Blue Plaques Research Articles

4-Chloro- o-phenylenediamine: a 26-week oral (in feed) mutagenicity study in Big Blue™ mice

4-Chloro- o-phenylenediamine (4-C- o-PDA) is a liver carcinogen in mice and was found to be weakly mutagenic in the liver of female Big Blue™ mice after short term treatment. In the present study the test compound was given subchronically in the diet for 26 weeks at doses of 0, 5000 and 10,000 ppm. The corresponding average test substance intake was 2166 mg kg −1 day −1 (males: 1794 mg kg −1 day −1; females: 2539 mg kg −1 day −1) and 4610 mg kg −1 day −1 (males: 3926 mg kg −1 day −1; females 5925 mg kg −1 day −1) at the low and high dose, respectively. After sacrifice, tissues were flash frozen in liquid nitrogen. The lacI mutant frequency in the liver was determined from three male and three female mice per dose group. The genomically integrated transgene was recovered by packaging into λ phage using Transpack™ packaging extract (Stratagene, La Jolla, USA) followed by infection of Escherichia coli strain SCS-8. Blue mutant plaques were scored against a background of clear non-mutant plaques. Food consumption decreased initially at 10,000 ppm, while no treatment related effect on food intake was observed at 5000 ppm. Body weight gain was found to be decreased in all treated animals. Absolute and relative liver weight increased in a dose-related manner, but only the latter effect was statistically significant. A clear dose dependent increase in lacI mutant frequencies was observed in the liver of both sexes. The following mutant frequencies (×10 −5) were observed: 2.73±1.01 (males, untreated), 7.24±1.50 (females, untreated), 18.91±5.30 (5000 ppm, males), 24.91±7.58 (5000 ppm, females), 20.47±6.68 (10,000 ppm, males) and 36.17±14.98 (10,000 ppm, females). It is therefore concluded that 4-C- o-PDA is a strong mutagen in the liver of mice treated subchronically for 26 weeks.

Recombinant Swinepox Virus Expressing β-Galactosidase: Investigation of Viral Host Range and Gene Expression Levels in Cell Culture

Swinepox virus (SPV) has been proposed as a potential vector for generating recombinant vaccines for swine. However, little is known about important aspects of SPV biology, such as the functionality of SPV promoters or the host range of SPV. Using a transient expression assay, well-characterized vaccinia virus promoters were shown to be active in cells infected with SPV. A recombinant SPV expressing β-galactosidase (β-gal) was constructed and characterized. TheE. coli LacZgene was placed under the control of a strong vaccinia synthetic early/late promoter and was inserted by homologous recombination in a noncoding region of the SPV genome. The recombinant SPV expressing β-gal was used to characterize the host range of the virus by measuring protein expression and virus production in different cell lines. In general, SPV expressed more protein and grew more efficiently than vaccinia virus in porcine cell lines. Surprisingly, the recombinant SPV was able to infect and replicate in several cell lines of nonswine origin. The virus directed regulated early and late gene expression of β-gal in those cells and formed blue plaques in cell monolayers in the presence of X-gal. Upon infection with the recombinant SPV, there was a significant level of viral replication, and the virus can be serially passaged in some nonswine cell lines. The data presented suggest that despite the strict host tropism of SPV, the virus exhibits a relatively broad host range in cell culture.

Construction of canine herpesvirus vector expressing foreign genes using a lacZ-TK gene cassette as a double selectional marker.

An improved method for constructing canine herpesvirus (CHV) recombinants expressing foreign genes by using the lacZ-TK gene cassette as a double selectional marker was developed. A recombinant CHV carrying the lacZ-TK gene at a targeted gene locus was constructed and used as a parental virus for generating new recombinants. The parental virus formed blue plaques and was sensitive to TK-specific drugs, while newly generated recombinants, in which the lacZ-TK gene was replaced with the desired foreign gene, become both resistant to the TK-specific drugs and formed white plaques. Recombinants were isolated by using the combination of drug selection and color selection. This improved method allows construction of recombinant CHV with great ease, because the drug selection can enrich the frequency of recombinant CHV from 0.01-0.1% to 10-80%. This method was employed to construct a recombinant CHV that expressed rabies virus (RV) glycoprotein (G protein).

