The penicillin method (Davis, 1948; Lederberg and Zinder, 1948) has permitted convenient isolation of auxotrophicl mutants of Escherichia coli with specific growth requirements for most of the known water-soluble vitamins, as well as amino acids, purines, and pyrimidines. Accordingly, when crystalline vitamin B12 became available, a search was made for mutants requiring this nutrilite. Several strains of the desired type were promptly recovered. In all cases methionine, but not homocysteine, could be substituted for the vitamin. This paper describes certain biochemical properties of these mutants, as well as of others responding to methionine but not to B,2. EXPERIMENTAL RESULTS Methods. The mutants were isolated by the penicillin method (Davis, 1949) from the W strain of E. coli (ATCC 9637) following ultraviolet irradiation. The minimal medium, improved over that previously reported (Davis, 1949), had the following composition: K2HPO4, 7.0; KH2PO4, 3.0; Na3-citrate*3H20, 0.5; MgS04-7H20, 0.1; (NH4)2504, 1.0; glucose (autoclaved separately), 2; H20, 1,000; pH 7.0. Solid media contained Difco agar 1.5 per cent. Cultures were incubated at 37 C. Response to methionine and homocysteine. By the use of minimal medium supplemented with 10 mpg per ml of vitamin Bn, three B1,2-requiring strains were obtained in one experiment. They were all found to grow rapidly on methionine, but not on homocysteine, the known precursor of methionine in E. coli (Lampen et al., 1947b; Simmonds, 1948) as well as in Neurospora (Horowitz, 1947). This response led to the testing of a number of previously isolated methionine auxotrophs for their response to B,,. The results are presented in table 1. The mutants blocked before homocysteine did not respond to B1n, whereas, with one exception, those blocked between homocysteine and methionine did respond to B1,. The exception, 137-113, showed no perceptible growth on Bn, alone, even after 3 days of incubation on solid medium, but its growth on a limited amount of methionine was definitely increased by B1,, suggesting a very limited ability to use B12. I The terms "auxotrophic" (Lat. auxilium = "aid"; Gr. troph = "food") and the corresponding noun "auxotroph" are suggested for convenience in denoting biochemical mutants with increased nutritional requirements.

Tropical spastic paraparesis (TSP) is a slowly progressive myelopathy associated with increased serum and cerebrospinal fluid antibodies to the human T-lymphotropic retrovirus type I (HTLV-I) (ref. 1), and has been observed in many regions of the world. A similar condition known as HTLV-I-associated myelopathy occurs in the Kagoshima prefecture of Japan. Recent but controversial reports suggest involvement of virus related to HTLV-I in multiple sclerosis. Magnetic resonance imaging and electrophysiological studies indicate that TSP lesions are like multiple sclerosis in that they are disseminated throughout the nervous system. Complete virus from patients with TSP has proved difficult to isolate using techniques successful in adult T-cell leukaemia cases associated with HTLV-I. Here we report the isolation of an HTLV-I-like virus from T-cell lines derived from the peripheral blood and cerebrospinal fluid of TSP patients. The monoclonal antibody OKT3 was used to generate non-transformed T-cell lines that express HTLV-I antigens. Infectious virus was demonstrated by co-cultivation and complete, replicating virions were visualized ultrastructurally.

Tropical spastic paraparesis (TSP) and other chronic-progressive myelopathies have been clearly associated with increased serum and cerebrospinal fluid antibody titers to human T-lymphotropic virus type I (HTLV-I). However, little is known about the cellular immune function in TSP. In the present study, activated T lymphocytes were found in the peripheral blood of patients with TSP. Specifically, there were increased numbers of large CD3+ cells that also expressed HLA-DR and interleukin-2-receptor molecules. A significantly elevated spontaneous lymphoproliferative response was demonstrated in all patients tested. Generation of measles virus-specific cytotoxic T-cell response was reduced in 4 of 4 patients. This was similar to previous findings in patients with multiple sclerosis. However, unlike multiple sclerosis, reduced generation of cytotoxic T-cell response to influenza and mumps viruses was observed in 2 of 4 patients. These observations confirm further the strong association between TSP and an HTLV-I-like virus and suggest that the observed abnormalities of the cellular immune response in TSP are related to infection of lymphocytes by the retrovirus.