Comparative mutagenesis in human cells in vitro and in vivo: First progress report for the period 15 July 1986-30 June 1987
We used the technique of denaturing gradient electrophoresis to produce a pure mutant sequence so that we were able to directly sequence the mutant without bacterial cloning. As part of our continuing studies of the hprt locus in human cells, we have isolated a series of chemically induced mutant DNA sequences in HPRT exon 3 which had been selected by 6-thioguanine resistance. Using denaturing gradient gels to study the amount and kinds of mutations induced in vitro by certain DNA polymerases, we modified polymerase chain reaction (PCR) DNA amplification to our studies of mutation in the human hprt gene. We studied the nature of T/sub 4/ DNA polymerase errors and discovered a means to overcome the principle source of noise in amplifying sequences from the human genome. We initiated studies of multi-copy sequences in the human genome so that mutational spectra may be obtained from small numbers of human cells. We worked out novel statistical modes of analysis for planning and evaluating long term low dose human cell mutation studies, and mutational spectra from cell-based or DNA sequence-based studies. We have characterized the positions of large deletion mutations, confirming 6TG resistance in human cells and have found an intron of hprt which has an apparently high density of deletion endpoints within it. We used exon specific probes to discover which exons are present in each of the four pseudogenes of hprt which are scattered over three human autosomes. 41 refs., 8 figs., 10 tabs.
- Research Organization:
- Massachusetts Inst. of Tech., Cambridge (USA). Dept. of Applied Biological Science
- DOE Contract Number:
- FG02-86ER60448
- OSTI ID:
- 5603649
- Report Number(s):
- DOE/ER/60448-02; ON: DE88001811
- Resource Relation:
- Other Information: Portions of this document are illegible in microfiche products
- Country of Publication:
- United States
- Language:
- English
Similar Records
Determination of point mutational spectra of benzo[a]pyrene-diol epoxide in human cells
Fidelity of DNA polymerases in DNA amplification
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
DNA
ELECTROPHORESIS
DNA BASE TRANSITIONS
MUTAGEN SCREENING
HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE
BLOOD CELLS
DNA SEQUENCING
DNA-CLONING
GENE AMPLIFICATION
IN VITRO
IN VIVO
LOW DOSE IRRADIATION
LYMPHOCYTES
MAN
PHOSPHORUS 32
PROGRESS REPORT
RESPONSE MODIFYING FACTORS
THYMUS CELLS
TRACER TECHNIQUES
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BODY FLUIDS
CLONING
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DOCUMENT TYPES
ENZYMES
GLYCOSYL TRANSFERASES
IRRADIATION
ISOTOPE APPLICATIONS
ISOTOPES
LEUKOCYTES
LIGHT NUCLEI
MAMMALS
MATERIALS
NUCLEI
NUCLEIC ACIDS
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PENTOSYL TRANSFERASES
PHOSPHORUS ISOTOPES
PRIMATES
RADIOISOTOPES
SCREENING
SOMATIC CELLS
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
VERTEBRATES
560120* - Radiation Effects on Biochemicals
Cells
& Tissue Culture
560300 - Chemicals Metabolism & Toxicology
550200 - Biochemistry