Prospects for applying genotypic selection of somatic oncomutation to chemical risk assessment.

Genotypic selection methods detect rare sequence changes in populations of DNA molecules. These methods have been used to investigate the chemical induction of mutation and for the detection and diagnosis of cancer. The possible use of genotypic selection for improving current risk assessment practices is based on the premise that the frequency of somatic mutation is of critical importance in understanding and modeling carcinogenesis. If genotypic selection can measure the induction of specific mutations that disrupt normal cell/tissue homeostasis, then it could provide key mechanistic information for cancer risk assessment. For example, genotypic selection data might support a particular low-dose extrapolation method or characterize the relationship between rodent and human cancer risk. Strategies for evaluating the use of genotypic selection in cancer risk assessment include the concept of developing a battery of targets that detect a range of agent-specific effects. Ideal targets to examine by genotypic selection are the oncogene and tumor suppressor gene mutations frequently detected in human tumors because these are thought to represent tumor-initiating events. The most commonly occurring basepair (bp) substitutions within the ras and p53 genes are identified. Also, the battery of genotypic selection methods is defined in terms of the most important mutational specificities to include. In theory, the major basepair substitution mutations induced by 29 of 31 chemical carcinogens could be detected by analyzing three different mutations: G:C-->T:A, G:C-->A:T, and A:T-->T:A. Genotypic selection will have the greatest impact on risk assessment if measurement of spontaneous mutation is possible. Data from phenotypic selection assays suggest this corresponds to detection of mutant fractions of approximately 10(-7), and this would necessitate examining DNA samples containing >10(7) target molecules. Despite its apparent potential, considerable development and validation is needed before genotypic selection data can be applied to cancer risk assessment.

Detection of a mouse H-ras codon 61 mutation using a modified allele-specific competitive blocker PCR genotypic selection method.

A modified allele-specific competitive blocker PCR (ACB-PCR) has been developed as an approach for genotypic selection, the detection of a rare mutant allele based solely upon its altered nucleotide sequence. ACB-PCR genotypic selection operates through the preferential PCR amplification of mutant DNA using a primer that has more mismatches to the wild-type allele than the mutant allele. In addition, a blocker-primer with a 3'-terminal dideoxynucleotide and more mismatches to the mutant allele than the wild-type allele is incorporated to reduce the background and increase sensitivity. Using ACB-PCR, the CAA-->AAA base substitution at codon 61 of the mouse H-ras gene was detected regularly at mutant fractions of 10(-5). To accurately quantify the occurrence of this particular mutation, an internal amplification standard (AS) DNA was constructed. The H-ras and AS DNAs were subject to the same genotypic selection but were amplified using different upstream primers to give PCR products that can be distinguished by size. Defined mixtures of mutant and wild-type AS DNAs were used to study the effects of various components of the ACB-PCR. The concentration of dNTPs, blocker primer and Perfect Match Polymerase Enhancer, as well as the choice of thermostable DNA polymerase and annealing temperature were examined. Conditions were identified for the concurrent detection of the CAA-->AAA mutation in the H-ras and AS DNAs. Using the identified conditions, approximately equal signals were obtained from equivalent amounts of the two DNA templates over a wide range of mutant fractions (1 in 10 to 1 in 10(5)). This ACB-PCR method can be used for any application where it is necessary to quantify relatively small mutant fractions.

Detection of basepair substitution mutation at a frequency of 1 x 10(-7) by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR.

The detection of rare mutations has many important applications, including risk assessment of drugs and chemicals, measuring environmental exposures to genotoxins, and cancer cell detection. A sensitive genotypic selection method has been developed that combines two different mutant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evaluated for the detection of a CAA --> AAA mutation at codon 61 of the mouse H-ras gene. The MutEx enrichment is based on MutS binding to a mismatched basepair in heteroduplex DNA. The bound MutS protects the mutant allele from degradation during subsequent exonuclease treatment. ACB-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mutant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 10(7). This sensitivity was achieved with the thermostable Thermus aquaticus MutS protein but not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate +/- the standard error of the mean for this particular basepair was estimated to be 8 +/- 3 x 10(-7) errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods for the detection of mutation.

Genotypic selection methods for the direct analysis of point mutations.

