Search

The carboxy-terminal region of dystrophin has previously been shown to interact directly with alpha1 syntrophin, a cytoplasmic component of the dystrophin-glycoprotein complex, by in vitro biochemical studies such as overlay assay or immunoprecipitation. Using the two-hybrid system, we have isolated from a human heart cDNA library the entire coding sequence of human alpha1 syntrophin, therefore confirming for the first time this interaction via an in vivo approach. In addition, we have reduced the interaction domain to the distal half of alpha1 syntrophin.

Glucose catabolism induces the expression of the L-type pyruvate kinase (L-PK) gene through the glucose response element (GIRE). The metabolic pathway used by glucose after its phosphorylation to glucose 6-phosphate by glucokinase to induce L-PK gene expression in hepatocytes remains unknown. The sugar alcohol xylitol is metabolized to xylulose 5-phosphate, an intermediate of the nonoxidative branch of the pentose phosphate pathway. In this study, we demonstrated that xylitol at low concentration (O.5 mM) induced the expression of the L-PK/CAT construct in glucose-responsive mhAT3F hepatoma cells at the same level as 20 mM glucose, while it did not affect intracellular concentration of glucose 6-phosphate significantly. The effect of xylitol on the induction of the L-PK gene expression was noncumulative with that of glucose since 20 mM glucose plus 5 mM xylitol induced the expression of the L-PK/CAT construct similarly to 20 mM glucose alone. In hepatocytes in primary culture, 5 mM xylitol induced accumulation of the L-PK mRNA even in the absence of insulin. Furthermore, the response to xylitol as well as glucose required the presence of a functional GIRE. It can be assumed from these results that glucose induces the expression of the L-PK gene through the nonoxidative branch of the pentose phosphate pathway. The effect of xylitol at low concentration suggests that the glucose signal to the transcriptional machinery is mediated by xylulose 5-phosphate.

Expression in mice of transgenes directed by regulatory regions of the rat aldolase B gene requires the presence of a B element located in the first intron, while constructs devoid of this intronic enhancer are silent. Histo- and immunochemical staining of transgenic tissue sections showed that the longer transgene was expressed in the proximal tubular cells of the kidney, enterocytes located in small intestine villi and liver parenchymal cells. In the liver, a maximal expression was observed in perivenous hepatocytes, while the transgene was weakly active in periportal hepatocytes, which reproduced the pattern of functional zonation already reported for other glycolytic and gluconeogenic genes in the liver. We also established that the transgene retained the necessary elements for a correct chronological expression during development but was lacking elements necessary for activation by high carbohydrate diet. Instead, transgene expression was paradoxically stimulated in fasted animals, suggesting that the endogenous gene, which must be active under both glycolytic and gluconeogenic conditions, could possess distinct elements activating it in fasted as well as in carbohydrate-fed animals; the former element might be conserved in the transgene and the latter one might be lost.

Medecin, chercheur a l'Inserm, membre du Comite consultatif national d'ethique, Axel Kahn fait le point sur la dimension ethique des problemes que pose l'avenement du genie genetique, en particulier dans le champ de la medecine humaine. En qualite d'ancien president de la Commission du genie biomoleculaire instituee aupres du ministre de l'Agriculture, il est aussi un acteur privilegie des debats relatifs au developpement des biotechnologies dans le secteur agricole. L'actualite confere une singuliere resonance a cette reflexion en forme de plaidoyer pour une recherche plus respectueuse de la relation entre l'Homme et la Nature.

The human aldolase A gene is expressed in several tissues through the use of three alternative promoters. The activity of one of the promoters, pM, is restricted to skeletal muscle. We reported previously that a proximal 280 bp pM fragment confers tissue-specific expression to a CAT reporter gene in transgenic mice. This small regulatory region directs expression to muscle composed mainly of fast-twitch fibers. Here we show that a minimal promoter fragment from base-pairs −164 to +45 is sufficient to highly active pM during myoblast differentiation in cell culture and demonstrate that two DNA elements play a major role in this activation. These elements consist of a binding site (M1) for unknown ubiquitous proteins and an overlapping binding site for MEF2 and NF1 families of transcription factors. The NF1 factor constitute the main binding activity on the MEF2/NF1 site and, interestingly, some of the DNA-protein complexes that form with muscle nuclear extracts on the NF1 element differ from those that form with non-muscular extracts.