Mutagenic consequences of the incorporation of 6-thioguanine into DNA

6-Thioguanine (S 6G) has been used in the treatment of acute leukemias because of its cytotoxic effect on proliferating leukemic cells. The cytotoxicity of S 6G is thought to derive from its incorporation into DNA in place of guanine. The deoxyribonucleoside triphosphate of S 6G, SdGTP, is a good substrate for bacterial and human DNA polymerases (Ling et al., Mol Pharmacol 40: 508–514, 1991). Since SdGTP was observed to misincorporate in place of adenine at a greater frequency than did dGTP, it appeared plausible that this analog could produce more subtle effects (mutations) due to mispairing with thymine. To assess whether such mutations occur, SdGTP was incorporated into the lacI gene of phage M131acISaXb in reactions that omitted dGTP ( −G) or dATP (−A). LacI mutation frequency was determined by β-galactosidase colorimetric staining (inactivation of the lac repressor results in blue plaques in the absence of inducet). When a high concentration of SdGTP (24 μM) was used in the DNA polymerase reaction, phage infectivity was inhibited. When a relatively low concentration (2.4 nM) was added to the −G and −A reactions, mutagenic effects were observed. DNA sequencing of mutant progeny arising from the −G + S 6G reaction revealed C-to-T base transitions and some C-to-A transversions. Similarly, the presence of SdGTP in the −A reactions led to mutants with T-to-C transitions. No insertions or deletions were observed. These data indicate that mispairing of S 6G with thymine leads to mutagenic effects in this assay.

On the origin of spontaneous somatic mutations and sectored plaques detected in transgenic mice

The use of transgenic mice with bacterial genes that can be readily recovered and analysed for mutation has made it possible to measure mutant frequencies in many tissues. The mutations are detected by packaging the murine DNA into λ phage and then growing these phage on a bacterial lawn under conditions such that the mutants are distinguishable from nonmutants. In the lacI mouse assay, the mutant plaques are blue whereas the nonmutant plaques are clear. The mutations detected in the lacI Big Blue™ Mouse are, in principle, a mixture of mutations that arose in the mouse (in vivo mutations), mutations that arose in the bacterium from lesions pre-existing in the murine DNA (ex vivo mutations), and mutations that arose during growth of the phage on the bacteria (in vitro mutations). It has been suggested that plaque morphology can be used to visually distinguish in vivo mutations (which would be seen as wholly blue plaques) from ex vivo mutations (which would produce blue and white sectored plaques) and in vitro mutations (which would produce sectored or pin-point plaques). We show here that this is not the case: ex vivo mutations produce plaques that are a homogeneous blue. By superinfection of bacteria with mutant and nonmutant phage and by in vitro mutagenesis, we found that blue plaques may contain large proportions of nonmutant phage. None of the mutant plaques seen after in vitro mutagenesis were sectored but most contained nonmutant phage. In addition, we show that most spontaneous mutations from the small intestine, which has a higher than normal mutant frequency, arose in vivo, since 17 of 17 mutants were homogeneous mutants. Sectored plaques must arise in some way other than by ex vivo mutation, like perhaps by the confluence of a mutant and nonmutant plaque.

Blue Plaques and Farther Shores: Derek Mahon's "A Kensington Notebook"

Published in 1984, a few months before Derek Mahon's return to Ireland at the midpoint of a career spent for the most part in England, A Kensington Notebook has been called his goodbye to London.1 The phrase recalls Pound's footnote (...distinctly a farewell to London) to the title page of Hugh Selwyn Mauberley, the work on which Mahon's poem is broadly modeled, and alerts us to the possibility of a shared agenda beyond their more obvious rela tionship. Mahon has never acknowledged this valedictory function but it is at least implicit in his choice of the poem to head his next volume, Antarctica, a sombre collection of farewells and leavetakings, while with its next appearance in Selected Poems, in pointed juxtaposition with Going Home, the handker chief waving had become routine. Thirteen years from its first appearance, A Kensington Notebook can bee seen as a watershed in his work from which we can look back at his long career as an Emigre poet beginning with that resonant ly titled first volume, Night-Crossing, and simultaneously forward to the new bearings his work would take on his arrival in Dublin. Again, what seemed in 1984 a superficial indebtedness to its Poundian model has evolved into a more profound resemblance.

