Molecular cloning of troponin i from the two-spotted cricket, Gryllus bimaculatus, and its expression pattern during starvation / Clonagem molecular da Troponina I do grilo africano, Gryllus bimaculatus, e seu padrão de expressão durante a inanição

There is few information about troponin gene expression by starvation in insect skeletal muscle and digestive tracts. The objective of this study was to perform molecular cloning of troponin I from the two-spotted cricket, Gryllus bimaculatus (GrybiTnI) and determine its expression patterns in three different skeletal muscles and digestive tracts during starvation. GrybiTnI was translated into a protein encoding 198 amino acids with a theoretical isoelectric point of 9.78 and a molecular weight of 23671.46 Da. The GrybiTnI has both the TnC-binding site and actin/TnC-binding site shown in the typical TnI amino acid sequences. Homology analysis revealed that GrybiTnI exhibited high similarity at the amino acid level to those of other insects already reported; 89~77% identity with those of other insects. Expression of GrybiTnI by starvation did not change in dorsal wing flight muscle and dorsal ventral flight muscle, but showed up-expression in dorsal longitudinal flight muscle. In the digestive tracts, the up-expression of GrybiTnI by starvation was observed only in the hindgut but not in the rest parts including Malpighian tubules. Re-feeding following starvation restored those expressions about the level before starvation in the dorsal longitudinal flight muscle and hindgut. In conclusion, troponin modulates gene expression not only to muscle elements but also to physiological changes such as strains.

Existe pouca informação sobre a expressão gênica da troponina por inanição no músculo esquelético de insetos e no trato digestório. O objetivo deste estudo foi realizar clonagem molecular da troponina I do grilo africano, Gryllus bimaculatus (GrybiTnI) e determinar seus padrões de expressão em três diferentes músculos esqueléticos e tratos digestivos durante a inanição. GrybiTnI foi traduzido em uma proteína codificando 198 aminoácidos com um ponto isoelétrico teórico de 9,78 e um peso molecular de 23671,46 Da. O GrybiTnI tem o local de ligação a TnC e o local de ligação a actina/TnC mostrado nas sequências de aminoácidos TnI típicas. A análise de homologia revelou que GrybiTnI exibiu alta similaridade no nível de aminoácidos em relação àqueles de outros insetos já relatados; 89~77% de identidade com os de outros insetos. A expressão de GrybiTnI pela inanição não se alterou no músculo de vôo da asa dorsal e no músculo de vôo ventral dorsal, mas mostrou expressão positiva no músculo de vôo longitudinal dorsal. Nos tratos digestivos, a expressão positiva de GrybiTnI por inanição foi observada apenas no intestino grosso, mas não nas partes de repouso, incluindo os túbulos de Malpighi. A realimentação após a inanição restaurou as expressões aproximadamente ao nível antes da inanição no músculo de vôo longitudinal dorsal e no intestino grosso. Em conclusão, a troponina modula a expressão gênica não apenas em elementos musculares, mas também em alterações fisiológicas, como as cepas.

Identifi cation of novel variants in the COL4A4 gene in Korean patients with thin basement membrane nephropathy.

Background & objectives: The α4 chain of the type 4 collagen family is an important component of the glomerular basement membrane (GBM) in the kidney. It is encoded by the COL4A4 gene, and mutations of this gene are known to be associated with thin basement membrane nephropathy (TBMN). To better understand the contribution of variants in the COL4A4 gene to TBMN, we investigated the sequence of the complete COL4A4 gene in 45 Korean patients with TBMN. Method: Genomic DNA was obtained from the peripheral blood lymphocytes. For the analysis of the COL4A4 gene, all the exons including splicing sites were amplififi ed by PCR and screened by direct sequencing analysis. Results: Eight novel COL4A4 sequence variants were found in these patients. Two of these variants, G199R and G1606E, were possibly pathogenic variants affecting the phenotype. None of these variants were observed in 286 chromosomes from normal Korean control subjects. In addition, 39 polymorphisms including 7 novel SNPs were identifi ed in this study. Interpretation & conclusion: The frequency of COL4A4 mutations in Korean patients with TBMN is low and the other cases may have mutations in other genes like COL4A3. Screening of the COL4A3 gene and fi nding a novel causative gene for TBMN will help clarify the pathogenesis of this disorder and perhaps for distinguishing TBMN from Alport syndrome.