| The human zinc finger protein ZNF191, a Krüppel-like transcription factor, which can specifically bind the TCAT repeats (HUMTH01) in the first intron of the human tyrosine hydroxylase (TH) gene. HUMTH01 may regulate transcription of TH gene, which encodes the rate-limiting enzyme in the synthesis of catecholamines. The disturbances of catecholaminergic neurotransmission have been implicated in the neuropsychiatric and cardiovascular diseases. ZNF191 may be relevant to these diseases. The transcript of ZNF191 is abundant in liver and the transcriptional level increases in the liver cancer, which suggests the expression of ZNF191 may be relevant to this disease. In the rat embryo, the ZF-12 mRNA is present at high levels in total embryonic head, which may reflect a role in brain development. The data on mice suggest that ZF-12 might play an important role not only in cartilage differentiation, but also in basic cellular processes. We tried to elucidate the functions of ZNF191 with the models of ZNF191 transgenic mice and ZF-12-/- mice.Firstly, we screened a mouse lambda genomic library with a human ZNF191 cDNA probe and isolated a ZF-12-like gene for the first time, named ZF12p (GenBank AY040222). This intronless gene closely resembles ZF-12 but displays several mutations, suggesting that ZF12p represents a ZF-12-related pseudogene. RT-PCR analysis on total RNA from mouse tissue and bioinformatics analysis on promoter region of ZF12p gene, suggest the transcripts of ZF12p may be not synthesized. BLAST on the data of the human genome in the GenBank with ZNF191 cDNA and Southern blotting show there is no any pseudogene related to ZNF191 gene in the human genome. The high similarity of ZF12p to ZF-12 might be of considerable importance for mutation and evolution analysis of ZF-12.Additionally, we screened a mouse lambda genomic library by using the probe of the Hind III fragment of ZF-12p. A 18 093 bp fragment of the mouse zinc finger protein ZF-12 gene was cloned and sequenced for the first time, the GenBank accession number is AY052495. It contains four exons and three introns; all intronic splice sites exhibited consensus gt/ag sequences. The single nucleotide polymorphisms (SNPs) in exon 2 and the alternative length of 3'-untranslated region (3'-UTR) of the have been found. The linkage of the ZF-12 gene and the zinc finger protein gene Zfp-35 has been found, so the ZF-12 gene can be localized to B3 to C or beside of chromosome 18. We assessed approximately 1.1 kb of 5'-flanking region of the ZF-12 gene for basal promoter activity. A series of deletion mutants of 5'-flanking region linked to the luciferase gene was constructed. Basal level expression of these constructs was tested in COS-7 cells,NIH3T3 cells and HeLa cells. By measuring luciferase activity, which was transiently expressed in the transfected cells, we found that regulatory elements sufficient for basal expression lie between -762 and +70 bp relative to the transcription start site and that a negative regulatory region lie between -824 and -762 bp. By using the mouse zinc finger protein gene ZF-12 genomic DNA fragment, pSSC-TV-10.5 was designed and constructed as a replacement vector. Structure of pSSC-TV-10.5 was identified by restrictive digestion analysis and partly sequencing. Then linearized vector was electroporated into ES cells, and transfected cells were screened by G418 and GANC selection. Among G418r/GANCr colonies, four were proved to have taken place the homologous recombination of ZF-12 by PCR and southern blotting analysis. We obtained four chimeric mice by injecting ZF-12+/- ES cells into the C57BL/6J blastocysts. Among chimeras, three are male mice and one is female. About 50% of the coat is chimeric in a male chimera and about 30% is chimeric in the others. We achieved 81 potential transgenic mice by microinjection. Four mice were proved to have taken place the integration of ZNF191 by PCR and southern blotting analysis. These studies lay the foundations of elucidating the functions of ZNF191 o...
|
PDF Full Text Request