AbstractGenomic DNA of zebrafish was first digested incompletely with Msp I, and then the fragments were joined to form rearranged genomic DNA. This rearranged genomic DNA was incompletely digested with EcoR I, and the fragments were linked with a long adaptor. Two primers (Gmprimer1 and Gmprimer2) were designed according to the adaptor sequence for two-step amplification. The Gmprimer1-amplified products were microinjected into fertilized zebrafish eggs after purification and a red flesh mutant was observed among 42 surviving zebrafish. We obtained several introduced sequences by two-step amplification. The second set of Gmprimer2-amplified products were purified and microinjected into fertilized zebrafish eggs; all 37 surviving fish were red flesh mutants. We found that the largest amplified band from the mutant from the first microinjection was also present in the amplified pattern from six mutants from the second microinjection. The length of the sequence was 2,565 bp, but it did not encode any proteins. Microinjecting this sequence into fertilized zebrafish eggs produced the red flesh mutant. The sequences differed slightly among different individuals from the second microinjection. Most regions of these sequences were the same, with the exception of a hypervariable region. We mixed 10 such sequences equally and microinjected them into zebrafish zygotes; the findings showed that most zygotes died and the surviving zebrafish were almost all mutants. By genome walking, we found that the site of insertion of the fragment was the same, beginning at position 41,365,003 of the eighth chromosome, and that downstream of the introduced fragment is a conservative sequence of 6,536 bp (named Cao-sequence), starting from a small reverse repeat sequence, not encoding any gene, nor similar to any known regulatory sequence. It has 322 homologous sequences in the zebrafish genome, which are distributed in all chromosomes. We designed two primers within Cao-sequence and several primers specific for different locations upstream of it. Compared with normal zebrafish, we found that the amplified patterns of all mutants in Cao-sequence regions changed to varying degrees. To further understand the effect of the introduced sequence on the zebrafish genomes, we selected six mutants for whole-genome resequencing. The results showed that numerous Cao-sequences from these six mutants were partially deleted and the lengths of the deletions was mostly approximately 6,100 bp, being located at the 5′ end of Cao-sequences. Among them, 43 Cao-sequence loci were commonly deleted from the six mutants (with slightly different locations), and the other deletion sites were not identical. We think that different deletion combinations of Cao-sequence may show different mutation characteristics. The tail part from four red flesh mutants and three individuals of wild type were collected for transcriptome sequencing. TopGO analysis showed that the 4 most significant enrichment nodes were sequence specific DNA binding proteins, sequence specific transcription factors, chromatin proteins and zinc binding proteins. The results of KEGG enrichment analysis showed that the top four affected KEGG-pathways were metabolic pathways, oxidative phosphorylation, citrate cycle and 2-oxocarboxylic acid metabolism.We conclude that deletion of Cao-sequence can affect the expression of a series of transcription regulators and specific DNA binding proteins, then many basic metabolic processes were disturbed which led to mutations.
Single Molecule Studies of DNA-Binding Proteins: Development of New Covalent DNA Anchoring Techniques for the Study of Rupture Forces of Replication Blocks