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1.
Somatic excision of the Mu1 transposable element of maize.   总被引:8,自引:1,他引:7       下载免费PDF全文
The Mu transposons of the Robertsons's Mutator transposable element system in maize are unusual in many respects, when compared to the other known plant transposon systems. The excision of these elements occurs late in somatic tissues and very rarely in the germ line. Unlike the other plant transposons, there is no experimental evidence directly linking Mu element excision and integration. We have analyzed the excision products generated by a Mu1 transposon inserted into the bronze 1 locus of maize. We find that the excision products or 'footprints' left by the Mu1 element resemble those of the other plant transposable elements, rather than those of the animal transposable element systems. We also find some novel types of footprints resembling recombinational events. We suggest that the Mu1 element can promote intrachromosomal crossovers and conversions near its site of insertion, and that this may be another mechanism by which transposons can accelerate the evolution of genomes.  相似文献   

2.
The Mu transposon of maize exists in a highly mutagenic strain called Robertson's Mutator. Plants of this strain contain 10-50 copies of the Mu element, whereas most maize strains and other plants have none. When Mutator plants are crossed to plants of the inbred line 1S2P, which does not have copies of Mu, the progeny plants have approximately the same number of Mu sequences as did their Mutator parent. Approximately one-half of these copies have segregated from their parent and one-half have arisen by transposition and are integrated into new positions in the genome. This maintenance of copy number can be accounted for by an extremely high rate of transposition of the Mu elements (10-15 transpositions per gamete per generation). When Mutator plants are self-pollinated, the progeny double their Mu copy number in the first generation, but maintain a constant number of Mu sequences with subsequent self-pollinations. Transposition of Mu and the events that lead to copy number maintenance occur very late in the development of the germ cells but before fertilization. A larger version of the Mu element transposes but is not necessary for transposition of the Mu sequences. The progeny of crosses with a Mutator plant occasionally lack Mutator activity; these strains retain copies of the Mu element, but these elements no longer transpose.  相似文献   

3.
Insertional mutagenesis is a cornerstone of functional genomics. High-copy transposable element systems such as Mutator ( Mu ) in maize ( Zea mays ) afford the advantage of high forward mutation rates but pose a challenge for identifying the particular element responsible for a given mutation. Several large mutant collections have been generated in Mu -active genetic stocks, but current methods limit the ability to rapidly identify the causal Mu insertions. Here we present a method to rapidly assay Mu insertions that are genetically linked to a mutation of interest. The method combines elements of MuTAIL (thermal asymmetrically interlaced) and amplification of insertion mutagenized sites (AIMS) protocols and is applicable to the analysis of single mutants or to high-throughput analyses of mutant collections. Briefly, genomic DNA is digested with a restriction enzyme and adapters are ligated. Polymerase chain reaction is performed with TAIL cycling parameters, using a fluorescently labeled Mu primer, which results in the preferential amplification and labeling of Mu -containing genomic fragments. Products from a segregating line are analyzed on a capillary sequencer. To recover a fragment of interest, PCR products are cloned and sequenced. Sequences with lengths matching the size of a band that co-segregates with the mutant phenotype represent candidate linked insertion sites, which are then confirmed by PCR. We demonstrate the utility of the method by identifying Mu insertion sites linked to seed-lethal mutations with a preliminary success rate of nearly 50%.  相似文献   

4.
Edwards D  Coghill J  Batley J  Holdsworth M  Edwards KJ 《BioTechniques》2002,32(5):1090-2, 1094, 1096-7
The amplification of transposon insertionflanking sequences is the basis of a variety of techniques usedfor the detection and characterization of specific transposon insertion events. We have developed a method for the efficient size determination and quantification of amplified genomic sequences thatflank Mutator (Mu) transposon insertions in maize. Using this detection method, we have been able to optimize Mu insertion site amplification and to assess amplification from increasingly complex templates representing increasing numbers of Mu-active maize plants. This detection method should be applicablefor the characterization of transposon or transgene insertion events in a wide variety of organisms.  相似文献   

