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.

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.

N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts

1990 ◽  
Vol 10 (8) ◽  
pp. 4180-4191
Author(s):  
G W Krystal ◽  
B C Armstrong ◽  
J F Battey
Keyword(s):  
Rna Polymerase Ii ◽  
Antisense Transcription ◽  
Initiation Site ◽  
Antisense Transcripts ◽  
Rnase Protection ◽  
Transcriptional Initiation ◽  
Intron 1 ◽  
Exon 1 ◽  
Duplex Formation

Nuclear runoff transcription studies revealed nearly equivalent sense and antisense transcription across exon 1 of the N-myc locus. Antisense primary transcription initiates at multiple sites in intron 1 and gives rise to stable polyadenylated and nonpolyadenylated transcripts. This pattern of antisense transcription, which is directed by RNA polymerase II, is independent of gene amplification and cell type. The nonpolyadenylated antisense transcripts have 5' ends which are complementary to the 5' ends of the N-myc sense mRNA. We determined, by using an RNase protection technique designed to detect in vivo duplexes, that most of the cytoplasmic nonpolyadenylated antisense RNA exists in an RNA-RNA duplex with approximately 5% of the sense N-myc mRNA. Duplex formation appeared to occur with only a subset of the multiple forms of the N-myc mRNA, with the precise transcriptional initiation site of the RNA playing a role in determining this selectivity. Cloning of each strand of the RNA-RNA duplex revealed that most duplexes included both exon 1 and intron 1 sequences, suggesting that duplex formation could modulate RNA processing by preserving a population of N-myc mRNA which retains intron 1.

Analysis of cDNAs of the proto-oncogene c-src: heterogeneity in 5' exons and possible mechanism for the genesis of the 3' end of v-src

1991 ◽  
Vol 11 (8) ◽  
pp. 4165-4176
Author(s):  
T Dorai ◽  
J B Levy ◽  
L Kang ◽  
J S Brugge ◽  
L H Wang
Keyword(s):  
Cdna Library ◽  
Chicken Embryo ◽  
Termination Codon ◽  
Cdna Clones ◽  
Rnase Protection ◽  
Exon 2 ◽  
Brain Cdna Library ◽  
Exon 1 ◽  
Polymerase Chain

To further characterize the gene structure of the proto-oncogene c-src and the mechanism for the genesis of the v-src sequence in Rous sarcoma virus, we have analyzed genomic and cDNA copies of the chicken c-src gene. From a cDNA library of chicken embryo fibroblasts, we isolated and sequenced several overlapping cDNA clones covering the full length of the 4-kb c-src mRNA. The cDNA sequence contains a 1.84-kb sequence downstream from the 1.6-kb pp60c-src coding region. An open reading frame of 217 amino acids, called sdr (src downstream region), was found 105 nucleotides from the termination codon for pp60c-src. Within the 3' noncoding region, a 39-bp sequence corresponding to the 3' end of the RSV v-src was detected 660 bases downstream of the pp60c-src termination codon. The presence of this sequence in the c-src mRNA exon supports a model involving an RNA intermediate during transduction of the c-src sequence. The 5' region of the c-src cDNA was determined by analyzing several cDNA clones generated by conventional cloning methods and by polymerase chain reaction. Sequences of these chicken embryo fibroblast clones plus two c-src cDNA clones isolated from a brain cDNA library show that there is considerable heterogeneity in sequences upstream from the c-src coding sequence. Within this region, which contains at least 300 nucleotides upstream of the translational initiation site in exon 2, there exist at least two exons in each cDNA which fall into five cDNA classes. Four unique 5' exon sequences, designated exons UE1, UE2, UEX, and UEY, were observed. All of them are spliced to the previously characterized c-src exons 1 and 2 with the exception of type 2 cDNA. In type 2, the exon 1 is spliced to a novel downstream exon, designated exon 1a, which maps in the region of the c-src DNA defined previously as intron 1. Exon UE1 is rich in G+C content and is mapped at 7.8 kb upstream from exon 1. This exon is also present in the two cDNA clones from the brain cDNA library. Exon UE2 is located at 8.5 kb upstream from exon 1. The precise locations of exons UEX and UEY have not been determined, but both are more than 12 kb upstream from exon 1. The existence and exon arrangements of these 5' cDNAs were further confirmed by RNase protection assays and polymerase chain reactions using specific primers. Our findings indicate that the heterogeneity in the 5' sequences of the c-src mRNAs results from differential splicing and perhaps use of distinct initiation sites. All of these RNAs have the potential of coding for pp60c-src, since their 5' exons are all eventually joined to exon 2.

