Recognition and cleavage site of the intron-encoded omega transposase.

The optional group I intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae contains a 235-codon-long open reading frame the translation product of which (the omega transposase) catalyzes the formation of a double-strand break within the intron-minus (omega-) copies of the same gene. Purified omega transposase generates in vitro a 4-base-pair staggered cut with 3' hydroxyl overhangs at the exact position where the intron eventually inserts in the gene. Using randomly mutagenized synthetic oligonucleotides, single-base mutants were produced at 21 positions around the cleavage site. Experiments with these oligonucleotides show that the recognition site extends over an 18-base pair-long sequence within which minimal sequence degeneracy is tolerated. The intron-encoded omega transposase is, therefore, one of the most specific restriction endonucleases known to date.     

Gene conversion at the var1 locus on yeast mitochondrial DNA.

Alleles of the var1 locus on yeast mtDNA determine the apparent size of the mitochondrial translation product, var1 polypeptide. We have analyzed most of the different var1 alleles in our collection, which number at least 15, and have developed procedures and a genetic rationale for determining their origin and predicting their behavior in crosses. The var1 alleles are characterized by two genetically defined segments, designated a and b, which can move from one var1 allele to another by asymmetric gene conversion. We show that the a segment behaves as an entity in recombination; it is either present in or absent from different var1 alleles. The b segment usually, but not always, recombines as an entity; in some cases, only portions of the b segment recombine by gene conversion. Thus, the total number of electrophoretically resolvable var1 species we observe is explained by the assortment of a, b, and partial b segments. Each segment recombines at a characteristic frequency; however, one example is presented which shows that the recipient can modulate the frequency of gene conversion. Finally, we show that, like the 21S rDNA region (omega), there is polarity of gene conversion within var1.     

The orangutan adult alpha-globin gene locus: duplicated functional genes and a newly detected member of the primate alpha-globin gene family.

We have cloned and sequenced the complete alpha 1- and alpha 2-globin genes of the orangutan, and here we compare them to the homologous genes of the human. The pattern of similarity apparent among the genes is most consistent with a model of gene correction operating on the primate alpha-globin cluster. This correction breaks down in both human and orangutan in the 3'-untranslated region at 14 base pairs downstream from the termination codon. The unit evolutionary period values calculated for either the replacement substitution or the silent substitutions are only slightly higher than the previously established molecular clock predicts. The 7-base-pair insertion in intron 2 of the human alpha 1-globin gene is not present in either orangutan gene, suggesting that this insertion is not the cause of the sequence divergence in the 3'-untranslated regions of primate alpha 2- and alpha 1-globin genes. Finally, blotting hybridization and partial DNA sequencing reveal a newly detected member of the primate alpha-globin gene family, which is located downstream from the duplicated adult alpha-globin genes.     

Double-strand gap repair results in homologous recombination in mouse L cells.

Previous studies have demonstrated that the presence of double-strand breaks or double-strand gaps increases the frequency of homologous recombination between two cotransferred DNAs when they are introduced into cultured mammalian cells. Here we demonstrate that the repair of these double-strand gaps is a major mechanism for homologous recombination between exogenous DNAs. In particular, when a plasmid DNA containing a 104-base-pair (bp) gap in its tk gene (herpes simplex virus gene for thymidine kinase) undergoes recombination in mouse L cells to generate an intact gene, the majority of events result from direct repair of the double-strand gap using a cotransferred DNA as the template. We analyzed the recombination events by comparing the frequency of tk+ colonies, Southern blotting of cloned tk+ cell lines, and cloning recombined functional tk genes by plasmid rescue. In addition, by creating double-strand breaks within or adjacent to heterologous insertions in a mutant tk gene, we estimate that the L cell can generate a double-strand gap of between 152 and 248 bp and then can repair the gap to create a functional tk gene. We conclude that double-strand breaks and double-strand gaps are recombinogenic in transferred plasmid DNAs because they serve as intermediates in homologous recombination by double-strand gap repair, a nonreciprocal exchange of DNA or gene conversion event.     

Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites

A typical homing endonuclease initiates mobility of its group I intron by recognizing DNA both upstream and downstream of the intron insertion site of intronless alleles, preventing the endonuclease from binding and cleaving its own intron-containing allele. Here, we describe a GIY-YIG family homing endonuclease, I-BmoI, that possesses an unusual recognition sequence, encompassing 1 base pair upstream but 38 base pairs downstream of the intron insertion site. I-BmoI binds intron-containing and intronless substrates with equal affinity but can nevertheless discriminate between the two for cleavage. I-BmoI is encoded by a group I intron that interrupts the thymidylate synthase (TS) gene (thyA) of Bacillus mojavensis s87-18. This intron resembles one inserted 21 nucleotides further downstream in a homologous TS gene (td) of Escherichia coli phage T4. I-TevI, the T4 td intron-encoded GIY-YIG endonuclease, is very similar to I-BmoI, but each endonuclease gene is inserted within a different position of its respective intron. Remarkably, I-TevI and I-BmoI bind a homologous stretch of TS-encoding DNA and cleave their intronless substrates in very similar positions. Our results suggest that each endonuclease has independently evolved the ability to distinguish intron-containing from intronless alleles while maintaining the same conserved recognition sequence centered on DNA-encoding active site residues of TS.     

