Ig V region hypermutation in B cell hybrids mimics in vivo mutation and allows for isolation of clonal variants.

In order to investigate the regulation of Ig hypermutation, we have established a cell culture system in which reversion of a V region stop codon in a stably transfected Ig gene permits the quantitation of mutation rates by fluctuation analysis. Transfected heavy chain V regions associated with the mu constant region undergo low rates of mutation in the NSO plasmacytoma cell line and a moderate rate of mutation in the 18.81 pre-B cell line. Most of the hybrids created by fusing these two cell lines resembled the non-permissive NSO cell line, though a few hybrids had constitutive V region mutation rates that were even higher than 18.81 and similar to the high rates of mutation that occur in vivo (Green, N. S., Rabinowitz, J. L., Zhu, M., Kobrin, B. J. and Scharff, M. D. (1995) Proc. Nat. Acad. Sci. (USA) 92, 6304 6308). Characterization of these hybrids now demonstrates that the transfected genes were integrated outside of the Ig locus. Mutation was due to multiple single base pair replacements in the V region and not the C region, was ongoing and often arose in hot spot motifs described by V region hypermutation in vivo. Subcloning of unstable hybrids allowed for the isolation of highly related clones with 44-70-fold different mutation rates. These results suggest that V region hypermutation in this mode in vitro systems is under both positive and negative regulation.

Isolation and characterization of human casein kinase I epsilon (CKI), a novel member of the CKI gene family.

The casein kinase I (CKI) gene family is a rapidly enlarging group whose members have been implicated in the control of cytoplasmic and nuclear processes, including DNA replication and repair. We report here the cloning and characterization of a novel isoform of CKI from a human placental cDNA library. The cDNA for this isoform, hCKI epsilon, predicts a basic polypeptide of 416 amino acids and a molecular mass of 47.3 kDa. It encodes a core kinase domain of 285 amino acids and a carboxyl-terminal tail of 123 amino acids. The kinase domain is 53-98% identical to the kinase domains of other CKI family members and is most closely related to the delta isoform. Localization of the hCKI epsilon gene to chromosome 22q12-13 and the hCKI delta gene to chromosome 17q25 confirms that these are distinct genes in the CKI family. Northern blot analysis shows that hCKI epsilon is expressed in multiple human cell lines. Recombinant hCKI epsilon is an active enzyme that phosphorylates known CKI substrates including a CKI-specific peptide substrate and is inhibited by CKI-7, a CKI-specific inhibitor. A budding yeast isoform of CKI, HRR25, has been implicated in DNA repair responses. Expression of hCKI epsilon but not hCKI alpha rescued the slow-growth phenotype of a Saccharomyces cerevisiae strain with a deletion of HRR25. Human CKI epsilon is a novel CKI isoform with properties that overlap those of previously described CKI isoforms.

Genes encoding giant danio and golden shiner ependymin.

Ependymin (EPN) is a brain glycoprotein that functions as a neurotrophic factor in optic nerve regeneration and long-term memory consolidation in goldfish. To date, true epn genes have been characterized in one order of teleost fish, Cypriniformes. In the study presented here, polymerase chain reactions were used to analyze the complete epn genes, gd (1480 bp), and sh (2071 bp), from Cypriniformes giant danio and shiner, respectively. Southern hybridizations demonstrated the existence of one copy of each gene per corresponding haploid genome. Each gene was found to contain six exons and five introns. Gene gd encodes a predicted 218-amino acid (aa) protein GD 93 percent conserved to goldfish EPN, while sh encodes a predicted 214-aa protein SH 91 percent homologous to goldfish. Evidence is presented classifying proteins previously termed "EPNs" into two major categories: true EPNs and non-EPN cerebrospinal fluid glycoproteins. Proteins GD and SH contain all the hallmark, features of true EPNs.

Comparative genomic hybridization and its application to Wilms' tumorigenesis.

Eighty sporadic Wilms' tumor samples were analyzed by comparative genomic hybridization (CGH) to identify chromosomal regions involved in the etiology of the disease. Twenty percent of the samples showed chromosomal gains or losses. The majority of chromosomal gains and losses were similar to those identified through molecular and cytogenetic studies. Gains were observed on chromosomes 1q, 7q, 8, and 12, whereas losses were found on chromosomes 1p, 4p, 4q, 7p, 16q, 18q, 21q, and 22q. Other genetic aberrations identified in this study included deletions of chromosomes 5p and 15q, as well as gains of discrete loci on chromosomes 3p and 3q. These latter regions have not been previously implicated in Wilms' tumorigenesis and may contain novel genes relevant to the development and/or progression of this disease.