Towards validation of the Big Blue ® transgenic mouse mutagenesis assay: The mutational spectrum of ex vivo pinpoint mutant plaques

To explore further the origin of spontaneous mutations recovered with the Big Blue ® transgenic mouse mutagenesis assay, the spectrum of ex vivo mutations from pinpoint mutant plaques was determined and compared with the spectrum of putatively mouse-derived mutations from circular, mutant plaques. The entire lacI gene and lacZ operator region from 62 pinpoint blue plaques was sequenced. The observed mutational spectrum of pinpoint mutants differed significantly from that seen in circular mutants ( p < 0.0001). Only four percent of the mutations were transitions at CpG sites whereas this type of mutation was the most common (35%) in circular mutants. Microdeletions/microinsertions were seen more frequently in pinpoint mutants relative to circular mutants. Four base pair deletion/insertion events at the E. coli hotspot tandem repeats were seen in 10 of 62 (16%) pinpoint mutants and minor hotspots of mutation were observed at bp 141 and 1110. The mutational spectrum of pinpoint mutants provides further evidence that most circular mutants originate in mouse.

A vaccinia virus transfer vector using a GUS reporter gene inserted into the I4L locus

A vaccinia virus (VV) transfer vector is described which enables integration of heterologous sequences into the I4L locus (ribonucleotide reductase-encoding gene) through co-insertion of a GUS selection marker. I4L- VV recombinants formed blue plaques when an agarose overlay containing XGluc (5-bromo-4-chloro-3-indolyl-β-glucuronide) was added to the infected cell monolayer. Viruses already containing a lacZ reporter gene were also suitable recipients for the selection procedure since infection with a VV lacZ recombinant did not produce any blue plaques with XGluc. The addition of a synthetic early promoter downstream from the GUS cassette initiated the predicted-size transcript during an infection. Insertion of genes with VV p7.5-promoters into the I4L, J2R and K1L loci of the same virus produced viable virus recombinants even though recombination between these loci could be demonstrated. These techniques should be valuable for the further development of VV as a polyvalent vector.

Recombinant channel catfish virus (Ictalurid herpesvirus 1) can express foreign genes and induce antibody production against the gene product

AbstractThe potential use of channel catfish virus (CCV) (Ictalurid herpesvirus 1) as a vaccine vector for the channel catfish industry was investigated by inserting the Escherichia coli lacZ gene into the CCV genome and evaluating the immune response to the foreign gene product in catfish exposed to the recombinant. The recombinant virus was produced by inserting the lacZ in reading frame with the ATG start codon of the CCV thymidine kinase (TK) gene in the recombinant transfer plasmid pBSCV457 described previously. The plasmid was then cotransfected with CCV DNA in a TK gene‐mediated selectable homologous recombination. The recombinant progeny were selected by resistance to 0.1 mM acycloguanosine (acyclovir) and the production of blue plaques in the presence of 5‐bromo‐4‐chloro‐3‐indolyl‐β‐D‐galactopyranoside (X‐gal). The resultant construct (CCVlacZ) was TK−, and contained the lacZ gene at both TK loci in the genome. β‐Galactosidase expression in infected CCO ceils reached 0.53 μg per 106 CCO cells at 12 h post‐infection. When channel catfish fingerlings were immersion exposed to CCVlacZ, these developed an antibody response to the inserted foreign gene product which peaked at approximately 15–20 days post‐infection. Additionally, the anti‐β‐galactosidase response was significantly enhanced when the fingerlings were re‐exposed to the virus 20 days after the initial exposure. These results demonstrate that foreign genes can be inserted into and expressed by CCV and that such constructs could be used as vaccine vectors.

Sequence analysis of flanking regions of the pfoA gene of Clostridium perfringens: beta-galactosidase gene (pbg) is located in the 3'-flanking region.