5.
The Mutator transposable element system of maize was originally identified through its induction of mutations at an exceptionally high frequency and at a wide variety of loci. The Mu1 subfamily of transposable elements within this system are responsible for the majority of Mutator-induced mutations. Mu 1-related elements were isolated from active Mutator plants and their flanking DNA was characterized. Sequence analyses revealed perfect nine base target duplications directly flanking the insert for 13 of the 14 elements studied. Hybridizational studies indicated that Mu1-like elements insert primarily into regions of the maize genome that are of low copy number. This preferential selection of low copy number DNA as targets for Mu element insertion was not directed by any specific secondary structure(s) that could be detected in this study, but the 9-bp target duplications exhibited a discernibly higher than random match with the consensus sequence 5'-G-T-T-G-G/C-A-G-G/A-G-3'.  相似文献   

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The high frequency of mutations in Mutator stocks of maize is the result of transposition of Mu elements. Nine different Mu elements that share the 220 bp Mu terminal inverted repeats have been described. Mu1 elements have been found inserted into most of the molecularly characterized mutant alleles isolated from Mutator stocks, and most Mutator stocks contain a high number of Mu1 elements (10-60). However, it is clear that additional Mu elements, which share the Mu1 termini but have unrelated internal sequences, can also transpose in Mutator stocks. We were interested in comparing the mutation frequency and type of elements that inserted into a particular locus when Mutator stocks with differing numbers of Mu1 elements were utilized. Furthermore, previous studies with Mu-induced mutations have demonstrated that the element that inserted most frequently was Mu1. Therefore, to try to obtain Mu elements different from Mu1 we utilized a stock that had a low number (3-6) of Mu1 elements as well as a Mutator stock with a more typical number of Mu1 elements (20-60). Utilizing both stocks, we isolated numerous mutants at one gene, Bronze 1 (Bz1), and compared the type of elements inserted. In this paper we report that both the high and low Mu1 stocks produced bz1 mutants at frequencies characteristic of Mutator stocks, 6.6 and 4.3 x 10(-5), respectively. We describe the isolation of 20 bz1 mutations, and the initial molecular characterization of eight unstable mutations: two from the high Mu1 stock and six from the low Mu1 stock. The six alleles isolated from the low Mu1 stock appear to contain deleted Mu1 elements, and the two alleles isolated from the high Mu1 stock contain elements very similar to Mu1. When the mutants from the low Mu1 stocks were examined, it was found that the Mu1-related elements increased from 3-6 copies to 9-20 copies in one generation. The high number of Mu1-related elements was maintained in subsequent outcrosses. This spontaneous activation and amplification of Mu1-related elements occurred in at least 1% of the low Mu1 plants.  相似文献   

8.
9.
The autonomous MuDR element of the Mutator (Mu) transposable element family of maize encodes at least two proteins, MURA and MURB. Based on amino acid sequence similarity, previous studies have reported that MURA is likely to be a transposase. The functional characterization of MURA has been hindered by the instability of its cDNA, mudrA, in Escherichia coli. In this study, we report the first successful stabilization and expression of MURA in Saccharomyces cerevisiae. Gel mobility shift assays demonstrate that MURA is a DNA-binding protein that specifically binds to sequences within the highly conserved Mu element terminal inverted repeats (TIRs). DNase I and 1,10-phenanthroline-copper footprinting of MURA-Mu1 TIR complexes indicate that MURA binds to a conserved approximately 32-bp region in the TIR of Mu1. In addition, MURA can bind to the same region in the TIRs of all tested actively transposing Mu elements but binds poorly to the diverged Mu TIRs of inactive elements. Previous studies have reported a correlation between Mu transposon inactivation and methylation of the Mu element TIRs. Gel mobility shift assays demonstrate that MURA can interact differentially with unmethylated, hemimethylated, and homomethylated TIR substrates. The significance of MURA's interaction with the TIRs of Mu elements is discussed in the context of what is known about the regulation and mechanisms of Mutator activities in maize.  相似文献   

