Understanding and harnessing cellular potency are fundamental in biology and are also critical to the future therapeutic use of stem cells. Transcriptome analysis of these pluripotent cells is a first step towards such goals. Starting with sources that include oocytes, blastocysts, and embryonic and adult stem cells, we obtained 249,200 high-quality EST sequences and clustered them with public sequences to produce an index of approximately 30,000 total mouse genes that includes 977 previously unidentified genes. Analysis of gene expression levels by EST frequency identifies genes that characterize preimplantation embryos, embryonic stem cells, and adult stem cells, thus providing potential markers as well as clues to the functional features of these cells. Principal component analysis identified a set of 88 genes whose average expression levels decrease from oocytes to blastocysts, stem cells, postimplantation embryos, and finally to newborn tissues. This can be a first step towards a possible definition of a molecular scale of cellular potency. The sequences and cDNA clones recovered in this work provide a comprehensive resource for genes functioning in early mouse embryos and stem cells. The nonrestricted community access to the resource can accelerate a wide range of research, particularly in reproductive and regenerative medicine.

We review our studies on the mechanism of somatic hypermutation of immunoglobulin genes. Most experiments were carried out using Ig transgenes. We showed in these experiments that all required cis-acting elements are present within the 10-16 kb of a transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region. Several Ig gene enhancers are permissive for somatic mutation. Association of the enhancer with its natural Ig promoter is not necessary. However, the mutation process seems specific for Ig genes. No mutations were found in housekeeping genes from cells with high levels of somatic hypermutation of their Ig genes. The Ig enhancers may provide the Ig gene specificity. An exception may be the BCL6 gene, which was mutated in human but not in mouse B cells. Transcription of a region is required for its mutability. When the transcriptional promoter located upstream of the variable region is duplicated upstream of the constant region, this region also becomes mutable. This suggests a model in which a mutator factor associates with the RNA polymerase at the promoter, travels with the polymerase during elongation, and causes mutations during polymerase pausing. The DNA repair systems, nucleotide excision repair and DNA mismatch repair, are not required. Our recent data with an artificial substrate of somatic mutation suggest that pausing may be due to secondary structure of the DNA or nascent RNA, and the specific mutations to preferences of the mutator factor.

Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57–65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.

No abstract

No abstract