Targeting vectors for gene diversification by meiotic recombination in Neurospora crassa.

We have constructed a pair of vectors, pDV2 and pDV3, that enable targeted insertion of exogenous DNA into Linkage Group I of Neurospora crassa at the his-3 locus. Transplaced sequences are inserted between his-3 and the cog(L) recombination hot spot and include his-3 mutations that allow meiotic recombination initiated by cog(L) to be monitored. Selection of correctly placed transforming DNA is based on complementation between different his-3 alleles borne by the plasmids and transformation hosts. The system allows investigation of the effect of any given sequence on recombination as well as diversification of sets of related sequences in vivo for directed evolution of genes.     

Alleles of the hotspot cog are codominant in effect on recombination in the his-3 region of Neurospora

There are two naturally occurring functional alleles of the recombination hotspot cog, which is located 3.5 kb from the his-3 locus of Neurospora crassa. The presence of the cog+ allele in a cross significantly increases recombination in the his-3 region compared to a cross homozygous for the cog allele. Data obtained shortly after discovery of cog+ suggested that it was fully dominant to cog. However, a dominant cog+ conflicts with observations of hotspots in Saccharomyces cerevisiae and Schizosaccharomyces pombe, in which recombination is initiated independently of homolog interactions, and suggests recombination mechanisms may differ in Neurospora and yeast. We present evidence that cog alleles are codominant in effect on both allelic recombination in his-3 and crossing over between loci flanking his-3. In addition, we show that genetic background variation has at least a twofold effect on allelic recombination. We speculate that variation in genetic background, together with the complexities of recombination in crosses bearing close mutant alleles, accounts for the previous conclusion that cog+ is dominant to cog.     

Long,interrupted conversion tracts initiated by cog in Neurospora crassa.

Multiple polymorphisms distinguish Emerson and Lindegren strains of Neurospora crassa within the histidine-3 gene and in its distal flank. Restriction site and sequence length polymorphism in a set of 14 PCR products covering this 6.9-kb region were used to identify the parental origin of DNA sequence information in prototrophic progeny of crosses heterozygous for auxotrophic mutations in his-3 and the silent sequence differences. Forty-one percent of conversion tracts are interrupted. Where the absence of rec-2+ permits activity of the recombination hotspot cog, conversion appears to originate at cog and conversion tracts are up to 5.9 kb long. The chromosome bearing cog(L), the dominant allele that confers a high frequency of recombination, is almost invariably the recipient of information. In progeny from crosses heterozygous rec-2/rec-2+, conversion tracts are much shorter, most are not initiated at cog and either chromosome seems equally likely to be converted. Although 32% of his-3 prototrophs have a crossover that may be associated with conversion, it is suggested that the apparent association between conversion and crossing over at this locus may be due to confounding of coincidental events rather than to a mechanistic relationship.     

Recombination events in Neurospora crassa may cross a translocation breakpoint by a template-switching mechanism

To assist investigation of the effect of sequence heterology on recombination in Neurospora crassa, we inserted the Herpes simplex thymidine kinase gene (TK) as an unselected marker on linkage group I, giving a gene order of Cen-his-3-TK-cog-lpl. We show here that in crosses heterozygous for TK, conversion of a his-3 allele on one homolog is accompanied by transfer of the heterologous sequence between cog and his-3 from the other homolog, indicating that recombination is initiated centromere-distal of TK. We have identified a 10-nucleotide motif in the cog region that, although unlikely to be sufficient for hotspot activity, is required for high-frequency recombination and, because conversion of silent sequence markers declines on either side, may be the recombination initiation site. Additionally, we have mapped conversion tracts in His(+) progeny of a translocation heterozygote, in which the translocation breakpoint separates cog from the 5' end of his-3. We present molecular evidence of recombination on both sides of the breakpoint. Because recombination is initiated close to cog and the event must therefore cross the translocation breakpoint, we suggest that template switching occurs in some recombination events, with repair synthesis alternating between use of the homolog and the initiating chromatid as template.     

Recombination at his-3 in Neurospora declines exponentially with distance from the initiator,cog

By deletion of 1.8 kb of sequence between cog(L) and his-3 and replacement with sequences of different lengths, we have generated a set of Neurospora strains in which the distance between cog(L) and the site at which recombination is selected varies from 1.7 to nearly 6 kb. Each of the manipulated strains includes cog(L), a highly active recombination hotspot, and rec-2, thus allowing high-frequency recombination. In addition, each is a his-3 mutant, either K26 or K480. The frequency of His(+) recombinants in progeny of these crosses is inversely proportional to the distance between his-3 and cog. Specifically, there is a linear relationship between log(10) (recombination frequency) and the distance in base pairs, indicating that as distance decreases, the rate of interallelic recombination increases exponentially. An exponential relationship between distance separating markers and the chance of co-conversion has been found in both Drosophila and fission yeast, indicating that the extension of recombination events may be a stochastic process in most organisms. On the basis of these and additional data presented in this article, we conclude that recombination is initiated at cog(L) in >17% of meioses, that most conversion tracts are very short, and that few extend >14 kb.     

