A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of ammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA framents distributed along a given genomic region. Terminal differentiation of HL-60 was achieved with retinoic acid and dimethylformamide, as described (17).Transfection. Plasmid pAWTSV (=9 kb), a kind gift of Cesare Vesco (Institute of Cell Biology, Rome), carries the whole simian virus 40 (SV40) genome inserted in the BamHI site of pAT153 (18). Six 10-cm tissue culture plates, containing about 106 COS-1 cells each, were transfected with 10 pg of pAWTSV by the calcium phosphate precipitation technique. After 10 hr of incubation in calcium phosphate solution, cells were extensively washed and fresh medium was added, containing 10 nCi of [14C]thymidine per ml. After 18 hr of incubation, BrdUrd (100 uM final concentration) and[3H]deoxycytidine (1 jAM final concentration) were added.After 1 min of incubation, cells were killed by addition of sodium azide and DNA was extracted as described below.Extrction and Purification of Newly Syntez DNA.Total DNA was extracted, denatured, and size-fractionated by sedimentation through neutral sucrose gradients as described (15).In the experiment involving transfection of plasmid pAW-TSV, DNA (700 /4 final volume) was fractionated on four 5-20%6 (wt/vol) linear sucrose gradients (5 ml each) for 210 min at 200C in a Beckman SW55Ti rotor at 55 krpm; 24 fractions of 200 j4 were collected.In the experiment with synchronized HL-60 cells, DNA (2 ml final volume) was fractionated on eight 5-30% sucrose Abbreviations: DHFR, dihydrofolate reductase; SV40, simian virus 40. tTo whom reprint requests should be addressed. 7119The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
In eukaryotic cells, messenger RNAs are formed by extensive post-transcriptional processing of primary transcripts, assembled with a large number of proteins and processing factors in ribonucleoprotein complexes. The protein moiety of these complexes mainly constitutes a class of about 20 major polypeptides called heterogeneous nuclear ribonucleoproteins or hnRNPs. The function and the mechanism of action of hnRNPs is still not fully understood, but the identification of RNA binding domains and RNA binding specificities, and the development of new functional assays, has stimulated interest in them. In contrast to previous models that hypothesised a mere structural (histone-like) function, a more diversified and dynamic role for these proteins is now emerging. In fact, they can be viewed as a subset of the trans-acting pre-mRNA maturation factors. They might actively participate in post-transcriptional events such as regulated splicing and mRNA export. Moreover, recent data suggest an involvement of some of these proteins in molecular diseases. Here we present an overview of the most relevant properties of hnRNPs and discuss some emerging ideas on their roles.
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...
DNA replication in mammalian cells occurs in discrete nuclear foci called ‘replication factories’. Here we show that DNA ligase I, the main DNA ligase activity in proliferating cells, associates with the factories during S phase but displays a diffuse nucleoplasmic distribution in non‐S phase nuclei. Immunolocalization analysis of both chloramphenicol acetyltransferase (CAT)‐DNA ligase I fusion proteins and epitope tagged DNA ligase I mutants allowed the identification of a 13 amino acid functional nuclear localization signal (NLS) located in the N‐terminal regulatory domain of the protein. Furthermore, the NLS is immediately preceded by a 115 amino acid region required for the association of the enzyme with the replication factories. We propose that in vivo the activity of DNA ligase I could be modulated through the control of its sub‐nuclear compartmentalization.
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