Activation of the mouse mdr3 gene by insertion of retroviruses in multidrug-resistant P388 tumor cells

1993 ◽  
Vol 13 (12) ◽  
pp. 7380-7392
Author(s):  
P Lepage ◽  
A Devault ◽  
P Gros
Keyword(s):  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Integration Site ◽  
Multidrug Resistant ◽  
Gene Copy ◽  
Nucleotide Sequencing ◽  
Intact Mouse ◽  
Exon 2 ◽  
Intron 1 ◽  
Exon 1

In multidrug-resistant (MDR) derivatives of the mouse lymphoid tumor P388, the emergence of MDR is associated with overexpression and transcriptional activation of the mdr3 gene, either in the absence of (P388/VCR-10) or concomitant with (P388/ADM-2) gene amplification. In both instances, Northern (RNA) blotting analyses have suggested the presence of altered mdr3 transcripts in these cells, possibly originating from novel transcription initiation sites. The mechanisms underlying mdr3 overexpression in these cells have been investigated. In P388/VCR-10 cells, Southern blotting analyses together with genomic DNA cloning and nucleotide sequencing have demonstrated the presence of an intact mouse mammary tumor virus (MMTV) within the boundaries of intron 1 of mdr3. cDNA cloning and nucleotide sequencing indicated that this integration event results in the synthesis and overexpression of a hybrid MMTV-mdr3 mRNA which initiates within the U3 region of the 5' long terminal repeat (LTR) of the provirus. Consequently, this mRNA lacks the normal exon 1 of mdr3 but contains (i) MMTV LTR-derived sequences at its 5' end, (ii) a novel mdr3 exon, mapping within the boundaries of intron 1 downstream of the MMTV integration site and generated by alternative splicing, and (iii) an otherwise intact 3' portion of mdr3 starting at exon 2. A similar type of analysis of P388/ADM-2 cells revealed that mdr3 overexpression in these cells is associated with the integration of an intracisternal A particle (IAP) within an L1Md repetitive element, immediately upstream of mdr3. The IAP insertion results in the overexpression of hybrid IAP-mdr3 mRNA transcripts that initiate within the 3' LTR of the IAP and which contain IAP LTR-derived sequences at the 5' end spliced 14 nucleotides upstream of the normal exon 1 of mdr3. Taken together, these results indicate that independent retroviral insertions were the initial mutagenic event responsible for mdr3 overexpression and survival during drug selection of these cell lines. Amplification of the rearranged and activated mdr3 gene copy occurred during further selection for high-level drug resistance in P388/ADM-2 cells.

scholarly journals Activation of the mouse mdr3 gene by insertion of retroviruses in multidrug-resistant P388 tumor cells.

1993 ◽  
Vol 13 (12) ◽  
pp. 7380-7392 ◽  
Author(s):  
P Lepage ◽  
A Devault ◽  
P Gros
Keyword(s):  
Transcriptional Activation ◽  
Transcription Initiation ◽  
Integration Site ◽  
Multidrug Resistant ◽  
Gene Copy ◽  
Nucleotide Sequencing ◽  
Intact Mouse ◽  
Exon 2 ◽  
Intron 1 ◽  
Exon 1

In multidrug-resistant (MDR) derivatives of the mouse lymphoid tumor P388, the emergence of MDR is associated with overexpression and transcriptional activation of the mdr3 gene, either in the absence of (P388/VCR-10) or concomitant with (P388/ADM-2) gene amplification. In both instances, Northern (RNA) blotting analyses have suggested the presence of altered mdr3 transcripts in these cells, possibly originating from novel transcription initiation sites. The mechanisms underlying mdr3 overexpression in these cells have been investigated. In P388/VCR-10 cells, Southern blotting analyses together with genomic DNA cloning and nucleotide sequencing have demonstrated the presence of an intact mouse mammary tumor virus (MMTV) within the boundaries of intron 1 of mdr3. cDNA cloning and nucleotide sequencing indicated that this integration event results in the synthesis and overexpression of a hybrid MMTV-mdr3 mRNA which initiates within the U3 region of the 5' long terminal repeat (LTR) of the provirus. Consequently, this mRNA lacks the normal exon 1 of mdr3 but contains (i) MMTV LTR-derived sequences at its 5' end, (ii) a novel mdr3 exon, mapping within the boundaries of intron 1 downstream of the MMTV integration site and generated by alternative splicing, and (iii) an otherwise intact 3' portion of mdr3 starting at exon 2. A similar type of analysis of P388/ADM-2 cells revealed that mdr3 overexpression in these cells is associated with the integration of an intracisternal A particle (IAP) within an L1Md repetitive element, immediately upstream of mdr3. The IAP insertion results in the overexpression of hybrid IAP-mdr3 mRNA transcripts that initiate within the 3' LTR of the IAP and which contain IAP LTR-derived sequences at the 5' end spliced 14 nucleotides upstream of the normal exon 1 of mdr3. Taken together, these results indicate that independent retroviral insertions were the initial mutagenic event responsible for mdr3 overexpression and survival during drug selection of these cell lines. Amplification of the rearranged and activated mdr3 gene copy occurred during further selection for high-level drug resistance in P388/ADM-2 cells.