Transcription of hepatocyte-specific genes requires the interaction of their regulatory regions with several nuclear factors. Among them is the hepatocyte nuclear factor 3 (HNF3) family, composed of the HNF3 alpha, HNF3 beta, and HNF3 gamma proteins, which are expressed in the liver and have very similar fork head DNA binding domains. The regulatory regions of numerous hepatocyte-specific genes contain HNF3 binding sites. We examined the role of HNF3 proteins in the liver-specific phenotype by turning off the HNF3 activity in well-differentiated mhAT3F hepatoma cells. Cells were stably transfected with a vector allowing the synthesis of an HNF3 beta fragment consisting of the fork head DNA binding domain without the transactivating amino- and carboxy-terminal domains. The truncated protein was located in the nuclei of cultured hepatoma cells and competed with endogenous HNF3 proteins for binding to cognate DNA sites. Overproduction of this truncated protein, lacking any transactivating activity, induced a dramatic decrease in the expression of liver-specific genes, including those for albumin, transthyretin, transferrin, phosphoenolpyruvate carboxykinase, and aldolase B, whereas the expression of the L-type pyruvate kinase gene, containing no HNF3 binding sites, was unaltered. Neither were the concentrations of various liver-specific transcription factors (HNF3, HNF1, HNF4, and C/EBP alpha) affected. In partial revertants, with a lower ratio of truncated to full-length endogenous HNF3 proteins, previously extinguished genes were re-expressed. Thus, the transactivating domains of HNF3 proteins are needed for the proper expression of a set of liver-specific genes but not for expression of the genes encoding transcription factors found in differentiated hepatocytes.

The rat L-type pyruvate kinase gene is transcribed either from promoter L in the liver or promoter L' in erythroid cells. We have now cloned and functionally characterized an erythroid-specific enhancer, mapped in the fetal liver as hypersensitive site B (HSSB) at 3.7 kilobases upstream from the promoter L'. Protein-DNA interactions were examined in the 200-base pair core of the site by in vivo footprinting experiments. In the fetal liver, footprints were revealed at multiple GATA and CACC/GT motifs, whose association is the hallmark of erythroid-specific regulatory sequences. Functional analysis of the HSSB element in transgenic mice revealed properties of a cell-restricted enhancer. Indeed, this element was able to activate the linked ubiquitous herpes simplex virus thymidine kinase promoter in erythroid tissues. The activation was also observed in a variety of nonerythroid tissues known to synthesize GATA-binding factors. In the context of L'-PK transgenes, HSSB was not needed for an erythroid-specific activation of the L' promoter, while it was required to stimulate the L' promoter activity to a proper level. Finally, HSSB cannot be replaced by strong ubiquitous viral or cellular enhancers, suggesting a preferential interaction of the HSSB region with the L' promoter.

Renal expression of the aldolase B isoenzyme and transcription factors previously shown to regulate the aldolase B gene promoter in the liver were analyzed in whole kidney, microdissected tubules, and the two PKSV-PCT and PKSV-PR proximal tubule cell lines derived from transgenic mice. Aldolase B gene expression appeared restricted to the proximal tubule, the site where HNF1 alpha, HNF1 beta, C/EBP alpha, and DBP transcripts were also abundant. Compared to the liver, another organ synthesizing aldolase B, proximal tubules from the kidney were characterized by the absence of HNF3 and the presence of higher ratio of HNF1 beta/HNF1 alpha transcripts. The same features were conserved in both PKSV-PCT and PKSV-PR proximal tubule cell lines. Transactivation experiments in PKSV-PCT cultured cells showed that HNF1 alpha, C/EBP alpha, and DBP behave as transactivators of the 190-bp aldolase B gene promoter, and that HNF1 beta had a low transactivating efficiency. HNF1 beta, as well as HNF3, antagonized the HNF1 alpha-dependent transactivation of the aldolase B promoter. The fact that both HNF1 beta and HNF3 factors play similar negative roles by competitively binding close to or on the HNF1 site could suggest that, in proximal tubule renal cells, HNF1 beta has the same attenuator effect on the aldolase B gene promoter as HNF3 in hepatocytes. Thus, these results indicate that such models of established renal tubule cell lines, which have conserved the same features of parental cells, represent valuable tools for studies of the regulation of genes expressed in proximal tubules of the kidney.

International audience; Aldolase C mRNA is detected by Northern blot in all fetal tissues in rat; it is very abundant in the adult brain and undetectable in the other adult tissues. However, reverse transcriptase polymerase chain reaction amplification indicates that this gene is not totally repressed in these tissues. A DNase-I hypersensitivity site located in a 115-base pair proximal promoter fragment is detectable in the brain as well as in other adult tissues. Two MspI/HpaII restriction sites located at -3800 and -450 base pairs are demethylated in the brain and totally or partially methylated in other tissues. In transgenic mice, a 12.5-kilobase genomic fragment is strongly and tissue specifically expressed in different lines, with conservation of a methylation pattern similar to that of the endogenous gene. A chloramphenicol acetyltransferase gene directed by either 800 or 115 base pairs of aldolase C 5'-flanking sequences is tissue specifically expressed in transgenic mice, but the level of expression is very low. This level is greatly increased when the transgene consists of a chloramphenicol acetyltransferase hybrid gene directed by 5.5 kilobases of aldolase C 5'-flanking sequences. We propose therefore that the chromatin structure around the aldolase C promoter is accessible in fetal tissues, then remains open in the adult brain, where the gene is very active, as well as in tissues in which it is practically inactive. The specificity of expression in the brain is conferred by a short 115-base pair proximal promoter fragment that needs more upstream sequences to be fully active.