Genotypic selection enriches a particular DNA sequence relative to another closely-related DNA sequence based only on a change of one or a few bases. This review is a survey of the genotypic selection methods that have the sensitivity to detect rare point mutations. These methods are primarily being used to study mutations caused by environmental mutagens; however, the ability to detect and measure very minor DNA sequence populations is likely to further research efforts in many fields. The approaches for allele-selection have intrinsic strengths and weaknesses, and vary greatly in sensitivity. The most sensitive method is Restriction Fragment Length Polymorphism/Polymerase Chain Reaction (RFLP/PCR) by which mutant fractions as low as 1 mutant allele in 10(8) wild-type alleles can be detected. The RFLP/PCR approach is presented as a prototype genotypic selection method. Genotypic selection methods are categorized in terms of those that (1) selectively destroy the abundant or wild-type allele, (2) selectively amplify the rare or mutant allele, or (3) spatially separate the alleles. Issues relevant to the further development of genotypic selection methods include initial DNA pool size, strategies to eliminate the bulk of extraneous DNA, the use of an internal copy number standard in quantitative PCR, the fidelity of thermostable DNA polymerases, and the effective use of PCR in linking two or more genotypic selection techniques. We conclude that proficient genotypic selection requires more than one allele-enrichment technique with at least one of these preceding a high-fidelity PCR amplification step.

Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA.

The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H-ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA). The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences were amplified by PCR. When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.

A wound-repressible glycine-rich protein transcript is enriched in vascular bundles of tomato fruit and stem.

The deduced protein sequence of a partial tomato cDNA clone is rich in the amino acid glycine and contains repeats of the amino acid sequence, (Gly)5Tyr(Gly)4-5Tyr(Gly)3ArgArgGlu. This protein sequence has significant similarity to a sorghum glycine-rich protein [S1, Cretin and Puigdomenech (1990) Plant Mol. Biol. 15: 783] and a maize embryo, abscisic acid-induced glycine-rich protein [Gomez et al. (1988) Nature 334: 262]. Tissue printing was used to localize the glycine-rich protein transcript in tomato fruit, stem and hypocotyl sections. The transcript is present throughout the tomato fruit pericarp but is enriched in the vascular bundles. In tomato hypocotyl tissue prints, the glycine-rich protein transcript as well as rRNA were localized within the vascular tissue. This shows that differential loading of RNA may occur when using the tissue printing technique on hypocotyl sections. Direct isolation and comparison of RNA from vascular-containing and non-vascular stem tissue confirmed, however, that the glycine-rich protein transcript is accumulated abundantly in the vascular tissue of tomato stem.

Accumulation of wound-inducible ACC synthase transcript in tomato fruit is inhibited by salicylic acid and polyamines.

Regulation of wound-inducible 1-aminocyclopropane-1-carboxylic acid (ACC) synthase expression was studied in tomato fruit (Lycopersicon esculentum cv. Pik-Red). A 70 base oligonucleotide probe homologous to published ACC synthase cDNA sequences was successfully used to identify and analyze regulation of a wound-inducible transcript. The 1.8 kb ACC synthase transcript increased upon wounding the fruit as well as during fruit ripening. Salicylic acid, an inhibitor of wound-responsive genes in tomato, inhibited the wound-induced accumulation of the ACC synthase transcript. Further, polyamines (putrescine, spermidine and spermine) that have anti-senescence properties and have been shown to inhibit the development of ACC synthase activity, inhibited the accumulation of the wound-inducible ACC synthase transcript. The inhibition by spermine was greater than that caused by putrescine or spermidine. The transcript level of a wound-repressible glycine-rich protein gene and that of the constitutively expressed rRNA were not affected as markedly by either salicylic acid or polyamines. These data suggest that salicylic acid and polyamines may specifically regulate ethylene biosynthesis at the level of ACC synthase transcript accumulation.

Wound-regulated accumulation of specific transcripts in tomato fruit: interactions with fruit development, ethylene and light.

Regulation of three cDNA clones (pT52, pT53, and pT58) was analyzed in terms of wounding alone and wounding in conjunction with developmental and environmental cues (ripening, ethylene, and light) in tomato fruit tissue. The pT52-specific transcript level is induced by wounding in early-red and red stage fruit and by ethylene. The pT58-specific transcript level is also induced by wounding and ethylene in early-red stage fruit but is not induced by wounding in red fruit. The pT53-specific transcript level is repressed by wounding in early-red and red stage fruit. Like the pT52- and pT58-specific transcripts, the pT53-specific transcript is induced by ethylene. Furthermore, the level of the pT52-specific transcript is regulated by light. Analysis of unwounded tissue showed that the abundance of each cDNA-specific transcript changes during fruit ripening and that each of the transcripts is present in other plant organs as well. This analysis provides information about the interactions between developmental and environmental factors affecting these genes.

Transcription of orthopoxvirus telomeres at late times during infection.

