As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defmed the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions . Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000-19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000-250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt 40,000 . These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles ; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pl 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins . It will also be useful in future studies of trichocysts and mating reactions. J. CELL BIOLOGY

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123lo, Thy-1+, and CD38−/lo CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.