Follicular lymphoma with t(8;14)(q24;q32): a distinct clinical and molecular subset of t(8;14)-bearing lymphomas

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2124-2130 ◽  
Author(s):  
M Ladanyi ◽  
K Offit ◽  
NZ Parsa ◽  
MR Condon ◽  
N Chekka ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
Point Mutations ◽  
Consecutive Series ◽  
Target Area ◽  
Intron 1 ◽  
Exon 1 ◽  
Myc Gene ◽  
Hodgkin's Lymphomas ◽  
Ig Heavy Chain ◽  
Histologic Subtypes

Abstract The presence of the translocation t(8;14)(q24;q32) has not been well described in follicular lymphoma (FL). In a consecutive series of 278 karyotypically abnormal non-Hodgkin's lymphomas (NHL), six patients with FL showing a t(8;14) without a t(14;18)(q32;q21) were identified. They ranged in age from 45 to 73 years. The cell type was mixed in four patients, small-cleaved in one, and large-cleaved in one; four cases also contained diffuse areas. All cases tested displayed monoclonal surface Ig. The clinical courses were consistent with the histologic subtypes, being less aggressive than other t(8;14)-bearing NHL. In five cases, frozen tissue was available for Southern blotting. The BCL2 gene showed a germline configuration when studied with the MBR, MCR, and 5′ cDNA probes. The MYC gene also appeared unrearranged using an exon-1 probe with EcoRI or HindIII digestion. Analysis of the Ig heavy chain (IgH) gene with a JH region probe and BamHI or EcoRI digestion showed only one rearranged band in all cases, indicating that the 14q32 breakpoint did not lie in either the J or switch-mu (SM) regions. In four cases, the exon-1/intron-1 border of the MYC gene, a target area for point mutations in cases of t(8;14) that do not display rearrangements of the MYC gene, was enzymatically amplified and sequenced; no point mutations were identified. The indolent behavior of our six cases, and the finding that the molecular structure of the t(8;14) in these cases does not follow the pattern of breakpoint sites and point mutations defined in other histologic subtypes of NHL with this translocation, suggest that the t(8;14) in these cases is cytogenetically and molecularly distinct from the t(8;14) seen in high- grade NHLs, and is relatively ineffectual in terms of MYC deregulation, or that other genetic elements at these chromosomal sites may be involved. Further analysis of these tumors may provide insights into MYC deregulation and BCL2-independent FL.

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.

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 5′-Heterogeneity of Glucocorticoid Receptor Messenger RNA Is Tissue Specific: Differential Regulation of Variant Transcripts by Early-Life Events

2000 ◽  
Vol 14 (4) ◽  
pp. 506-517 ◽  
Author(s):  
J. A. McCormick ◽  
V. Lyons ◽  
M. D. Jacobson ◽  
J. Noble ◽  
J. Diorio ◽  
...  
Keyword(s):  
Glucocorticoid Receptor ◽  
Stress Responses ◽  
Early Life ◽  
Messenger Rna ◽  
Promoter Activity ◽  
Cpg Island ◽  
Rnase Protection ◽  
Exon 2 ◽  
Tissue Specific ◽  
Exon 1

Abstract Glucocorticoid receptor (GR) gene expression is regulated in a complex tissue-specific manner, notably by early-life environmental events that program tissue GR levels. We have identified and characterized several new rat GR mRNAs. All encode a common protein, but differ in their 5′-leader sequences as a consequence of alternate splicing of, potentially, 11 different exon 1 sequences. Most are located in a 3-kb CpG island, upstream of exon 2, that exhibits substantial promoter activity in transfected cells. Ribonuclease (RNase) protection analysis demonstrated significant levels of six alternate exons 1 in vivo in rat, with differences between liver, hippocampus, and thymus reflecting tissue-specific differences in promoter activity. Two of the alternate exons 1 (exons 16 and 110) were expressed in all tissues examined, together present in 77–87% of total GR mRNA. The remaining GR transcripts contained tissue-specific alternate first exons. Importantly, tissue-specific first exon usage was altered by perinatal environmental manipulations. Postnatal handling, which permanently increases GR in the hippocampus, causing attenuation of stress responses, selectively elevated GR mRNA containing the hippocampus-specific exon 17. Prenatal glucocorticoid exposure, which increases hepatic GR expression and produces adult hyperglycemia, decreased the proportion of hepatic GR mRNA containing the predomin-ant exon 110, suggesting an increase in a minor exon 1 variant. Such tissue specificity of promoter usage allows differential GR regulation and programming.

scholarly journals N-myc mRNA forms an RNA-RNA duplex with endogenous antisense transcripts.

