Simultaneous targeted exchange of two nucleotides by single-stranded oligonucleotides clusters within a region of about fourteen nucleotides.

BACKGROUND: Transfection of cells with gene-specific, single-stranded oligonucleotides can induce the targeted exchange of one or two nucleotides in the targeted gene. To characterize the features of the DNA-repair mechanisms involved, we examined the maximal distance for the simultaneous exchange of two nucleotides by a single-stranded oligonucleotide. The chosen experimental system was the correction of a hprt-point mutation in a hamster cell line, the generation of an additional nucleotide exchange at a variable distance from the first exchange position and the investigation of the rate of simultaneous nucleotide exchanges. RESULTS: The smaller the distance between the two exchange positions, the higher was the probability of a simultaneous exchange. The detected simultaneous nucleotide exchanges were found to cluster in a region of about fourteen nucleotides upstream and downstream from the first exchange position. CONCLUSION: We suggest that the mechanism involved in the repair of the targeted DNA strand utilizes only a short sequence of the single-stranded oligonucleotide, which may be physically incorporated into the DNA or be used as a matrix for a repair process.

Concurrent targeted exchange of three bases in mammalian hprt by oligonucleotides.

The repair of point mutations in hprt gene by single-stranded oligonucleotides represents a model to test targeted nucleotide exchange. We studied the concurrent nucleotide exchange of two or three nucleotides in the hprt deficient hamster cell line V79-151. The used oligonucleotides resulted in mismatches at two (151, 159) or three (151, 144, and 159) hprt positions. The hprt point mutation at position 151 was repaired in about 2/10(6) cells as shown by hprt sequencing in clones surviving HAT selection. The second nucleotide exchange at hprt position 159 was found in 7% of these HAT selected clones. Using oligonucleotides resulting in three mismatches, 29% of the clones showed nucleotide exchanges at the two hprt positions (151, 144) and about 4% at three positions (151, 144, and 159). These results indicate that single-stranded oligonucleotides can generate two or three nucleotide exchanges in a mammalian chromosomal gene.

In NF1, CFTR, PER3, CARS and SYT7, alternatively included exons show higher conservation of surrounding intron sequences than constitutive exons.

It is still not fully understood to what extent intronic sequences contribute to the regulation of the different forms of alternative splicing. We are interested in the regulation of alternative cassette exon events, such as exon inclusion and exon skipping. We investigated these events by comparative genomic analysis of human and mouse in five experimentally well-characterized genes, neurofibromatosis 1 (NF1), cystic fibrosis transmembrane conductance regulator (CFTR), period 3 (PER3), cysteinyl-tRNA synthetase (CARS) and synaptotagmin 7 (SYT7). In NF1, high intron identity around the 52 constitutive and four alternatively skipped NF1 exons is restricted to the close vicinity of the exons. In contrast, we found on average high conservation of intron sequences over 300 base pairs up- and downstream of the five alternatively included NF1 exons. The investigation of alternatively included exons in CFTR, PER3, CARS and SYT7 supported this finding. In contrast, the mean intron identities around the alternatively skipped exons in CTFR and NF1 do not differ considerably from those around the constitutive exons. In these genes, the difference in intron conservation could point to a difference between the regulation of alternative exon inclusion and alternative exon skipping or constitutive exon splicing. Additional genome-wide investigations are necessary to elucidate to what extent our finding can be generalized.

Targeted gene correction of hprt mutations by 45 base single-stranded oligonucleotides.

Targeted correction of a single base in a gene of an eucaryotic cell by specific oligonucleotides is a yet controversial technique. Here, we introduce the correction of point mutations in the hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) gene as an additional model system to test targeted gene correction. In human, Hprt mutations cause Lesch-Nyhan syndrome. Using hamster V79 cells, we generated three cell lines with one hprt point mutation each. These cell lines were treated with specific single-stranded 45 base phosphothioate modified oligonucleotides and selected by HAT medium. The surviving clones were investigated for the correction of the respective hprt mutation. Treatment with the oligonucleotides was successful in repairing all three hprt mutations (hprt cDNA position 74, C --> T; position 151, C --> T; and position 400, G --> A). The correction efficiency was very low but reproducible. We suggest that this system allows one to investigate targeted gene correction in dependence on the target sequence and the oligonucleotides used.

The N-terminal splice product NF1-10a-2 of the NF1 gene codes for a transmembrane segment.

One important function of the neurofibromatosis type 1 (NF1) product neurofibromin is the negative regulation of Ras activity on the cell membrane. Here, we describe an alternative splice product of the N-terminus of the NF1 gene. In this splice product, termed NF1-10a-2, the 45 bp exon 10a-2 is inserted between exons 10a and 10b. Amino acid sequence analysis for motifs showed that the new splice product contains a transmembrane segment not found in wild-type neurofibromin. The overall expression was found to be very low in comparison to the expression of the wild-type mRNA in all human primary and tumor cells examined. Because transcripts were found in the majority of human tissues examined, we assume a housekeeping function of this splice product. Investigation of the intracellular localization of an NF1-10a-2-EGFP fusion protein in HeLa cells revealed a preferential localization in perinuclear granular structures. We therefore assume that NF1-10a-2 has a function on an intracellular membrane.

Aberrant splicing in several human tumors in the tumor suppressor genes neurofibromatosis type 1, neurofibromatosis type 2, and tuberous sclerosis 2.

Mutations at splice sites or surrounding sequences have been reported to cause aberrant splicing. However, splicing errors can also occur without sequence alterations. We investigated three tumor suppressor genes for aberrant splicing in tumors. At a low frequency per exon it was found in five of seven of the investigated in-frame exons of the neurofibromatosis type 1 (NF1) gene, in two of three exons of the neurofibromatosis type 2 (NF2) gene, and in one of three exons of the tuberous sclerosis 2 gene. It was detectable in all of the human tumor tissues tested (NF1 neurofibroma, sporadic intramedullar neurinoma, sporadic meningiomas, NF2 schwannoma, NF2 meningioma, basalioma, and naevus) as well as in cultured tumor cell lines and cultured primary cells. Hence, our data show that aberrant splicing is a very common process. According to simulations of the secondary structures of the pre-mRNA, we suggest that aberrant splicing is attributable to the rare occurrence of alternative structures at the splice donor site, which are not recognized by the splice machinery. In HeLa cells, aberrant splicing is found to be increased at elevated temperatures and low pH in vitro, conditions often found in tumor tissues. In three tumor tissues tested for one NF1 exon, we found approximately twice the amount of aberrant transcript as in normal tissues. Therefore, we suggest that the increase in aberrant splicing caused by environmental factors represents an additional mechanism for the reduction of the amount of tumor suppressor mRNA in the absence of relevant mutations in the tumor.