785Prime plasmids have been isolated from Methylophilus methylotrophus AS1 using the IncP-1 plasmid pMOl72. Such plasmids can complement mutant function when transferred to appropriate strains of Pseudomonas aeruginosa PAO. From the range of particular functions complemented by each prime plasmid, a preliminary map of the M. methylotrophus AS1 genome with four groups of linked markers was obtained. Physical examination of two of these prime plasmids showed that each had acquired an additional DNA segment. Southern hybridization experiments showed there was sequence homology between the additional DNA segments and M . methylotrophus AS1 DNA, confirming that these prime plasmids carried a segment of the M. methylotrophus AS1 genome. Mapping by complementation may be applicable to other bacteria in which mutants suitable for selecting recombinants are not readily available. I N T R O D U C T I O NThere is an increasing interest in genetic studies of bacteria other than those traditionally used (Levinthal, 1974), because of the availability of plasmids with chromosome-mobilizing ability in a variety of species (Holloway, 1979) and the need for genetic manipulation of bacteria used in biotechnology. The development of a conventional genetic recombination system in bacteria involves the use of an effective chromosome mobilization agent, the construction of multiply marked strains and a means of selecting rare recombinants. Methylophilus methylotrophus AS1 is a Gram-negative bacillus capable of growth on methanol as sole carbon source. It is used by Imperial Chemical Industries for the commercial production of 'Pruteen', a dried protein, animal feed additive. Our attempts to isolate an adequate range of auxotrophically marked strains of M. methylotrophus were unsuccessful, in agreement with the results of O'Connor & Hanson (1978) who found that auxotrophs of methylotrophs are difficult to isolate.Enhanced chromosome mobilizing (ECM) plasmids such as R68.45 are able to generate plasmid primes in a variety of bacterial genera (reviewed by Holloway, 1979). Primes carrying fragments of chromosomal DNA from Pseudomonas putida, Escherichia coli and Rhizobium spp. can complement the phenotype of P . aeruginosa mutants to produce a wild-type phenotype . Morgan, unpublished). Although ECM plasmids such as R68.45 can promote chromosome transfer in M. methylotrophus ASl, the frequency of recombinants and the range of suitable selective mutants are inadequate for an effective mapping system to be established (Holloway, 1981). Accordingly we set out to establish an alternative system of mapping, which we have called complementation mapping. An ECM plasmid was used to generate a library of prime plasmids carrying fragments of the M. methylotrophus AS1 genome. The genes carried by these primes were identified by complementation after transfer to suitably marked P . aeruginosa P A 0 strains. The prime plasmids were further tested for their ability to complement a range of P . aeruginosa mutants to indicate which genes were carried b...

To evaluate whether marrow contributes to relapse after autologous bone marrow transplantation (AuBMT) for acute leukemia, transplanted marrow was marked with the G1N retroviral vector (Genetic Therapy Inc.) containing the neomycin phosphotransferase gene (neo). Between April 1992 and August 1993, 4 patients were transplanted for acute myeloid leukemia (AML) in second complete remission (CR) and 1 patient for acute lymphoid leukemia in first CR. An average of 12.4% (range 5-19%) of transplanted marrow mononuclear cells were exposed to G1N vector for 4 hr. In the vector-treated portion of the marrow, 4.9% of GM-CFU and 3.6% of erythroid burst-forming units (BFU-E) were resistant to G418 in vitro. In the 5 patients, the polymerase chain reaction (PCR) detected the neo sequence on only two occasions after AuBMT. Of 4 patients surviving 1 year after transplantation, only 1 had evidence of gene marked cells by PCR. Two AML patients have relapsed, one of whom had evidence of neo sequences in the bone marrow at day 100 but not at relapse 11 months after AuBMT. The second patient relapsed 18 months after AuBMT but never had PCR evidence of neo sequences before or after relapse. Our results indicate vector-transduced autologous bone marrow from heavily pretreated adults with acute leukemia mark with low efficiency, although vector sequences have been detected in bone marrow and peripheral blood up to 1 year after transplant. Of the 2 relapsed patients, no evidence of vector-marked leukemic blasts have been detected.

Gene therapy for chronic myelogenous leukaemia (CML) may provide a therapeutic option for patients who are ineligible for bone marrow transplantation. To determine the feasibility of such an approach we evaluated the transduction efficiency of CML progenitor colonies from seven patients in chronic phase. Vector transduction was optimized using the CML-derived K562 cell line and applied to CML mononuclear cells. After vector exposure, optimal gene transfer was noted when CML mononuclear cell cultures contained stem cell factor, IL-3, GM-CSF and erythropoietin. The addition of IL-6 to this combination decreased transduction efficiency. Using these conditions, 20.4% +/- 2.4 (SE) of erythroid colonies (CFU-GEMM and BFU-E) and 20.2% +/- 4.7 of CFU-GM colonies were G418 resistant. This compares with a transduction efficiency of 5.9% +/- 1.1 and 6.4% +/- 1.5, respectively, for erythroid and CFU-GM colonies using marrow obtained from normal donors. Only a modest increase in gene transfer was noted when CML cells were stimulated with cytokines for the 24 h preceding vector exposure. Vector DNA in colonies expressing the BCR/ABL transcript was documented by performing PCR analysis on individual colonies. The relatively high gene transfer rate in CML suggests that this disease might be very suitable for gene therapy.

Human breast cancer cell lines which grow in athymic (nude) mice provide a model of tumor cell growth and metastasis. Marking transplanted tumor cell populations with retroviral vectors provides a means of studying the dynamics of tumor cell growth in vivo. We evaluated three human breast cancer cell lines, MDA-MB-435, MDA-MB-231 and MCF-7, and found the cells were highly susceptible to retroviral gene transfer after a single 2-h exposure (90.9%, 62.7% and 72.3%, respectively). MDA-MB-435 cells (5 x 10(5)) marked with a retroviral vector containing the beta-galactosidase gene (approximately 10(4) uniquely marked clones) were injected into the mammary fat pad of athymic mice to study clonal dominance. Primary tumors resected 10 weeks after injection expressed beta-galactosidase, demonstrating persistent vector expression in vivo. Southern blot analysis did not reveal clonal dominance in the primary tumors of the five mice studied. In contrast, pulmonary metastases in each animal were monoclonal or biclonal. These results demonstrate clonal dominance in pulmonary metastases but not primary tumors of retrovirally marked MDA-MB-435 cells. Our findings suggest that this model may also be used to introduce retroviral vectors expressing oncogenes, and anti-sense oncogenes, to determine their effect on tumor cell proliferation and metastasis in vivo.

Forty‐four women with advanced breast cancer participated in a prospective clinical trial to evaluate the efficacy and toxicity of a regimen consisting of cytosine arabinoside and cisplatin. All patients had previously received chemotherapy. Three patients (7%) responded to therapy with response durations of 153, 160, and 441 days. The median time to disease progression and median survival time in all 44 patients were 2.3 and 5 months, respectively. This regimen had significant toxicity, with most patients experiencing severe or life‐threatening hematologic, renal, or infectious complications. This regimen cannot be recommended for previously treated patients with advanced breast cancer.