The application of the polymerase chain reaction technique (PCR) to the study of gene expression, variously referred in the literature to as cDNA-PCR reverse transcription-PCR (RT-PCR) (Chelly et al., 1988; Rappolee et al., 1988a) and sometimes as Patty (PCR aided transcript titration assay, Becker-Andre and Hahlbrock, 1989), represents a dramatic technical innovation. The RT-PCR procedure has proven more sensitive and discriminating than Northern blot analysis, nuclease protection assay, and in situ hybridization. It is rapid and easy to handle, allows simultaneous analysis of several transcripts from total RNA, and can be used for relative or absolute quantification of mRNAs (Chelly et al., 1990a; Rappolee et al., 1989; Becker-Andre and Hahlbrock, 1989; Wang et al., 1989; Singer-Sam et al., 1990; Gilliland et al., 1990). This technique is very powerful in detecting transcripts that have a low copy number because of their short half-life or low rate of transcription and in detecting transcripts from a small number of cells (even from a single cell) or a small amount of tissue (even from a tissue section).

Quantitative analysis of RNA is an important aspect of gene expression studies and central to the understanding of the mechanisms that regulate gene activity. Northern gels, dot or slot blots, are currently used for analyzing RNA levels, however, Northern analysis requires large quantities of RNA and is not very sensitive for quantitative use in detecting low abundance mRNA. S1 nuclease assays, RNase A protection, and in situ hybridization methods are more sensitive and quantitative, however, these methods are time consuming, can be technically difficult, and are not useful for a simultaneous processing of a large number of samples. Although these methods are more sensitive than Northern analysis, for many practical purposes, i.e., analyzing low copy number of transcripts or small amount of cells, the very low levels of mRNA limit their widespread use.

Hepatocyte-like mhAT3F cells have been derived from the hepatoma of a transgenic mouse expressing the SV40 large T antigen under the control of the antithrombin III gene regulatory region (Antoine, B., Levrat, F., Vallet, V., Berbar, T., Cartier, N., Dubois, N., Briand, P., and Kahn, A. (1992) Gene expression in hepatocyte-like lines established by targeted carcinogenesis in transgenic mice. Exp. Cell. Res. 200, 175-185; F. Levrat et al., unpublished results). In these cells, the L-PK gene is transcriptionally activated by glucose, as it is in vivo and in cultured hepatocytes. However, in contrast to the L-PK gene regulation in the liver and isolated hepatocytes, the glucose responsiveness does not require insulin and is not blocked by cyclic AMP. In mhAT3F cells, the insensitivity to insulin might be due to the replacement of insulin-dependent glucokinase by insulin-independent hexokinases able to phosphorylate glucose in the absence of the hormone. The glucose-dependent activation of the L-PK gene is delayed, requires ongoing protein synthesis, and is mediated by the same glucose response element as in vivo and in isolated hepatocytes. These results suggest that the glucose-dependent signaling pathway responsible for the transcriptional activation of glycolytic and lipogenic genes requires glucose phosphorylation, a phenomenon that is insulin-dependent in the liver but insulin-independent in cultured hepatoma cells. Nevertheless, the action of glucose 6-phosphate is most likely indirect.

Glucose can modulate the transcription of many genes, particularly those encoding enzymes of liver metabolism. The transcriptional effect of glucose can be indirect, being mediated in vivo by hormonal variations, especially increase in insulin and decrease in glucagon secretion. Whereas the transcription of the glucokinase gene, for example, is stimulated by insulin without the aid of glucose, the transcriptional activation of most glycolytic and lipogenic genes in hepatocytes requires the presence of both glucose and insulin. The role of insulin in the activation of these genes seems mainly to stimulate glucokinase synthesis, and thus to permit glucose phosphorylation. In some cells in which hexokinase activity is constitutive, the glucose-dependent activation of the same genes does not require insulin and, in addition, can be produced by the nonmetabolisable analog, 2-deoxyglucose. In hepatocytes, the insulin effect on the glucose-dependent activation of the L-pyruvate kinase gene can be reproduced by fructose at low concentrations. Fructose probably acts through the fructose 1-phosphate dependent deinhibition of glucokinase activity. A glucose/carbohydrate element has been identified on the L-type pyruvate kinase and spot 14 gene promoters. It is able to bind, in vitro, transcriptional factors of the MLTF/USF family and could act in cooperation with tissue-specific contiguous elements, such as the HNF4 binding site in the L-type pyruvate kinase gene.

We have described new well-differentiated mouse hepatocyte-like cell lines (mhAT) derived from transgenic mice expressing simian virus 40 large T antigen under the control of antithrombin III gene promoter (Exp. Cell Res. (1992) 200, 175-185). In an attempt to understand the phenotypic variations of the different cell lines, we analyzed their content in liver-specific transcription factors at the level of both the proteins, by gel shift analysis, and the mRNA, by quantitative reverse-transcription PCR. Moreover, the transactivating ability of endogenous HNF1 alpha and C/EBP alpha was also evaluated by measuring the activity of transfected synthetic promoters consisting of DNA element homopolymers upstream of a TATA box. High levels of HNF1, HNF3, and HNF4 transcription factors were maintained in mhAT cells. In contrast, C/EBP alpha was much more variable between the different cell lines and was less abundant than it was in vivo, in the liver. We investigated the influence of HNF1 alpha and C/EBP alpha on the activity of transfected liver-specific promoters. HNF1 alpha was not limiting for the activity of transfected liver-type pyruvate kinase and albumin promoters. In contrast, the activity of the albumin promoter in the different lines was clearly dependent on the C/EBP alpha content, which seems, therefore, to be an essential factor modulating the expression of this gene in HNF1 alpha-containing cells. This work shows that the correlations between promoter activities and transacting factor contents in a panel of well-differentiated cultured cells can be used to determine the respective role of transcription factors on the strength of some promoters.

