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1.
Identification of a family of bacteriophage T4 genes encoding proteins similar to those present in group I introns of fungi and phage.
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M Sharma R L Ellis D M Hinton 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(14):6658-6662
The bacteriophage T4 segA gene lies in a genetically unmapped region between the gene beta gt (beta-glucosyltransferase) and uvsX (recombination protein) and encodes a protein of 221 amino acids. We have found that the first 100 amino acids of the SegA protein are highly similar to the N termini of four other predicted T4 proteins, also of unknown function. Together these five proteins, SegA-E (similar to endonucleases of group I introns), contain regions of similarity to the endonuclease I-Tev I, which is encoded by the mobile group I intron of the T4 td gene, and to putative endonucleases of group I introns present in the mitochondria of Neurospora crassa, Podospora anserina, and Saccharomyces douglasii. Intron-encoded endonucleases are required for the movement (homing) of the intron DNA into an intronless gene, cutting at or near the site of intron insertion. Our in vitro assays indicate that SegA, like I-Tev I, is a Mg(2+)-dependent DNA endonuclease that has preferred sites for cutting. Unlike the I-Tev I gene, however, there is no evidence that segA (or the other seg genes) resides within introns. Thus, it is possible that segA encodes an endonuclease that is involved in the movement of the endonuclease-encoding DNA rather than in the homing of an intron. 相似文献
2.
R D Palmiter E P Sandgren M R Avarbock D D Allen R L Brinster 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(2):478-482
In a previous study we showed that genomic constructs were expressed more efficiently in transgenic mice than constructs that were identical except for the lack of introns. Using the mouse metallothionein promoter-rat growth hormone gene construct as a model, we show that the first intron of the rat growth hormone gene is essential for high-level expression, whereas the other three introns are less effective. Several heterologous introns placed 3' of the coding region of an intronless rat growth hormone gene are also ineffective. However, insertion of some heterologous introns between the metallothionein promoter and the growth hormone gene improves expression. To determine whether addition of heterologous introns would provide a general strategy for improving expression, we have tested them in conjunction with other intronless genes and with different promoters. 相似文献
3.
Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites
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Lorna Dickson Hon-Ren Huang Lu Liu Manabu Matsuura Alan M. Lambowitz Philip S. Perlman 《Proceedings of the National Academy of Sciences of the United States of America》2001,98(23):13207-13212
4.
Related homing endonucleases I-BmoI and I-TevI use different strategies to cleave homologous recognition sites
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David R. Edgell David A. Shub 《Proceedings of the National Academy of Sciences of the United States of America》2001,98(14):7898-7903
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. 相似文献
5.
Fanglei Zhuang Marta Mastroianni Travis B. White Alan M. Lambowitz 《Proceedings of the National Academy of Sciences of the United States of America》2009,106(43):18189-18194
Mobile group II introns retrohome by an RNP-based mechanism in which the excised intron lariat RNA fully reverse splices into a DNA site via 2 sequential transesterification reactions and is reverse transcribed by the associated intron-encoded protein. However, linear group II intron RNAs, which can arise by either hydrolytic splicing or debranching of lariat RNA, cannot carry out both reverse-splicing steps and were thus expected to be immobile. Here, we used facile microinjection assays in 2 eukaryotic systems, Xenopus laevis oocyte nuclei and Drosophila melanogaster embryos, to show that group II intron RNPs containing linear intron RNA can retrohome by carrying out the first step of reverse splicing into a DNA site, thereby ligating the 3′ end of the intron RNA to the 5′ end of the downstream exon DNA. The attached linear intron RNA is then reverse transcribed, yielding an intron cDNA whose free end is linked to the upstream exon DNA. Some of these retrohoming events result in the precise insertion of full-length intron. Most, however, yield aberrant 5′ junctions with 5′ exon resections, 5′ intron truncations, and/or extra nucleotide residues, hallmarks of nonhomologous end-joining. Our findings reveal a mobility mechanism for linear group II intron RNAs, show how group II introns can co-opt different DNA repair pathways for retrohoming, and suggest that linear group II intron RNAs might be used for site-specific DNA integration in gene targeting. 相似文献
6.
