SP6
Transcription factor Sp6 (Krueppel-like factor 14) [KLF14]
PublicationsПравить
The Long-Evans Cinnamon (LEC) rat is a mutant strain characterized by abnormal copper metabolism and a high incidence of hepatitis and hepatoma. Using a yeast-based assay which scores mutants in p53 gene transcripts as red colonies, we detected frequent mutations in the liver of LEC rats. The majority (50-60%) of these were frameshift mutations caused by the insertion of an extra adenine (A) in the regions containing six consecutive adenines. The rate of A insertion was calculated to be 6.9-9.0% of the total p53 cDNA. Insertions of an extra adenine were found almost exclusively in the mRNA (cDNA), especially in the (A)(6) tract located at the most 5'-side (exon 4) among the three (A)(6) tracts (exons 4, 7, and 8), but rarely in the corresponding sites of genomic DNA. Wild-type p53 cDNA was transcribed in vitro into mRNA with the use of SP6 RNA polymerase and tested by the yeast functional assay. Subsequent sequencing detected A insertions at an overall rate of 1.6% in exons 7 and 8 but none in exon 4. This indicates that the A insertion in the exon 4 (A)(6) tract was an in vivo phenomenon rather than an artifact in reverse transcription or polymerase chain reaction. The percentage of red colonies increased sharply to about 20% of the liver samples in the acute hepatitis stage, and returned to control level of those in the chronic hepatitis stage, and increased again slightly to those in the neoplastic stage. The percentage of red colonies correlated with the serum GOT level (r=0.96, p<0.001) but not with the contents of copper and 8-hydroxydeoxyguanosine in the liver of LEC rats. Ethanol treatment of hepatic cell lines also increased the rate of transcriptional slippage at the (A)(6) tract. These findings indicate that cellular damage is responsible for the increase in the rate of mutation at the transcriptional level, and suggest that cellular damage degrades transcriptional fidelity, thereby further impairing cellular functions.
MeSH Terms
- 8-Hydroxy-2'-Deoxyguanosine
- Adenine
- Aging
- Animals
- Aspartate Aminotransferases
- Copper
- DNA, Complementary
- Deoxyguanosine
- Ethanol
- Genes, p53
- Hepatitis
- Liver
- Mutagenesis, Insertional
- Mutation
- RNA, Messenger
- Rats
- Rats, Inbred LEC
- Reverse Transcriptase Polymerase Chain Reaction
- Saccharomyces cerevisiae
- Transcription, Genetic
- Transfection
Transcriptional slippage was previously found in Escherichia coli during RNA elongation at runs of 10 or more As or Ts, resulting in the addition of untemplated A or U residues. To evaluate the incidence of transcriptional slippage in vivo, we employed a yeast functional assay, and analyzed the frequency and spectrum of mutations in mRNA of the tumor suppressor p53 in rat tissues. In this assay, yeast are transfected with p53 PCR products and a gapped p53 expression vector, which allow homologous recombination in vivo and yield a percentage of red colonies which reflects the proportion of mutant PCR products. Insertion mutations of single base of adenine (A) at stretches of 6 As were frequently detected in the liver samples of LEC rats which develop spontaneous hepatitis and hepatocellular carcinoma. For excluding the possibility of artifacts involvement, p53 cDNA was amplified by PCR from plasmids containing wild-type p53 and tested with the yeast functional assay, which resulted in no A insertion after sequencing 23 mutant clones. Furthermore, in vitro transcript of wild-type p53 was synthesized by SP6 RNA polymerase, and then, reverse-transcribed, PCR-amplified, and tested with the yeast functional assay. The overall rate of A insertion was much lower than that in the LEC rat liver. Since A insertions were found predominantly at nucleotides 293-298 in exon 4, an exon 4-specific yeast functional assay was developed. A insertion was detected in 4.8% of the PCR product of mRNA but 0-0.1% from genomic DNA, which suggested that such A insertion was caused by transcriptional slippage in vivo. The A insertion rate abruptly increased in acute hepatitis stage in the LEC rat liver, while the rate slowly increased by aging in control WKAH rat liver. It was suggested that cell damage and aging were primarily responsible for the increased rate of transcriptional slippage.
MeSH Terms
- Adenine
- Aging
- Animals
- Cells, Cultured
- DNA
- DNA Mutational Analysis
- Genes, p53
- Mutation
- Polymerase Chain Reaction
- RNA
- Rats
- Rats, Inbred LEC
- Rats, Wistar
- Saccharomyces cerevisiae
- Transcription, Genetic