International audience; A 115-bp promoter fragment of the aldolase C gene is sufficient for conferring neural cell specificity on a reporter gene, in cultured PC12 cells and in transgenic mice. In vitro DNase I protection experiments detected two footprints on the promoter, termed boxes A/A', and B. The 5' A/A' box contains overlapping Sp1 and Krox20/Krox24 binding sites; it binds Sp1 in fibroblasts (box A') and a different complex in brain (box A). Any deletion or mutation of this box that impairs protein recognition also suppresses promoter activity. The replacement of box A/A' by a Sp1 consensus binding site results in the loss of the brain specificity of expression in transgenic mice. Further 3', box B is composed of a 5' direct repeat and a 3' GC box consisting of overlapping Sp1 and Krox20/Krox24 binding sites. Mutation of the direct repeat subregion appears to be more deleterious for the promoter activity than mutation of the G+C-rich subregion.

Duchene muscular dystrophy (DMD) is a fatal progressive X-linked muscle disorder, caused by mutations in the dystrophin gene. We have investigated adenovirus-mediated transfer of a dystrophin minigene in a mutant mouse lacking dystrophin, the mdx mouse. We report here that six months after a single intramuscular injection of a recombinant adenovirus containing a human dystrophin minigene, a large number of dystrophin-positive fibres are still detected in the injected muscles. Moreover, although the minigene encodes a truncated protein, its expression is able to protect the fibres efficiently against the degeneration process that affects the dystrophin-deficient mdx myofibres.

The functional role of the different sites binding transcriptional factors on the tissue-specific, glucose-responsive promoter of the L type pyruvate kinase gene (L-PK) has been investigated in transgenic mice. These sites are able to bind, from 3' to 5', HNF1, NF1, HNF4, and MLTF/USF, respectively. We have compared the level of chloramphenicol acetyltransferase reporter transgene expression when driven by a L-PK promoter fragment of either -96 base pairs (bp) (containing only the HNF1 binding site) or -150 bp (lacking the MLTF/USF binding site) or driven by a -183-bp L-PK promoter fragment with or without the NF1 binding site. Our results demonstrate that: 1) HNF1 alone is not sufficient to promote an efficient L-PK gene transcription in vivo; 2) with only binding sites for HNF1, NF1, and HNF4, though the tissue-specific pattern of expression is respected, the level of the gene transcription is low and the hormonal control is lost; 3) the MLTF/USF binding site is the target of the hormonal control, required for both positive response to carbohydrates and negative response to glucagon; 4) the role of NF1 in the promoter activity could be to negatively modulate the L-PK gene expression in the different tissues, without interfering with the glucose and hormone responsiveness.

International audience; The dystrophin whose defect is responsible for Duchenne and Becker muscular dystrophies is present in muscle, brain and cerebellum. We describe here the detection of dystrophin in human cultured skin fibroblasts, L809 cells and murine 3T6 cell line. Dystrophin transcripts initiated at the muscle specific first exon can also be amplified by cDNA-PCR from various fibroblastic cells. The expression of the dystrophin gene in fibroblasts could account for some abnormalities observed in patient's fibroblast cultures.

DUCHENNE progressive muscular dystrophy is a lethal and common X-linked genetic disease1 caused by the absence of dystrophin2,3, a 427K protein encoded by a 14 kilobase transcript4. Two approaches have been proposed to correct the dystrophin deficiency in muscle. The first, myoblast transfer therapy, uses cells from normal donors5–7, whereas the second involves direct intramuscular injection of recombinant plasmids expressing dystrophin8. Adenovirus is an efficient vector for in vivo expression of various foreign genes9–13. It has recently been demonstrated that a recombinant adenovirus expressing the lac-Z reporter gene can infect stably many mouse tissues, particularly muscle and heart12,13. We have tested the ability of a recombinant adenovirus, containing a 6.3 kilobase pair Becker-like dystrophin complementary DNA14 driven by the Rous sarcoma virus promoter to direct the expression of a 'minidystrophin' in infected 293 cells and C2 myoblasts, and in the mdx mouse15,16, after intramuscular injection. We report here that in vivo, we have obtained a sarcolemmal immunostaining in up to 50% of fibres of the injected muscle.

The molecular basis of null alleles was investigated by cDNA polymerase chain reaction (PCR) in seven Tay-Sachs patients. Although mRNAs were undetectable by Northern blot, cDNA-PCR amplification allowed us to get a sufficient amount of cDNA to characterize abnormal transcripts. In two French patients (one homozygote and one compound heterozygote with a 4-bp insertion in exon 11 of the second allele) suffering an infantile form of the disease, we detected abnormal RNAs with a 17-bp insertion due to a GT to AT transition at the donor site of intron 9, resulting in the activation of a cryptic donor site in the intron. This mutation has been found in 9 out of 82 Tay-Sachs chromosomes (11%) in association with alleles responsible from different clinical courses. In the other five patients we found the 4-bp insertion in exon 11 and two nonsense mutations.