Correcting errors in synthetic DNA through consensus shuffling

Although efficient methods exist to assemble synthetic oligonucleotides into genes and genomes, these suffer from the presence of 1–3 random errors/kb of DNA. Here, we introduce a new method termed consensus shuffling and demonstrate its use to significantly reduce random errors in synthetic DNA. In this method, errors are revealed as mismatches by re-hybridization of the population. The DNA is fragmented, and mismatched fragments are removed upon binding to an immobilized mismatch binding protein (MutS). PCR assembly of the remaining fragments yields a new population of full-length sequences enriched for the consensus sequence of the input population. We show that two iterations of consensus shuffling improved a population of synthetic green fluorescent protein (GFPuv) clones from ~60 to >90% fluorescent, and decreased errors 3.5- to 4.3-fold to final values of ~1 error per 3500 bp. In addition, two iterations of consensus shuffling corrected a population of GFPuv clones where all members were non-functional, to a population where 82% of clones were fluorescent. Consensus shuffling should facilitate the rapid and accurate synthesis of long DNA sequences.     

A complete mitochondrial genome sequence of Ogura-type male-sterile cytoplasm and its comparative analysis with that of normal cytoplasm in radish (Raphanus sativus L.)

ABSTRACT: BACKGROUND: Plant mitochondrial genome has unique features such as large size, frequent recombination and incorporation of foreign DNA. Cytoplasmic male sterility (CMS) is caused by rearrangement of the mitochondrial genome, and a novel chimeric open reading frame (ORF) created by shuffling of endogenous sequences is often responsible for CMS. The Ogura-type male-sterile cytoplasm is one of the most extensively studied cytoplasms in Brassicaceae. Although the gene orf138 has been isolated as a determinant of Ogura-type CMS, no homologous sequence to orf138 has been found in public databases. Therefore, how orf138 sequence was created is a mystery. In this study, we determined the complete nucleotide sequence of two radish mitochondrial genomes, namely, Ogura- and normal-type genomes, and analyzed them to reveal the origin of the gene orf138. RESULTS: Ogura- and normal-type mitochondrial genomes were assembled to 258,426-bp and 244,036-bp circular sequences, respectively. Normal-type mitochondrial genome contained 33 protein-coding and three rRNA genes, which are well conserved with the reported mitochondrial genome of rapeseed. Ogura-type genomes contained same genes and additional atp9. As for tRNA, normal-type contained 17 tRNAs, while Ogura type contained 17 tRNAs and one additional trnfM. The gene orf138 was specific to Ogura-type mitochondrial genome, and no sequence homologous to it was found in normal-type genome. Comparative analysis of the two genomes revealed that radish mitochondrial genome consists of 11 syntenic regions (length >3kb, similarity >99.9%). It was shown that short repeats and overlapped repeats present in the edge of syntenic regions were involved in recombination events during evolution to interconvert two types of mitochondrial genome. Ogura-type mitochondrial genome has four unique regions (2,803 bp, 1,601 bp, 451 bp and 15,255 bp in size) that are non-syntenic to normal-type genome, and the gene orf138 was found to be located at the edge of the largest unique region. Blast analysis performed to assign the unique regions showed that about 80% of the region was covered by short homologous sequences to the mitochondrial sequences of normal-type radish or other reported Brassicaceae species, although no homology was found for the remaining 20% of sequences. CONCLUSIONS: Ogura-type mitochondrial genome was highly rearranged compared with the normal-type genome by recombination through one large repeat and multiple short repeats. The rearrangement has produced four unique regions in Ogura-type mitochondrial genome, and most of the unique regions are composed of known Brassicaceae mitochondrial sequences. This suggests that the regions unique to the Ogura-type genome were generated by integration and shuffling of pre-existing mitochondrial sequences during the evolution of Brassicaceae, and novel genes such as orf138 could have been created by the shuffling process of mitochondrial genome.     

Analysis of two genes encoding heat-labile toxins and located on a single Ent plasmid from Escherichia coli.

