RESUMO
A complementary DNA (cDNA) corresponding to sheep immunoglobulin E heavy (IgE H) chain messenger RNA (mRNA) was isolated and sequenced. A fragment of sheep IgE constant H (epsilon)-chain was initially synthesized using the polymerase chain reaction (PCR) and single-stranded cDNA prepared from the mRNA of parasite-stimulated sheep lymph nodes. This fragment was then used to probe a cDNA library, again prepared from a parasite-stimulated lymph node. A recombinant clone containing cDNA encoding a sheep IgE H-chain of 1802 base pairs was isolated and the H-chain cDNA-sequenced. The nucleotide sequence was found to code for a complete sheep IgE H-chain, consisting of both variable (V) and constant (C) regions. When the amino acid sequence derived from the nucleotide sequence was compared to other species epsilon-chains, interesting homologies and differences between corresponding domains were found, including conservation in cysteine and tryptophan residues and variable glycosylation sites.
Assuntos
DNA/isolamento & purificação , Imunoglobulina E/genética , Cadeias épsilon de Imunoglobulina/genética , Ovinos/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA/química , DNA/genética , Humanos , Dados de Sequência Molecular , Poli A/genética , Reação em Cadeia da Polimerase , RNA/genética , RNA Mensageiro , Homologia de Sequência de Aminoácidos , Ovinos/genéticaRESUMO
The sequencing and comparison of the genes encoding the glyoxylate bypass enzyme malate synthase of Aspergillus nidulans (acuE) and Neurospora crassa (acu-9) are presented. The predicted amino acid sequences of the A. nidulans and N. crassa enzymes are 538 and 542 residues respectively and the proteins are 87% homologous. In fungi, the malate synthase proteins are located in glyoxysomes and the deduced acuE and acu-9 proteins both contain a C-terminal S-K-L sequence, which has been implicated in transport into peroxisomes. The acuE coding region is interrupted by four introns and the acu-9 coding region is interrupted by one intron which occurs at the same position as the C-terminal acuE intron. The 5' non-coding regions of the two genes were examined for short homologous sequences that may represent the binding sites for regulatory proteins. Pyrimidine-rich sequences with weak homology to the amdI9 sequence, which has been implicated in facB-mediated acetate regulation of the amdS gene, were found but their functional significance remains to be determined.
Assuntos
Aspergillus nidulans/enzimologia , Genes Fúngicos , Malato Sintase/genética , Neurospora crassa/enzimologia , Sequência de Aminoácidos , Aspergillus nidulans/genética , Sequência de Bases , Dados de Sequência Molecular , Neurospora crassa/genética , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The genes encoding the acetate-inducible enzyme acetyl-coenzyme A synthetase from Neurospora crassa and Aspergillus nidulans (acu-5 and facA, respectively) have been cloned and their sequences compared. The predicted amino acid sequence of the Aspergillus enzyme has 670 amino acid residues and that of the Neurospora enzyme either 626 or 606 residues, depending upon which of the two possible initiation codons is used. The amino acid sequences following the second alternative AUG show 86% homology between the two species; the extended N-terminal sequences show no homology. The Neurospora protein is characterized by the appearance of the S(T)PXX sequence motif where the amino acid homologies break down. The codon usage is biased in both genes, with a marked deficiency, especially in Neurospora, of codons with A in the third position. The facA transcribed sequence contains six introns: one in the long leader sequence, one in the 5' coding sequence not homologous with acu-5, and four within the sequence that is largely similar to that of acu-5. Only one intron, corresponding in size and position to the furthest downstream of the facA introns, is found in acu-5. The evolution of introns during the divergence of these two Ascomycete fungi is discussed. Each of the two genes has been transferred by transformation into the other species. Each species is evidently able to splice out the other's introns. Most transformants have normal acetate-induction of acetyl-CoA synthetase, implying that the two genes respond to transcriptional control signals common to both species, in spite of the striking divergence of their 5' ends.
Assuntos
Acetato-CoA Ligase/genética , Aspergillus nidulans/genética , Coenzima A Ligases/genética , Genes Fúngicos , Neurospora crassa/genética , Neurospora/genética , Acetato-CoA Ligase/biossíntese , Sequência de Aminoácidos , Aspergillus nidulans/enzimologia , Sequência de Bases , Evolução Biológica , Íntrons , Dados de Sequência Molecular , Neurospora crassa/enzimologia , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Transformação GenéticaRESUMO
Acetate inducible genes of Aspergillus nidulans were cloned via differential hybridization to cDNA probes. Using transformation of mutant strains the genes were identified as facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase). The levels of RNA encoded by these genes were shown to be acetate inducible and subject to carbon catabolite repression. Induction is abolished in a facB mutant and carbon catabolite repression is relieved in a creA mutant.
