Cell adhesiveness is fundamental to a variety of biological phenomena, including tumour development and metastasis. Recently, Bubenik et al. have reported that in various malignant fibroblastoid cell lines those cells which demonstrated less adhesiveness were more tumorigenic. The relationship between cell adhesiveness, transformation and metastasis has been studied extensively in cells (fibroblastoid) grown as monolayers attached to their substratum and to each other, but, to our knowledge, there has been no report describing this relationship in suspension-borne (lymphoid) cells that grow free of each other and their substratum. We report here that substrate-adhering variant cells, selected from the tumorigenic, suspension-growing S49 mouse lymphoma, have impaired tumorigenicity. Furthermore, the substrate-adhering cells also have increased immunogenicity, as their inoculation into mice protects the mice from subsequent challenges with parental, non-adherent tumorigenic S49 cells. These findings suggest a new approach for the selection of immunogenic cells from suspension-borne tumorigenic cell populations.

Mouse lymphoma cells ($49) that grow in suspension culture were selected for increased tumorigenicity through continuous passages in syngeneic BALB/c mice. Developing tumors were classified as high grade malignant lymphoma, small noncleaved type. Variants were selected from these tumorigenic cells that were able to grow as a monolayer attached to their substrate, resembling, in this respect, fibroblastoid cells. Whereas the tumorigenic suspension-growing parental cells were able to induce progressive tumors with an inoculum as low as 100 cells per mouse, the adherent cells were unable to develop as tumors even at an inoculum of 1 x 108 cells per mouse. In addition, mice inoculated once with live adherent cells were immunized against 1 x 107 suspension-growing cells. Involvement of an immune response in the rejection of tumorigenic $49 cells was suggested by (a) adoptive transfer experiments in which spleen cells from immunized mice protected naive mice and (b) the appearance of antibodies in the sera of immunized syngeneic mice that specifically recognized both adherent and suspension-growing $49 cells and detected differences in [35S]methioninelabeled antigens from these cells. Antibodies raised in rabbits against adherent cells recognized three proteins of 34,000, 61,000, and 72,000 apparent molecular weight in radiolabeled adherent cell extracts that are either absent or present in small amounts in extracts of suspension-growing tumorigenic $49 cells. These findings, taken together with our previous report (Hochman, J., A. Katz, E. Levy, and S. Eshel, 1981, Nature (Lond.), 290:248-249), suggest the $49 system as a novel system for studying growth control in malignant lymphoid cells.The relationship among cell adhesiveness (the ability to adhere to other cells and to extracellular substrata), transformation, tumor development, and the metastatic behavior of malignant cells is a well-known but complex phenomenon that has been studied extensively (l, 3, 5, 9, 18, 20, 24) since Ludford (17) and Cowdry (6) first suggested that membranes of tumor cells had lower general adhesive properties than the normal cells from which they were derived. These previous studies have all been carried out on cells grown as monolayers (i.e., fibroblastoid) attached to their substratum and to each other.We have undertaken to study this relationship in malignant lymphoid cells that grow attached neither to each other nor to their substratum. Our working hypothesis is that if in fibroblasts decreased cell adhesiveness correlates with in-1282 creased tumorigenicity (3, 18) and vice versa, then isolation of adherent variants from malignant lymphoid cells may result in decreased tumorigenicity. Such a model would be of potential significance in the study of in vitro growth regulation of malignant lymphoid cells.To test this approach, we have used $49 cells--a mouse lymphoma cell line of BALB/c origin (13). We have first demonstrated that stable variants can be isolated, characterized by their ability to adhere to their substratu...

Mutagenesis of Azospirillum brasilense with nitrosoguanidine and selection on ethylenediamine yielded prototrophs which fixed nitrogen in the presence of ammonia. Nitrogenase activity in mutant strains exceeded that of the wild type three-to sixfold. The same mutants were also constitutive for histidine transport. Enzyme activities involved in ammonia assimilation were not affected by the mutation. The data suggest that the mutation occurred at a site which regulates nif and histidine tra'nsport functions.

Computational approaches for detecting co-evolution in proteins allow for the identification of protein-protein interaction networks in different organisms and the assignment of function to under-explored proteins. The detection of co-variation of amino acids within or between proteins, moreover, allows for the discovery of residue-residue contacts and highlights functional residues that can affect the binding affinity, catalytic activity, or substrate specificity of a protein. To explore the functional impact of co-evolutionary changes in proteins, a combined experimental and computational approach must be recruited. Here, we review recent studies that apply computational and experimental tools to obtain novel insight into the structure, function, and evolution of proteins. Specifically, we describe the application of co-evolutionary analysis for predicting high-resolution three-dimensional structures of proteins. In addition, we describe computational approaches followed by experimental analysis for identifying specificity-determining residues in proteins. Finally, we discuss studies addressing the importance of such residues in terms of the functional divergence of proteins, allowing proteins to evolve new functions while avoiding crosstalk with existing cellular pathways or forming reproductive barriers and hence promoting speciation.

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