1990 ◽  
Vol 10 (8) ◽  
pp. 4180-4191 ◽  
Author(s):  
G W Krystal ◽  
B C Armstrong ◽  
J F Battey
Keyword(s):  
Rna Polymerase Ii ◽  
Antisense Transcription ◽  
Initiation Site ◽  
Antisense Transcripts ◽  
Rnase Protection ◽  
Transcriptional Initiation ◽  
Intron 1 ◽  
Exon 1 ◽  
Duplex Formation

Nuclear runoff transcription studies revealed nearly equivalent sense and antisense transcription across exon 1 of the N-myc locus. Antisense primary transcription initiates at multiple sites in intron 1 and gives rise to stable polyadenylated and nonpolyadenylated transcripts. This pattern of antisense transcription, which is directed by RNA polymerase II, is independent of gene amplification and cell type. The nonpolyadenylated antisense transcripts have 5' ends which are complementary to the 5' ends of the N-myc sense mRNA. We determined, by using an RNase protection technique designed to detect in vivo duplexes, that most of the cytoplasmic nonpolyadenylated antisense RNA exists in an RNA-RNA duplex with approximately 5% of the sense N-myc mRNA. Duplex formation appeared to occur with only a subset of the multiple forms of the N-myc mRNA, with the precise transcriptional initiation site of the RNA playing a role in determining this selectivity. Cloning of each strand of the RNA-RNA duplex revealed that most duplexes included both exon 1 and intron 1 sequences, suggesting that duplex formation could modulate RNA processing by preserving a population of N-myc mRNA which retains intron 1.

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 Follicular lymphoma with t(8;14)(q24;q32): a distinct clinical and molecular subset of t(8;14)-bearing lymphomas

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2124-2130
Author(s):  
M Ladanyi ◽  
K Offit ◽  
NZ Parsa ◽  
MR Condon ◽  
N Chekka ◽  
...  
Keyword(s):  
Follicular Lymphoma ◽  
Point Mutations ◽  
Consecutive Series ◽  
Target Area ◽  
Intron 1 ◽  
Exon 1 ◽  
Myc Gene ◽  
Hodgkin's Lymphomas ◽  
Ig Heavy Chain ◽  
Histologic Subtypes

The presence of the translocation t(8;14)(q24;q32) has not been well described in follicular lymphoma (FL). In a consecutive series of 278 karyotypically abnormal non-Hodgkin's lymphomas (NHL), six patients with FL showing a t(8;14) without a t(14;18)(q32;q21) were identified. They ranged in age from 45 to 73 years. The cell type was mixed in four patients, small-cleaved in one, and large-cleaved in one; four cases also contained diffuse areas. All cases tested displayed monoclonal surface Ig. The clinical courses were consistent with the histologic subtypes, being less aggressive than other t(8;14)-bearing NHL. In five cases, frozen tissue was available for Southern blotting. The BCL2 gene showed a germline configuration when studied with the MBR, MCR, and 5′ cDNA probes. The MYC gene also appeared unrearranged using an exon-1 probe with EcoRI or HindIII digestion. Analysis of the Ig heavy chain (IgH) gene with a JH region probe and BamHI or EcoRI digestion showed only one rearranged band in all cases, indicating that the 14q32 breakpoint did not lie in either the J or switch-mu (SM) regions. In four cases, the exon-1/intron-1 border of the MYC gene, a target area for point mutations in cases of t(8;14) that do not display rearrangements of the MYC gene, was enzymatically amplified and sequenced; no point mutations were identified. The indolent behavior of our six cases, and the finding that the molecular structure of the t(8;14) in these cases does not follow the pattern of breakpoint sites and point mutations defined in other histologic subtypes of NHL with this translocation, suggest that the t(8;14) in these cases is cytogenetically and molecularly distinct from the t(8;14) seen in high- grade NHLs, and is relatively ineffectual in terms of MYC deregulation, or that other genetic elements at these chromosomal sites may be involved. Further analysis of these tumors may provide insights into MYC deregulation and BCL2-independent FL.

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