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In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.

We have previously described HAP, a novel hnRNP protein that is identical both to SAF-B, a component of the nuclear scaffold, and to HET, a transcriptional regulator of the gene for heat shock protein 27. After heat shock, HAP is recruited to a few nuclear bodies. Here we report the characterisation of these bodies, which are distinct from other nuclear components such as coiled bodies and speckles. The formation of HAP bodies is part of a general cell response to stress agents, such as heat shock and cadmium sulfate, which also affect the distribution of hnRNP protein M. Electron microscopy demonstrates that in untreated cells, similar to other hnRNP proteins, HAP is associated to perichromatin fibrils. Instead, in heat shocked cells the protein is preferentially associated to clusters of perichromatin granules, which correspond to the HAP bodies observed in confocal microscopy. Inside such clusters, perichromatin granules eventually merge into a highly packaged 'core'. HAP and hnRNP M mark different districts of these structures. HAP is associated to perichromatin granules surrounding the core, while hnRNP M is mostly detected within the core. BrU incorporation experiments demonstrate that no transcription occurs within the stress-induced clusters of perichromatin granules, which are depots for RNAs synthesised both before and after heat shock.

A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.

The hnRNP A1 transcript has a relatively short 5'- untranslated region (UTR) starting with a pyrimidine tract similar to that of mRNAs encoded by the TOP [terminal oligo(pyrimidine)] genes in vertebrates. Such genes code for ribosomal proteins and for other proteins directly or indirectly involved in the production and function of the translation apparatus. As expected from the role of the pyrimidine tract in the translational regulation of TOP mRNAs, the A1 mRNA is more efficiently loaded onto polysomes in growing than in resting cells. On the other hand, a less stringent regulation with respect to that of other TOP mRNAs is observed, partially due to the presence of multiple transcription start sites within the pyrimidine tract, where transcripts with shorter TOP sequences are less sensitive to regulation. Thus, from the point of view of structural features and translation behaviour the A1 mRNA can be included in the class of TOP genes, suggesting a possible role of A1 in translation. Interestingly, a TOP-like behaviour was observed for hnRNP I mRNA but not for hnRNP C1/C2 and A2/B1 mRNAs, indicating the existence of two classes of hnRNPs with different translational regulation.

hnRNP I, also referred to as polypyrimidine tract binding protein, is one of the proteins associated with nascent pre-mRNA in the heterogeneous nuclear ribonucleoprotein (hnRNP) complexes. As for all karyophilic proteins, the nuclear import of hnRNP proteins requires specific sequence determinants that in many instances differ from the canonical import signal. In order to identify the sequences responsible for the nuclear localization, various hnRNP I portions were fused to a reporter protein (bacterial chloramphenicol acetyl transferase) and used in transient transfection assay. By this approach we identified a 60-amino-acid sequence located at the amino terminus of hnRNP I (designated NLD-I) that is both necessary and sufficient for nuclear localization. NLD-I represents a novel bipartite type of nuclear localization signal that bears no resemblance to other nuclear localization determinants so far identified in hnRNP proteins.

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.

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.

hnRNP A1 (34 kDa) is an RNA binding protein consisting of two tandemly arranged RNA binding domains C-terminally linked to a glycine-rich auxiliary domain (2 x RBD-Gly). A1 belongs to the set of polypeptides that bind nascent hnRNA in the nucleus to form the so called hnRNP complexes. These complexes seem to be involved both in pre-mRNA processing and in the nuclear export of mRNA. In fact A1, along with other hnRNP proteins, is exported from the nucleus probably bound to mRNA and is immediately re-imported. A1 nuclear re-import, which requires active transcription, is not mediated by a canonical nuclear localisation signal (NLS). To identify the determinants of A1 subcellular localisation we developed an expression vector for studying the localisation, in transiently transfected cells, of the different structural motifs of A1 fused to a small reporter protein (chloramphenicol acetyltransferase, CAT; 26 kDa). We demonstrate that a 30 amino acid sequence in the glycine-rich domain (YNDFGNYNNQSSNFGPMKGGNFGGRSSGPY), which bears no resemblance to canonical NLS, is necessary and sufficient to target the protein to the nucleus. Our data suggest that this targeting sequence might act by mediating the interaction of A1 with a NLS-containing nuclear import complex. On the other hand, the nuclear export of A1 requires at least one RNA binding domain in accord with the hypothesis that A1 exits from the nucleus bound to mRNA. We propose a mechanism for the nucleo-cytoplasmic shuttling of A1 that envisages a specific role for the different structural domains and can explain the dependence of nuclear import from active transcription.

A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian 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 fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one. This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.

A previously described human DNA fragment which is replicated early in S-phase of HL-60 cell DNA (C. Tribioli, G. Biamonti, M. Giacca, M. Colonna, S. Riva, and A. Falaschi, Nucleic Acids Res. 15:10211-10232, 1987) was used to screen a genomic library in lambda Ch28. A clone which contained a 13.7-kb insert (L30E) found to code for several transcripts was isolated. The transcription of L30E DNA exhibited a complex pattern and a tissue-specific and proliferation-dependent type of regulation. The data were consistent with two tandemly arranged transcription units, the 3' end of one separated from the 5' end of the other by a sequence of about 600 bp containing an active promoter. The isolation and sequencing of L30E-specific cDNAs permitted identification of two genes, one of which encoded a B-type human lamin (analogous to mouse lamin B2). L30E DNA was mapped by in situ hybridization at the G-negative subtelomeric band p13.3 of chromosome 19. Interestingly, in synchronized HL-60 cells, L30E DNA is replicated in the first minute of S-phase. Replication of the lamin gene early in S-phase may reflect a coupling between early replication and transcription of genes for S-phase-specific proteins such as lamins.

A single-copy 13.7 kb human DNA region (L30E) located on Ch. 19 p13.3 contains an origin of DNA replication in myeloid HL-60 cells. The origin was localized, by means of quantitative PCR within approximately 3000 bp, in a highly transcribed region containing at least two closely spaced genes with the same polarity of transcription, one encoding lamin B2 and the other an unidentified protein. The origin region overlaps an undermethylated "CpG island" at the 5'-end of the second transcription unit. A binding site (CACGTG) for basic helix-loop-helix (bHLH) DNA binding proteins such as USF/MLTF or MYC-MAX was located by DNase I footprinting analysis in the promoter of the second gene. DMSO differentiation of HL-60 cells, that completely shuts off replication, also drastically reduces the transcription of L30E region. On the other hand such treatment does not modify the methylation pattern of the CpG island and does not abolish the DNase I protection of the bHLH binding site.