Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1991-06-06
2002-01-08
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C435S455000
Reexamination Certificate
active
06337182
ABSTRACT:
The invention relates to a method for the quantitative determination of DNA sequences containing at least one mutationally eliminated restriction site in which the PCR-technology is applied.
The study of the formation and processing of chromosomal damage is of fundamental importance to many aspects of the life sciences including evolution, hereditary disease, carcinogenesis and possibly aging. Mutations in the form of deletions, insertions, rearrangements and base pair substitutions are hereditary consequences of spontaneous processes and the exposure to DNA damaging agents. Since mutation rates at low or no toxicity are in the range of 10
−5
to 10
−8
(for an average size target gene) the isolation of a mutated cell requires the selection of an altered phenotype. In most cases cells are isolated which have acquired the ability to grow in the presence of a particular drug. In all systems using phenotypic selection only mutations are recovered which have resulted in a selectable change in the function of the target protein or in its elimination altogether. This represents a severe limitation since many mutations remain functionally silent. Furthermore, only a few genes are suitable targets for drug selection. Drug resistance can be the consequence of a missense mutation or small in-frame deletion/insertion which renders the target protein inert to drug toxicity (e.g. resistance to cuabain). More useful are non-essential target genes/proteins which upon inactivation allow cell growth in the presence of a particular selecting drug because a wider spectrum of mutations can be obtained under these conditions. Target genes in this second category are X-linked hypoxanthineguanine-phosphoribosyl transferase (hgprt), bacterial xanthine-guanine-phosphoribosyl transferase (gpt), chromosomally integrated into hgprt
3
mammalian cells and adenine-phosphoribosyl transferase (aprt): hgprt and gpt-mutants are selectable with 6-thioguanine (6-TG) and aprt-mutants with 8-azaadenine (8-aza-A). By simply measuring the frequency of the generation of drug-resistant phenotypes, preferably coupled with the demonstration of a loss in the activity of the target enzyme, overall mutation frequencies can be obtained albeit without any information regarding the molecular event by which they arose.
The sophisticated protocols using molecular cloning of target genes from drug-resistant cells furnished insights into mutagenic mechanisms first in bacteria and then in mammalian cells over the last decade. Considerable progress was made with the help of shuttle vectors which allow the rescue of an extrachromosomal plasmid from mammalian cells, its amplification in bacteria and finally its sequencing by standard methodology. While important information has been gathered with this approach it has several serious disadvantages (e.g. the presence of variable numbers of copies of the vector per cell and its extrachromosomal location do not allow conclusions about the role of local chromosome- and DNA-structure on lesion-processing in mutagenesis). Work with bona fide shuttle vectors has been reviewed (Banbury Report 28 “Mammalian Mutagenesis” (1987); see in Friedberg and Hanawalt, eds. “Mechanisms and Consequences of DNA Damage Processing” (1989)) and is not further discussed.
An advanced shuttle vector system has been developed by Davidson et al. (Ashman and Davidson, (1987); Davidson et al. (1988); Greenspan et al. (1988)) who constructed a hgprt
−
L-cell line with a chromosomally integrated bacterial gpt-gene (as part of a vector containing an SV40 origin). Cells mutated in the gpt-gene are selectable with 6-TG, the plasmid can be rescued and amplified by fusion with COS cells and then sequenced. Limitations of this system are relatively high spontaneous mutation frequencies (>10
−5
) and the formation of gross rearrangements during vector recovery which necessitates the isolation of a. “majority plasmid” (with a “normal” restriction pattern); advantageous is the good recovery of deletions. Of particular interest is the study of the reversion of specific gpt mutations induced by ethylmethanesulfonate (EMS). This ethylating agent which modifies preferentially guanine residues specifically reverts mutations which have resulted from A•T →→→>G•C transitions or G•C→→→>C•G transversions. Reversion frequencies with 3.3mM EMS of a specific bp were 1-4×10
−5
(Greenspan et al. (1986)).
An analogous approach has also been taken by Tindall & Stankowski (Stankowski and Tindall (1987); Tindall and Stankowski (1987)) who compared the mutability of a chromosomally integrated bacterial gpt-gene to that of the endogenous hgprt-gene in chinese hamster ovary (CHO) cells. Much higher mutability of gpt was noted for clastogens, possibly because the flanking sequences at the insertion site of the gpt-gene are non-essential for viability.
Meuth et al. selected spontaneous (Nalbantoglu et al. (1986); Nalbantoglu et al. (1987)) or radiation induced (Breimer et al. (1987)) aprt mutants (with 8-aza-A) from a CHO line which is hemizygous for this small gene (3.8 kb).
Either large deletions (detectable on Southern blots) or mutants which had gained or lost a restriction site (Southern/RFLP) were cloned and sequenced. Only relatively few mutations induced by ionizing radiation contained large deletions (6 out of 25); several deletions were flanked by direct repeats or at least one terminus was associated with a region of dyad symmetry. Spontaneous point mutations in aprt were mostly simple transitions and transversions. In contrast, ionizing radiation induced preferentially massive deletions in the hgprt-gene documenting locus specificity for mutations which are caused by the same mutagen.
An ingenious protocol for the analysis of 8-aza-A selected aprt mutants in CHO cells has been devised by Glickman et al.: Size-fractionated DNA is cloned into lambda which is then grown in a host containing a plasmid with flanking sequences of the hamster aprt-gene. This results in efficient recombinational transfer of the aprt-gene from lambda to the plasmid. The plasmid is rescued and sequenced using the M13 vector system. Spontaneous mutations were mostly G•C→A•T transitions. This could be due to cytosine deamination or reflect the fidelity of mammalian polymerases; all (except one) base-pair (bp) substitutions resulted in an amino acid change suggesting that protein structure and function co-determine the spectrum of mutants which are selected by 8-aza-A (De Jong et al. (1988)). Point mutations (i.e. no detectable changes on Southern blots because no relevant restriction sites were affected) induced by ionizing radiation were mostly simple transversions and transitions and small deletions. The latter were in some cases flanked by direct repeats (Grosovsky et al. (1988)). Ultraviolet light induced mutations were mostly targeted to dipyrimidine sites and consisted of G•C→→A•T transitions (Drobetsky et al. (1987)). Important general insights derive from this work. The fact that mutations in aprt were characteristic for a particular mutagen and distributed non-randomly points to a role for a chromatin/DNA “context”. “Micro-environment” determines damage distribution, relative efficiency of error-free and error-prone repair, region- and site-specific repair, site specificity of polymerase fidelity, transcriptional activity etc. (Bohr et al. (1987)). However, mutation distribution in phenotypically selected systems is also affected by protein selectable sites and protein functional hot spots.
A breakthrough in mutagenesis research (and many other aspects of molecular biology) arrived with the introduction of the polymerase chain reaction (PCR) which allows the potent amplification of single copy genes (or their transcripts) in unfractionated cellular DNA or even in crude cell lysates (Saiki et al. (1988); Mullis and Fallona (1987) and EP-A1 201 184, EP-A2 200 362, and EP-A1 237 362). PCR was also used to amplify gpt sequences i
Amstad Paul
Cerutti Peter A.
Felley-Bosco Emanuela
Pourzand Charareh
Sandy Martha
Dade Behring Marburg GmbH
Finnegan, Henderson Farabow, Garrett and Dunner L.L.P.
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