The 3'-flanking region of the perfringolysin O (theta-toxin) gene (pfoA) of Clostridium perfringens was analyzed by chromosome walking. A total of 5,363 bp of the downstream region of the pfoA gene was sequenced and four open reading frames were found. ORF54 and ORF80 were found to be homologous to genes coding for membrane-bound transporter proteins of other bacteria and the beta-galactosidase gene (bgaB) of Bacillus stearothermophilus, respectively. ORF80 was named the pbg gene. Clones which showed beta-galactosidase activities were selected from a lambda FIXII genomic library of C. perfringens by blue plaque screening using X-Gal as a substrate. Four clones whose plaques showed blue appearances were obtained. Two of the four clones hybridized with the pbg probe but the others did not, indicating that there are two distinct beta-galactosidase genes in C. perfringens. The pbg gene was subcloned into pBR322 and was successfully expressed in Escherichia coli, suggesting that the pbg gene codes for a beta-galactosidase of C. perfringens.

Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase.

Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.

Natural DNA Precursor Pool Asymmetry and Base Sequence Context as Determinants of Replication Fidelity

Previous studies showed a complex relationship between nucleotide composition of a gene and the rate of the gene's evolutionary variation. We have investigated mechanisms by constructing M13 phagemids containing part of the Escherichia coli lacZ gene, in which an opal codon is flanked either by nine adenine-thymine base pairs on each side, or by nine guanine-cytosine pairs, or by its wild-type sequence context. Reversions or pseudoreversions within the opal codon yield a lacZ alpha-peptide that can undergo alpha-complementation and yield a blue plaque when plated with a chromogenic substrate. When these constructs were replicated in HeLa cell extracts, in the presence of equimolar deoxyribonucleoside triphosphate (dNTP) mixtures, reversion was near background levels in both the AT-rich and GC-rich contexts. By contrast, when the DNAs were replicated at dNTP concentrations approximating those in HeLa cell nuclei, increases over background were seen in all three contexts. Replication of the phagemids in vivo led to even higher mutation frequencies. Replication in the presence of dGMP, added to inhibit proofreading, caused extraordinarily high reversion frequencies in the GC-flanked opal codon. Apparently, dNTP concentrations approximating intracellular concentrations are mildly but significantly mutagenic, and pool asymmetries and base sequence context both contribute to the natural fidelity of DNA replication.

Construction of Recombinant Avian Infectious Laryngotracheitis Virus Expressing the β-Galactosidase Gene and DNA Sequencing of the Insertion Region

Avian infectious laryngotracheitis virus (ILTV), a herpesvirus, is a highly contagious pathogen that causes an upper respiratory tract infection in chickens. It is one of the major problems in the poultry industry worldwide. Current vaccines are not satisfactory due to the induction of latent infection. Here we describe a system for the construction of recombinant ILTV. A 4-kbp ILTV EcoRI DNA fragment was cloned into plasmid pUC13 and sequenced. Computer prediction revealed two potential open reading frames with 216 and 259 amino acid residues, respectively. The 259-residue polypeptide was serine-rich. The β-galactosidase (β-gal) gene of E. coli was cloned into the XhoI/BglII site of this DNA fragment, integrated into the ILTV genome via homologous recombination, expressed under the control of the immediate-early cytomegalovirus promoter, and caused the formation of blue plaques in the presence of X-gal. The insertion of a foreign gene into the ILTV genome and the successful expression of the incorporated gene demonstrated the potential for the construction of attenuated recombinant ILTV vaccines and the development of ILTV as vectors for polyvalent vaccines against avian upper respiratory tract infections.

Studies of fowlpox virus recombination in the generation of recombinant vaccines

A p7.5/β-galactosidase (7.5 lacZ) gene construct, cloned adjacent to the fowlpox virus (FPV) thymidine kinase ( tk) gene was used as a marker to identify the products of recombination as ‘blue’ FPV plaques. The rFPVs were detected as early as 4 h after the introduction of plasmid DNAs and by 72 h post-infection (p.i.) for one transfer vector comprised 0.48% of the viral population. The proportion of rFPV increased linearly from 0.073% to 0.62% as the cumulative length of homologous sequences in the transfer vector increased from 0.73 to 4.5 kb. Two approaches using a second reporter gene, the Newcastle disease virus haemagglutinin-neuraminidase (NDV HN) gene were tested to differentiate between single and double cross-over events. In one, the HN gene was cloned into the FPV tk gene and the 7.5 lacZ cloned outside of the homologous region. Progeny of a single cross-over with FPV DNA generated an unstable blue plaque containing the HN gene and subsequent intramolecular recombination resulted in excision of the 7.5 lacZ and the generation of a stable ‘white’ plaque. For virus grown in CEF cells (tk +) in the presence of 5-bromo-deoxyuridine, only those viruses which contained a tk gene disrupted by the HN gene formed plaques. This approach allowed us to easily identify rFPV containing the HN gene but lacking 7.5 lacZ or other bacterial sequences. In a second approach, a double cross-over between rFPV DNA containing a stably expressed β-galactosidase gene cloned into the tk gene (blue plaque) and plasmid DNA containing the HN gene flanked by tk sequences would allow transplacement of the 7.5 lacZ gene with the HN gene, and generating a white plaque. We were unable to generate recombinant viruses with the HN gene and which generated a white plaque, indicating that double cross-over events do not occur at a sufficiently high frequency in FPV.

