093] and reports the first chemical synthesis of an oligonucleotide carrying an O2-alkylthymine. The oligonucleotides synthesized have a sequence corresponding to the minus strand that is complementary to the viral strand at the start of gene G in bacteriophage phi X174 replicative form DNA where the normal third codon has been replaced with the ocher codon, TAA.

On the nature of suppression by Escherichia coli HF4714.

The growth of 2 well characterized bacteriophage phi X174 mutants, phi XGam3 and phi XGms3 (TCG)Ser, was examined on 3 suppressor strains of Escherichia coli: CQ3 Su1, which inserts serine at amber codons; HF4714, a well known amber suppressor that has been used as the permissive strain for a number of phi X mutants; and CQ2 Su3, which inserts tyrosine. The data demonstrate HF4714 inserts glutamine and is Su2, not Su1 as has been reported in several recent papers.

In vivo effect of DNA repair on the transition frequency produced from a single O6-methyl- or O6-n-butyl-guanine in a T:G base pair.

We have previously reported some effects of DNA repair on the transition frequencies produced by an O6-methyl-guanine (MeG) or an O6-n-butyl-guanine (BuG) paired with C at the first position of the third codon in gene G of bacteriophage phi X174 form I' DNA (Chambers et al. 1985). We now report experiments in which the transition is produced from T:MeG or T:BuG, instead of C:MeG or C:BuG, located at this site. The site-modified DNAs were transfected into cells with normal DNA repair as well as into cells with repair defects (uvrA, uvrB, uvrC, recA, uvrArecA). The lysates were screened for phage carrying the expected transition using a characteristic change in phenotype. The data demonstrate that the transition frequency from T:BuG is low (0.3% of total phage progeny) in cells with normal repair (Escherichia coli AB1157) and increases 7-fold in uvrA cells (E. coli AB1886). A similar increase is seen in uvrB and uvrC cells (AB1885, AB1884). These data, like our previous data, indicate BuG is repaired primarily by excision. In contrast to this, the transition frequency from T:MeG is high (5 +/- 2%) in cells with normal repair. After induction of alkyl transfer repair in E. coli AB1157, the transition frequency goes up 5-fold. Compared with cells with normal repair, the transition frequency goes up 2-fold in uvrA, uvrB and uvrC cells; it goes up 1.5-fold in recA cells (E. coli AB2463). The data reinforce our earlier conclusion that MeG is repaired primarily by alkyl transfer, but the ABC excinuclease as well as RecA protein inhibit this repair process.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and properties of oligodeoxynucleotides with an AP site at a preselected position.

A general synthesis of a deoxyoligonucleotide with an AP site at a preselected sequence is described. Deoxyuridine is introduced during routine oligonucleotide syntheses of d(TTTUTTTT) and d(AAAAGTTUAAAACAT). Treatment with uracil DNA-glycosylase produces d(TTTrTTTT), where r = deoxyribose, and d(AAAAGTTprpAAAACAT). KM and Vmax are: d(TTTUTTTT), 7.3 X 10(-9)M and 2.0 X 10(-9) mumol/min; d(AAAAGTTUAAAACAT), 1.5 X 10(-8) M and 6.4 X 10(-9) mumol/min. Both d(AAAAGTTprpAAAACAT) and d(TTTprpTTTT) undergo rapid beta-elimination in 1 M piperidine at 25 degrees giving two oligonucleotide fragments, d(AAAAGTTpr') and d(pAAAACAT), where r' = -O-CH2-CHOH-CH=CH-CHO (or its hemiacetal form). The fragment, d(AAAAGTTpr'), which can be isolated by reverse phase chromatography, is resistant to the 3'----5' exonuclease activity of snake venom phosphodiesterase. Endonucleolytic hydrolysis of the penultimate phosphodiester occurs removing pTpr' and generating a normal 3'-OH end. In 1 M piperidine at 90 degrees two beta-eliminations occur producing the oligonucleotides d(AAAAGTTp) and d(pAAAACAT) from d(AAAAGTTprpAAAACAT); d(TTTp) and d(pTTTT) from d(TTTprpTTTT).

