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1.
Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila 下载免费PDF全文
During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena. 相似文献
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Evolutionary Conservation of Sequences Directing Chromosome Breakage and Rdna Palindrome Formation in Tetrahymenine Ciliates 总被引:2,自引:0,他引:2 下载免费PDF全文
Extensive, programmed chromosome breakage occurs during formation of the somatic macronucleus of ciliated protozoa. The cis-acting signal directing breakage has been most rigorously defined in Tetrahymena thermophila, where it consists of a 15-bp DNA sequence known as Cbs, for chromosome breakage sequence. We have identified sequences identical or nearly identical to the T. thermophila Cbs at sites of breakage flanking the germline micronuclear rDNA locus of six additional species of Tetrahymena as well as members of two related genera. Other general features of the breakage site are also conserved, but surprisingly, the orientation and number of copies of Cbs are not always conserved, suggesting the occurrence of germline rearrangement events over evolutionary time. At one end of the T. thermophila micronuclear rDNA locus, a pair of short inverted repeats adjacent to Cbs directs the formation of a giant palindromic molecule. We have examined the corresponding sequences from two other Tetrahymena species. We find the sequence to be partially conserved, as previously implied from analysis of macronuclear rDNA, but of variable length and organization. 相似文献
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Diverse sequences within Tlr elements target programmed DNA elimination in Tetrahymena thermophila 下载免费PDF全文
Tlr elements are a novel family of ~30 putative mobile genetic elements that are confined to the germ line micronuclear genome in Tetrahymena thermophila. Thousands of diverse germ line-limited sequences, including the Tlr elements, are specifically eliminated from the differentiating somatic macronucleus. Macronucleus-retained sequences flanking deleted regions are known to contain cis-acting signals that delineate elimination boundaries. It is unclear whether sequences within deleted DNA also play a regulatory role in the elimination process. In the current study, an in vivo DNA rearrangement assay was used to identify internal sequences required in cis for the elimination of Tlr elements. Multiple, nonoverlapping regions from the ~23-kb Tlr elements were independently sufficient to stimulate developmentally regulated DNA elimination when placed within the context of flanking sequences from the most thoroughly characterized family member, Tlr1. Replacement of element DNA with macronuclear or foreign DNA abolished elimination activity. Thus, diverse sequences dispersed throughout Tlr DNA contain cis-acting signals that target these elements for programmed elimination. Surprisingly, Tlr DNA was also efficiently deleted when Tlr1 flanking sequences were replaced with DNA from a region of the genome that is not normally associated with rearrangement, suggesting that specific flanking sequences are not required for the elimination of Tlr element DNA. 相似文献
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A programmed site-specific DNA rearrangement in Tetrahymena thermophila requires flanking polypurine tracts 总被引:24,自引:0,他引:24
During macronuclear development in ciliates, precise deletion events eliminate thousands of specific DNA segments. Each segment is bounded by a unique pair of short direct repeats, but no other common feature has been reported. To determine the critical cis-acting sequences, we developed an in vivo system for analyzing this process in Tetrahymena. We show that sequences essential for recognition and excision of one such region are located within the 70 bp of DNA flanking either side of it. Three authentic splice sites and one cryptic site are each adjacent to an unusual polypurine tract (5'-A5G5) situated 40-50 bp distal to each terminal repeat. Removal of this tract or substitution of 3 bp within it abolishes splicing to the adjacent site. The normal chromosomal environment and the integrity of the eliminated sequence are not required for its removal. We believe the polypurine tract is a signal essential for excision of this sequence. 相似文献
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A pathway for the evolution of the plasmid NTP16 involving the novel kanamycin resistance transposon Tn4352 总被引:7,自引:0,他引:7
The kanamycin resistance determinant of the drug resistance plasmid NTP16 has been characterized by DNA sequencing and has been shown to possess all of the structural features of a transposable element. It is made up of a 1040-bp central region encoding a protein identical to the aminoglycoside 3'-phosphotransferase of Tn903, flanked by direct repeats of an element identical to IS26. This novel transposon has been designated Tn4352. Analysis of the host sequences flanking the transposon reveal that they are derived from a Tn3-like element, and contain no 8 base pair target size duplications which are normally created by the insertion of IS26-like elements. Comparison to the Tn3 sequence shows that the flanking sequences are noncontiguous within Tn3, with the clear implication that NTP16 has evolved from a similar plasmid encoding only ampicillin resistance (presumably NTP1) by the insertion of Tn4352 into the Tn3-like element, followed by a substantial deletion. The sequence analysis suggests that the initial insertion was into the tnpR gene of the ampicillin transposon, followed by a deletion extending to a specific site within tnpA. 相似文献
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Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA 总被引:2,自引:0,他引:2
The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA
has been cloned and sequenced. The A+T region is organized in two large
arrays of tandemly repeated DNA sequence elements, with nonrepetitive
intervening and flanking sequences comprising only 22% of its length. The
first repeat array consists of five repeats of 338-373 bp. The second
consists of four intact 464-bp repeats and a fifth partial repeat of 137
bp. Three DNA sequence elements are found to be highly conserved in D.
melanogaster and in several Drosophila species with short A+T regions.