Comparative analysis of DNA mutations in lacI transgenic mice with age

Transgenic mice have been developed recently that contain copies of the well-defined mutagenesis reporter gene, lacI, in an integrated bacteriophage-based shuttle vector. The lacI gene, which is present in all cells of the mouse, can be excised specifically from isolated genomic DNA and efficiently packaged into bacteriophage particles after the addition of packaging extracts. Mutations of the lacI gene are easily detected by the derepression of beta-galactosidase resulting in blue plaques in the presence of X-gal. Originally developed as a short-term in vivo mutagenesis assay to screen genotoxic agents, we have used this system to measure naturally occurring DNA mutations as a function of chronological age. There was a linear increase in the presence of phenotypic lacI mutants from birth to 24 months (r = 0.731, P < 0.001), such that 24-month-old mice have accumulated approximately fourfold more mutants than newborn pups. Molecular analysis of these spontaneously arising DNA mutations showed them to result predominantly from base substitutions (80.6-98.5%) that were equally distributed between transitions and transversions. However, lacI mutations in animals > 3 months of age demonstrated a higher percentage of mutations (12-19.4% vs. 1.2%) resulting from discriminable size changes (> 20 bp) than was observed for mice 1-2 months old. Sequence analysis of mutations resulting from a > 20 bp size change revealed them to be due to a duplication of adjacent lacI sequence. These results indicate that there is a gradual accumulation of DNA mutations with age and that the types of mutations also are influenced by the age of the animal.

Propylene oxide mutagenesis at template cytosine residues

Propylene oxide (PO) is a widely used industrial reagent which is mutagenic and carcinogenic. We have recently shown that a variety of aliphatic epoxides, including propylene oxide, can react with DNA to form hydroxyalkyl adducts at N-3 of cytosine which rapidly undergo hydrolytic deamination to produce uracil adducts. These 3-hydroxyalkyl uracil adducts are stable in DNA and are postulated to be an important class of potentially mutagenic lesions. Mutagenesis at cytosine residues due to PO modification of single-stranded M13mp2/C141 DNA was studied by transfection of modified DNA into SOS and non-SOS induced E. coli host cells. Mutations of the proline (CCC) codon at C141 which result in reversion of the lacZ phenotype (blue plaques) were scored. It was found that PO treatment of single-stranded DNA results in dose-dependent mutagenesis that is highly SOS dependent. The spectrum of base-substitution mutations found at this site differed when PO-modified DNA was transfected into E. coli with different DNA repair backgrounds. These results indicate that propylene oxide induced DNA adducts at template cytosine residues are mutagenic in E. coli and that this mutagenesis is greatly increased by SOS processing. They also show that these lesions may be repaired by one or more mechanisms.

Sequence spectra of spontaneous lacZ gene mutations in transgenic mouse somatic and germline tissues.

As a critical step in determining whether transgenic mouse gene mutation systems are suitable models for the detection and quantification of induced gene mutations in vivo, spontaneous mutant frequencies and mutation spectra have been characterized for liver, bone marrow, and male germ cells of the lacZ transgenic mouse strain 40.6. The lacZ transgene is carried on a recombinant bacteriophage lambda shuttle vector that is recovered from mouse genomic DNA, and analysed in vitro for mutations that occurred in the mouse tissues. Mutations are detected visually as clear or pale blue plaques when X-gal is the substrate for beta-galactosidase; whereas, the wild-type plaques are dark blue. There was no statistical difference in the mutant frequency among the three tissues studied, the pooled mutant frequency being 2.23 +/- 0.41 per 10(5) pfu. The predominant type of mutation was GC-->AT transitions, with most occurring in 5'-CpG dinucleotides, suggesting that the deamination of 5-methylcytosine is the main mechanism of mutagenesis. There was, however, a statistically significant difference in the base pair substitution mutation spectrum for the liver and bone marrow when mutations were grouped according to GC or AT base-pairs. The proportion of transition versus transversion mutations was also statistically different among the three tissues, resulting mainly from the fact that germ cells were different from both bone marrow and liver. A lower number of spontaneous transitions in male germ cells was accompanied by an increase in transversions, with the proportion of GC-->AT transitions in 5'-CpG sites also declining.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosine deamination in mismatched base pairs.

