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Many of the models of hemopoiesis that have been proposed (see, for example, Cronkite et al., '59) are based on the assumption that the continued production of blood cells requires the presence of progenitor cells with the capacity for continued proliferation. From this point of view, hemopoietic tissue may be considered to consist of two compartments; the first, or stem cell compartment, consists of progenitor cells with the capacity to give rise to progeny consisting of both differentiated cells and new stem cells; the second, or differentiated cell compartment, contains cells with limited capacity for cell division, giving rise only to fully differentiated cells. It follows that studies on the processes involved in hemopoiesis require methods for determining the composition of the two compartments. Members of the differentiated cell compartment can frequently be recognized by clear functional markers, for example, the ability to incorporate radioiron (Alpen and Cranmore, '59). In contrast, recognition and assay of stem cells must involve a procedure in which the descendants of the stem cell are examined. Ideally, such a procedure should test the stem cell not only for its ability to give rise to differentiated descendants (which is the basis for the assay for stem cells described by Gurney and co-workers (Gurney et al., '62)), but should also test for other key properties of stem cells. These include the capacities for self-renewal and extensive proliferation, both of which are required for the maintenance of the stem cell compartment. Recently, a method has been developed which may fulfill these requirements for studies of stem cells. The method depends on the observation that mouse hemopoietic tissue contains a population of cells that have the capacity to give rise to macroscopic colonies in the spleens of 327 ___________

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Three mechanisms for resistance to methotrexate (Mtx) have been identified in Chinese hamster ovary (CHO) cells selected from resistance to this drug. First-step selections produce cells with either an apparent structural alteration in the enzyme dihydrofolate reductase (class I), or a decreased permeability to the drug (class II). Mutagenesis with ethyl methanesulfonate increases the proportion of Mtx-resistant cells 5-10-fold. Second-step selections to higher resistance using class I resistant cells as parents results in cells with an increased activity of the reductase enzyme (class III) with no apparent further qualitative alterations in the enzyme. All three classes of resistant cells retain their Mtx-resistant phenotype when cultured under nonselectivve conditions.

A favorable system which is amenable to frequent and reproducible sampling, consisting of suspension cultures of strain L cells and vaccinia virus, was employed to study the animal virus-mammalian host cell relationship. The three principal aspects investigated concerned the adsorption and penetration of vaccinia into the host, the relationship between the sequence of virus development and the production of infectious particles, and the changes in the fine structure of the host cells. Experiments in which a very high multiplicity of infection was used revealed that vaccinia is phagocytized by L cells in less than 1 hour after being added to the culture, without any apparent loss of its outer limiting membranes. Regions of dense fibrous material, thought to be loci of presumptive virus multiplication, appear in the cytoplasm 2 hours after infection. A correlation between electron microscope studies and formation of infectious particles shows that although immature forms of the virus appear 4 hours after infection, infectious particles are produced 6 hours after infection of the culture, at the time when mature forms of vaccinia appear for the first time in thinly sectioned cells. Spread of the infection is gradual until eventually, after 24 hours, virus is being elaborated throughout the cytoplasm. Addition of vaccinia to monolayer cultures induced fusion of L cells and rapid formation of muhinucleate giant forms. In both suspension and stationary cultures infected cells elaborate a variety of membranous structures not present in normal L cells. These take the form of tube-like lamcllar and vesicular formations, or appear as complex reticular networks or as multi-laminar membranes within degenerating mitochondria.

PLATES 61 AND 62

Chinese hamster cell mutants resistant to the lectins PHA, WGA, RIC, LCA, and CON A were previously grouped into 8--10 distinct phenotypes on the basis of their unique patterns of lectin resistance and lectin-binding properties. All but one of these classes of lectin-resistant (LecR) mutants behave recessively in somatic cell hybrids. One ricin-resistant class (RicRII) behaves dominantly. Tests for complementation, by measuring the lectin-resistant properties of appropriate hybrids, show that seven distinct complimentation groups can be delineated among the phenotypically recessive mutants.