A clinical isolate of Escherichia coli harbored two copies of the heat-labile toxin (LT)-encoding gene (elt) on a 157-kb plasmid. The arrangement of the gene copies is different from the cholera toxin-encoding gene duplication described for some strains of Vibrio cholerae. The nucleotide sequences of the elt alleles are not identical (differing by 2 bp) and the duplicated region flanking the alleles extends 314 bp on one side and 1122 bp on the other side of each copy. Different partial copies of IS600 were identified 280 bp 3' to the stop codon of each allele. Partial and complete copies of other IS were also identified near the elt alleles. The data suggest that the regions surrounding the genes are hot spots for IS which would account for the observed heterogeneity in DNA flanking elt genes.     

Structure and evolution of the bovine prothrombin gene

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.     

中华菊头蝠MHCⅡ-DRB 基因遗传多态性及进化

采样自云南同一种群的中华菊头蝠共16 个个体,用于DRB 基因的分子进化和多态性研究。利用翼膜组织提取DNA 基因组,并PCR 克隆测序分析。获得了相差3 bp 的两种不同长度序列类型,A 序列类型263 bp,在研究群体中有15 个等位基因;B 序列类型260 bp,在研究群体中有8 个等位基因。在分析的74 个氨基酸变异位点上检测到12 个正向选择位点。在9 个个体中检测到分布频率最高的等位基因,也有多个等位基因只存在一个个体中。单个个体中最多存在6 个等位基因。遗传多态性分析表明中华菊头蝠DRB 基因具有较高的多态性。中华菊头蝠DRB 基因可能至少存在3 个重复座位。利用已发表的翼手目DRB 第二外显子序列构建的系统进化树表明中华菊头蝠MHCⅡ-DRB 基因处于独立进化支。     

Mutation of msh-2 in Neurospora crassa does not reduce the incidence of recombinants with multiple patches of donor chromosome sequence

The Neurospora homologue msh-2 of the Escherichia coli mismatch repair gene mutS was mutated by repeat-induced point mutation (RIP) of a 1.9-kb duplication covering 1661bp of the coding sequence and 302 bp 5' of the gene. msh-2(RIP-LK1) exhibited a mutator phenotype conferring a 17-fold increase in the frequency of spontaneous mitotic reversion of his-3 allele K458. In msh-2(RIP-LK1) homozygotes, recombination frequency at the his-3 locus increased up to 2.9-fold over that in msh-2(+) diploids. Progeny of crosses homozygous msh-2(RIP-LK1), like those from crosses homozygous msh-2(+) frequently had multiple patches of donor chromosome sequence, suggesting that patchiness in msh-2(+) crosses is not explained by incomplete repair of heteroduplex DNA by MSH-2. These findings are consistent with data from the analysis of events in a Neurospora translocation heterozygote that suggested multiple patches of donor chromosome sequence arising during recombination reflect multiple template switches during DNA repair synthesis.     

Predicting out-of-sequence reassembly in DNA shuffling

We present an analysis for calculating the frequency of out-of-sequence reassembly in DNA shuffling experiments. Out-of-sequence annealing events are undesirable since they typically encode non-functional proteins with missing or repetitive regions. The approach builds on the e Shuffle framework for the prediction of crossover formation using equilibrium thermodynamics and complete sequence information to model the reassembly process. An in silico case study of a set of subtilases reveals that, as expected, the presence of significant sequence identity between distant portions of the parental sequences gives rise to out-of-sequence annealing events that upon reassembly generate sequences with missing or repetitive DNA segments. The frequency of these events increases as the fragment length decreases. Interestingly, out-of-sequence annealing events are at a minimum near the annealing temperature of 55 degrees C used in the original DNA shuffling protocol. Neither parental sequence identity nor number of shuffled parents significantly alter the extent of out-of-sequence reassembly     

一种检测转1Dx5基因小麦植株的新方法

目的:建立一种有效区分Glu-1D等位基因的Multiplex-PCR体系,快速检测转1Dx5小麦植株。方法:根据1Dx5和1Dx2亚基基因的区别设计特异的PCR引物,通过多重PCR技术检测花粉管通道法转化1Dx5核心片段的转基因T1代植株。结果:Multiplex-PCR能够在转基因T1代材料中扩增Glu-1D等位基因的特征条带,分别为343bp、320bp的1Dx5基因特异片段以及361bp的1Dx2基因特异片段,与预期结果一致。结论:该技术能够检测多个靶基因,有效地区分转基因Glu-1D上的等位基因,证实外源1Dx5基因已整合到受体基因组中,对检测基因组庞大、外源基因序列GC含量高且与内源基因同源性高的转基因小麦十分有效。     

Analysis of shuffled gene libraries

In vitro recombination of homologous genes (family shuffling) has been proposed as an effective search strategy for laboratory evolution of genes and proteins. Few data are available, however, on the composition of shuffled gene libraries, from which one could assess the efficiency of recombination and optimize protocols. Here, probe hybridization is used in a macroarray format to analyze chimeric DNA libraries created by DNA shuffling. Characterization of hundreds of shuffled genes encoding dioxygenases has elucidated important biases in the shuffling reaction. As expected, crossovers are favored in regions of high sequence identity. A sequence-based model of homologous recombination that captures this observed bias was formulated using the experimental results. The chimeric genes were found to show biases in the incorporation of sequences from certain parents, even before selection. Statistically different patterns of parental incorporation in genes expressing functional proteins can help to identify key sequence-function relationships.     