Yeast recombination: the association between double-strand gap repair and crossing-over.

In previous experiments, we have used yeast transformation to study the recombinogenic repair of double-strand breaks and gaps. A plasmid containing a double-strand gap within sequences homologous to the yeast genome integrates efficiently by crossing-over. During the process of integration, the double-strand gap is repaired, using chromosomal information as a template. This repair reaction results in the transfer of genetic information from one DNA duplex to another and is therefore a pathway for gene conversion. Because meiotic gene conversion is associated with a high frequency (up to 50%) of crossing-over, we wished to determine the degree of association of double-strand gap repair with crossing-over. Only the class of repair events resulting in crossovers (plasmid integration) were detected in our earlier experiments with nonreplicating plasmids. In this paper, we describe the outcome of double-strand gap repair in plasmids that are capable of autonomous replication and therefore allow recovery of both crossover and noncrossover products. After the correct repair of a double-strand gap, we recover approximately equal numbers of integrated and nonintegrated plasmids. Thus, gene conversion by double-strand gap repair can occur either with or without crossing-over, and it is similar in this respect to meiotic gene conversion. Circularization of linear plasmid DNA by ligation is also observed, suggesting that yeast has an additional repair pathway for double-strand breaks that is independent of recombination. Gap repair on replicating plasmids permits rapid cloning of chromosomal alleles.     

Insertion in the mRNA of a metachromatic leukodystrophy patient with sphingolipid activator protein-1 deficiency.

The lysosomal catabolism of sulfatide requires arylsulfatase A and a specific sphingolipid activator protein, SAP-1. While most patients with metachromatic leukodystrophy have mutations in the gene for arylsulfatase A, some patients have deficient SAP-1, as determined by immunological techniques. We now describe the molecular findings in a patient who died at 22 years of age with SAP-1 deficiency. The DNA polymerase chain reaction was used to amplify regions of cDNA which were subcloned in M13 phage DNA and sequenced by the dideoxy chain-termination method. The patient was found to have a 33-base-pair insertion between nucleotides 777 and 778 (numbered from the A of the ATG initiation codon). No other changes were found in the coding sequence of the cDNA from this patient. At the site of the insertion some normal people have an additional 9 base pairs, which correspond to the last 9 nucleotides at the 3' end of the insertion. The cDNAs from the second-cousin parents were amplified and sequenced, and in both two alleles were identified, one with the 33-base-pair insertion and one with no insertion. Two brothers were found to have only the normal alleles and a sister was found to have the 33-base-pair insertion and a normal allele. The findings confirm studies performed on leukocyte extracts demonstrating normal antigen levels in the two brothers and a lower level in the sister. The presence of 11 additional amino acids in the coding region of mature SAP-1 in this patient causes significant changes in the hydropathy profile compatible with the previous findings at the protein level.     

Characterization of the restriction site of a prokaryotic intron-encoded endonuclease.

The 1016-base-pair (bp) intron in the T4 bacteriophage thymidylate synthase gene (td) contains a 735-bp open reading frame that encodes a protein product with endonucleolytic activity. The endonuclease shows specificity for the intronless form of the td gene. Highly purified endonuclease cleaves the DNA of the intronless form of the td gene in vitro at 24 bp upstream of the exon 1-exon 2 junction, generating a 2-base staggered cut with 3'-hydroxyl overhangs. Although the endonuclease cleaves in exon 1, it requires some exon 2 sequence for recognition. The maximum recognition sequence lies in an 87-bp stretch, from 52 bp upstream to 35 bp downstream of the cleavage site, ending at 11 bp into exon 2. The td intron endonuclease appears involved in the conversion of the intronless form of td to intron-containing td gene in the T-even phages. A role for intron mobility is discussed.     

Sequences homologous to ribosomal insertions occur in the Drosophila genome outside the nucleolus organizer

Many repeating units of Drosophila melanogaster rDNA contain a DNA sequence within the gene for 28S rRNA that does not code for rRNA. This sequence has been called the ribosomal insertion [Wellauer, P. K. & Dawid, I. B. (1977) Cell 10, 193-212]. We report here that members of the same sequence family occur outside the ribosomal locus. "Non-rDNA insertion DNA" was separated from rDNA by density gradient centrifugation, and sequences homologous to the ribosomal insertion were detected by hybridization with restriction endonuclease fragments derived from a cloned rDNA repeating unit. Pure insertion sequences from cloned rDNA separated from main band DNA and behaved like a component with high G + C content. Non-rDNA components hybridizing to the insertion also separated from main band DNA but less so than pure insertion sequences, suggesting that non-rDNA insertion sequences are linked to DNA of different nucleotide composition. Restriction endonuclease analysis of non-rDNA insertion DNA showed many fragments of different sizes. The patterns obtained were similar in embryonic, larval, pupal, and adult DNA and DNA from cultured cells (Schneider cell line 3). Non-rDNA insertion sequences account for about 0.2% of the genome or about 400 kbases of DNA per haploid complement.     