A study of side reactions occurring during synthesis of oligodeoxynucleotides containing O6-alkyldeoxyguanosine residues at preselected sites.

As part of our studies on the molecular mechanisms of mutation by carcinogens we have synthesized 12 oligonucleotides (15-mers) containing an O6-alkylguanine residue at a preselected position for use as primers in the enzymatic synthesis of biologically active DNA. Ten of these oligonucleotides are derived from a minus strand sequence carrying the modified nucleotide in the third codon of gene G of bacteriophage phi X174 DNA. Two others are derived from plus strand sequences carrying the modification in the 12th codon of the human Ha-ras protooncogene. During this work several potentially serious side reactions, which could complicate interpretation of mutagenesis data, were observed. This paper describes a detailed study of these reactions. Since we were unable to avoid undesirable side products, we developed simple chromatographic methods for detecting and removing them.

uvrA and recA mutations inhibit a site-specific transition produced by a single O6-methylguanine in gene G of bacteriophage phi X174.

Using site-specific mutagenesis, we have examined the mutagenic activity in vivo of O6-methylguanine or O6-n-butylguanine located at a preselected site in gene G of bacteriophage phi X174. The experiments were designed so that the phage mutant produced by a targeted transition from either of these alkylated derivatives would be recognizable by a simple plaque assay. Spheroplasts derived from normal and repair-deficient cells were transfected, and the lysates were screened for mutant virus. In cells with normal repair, DNA carrying the methylguanine produced the expected transition in 15% of the total phage; DNA carrying the butylguanine produced the same mutation in 0.3% of the phage. In cells deficient in excision repair (uvrA) the transition frequency went up by a factor of 8 for O6-butylguanine and down by a factor of 40 for O6-methylguanine. In cells deficient in recombination (recA), the transition frequency increased 1.5-fold for butylguanine and decreased by a factor of 8 for methylguanine. The data show that both methyl- and butylguanine produce site-directed transitions in phi X174; the transition occurs in recA cells; the frequency of the transition is influenced by both recA and uvrA mutations; the recA and uvrA mutations alter the transition frequency for methylguanine and butylguanine in opposite directions.

Chemical carcinogenesis: a biochemical overview.

In principle, it is possible to identify and eliminate chemicals in our environment that cause cancer, but practical considerations make it very difficult to achieve this goal. Since we will be exposed to carcinogens for some time to come, we must try to understand how known carcinogens work at the molecular level if we are to develop more rational approaches for preventing their action. This knowledge is also important if we are to proceed logically in developing better procedures for early diagnosis and treatment. Since somatic cell mutations produced by exposure to chemical carcinogens represent key events in the initiation of tumors, we must try to understand which genes are involved, what kind of mutations trigger transformation and how carcinogens produce these alterations of DNA. In this paper I discuss metabolic activation of chemicals to produce carcinogens that react with DNA, consider repair of the covalent adducts that are produced when carcinogens react with DNA, and point out some key questions that must be answered before we can understand how carcinogens work. Then I outline some recent progress in the molecular biology of human oncogenes and indicate how we are using site-specific mutagenesis in preselected codons of the c-Ha ras protooncogene in an attempt to define which carcinogen-induced DNA adducts transform normal protooncogenes into active oncogenes.

Lumbar nerve root compression by synovial cysts of the ligamentum flavum. Report of four cases.

Synovial cysts of the ligamentum flavum, measuring 1 cm in diameter, caused compression of the lumbar nerve roots in four patients. The authors discuss the association of these cysts with advanced focal spondylosis, and speculate on their etiology.

Photochemical cleavage of phosphodiester bonds in oligoribonucleotides.

