Identification of centrosomal proteins in a human lymphoblastic cell line.

Highly enriched preparations of centrosomes from human T-lymphoblasts KE 37 were analyzed for their protein content. The specific pattern of polypeptides was characterized by an abundant subset of high mol. wt proteins and a major group of proteins with mol. wt ranging from 50 to 65 kd. Several immunoreactive proteins were identified, using a rabbit serum spontaneously reacting with human centrosomes. They include a family of high mol. wt ranging from 180 to 250 kd, a 130-kd protein and a 60-65 kd doublet. These antigens have the following properties: they are localized within the pericentriolar material; their abundance, as judged by centrosome labelling, changes significantly during the cell cycle, the maximum being observed at the pole of the metaphasic spindle; in Taxol-treated cells where the centrosome is no longer acting as a nucleating center, they redistribute at one end of the microtubule arrays in both mitotic and interphasic cells, as expected for nucleating, or capping, proteins. All these properties are compatible with their involvement in microtubule nucleation.

A protein of Mr 80,000 is associated with the nucleolus organizer of human cell lines.

A rabbit serum which had previously been reported to have an immunological affinity for centrosomes of human cell lines was shown also to be specific for the nucleus. Optical and ultrastructural immunolocalization in HeLa cells showed that this specificity is restricted to the fibrillar centre of nucleoli either in untreated or actinomycin D treated interphase cells. In mitotic cells discrete labelling was observed on chromosomes and shown to correspond, on spread metaphase plates, to the short arms of acrocentric chromosomes, i.e. to the nucleolar organizer regions (NORs). Using independent cell fractionation procedures in the human T-lymphoblastic KE 37 cell line and purification of immunoglobulins by affinity to antigens detected by electrophoresis and blotting, a strict correlation between immunoreactive proteins and cytological staining was established. The nucleolar specificity was shown to correspond to a protein with an Mr of 80,000 while the centrosomal specificity corresponded principally to a protein doublet of 60,000-65,000. These antigens share common epitopes as shown by the staining of both NOR and centrosome by immunoglobulins purified by affinity to either type of protein.

Restriction map of Tn7.

Tn7, a transposon of 14 kb, encodes resistance to trimethoprim (Tp) and streptomycin (Sm). A cleavage site map of this transposon for twenty-two different restriction enzymes as determined by comparison of restriction enzyme cleavage patterns of the plasmids ColE1 and ColE1::Tn7 is presented. The precise localization of these sites was facilitated by the use of two deletion derivatives of ColE1::Tn7: pGB2 and ColE1::Tn7 delta 6, and by the use of pOB14 and pOB15 which contain a part of Tn7 cloned into the plasmid pBR322. This map should aid in the study of the structural and genetic organization of this transposon.