当前位置： SCI文献检索 > BIOCHEMICAL PHARMACOLOGY期刊下所有文献 > Saturation mutagenesis at dihydrofolate reductase codons 22 and 31. A variety of amino acid substitutions conferring methotrexate resistance.

Saturation mutagenesis at dihydrofolate reductase codons 22 and 31. A variety of amino acid substitutions conferring methotrexate resistance.

Abstract：

:Naturally occurring amino acid substitutions conferring resistance to methotrexate (MTX) have been reported previously at codon positions 22 (leu-->arg, phe) and 31 (phe-->ser, trp) of mammalian dihydrofolate reductases (DHFR). To explore the character of other substitutions, a polymerase chain reaction (PCR)-assisted saturation mutagenesis protocol was devised to introduce all possible codon sequences at positions 22 and 31 of the murine DHFR coding sequence in an expressible simian virus 40 (SV40)-regulated transcription unit. Nucleotide sequencing confirmed the presence of all four nucleotides at each of the three codon positions in the mutagenized material. Transfection of these "codon libraries" into DHFR-deficient Chinese hamster ovary cells resulted in an increased frequency of MTX-resistant colony formation in comparison with wild-type DHFR transfected cells. DHFR variants contained in different clones were characterized by PCR amplification and DNA sequencing, identifying six different amino acid substitutions at position 22 and seven substitutions at position 31. DHFR variants were extracted for determination of MTX inhibition character and catalytic activity, normalizing for the amount of DHFR protein by western blot analysis. A wide range of MTX sensitivities and catalytic activities were observed which is consistent with the role of these side chains in DHFR catalytic function. We observed that codon 22 variants were generally more resistant to MTX, but codon 31 variants retained substantially more catalytic activity (about 2.5-fold) at a given level of MTX resistance. This heterogeneity in catalytic and inhibition character has important implications for the function of different DHFR variants as mediators of drug resistance.

journal_name

Biochem Pharmacol

journal_title

Biochemical pharmacology

authors

Morris JA,McIvor RS

doi

10.1016/0006-2952(94)90393-x

subject

Has Abstract

pub_date

1994-03-29 00:00:00

pages

1207-20

issue

eissn

0006-2952

issn

1873-2968

pii

0006-2952(94)90393-X

journal_volume

pub_type

杂志文章

在线工具

Saturation mutagenesis at dihydrofolate reductase codons 22 and 31. A variety of amino acid substitutions conferring methotrexate resistance.