The release of inorganic phosphate from a variety of mononucleotides and the generation of new phosphomonoester end groups as a result of chain cleavage in a number of oligoribonucleotides have been studied quantitatively as a function of irradiation with 254-nm light. The reaction cross sections for adenosine 5'-phosphate, guanosine 2'(3')-phosphate, cytidine 5'-phosphate, cytidine 3'-phosphate, cytidine 2'(3')-phosphate, uridine 5'-phosphate, uridine 2'(3')-phosphate, dihydrouridine 5'-phosphate, and ribose 5-phosphate are close to 2 X 10(-7) m2/J. The value for UpU is similar. The reaction cross sections, sigma, for (Ap)n where n = 3-10 as well as for the oligonucleotides ApUpGp, m1ApCpUpCpGp, CpCpCpCpCpGp, and DpDpDpApApGp increased linearly as a function of the number of phosphodiester bonds and gave values close to 6.4 X 10(-7) m2/J per bond. The cross sections for (Up)n were also about 6.4 X 10(-7) m2/J per bond for n = 2-5 and then, unexpectedly, increased rapidly for n = 6-10. By analogy to the carefully studied release of phosphate from ethyl phosphate and several sugar phosphates by 254-nm light [Halmann, M., & Platzner, I. (1965) J. Chem. Soc., 5380-5385], we conclude that the photolysis reactions we have observed are induced by absorption of photons by the sugar phosphate groups rather than the purine or pyrimidine rings. It follows that the quantum yields for chain cleavage of both RNA and DNA have been seriously underestimated since these calculations were based on the assumption that the observed photochemistry is due to absorption of photons by the purine and pyrimidine rings, and the absorption cross section of these rings is roughly 10 000 times greater than that of the sugar phosphate group itself.

Isolation and characterization of phi X174 mutants carrying lethal missense mutations in gene G.

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.

A new system for studying molecular mechanisms of mutation by carcinogens.

A new system for studying the molecular mechanisms of mutation by carcinogens is described. The system involves (a) site-specific modification of the essential gene G in phi X174 replicative form DNA by a combination of chemical and enzymatic steps; (b) production of mutant virus carrying a change at a single preselected site by transfection of spheroplasts with the site modified phi X174 DNA; (c) detection and propagation of mutants using a host carrying the plasmid, p phi XG, that rescues all type of gene G mutants by complementation; (d) identification of the mutation in the progeny virus by isolating and sequencing mutant phi X174 DNA in the region that carried the parental, site-specific change. To demonstrate that this system is operational, we have produced a previously unknown phi X174 gene G mutant carrying a C leads to T base change at position 2401 of the viral (plus) strand. This preplanned, nonsense (amber) mutant was obtained by changing G to A at the appropriate position in a chemically synthesized, octadeoxynucleotide, minus strand primer; elongating this enzymatically with Escherichia coli DNA polymerase I (larger fragment) (lacking 5' leads to 3' exonuclease activity) to a 17-mer; and repriming to obtain the site-modified phi X174 replicative form DNA enzymatically with E. coli DNA polymerase I (large fragment) and T4 DNA ligase. After transfection of spheroplasts with the heteroduplex DNA, the lysate was screened for mutant virus with permissive (carrying p phi XG) and nonpermissive (without p phi XG) host cells. About 1% of the progeny virus were mutants. Out of 15 isolates, 11 were suppressible by an amber Su1+ (serine) or an ochre Su8+ (glutamine) suppressor. The other 4 isolates were not suppressed at all. Replicative form DNA produced from one of the suppressible mutants was shown (by sequencing) to contain the expected C leads to T change at the preselected site in the viral strand. Replicative form DNA from one of the nonsuppressible mutants was partially sequenced. No change was found at or around position 2401. The nature of the mutation(s) in these isolates is still unknown. The occurrence of mutations outside the preselected sites represent a potential problem for our projected studies, but additional data is required before the problem can be fully evaluated. In spite of this, it should be possible to study, in vivo, the biological effects of any site-specific modification (including covalent modifications by carcinogens) that can be introduced into gene G of phi X174 DNA via a synthetic, oligonucleotide primer.

Construction of a site-specific, deletion-frameshift mutation in an essential gene of bacteriophage phiX174.

