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An X-linked gene affecting mouse cell DNA synthesis also affects production of unintegrated linear and supercoiled DNA of murine leukemia virus. 总被引:9,自引:1,他引:8
To identify specific cellular factors which could be required during the synthesis of retroviral DNA, we have studied the replication of murine leukemia virus in mouse cells temperature sensitive for cell DNA synthesis (M. L. Slater and H. L. Ozer, Cell 7:289-295, 1976) and in several of their revertants. This mutation has previously been mapped on the X chromosome. We found that a short incubation of mutant cells at a nonpermissive temperature (39 degrees C) during the early part of the virus cycle (between 0- to 20-h postinfection) greatly inhibited virus production. This effect was not observed in revertant or wild-type cells. Molecular studies by the Southern transfer procedure of the unintegrated viral DNA synthesized in these cells at a permissive (33 degrees C) or nonpermissive temperature revealed that the levels of linear double-stranded viral DNA (8.8 kilobase pairs) were nearly identical in mutant or revertant cells incubated at 33 or 39 degrees C. However, the levels of two species of supercoiled viral DNA (with one or two long terminal repeats) were significantly lower in mutant cells incubated at 39 degrees C than in mutant cells incubated at 33 degrees C or in revertant cells incubated at 39 degrees C. Pulse-chase experiments showed that linear viral DNA made at 39 degrees C could not be converted into supercoiled viral DNA in mutant cells after a shift down to 33 degrees C. In contrast, such conversion was observed in revertant cells. Restriction endonuclease analysis did not detect differences in the structure of linear viral DNA made at 39 degrees C in mutant cells as compared to linear viral DNA isolated from the same cells at 33 degrees C. However, linear viral DNA made at 39 degrees C in mutant cells was poorly infectious in transfection assays. Taken together, these results strongly suggest that this X-linked gene, affecting mouse cell DNA synthesis, is operating in the early phase of murine leukemia virus replication. It seems to affect the level of production of unintegrated linear viral DNA only slightly while greatly reducing the infectivity of these molecules. In contrast, the accumulation of supercoiled viral DNA and subsequent progeny virus production are greatly reduced. Our pulse-chase experiments suggest that the apparent, but not yet identified, defect in linear viral DNA molecules might be responsible for their subsequent impaired circularization. 相似文献
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Tissue specific trans-acting factor interaction with proximal rat prolactin gene promoter sequences. 总被引:2,自引:0,他引:2
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Using an exonuclease III protection assay, strong, reversible and tissue-specific binding of GH3 cell nuclear factors to proximal regions of the rat prolactin (rPrl) promoter (-31 to -77) has been detected. A second less prominent region of factor binding, that may have a correlate in HeLa cell extracts, was detected in the region (-155 to -180). The binding is eliminated in the presence of excess unlabelled rPrl promoter sequences (-423 to +38), excess unlabelled distal rPrl 5'-flanking sequences (-1960 to -1260) and SV40-enhancer/promoter sequences; it is largely unaffected by growth hormone (rGH) promoter and RSV-LTR sequences. A plasmid containing the proximal rPrl promoter sequences (-75 to +38) was also shown to be an avid inhibitor, at low concentrations, of rPrl promoter driven chloramphenicol acetyl transferase (CAT) gene expression in transient cotransfection competition studies; under these assay conditions distal rPrl 5'-flanking sequences and RSV and rGH promoter plasmids do not compete. The results emphasize the critical importance of proximal rPrl promoter sequences for prolactin gene expression in GH3 cells but recognize the related functional potential of more distal sequences. 相似文献
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Background: In the human genome, distal enhancers are involved in regulating target genes through proximal promoters by forming enhancer-promoter interactions. Although recently developed high-throughput experimental approaches have allowed us to recognize potential enhancer-promoter interactions genome-wide, it is still largely unclear to what extent the sequence-level information encoded in our genome help guide such interactions. Methods: Here we report a new computational method (named “SPEID”) using deep learning models to predict enhancer-promoter interactions based on sequence-based features only, when the locations of putative enhancers and promoters in a particular cell type are given. Results: Our results across six different cell types demonstrate that SPEID is effective in predicting enhancer-promoter interactions as compared to state-of-the-art methods that only use information from a single cell type. As a proof-of-principle, we also applied SPEID to identify somatic non-coding mutations in melanoma samples that may have reduced enhancer-promoter interactions in tumor genomes. Conclusions: This work demonstrates that deep learning models can help reveal that sequence-based features alone are sufficient to reliably predict enhancer-promoter interactions genome-wide. 相似文献
