Analysis of the CC chemokine receptor 3 gene reveals a complex 5′ exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2346-2354 ◽  
Author(s):  
Nives Zimmermann ◽  
Bruce L. Daugherty ◽  
Jessica L. Kavanaugh ◽  
Faisal Y. El-Awar ◽  
Elizabeth A. Moulton ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Messenger Rna ◽  
Direct Sequencing ◽  
Cell Types ◽  
Northern Analysis ◽  
Strong Activity ◽  
Exon 2 ◽  
Cc Chemokine ◽  
Untranslated Exon ◽  
Exon 1

Abstract To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5′ untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5′ flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the humanCCR3 gene has a complex 5′ exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5′ untranslated exon.

Analysis of the CC chemokine receptor 3 gene reveals a complex 5′ exon organization, a functional role for untranslated exon 1, and a broadly active promoter with eosinophil-selective elements

Blood ◽  
2000 ◽  
Vol 96 (7) ◽  
pp. 2346-2354
Author(s):  
Nives Zimmermann ◽  
Bruce L. Daugherty ◽  
Jessica L. Kavanaugh ◽  
Faisal Y. El-Awar ◽  
Elizabeth A. Moulton ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Messenger Rna ◽  
Direct Sequencing ◽  
Cell Types ◽  
Northern Analysis ◽  
Strong Activity ◽  
Exon 2 ◽  
Cc Chemokine ◽  
Untranslated Exon ◽  
Exon 1

To understand the regulation of CC chemokine receptor 3 (CCR3) expression, its gene structure and promoter have been characterized. The CCR3 gene contains 4 exons that give rise to multiple messenger RNA (mRNA) species by alternative splicing. Exon 1 is present in all transcripts, whereas exon 2 or 3 is present at low frequency (< 10%). Exon 4 contains the open reading frame and 11 bp of the 5′ untranslated region. Northern analysis revealed 4 species of CCR3 mRNA. Direct sequencing revealed that the first 1 kb of the promoter and exon 1 contained only one mutation in 19 individuals, indicating that the CCR3 promoter and exon 1 are conserved between individuals. The first 1.6 kb of the 5′ flanking region of exon 1 contained promoter elements including a TATA box and motifs for myeloid transcription factors and had strong promoter activity in eosinophilic, lymphoid, myeloid, and respiratory epithelial cell lines. Deletion analysis revealed differential regulation of the CCR3 promoter in eosinophilic and epithelial cells suggesting the presence of lineage-specific elements. Interestingly, exon 1 enhanced the activity of the promoter and this effect was especially prominent in eosinophilic cells. Thus, the humanCCR3 gene has a complex 5′ exon structure, a conserved promoter with strong activity in multiple cell types, and a functional 5′ untranslated exon.

scholarly journals Complete Thyroxine-Binding Globulin (TBG) Deficiency Produced by a Mutation in Acceptor Splice Site Causing Frameshift and Early Termination of Translation (TBG-Kankakee)12

1998 ◽  
Vol 83 (10) ◽  
pp. 3604-3608
Author(s):  
Gisah A. Carvalho ◽  
Roy E. Weiss ◽  
Samuel Refetoff
Keyword(s):  
Splice Site ◽  
Messenger Rna ◽  
Restriction Site ◽  
Splice Junction ◽  
Early Termination ◽  
Coding Region ◽  
Acceptor Splice Site ◽  
Exon 2 ◽  
Gene Level ◽  
Exon 1

Fourteen T4-binding globulin (TBG) variants have been identified at the gene level. They are all located in the coding region of the gene and 6 produce complete deficiency of TBG (TBG-CD). We now describe the first mutation in a noncoding region producing TBG-CD. The proband was treated for over 20 yr with L-T4 because of fatigue associated with a low concentration of serum total T4. Fifteen family members were studied showing low total T4 inherited as an X chromosome-linked trait, and affected males had undetectable TBG in serum. Sequencing of the entire coding region and promoter of the TBG gene revealed no abnormality. However, an A to G transition was found in the acceptor splice junction of intron II that produced a new HaeIII restriction site cosegregating with the TBG-CD phenotype. Sequencing exon 1 to exon 3 of TBG complementary DNA reverse transcribed from messenger RNA of skin fibroblasts from an affected male, confirmed a shift in the ag acceptor splice site. This results in the insertion of a G in exon 2 and causes a frameshift and a premature stop at codon 195. This early termination of translation predicts a truncated TBG lacking 201 amino acids.