By in vitro methods, we have deleted 80 nucleotides from a preselected site in the coding sequence of gene G of bacteriophage phiX174. This mutant (Gdelta/FS ZH1) also carries a--2 frameshift. The mutation is lethal, but mutant virus can be stably maintained on host strains bearing plasmids carrying phiX gene G.

Construction and characterization of an Escherichia coli plasmid bearing a functional gene G of bacteriophage phiX174.

In order to study the mutagenic effects of site-specific, covalent modifications of biologically active DNA, we need host cells that are permissive for any type of mutation that might be produced in vivo from the modified DNA. Specifically, we require a general, in vivo complementation system for the bacteriophage phiX174 gene G, an essential gene that we have chosen for our initial studies of chemical mutagenesis. Toward this end, we have constructed a plasmid (pphiXG) that carries a functional copy of phiX174 gene G. Three different bacterial strains that are nonpermissive for am9, a gene G amber mutant, have been transformed with pphiXG. The transformants are now permissive for this gene G mutant, but not for the gene A or E mutants that have been tested. This paper describes the construction and the biochemical characterization of this plasmid, pphiXG, and describes some of the biological properties exhibited by the pphiXG-bearing strains.

Automated donor-recipient identification systems as a means of reducing human error in blood transfusion.

Clerical and administrative errors are the most common mistakes in blood banking, with errors of sample or patient identification accounting for the majority of mistakes. In an effort to reduce the incidence of human errors, three automated donor-recipient identification systems were developed and were evaluated under clinical conditions. After two years of development and bench testing, each set of equipment was tested, along with established procedures, at a large clinical center, a medium-sized community hospital and a regional donor center. Although none of the systems, as tested, proved acceptable under clinical conditions, a great deal was learned about prototype equipment testing in the clinical setting. Future development of identification systems for clinical use should respond to carefully defined problems, apply in all areas of the hospital or donor center, and be convenient and reliable. The development of the equipment should involve established manufacturers and be subject to exacting preclinical testing.

Techniques for typing Herpesvirus hominis antibody: a comparison of inhibition of peroxidase-labelled antibody staining with inhibition of indirect haemagglutination and with microneutralisation.

A new technique based upon the inhibition of the peroxidase-labelled antibody staining (PLAS) has been used to type Herpesvirus hominis (HVH) antibodies in four groups of human sera taken from patients with one or both types of HVH infection and in mixtures of different proportions of type 1 and type 2 antisera. The results were compared with those of the microneutralisation (MN) test and the indirect haemagglutination (IHA) inhibition test. The sensitivity and specificity of the three methods were identical for sera containing only one type of HVH antibody. The MN test was slightly more sensitive than the other tests for detecting small amounts of HVH-1 antibody mixed with large amounts of HVH-2 antibody. Nevertheless, the PLAS inhibition technique was far more rapid and it would seem a satisfactory alternative to the IHA inhibition test for HVH antibody typing.

Bisulfite-induced C changed to U transitions in yeast alanine tRNA.

The reaction of yeast tRNAAla1ab with NaHSO3 at 25 degrees and pH 5.8 has been studied. Five reactive residues have been located. Four of these (C-17 in Loop I, C-36 in the anticodon, C-74 and C-75 near the acceptor end) react to the same extent (42%) under the conditions of the experiment. The other (C-72 in the first base pair of the acceptor stem) reacts much more slowly (8%). No other changes were detected, but kinetic data suggest two or more additional residues may react very slowly. The C changed to U change in the anticodon (igc changed to igu) is a missense change (Ala changed to Thr). Both mechanistic considerations and experimental data from the literature show that HSO3--induced deamination of cytosine residues occurs only at unstacked residues. The quantitative changes for tRNAAla indicate that the stacking lifetimes of C-17, C-36, C-74, and C-75 are about equal. All other cytidine residues are much more tightly stacked. These results are consistent with the folded cloverleaf models that have been proposed from x-ray diffraction studies of yeast tRNAPhe. Residues 48 and 56, which are in single-stranded regions in the unfolded cloverleaf structure, do not react suggesting that they are tightly stacked in solution under the conditions of this experiment. The data also indicate that the anticodon loop is flexible in solution.