RESUMO
We undertook cloning and sequencing of the 5' portion of the human aldolase A gene to elucidate the mechanisms that govern synthesis of its different mRNAs. The sequenced gene is the only active gene in human-rodent fibroblastic somatic hybrids, while the other aldolase A-related sequences are inactive. S1 mapping and primer extension analysis enabled us to demonstrate that three promoter regions were implicated in the initiation of different aldolase A mRNAs, differing only in their 5' non-coding extremities. A distal promoter, N (non-specific), governs the synthesis of a 5' non-coding region of 142 bases composed of two exons, N1 and N2, which are found in a variety of tissues. A median promoter, M (muscle), is only active in skeletal muscle, and initiates the transcription by a 5' non-coding exon of 45 bases. Finally, a proximal promoter, H (housekeeping), contained in a "G + C-rich island", permits transcription of three colinear mRNAs containing 172, 126 or 112 bases of 5' non-coding sequence; their expression seems ubiquitous. These three promoters are arranged in 1.5 X 10(3) base-pairs of DNA. Homologies between rat and human genomic sequences and the absence of homology between promoters or 5' non-coding exons of the same species exclude a recent duplication of the promoter regions.
Assuntos
Frutose-Bifosfato Aldolase/genética , Regiões Promotoras Genéticas , Sequência de Aminoácidos , Animais , Sequência de Bases , Genes , Humanos , Células Híbridas , Dados de Sequência Molecular , RNA Mensageiro/genética , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
Lack of muscle glycogen phosphorylase activity leads to McArdle's disease, a rare metabolic myopathy. To investigate its molecular basis at the nucleic acid level, we isolated muscle phosphorylase cDNA clones from a human cDNA library in Escherichia coli plasmid pBR 322. Subcloning of one insertion of M13 bacteriophage permitted its definite identification by sequencing. Northern blot experiments revealed one specific messenger RNA of 3.4 kilobases found uniquely in tissues expressing muscle phosphorylase. We show that McArdle's disease exhibits a molecular heterogeneity at the messenger RNA level. In eight unrelated cases of McArdle's disease in which no inactive proteins had been detected, we assayed muscle biopsies for phosphorylase mRNA by Northern blotting. In five cases, no muscle phosphorylase mRNA could be detected, while in three other cases, normal length mRNA was present in lower amounts. Moreover, Southern blot analysis of DNA isolated from white blood cells in four McArdle patients revealed no major deletion or rearrangements of the phosphorylase gene as compared with controls.
Assuntos
Doença de Depósito de Glicogênio Tipo V/genética , Doença de Depósito de Glicogênio/genética , Fosforilases/deficiência , Sequência de Bases , Clonagem Molecular , DNA/genética , Regulação da Expressão Gênica , Genes , Humanos , Músculos/enzimologia , Músculos/fisiologia , Fosforilases/genética , RNA Mensageiro/genéticaRESUMO
3 specific cDNA clones for human aldolase A were isolated from a human muscle library. One of them was subcloned in M 13 phage, then used as a probe to investigate the patterns and the levels of aldolase A mRNA in various human tissues. Two mRNA species differing in length were observed. The lighter one -1550 bases- was found specific to skeletal muscle; its amount increased during muscle development. The heavier aldolase A mRNA -1650 bases- accounted for foetal and ubiquitous presence of aldolase A isozyme. The resurgence of aldolase A in hepatomas occurred through this latter mRNA species.
Assuntos
Carcinoma Hepatocelular/enzimologia , Frutose-Bifosfato Aldolase/genética , Neoplasias Hepáticas/enzimologia , RNA Mensageiro/fisiologia , Animais , DNA/análise , Enzimas de Restrição do DNA , Feto/enzimologia , Frutose-Bifosfato Aldolase/biossíntese , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Músculos/enzimologia , Hibridização de Ácido Nucleico , RatosRESUMO
Crude hemolysates, partially purified aldolase and aldolase purified to homogeneity from reticulocytes and mature erythrocytes, were incubated with a specific antiserum raised against crystalline rabbit muscle aldolase. We show that the same aldolasic activity corresponds to a greater amount of antigen in older than in younger cells, in crude hemolysates as well as in the above mentioned preparations; that is to say, old-cell aldolase contains cross-reacting material (CRM). Properties of purified enzyme from reticulocytes and mature erythrocytes were compared to those of muscle crystalline aldolase: -- the molecular specific activity of purified aldolase from erythrocytes is lower than with crystalline muscle aldolase, i.e. CRM is maintained throughout the purification steps. -- the specific activity of red cell aldolase towards both substrates (FDP and F1P) is lower than that of crystalline muscle aldolase. However, the ratio of activity towards the two substrates FDP/F1P is decreased in erythrocytes and reticulocytes. -- no other difference was found: Michaelis constant towards FDP, thermodenaturation constant and C terminal extremities are identical as are the molecular weights.
Assuntos
Envelhecimento Eritrocítico , Eritrócitos/enzimologia , Frutose-Bifosfato Aldolase/sangue , Aminoácidos/análise , Animais , Carboxipeptidases , Reações Cruzadas , Imunodifusão , Cinética , Desnaturação Proteica , Coelhos , Reticulócitos/enzimologiaRESUMO
An isolation procedure for rat brain aldolase C has been developed which also permits the isolation of aldolase C from experimental hepatomas. Certain enzymatic properties (specific activity and Michaelis constant towards the two specific substrates: fructose 1,6-biphosphate and fructose 1-phosphate) and physico-chemical properties (molecular weight, N-terminal amino-acid) of the two enzymes have been studied and compared. Moreover, an amino-acid analysis has been carried out for rat brain aldolase C. Within experimental errors, the two enzymes appear to be identical.