10.
K. J. Hardeman  V. L. Chandler 《Genetics》1993,135(4):1141-1150
The Mutator transposable element system of maize has been used to isolate mutations at many different genes. Six different classes of Mu transposable elements have been identified. An important question is whether particular classes of Mu elements insert into different genes at equivalent frequencies. To begin to address this question, we used a small number of closely related Mutator plants to generate multiple independent mutations at two different genes. The overall mutation frequency was similar for the two genes. We then determined what types of Mu elements inserted into the genes. We found that each of the genes was preferentially targeted by a different class of Mu element, even when the two genes were mutated in the same plant. Possible explanations for these findings are discussed. These results have important implications for cloning Mu-tagged genes as other genes may also be resistant or susceptible to the insertion of particular classes of Mu elements.  相似文献   

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13.
Developmental and genetic aspects of Mutator excision in maize   总被引:4,自引:0,他引:4  
The regulation of excision of Mu elements of the Mutator transposable element family of maize is not well understood. We have used somatic instability of Mu receptor elements from the Bronze 1 and Bronze 2 loci to monitor the frequency and the timing of excision of Mu elements in several tissues. We show that spot size in the aleurone of a bz2::mu1 stock varies between one to approximately 256 cells. This indicates that excision events begin eight divisions prior to full aleurone differentiation and end after the last division of the aleurone. We show that excision is equally biased for late events in all other tissues studied. A locus on chromosome 5 has been identified that affects spot size, possibly by altering the timing of Mu excision. Using somatic excision as an assay of Mutator activity, we found that activity can change in small sectors of the tassel; however, there are no overall activity changes in the tassel during the period of pollen shedding. We also report the recovery of germinal revertants for the bz1::mu1 and bz2::mu1 alleles. One of these revertant alleles was characterized by Southern blot analysis and found to be similar to the progenitor of the mutable allele.  相似文献   

14.
Vicki Chandler  Carol Rivin    Virginia Walbot 《Genetics》1986,114(3):1007-1021
Mutator stocks of maize produce mutants at many loci at rates 20- to 50-fold above spontaneous levels. Current evidence suggests that this high mutation rate is mediated by an active transposable element system, Mu. Members of this transposable element family are found in approximately 10-60 copies in Mutator stocks. We report here an initial characterization of previously undetected sequences homologous to Mu elements in eight non-Mutator inbred lines and varieties of maize that have a normal low mutation rate. All stocks have approximately 40 copies of sequences homologous only to the terminal repeat and show weak homology to an internal probe. In addition, several of the stocks contain an intact Mu element. One intact Mu element and two terminal-specific clones have been isolated from one non-Mutator line, B37. The cloned sequences have been used to demonstrate that in genomic DNA the intact element, termed Mu1.4B37, is modified, such that restriction sites in its termini are not accessible to cleavage by the HinfI restriction enzyme. This modification is similar to that observed in Mutator lines that have lost activity. We hypothesize that the DNA modification of the Mu-like element may contribute to the lack of Mutator activity in B37.  相似文献   

15.
V. L. Chandler  L. E. Talbert    F. Raymond 《Genetics》1988,119(4):951-958
The increased mutation rate of Mutator stocks of maize has been shown to be the result of transposition of Mu elements. One element, Mu1, is present in 10-60 copies in Mutator stocks and approximately 0-3 copies in non-Mutator stocks. The sequence, structure and genomic distribution of an intact Mu1 element cloned from the non-Mutator inbred line B37 has been determined. The sequence of this element, termed Mu1.4-B37, is identical to Mu1 and it is flanked by 9-bp direct repeats indicative of a target site duplication. Mu1.4-B37 is not in the same genomic location in all stocks, which further suggests that it transposed into its genomic location in B37. We previously reported that in genomic DNA this element is modified such that certain methylation-sensitive restriction enzymes will not cut sites within the element. This is similar to that observed for Mu elements in Mutator stocks that have lost activity. We report herein that the Mu1.4-B37 element loses its modification and becomes accessible to digestion when placed in an active Mutator stock by genetic crosses. This suggests that factors conditioning unmodified elements are dominant in the initial cross between Mutator and non-Mutator stocks. In F2 individuals that have subsequently lost Mutator activity the Mu1.4-B37 element again becomes modified as do most of the Mu elements in the stock. Thus, the modification state of the Mu1.4-B37 element and the other Mu1-like elements correlates with Mutator activity. We hypothesize that factor(s) within an active Mutator stock may inhibit the modification of Mu elements, and that this activity is missing in non-Mutator stocks and may become limiting in certain Mutator stocks resulting in DNA modification.  相似文献   