In-Vitro Cytotoxicity of Tipifarnib (Zarnestra TM) in Pediatric AML and ALL Samples Is Independent of RAS Mutations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1172-1172 ◽  
Author(s):  
Bianca F. Goemans ◽  
Christian M. Zwaan ◽  
Gertjan J.L. Kaspers ◽  
Karel Hählen ◽  
Dirk Reinhardt ◽  
...  
Keyword(s):  
Clinical Studies ◽  
Direct Sequencing ◽  
Farnesyltransferase Inhibitors ◽  
Exon 2 ◽  
Ras Mutations ◽  
Lc50 Value ◽  
Significant Difference ◽  
Exon 1 ◽  
Pediatric Aml

Abstract The farnesyltransferase inhibitor tipifarnib (Zarnestra™) was originally developed to target malignancies harbouring RAS mutations. In the first clinical studies with tipifarnib, in adults with leukemia, it was found that patients who responded did not harbour any RAS mutations, suggesting a different mechanism of response. In a previous study we showed that 18% of 150 untreated pediatric AML patients harbour mutations in RAS, of which 30% were CBF-AML. We now studied 44 untreated and 13 relapsed pediatric AML, as well as 22 untreated ALL samples for mutations in RAS, using D-HPLC and direct sequencing. In vitro tipifarnib resistance was determined by a 4-day MTT assay (concentration 0.016-51μM, kindly provided by Janssen Research). The LC50 value, the concentration at which 50% of cells are killed by tipifarnib, was used as a measure of resistance. Patient characteristics were; for untreated AML: 64% boys; median age 9.3 years; median WBC 74.8x109/L; FAB 2xM0, 2xM1, 8xM2, 3xM3, 16xM4, 8xM5, 5x unclassified; for relapsed AML: 77% boys; median age 4.0 years; median WBC 41.6x109/L; FAB 2xM0, 2xM2, 3xM4, 2xM5, 2xM7, 2x unclassified; for untreated ALL: 73%boys; median age 6.0 years; median WBC 10.2x109/L; 15 B-cell precursor (BCP) ALL and 7 T-ALL. We found RAS mutations in 14 (32%) untreated AML samples (N-RAS : 8 samples exon 1, 1 sample exon 2; K-RAS: 5 samples exon 1 mutations). In relapsed AML 2 samples showed an N-RAS exon 1 mutation (15.4%). In ALL 18.2% had a RAS mutation: an N-RAS exon 1 mutation was found in 2 patients (9.1%) and a K-RAS exon 1 mutation in another 2 patients (9.1%). The distribution of tipifarnib sensitivity was similar in RAS mutated- and non-mutated untreated AML patients [median LC50 RAS mutated 7.1μM (P25-P75: 6.0-9.6μM) vs. non-mutated 4.9μM (P25-P75 2.3-8.2μM); p=0.199]. When we compared N-RAS mutated samples with K-RAS mutated samples there was no statistically significant difference in sensitivity to tipifarnib (median LC50 [p25-p75] 3.2μM [2.9-3.9μM] and 4.9μM [3.7-23.1μM], p=0.20), and comparing them separately with non-mutated AML did not show differences in sensitivity to tipifarnib (p=0.172 and p=0.463 respectively). One out of 9 (11%) N-RAS mutated and 3 out of 5 (60%) K-RAS mutated samples had an LC50 value above the 75th percentile for non-mutated AML and were considered resistant. Within relapsed AML the 2 RAS mutated samples had LC50 values of 0.83 and 6.3μM, versus a median value of 6.9μM for non-mutated relapsed AML. In ALL, we found similar results [median LC50 RAS mutated 7.8μM (P25-75: 4.1-12.8μM) vs. non-mutated 17.4μM (P25-75: 4.5-22.9μM), p=0.3], but the groups were very small. In conclusion, primary pediatric AML and ALL samples withRAS mutations show similar distributions of tipifarnib sensitivity as samples withoutRAS mutations. Hence, some RAS mutated samples may be relatively in-vitro resistant to tipifarnib, and some non-mutated samples may be relatively sensitive. Therefore, clinical studies with these compounds should not be restricted to RAS-mutated leukemia. Further studies are necessary to determine the molecular targets of farnesyltransferase inhibitors.