The telomeres of orthopoxvirus DNAs consists largely of short repeated sequences organized into at least two separate sets. Although the sequence composition of the orthopoxvirus telomeres is highly conserved, these regions do not appear to encode any proteins. At late times during infection, the telomeres of vaccinia virus are transcribed. A promoter in the region between the two sets of repeats directs transcription towards the hairpin-loop end of the viral DNA. This promoter resembles the promoters of other poxvirus late genes, and directs the synthesis of RNAs whose structure is consistent with the presence of 5' poly(A) sequences typical of late RNAs. The lengths of these late transcripts suggest that some transcription extends through the hairpin-loop region. This might occur either when the genome is in a monomeric form or when the genome is in the concatemeric form of the DNA replication intermediate. The function of late transcription of the telomeres is unclear, but similar transcription of the telomeres of vaccinia virus, cowpox virus, and raccoonpox virus suggests that such transcription may have a role in viral replication.

Tandemly repeated sequences are present at the ends of the DNA of raccoonpox virus.

The DNA of raccoonpox virus (RCN) has been characterized by restriction enzyme analysis. DNA hybridization studies showed that all HindIII fragments of the 215-kbp RCN DNA share some nucleotide sequence similarity with fragments of the DNA of cowpox virus (CPV). This information was used to construct a HindIII restriction map of the RCN DNA. The nucleotide sequence of the 2.2-kbp Sal 1 end fragment of the RCN DNA has been determined from a cloned copy of the HindIII O fragment. Of this 2.2-kb region 75% consists of short, tandemly repeated sequences. It does not contain any open reading frames capable of encoding polypeptide chains of more than 62 amino acids. There are six related types of repeated sequence, and these are arranged into two separate sets, each flanked by nonrepeated sequences. The nucleotide sequences of both repeated and nonrepeated sequences within this Sal 1 fragment are extremely similar to those of the Sal 1-generated end fragments of the DNas of CPV and vaccinia virus. The arrangements of the repeated and nonrepeated sequences are also similar in the DNAs of these three viruses. In contrast, the remainder of the RCN DNA is markedly different from the DNAs of other orthopoxviruses. The high degree of similarity between the ends of the RCN DNA and the ends of the other orthopoxvirus DNAs suggest that the complex arrays of repeated and nonrepeated sequences have been conserved because they have a role in virus multiplication.

Spontaneous deletions and duplications of sequences in the genome of cowpox virus.

Examination of the genomes of 10 white-pock variants of cowpox virus strain Brighton red (CPV-BR) revealed that 9 of them had lost 32 to 38 kilobase pairs (kbp) from their right-hand ends and that the deleted sequences had been replaced by inverted copies of regions from 21 to 50 kbp long from the left-hand end of the genome. These variants thus possess inverted terminal repeats (ITRs) from 21 to 50 kbp long; all are longer than the ITRs of CPV-BR (10 kbp). The 10th variant is a simple deletion mutant that has lost the sequences between 32 and 12 kbp from the right-hand end of the genome. The limits of the inner ends of the observed deletions (between 32 and 38 kbp from the right-hand end of the CPV-BR genome) appear to be defined by the location of the nearest essential gene on the one hand and the location of the gene that encodes "pock redness" on the other. The genomes of the deletion/duplication white-pock variants appear to have been generated either by single crossover recombinational events between two CPV-BR genomes aligned in opposite directions or by the nonreciprocal transfer of genetic information. The sites where such recombination/transfer occurred were sequenced in four variants. In all of them, the sequences adjacent to such sites show no sequence homology or any other unusual structural feature. The analogous sites at the internal ends of the two ITRs of CPV-BR also were sequenced and also show no unusual features. It is likely that the ITRs of CPV-BR and of its white-pock variants, and probably those of other orthopox-virus genomes, arise as a result of nonhomologous recombination or by random nonreciprocal transfer of genetic information.

Nucleotide sequences from the adenovirus-2 genome.

The sequence of 15,441 nucleotides from the adenovirus-2 genome has been determined and includes the regions between coordinates 0-32% and 89-100%. These regions contain the early (E) transcription units E1A, E1B, E2B, and E4, the genes for polypeptides IVa2 and IX, the COOH terminus of fiber polypeptide, as well as the two virus-associated RNAs and the leader sequences for the major late mRNAs. Analysis of tryptic peptides from the terminal protein and its precursor (Smart, J. E., and Stillman, B. W. (1982) J. Biol. Chem. 257, 13499-13506) has allowed the gene for the precursor terminal protein to be positioned between coordinates 28.0 and 23.5 on the 1-strand. A minimum Mr = 74,500 is predicted. A second, longer open reading frame is also found on the 1-strand between coordinates 22.9 and 14.2 and predicts a polypeptide of at least Mr = 120,000. Many open reading frames longer than 10,000 exist within this sequence although less than half of them can be assigned to previously characterized polypeptides. As with other viral genomes, the available coding information is highly compressed. Intergenic distances are very short and examples are found of genes which overlap either on the same strand or the complementary strand.