Special factors in biological strings

Biological macromolecules such as DNA, RNA, and proteins can be regarded as finite sequences of symbols (or words) over a finite alphabet. In this paper, we refer to DNA (RNA) sequences which are words on a four-letter alphabet. A comparison is made between some "genes", or fragments of them, with random sequences or random reshuffled sequences on the same alphabet and having the same length. Some combinatorial techniques of analysis of finite words are developed. A crucial role in the comparison is played by the so-called special factors of a given word. In all the analysed DNA (RNA) fragments the distribution on the length of the number of right (left) special factors differs, in a very typical way, from the corresponding distribution in a string on the same alphabet and having the same length generated by a random source or obtained by making a random alteration (=shuffling) of the original string. This kind of change is irrespective of the length in the range that we have considered <2650 bp and of the phylogenetic origin of the fragment.     

Internal eliminated sequences interrupting the Oxytricha 81 locus: allelic divergence, conservation, conversions, and possible transposon origins

Internal eliminated sequences (IESs) often interrupt ciliate genes in the silent germline nucleus but are exactly excised and eliminated from the developing somatic nucleus from which genes are then expressed. Some long IESs are transposons, supporting the hypothesis that short IESs are ancient transposon relics. In light of that hypothesis and to explore the evolutionary history of a collection of IESs, we have compared various alleles of a particular locus (the 81 locus) of the ciliated protozoa Oxytricha trifallax and O. fallax. Three short IESs that interrupt two genes of the locus are found in alleles from both species, and thus must be relatively ancient, consistent with the hypothesis that short IESs are transposon relics. In contrast, TBE1 transposon interruptions of the locus are allele-specific and probably the results of recent transpositions. These IESs (and the TBE1s) are precisely excised from the DNA of the developing somatic macronucleus. Each IES interrupts a highly conserved sequence. A few nucleotides at the ends of each IES are also conserved, suggesting that they interact critically with IES excision machinery. However, most IES nucleotide positions have evolved at high rates, showing little or no selective constraint for function. Nonetheless, the length of each IES has been maintained (+/- 3 bp). While one IES is approximately 33 bp long, three other IESs have very similar sizes, approximately 70 bp long. Two IESs are surrounded by direct repeats of the sequence TTCTT. No other sequence similarities were found between any of the four IESs. However, the ends of one IES do match the inverted terminal repeat consensus sequence of the "TA" IESs of Paramecium. Three O. trifallax alleles appear to have been recipients in recent conversion events that could have been provoked by double-strand breaks associated with IES ends subsequent to IES transposition. Our findings support the hypothesis that short IESs evolved from ancient transposons that have lost most of their sequences, except those necessary for precise excision during macronuclear development.      

Digestion-after-shuffling: a method for combinatorial library construction

We have developed a convenient method for family shuffling of amino acid sequences, termed digestion-after-shuffling. After DNA shuffling of homologous genes, plasmid mixture is extracted from a library and used for several double digestions with restriction enzymes. For each double digestion, two restriction enzymes are selected, corresponding to the single restriction sites of different parental genes. After digestions, fragments with expected sizes are obtained by gel purification and religated to construct recombinant plasmids. Thus, the obtained genes should be chimeras and have at least two restriction sites originating from different parental sequences.     

Prospecting metagenomic enzyme subfamily genes for DNA family shuffling by a novel PCR-based approach

DNA family shuffling is a powerful method for enzyme engineering, which utilizes recombination of naturally occurring functional diversity to accelerate laboratory-directed evolution. However, the use of this technique has been hindered by the scarcity of family genes with the required level of sequence identity in the genome database. We describe here a strategy for collecting metagenomic homologous genes for DNA shuffling from environmental samples by truncated metagenomic gene-specific PCR (TMGS-PCR). Using identified metagenomic gene-specific primers, twenty-three 921-bp truncated lipase gene fragments, which shared 64-99% identity with each other and formed a distinct subfamily of lipases, were retrieved from 60 metagenomic samples. These lipase genes were shuffled, and selected active clones were characterized. The chimeric clones show extensive functional and genetic diversity, as demonstrated by functional characterization and sequence analysis. Our results indicate that homologous sequences of genes captured by TMGS-PCR can be used as suitable genetic material for DNA family shuffling with broad applications in enzyme engineering.