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18.
Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics.Isolation of genomic seg-ments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies.Herein,we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize.The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl ⅡMsp Ⅰ and ligation of digested fragments to the Bgl Ⅱ- and Msp Ⅰ-adaptors; 2) enrichment of a subset of Bgl Ⅱ/Msp Ⅰ fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking re-gions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions.Using this approach,we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-indueed mutant,and one such fragment,M196-FF,is found to contain a partial sequence of the DNA topoisomerase Ⅰ gene Topl.Moreover,the modified AFLP method including all restriction enzymes,adaptors and primers has been optimized in this study.The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.  相似文献   

19.
Mutator transposase is widespread in the grasses   总被引:5,自引:0,他引:5  
Although the Mutator (Mu) system is well characterized in maize (Zea mays), very little is known about this highly mutagenic system of transposons in other grasses. Mutator is regulated by the MuDR class of elements, which encodes two genes, one of which, mudrA, has similarity to a number of bacterial transposases. Experiments in our laboratory, as well as database searches, demonstrate that mudrA sequences are ubiquitous and diverse in the grasses. In several species it is clear that multiple paralogous elements can be present in a single genome. In some species such as wheat (Triticum aestivum) and rice (Oryza sativa), mudrA-similar sequences are represented in cDNA databases, suggesting the presence of active Mu transposon systems in these species. Further, in rice and in sorghum, mudrA-like genes are flanked by long terminal inverted repeats, as well as the short host sequence direct repeats diagnostic of insertion. Thus, there is ample evidence that systems related to Mu in maize are at least potentially active in a wide variety of grasses. However, the mudrB gene, though important for Mu activity in maize, is not necessarily a component of Mu elements in other grasses.  相似文献   

20.
The mobile DNAs of the Mutator system of maize (Zea mays) are exceptional both in structure and diversity. So far, six subfamilies of Mu elements have been discovered; all Mu elements share highly conserved terminal inverted repeats (TIRs), but each sub-family is defined by internal sequences that are apparently unrelated to the internal sequences of any other Mu subfamily. The Mu1/Mu2 subfamily of elements was created by the acquisition of a portion of a standard maize gene (termed MRS-A) within two Mu TIRs. Beside the unusually long (185–359 bp) and diverse TIRs found on all of these elements, other direct and inverted repeats are often found either within the central portion of a Mu element or within a TIR.Our computer analyses have shown that sequence duplications (mostly short direct repeats interrupted by a few base pairs) are common in non-autonomous members of the Mutator, Ac/Ds, and Spm(En) systems. These duplications are often tightly associated with the element-internal end of the TIRs. Comparisons of Mu element sequences have indicated that they share more terminal components than previously reported; all subfamilies have at least the most terminal 215 bp, at one end or the other, of the 359-bp Mu5 TIR. These data suggest that many Mu element subfamilies were generated from a parental element that had termini like those of Mu5. With the Mu5 TIRs as a standard, it was possible to determine that elements like Mu4 could have had their unusual TIRs created through a three-step process involving (1) addition of sequences to interrupt one TIR, (2) formation of a stem-loop structure by one strand of the element, and (3) a subsequent DNA repair/gene conversion event that duplicated the insertion(s) within the other TIR. A similar repair/conversion extending from a TIR stem into loop DNA could explain the additional inverted repeat sequences added to the internal ends of the Mu4 and Mu7 TIRs. This same basic mechanism was found to be capable of generating new Mu element subfamilies. After endonucleolytic attack of the loop within the stem-loop structure, repair/conversion of the gap could occur as an intermolecular event to generate novel internal sequences and, therefore, a new Mu element subfamily. Evidence supporting and expanding this model of new Mu element subfamily creation was identified in the sequence of MRS-A.  相似文献   

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