The mouse U2af1-rs1(SP2) gene, which was cloned by a two-dimensional genome scanning method, is expressed exclusively from the paternally inherited chromosome. This gene has significant similarity to U2AF and located in chromosome 11, of which maternal duplication/paternal deficiency results in a small body. In this report, we cloned genomic U2af1-rs1(SP2) and found its promoter was methylated in a maternal-allele-specific manner. This allelic methylation was not established in parental gametes, but established between 1.5 d.p.c. and 12.5 d.p.c. on the contrary, the allele-specific expression occurred in the two-cell stage when transcription initiates. Absence of the methylation of the upstream region in this stage indicates that methylation is not necessary for inactivation of the expression.
R2Bm is a non-long-terminal-repeat (non-LTR) retrotransposon that was identified at a specific target site in the 28S rRNA genes of the silkworm, Bombyx mori. Although in vitro analysis has revealed that the 3' end of R2Bm is integrated into the target site by means of target-primed reverse transcription (TPRT), the mechanism of the 5' end integration is not well understood. We established a novel in vivo system to assay the insertion mechanism of R2Bm using a cultured cell line, C65, and a baculovirus, AcNPV, as host and vector, respectively. The 3' end of R2Bm integrated at the target site in the rRNA genes of C65 cells when an AcNPV containing both the full-length 3' UTR and the entire open reading frame (ORF) of R2Bm was introduced while the 5' end integration was incorrect. The 5' end of R2Bm was integrated, however, when the 28S gene sequence upstream of the R2Bm target site was added to the R2Bm sequence. Thus, in our assay, homologous sequences were likely essential for the successful integration of the entire R2Bm into the host cell genome. We also demonstrated that the failure to integrate caused by a frame-shifted ORF was rescued by co-infection with a helper virus that contained only the R2Bm ORF. This indicates that R2 retrotransposition can be complemented in trans. These findings suggest that the host's mechanism for DNA repair may be necessary for the integration of the 5' end of R2Bm and that R2Bm protein may only have the ability to integrate the 3' end of the element by TPRT.
A 13-kb fragment of the rat aldolase C gene contains sufficient information for gene expression. Transgenic mice carrying the 13-kb fragment showed restoration of chromatin structure and tissue-specific, copy number-dependent expression. To localize the regulatory elements responsible for restoring chromatin structure, several mutated constructs were used to produce transgenic mice. Three activities were examined: recreation of DNase hypersensitive sites, restoration of methylation status, and copy number-dependent expression. Deletions of the 3'-flanking region did not affect those activities. Deletion of seven introns affected the mRNA levels but not the restoration of the chromatin structure. The insertion of the LacZ gene into the first exon of the transgene interfered with both the restoration of the chromatin structure and the copy number-dependent expression in transgenic mice. DNase I footprinting assays revealed that brain-specific factors bind to the sequence disrupted by the LacZ insertion. These results suggest that the sequence in the first exon is essential for restoring the chromatin structure of the rat aldolase C gene.
A clone of a DNA-mediated mobile element (transposon) corresponding to a mariner-like element (MLE) was obtained by carrying out the polymerase chain reaction with genomic DNA of Bombyx mori using a Hyalophora cecropia MLE sequence as a primer. This clone had a size of about 4.2 kb and, after sequencing, was found to contain an RNA-mediated, shorter retrotransposon named L1Bm, which was in turn integrated with a much longer retrotransposon named BMC1. Thus, the mobile elements made a novel tripartite structure. The BMC1 and L1Bm moieties of the composite structure each contained a 63-bp conserved sequence which was subsequently found to be highly conserved in all BMC1 and L1Bm elements registered so far. We propose that the 63-bp stretch may be a recognition site for a retrotransposition mechanism conducted by a reverse transcriptase and an endonuclease complex. On the basis of this inference, we propose a model that predicts how different types of BMC1 and L1Bm elements are dispersed in the genome. In addition, a phylogenetic tree made from the current and extant BMC1 and L1Bm sequences indicated that these elements can be classified into Subfamilies I and II.
In order to understand the molecular mechanisms of the temporal and spatial differences of gene expression in higher organisms, rat aldolase A gene carrying two distinct promoters was introduced into fertilized eggs and the resulting transgenic mice were analyzed. The transgene expression is tissue-specific and is developmentally regulated. In addition, the expression is regulated in a copy-dependent manner irrespective of where the transgene is integrated, suggesting that a mechanism excluding the effect of the integration site exists within the transgene itself. To explore the conformational change of this gene in the genome, the DNase I hypersensitive sites of the gene were examined. Three sites (DHS-1,2, and 3) were identified upstream and downstream of the gene and these sites were retained in the transgene as well as in the gene observed endogenously.
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