Identification and order of sequential mutations in beta-actin genes isolated from increasingly tumorigenic human fibroblast strains.

We have sequenced the mutant beta-actin gene of a tumorigenic human fibroblast cell line (HuT-14T) and found that it carries three mutations that alter the amino acids at positions 36, 83, and 244 as well as a 22-base-pair "insertion" sequence, in the 5' intron, not present in a wild-type gene. The less tumorigenic cell line HuT-14, a progenitor of HuT-14T, has the same codon-244 mutation and the insertion sequence but not the other two mutations. A nontumorigenic cell line that is related to HuT-14 but that has no beta-actin mutations does carry the intron-length polymorphism. We conclude that the mutation at codon 244 occurred first in a beta-actin allele already bearing the 22-base-pair intron insert and that mutations at codons 36 and 83 arose subsequently during the selection for the HuT-14T phenotype. Rat-2 cells synthesize the appropriate charge-variant species of mutant actin protein when transfected with either the singly or the triply mutated beta-actin gene.     

Sequence specific cleavage of DNA by the antitumor antibiotics neocarzinostatin and bleomycin.

We have investigated the sites of DNA damage by the antitumor antibiotics neocarzinostatin and bleomycin by using a 5'-end-labeled DNA fragment of defined sequence as a substrate. At the high drug concentrations used here, neocarzinostatin creates single-strand breaks in DNA at positions of adenine and thymine in the presence of 2-mercaptoethanol, and bleomycin cleaves DNA at GC and GT sequences and to a lesser extent at TA sequences with its degradative activity enhanced by 2-mercaptoethanol. In the presence of ferrous ions, bleomycin cleaves DNA at TT, AT, and TA, as well as at GC and GT sequences. Both antibiotics make double-strand breaks in DNA at specific sites and it is likely that these result from two independent single-strand breaks at nearby sites on opposite strands of the DNA.     

Inactivation of the cholinesterase gene by Alu insertion: possible mechanism for human gene transposition.

The human cholinesterase (ChE) gene from a patient with acholinesterasemia was cloned and analyzed. By using ChE cDNA as a probe, four independent clones were isolated from a genomic library constructed from the patient's DNA. Sequencing analysis of all of the four clones revealed that exon 2 of the ChE gene was disrupted by a 342-base-pair (bp) insertion of Alu element, including a poly(A) tract of 38 bp, which showed 93% sequence homology with a current type of human Alu consensus sequence. Southern blot analysis showed that the Alu insertion occurred in both alleles of the patient and was inherited in the patient's family. This Alu insertion was flanked by 15-bp of target site duplication in exon 2 corresponding to positions 1062-1076 of ChE cDNA, indicating that an Alu element could have been integrated by retrotransposition. Thus, this case provides an important clue to the mechanism of inactivation of a gene by integration of a retrotransposon.     

The fate of 8-methoxypsoralen photoinduced crosslinks in nuclear and mitochondrial yeast DNA: comparison of wild-type and repair-deficient strains.

In Saccharomyces cerevisiae, after 8-methoxypsoralen [8-(OMe)Ps] photoaddition, more crosslinks are induced per unit dose in mitochondrial DNA than in nuclear DNA. In wild-type cells treated in the exponential phase of growth, single- and double-strand breaks are produced during crosslink removal and then are rejoined upon postexposure incubation. The incision step is almost blocked in the rad 3-2 mutant, which is also defective in excision-repair of UV-induced (254 nm) pyrimidine dimers. The cutting of crosslinks from nuclear DNA is depressed in wild-type stationary-phase cells. This is correlated with a higher sensitivity of such cells to 8-(OMe)Ps photoinduced cell killing. The incision of crosslinks is dramatically reduced in mitochondrial DNA. The rejoining of single- and double-strand breaks is not only dependent on the product of the RAD51 gene (as shown by others) but also of the PSO2 gene. A correlation was found between the ability to recombine and strand rejoining. Therefore, as in bacteria, both the excision and the recombinational repair systems are involved in crosslink repair in yeast. However, double-strand breaks in yeast constitute repair intermediates which are not detected in Escherichia coli. The LD37 (dose necessary to induce a mean of one lethal hit per cell) corresponds to about 120 crosslinks per genome in exponential-phase cells of the wild type and to 1-2 crosslinks in the pso2-1 mutant.