DNA sequences of internal deletion breakpoints of in vivo and in vitro derived human T-lymphocytes hprt mutations

Journal Article · Sat Dec 31 04:00:00 EST 1994 · Environmental and Molecular Mutagenesis

OSTI ID:88909

Rainville, I R; Albertini, R J; Nicklas, J A ^[1]

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1. Univ. of Vermont, Burlington, VT (United States)

Studies of over 1600 human T-lymphocyte hprt mutations by Southern blotting determined 30% have gross structural alterations of the hprt gene. Of these 64% are deletions with 34% of those internal deletions. The breakpoints of these mutations were sequenced to ascertain possible DNA structures and mechanisms important in the processes leading to DNA deletion. Breakpoints were isolated for sequencing as PCR products in 2 ways: either as novel fragments on multiplex PCR or by performing PCR utilizing a large set of hprt primers. A set of oligos spaced at {approximately}1kb intervals were designed and used pairwise as PCR primers. Deletion intervals were designed and used pairwise as PCR primers. Deletion mutations give a product with primers flanking the deletion breakpoints while these primers are, using these conditions, generally too far apart to give a PCR product in wildtype DNA. To date, 13 breakpoints have been sequenced. One mutation was from a normal adult, 3 from platinum treated patients, 4 from radioimmunotherapy patients (RIT), 3 from children and 2 in vitro mutations. Deletion sizes range form 18 to 139445bp (mean 3091bp, median 974bp). One mutant has a 4bp (RIT), one mutant has a 3bp (RIT) and one mutant has a 2bp (platinum) repeat at the breakpoint. Several 5{prime} or 3{prime} breakpoints are in AT rich regions and several are in Alu repeats but because of the high frequency of these features in hprt gene, the significance of this is unclear. No mutation has both breakpoints in Alu sequences giving no evidence for homologous recombination. One 22bp exon 6 deletion has an insert of 9 non-templated nucleotides and is located very closely to another 18bp deletion. Examination of the hprt database indicates this exon 6 region may be prone to small deletions. Sequencing of deletion breakpoints is continuing and the breakpoint regions are being searched for sequences or structures which may indicate mutational mechanisms.

• OSTI ID: 88909
• Report Number(s): CONF-9405324--
• Journal Information: Environmental and Molecular Mutagenesis, Journal Name: Environmental and Molecular Mutagenesis Journal Issue: Suppl.23 Vol. 23; ISSN EMMUEG; ISSN 0893-6692
• Country of Publication: United States
• Language: English

Related Subjects

55 BIOLOGY AND MEDICINE
BASIC STUDIES
DNA SEQUENCING
GENES
HUMAN CHROMOSOMES
HYPOXANTHINE PHOSPHORIBOSYLTRANSFERASE
LYMPHOCYTES
MUTATIONS