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Although much of the information regarding genes'' expressions is encoded in the genome, deciphering such information has been very challenging. We reexamined Beer and Tavazoie''s (BT) approach to predict mRNA expression patterns of 2,587 genes in Saccharomyces cerevisiae from the information in their respective promoter sequences. Instead of fitting complex Bayesian network models, we trained naïve Bayes classifiers using only the sequence-motif matching scores provided by BT. Our simple models correctly predict expression patterns for 79% of the genes, based on the same criterion and the same cross-validation (CV) procedure as BT, which compares favorably to the 73% accuracy of BT. The fact that our approach did not use position and orientation information of the predicted binding sites but achieved a higher prediction accuracy, motivated us to investigate a few biological predictions made by BT. We found that some of their predictions, especially those related to motif orientations and positions, are at best circumstantial. For example, the combinatorial rules suggested by BT for the PAC and RRPE motifs are not unique to the cluster of genes from which the predictive model was inferred, and there are simpler rules that are statistically more significant than BT''s ones. We also show that CV procedure used by BT to estimate their method''s prediction accuracy is inappropriate and may have overestimated the prediction accuracy by about 10%. 相似文献
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Novel genomic rearrangement that affects expression of the Streptococcus pyogenes streptolysin O (slo) gene
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A RecA-independent chromosomal rearrangement in the upstream region of the streptolysin O (slo) gene of Streptococcus pyogenes which affects slo expression was identified. PCR analysis was used to demonstrate that this kind of rearrangement was found in several strains of different lineages. Chromosomal loci involved in the recombination were found to be 746 kb apart on the 1.85-Mb-long chromosome. The primary structure of the splicing region, the reproducibility of the rearrangement, and the fact that reconstructed recombinant molecules fused to erm and lacZ reporter genes affected their expression indicate that this event is not accidental but may play a role in the expression of the slo gene. In addition, the product of the recombining DNAs, including the splicing site, does not follow any example of a known recombination mechanism. The implications of this rearrangement for slo expression are discussed. 相似文献
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A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in "unigenes" that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family. 相似文献
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A cis-acting element and a trans-acting factor involved in the wound-induced expression of a horseradish peroxidase gene 总被引:2,自引:0,他引:2
Akiyoshi Kawaoka Tomohiro Kawamoto Masami Sekine Kazuya Yoshida Mitsuo Takano Atsuhiko Shinmyo 《The Plant journal : for cell and molecular biology》1994,6(1):87-97
The mechanisms that control the wound-induced expression of the prxC2 gene for horseradish peroxidase (HRP) have been investigated. Analysis of the regulatory properties of 5′-deleted promoters showed that a positive element involved in the response to wounding was located between −307 and −99 bp from the site of initiation of translation. In in vitro binding assays of tobacco nuclear proteins and DNA fragments of prxC2 promoter, the binding site was the Box 1 from −296 to −283 containing the CACGTG motif. To identify the functional role of Box 1, the prxC2 promoter that has been digested from the 5′ end to −289 with a disrupted Box 1 was fused to a reporter gene for β-glucuronidase (GUS). No induction of GUS activity was observed in transgenic tobacco plants with the prxC2(−289)/GUS construct. These data indicated that the expression of prxC2 in response to wounding required the Box 1 sequence from −296 to −283. Furthermore, a tobacco cDNA expression library was screened and a cDNA clone for a protein, designated TFHP-1, that bound specifically to the Box 1 sequence was identified. The putative TFHP-1 protein contains a basic region and leucine zipper (bZip) motif and a helix—loop—helix (HLH) motif. The mRNA for TFHP-1 was abundant in roots and stems, and it was not induced by wounding in leaves. In tobacco protoplasts, antisense TFHP-1 suppressed the expression of prxC2 (−529)/GUS. 相似文献
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A mouse U3 RNA pseudogene has been identified; it corresponds to a U3B full length coding sequence with a 3'-oligo(A) tail, precisely flanked at both ends by a pair of 15 bp direct repeats. These structural features suggest that it has arisen through an RNA-mediated mechanism involving an insertion at staggered nicks in the genome. Sequence data indicate that this mouse specimen has been generated by a different event as compared to the recently described rat pseudogenes. It represents the first reported case, for a pseudogene of this class, to be present at more than one copy per genome. 相似文献
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Functional expression of the genomic DNA sequences encoding mouse Na,K-ATPase alpha 1 gene by cotransfection of overlapping genomic DNA segments. 总被引:2,自引:0,他引:2
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The entire 33-kb coding region of the mouse Na,K-ATPase alpha 1 subunit gene was cloned in two overlapping cosmids which contain inserts of 40 kb. To assess the functional expression of the mouse alpha 1 gene, the two cosmids were cotransfected into ouabain-sensitive CV-1 monkey cells yielding an average of 64 resistant colonies per 10(6) cells per microgram of DNA. Analysis of the DNA transferred to the ouabain-resistant transformants by the two cosmids suggests that the generation of a functional gene can occur by homologous recombination between the two introduced segments, as demonstrated by generation of a novel diagnostic restriction fragment. The ability to reconstruct the intact mouse alpha 1 gene in a heterologous host cell and to monitor its functional expression with a selection protocol permits direct identification and isolation of regulatory sequences for the gene. 相似文献