The gene for a novel human lamin maps at a highly transcribed locus of chromosome 19 which replicates at the onset of S-phase.

Abstract

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...

Fine mapping of a replication origin of human DNA.

“…This region was shown to contain a strong promoter for the downstream transcription unit; a key feature ofthis promoter is the presence of a basic helix-loop-helix protein binding motif that was shown to bind in vitro to the USF/MLTF protein (23); interestingly, such motif also fits the binding consensus for the Myc/Max complex (24), whose function in regulating cell proliferation is suggested by several studies (25) [incidentally, the c-myc gene domain is amplified in HL-60 cells (26)]. An evolutionary conserved (A+T)-rich region (22) can be envisaged in the proximity of the origin, as is the case for well-defined replication origins of different organisms (27)(28)(29).…”

“…In the previous years, we demonstrated that a 1560-bp DNA fragment [B48, selected from a mini-library of HL-60 cell DNA containing fragments derived from the replicons activated at the beginning of the S phase (16)] was in fact replicated in the very first minute of the S phase in HL-60 synchronized cells (22) and, thus, presumably very close to, or even coincident with, an origin. We sequenced 13.7 kb around the initial isolate, and we observed that this genomic region, mapping on band p13.3 of chromosome 19, contains the final portion of the lamin B2 gene and, downstream of it, another gene (ppvl), transcribed in the same direction and coding for a still unidentified product [ Fig.…”

“…The pair of primers (B48BIS and B48TER) flanking the one giving the strongest signal (B48, Fig. 2B) confine the replication origin within a 474-bp region roughly corresponding to the nontranscribed spacer between two transcription units arranged head to tail (22). This region was shown to contain a strong promoter for the downstream transcription unit; a key feature ofthis promoter is the presence of a basic helix-loop-helix protein binding motif that was shown to bind in vitro to the USF/MLTF protein (23); interestingly, such motif also fits the binding consensus for the Myc/Max complex (24), whose function in regulating cell proliferation is suggested by several studies (25) [incidentally, the c-myc gene domain is amplified in HL-60 cells (26)].…”

Both lamin A and lamin C mutations cause lamina instability as well as loss of internal nuclear lamin organization

“…Therefore, it is suggested that lamins play a key role in providing structural support to the nuclear membrane. Next to the ubiquitously expressed B-type lamins, lamins B1 and B2 [3,4], most highly differentiated cells express A-type lamins. These proteins, lamin A, lamin AD10, and lamin C, are encoded by the A-type lamin (LMNA) gene [5,6].…”

The Nuclear Envelope: An Intriguing Focal Point for Neurogenetic Disease

“…Human LMNB1 encodes lamin B1 and LMNB2 encodes lamin B2. These "B-type" lamins are expressed in all or most somatic cells [19,20]. All of these proteins, except lamin C, are posttranslationally modified by farnesylation and carboxymethylation at a carboxyl-terminal cysteine-aliphatic-aliphatic-any ("CAAX") amino acid motif.…”