Effect of 5' splice site mutations on splicing of the preceding intron

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals Transcriptional activity of the human tissue inhibitor of metalloproteinases 1 (TIMP-1) gene in fibroblasts involves elements in the promoter, exon 1 and intron 1

1997 ◽  
Vol 324 (2) ◽  
pp. 611-617 ◽  
Author(s):  
Ian M. CLARK ◽  
Andrew D. ROWAN ◽  
Dylan R. EDWARDS ◽  
Torben BECH-HANSEN ◽  
Derek A. MANN ◽  
...  
Keyword(s):  
Point Mutations ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Minor Role ◽  
Detailed Knowledge ◽  
Rnase Protection ◽  
Exon 2 ◽  
Promoter Fragment ◽  
Basal Expression ◽  
Intron 1 ◽  
Exon 1

The active forms of all of the matrix metalloproteinases (MMPs) are inhibited by a family of specific inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). Inhibition represents a major level of control of MMP activity. A detailed knowledge of the mechanisms controlling TIMP gene expression is therefore important. We have isolated a genomic clone of the human TIMP-1 gene. A 3 kbp XbaI fragment has been sequenced; this fragment contains 1718 bp 5′ flanking sequences, exon 1, a 929 bp intron 1 and part of exon 2. Computer analysis reveals 10 consensus sequences for Sp1, six for activating protein 1 (AP-1), six for polyoma enhancer A3 (PEA3), 12 for AP-2 and five CCAAT boxes. The region hybridizing with a murine TIMP-1 promoter fragment has been subcloned and analysed further. RNase protection identifies six transcription start points, making exon 1 up to 48 bp in length. Transient transfection of promoter–chloramphenicol O-acetyltransferase reporter constructs into primary human connective tissue fibroblasts shows that a 904 bp fragment that hybridizes to a murine TIMP-1 promoter fragment contains a functional promoter. Constructs of -738/+95 to -194/+21 are inducible with serum or phorbol ester to a similar extent to the endogenous TIMP-1 gene. These results and further mapping with 5′ deletion mutants from the -738/+95 region have demonstrated that an AP-1 site at -92/-86 is essential for basal expression of the gene. Point mutations within this region have further confirmed the role of this site, along with a more minor role for a neighbouring PEA3 site, in basal expression. Deletions from the 3′ end also implicate a region across the exon 1/intron 1 boundary and especially +21 to +58 in basal expression. The +21/+58 region contains a putative binding site for the transcription factor leader-binding protein 1 (LBP-1). Gel-shift analysis shows that protein binds specifically to this region, but competition studies suggest that it is unlikely to be LBP-1.

scholarly journals Functional analysis of the AUG- and CUG-initiated forms of the c-Myc protein.

1994 ◽  
Vol 5 (5) ◽  
pp. 597-609 ◽  
Author(s):  
E M Blackwood ◽  
T G Lugo ◽  
L Kretzner ◽  
M W King ◽  
A J Street ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Chromosomal Translocation ◽  
Regulatory Elements ◽  
Genetic Alterations ◽  
Coding Region ◽  
Exon 2 ◽  
Translational Initiation ◽  
Selective Growth Advantage ◽  
Exon 1 ◽  
Normal Human

