Biomedical subjects

F C Christians

Publications and source records attributed to F C Christians.

9 recordsLinked to original sources

PubMed1999

Directed evolution of thymidine kinase for AZT phosphorylation using DNA family shuffling.

The thymidine kinase (TK) genes from herpes simplex virus (HSV) types 1 and 2 were recombined in vitro with a technique called DNA family shuffling. A high-throughput robotic screen identified chimeras with an enhanced ability to phosphorylate zidovudine (AZT). Improved clones were combined, reshuffled, and screened on increasingly lower concentrations of AZT. After four rounds of shuffling and screening, two clones were isolated that sensitize Escherichia coli to 32-fold less AZT compared with HSV-1 TK and 16,000-fold less than HSV-2 TK. Both clones are hybrids derived from several crossover events between the two parental genes and carry several additional amino acid substitutions not found in either parent, including active site mutations. Kinetic measurements show that the chimeric enzymes had acquired reduced K(M) for AZT as well as decreased specificity for thymidine. In agreement with the kinetic data, molecular modeling suggests that the active sites of both evolved enzymes better accommodate the azido group of AZT at the expense of thymidine. Despite the overall similarity of the two chimeric enzymes, each contains key contributions from different parents in positions influencing substrate affinity. Such mutants could be useful for anti-HIV gene therapy, and similar directed-evolution approaches could improve other enzyme-prodrug combinations.

Amino Acid Sequence↗

PubMed1997

Creation of human alkyltransferases resistant to O6-benzylguanine.

O6-benzylguanine (BG), an inhibitor of O6-alkylguanine-DNA alkyltransferase, is being tested clinically for its ability to chemosensitize tumors to alkylating agents. Although this drug may increase the killing of tumors that express high levels of alkyltransferase, it would also be expected to reduce the already low alkyltransferase levels of hematopoietic stem cells and, thus, exacerbate the dose-limiting side effect of myelosuppression. One way to overcome this problem would be to transduce hematopoietic stem cells with a gene encoding a BG-resistant alkyltransferase prior to BG/alkylation treatment. We used the technique of random mutagenesis followed by positive genetic selection to create such a mutant gene. A pool of 6.5 x 10(6) human alkyltransferases that were randomly mutated at six amino acids near the alkyl-accepting cysteine was transformed into alkyltransferase-deficient Escherichia coli. Five mutants were selected based on their ability to provide the bacteria with resistance to both N-methyl-N'-nitro-N-nitrosoguanidine and BG. One mutant, V139F/P140R/L142M, not only had the highest BG resistance (50% inhibitory concentration, >500 microM) but also offered E. coli the best protection from N-methyl-N'-nitro-N-nitrosoguanidine and, thus, is a promising gene therapy candidate.

Binding Sites↗

PubMed1996

Novel human DNA alkyltransferases obtained by random substitution and genetic selection in bacteria.

DNA repair alkyltransferases protect organisms against the cytotoxic, mutagenic, and carcinogenic effects of alkylating agents by transferring alkyl adducts from DNA to an active cysteine on the protein, thereby restoring the native DNA structure. We used random sequence substitutions to gain structure-function information about the human O6-methylguanine-DNA methyltransferase (EC 2.1.1.63), as well as to create active mutants. Twelve codons surrounding but not including the active cysteine were replaced by a random nucleotide sequence, and the resulting random library was selected for the ability to provide alkyltransferase-deficient Escherichia coli with resistance to the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Few amino acid changes were tolerated in this evolutionarily conserved region of the protein. One mutation, a valine to phenylalanine change at codon 139 (V139F), was found in 70% of the selected mutants; in fact, this mutant was selected much more frequently than the wild type. V139F provided alkyltransferase-deficient bacteria with greater protection than the wild-type protein against both the cytotoxic and mutagenic effects of N-methyl-N'-nitro-N-nitrosoguanidine, increasing the D37 over 4-fold and reducing the mutagenesis rate 2.7-5.5-fold. This mutant human alkyltransferase, or others similarly created and selected, could be used to protect bone marrow cells from the cytotoxic side effects of alkylation-based chemotherapeutic regimens.

Amino Acid Sequence↗

PubMed1996

Multiple mutations in human cancers.

Increasing evidence indicates that most human cancers contain multiple mutations. The exact number of mutations, their origin, and types remain to be determined. An over-riding question is whether the multiple mutations that accumulate in cancers is rate-limiting for the carcinogenic process. In this review we consider the argument that the large numbers of mutations routinely reported in human cancers cannot be accounted for by the rate of spontaneous mutation observed in normal human cells. We will analyze different mechanisms that might account for the accumulation of mutations in cancer cells. We conclude that cancer cells are genetically unstable; i.e., they exhibit a mutator phenotype. The recent reports of microsatellite instability in a variety of human cancers have provided the first strong evidence for the presence of a mutator phenotype in human cancers. However, we still lack information about the relationship between microsatellite instability and mutations that allow cancer cells to proliferate, invade, and metastasize.

Carcinoma, Hepatocellular↗

PubMed1996

Tolerance of different proteins for amino acid diversity.