Exon 2 of the TFPI Gene Is a Translational Repressor of TFPIβ Protein Expression

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3358-3358
Author(s):  
Paul E. R. Ellery ◽  
Susan A Maroney ◽  
Alan E. Mast
Keyword(s):  
Alternative Splicing ◽  
Protein Expression ◽  
Northern Analysis ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Exon 2 ◽  
Translational Repressor ◽  
Exon 1 ◽  
Reporter Assays ◽  
Protein Isoform

Abstract Abstract 3358 Background: Tissue factor pathway inhibitor (TFPI) is an essential anticoagulant protein located on endothelium and within platelets. Two TFPI isoforms, termed TFPIα and TFPIβ, are generated through alternative splicing toward the 3' end of the TFPI gene. Alternative splicing also occurs within the 5' untranslated region (UTR) of TFPI mRNA, resulting in the removal of exon 2 (X2). Previous studies have demonstrated that TFPIα and TFPIβ mRNA are produced, on average, in a ratio of 10:1 (TFPIα:TFPIβ) in human tissues. However, this does not appear to correlate with protein expression, suggesting that TFPI protein isoform production is regulated during translation. Biochemical studies were undertaken to determine how alternative splicing within the 5' or 3' UTRs of TFPI mRNA may function in the translational regulation of TFPI protein isoform production. Methods: Northern analysis of human lung RNA using probes directed toward Exon 1 (X1) or Exon 2 (both 5' UTR), Exon 6 (common to both TFPIα and TFPIβ), Exon 8 (TFPIβ specific) or Exons 9 and 10 (TFPIα specific), and RT-PCR analysis of multiple human tissue cDNAs using an exon 1 specific forward primer and TFPIα or TFPIβ specific reverse primers, were used to investigate isoform specific alternative splicing of exon 2. Polysome profiling of HUVEC and EA.hy926 cellular RNA was performed to investigate translational control of TFPI isoform expression. Luciferase (Luc) reporter assays were established to quantify the effect of exon 2 on TFPI isoform protein expression. Constructs were produced where both splice variants of the 5' UTR, and the TFPIα or TFPIβ specific 3' UTRs, were inserted at their respective ends of a Gaussia Luc expression cassette. Luc activity was assessed in stably transfected CHO or EA.hy926 cells and normalized to GFP production or Luc mRNA. Results: Northern analysis revealed the presence of two TFPIα specific bands at 4.1 and 1.4 kb, produced via alternative polyadenylation, and one TFPIβ specific band at 1.1 kb. Exon 1 and 2 probes bind both TFPIα species, demonstrating that 5' UTR alternative splicing occurs in TFPIα mRNA. RT-PCR analysis of human placenta, lung, and heart cDNA confirmed this, and demonstrated that exon 2 is also alternatively spliced in TFPIβ message. Polysome analysis identified TFPIβ-specific translational repression by exon 2. Luciferase reporter assays quantified this repression at 93% and 86% in CHO and EA.hy926 cells, respectively (see Table 1 for normalized data). In contrast, exon 2 had minimal effect on luciferase expression when the TFPIα 3' UTR was present. RT-PCR analysis of 15 human tissues revealed that testis, thymus, brain, and spleen had the highest relative amount of exon 2 containing TFPIβ message, although all tissues had more exon 2-absent TFPIβ message compared to that containing exon 2. Conclusion: Exon 2 is a strong translational repressor of TFPIβ production, and a much weaker repressor of TFPIα. To our knowledge, this is the first time that alternative splicing within a 5' UTR has been demonstrated to affect a specific protein isoform produced via alternative splicing at the 3' end of the same gene. While the mechanism of repression has yet to be elucidated, the repression of TFPIβ, and not TFPIα, by exon 2 suggests that regions within both exon 2 and the 3' UTR are required for this effect and is consistent with the hypothesis of mRNA circularization via interactions between the 5' and 3' UTRs. We hypothesize that the physiological relevance of the demonstrated repression will include tissue-specific regulation of TFPIβ expression and cellular regulation of TFPIβ expression in inflammatory disease. Disclosures: Mast: Novo Nordisk A/S: Research Funding.