Activation of the c-myc proto-oncogene by chromosomal translocation or proviral insertion frequently results in the separation of the c-myc coding region from its normal regulatory elements. Such rearrangements are often accompanied by loss or mutation of c-myc exon 1 sequences. These genetic alterations do not affect synthesis of the major c-myc protein, p64, which is initiated from the first AUG codon in exon 2. However they can result in mutation or loss of the CUG codon located in exon 1 that normally serves as an alternative translational initiation codon for synthesis of an N-terminally extended form of c-Myc (p67). It has been hypothesized that p67 is a functionally distinct form of c-Myc whose specific loss during c-myc rearrangements confers a selective growth advantage. Here we describe experiments designed to test the functional properties of the two c-Myc protein forms. We introduced mutations within the translational initiation codons of a normal human c-myc cDNA that alter the pattern of Myc protein synthesis (p64 vs. p67). The functions of each of these proteins were experimentally addressed using co-transformation and transcriptional activation assays. Both the p64 and p67 c-Myc proteins were independently able to collaborate with bcr-abl in the transformation of Rat-1 fibroblasts. In addition, both the exon 1- and exon 2-initiated forms of the c-Myc protein stimulated transcription of a Myc/Max-responsive reporter construct to a similar level. Given the apparent absence of functional differences between p64 and p67, we conclude that the basis for c-Myc oncogenic activation lies primarily in the overall deregulation of its expression and not in alterations in the protein. The existence of the CUG translational initiator may reflect a mechanism for the continued synthesis of c-Myc protein under conditions where AUG initiation is inhibited.

Transcription initiation of the rat insulin-like growth factor-I gene in hepatocyte primary culture

1996 ◽  
Vol 151 (2) ◽  
pp. 215-223 ◽  
Author(s):  
A Y Krishna ◽  
C-I Pao ◽  
P M Thulé ◽  
B C Villafuerte ◽  
L S Phillips
Keyword(s):  
Transcription Initiation ◽  
Rat Hepatocytes ◽  
Regulatory Mechanisms ◽  
Exon 2 ◽  
Factor I ◽  
Igf I ◽  
Exon 1 ◽  
Normal Rat

Abstract Transcription initiation in the insulin-like growth factor-I (IGF-I) gene is complex, involving multiple sites in two exons. While most transcripts are initiated in exon 1 in vivo, critical regulatory mechanisms are difficult to assess in intact animals. To examine the impact of insulin and growth hormone (GH) under more controlled conditions, we have studied the utilization of different exon 1 and exon 2 transcription-initiation sites in normal rat hepatocytes in primary culture. Normal rat hepatocytes were cultured for 48 h in serum-free medium, with insulin at 10−6 or 10−11 m, and with or without human GH 200 ng/ml. Relative abundance of IGF-I transcripts was evaluated by the RNase-protection assay, using a probe which permitted identification of initiation in exon 1 (site 1 (−380 bp from the 3′ end of exon 1), site 2 (−343 bp), site 3 (−242 bp), sites 1 and 2 spliced, and site 4 (−32 bp)), as well as in exon 2. After normalization of signal intensity to adjust for differences in length of protected probe, the utilization of initiation sites in vitro was remarkably similar to that in vivo: 1, 14, 6, 23, 19 and 37% for sites 1, 2, 3, 1 and 2 spliced, 4 and exon 2 respectively in the cultured hepatocytes, compared with 1, 12, 8, 21, 18 and 40% for these sites in normal liver. Insulin alone increased transcripts initiated from exon 1, site 2 by over 3 times, and both sites 1 and 2 spliced and exon 2 transcripts by about 5 times. GH alone had similar effects, producing a 4–5 times increase in transcripts from these initiation sites. Addition of both insulin and GH had additive effects, increasing transcripts from exon 1, sites 2, 3 and 4 by 4–6 times, and from exon 1, sites 1 and 2 spliced, and exon 2 by over 8 times. Of the total IGF-I mRNA transcripts, 37% were initiated from sites 2 and/or sites 1 and 2 spliced, and 37% from exon 2. Analysis of the relative contribution of individual initiation sites revealed hormone-induced increases which were statistically significant only for exon 2, in the presence of insulin alone and in combination with GH. In conclusion, in cultured hepatocytes, insulin or GH alone produced a coordinated increase in all exon 1 transcripts, and the effect of the combination of insulin and GH was additive for these transcripts. Exon 2 appeared to be more sensitive to insulin alone, and to GH in the presence of insulin, than exon 1. Since utilization of initiation sites in hepatocytes mimics that found in liver, this in vitro system should be useful for examining underlying transcriptional regulatory mechanisms. Journal of Endocrinology (1996) 151, 215–223

scholarly journals Effect of 5' splice site mutations on splicing of the preceding intron.

