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The past several years have seen an increasing interest in the peroxisome proliferator-activated receptors (PPARs). These transcriptional factors belong to the superfamily of the steroid/thyroid/retinoid receptors. They are activated by fatty acids or their metabolites as well as by different xenobiotic peroxisome proliferators. These receptors are expressed in both the embryo and the adult organism. They have been implicated in cell proliferation, differentiation and apoptosis. In this review, we will attempt to point out some of the more salient features of this expression pattern during development and the different steps of cell life. The current understanding of how PPARs are involved in some human diseases will also be described.

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Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance. Besides their involvement in the control of nuclear gene expression by activating several peroxisome proliferator-activated receptors (PPARs), peroxisome proliferators influence mitochondrial activity. By analogy with the previous characterization of a mitochondrial T3 receptor (p43), we searched for the presence of a peroxisome proliferator target in the organelle. Using several antisera raised against different domains of PPARs, we demonstrated by Western blotting, immunoprecipitation and electron microscopy experiments, that a 45 kDa protein related to PPARgamma2 (mt-PPAR) is located in the matrix of rat liver mitochondria. In addition, we found that the amounts of mt-PPAR are increased by clofibrate treatment. Moreover, in EMSA experiments mt-PPAR bound to a DR2 sequence located in the mitochondrial D-loop, by forming a complex with p43. Last, studies of tissue-specific expression indicated that mt-PPAR is detected in mitochondria of all tissues tested except the brain in amounts positively related to p43 abundance.

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We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (alpha, beta, and gamma) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPARgamma, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPARgamma was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPARalpha, PPARbeta, and PPARgamma exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPARgamma is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract.

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Using in vitro splicing assays with HeLa cell nuclear extracts, we showed that the HIV-1 pairs of splice sites D1-A2 and D2-A2 are efficiently used in vitro, as compared to the control D1-A2 pair of sites from the E3 transcription unit of human adenovirus-2. The strong efficiency of the two HIV-1 pairs of sites is surprising, as we also showed by primer extension analysis that the branch-site sequence used at the HIV-1 acceptor site A2 is UAGCAGA, with a dominant utilization of the ultimate G as the branched residue. No significant increase of the splicing efficiency was observed upon replacement of the wild-type branch-site sequence by a canonical sequence, in spite of the utilization of an A residue as the branched nucleotide. Results are discussed taking into account the present knowledge on branch-site selection.

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In order to establish the structural features of the cis-elements involved in splicing and in its regulation, we have analyzed a synthetic premessenger RNA, derived from the E3 transcription unit of adenovirus-2 and previously shown to be a good substrate for in vitro splicing. The transcript was probed by enzymatic and chemical methods and we present the structure in solution of the upstream exon and of the 5' part of the intron. This 417 nucleotide long fragment, which overlaps the exon-intron junction harbors the natural 5' splice site D1 and an intronic cryptic site, Dcr1, used when D1 is suppressed. The 5' exon is folded in three stem-loop structures and D1 is located in a free single-stranded region close to the foot of the most stable of these structures (ex1-HP1). The 5' part of the intron also contains a stable hairpin structure (IVS1-hp1), which sequesters Dcr1. The different structural context of the two 5' splice sites may partly explain the selection of D1 and the silencing of Dcr1. We also found a long-range, 20 base-pair, exon-intron interaction, which agrees with the enzymatic and chemical probings and was further demonstrated by the study of the colinear messenger RNA, lacking the intron and of 5' deletion transcripts, lacking the 5' part of the exon. This folding creates a three-branched structure, including IVS1-hp1 and divides the 5' part of the transcript into two domains. Finally, only a few sequences are not involved in folded structures. Free single-stranded fragments are found between the exonic hairpins and at the beginning of the intron and are mostly U-rich. All the structural features of the adenovirus-2 transcript are conserved in adenovirus-5, in spite of 37 nucleotide substitutions.

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To investigate the mechanism by which a 5' splice site (D1) is selected, while a nearby potentially functional site (Dcr1) is silenced, we have studied the importance of the 9-nucleotide sequence of these 5' splice sites for their respective usage. Our model system uses a transcript derived from the early transcription unit 3 of adenovirus-2. Transcripts, harboring an exonic element previously shown to be required for the selection of D1 in the presence of Dcr1, were mutated in the D1 and Dcr1 sequences and assayed for splicing in vitro. We first show that an increased ability of D1 to pair with U1 small nuclear (sn) RNA correlates with an increased accumulation of splicing intermediates, independently of the presence of Dcr1. This variation of efficiency of the first splicing reaction does not significantly affect the overall splicing efficiency except when the potential D1-U1 snRNA hybrid is less than 6 base pairs. Equally, the selector activity of the upstream exon element requires a D1 sequence that is able to form hybrids of 6 base pairs or more with U1 snRNA. This indicates that the cis-acting selector of D1 includes the exonic element (a potential stem-loop structure) and a D1 sequence of sufficient strength.

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To study the mechanism of selection of 5' splice sites, we first analyzed the in vitro time course of appearance of intermediates and products of splicing at a natural and at a cryptic 5' splice site. Our model system was a transcript derived from the early transcription unit 3 of adenovirus-2 harboring a cryptic 5' splice site Dcr1, 74 nucleotides downstream of the natural site D1. When studied in isolation, the two sites have different kinetics of splicing, Dcr1 being spliced markedly more slowly than D1. The upstream exon, shown elsewhere to have a positive effect on the selection of D1, has no influence on these kinetics; thus, it does not affect selection by modifying the kinetics of splicing. Nevertheless, this exon is of crucial importance for the exclusive selection of D1. We demonstrate that the cryptic site is recognized in all cases, but that exons harboring a potential stem-loop structure (HP1) prevent Dcr1 usage. The data suggest that the upstream exon sequences play the role of a cis-acting selector for the natural 5' splice site. The intrinsically rapid and efficient kinetics of splicing at the natural site and the selector function of the exon sequence may result in the exclusive use of the D1 site in the natural context.