Chromosomal organization of the human dihydrofolate reductase genes: dispersion, selective amplification, and a novel form of polymorphism. 总被引:12,自引:3,他引:9
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N P Anagnou S J O''''Brien T Shimada W G Nash M J Chen A W Nienhuis 《Proceedings of the National Academy of Sciences of the United States of America》1984,81(16):5170-5174
The human dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) gene family includes a functional gene (hDHFR) and at least four intronless genes. Three intronless genes (hDHFR-psi 2, hDHFR-psi 3, and hDHFR-psi 4) are identifiable as pseudogenes because of DNA sequence divergence from the functional gene with introns, while one intronless gene (hDHFR-psi 1) is completely homologous to the coding sequences of the functional gene. Analysis of genomic DNA from two panels of somatic human-rodent cell hybrids with specific molecular probes provide insight into the chromosomal organization and assignment of these genes. The five genes are dispersed in that each one is found on a different chromosome. The functional gene hDHFR has been assigned to chromosome 5, and one pseudogene (hDHFR-psi 4), to chromosome 3. In a human cell line (HeLa) that was selected for methotrexate resistance, the functional locus became amplified, while there was no amplification of the four intronless pseudogenes. hDHFR-psi 1 was found to be present in DNA of some individuals and absent from DNA of others, consistent with a recent evolutionary origin of this gene originally suggested by its sequence identity to the coding portions of the functional gene. The presence or absence of this intronless pseudogene represents a previously unreported form of DNA polymorphism. 相似文献
7.
De novo insertion of an intron into the mammalian sex determining gene, SRY 总被引:5,自引:0,他引:5
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Rachel J. Waugh ONeill Francine E. Brennan Margaret L. Delbridge Ross H. Crozier Jennifer A. Marshall Graves 《Proceedings of the National Academy of Sciences of the United States of America》1998,95(4):1653-1657
Two theories have been proposed to explain the evolution of introns within eukaryotic genes. The introns early theory, or “exon theory of genes,” proposes that introns are ancient and that recombination within introns provided new exon structure, and thus new genes. The introns late theory, or “insertional theory of introns,” proposes that ancient genes existed as uninterrupted exons and that introns have been introduced during the course of evolution. There is still controversy as to how intron–exon structure evolved and whether the majority of introns are ancient or novel. Although there is extensive evidence in support of the introns early theory, phylogenetic comparisons of several genes indicate recent gain and loss of introns within these genes. However, no example has been shown of a protein coding gene, intronless in its ancestral form, which has acquired an intron in a derived form. The mammalian sex determining gene, SRY, is intronless in all mammals studied to date, as is the gene from which it recently evolved. However, we report here comparisons of genomic and cDNA sequences that now provide evidence of a de novo insertion of an intron into the SRY gene of dasyurid marsupials. This recently (approximately 45 million years ago) inserted sequence is not homologous with known transposable elements. Our data demonstrate that introns may be inserted as spliced units within a developmentally crucial gene without disrupting its function. 相似文献
8.
Intronless human dihydrofolate reductase genes are derived from processed RNA molecules. 总被引:16,自引:5,他引:16
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M J Chen T Shimada A D Moulton M Harrison A W Nienhuis 《Proceedings of the National Academy of Sciences of the United States of America》1982,79(23):7435-7439
Three groups of recombinant bacteriophage containing coding sequences for dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) were isolated from two human DNA clone libraries. One recombinant (lambda hDHFR-1) contains three exons that encode the COOH-terminal portion of human DHFR. The other two human DHFR genes (hDHFR-psi 1 and hDHRF-psi 2) lack introns. hDHFR-psi 2 contains several in-phase termination codons and is only 93% homologous to the normal human DHFR coding sequences, whereas hDHFR-psi 1 has an open reading frame and is virtually identical to the coding sequence of the normal DHFR gene. The region of DNA sequence homology between each intronless gene and the normal DHFR gene extends 2.9 kilobases beyond the end of the coding sequences. At the 3' end of this homologous sequence, each intronless gene has an A-rich tract. The lack of introns and the presence of the 3' A-rich tract suggest that hDHFR-psi 1 and hDHFR-psi 2 were derived from processed RNA molecules. A short DNA sequence, 60 nucleotides 5' to the ATG start codon in lambda hDHFR-psi 2, is directly repeated immediately after the 3' A-rich tract; such terminal direct repeats also flank integrated proretroviruses and transposable DNA elements and are thought to be the hallmark of inserted DNA sequences. 相似文献
9.
10.
11.
Characterization of the restriction site of a prokaryotic intron-encoded endonuclease. 总被引:8,自引:2,他引:6
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F K Chu G Maley J Pedersen-Lane A M Wang F Maley 《Proceedings of the National Academy of Sciences of the United States of America》1990,87(9):3574-3578
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. 相似文献
12.
13.