1990 ◽  
Vol 10 (12) ◽  
pp. 6299-6305 ◽  
Author(s):  
M Talerico ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Exon Skipping ◽  
Splicing Factors ◽  
Exon 2 ◽  
Intron 1 ◽  
Direct Assembly ◽  
Internal Exon ◽  
Exon 1 ◽  
Lariat Rna

Three exon constructs containing identical intron and exon sequences were mutated at the 5' splice site beginning intron 2 and assayed for the effect of the mutation on splicing of the upstream intron in vitro. Alteration of two or six bases within the 5' splice site reduced removal of intron 1 at least 20-fold, as determined by quantitation of either spliced product or released lariat RNA. The prominent product was skip splicing of exon 1 to exon 3. Examination of complex formation indicated that mutation of the 5' splice site terminating exon 2 depressed the ability of precursor RNAs containing just the affected exon to direct assembly in vitro. These results suggest that mutation at the end of an internal exon inhibits the ability of the exon to be recognized by splicing factors. A comparison of the known vertebrate 5' splice site mutations in which the mutation resides at the end of an internal exon indicated that exon skipping is the preferred phenotype for this type of mutation, in agreement with the in vitro observation reported here. Inhibition of splicing by mutation at the distal and of the exon supports the suggestion that exons, rather than splice sites, are the recognition units for assembly of the spliceosome.

scholarly journals Characterization and Regulation of the Rat and Human Ghrelin Promoters

Endocrinology ◽  
2005 ◽  
Vol 146 (3) ◽  
pp. 1611-1625 ◽  
Author(s):  
Wei Wei ◽  
Guiyun Wang ◽  
Xiang Qi ◽  
Ella W. Englander ◽  
George H. Greeley
Keyword(s):  
Transcription Initiation ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Core Promoter ◽  
Gh3 Cells ◽  
Ags Cells ◽  
The Core ◽  
Intron 1 ◽  
Proximal Site ◽  
Exon 1

Ghrelin is a recently discovered stomach hormone and endogenous ligand for the GH secretagogue receptor. The aim of these studies is to elucidate molecular mechanisms underlying regulation of the ghrelin gene. Distal and proximal transcription initiation sites are present. A short transcript, a product of the proximal site, showed a more widespread distribution. Two sets of 5′-upstream segments of the rat and human ghrelin genes were cloned and sequenced. Rat promoter segments upstream of the distal site showed highest activity in kidney (COS-7) and stomach (AGS) cells, whereas human promoter segments upstream of the proximal site showed highest activity in AGS and pituitary (GH3) cells in transient transfection assays. For the human, the core promoter spanned −667 to −468 bp, including the noncoding exon 1 and a short 5′ sequence of intron 1. For the rat, the core promoter spanned −581 to −469 bp, and inclusion of exon 1 and a short 5′-sequence of intron 1 reduced activity by 67%. Mutation of initiator-like elements in the rat lowered activity by 20–50%, whereas in the human, all activity was abolished. Overexpression of upstream stimulatory factors increased ghrelin core promoter activity. Fasting increases stomach ghrelin expression, glucagon-a fasting-induced hormone, increased ghrelin expression in vivo in rats, and promoter activity by approximately 25–50%. Together, these findings indicate that structural differences between the rat and human ghrelin core promoters may account in part for the differences in their transcriptional regulation. Nonetheless, upstream stimulatory factor and glucagon exert similar effects on regulation of rat and human ghrelin promoters.

Association analyses of polymorphisms in porcine MYF5 and MYOD1 genes with carcass traits

2007 ◽  
Vol 58 (11) ◽  
pp. 1040 ◽  
Author(s):  
M. Liu ◽  
J. Peng ◽  
D. Q. Xu ◽  
R. Zheng ◽  
F. E. Li ◽  
...  
Keyword(s):  
Myogenic Differentiation ◽  
Carcass Traits ◽  
Large White ◽  
Exon 2 ◽  
Association Analyses ◽  
Intron 1 ◽  
Pcr Rflp ◽  
Exon 1 ◽  
Myogenic Factor ◽  
Fat Thickness

The objective of this study was to assess the effect of polymorphisms of myogenic factor 5 (MYF5) and myogenic differentiation 1 (MYOD1) genes on carcass traits in pigs. PCR-RFLP was used to identify three and one SNP(s) from the MYF5 and the MYOD1 gene, respectively. Association analysis performed on the four polymorphisms in a series of three Large White × Meishan F2 populations totalling near 400 pigs showed: (1) an MYF5 exon 1 Hsp92II polymorphism causing a Met→Leu substitution was significantly associated with fat meat percentage, shoulder fat thickness, thorax-waist fat thickness, average backfat thickness and carcass length to 1st rib (P < 0.05); (2) an MYF5 exon 2 MspI polymorphism and an intron 1 HaeIII polymorphism, which were completely linked, were significantly associated with thorax-waist fat thickness, 6–7th rib fat thickness and carcass length to 1st rib (P < 0.05); (3) an MYOD1 intron 1 DdeI polymorphism was significantly associated with carcass length to 1st rib.