The human aldolase A gene is transcribed from three different promoters, pN, pM, and pH, all of which are clustered within a small 1.6-kbp DNA domain. pM, which is highly specific to adult skeletal muscle, lies in between pN and pH, which are ubiquitous but particularly active in heart and skeletal muscle. A ubiquitous enhancer, located just upstream of pH start sites, is necessary for the activity of both pH and pN in transient transfection assays. Using transgenic mice, we studied the sequence controlling the muscle-specific promoter pM and the relations between the three promoters and the ubiquitous enhancer. A 4.3-kbp fragment containing the three promoters and the ubiquitous enhancer showed an expression pattern consistent with that known in humans. In addition, while pH was active in both fast and slow skeletal muscles, pM was active only in fast muscle. pM activity was unaltered by the deletion of a 1.8-kbp region containing the ubiquitous enhancer and the pH promoter, whereas pN remained active only in fast skeletal muscle. These findings suggest that in fast skeletal muscle, a tissue-specific enhancer was acting on both pN and pM, whereas in other tissues, the ubiquitous enhancer was necessary for pN activity. Finally, a 2.6-kbp region containing the ubiquitous enhancer and only the pH promoter was sufficient to bring about high-level expression of pH in cardiac and skeletal muscle. Thus, while pH and pM function independently of each other, pN, remarkably, shares regulatory elements with each of them, depending on the tissue. Importantly, expression of the transgenes was independent of the integration site, as originally described for transgenes containing the beta-globin locus control region.

Five Dnase 1 footprints, termed boxes L1 to L5, have been characterized in the 280 bp upstream from the cap site of the L-type pyruvate kinase (L-PK) gene. They bind from 3′ to 5′, the factors HNF1, HNF4, MLTF, and a non-identified protein referred to as L5 binding factor (L5-BF). These elements, individually or variably combined, were tested by cell-free transcription. The L1 box stimulates both L-PK and TK promoters, but in the context of L-PK promoter, it cooperates with the L3 element to reach a high level of transcriptional activation. The L3 and L4 elements exhibit weaker influences, increased by homologous or heterologous interactions. The L5 box behaves as a promoter-dependent negative regulatory element.

A transcript generated by the distal part of the Duchenne Muscular Dystrophy (DMD) gene was initially detected in cells where the full size 14-kilobase (kb) messenger RNA is not found at a significant level. This transcript, approximately 4.5 kb long, corresponds to the cysteine-rich and carboxyl-terminal domains of dystrophin. It begins with a novel 80- to 100-nucleotide exon containing an ATG start site for a new coding sequence of 17 nucleotides in-frame with the consecutive dystrophin cDNA sequence from exon 63. This result suggests the existence of a third promoter that would be localized about 8 kilobases upstream from exon 63 of the DMD gene. The distal transcript is widely distributed but is absent in adult skeletal and myometrial muscle. It is much more abundant in fetal tissues. With an antibody directed against the dystrophin carboxyl terminus, the protein corresponding to this transcript was detected as a 70- to 75-kDa entity on Western blots. It was found in all tissues analyzed except in skeletal muscle. It was not found in lymphoblastoid cells from a Duchenne patient with a complete deletion of the dystrophin gene. The role and subcellular localization of this protein is not known. It may explain extramuscular symptoms exhibited by some Duchenne patients.

The rat L-type pyruvate kinase gene possesses two promoters located 500 bp apart. The L' promoter is specific to erythroid cells. The L promoter is specific to liver and is regulated by diet and hormones; positively by glucose and insulin and negatively by glucagon via cAMP. The DNA elements involved in this tissue-specific and hormone-regulated gene expression are located within 3.2 kbp of 5' flanking region as previously demonstrated by transgenic mice analysis [Tremp, G. L., Boquet, D., Ripoche, M. A., Cognet, M., Yu-Chun, L., Jami, J., Kahn, A. and Daegelen, D. (1989) J. Biol. Chem. 264, 19,904-19,910]. Moreover, we have observed in these mice that gene expression was dependent on the transgene copy number and independent of the integration site. We present here DNase-I-hypersensitivity analysis of the endogenous rat L-type pyruvate kinase gene and of two transgene constructs in relation to development, tissue differentiation, nutritional and hormonal status. In rats, two groups of proximal sites were detected on the endogenous gene; hypersensitive site (HSS) HSS-1 in adult liver and HSS-A in fetal liver (a major erythropoietic tissue). Both groups are probably related to the transcriptional initiation complexes at either the L or L' promoter. Two other distal groups were detected; HSS-2 at -3 kbp (with respect to the liver-specific cap site) in adult liver and HSS-B around -4 kbp in fetal liver. These sites are thought to correspond to activating sequences; in adult liver, deletion of a fragment encompassing HSS-2 provokes a dramatic reduction of transcription starting at the L promoter of the transgene. In adult liver, HSS-1 appears to be a transcription-associated site, being greatly weakened in fasted rats, while HSS-2 is transcription independent. The pattern of DNase-I hypersensitivity is similar for the rat endogenous gene and for the complete rat transgene; the liver-specific HSS-1 and HSS-2 are present and the intensity of the sites is correlated to the number of integrated copies. Interestingly, HSS-1 is still detectable and its intensity remains proportional to the number of integrated copies in a truncated transgene with HSS-2 deletion, while this transgene is very weakly (but nevertheless tissue-specifically) expressed. These results strongly suggest that each transgene copy possesses a complete set of specific nucleoprotein complexes and that, with or without HSS-2, the DNA is in a potentially active configuration.

