A system for assaying homologous recombination at the endogenous human thymidine kinase gene

Journal Article · Thu Aug 01 00:00:00 EDT 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.15.6652· OSTI ID:5602443

Benjamin, M B; Little, J B ^[1]; Potter, H ^[2]; Yandell, D W ^[3]

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1. Harvard School of Public Health, Boston, MA (United States)
2. Harvard Medical School, Boston, MA (United States)
3. Harvard School of Public Health, Boston, MA (United States) Massachusetts Eye and Ear Infirmary, Boston (United States) Harvard Medical School, Boston, MA (United States)

A system for assaying human interchromosomal recombination in vitro was developed, using a cell line containing two different mutant thymidine kinase genes (TK) on chromosomes 17. Heteroalleles were generated in the TK{sup +/+} parent B-lymphoblast cell line WIL-2 by repeated exposure to the alkylating nitrogen mustard ICR-191, which preferentially causes +1 or {minus}1 frameshifts. Resulting TK{sup {minus}/{minus}} mutants were selected in medium containing the toxic thymidine analog trifluorothymidine. In two lines, heterozygous frameshifts were located in exons 4 and 7 of the TK gene separated by {approx}8 kilobases. These lines undergo spontaneous reversion to TK{sup +} at a frequency of < 10{sup {minus}7}, and revertants can be selected in cytidine/hypoxanthine/aminopterin/thymidine medium. The nature and location of these heteroallelic mutations make large deletions, rearrangements, nondisjunction, and reduplication unlikely mechanisms for reversion to TK{sup +}. The mode of reversion to TK{sup +} was specifically assessed by DNA sequencing, use of single-strand conformation polymorphisms, and analysis of various restriction fragment length polymorphisms (RFLPs) linked to the TK gene on chromosome 17. The data suggest that a proportion of revertants has undergone recombination and gene conversion at the TK locus, with concomitant loss of frameshifts and allele loss at linked RFLPs. Models are presented for the origin of two recombinants.

• OSTI ID: 5602443
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 88:15; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
HEREDITARY DISEASES
MOLECULAR BIOLOGY
HUMAN CHROMOSOME 17
GENE RECOMBINATION
PHOSPHOTRANSFERASES
ENZYME ACTIVITY
DNA SEQUENCING
GENE AMPLIFICATION
LYMPHOCYTES
PHOSPHORUS 32
REVERTANTS
RFLPS
STRUCTURAL MODELS
THYMIDINE
ANIMAL CELLS
AZINES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CHROMOSOMES
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DISEASES
ENZYMES
HETEROCYCLIC COMPOUNDS
HUMAN CHROMOSOMES
ISOTOPES
LEUKOCYTES
LIGHT NUCLEI
MATERIALS
MUTANTS
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
PYRIMIDINES
RADIOISOTOPES
RIBOSIDES
SOMATIC CELLS
STRUCTURAL CHEMICAL ANALYSIS
TRANSFERASES
550400* - Genetics