A previously described human DNA fragment which is replicated early in S-phase of HL-60 cell DNA (C. (7,19). Elucidation of the underlying mechanisms will constitute a major step in understanding the nuclear dynamics of the process, cell growth, and differentiation.We have previously described the isolation and cloning of a human DNA fragment (pB48; 1,560 bp) that belongs to the fraction of DNA replicated at the onset of S-phase in synchronized HL-60 cells (35). Analysis of this fragment indicated that it derives from an expressed region of the human genome, since in Northern (RNA) blot experiments, it hybridized to two mRNAs; moreover, it contains a binding site for the transcription factor USF/MLTF and an active promoter overlapping a putative CpG island (8,13). This finding is consistent with the notion that actively transcribed regions of the human genome are preferentially replicated early in S-phase (7,14,15,19,36). In this context, elucidation of the structural features of an early-replicating DNA and identification of the encoded genes could help us to understand the biological significance of this phenomenon. pB48 DNA represents an opportunity to explore this approach. In this work, we have extended our studies to a chromosomal region of 13.7 kb encompassing pB48, which was mapped by in situ hybridization to the subtelomeric G-negative band p13.3 of chromosome 19. The 13.7-kb region was found to code for several transcripts and to contain at least two closely spaced and nonoverlapping genes, one of which corresponded to a B-type nuclear lamin. Lamin proteins line the inner side of the nuclear envelope * Corresponding author.with a meshwork of filamentous structures and are thought to play a role in nuclear stability, chromatin structure, and gene expression (12,23,29). Several reports indicate that lamin B proteins are preferentially synthesized during S-phase (3, 9), in accord with the notion that the nuclear lamina may be related to changes in the dynamics of the nuclear envelope or chromatin (12). Our findings tend to support the model relating early replication to actively transcribed genes.
MATERIALS AND METHODSCells, cell lines, and probes. Peripheral blood lymphocytes were obtained from healthy donors and prepared according to the Ficoll-metrizoate method as described elsewhere (10). Lymphocytes were grown in RPMI 1640 (GIBCO), 10% fetal calf serum, 50 ,g of gentamicin per ml, 2 mM L-glutamine, and 50 p,g of phytohemagglutinin per ml. Human primary fibroblasts were cultured in Dulbecco modified Eagle medium (DMEM; GIBCO) supplemented with 15% fetal calf serum, 50 jig of gentamicin per ml, and 2 mM L-glutamine.To obtain resting cells, confluent fibroblasts were grown for 7 days in DMEM supplemented with 0.25% fetal calf serum. Human epidermoid carcinoma A-431 cells were obtained from the American Type Culture Collection and maintained in DMEM (GIBCO) supplemented with 10% fetal calf serum.The rDNA probe (plasmid LS6BB) (32) was kindly provided by G. N. Ranzani. The human 3-globin DNA probe was pr...