The muscle-specific promoter of the dystrophin gene is active in skeletal, cardiac, and smooth muscles and is specifically stimulated during differentiation of myoblasts into multinucleated myotubes. An 850-base pair (bp) DNA fragment upstream from the cap site is able to confer a partial muscle specificity to a reporter gene. The region between -850 and -140 bp includes nonspecific negative and positive regulatory sequences. A continuous stretch of 140 bp upstream from the cap site exhibits a striking conservation between rodents and human (93% homology) and still retains muscle preference of expression. It contains two putative binding sites for factors involved in regulation of other muscle-specific genes, a CCArGG box and an E box. This latter element, however, is unable to confer the ability to be transactivated by MyoD1 to the dystrophin promoter. The -140-bp promoter fragment exhibits antagonist effects contributed by one inhibiting sequence (nucleotide -140/-96), active in all cell types, and one activating region, from nucleotide -96 to the cap site, sufficient to confer a muscle preference of expression, in which the CCArGG box seems to play a major role.

New hepatocyte-like cell lines (mhAT) were derived from the liver of a transgenic mouse expressing SV40 early genes under the direction of the liver-specific antithrombin III gene promoter (ATIII-TSV40). Their differentiated phenotypes were improved and stabilized by the use of liver-specific growth media (arginine-free, glucose-free, or low-fructose/glucose-free medium). The best differentiated lines display a very high level of albumin, transferrin, and L-type pyruvate kinase (L-PK) gene expression that is comparable to that observed in the mouse liver. Abundance of the aldolase B and phosphoenolpyruvate carboxykinase (PEPCK) transcripts varied from 5 to 35% of the in vivo concentrations while abundance of the alpha-fetoprotein and phenylalanine hydroxylase transcripts remained very low. Hormonal (cAMP and insulin) and nutritional (glucose) gene controls of PEPCK and L-PK were, at least partially, conserved. mhAT cells are readily transfectable by the calcium phosphate coprecipitation technique and exhibit a liver-specific pattern of expression of exogenous genes. Thus, mhAT cells seem suitable for the analysis of the regulatory regions involved in the tissue-specific transcription of genes. This work demonstrates, therefore, the great efficiency of targeted carcinogenesis in transgenic mice to create new differentiated cell lines. The availability of various lines of liver-specific cells with different phenotypes will constitute useful tools to establish correlations between expression of trans-acting factors and control of the phenotype.

Publisher Summary This chapter presents a quantitative polymerase chain recation (PCR) strategy that allows one to accurately compare amounts of a specific mRNA in different samples. The strategy involves coamplification of a known amount of an exogenous mRNA used as an internal standard together with the investigated mRNA. Specific amplification of mRNA sequences by the PCR procedure after a first step of reverse transcription is a powerful method for detecting low-abundance mRNA. In addition, this method is so sensitive that it allowed researchers to demonstrate a basal level of transcription of any highly tissue-specific genes in any nonspecific cells. As it is possible to amplify any transcript, whatever its initial amount, analysis of gene expression using the efficient in vitro amplification of transcripts should be concurrently completed by a quantitative estimation of these transcripts. The data obtained indicates that the huge dystrophin gene harbors information for a variety of transcripts that are expressed in a wide range of tissues, including the brain.

PDX-1 encodes a homeodomain protein involved in pancreas development and islet-cell function, and transfer of this gene to liver cells induces them to produce insulin in diabetic mice. Therapeutic strategies for diabetes should focus not only on transplantation approaches, but also on the induction of surrogate β cells to replace impaired islet-cell function in diabetics (pages 568–572).

La genetique est une science neuve puisque ses premisses n’ont ete jetees qu’en 1865 par Johann Gregor Mendel. Discipline se fixant pour objet la transmission des caracteres hereditaires, elle est, associee a la theorie de l’evolution qui lui est presque contemporaine, a l’origine de bouleversements scientifiques, sociologiques, philosophiques et politiques considerables. En ce sens, elle est par excellence une science des crises, passees, presentes et futures.