scholarly journals The promoter and first, untranslated exon of the human glucocorticoid receptor gene are GC rich but lack consensus glucocorticoid receptor element sites.

1990 ◽  
Vol 10 (10) ◽  
pp. 5580-5585 ◽  
Author(s):  
J Zong ◽  
J Ashraf ◽  
E B Thompson
Keyword(s):  
Glucocorticoid Receptor ◽  
Receptor Gene ◽  
Flanking Sequence ◽  
Exon 2 ◽  
Transcription Start Sites ◽  
Untranslated Exon ◽  
Glucocorticoid Response ◽  
Glucocorticoid Receptor Gene ◽  
Exon 1 ◽  
Glucocorticoid Receptor Mrna

Glucocorticoid receptor mRNA is regulated by glucocorticoids. We found no consensus glucocorticoid response element, TATA box, or CAAT box but many GC boxes in approximately 3 kilobases of the 5'-flanking sequence of the human glucocorticoid receptor gene. We identified several transcription start sites, an untranslated exon 1, and the coding content of exon 2.

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 Identification of single nucleotide polymorphism c.957A>C of PLAG1 gene and its association with growth traits in Bali cattle

2021 ◽  
Vol 46 (3) ◽  
pp. 199-208
Author(s):  
I. G. R. Putra ◽  
D. A. Sari ◽  
S. M. Rachmawati ◽  
R. Oktaviani ◽  
R. R. Noor ◽  
...  
Keyword(s):  
Growth Traits ◽  
Direct Sequencing ◽  
Average Daily Gain ◽  
Single Nucleotide ◽  
Exon 2 ◽  
Sequencing Method ◽  
Pcr Rflp ◽  
Exon 1 ◽  
Bali Cattle ◽  
Daily Gain

The PLAG1 gene is one of the genes that affect the growth traits located on chromosome 14 in cattle. This study aims to obtain SNP of the PLAG1 gene in exon 1 and exon 2 and their association with growth traits in Bali cattle. The number of samples used was 52 samples of Bali cattle, 10 samples of Peranakan Ongole (PO), and 8 samples of Limousine cattle. Identification of SNPs PLAG1 gene was analyzed by direct sequencing method and genotyping of selected SNPs was carried out using PCR-RFLP. Association of genotypes of SNP c.957A>C with growth using t-test. There were 7 SNPs in exon 2 of the PLAG1 gene, namely SNP c.339A>G, c.489C>T, c.795A>G, c.957A>C, c.1023C>T, c.1056A>G, and c.1353A>G. SNP c.957A>C was validated by PCR-RFLP using TaqI enzyme and obtained three genotypes, namely genotypes AA, AC, and CC with allele frequency A and C, respec-tively 0.10 and 0.90 in Bali cattle, while in PO and Limousine cattle were monomorphic. Genotype association of SNP c.957A>C PLAG1 gene were not associated with birth weight (BW0), weaning weight at 205 days of age (WW205), yearling weight at 365 days of age (YW365), yearling weight at 730 days of age (YW730), and average daily gain (ADG). SNP c.957A>C as a specific SNP for Bali cattle needs to be investigated in further research as a candidate marker for growth traits in Bali cattle.

