Heterogeneity of DNA polymerase a [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] has commonly been observed during its purification from a variety of cell systems (1-5). This has hampered the establishment of the physical structure of DNA polymerase a and there is, as yet, no general agreement on the physical properties ofthe core enzyme (6-10). The physiological significance, if any, of this heterogeneity is not clear at this time. Johnston and coworkers (11) showed that mild treatment of a high molecular weight form (200,000-250,000 daltons) of calf thymus DNA polymerase a with 2.5 M urea converts the enzyme to a 155,000-dalton form with the release of a 50,000-to 70,000-dalton protein(s). McKune and Holmes (12) reconstituted the high molecular weight form of polymerase a from the combination of the 155,000-dalton and the 50,000-to 70,000-dalton protein (s). They report that the 50,000-to 70,000-dalton protein(s) enhances the catalytic activity of the polymerase with synthetic polydeoxyribonucleotide templates. Villani et al. (9) have recently shown a similar conversion of DNA polymerase a from Drosophila melanogaster embryos in the presence of2.8 M urea (9). The catalytic activity of the. 148,000-dalton form of DNA polymerase a from Drosophilalembryos (9) and a 156,000-dalton form from rat liver (8) were enhanced when associated with four separable proteins of 54,000-64,000 daltons.We have previously reported the isolationwof a protein (C1) from HeLa cells thattspecifically stimulates the catalytic activity of HeLa DNA polymerase a 20-fold with DNA templateprimers that contain extended single-strand regions (13). In this paper we report the isolation and purification of three forms of DNA polymerase a from synchronized HeLa cells, one ofwhich has equal catalytic activity with activated DNA and with DNA template-primers that contain extended single-strand regions. Two proteins (Cl and C2) that are necessary for its catalytic activity with the latter template-primer are resolved from the DNA polymerase a.MATERIALS AND METHODS Materials. 3H-Labeled deoxyribonucleoside triphosphates were purchased from New England Nuclear. Unlabeled deoxyribonucleoside triphosphates and poly-and oligodeoxyribonucleotide homopolymers were from P-L

Tregs are absolutely required for the maintenance of self tolerance in mouse and man. Major abnormalities in Treg number or function cause rare but fatal syndromes with autoimmune, allergic and inflammatory features. Whether subtle Treg abnormalities contribute to the pathogenesis of sporadic autoimmune, allergic and immunoinflammatory diseases in man remains controversial. Robust methods for identifying and isolating human Tregs in patients and healthy controls are essential if we are to understand their role in these increasingly common diseases. We have outlined below a flow cytometric technique to detect and isolate the entire human Treg population based on expression of CD4, CD25, and CD127. Use of a number of additional antibodies for defining subsets within the Treg compartment is described. For analysis, anti-Foxp3 can be added to the cocktail, but the necessity for fixation and permeabilisation may reduce the signal from other antibodies.

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