E2A deficiency leads to abnormalities in alphabeta T-cell development and to rapid development of T-cell lymphomas.

The E2A gene products, E12 and E47, are critical for proper early B-cell development and commitment to the B-cell lineage. Here we reveal a new role for E2A in T-lymphocyte development. Loss of E2A activity results in a partial block at the earliest stage of T-lineage development. This early T-cell phenotype precedes the development of a T-cell lymphoma which occurs between 3 and 9 months of age. The thymomas are monoclonal and highly malignant and display a cell surface phenotype similar to that of immature thymocytes. In addition, the thymomas generally express high levels of c-myc. As assayed by comparative genomic hybridization, each of the tumor populations analyzed showed a nonrandom gain of chromosome 15, which contains the c-myc gene. Taken together, the data suggest that the E2A gene products play a role early in thymocyte development that is similar to their function in B-lineage determination. Furthermore, the lack of E2A results in development of T-cell malignancies, and we propose that E2A inactivation is a common feature of a wide variety of human T-cell proliferative disorders, including those involving the E2A heterodimeric partners tal-1 and lyl-1.

Both E12 and E47 allow commitment to the B cell lineage.

The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.

Disruption of the mouse mdr1a P-glycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs.

We have generated mice homozygous for a disruption of the mdr1a (also called mdr3) gene, encoding a drug-transporting P-glycoprotein. The mice were viable and fertile and appeared phenotypically normal, but they displayed an increased sensitivity to the centrally neurotoxic pesticide ivermectin (100-fold) and to the carcinostatic drug vinblastine (3-fold). By comparison of mdr1a (+/+) and (-/-) mice, we found that the mdr1a P-glycoprotein is the major P-glycoprotein in the blood-brain barrier and that its absence results in elevated drug levels in many tissues (especially in brain) and in decreased drug elimination. Our findings explain some of the side effects in patients treated with a combination of carcinostatics and P-glycoprotein inhibitors and indicate that these inhibitors might be useful in selectively enhancing the access of a range of drugs to the brain.

Heterospecific transformation in Bacillus subtilis: protein composition of a membrane-DNA complex containing unstable heterologous donor-recipient complex.

Previously it was demonstrated that, in contrast to the homologous donor-recipient complex, the unstable heterologous donor-recipient complex remains bound to the cellular membrane. To examine whether proteins known to be involved in the processing of transforming DNA in Bacillus subtilis are associated with membrane fragments which carry chromosomal DNA, a crude membrane-DNA complex was subjected to electrophoresis through a sucrose gradient. This resulted in the separation of membrane fragments associated with DNA and free membrane fragments. By means of two-dimensional gel electrophoresis several proteins, either uniquely present or considerably enriched in the purified membrane-DNA complex, were detected. Among these proteins we identified the 45 kD recE gene product, required for recombination, the 18 kD binding protein involved in the binding of transforming DNA and a 17 kD nuclease involved in the entry of transforming DNA. These results suggest that the membrane sites at which donor DNA integrates into the recipient chromosome are in the vicinity of the sites of entry of donor DNA through the membrane.

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane.

Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5',8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm = 48 degrees C), we presume that donor and recipient DNA are associated either by several short sequences of 15-25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation. The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis. II. Unstable association of heterologous DNA with the recipient chromosome.

In CsCl density gradients of lysates from competent Bacillus subtilis cells, which had been exposed to heterologous bacterial DNA, very little donor-recipient complex (DRC) formation could be detected. The present study demonstrates that photocrosslinking of such lysates by irradiation with long-wave UV light in the presence of 4,5',8-trimethylpsoralen results in a dramatic increase in the amount of heterologous DRC. This phenomenon may be interpreted as the stabilization of a pre-existing weak association between entered heterologous donor DNA and one recipient strand in unpaired regions of the chromosome. When a recombination-deficient mutant is used, the amount of stabilizable heterologous DRC is reduced to the same extent as the specific transforming activity of homologous DNA. Although the amount of stabilizable complex is related to the degree of homology between donor and recipient DNA, this relation is not a quantitative one. Probably the association is caused by very short regions of base pairing between the donor and recipient moieties in the complex. Heating of a lysate at 70 degrees prior to photocrosslinking prevents stabilization, apparently because the regions of base pairing are rapidly melted out. The results described in this paper can be best interpreted as the fixation of a process in which entered donor DNA in competent cells tries to find homologous stretches in the recipient chromosome.

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis. I. Processing of B. pumilus and B. licheniformis DNA in B. subtilis.

Competent Bacillus subtilis cells were exposed to radioactive and density labeled donor DNA extracted from B. pumilus and B. licheniformis. The DNA from these strains hybridized with B. subtilis DNA in vitro at a rate of 24% and 11%, respectively. After entry the vast majority of heterologous DNA was found at the single-strand DNA position in CsCl gradients, and was gradually degraded during incubation. Much less donor DNA than expected from the hybridization values participated in the formation of the donor-recipient complex (DRC). By subjecting the heterologous DRC to sonication and alkaline CsCl gradient centrifugation, it was established that the DRC consisted of three components: (1) recipient DNA in which breakdown products of donor DNA were incorporated through DNA synthesis, (2) recipient DNA in which donor DNA was covalently integrated and (3) recipient DNA in which the donor moiety was not covalently integrated.

Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis. Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G. Venema and U. Canosi, Mol. Gen. Genet. 179:1--11), TMP monoadducts have only a slight effect on entry. Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.