Discrete mutations introduced in the predicted nucleotide-binding sites of the mdr1 gene abolish its ability to confer multidrug resistance.

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.     

High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1.

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.     

Accumulation of viruslike particles in a yeast mutant lacking a mitochondrial pore protein.

The lack of mitochondrial porin is not lethal in Saccharomyces cerevisiae, but it impairs some respiratory functions and, therefore, growth on nonfermentable carbon sources such as glycerol. However, after a lag phase porinless mutant cells adapt to growth on glycerol, accumulating large amounts of an 86-kilodalton (kDa) protein (M. Dihanich, K. Suda, and G. Schatz, EMBO J. 6:723-728, 1987) and of a 5-kilobase RNA. Immunogold labeling localized the 86 kDa-protein exclusively to the cytosol fraction, although most of it cosedimented with the microsome fraction in earlier cell fractionations. This discrepancy was resolved when the 86-kDa protein was identified as the major coat protein in viruslike particles (VLPs) which is encoded by a double-stranded RNA (L-A RNA). Elimination of VLPs in the original porinless strain by introduction of the mak10 or the mak3 mutation increased the respiratory defect and prolonged its lag phase on nonfermentable carbon sources. The fact that the simultaneous loss of VLPs and respiratory functions are the introduction of mak10 or mak3 occurred even in some porin-containing wild-type strains suggests that there is a link between VLP and mitochondrial functions.     

Rates of mutation to growth factor autonomy and tumorigenicity differ in hematopoietic stem and precursor cells expressing the multilineage colony-stimulating factor gene.

At least two separate but interdependent events are required to attain autonomous growth as a consequence of ectopic expression of the multilineage colony-stimulating factor gene in hematopoietic progenitor cells. The rate at which the second event occurs is more than 3 orders of magnitude higher in precursor cell lines (FDC-P1 or FDC-P2) than in stem cell lines (FDC-Pmix). Autonomous, but not density-dependent, growth is tightly coupled to tumorigenicity in precursor cells; however, neither growth-factor-independent nor autonomously growing stem cell lines are tumorigenic.     

Mock retroviral infection alters the developmental potential of murine bone marrow stem cells.

Retroviral vectors were used to introduce an activated ras gene into murine pluripotent hemopoietic stem cells. We attempted to reconstitute the hemopoietic system of lethally irradiated mice with isolated spleen colonies obtained in vivo after injection of infected bone marrow cells. Spleen colonies derived from infected bone marrow were inefficient in promoting long-term survival of irradiated hosts. This loss of reconstitutive capacity of spleen colonies was not due to the retroviral infection per se but to the in vitro culture of spleen colony precursors. Incubation for 24 h in the presence of fetal calf serum and interleukin-3 without virus-producing cells was sufficient to abolish completely the reconstitutive capacity of spleen colonies while maintaining both self-renewal and pluripotential capacities of spleen colony precursors. These results show that the in vitro manipulation of stem cells that is included in current protocols for retroviral infection can modify the developmental potential of these cells. This finding clearly indicates that the use of retroviral vectors can introduce a bias in the analysis of hemopoiesis.     

Variation of tandem repeats in the developmentally regulated procyclic acidic repetitive proteins of Trypanosoma brucei.

The procyclic acidic repetitive proteins (PARPs) of Trypanosoma brucei are developmentally regulated surface proteins encoded by a family of polymorphic genes. We have determined the complete nucleotide sequence of a novel member of the PARP gene family and investigated its expression. The amino acid sequence deduced from the parpA alpha gene showed a marked conservation of both the amino- and carboxy-terminal regions compared with other PARPs but revealed the substitution of a pentapeptide for the dipeptide repeating unit that is characteristic of all other PARPs. Northern hybridization analysis indicated that expression of the parpA alpha gene, like that of other members of this gene family, is confined to the procyclic stage of the T. brucei life cycle. This result implies coordinate regulation of the unlinked genetic loci that encode PARPs.