Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdrl. Failure to reduce intracellular accumulation of [3Hlvinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdrl.

We have cloned a member of the fork head/HNF-3 family of transcription factors from the nematode Caenorhabditis elegans. Within the predicted DNA binding domain, this gene, called Ce-fkh-1, is 75-78% identical to the Drosophila fork head and rat liver HNF-3 alpha, beta, and gamma genes. Ce-fkh-1 mRNA is highly enriched in embryos. The Ce-fkh-1 gene produces three major transcripts: the longest mRNA retains its original 5'-end but two shorter mRNAs are trans-spliced at the beginning of exons 2 and 3, respectively. In situ hybridization and transgenic Ce-fkh-1::lacZ reporter constructs indicate that the Ce-fkh-1 gene is expressed in both pharynx and intestine of the embryo, beginning at the midproliferation stage. A second phase of Ce-fkh-1 expression occurs in cells of the larval somatic gonad. The pharynx-gut expression of Ce-fkh-1 in the C. elegans embryo is compared with expression of fork head throughout the gut of Drosophila embryos and with expression of HNF-3 (alpha beta gamma) in the endoderm of mammalian embryos. Such conserved patterns of gene expression point to universal features of gastrulation and of digestive tract formation.

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In cells stably transfected and overexpressing the mouse mdr1 gene, multidrug resistance is associated with an increased ATP-dependent drug efflux. Analysis of the predicted amino acid sequence of the MDR1 protein revealed the presence of two putative nucleotide-binding sites (NBS). To assess the functional importance of these NBS in the overall drug resistance phenotype conferred by mdr1, we introduced amino acid substitutions in the core consensus sequence for nucleotide binding, GXGKST. Mutants bearing the sequence GXAKST or GXGRST at either of the two NBS of mdr1 and a double mutant harboring the sequence GXGRST at both NBS were generated. The integrity of the two NBS was essential for the biological activity of mdr1, since all five mutants were unable to confer drug resistance to hamster drug-sensitive cells in transfection experiments. Conversely, a lysine-to-arginine substitution outside the core consensus sequence had no effect on the activity of mdr1. Failure to reduce intracellular accumulation of [3H]vinblastine paralleled the loss of activity in cell clones expressing mutant MDR1 proteins. However, the ability to bind the photoactivatable ATP analog 8-azido ATP was retained in the five inactive MDR1 mutants. This result implies that an essential step subsequent to ATP binding is impaired in these mutants, possibly ATP hydrolysis or secondary conformational changes induced by ATP-binding or hydrolysis. Our results suggest that the two NBS function in a cooperative fashion, since mutations in a single NBS completely abrogated the biological activity of mdr1.

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