Bidirectional activity and orientation-dependent specificity of the rat aldolase C promoter in transgenic mice.

We previously reported that the rat aldolase C 115 bp promoter is sufficient to ensure the brain specific expression of the chloramphenicol acetyltransferase reporter gene in transgenic mice. We identify in a further reduced 84 bp promoter several putative binding sites for the transcriptional factors Sp1, USF, AP1, and AP2. Deletion or mutation of these partially overlapping binding sites results in inactivation of the cognate transgenes. Moreover, we show that the 115 bp sequence is able to direct bidirectional transcription in vivo but, surprisingly, transcriptional activity in the opposite direction is no more brain specific.

Upstream elements involved in vivo in activation of the brain-specific rat aldolase C gene. Role of binding sites for POU and winged helix proteins.

The rat aldolase C gene encodes a glycolytic enzyme strongly expressed in adult brain. We previously reported that a 115-base pair (bp) promoter fragment was able to ensure the brain-specific expression of the chloramphenicol acetyltransferase (CAT) reporter gene in transgenic mice, but only at a low level (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). Here we show that in vivo activation of this promoter at a high level requires cooperation between an upstream 0.6-kilobase pair (kb) fragment and far upstream sequences. In the 0.6-kb region, a 28-bp DNA element is shown to include overlapping in vitro binding sites for POU domain regulatory proteins and for the Winged Helix hepatocyte nuclear factor-3beta factor. An hepatocyte nuclear factor-3beta-binding site previously described in the short proximal promoter fragment is also shown to interact in vitro with POU proteins, although with a lower affinity than the 28-bp motif. Additional binding sites for POU factors were detected in the upstream 0.6-kb sequences. Progressive deletion in this region resulted in decreased expression levels of the transgenes in mice, suggesting synergistic interactions between these multiple POU-binding sites. We propose that DNA elements characterized by a dual binding specificity for both POU domain and Winged Helix transcription factors could play an essential role in the brain-specific expression of the aldolase C gene and other neuronal genes.

Functional dissection of the brain-specific rat aldolase C gene promoter in transgenic mice. Essential role of two GC-rich boxes and an HNF3 binding site.

The aldolase C gene product is a glycolytic isoenzyme specifically detected in brain. We have previously defined a short 115-base pair promoter fragment able to confer on a reporter chloramphenicol acetyltransferase (CAT) gene a specific expression in brain of transgenic mice. In this promoter fragment, two GC-rich regions (A/A' and B boxes) were detected by in vitro DNase1 footprinting experiments with brain, fibroblast, or liver nuclear extracts. Both A/A' and B boxes, sharing structural homology, are able to interact with Sp1, Krox20/Krox24 factors and with other proteins (Thomas, M., Makeh, I., Briand, P., Kahn, A., and Skala, H. (1993) Eur. J. Biochem. 218, 143-151). In this paper, we describe a new ubiquitous factor termed Ub able to bind the A/A' box. We also delimit a third element (box C) binding a hepatocyte-enriched protein displaced by a hepatocyte nuclear factor 3-specific oligonucleotide. The functional involvement of each binding site in brain-specific transcription of the aldolase C gene has been tested in transgenic mice carrying different mutant promoters cloned in front of the CAT gene. A promoter containing only box C was totally inactive, suggesting an essential role of the region containing A/A' and B boxes. However, mutations or deletions of either the A/A' or the B box have no significant effect on the CAT gene expression. We therefore hypothesize that the A/A' and B sites may be functionally redundant. Indeed, constructs harboring only one of these two boxes (A/A' or B) linked to the C box displayed a brain-specific CAT activity similar to that obtained with the wild-type promoter. Furthermore, a transgene with disruption of the C box, keeping intact the A/A' and B boxes, was totally inactive, suggesting a crucial role of the hepatocyte nuclear factor 3 binding site in activation of the aldolase C gene.

Analysis of a brain-specific isozyme. Expression and chromatin structure of the rat aldolase C gene and transgenes.

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.

Determinants of the brain-specific expression of the rat aldolase C gene: ex vivo and in vivo analysis.

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.

Localization of aldolase C mRNA in brain cells.

The expression of aldolase C and aldolase A mRNA was assessed by Northern blot hybridization using RNAs purified from cultured rat and mouse brain neurons and astroglial cells. Neurons were found to contain about 4-fold more aldolase C mRNA and about twice as much aldolase A mRNA than astroglia. Analysis of the cellular localization of aldolase C mRNA by in situ hybridization to brain slices showed a predominantly neuronal labeling with an irregular distribution. A strong signal was observed in Purkinje cell somata and a weaker signal in subpopulations of neurons in cerebral cortex, striatum, hippocampus, hypothalamic nuclei and primary olfactory cortex.

The brain-specific gene for rat aldolase C possesses an unusual housekeeping-type promoter.

A DNA fragment encompassing the first exon and about 750 bp of the 5'-flanking sequence has been isolated and sequenced. The gene has multiple start sites of transcription which are dispersed over about 200 bp. The promoter lacks TATA and CAAT boxes and is very G + C-rich, with putative binding sites for the transcriptional factors Sp1 and AP2. Similar features are shared with two other brain-specific genes encoding thy-1 antigen and gamma-enolase. The existence of a conserved block of similarity upstream of the human and rat aldolase C genes suggests that this region could be involved in tissue-specific expression whose mechanism seem to be, at least in part, transcriptional.

Molecular cloning and expression of rat aldolase C messenger RNA during development and hepatocarcinogenesis.