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The first intron of the early region 3 from adenovirus type 2 contains a cryptic 5' splice site, Dcr1, 74 nucleotides downstream from the natural site D1. The cryptic site can be activated when the natural site is inactivated by mutagenesis. To investigate the basis for selection between a natural and a cryptic 5' splice site, we searched for cis-acting elements responsible for the exclusive selection of the natural site. We show that both the relative intrinsic strength of the sites and the sequence context affect the selection. A 120-nucleotide segment located at the 3' end of exon 1 enhances splicing at the proximal site D1; in its absence the two sites are used according to their strength. Thus, three cis-acting elements are involved in the silencing of the cryptic site: the sequence of D1, the sequence of Dcr1, and an upstream exonic sequence. We show that the exonic element folds, in solution, into a 113-nucleotide-long stem-loop structure. We propose that this potential stem-loop structure which is located 6 nucleotides upstream of the exon 1-intron junction is responsible for the preferential use of the natural 5' splice site.

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Protamines are low molecular weight, highly basic nuclear proteins involved in the condensation of sperm chromatin. cDNA clones for human protamine 1 and 2 (PRM1 and PRM2) were used for Northern blot experiments with RNA from different human tissues. Protamine transcripts, 0.6 kb and 0.9 kb in lengths for PRM1 and PRM2, respectively, were detected only in testicular RNA. The hybridization signals though did not produce sharp bands but enclosed a minor fraction of significant smaller transcripts. When polysomal RNA fractions were used in the hybridization, these shorter transcripts, 0.45 kb in length for PRM1 and 0.7 kb for PRM2, were specifically enriched. As demonstrated by in situ hybridization on human testis sections, the transcripts of both protamine genes are restricted to the central cell layer of the tubuli seminiferi corresponding to the spatial arrangement of postmeiotic cells. This result indicates that the protamine genes in the human are postmeiotically and haploid expressed. When cDNA clones of both protamines of the boar (BPrm-1 and BPrm-2) were used for hybridization experiments with the testicular RNA of those mammalian species which lack protamine 2 in their spermatozoa, the presence of transcripts for both protamines was detected. It can be assumed that mammals in general are endowed with at least two protamine genes which are both transcribed but are translationally regulated in a species-specific manner.

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Protamines are small, arginine-rich proteins involved in the condensation of sperm chromatin. Using cDNA clones, we have isolated the genes for both human protamines, i.e., protamine 1 (PRM1) and protamine 2 (PRM2), from a human cosmid library. Each of these genes contains a single intron consisting of 91 and 163 bp, respectively. From the 5'-noncoding region of PRM1 664 bp and from the 5'-noncoding region of PRM2 902 bp were determined. Both genes contain typical TATAA and CAAT boxes at conventional distances from the transcription start points, which by using primer extension experiments could be assigned to nucleotides -91 and -110 for PRM1 and PRM2 genes, respectively. Comparison of the 5'-noncoding regions of PRM1 and PRM2 genes reveals 12 different motifs in common, 8 of which are clustered in both genes and could reflect regulatory elements for testis- and spermatid-specific gene expression. Both human genes have been found to be clustered at a distance of 4.8 kb. Comparison of the genomic organization of human and mouse protamine genes revealed greater similarities between the two in the 5'-noncoding region.

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Protamines are sperm-specific proteins that replace histones in the nuclear chromatin of mature spermatozoa. A chromosomal localization of the genes coding for human protamines has been achieved by in situ hybridization. Two cDNA probes of 423 bp and 397 bp containing the entire coding sequence for human protamine 1 (HP1) and human protamine 2 (HP2), respectively, have been used. The genes, called PRM1 and PRM2, have been found, tightly linked, on band 16p13.3. Arguments are given for the existence of these two genes as single copies, PRM1 coding for the unique HP1 protamine and PRM2 coding for a precursor of several proteins belonging to the HP2 family.

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The nuclei of spermatozoa in all mammals examined so far contain P1 protamine. A second protamine variant, protamine P2, has to date been isolated only from human and murine spermatozoa where it represents the major fraction of basic nuclear protein. In order to elucidate the reason for this unusual distribution of the protamine variants among mammals we have investigated the expression of protamine P2 in boar and bull. It can be shown that also in these species protamine 2 is transcribed and translated on low levels. Various mutational events though have altered the primary structure of the protein: In boar, a deletion of 8 aminoacids has removed a sequence motif from the amino-terminus of the molecule, which highly probable is of functional relevance. The bovine sequence, as a consequence of numerous point mutations has accumulated neutral and hydrophobic aminoacids which reduce the affinity of the protamine 2 to DNA.

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To study the assembly of intermediate filaments in vivo we have transfected fibroblast cell lines with the cDNAs coding for keratins 8 and 18 under the control of the promoter of the SV40 early region and followed keratin expression by RNA hybridization, two-dimensional gel electrophoresis, and immunofluorescence analysis. When expressed individually, keratins 8 and 18 failed to polymerize into intermediate filaments but formed granular aggregates of variable size distributed throughout the cytoplasm as seen by staining with specific antibodies. The expression of one of these two keratins did not induce the synthesis of its partner or of any other keratin. Coexpression of the two keratins produced filamentous structures, frequently perinuclear, indicating that the two types of polypeptides were able to assemble into intermediate filaments but could not form the cytoskeleton characteristic of epithelial cells. These results demonstrate that assembly in heterocomplexes stabilizes keratins against cellular degradation, helping to explain why excess pools of simple keratins have never been detected.

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