Random mutagenesis of genes followed by positive genetic selection in bacteria requires that the variant molecules confer biological activity, and is thus the most demanding approach for generating new functionally active molecules. Furthermore, one can learn much about the protein in question by comparing the population of selected molecules to the library from which they were selected. Described here is a mathematical method designed to guide such comparisons. We use as examples the results of randomization-selection studies of four different proteins. There exists, in general, a positive correlation between the number of amino acid substitutions in a critical region of a protein and the likelihood of inactivation of that protein; a correlation long suspected, but developed here in detail. At this time, we are comparing regions in different proteins and our conclusions must be limited. However, the method presented can serve as a guideline for anticipating the yield of new active mutants in genetic complementation assays based on the extent of randomization.

Amino Acids↗

PubMed1995

Potential sources of multiple mutations in human cancers.

Increasing evidence indicates that most human cancers contain multiple chromosomal alterations. These aberrations are the result of mutations produced not only during the initiation of cancer but also during tumor progression. Since the rates of spontaneous mutations exhibited by normal human cells cannot account for the large numbers of mutations routinely reported in human cancers, we argued that cancer cells are genetically unstable; i.e., they express a mutator phenotype. In this review, we consider potential endogenous sources of these mutations and the recent evidence demonstrating that multiple mutations are present in human cancers. These studies, which connect mismatch repair, genomic instability, and cancer, support the mutator phenotype hypothesis. We conclude that, if multiple mutations are necessary for the progression of cancer, then agents designed to delay their accumulation could significantly reduce cancer deaths.

Chromosome Aberrations↗

PubMed1994

Repair in ribosomal RNA genes is deficient in xeroderma pigmentosum group C and in Cockayne's syndrome cells.

Previous studies have demonstrated transcription-coupled DNA repair in mammalian genes transcribed by RNA polymerase II but not in ribosomal RNA genes (rDNA), which are transcribed by RNA polymerase I. The removal of UV-induced cyclobutane pyrimidine dimers (CPD) from rDNA in repair-proficient human cells has been shown to be slow but detectable and apparently not coupled to transcription. We studied the induction and removal of CPD from rDNA in cultured cells from two repair-deficient human disorders. Primary xeroderma pigmentosum complementation group C (XP-C) cells, whether proliferating or nondividing, removed no CPD from either rDNA strand in 24 h post-UV, a result which supports earlier conclusions that XP-C cells lack the general, transcription-independent pathway of nucleotide excision repair. We also observed lower than normal repair of rDNA in Cockayne's syndrome (CS) cells from complementation groups A and B. In agreement with previous findings, the repair of both strands of the RNA polymerase II-transcribed dihydrofolate reductase gene was also deficient relative to that of normal cells. This strongly suggests that the defect in CS cells is not limited to a deficiency in a transcription-repair coupling factor. Rather, the defect may interfere with the ability of repair proteins to gain access to all expressed genes.

Animals↗

PubMed1993

Lack of transcription-coupled repair in mammalian ribosomal RNA genes.

We studied the induction and removal of UV-induced cyclobutane pyrimidine dimers (CPDs) in the ribosomal RNA genes (rDNA) in cultured hamster and human cells. In these genes, which are transcribed by RNA polymerase I, we found no evidence for transcription-coupled repair. The induction of CPDs was heterogeneous in rDNA due to nucleotide sequence: it was lower on the transcribed strand than on the nontranscribed strand and slightly lower in the coding region than in the nontranscribed spacer. Nevertheless, no dramatic difference in CPD induction was observed between rDNA and the dihydrofolate reductase (DHFR) gene. In Chinese hamster ovary cells, we observed no removal of CPDs from either rDNA strand within 24 h after UV irradiation. In these experiments, we did observe efficient repair of the transcribed, but not the nontranscribed, strand of the DHFR gene, in agreement with published results. In human cells, repair of rDNA was observed, but it showed no strand preference and was slower than that reported for the genome overall. No significant differences in repair were observed between restriction fragments from transcribed and nontranscribed regions or between growth-arrested and proliferating human cells, with presumably different levels of transcription of rDNA. We conclude that the modest level of rDNA repair is accomplished by a transcription-independent repair system and that repair is impeded by the nucleolar compartmentalization of rDNA. We discuss the possibility that recombination, rather than repair, maintains the normal sequence of rDNA in mammalian cells.

Animals↗

PubMed1992

Inhibition of transcription and strand-specific DNA repair by alpha-amanitin in Chinese hamster ovary cells.

Recent studies have shown preferential repair of UV-induced cyclobutane pyrimidine dimers (CPD) in the transcribed strand of the expressed dihydrofolate reductase (DHFR) gene in human and rodent cells. We have tested the hypothesis that the strand-specific repair of such transcription-blocking lesions is dependent upon concurrent transcription. Chinese hamster ovary (CHO) B11 cells with an amplified DHFR gene were treated with alpha-amanitin before irradiation with UV (254 nm) and during post-irradiation incubation. Nuclear run-off analysis verified inhibition of transcription in the DHFR gene. CsCl density gradient analysis showed that alpha-amanitin at the levels used does not significantly interfere with overall genomic repair replication or semiconservative replication. However, we did observe a dramatic reduction in the removal of CPD from the transcribed strand in the 14 kb KpnI fragment within the DHFR gene in treated cells. We conclude that strand-specific repair of an active gene in CHO cells is dependent upon the activity of the transcribing RNA polymerase. Our results support the model that transcription complexes stalled at CPD signal the repair machinery to achieve efficient repair of the transcribed strand in active genes.

Amanitins↗