The rate of deamination of cytosine in mismatched base pairs has been determined. Incubation of M13mp2 nicked heteroduplex DNA molecules containing T.C or C.C mispairs in the lacZ alpha-complementation gene results in deamination of cytosine to uracil, producing T.U or C.U mispairs. Strands which have undergone deamination at the target site to produce uracil will yield dark blue plaque revertants, while all other strands yield faint blue or colorless plaque phenotypes upon transfection of an ung- alpha-complementation Escherichia coli host strain. Rate constants were calculated from the reversion frequencies for several different heteroduplexes incubated at either 60 or 37 degrees C. For the 60 degrees C incubations, the hydrolytic deamination rate constants for mispairs in three different local sequence environments ranged from 8 x 10(-10) to 40 x 10(-10) s-1. For incubations at 37 degrees C, the rate constants were between 0.4 x 10(-10) and 1.3 x 10(-10) sec-1. At both temperatures and for all mispairs, these rate constants are significantly greater than deamination rate constants in properly matched Watson-Crick G.C base pairs and are similar to those constants determined for cytosine deamination in single-stranded DNA. Since deamination most likely occurs via a single-stranded intermediate, the data suggest that, at 37 degrees C, the T.C and C.C mispairs exhibit from 20% to 100% single-stranded character. We conclude that cytosine residues involved in a mispair in DNA are 1-2 orders of magnitude more prone to deaminate to uracil than are cytosines in double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Construction of a vector for site-specific frameshift mutagenesis containing the mutable hotspot of Salmonella typhimurium TA98 on an M13 bacteriophage.

Frameshift mutations demonstrate a high degree of sequence specificity. In order to provide a vector for site-specific frameshift mutagenesis experiments, a recombinant M13 phage (M13MB102) was constructed by substitution of 27 base pairs of the Salmonella typhimurium hisD3052 sequence for 27 base pairs of the polylinker region of M13mp19. The inserted sequence contains most of the hotspot for frameshift mutations in hisD3052 and its derivative strain TA98. Structural elements of the insert include reiterated bases, direct repeats, and palindromes, and four unique restriction endonuclease cleavage sites. The recombinant phage produced blue plaques when grown in Escherichia coli strain JM105 on X-Gal indicator plates and exhibited a spontaneous mutation frequency similar to that of M13mp19. Methodology is described for preparation, isolation, and purification of closed circular duplex M13MB102 genomes containing an adduct between the SphI and BssHII cleavage sites in the (-)-strand and uracil residues in the (+)-strand. The latter modification decreases replication of the (+)-strand by 4 orders of magnitude and maximizes use of the adducted (-)-strand for in vivo replication. The structure of M13MB102 and the procedures described for introducing adducts at defined positions in its hisD3052 insert provide a convenient approach for evaluating the potential of individual carcinogen-DNA adducts to induce frameshift mutations.

Big Blue transgenic mouse lacI mutation analysis.

In this report the authors describe a rapid general method for mutant blue plaque molecular analysis. The mutant analysis discussed here resulted from radon inhalation exposure. The described method circumvents Stratagene's plasmid isolation and ensuing sequence analysis of the entire lac1 gene. The authors have adapted the polymerase chain reaction (PCR) method and single-strand conformation polymorphism (SSCP) analysis for localizing mutations within the lac1 coding region. Three overlapping PCR products of approximately 450 bp representing the entire lac1 coding region are used for SSCP analysis. Those PCR products with an altered SSCP electrophoretic migration focus the mutation to a smaller region of the lac1 gene that is analyzed by direct cycle sequencing. 5 refs., 1 fig.