scholarly journals Convergence of RNA cis Elements and Cellular Polyadenylation Factors in the Regulation of Human Cytomegalovirus UL37 Exon 1 Unspliced RNA Production

2003 ◽  
Vol 77 (23) ◽  
pp. 12729-12741 ◽  
Author(s):  
Yan Su ◽  
Richard Adair ◽  
Candice N. Davis ◽  
Nancy L. DiFronzo ◽  
Anamaris M. Colberg-Poley
Keyword(s):  
Human Cytomegalovirus ◽  
Rna Splicing ◽  
Hcmv Infection ◽  
Human Fibroblasts ◽  
Rna Regulation ◽  
Exon 2 ◽  
Intron 1 ◽  
Exon 1 ◽  
Alternatively Spliced ◽  
Stimulatory Factor

ABSTRACT The human cytomegalovirus (HCMV) UL36-38 immediate early (IE) locus encodes proteins required for its growth. The UL37 promoter drives production of an unspliced and several alternatively spliced RNAs. The UL37 exon 1 (UL37x1) unspliced RNA is abundant from IE to late times of HCMV infection, whereas the UL37 spliced RNAs are markedly less abundant. Production of the UL37x1 unspliced RNA requires polyadenylation (PA) at nucleotide 50998, which lies within intron 1, upstream of the UL37 exon 2 (UL37x2) acceptor. The physical proximity of its cis elements suggests steric hindrance between PA and splicing machineries for UL37 pre-mRNA. To test this possibility, we generated site-specific mutants in Target 1 PA and RNA splicing cis elements and compared the PA and splicing efficiencies of mutant RNAs with those of wild-type RNA. The mutually exclusive processing events of UL37x1 PA and UL37x1-UL37x2 splicing have been accurately recapitulated in transfected permissive human fibroblasts (HFFs) expressing a Target 1 minigene RNA, which contains the required splicing and PA cis elements. Two mutants in the invariant PA signal dramatically decreased UL37x1 PA as expected and, concomitantly, increased the efficiency of UL37x1-UL37x2 RNA splicing. Consistent with these results, changes to consensus UL37x1 donor and UL37x2 acceptor sites increased the efficiency of UL37x1-UL37x2 RNA splicing but decreased the efficiency of UL37x1 PA. Moreover, HCMV infection of HFFs increased the abundance of the PA cleavage stimulatory factor CstF-64, the potent splicing suppressor PTB, and the hypophosphorylated form of the splicing factor SF2 at 4 h postinfection. Induction of these factors further favors production of the UL37x1 unspliced RNA over that of the spliced RNAs. Taken together, these results suggest that there is a convergence in UL37 RNA regulation by cis elements and cellular proteins which favors production of the UL37x1 unspliced RNA during HCMV infection at the posttranscriptional level.

scholarly journals Targeted intragenic demethylation initiates chromatin rewiring for gene activation

2020 ◽  
Author(s):  
Yanjing V. Liu ◽  
Mahmoud A. Bassal ◽  
Quy Xiao Xuan Lin ◽  
Chan-Shuo Wu ◽  
Junsu Kwon ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Ad Hoc ◽  
Gene Activation ◽  
Methyltransferase Activity ◽  
Transcription Start ◽  
Intron 1 ◽  
Long Range Interactions ◽  
Exon 1 ◽  
Cognate Gene ◽  
Aberrant Dna Methylation

AbstractAberrant DNA methylation in the region surrounding the transcription start site is a hallmark of gene silencing in cancer. Currently approved demethylating agents lack specificity and exhibit high toxicity. Herein we show, using the p16 gene as an example, that targeted demethylation of the promoter-exon 1-intron 1 (PrExI) region initiates an epigenetic wave of local chromatin remodeling and distal long-range interactions, culminating in gene-locus specific activation. Through development of CRISPR-DiR (DNMT1-interacting RNA), in which ad hoc edited guides block methyltransferase activity in a locus-specific fashion, we demonstrate that demethylation is coupled to epigenetic and topological changes. These results suggest the existence of a specialized “demethylation firing center (DFC)” which can be switched on by an adaptable and selective RNA-mediated approach for locus-specific transcriptional activation.One Sentence SummaryLocus demethylation via CRISPR-DiR reshapes chromatin structure and specifically reactivates its cognate gene.

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