c-myc activates RCC1 gene expression through E-box elements.

Proto-oncogene c-myc is implicated in proliferation of mammalian cells. Although c-Myc protein has been demonstrated to function as a transcription factor recognizing an E-box (CACGTG) element, few c-myc-regulated genes have been identified and the specific role of c-myc is still unclear. RCC1 is necessary for mammalian cells to proliferate. Four CACGTG elements exist within 1.3 kb downstream of the major transcription start site for the human RCC1 gene in HeLa cells. Stimulation of HeLa cells with serum increased c-myc expression and RCC1 expression. Therefore the relationship between the expression of RCC1 and c-myc was investigated. Rat 3Y1 cells overexpressing c-myc contained about twice as much RCC1 mRNA as control cells. When a chimeric protein comprised of c-myc and the estrogen binding domain of estrogen receptor was activated by addition of 4-hydroxytamoxifen (OHT), expression of RCC1 mRNA increased twofold. To examine whether c-Myc functions through the CACGTG elements, a DNA fragment of RCC1 intron 4, exon 5 and part of intron 5 was joined to firefly luciferase cDNA to construct a reporter plasmid. In transient expression experiments using HeLa cells, co-transfection with c-myc stimulated the luciferase activity up to 2.5-fold in a dose-dependent manner. When the CACGTG elements in the reporter plasmid were destroyed, stimulation by c-myc was not observed. The four CACGTG elements did not contribute equally to the stimulation by c-myc. Gel retardation experiments suggest that c-Myc with Max binds to the CACGTG elements in the context of the RCC1 gene sequence in vitro. These results indicate that c-Myc can regulate expression of RCC1 through the E-box elements.

Sequence-tagged NotI sites of human chromosome 21: sequence analysis and mapping.

We isolated and analyzed 19 NotI site-containing clones specific for human chromosome 21. The overall process consisted of selective isolation of NotI site-containing clones from flow-sorted chromosome 21 libraries, selection of independent clones by their restriction patterns and nucleotide sequences, and assignment of the clones to chromosome 21. Sequence analysis showed that the regions around the NotI sites had features typical of CpG islands, such as extremely high GC content, high-frequency appearance of CpG dinucleotide sequences, and lack of methylation of these CpGs. PCR conditions for these extremely G + C-rich templates were optimized to establish these NotI sites as sequence-tagged sites.

An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: isolation of SfiI linking clones from a chromosome 21-specific library.

We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking fragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.

Structure of the rat alpha 2-macroglobulin-coding gene.

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase protein, i.e., produced upon tissue inflammation. Genomic DNA clones covering the entire sequence of the alpha 2M gene were isolated and characterized by restriction mapping. Southern blotting and (partial) DNA sequencing. The rat alpha 2M gene is approx. 50 kb in length and consists of 36 exons ranging in size from 21 to 229 bp. Two functional domains, a bait region and a thiol ester site, are encoded by the exon 18 and 24, respectively. Several possible regulatory signals such as a TPA-inducible enhancer core, an identifier sequence, purine-pyrimidine alternative stretches and viral enhancer core sequences were identified. Several genomic DNA clones which cross-hybridized with the alpha 2M cDNA probe were also identified. Sequence analysis showed that they possessed sequences identical to a part of the rat alpha 1-inhibitor III cDNA and that they had a strikingly similar exon organization to the alpha 2M gene.