The glycosylation pattern of chCE7, an antineuroblastoma chimeric IgG1, was engineered in Chinese hamster ovary cells with tetracycline-regulated expression of beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC). Measurement of the ADCC activity of chCE7 produced at different tetracycline levels showed an optimal range of GnTIII expression for maximal chCE7 in vitro ADCC activity, and this activity correlated with the level of constant region-associated, bisected complex oligosaccharides determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The new optimized variants of chCE7 exhibit substantial ADCC activity and, hence, may be useful for treatment of neuroblastoma. The strategy presented here should be applicable to optimize the ADCC activity of other therapeutic IgGs.

The Schizosaccharomycespombe centromerelinked genes, LYS] and CYHI on chromosome I and TPS13 and RAN) on chromosome II, have been isolated. The genetic order of these markers with respect to their centromeres was determined to establish relative directionality on the genetic and physical maps. Chromosome walking toward the centromeres reveals a group of repetitive sequences that occur only in the centromere regions of chromosomes I and II and at one other specific location in the S. pombe genome, presumably the centromere of chromosome ITH. The major class of large repeated sequence elements is 6.4 kilobases (kb) long (repeat K), portions ofwhich occur at least twice on chromosome II and in several tandemly arranged intact copies at another centromeric location. Repeat K in turn contains groups of smaller repeats. Genetic recombination is strongly suppressed in the centromere II region, which contains at least 30 kb of repeated sequences. Centromeric DNA organization is much more complex in fission yeast than has been described in budding yeast (Saccharomyces cerevisiae), possibly because of the larger more condensed nature of the S. pombe chromosomes.The fission yeast, Schizosaccharomyces pombe, has a genetic complexity roughly equivalent to that of the budding yeast, Saccharomyces cerevisiae. Unlike the common yeast, however, which contains 17 small chromosomes, the DNA of S. pombe is organized into three larger chromosomes, which condense and are visible under light microscopy (1, 2). In S. cerevisiae there appears to be only a single microtubule attachment site per chromosome (3), an observation consistent with the lack of repeated DNA sequences and the overall small size [150-200 base and was propagated in E. coli strain JA221 (recA).Transformations and Isolation of the S. pombe Genes LYS), CYHi, TPS13, and RAN). S. pombe DNA transformations (5) and site-directed integrations of plasmids (20) were performed as described. The LYSJ and TPSJ3 genes (6) were cloned by complementation of appropriate markers (lysl and tpsl3 ura4) in S. pombe with plasmids pSpLYS1 and pSpTPS13 (Fig. 1) from the PCV4 and pFL20 libraries, respectively. These plasmids were recovered in E. coli strain JA226 as described (21). Plasmids or cosmids complementing ranl (9) and cyhl (6) were subsequently identified among those isolated by overlap hybridization screening (22), using hybridization probes isolated from pSpLYS1 and pSpTPS13 DNAs.Abbreviations: bp, base pair(s); kb, kilobase(s). 8253The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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