A rat brain cDNA library was screened at low stringency with an aldolase B cDNA probe corresponding to the coding sequence of the mRNA, then at high stringency with a 3' non-coding aldolase A cDNA probe. One clone, which hybridized only under the first conditions, was further characterized and used to screen the library again. Two overlapping clones, complementary to aldolase C mRNA, were obtained. They cover the 113 carboxy-terminal coding residues and the 3' non-coding region up to the poly(A) tail. Their nucleotide sequence was determined. In the coding region the overall homology with aldolase A was 67% at the nucleotide level and 76% at the protein level. With aldolase B these values were 63% and 65% respectively. The 3' non-coding region was 380 bases long and did not exhibit any homology with the untranslated 3' extension of aldolase A and B mRNAs. Southern blot analysis indicates that probably a single aldolase C gene exists per haploid genome. Aldolase C mRNA was detected at low concentration in practically all the foetal tissues and its expression markedly and rapidly decreased after birth. In brain the concentration of aldolase C mRNA remained high and stable even after birth. Aldolase C mRNA is approximately 50-fold more abundant in brain than in foetal tissues, which are the richest in messenger RNA. In the course of azo-dye hepatocarcinogenesis the aldolase C gene is re-expressed early, with a maximum at the 4th week of carcinogenic diet, which probably corresponds to the maximal proliferation of the oval cells.

Protein ADP-ribosylation in rat liver cytosol.

ADP and poly ADP-ribosylation are post-translational modifications of proteins which have been reported to occur essentially in eucaryotic nuclei. This phenomenon has been shown to interfere with a great variety of biological functions (cell differentiation, DNA repair, malignant transformation...). In this paper, we demonstrate for the first time that ADP-ribosylation occurs also in cytosol (120 000 g supernatant) and that several cytosolic proteins can be ADP-ribosylated in rat liver.

Elevated NAD(P) glycohydrolase activity: a possible enzymatic marker for malignancy in Burkitt's lymphoma cells.

A comparative analysis of enzymatic activities has been performed on 47 human continuous lymphoid lines: 22 tumors derived from Burkitt's lymphoma lines, 6 other lymphomatous long-term cultures, and 19 nonmalignant ties determined on the cell extracts. 4 showed no significant differences between the various lines. They included adenosine diphosphoribose incorporation, glucose-6-phosphate dehydrogenase, cyclic-AMP phosphodiesterase, and glutathione reductase. However, striking differences of activity were found for the enzyme, NAD(P) glycohydrolase (EC 3.2.2.6). Activity levels were, as a mean, four times higher in Burkitt's lymphoma-derived cell lines than in nonmalignant control lines, and the difference was highly significant (p less than 0.02). All Burkitt cell lines containing translocations of chromosome 8 with either chromosomes 2, 14 or 22 showed an increased activity. The specificity and significance of this possible enzymatic marker of Burkitt's lymphoma cells is discussed.

PK3: a new chromosome enzyme marker for gene dosage studies in chromosome 15 imbalance.

Gene dosage studies yielded results consistent with the assignment of the locus for pyruvate kinase (PK3) to chromosome 15. The activity of seven cytoplasmic enzymes has been determined in fibroblast extracts from six trisomy 15 lines and 16 normal control lines. The fibroblast extracts from the trisomic patients had pyruvate kinase activity 57% higher than fibroblast extracts from control lines, while other enzyme activities were within the normal range of activity.

Hyperanodic forms of human glucose-6-phosphate dehydrogenase.

Pure glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ 1-oxidoreductase, EC 1.1.1.49) is transformed into 'hyperanodic forms' when incubated at acidic pH and in the presence of NADP+ with excess of glucose-6-phosphate or with some 'NADP+ modifying proteins' purified from the same cells. The enzyme hyperanodic forms exhibit low isoelectric point, altered kinetic properties and high lability to heat, urea, and proteolysis. Differences between hyperanodic and native forms of glucose-6-phosphate dehydrogenase are also noted by microcomplement fixation analysis, ultraviolet absorbance difference spectrum and fluorescence emission spectrum. Drastic denaturation of the enzyme by urea and acid treatment did not suppress the difference of isoelectric point between native and hyperanodic forms of glucose-6-phosphate dehydrogenase. From our data we suggest that the conversion into hyperanodic forms could be due to the covalent binding on the enzyme of a degradation product of the pyridine nucleotide coenzyme. This modification could constitute a physiological transient step toward the definitive degradation of the enzyme.

Triosephosphate isomerase deficiency with hemolytic anemia and severe neuromuscular disease: familial and biochemical studies of a case found in Spain.

A 16-month-old girl of Spanish origin with chronic hemolytic anemia and severe neuromuscular disease was found to have markedly reduced triosephosphate isomerase (TPI) activity in her erythrocytes, leukocytes, and plateletes. Both parents and some other family members had moderately reduced erythrocyte TPI activity in accordance with the autosomal recessive mode of inheritance in this enzymopathy. Latex ingestion and latex-stimulated histochemical NBT reduction by the patient's granulocytes were normal. Zymosan-stimulated superoxide radical (O-.2) formation, not previously studied in TPI-deficient granulocytes, was also within normal limits. Starchgel electrophoresis of TPI in both erythrocytes and leukocytes of the proposita and her parents was normal. Molecular studies of deficient TPI showed a normal kinetic pattern with markedly reduced heat instability. Immunologic studies demonstrated no cross reacting material in proposita leukocytes and a normal molecular specific activity. These studies suggest that molecular instability might cause both enzymatic and antigenic degradation of the TPI molecule and, therefore, TPI deficiency in our patient.