These include a 300-bp DNA sequence element that overlaps the DNA
replication origin and two thymidylate stretches identified on opposite DNA
strands. We conclude that the length heterogeneity observed in the A+T
regulatory region in mitochondrial DNAs from the genus Drosophila results
from the expansion (and contraction) of the number of repeated DNA sequence
elements. We also propose that the 300-bp conserved DNA sequence element,
in conjunction with another primary sequence determinant, perhaps the
adjacent thymidylate stretch, functions in the regulation of mitochondrial
DNA replication.
相似文献
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Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene. 总被引:29,自引:16,他引:13 下载免费PDF全文
P G Quinn T W Wong M A Magnuson J B Shabb D K Granner 《Molecular and cellular biology》1988,8(8):3467-3475
Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene. 相似文献
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Role of micronucleus-limited DNA in programmed deletion of mse2.9 during macronuclear development of Tetrahymena thermophila 下载免费PDF全文
Extensive programmed DNA rearrangements occur during the development of the somatic macronucleus from the germ line micronucleus in the sexual cycle of the ciliated protozoan Tetrahymena thermophila. Using an in vivo processing assay, we analyzed the role of micronucleus-limited DNA during the programmed deletion of mse2.9, an internal eliminated sequence (IES). We identified a 200-bp region within mse2.9 that contains an important cis-acting element which is required for the targeting of efficient programmed deletion. Our results, obtained with a series of mse2.9-based chimeric IESs, led us to suggest that the cis-acting elements in both micronucleus-limited and macronucleus-retained flanking DNAs stimulate programmed deletion to different degrees depending on the particular eliminated sequence. The mse2.9 IES is situated within the second intron of the micronuclear locus of the ARP1 gene. We show that the expression of ARP1 is not essential for the growth of Tetrahymena. Our results also suggest that mse2.9 is not subject to epigenetic regulation of DNA deletion, placing possible constraints on the scan RNA model of IES excision. 相似文献
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The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene''s expression. 总被引:26,自引:7,他引:19 下载免费PDF全文
Expression of the allantoin system genes in Saccharomyces cerevisiae is induced by allophanate or its analog, oxalurate. This work provides evidence for the involvement of distinct types of cis-acting elements in the induction process. The first element was found to have the properties of an upstream activation sequence (UAS). This element was localized to a 16-base-pair (bp) DNA fragment containing a short 5-bp sequence that occurred repeatedly in the upstream region of DAL7. When present in two or more copies, the 16-bp fragment supported high-level beta-galactosidase production in a CYC1-lacZ expression vector; there was, however, no response to the allantoin pathway inducer. The second element had the properties of a negatively acting element or upstream repression sequence (URS). This element was localized to a 16-bp DNA fragment containing an 8-bp sequence that was repeated four times in the upstream region of DAL7. A fragment containing the 8-bp repeated sequence placed adjacent to the UAS-containing fragment mediated inhibition of the ability of the UAS to support lacZ expression regardless of whether inducer was present. A third element, designated an upstream induction sequence (UIS), was required for response to inducer. The UIS was localized to a small DNA fragment containing an approximately 10-bp sequence that was repeated twice in the upstream region of DAL7. When a fragment containing the 10-bp repeated sequence was placed adjacent to these UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of beta-galactosidase synthesis. These data are consistent with the suggestion that multiple, cis-acting elements participate in the induction process. 相似文献
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Genomic Organization and Developmental Fate of Adjacent Repeated Sequences in a Foldback DNA Clone of Tetrahymena thermophila 下载免费PDF全文
Almuth H. Tschunko Robert H. Loechel Ning C. McLaren Sally Lyman Allen 《Genetics》1987,117(3):451-466
DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed. 相似文献
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The very large amplifiable element AUD2 from Streptomyces lividans 66 has insertion sequence-like repeats at its ends. 下载免费PDF全文
In a spontaneous, chloramphenicol-sensitive (Cms), arginine-auxotrophic (Arg-) mutant of Streptomyces lividans 1326, two amplified DNA sequences were found. One of them was the well-characterized 5.7-kb ADS1 sequence, amplified to about 300 copies per chromosome. The second one was a 92-kb sequence called ADS2. ADS2 encoding the previously isolated mercury resistance genes of S. lividans was amplified to around 20 copies per chromosome. The complete ADS2 sequence was isolated from a genomic library of the mutant S. lividans 1326.32, constructed in the phage vector lambda EMBL4. In addition, the DNA sequences flanking the corresponding amplifiable element called AUD2 in the wild-type strain were isolated by using another genomic library prepared from S. lividans 1326 DNA. Analysis of the ends of AUD2 revealed the presence of an 846-bp sequence on both sides repeated in the same orientation. Each of the direct repeats ended with 18-bp inverted repeated sequences. This insertion sequence-like structure was confirmed by the DNA sequence determined from the amplified copy of the direct repeats which demonstrated a high degree of similarity of 65% identity in nucleic acid sequence to IS112 from Streptomyces albus. The recombination event leading to the amplification of AUD2 occurred within these direct repeats, as shown by DNA sequence analysis. The amplification of AUD2 was correlated with a deletion on one side of the flanking chromosomal region beginning very near or in the amplified DNA. Strains of S. lividans like TK20 and TK21 which are mercury sensitive have completely lost AUD2 together with flanking chromosomal DNA on one or both sides. 相似文献