The human aldolase A gene is transcribed from three different promoters, which are all clustered within a 1.6 kbp DNA domain. Two of these, PN and PH, are ubiquitous and seem to be co-regulated in most tissues while the third one, PM, is specific to adult skeletal muscle. We investigated the sequences involved in the ubiquitous activity of the PN and PH promoters of the human aldolase A gene. Deletion analysis, performed by transient expression assays of chloramphenicol acetyltransferase reporter genes in human HepG2 hepatoma cells, indicated that PH activity results from the interaction of an upstream activating region with two distinct core promoters. The upstream activating region was able to stimulate transcription from the HSV tk promoter as efficiently as the SV40 enhancer in all cell types tested. It appears, therefore, to be a strong ubiquitous enhancer. DNAsel footprinting revealed protections covering sequences scattered along the enhancer, including Sp1 and AP1 motifs. Importantly, we found that this enhancer was also necessary to activity of the other ubiquitous promoter of the aldolase A gene, PN. These studies demonstrate that expression of the human aldolase A gene is mediated by a complex interplay of enhancer and promoter elements.

Proopiomelanocortin (POMC) gene expression is not restricted to the pituitary corticotroph cell, but also takes place in many normal and tumoral nonpituitary tissues. In contrast, the ectopic ACTH syndrome is a rare event. Because it is most often associated with lung tumors, we specifically studied this tissue, analyzing the different forms of POMC RNAs in normal specimens as well as in various types of tumors. The endocrine nature of the tumors was assessed by both histological examination and measurements of secretogranin-I fragments in the tissue extracts. POMC RNA was first detected by Northern blot analysis; its absolute amounts and its various molecular forms were more precisely quantified and discriminated by S1 mapping studies using a single stranded DNA probe located at the 5' end of exon 3. In five bronchial carcinoid tumors associated with the ectopic ACTH syndrome, a highly predominant, if not single, POMC RNA identical to the 1200-nucleotide (nt) pituitary message was present, the high amounts of which were correlated with those of POMC peptides in the same tissues. In five bronchial carcinoid tumors not associated with the ectopic ACTH syndrome, the same message was detected (four of five), with a second, often predominant, short RNA of about 800 nt (five of five), and the overall amounts of POMC RNAs were low. Similar patterns of POMC RNAs were observed in squamous cell tumors, adenocarcinomas, and normal lung, where the short 800-nt RNA tended to be predominant. These results show that POMC gene expression can be demonstrated in normal lung tissue and in all types of lung tumors. The ectopic ACTH syndrome only occurs with tumors capable of generating high amounts of the pituitary-like message, a phenomenon that seems to be restricted to an occasional tumor with features of neuroendocrine differentiation.

Illegitimate transcription corresponds to the low level presence of specific transcripts in non-specific cells. This phenomenon allows to analyse any tissue-specific disease transcript in any easily accessible cell. We demonstrate here that the start sites of transcription are the same in specific and non-specific cells, which indicates that illegitimate transcription is due to a low level activity of the normal promoter. In addition, it is possible to increase about 10 fold the abundance of illegitimate trancripts through the use of cycloheximide. This treatment should, therefore, facilitate detection and qualitative analysis of illegitimate transcripts.

In primary culture of adult rat hepatocytes, vanadate in the presence of glucose stimulates the expression of the liver (L-type) pyruvate kinase gene. Glucose by itself was inactive, and vanadate, like insulin, was also inefficient in the absence of glucose. Similar results were obtained on glucokinase gene expression. An analogue of cAMP, 8-(4-chlorophenylthio)-cAMP, inhibited the production of L-type pyruvate kinase and glucokinase mRNAs in the presence of glucose plus vanadate.

The nature and location of the cis-acting DNA sequences regulating expression of the rat aldolase B gene has been investigated. Two liver-specific DNAse I hypersensitive sites were detected, one located just upstream from the cap site, the second in the middle of the first, 4.8-kbp-long, intron. A fragment of 190 bp 5' to the cap site behaved as a tissue-specific but weak core promoter: it directed a detectable reporter gene expression in the Hep G2 cells and hepatocytes, but not in fibroblasts. The tissue-specific expression was stimulated at least 16 fold when constructs contained the entire first intron. The intronic activating sequences could be ascribed to an inner 2 kbp fragment in which the downstream liver-specific DNAse I hypersensitive site was located.

The human aldolase A gene is transcribed from three distinct promoters, the two ubiquitous promoters PN and PH and the muscle specific promoter PM. In the present study, we investigate further aldolase A mRNA structure and expression. We demonstrate that the upstream N-type exon is, in fact, extremely heterogeneous. RNAse H mapping experiments permit quantification of relative abundance of N, M, and H type mRNAs and show that the level of transcripts containing the downstream H-type exon is at least 30 times higher than that of those containing N exon, in all tissues tested. Aldolase A level is up-regulated in proliferating cells. Here we show that both N and H type mRNAs, although barely detectable in normal liver, are highly expressed in human hepatomas biopsies. Furthermore, in human lymphocytes, N-type mRNA level is enhanced by serum treatment, while in cultured Hep G2 cells, both N-type and H-type mRNA levels are increased by serum and by the tumor promoting agent PMA. Using CAT constructs in transfection experiments, we demonstrate that the H exon plus its upstream region can function autonomously: the 420 base pairs upstream of the H exon are sufficient to confer to promoter PH an efficiency comparable that of the complete SV40 early promoter and enhancer in two cell lines.

