RESUMEN
OBJECTIVES: To study the in vitro expression of three phenylalanine hydroxylase (PAH) mutants (p.R243Q, p.R241C, and p.Y356X) and determine their pathogenicity. METHODS: Bioinformatics techniques were used to predict the impact of PAH mutants on the structure and function of PAH protein. Corresponding mutant plasmids of PAH were constructed and expressed in HEK293T cells. Quantitative reverse transcription polymerase chain reaction was used to measure the mRNA expression levels of the three PAH mutants, and their protein levels were assessed using Western blot and enzyme-linked immunosorbent assay. RESULTS: Bioinformatics analysis predicted that all three mutants were pathogenic. The mRNA expression levels of the p.R243Q and p.R241C mutants in HEK293T cells were similar to the mRNA expression level of the wild-type control (P>0.05), while the mRNA expression level of the p.Y356X mutant significantly decreased (P<0.05). The PAH protein expression levels of all three mutants were significantly reduced compared to the wild-type control (P<0.05). The extracellular concentration of PAH protein was reduced in the p.R241C and p.Y356X mutants compared to the wild-type control (P<0.05), while there was no significant difference between the p.R243Q mutant and the wild type control (P>0.05). CONCLUSIONS: p.R243Q, p.R241C and p.Y356X mutants lead to reduced expression levels of PAH protein in eukaryotic cells, with p.R241C and p.Y356X mutants also affecting the function of PAH protein. These three PAH mutants are to be pathogenic.
Asunto(s)
Fenilalanina Hidroxilasa , Humanos , Células HEK293 , Fenilalanina Hidroxilasa/genética , Western Blotting , Biología Computacional , ARN MensajeroRESUMEN
The chromosome samples of Rana nigromaculata were prepared by peripheral blood lymphocyte cultured in vitro. Karyotype, C-banding and silver-staining nucleolus organizer regions (Ag-NORs) were observed on chromosomes samples. The result showed that: (1) the chromosome number of Rana nigromaculata is 26 (2n=26), i.e., 10 large and 16 small chromosomes, the large chromosomes are matched into 5 homologous pairs and small chromosomes are matched into 8 homologous pairs, and further indicates the karyotype of Rana nigromaculata is bi-modal; (2) Metaphases of female and male were observed separately, the chromosome of No.11 has a pronounced secondary constriction in the middle of long arms. But the position of secondary constriction in the aberrant type individuals is in the middle of long arms of the No.8; (3) it has only one obvious C-banding close to the telomere, which is located in the long arms of the No.5; (4) The chromosome No.11 is a pair of homologous chromosomes with Ag-NORs, and the position of Ag-NORs in female and male is identical.