I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target. 总被引:7,自引:0,他引:7
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D Bell-Pedersen S M Quirk M Bryk M Belfort 《Proceedings of the National Academy of Sciences of the United States of America》1991,88(17):7719-7723
Mobility of the phage T4 td intron depends on activity of an intron-encoded endonuclease (I-TevI), which cleaves a homologous intronless (delta In) target gene. The double-strand break initiates a recombination event that leads to intron transfer. We found previously that I-TevI cleaves td delta In target DNA 23-26 nucleotides upstream of the intron insertion site. DNase I-footprinting experiments and gel-shift assays indicate that I-TevI makes primary contacts around the intron insertion site. A synthetic DNA duplex spanning the insertion site but lacking the cleavage site was shown to bind I-TevI specifically, and when cloned, to direct cleavage into vector sequences. The behavior of the cloned duplex and that of deletion and insertion mutants support a primary role for sequences surrounding the insertion site in directing I-TevI binding, conferring cleavage ability, and determining cleavage polarity. On the other hand, sequences around the cleavage site were shown to influence cleavage efficiency and cut-site selection. The role of cleavage-site sequences in determining cleavage distance argues against a strict "ruler" mechanism for cleavage by I-TevI. The complex nature of the homing site recognized by this unusual type of endonuclease is considered in the context of intron spread. 相似文献
14.
A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes. 总被引:8,自引:1,他引:8
B Davies S Feo E Heard M Fried 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(17):6691-6695
We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns. 相似文献
15.
16.
Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. 总被引:49,自引:12,他引:49
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B Sauer N Henderson 《Proceedings of the National Academy of Sciences of the United States of America》1988,85(14):5166-5170
The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra- and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro. Recombination occurs at a specific site, called lox, and does not require any other protein factors. The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line. A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter. DNA recombination was monitored with DNA substrates containing two directly repeated lox sites. One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(PO4)2 transformation. As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination. In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites. These results demonstrate the controlled site-specific synapsis of DNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes. 相似文献
17.
The FLP protein of the yeast 2-microns plasmid: expression of a eukaryotic genetic recombination system in Escherichia coli. 总被引:15,自引:7,他引:15
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M M Cox 《Proceedings of the National Academy of Sciences of the United States of America》1983,80(14):4223-4227
The FLP gene of the yeast 2-microns plasmid is involved in a site-specific recombination event that results in the inversion of a set of sequences within the plasmid. This gene has been cloned and expressed in Escherichia coli. Expression of the FLP gene results in efficient recombination within the bacterial cell, which is specific for plasmids containing at least one 2-microns plasmid recombination site. This work demonstrates that (i) FLP protein is actively involved in 2-microns plasmid recombination; (ii) no other factors specific to yeast are required for the reaction; (iii) FLP protein acts efficiently in trans; (iv) FLP protein will promote site-specific insertion and deletion reactions in addition to the inversion reaction; and (v) FLP-promoted recombination is not dependent upon any DNA structural features unique to yeast chromatin. 相似文献
18.
Coghlan A Wolfe KH 《Proceedings of the National Academy of Sciences of the United States of America》2004,101(31):11362-11367
The genomes of the nematodes Caenorhabditis elegans and Caenorhabditis briggsae both contain approximately 100,000 introns, of which >6,000 are unique to one or the other species. To study the origins of new introns, we used a conservative method involving phylogenetic comparisons to animal orthologs and nematode paralogs to identify cases where an intron content difference between C. elegans and C. briggsae was caused by intron insertion rather than deletion. We identified 81 recently gained introns in C. elegans and 41 in C. briggsae. Novel introns have a stronger exon splice site consensus sequence than the general population of introns and show the same preference for phase 0 sites in codons over phases 1 and 2. More of the novel introns are inserted in genes that are expressed in the C. elegans germ line than expected by chance. Thirteen of the 122 gained introns are in genes whose protein products function in premRNA processing, including three gains in the gene for spliceosomal protein SF3B1 and two in the nonsense-mediated decay gene smg-2. Twenty-eight novel introns have significant DNA sequence identity to other introns, including three that are similar to other introns in the same gene. All of these similarities involve minisatellites or palindromes in the intron sequences. Our results suggest that at least some of the intron gains were caused by reverse splicing of a preexisting intron. 相似文献
19.
Duplication-targeted DNA methylation and mutagenesis in the evolution of eukaryotic chromosomes. 总被引:7,自引:4,他引:7
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M C Kricker J W Drake M Radman 《Proceedings of the National Academy of Sciences of the United States of America》1992,89(3):1075-1079
Mammalian genomes are threatened with gene inactivation and chromosomal scrambling by recombination between repeated sequences such as mobile genetic elements and pseudogenes. We present and test a model for a defensive strategy based on the methylation and subsequent mutation of CpG dinucleotides in those DNA duplications that create uninterrupted homologous sequences longer than about 0.3 kilobases. The model helps to explain both the diversity of CpG frequencies in different genes and the persistence of gene fragmentation into exons and introns. 相似文献