Proopiomelanocortin is a polypeptide precursor molecule, the processing of which generates ACTH, beta-endorphin, the beta- and gamma-lipotropins, the joining peptide, and the NH2-terminal fragment. Anterior pituitary corticotrophs are the major site of proopiomelanocortin gene expression in man and the predominant, if not sole source of circulating ACTH. Recent data have established that proopiomelanocortin gene expression also occurs in various normal nonpituitary tissues, one of the best studied being the testis. In this latter organ the dominant gene products are short transcripts of approximately 800 nucleotides, which lack the first two exons of the gene and cannot encode a complete proopiomelanocortin molecule. In this report we show that the mode of proopiomelanocortin gene expression is occasionally modified in human Leydig cell tumors: a 1,200-nucleotide mRNA species identical to that in the pituitary is produced. It results from the usual (pituitary) start site of transcription and thus can encode the complete proopiomelanocortin molecule. In two out of six tumors, large amounts of the 1,200-nucleotide transcript led to a dramatic increase of approximately 1,000-fold in proopiomelanocortin peptide concentrations as compared with the normal and peritumoral testis. Proopiomelanocortin processing in these tumors generates various peptide fragments including ACTH. These results may help to understand the mechanism of proopiomelanocortin expression in nonpituitary tumors and have implications for the more general phenomenon of ectopic hormone secretion.

Tay-Sachs disease is an autosomal recessive genetic disease caused by a deficiency in beta-hexosaminidase A. We have characterized a new mutation in a Tunisian patient displaying a late infantile form of Tay-Sachs disease. Northern blot analysis of patient's fibroblast total RNAs shows a broad, fast migrating band in the region of the normal beta-hexosaminidase alpha transcripts. The mRNA coding for beta-hexosaminidase alpha subunit was first reverse transcribed and then amplified in four overlapping segments spanning the entire coding sequence by polymerase chain reaction. We found in the products of polymerase chain reaction (PCR) that amplify the segment spanning exons 2-7, in addition to a normal fragment, two smaller size fragments, one of which is also seen in normal control fibroblasts. The analysis of the patient's specific abnormal fragment by hybridization with exon-specific oligonucleotides and then sequencing allowed us to conclude that this fragment lacked exon 5. The other smaller species lacked exons 4 and 5 in both patient and normal control. The sequence of a genomic fragment containing exon 5 and of the patient's normal cDNA fragment spanning exons 2-7, revealed a point mutation G to A at the last nucleotide of exon 5. This mutation doesn't change the sense of the affected codon. Northern blot of patient's fibroblast poly(A+) RNAs allowed us to quantify two of the forms of transcripts seen by PCR. In the patient, the normal size transcript and the exon 5-deleted transcript represent, respectively, 3 and 7% of the normal control beta-hexosaminidase alpha mRNA. We propose that this point mutation is responsible for an inefficient and abnormal processing of the mutant transcript resulting in the appearance of two low abundance spliced mRNAs. One is lacking exon 5 and most likely codes for an inactive protein; the other is similar to normal beta-hexosaminidase alpha mRNA, except for the presence of the silent G to A mutation and codes therefore for a normal enzyme accounting for the 2.5% residual beta-hexosaminidase A activity measured in patient's fibroblasts by a fluorometric assay. The third form, without exons 4 and 5, is also evidenced in normal fibroblasts by PCR so that we think that it is not related to Tay-Sachs disease.

Le droit est-il condamne a courir apres la science et les techniques pour tenter, toujours en retard, de s’y adapter ? Ou bien fixe-t-il des principes d’une relative stabilite dans les limites desquels devraient se deployer les innovations du monde moderne ? Et, dans ce cas, quels sont les fondements de ces principes ? Ont-ils vocation a etre universels ? Prenant pour exemple l’interruption volontaire de grossesse, la jurisprudence de la Cour de cassation sur l’indemnisation des personnes handicapees nees apres une erreur de diagnostic prenatal, la recherche sur l’embryon et le clonage, cet article defend l’argument selon lequel la loi confrontee a ces questions n’est nullement tenue a changer de paradigme. Bien a l’inverse, la coexistence du rythme rapide des sciences et des techniques et du temps lent du droit et de l’ethique constituent la meilleure garantie d’une mobilisation des fruits du progres au profit d’une idee de l’homme aux racines robustes.

It has been shown that the dystrophin gene, which is defective in patients with Duchenne and Becker muscular dystrophy (reviewed in ref. 1), is transcribed in brain from a specific promoter that is different from the one used in muscle, and so the two types of transcripts differ at least in their first exon. We recently found that the dystrophin gene is expressed at a higher level in primary cultures of neuronal cells than in astro-glial cells derived from adult mouse brain. Here we investigate the use of two different promoters in each cell type. Our results demonstrate that the brain-type promoter of the dystrophin gene is highly specific to neurons, in which there is a significant increase in the amount of brain-specific messenger RNA during the course of in vitro maturation. By contrast, the muscle-type promoter is active in a wider range of cell types, including not only striated and smooth muscle, but also glial cells to a lesser extent, and probably neurons.