The chemokine receptor CCR8 mediates human endothelial cell chemotaxis induced by I-309 and Kaposi sarcoma herpesvirus-encoded vMIP-I and by lipoprotein(a)-stimulated endothelial cell conditioned medium

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 39-45 ◽  
Author(s):  
Nasreen S. Haque ◽  
John T. Fallon ◽  
Mark B. Taubman ◽  
Peter C. Harpel
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Conditioned Medium ◽  
Chemokine Receptor ◽  
Messenger Rna ◽  
Umbilical Vein ◽  
Human Herpesvirus ◽  
Kaposi Sarcoma ◽  
Cc Chemokine ◽  
Apolipoprotein A

Abstract The CC chemokine receptor 8 (CCR8) is expressed on monocytes and type 2 T lymphocytes. CCR8 is the sole receptor for the human CC chemokine I-309, as well as for viral monocyte inflammatory protein-I (vMIP-I), a human chemokine homologue induced in human cells by the Kaposi sarcoma-related human herpesvirus-8. Recently it was found that I-309 messenger RNA and protein are expressed by human umbilical vein endothelial cells (HUVECs) and that the secretion of endothelial I-309 is stimulated by apolipoprotein(a). I-309, vMIP-I, and the conditioned medium from apolipoprotein(a)-stimulated HUVECs induce endothelial chemotaxis. A polyclonal anti-CCR8 antibody and a newly developed murine monoclonal antibody against CCR8 inhibited this activity. The G-protein inhibitor pertussis toxin also inhibited endothelial chemotaxis, providing further evidence for a chemokine receptor-mediated effect. Endothelial cells contain CCR8 mRNA as shown by RNA blot analysis as well by direct sequence analysis. Immunohistochemical studies identified CCR8 and I-309 on the endothelium of human atherosclerotic plaques and in endothelial-derived spindle cells of Kaposi sarcoma. These results indicate that CCR8 is an endothelial receptor that may modulate endothelial function.

The promoter and first, untranslated exon of the human glucocorticoid receptor gene are GC rich but lack consensus glucocorticoid receptor element sites

1990 ◽  
Vol 10 (10) ◽  
pp. 5580-5585
Author(s):  
J Zong ◽  
J Ashraf ◽  
E B Thompson
Keyword(s):  
Glucocorticoid Receptor ◽  
Receptor Gene ◽  
Flanking Sequence ◽  
Exon 2 ◽  
Transcription Start Sites ◽  
Untranslated Exon ◽  
Glucocorticoid Response ◽  
Glucocorticoid Receptor Gene ◽  
Exon 1 ◽  
Glucocorticoid Receptor Mrna

Glucocorticoid receptor mRNA is regulated by glucocorticoids. We found no consensus glucocorticoid response element, TATA box, or CAAT box but many GC boxes in approximately 3 kilobases of the 5'-flanking sequence of the human glucocorticoid receptor gene. We identified several transcription start sites, an untranslated exon 1, and the coding content of exon 2.

Alternative Splicing Is Responsible for the Presence of Two Tissue Factor mRNA Species in LPS Stimulated Human Monocytes

1992 ◽  
Vol 67 (02) ◽  
pp. 272-276 ◽  
Author(s):  
C Paul ◽  
E van der Logt ◽  
Pieter H Reitsma ◽  
Rogier M Bertina
Keyword(s):  
Alternative Splicing ◽  
Tissue Factor ◽  
Stop Codon ◽  
Cell Types ◽  
Northern Analysis ◽  
Human Monocytes ◽  
Mrna Species ◽  
Protein Levels ◽  
Intron 1 ◽  
Tf Gene

SummaryAlthough normally absent from the surface of all circulating cell types, tissue factor (TF) can be induced to appear on circulating monocytes by stimulants like bacterial lipopolysaccharide (LPS) and phorbolesters. Northern analysis of RNA isolated from LPS stimulated human monocytes demonstrates the presence of 2.2 kb and 3.1 kb TF mRNA species. The 2.2 kb message codes for the TF protein. As demonstrated by Northern blot analysis with a variety of TF gene probes, the 3.1 kb message arises from an alternative splicing process which fails to remove 955 bp from intron 1. Because of a stop codon in intron 1 no TF protein is produced from the 3.1 kb transcript. This larger transcript should therefore not be taken into account when comparing TF gene transcription and TF protein levels.

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