Identification of upstream regulatory regions in the heart-expressed homeobox gene Nkx2-5

Development ◽  
1999 ◽  
Vol 126 (4) ◽  
pp. 839-849 ◽  
Author(s):  
J.M. Reecy ◽  
X. Li ◽  
M. Yamada ◽  
F.J. DeMayo ◽  
C.S. Newman ◽  
...  
Keyword(s):  
Homeobox Gene ◽  
Protein Isoforms ◽  
Proximal Promoter ◽  
Coding Region ◽  
Heart Tube ◽  
Regulatory Regions ◽  
Regulatory Domains ◽  
Exon 1 ◽  
Flanking Sequences ◽  
Upstream Exon

Nkx2-5 marks the earliest recognizable cardiac progenitor cells, and is activated in response to inductive signals involved in lineage specification. Nkx2-5 is also expressed in the developing foregut, thyroid, spleen, stomach and tongue. One approach to elucidate the signals involved in cardiogenesis was to examine the transcriptional regulation of early lineage markers such as Nkx2-5. We generated F0 transgenic mice, which carry Nkx2-5 flanking sequences linked to a lacZ reporter gene. We identified multiple regulatory regions located within the proximal 10.7 kb of the Nkx2-5 gene. In addition to a proximal promoter, we identified a second promoter and a novel upstream exon that could participate in the regulation of Nkx2-5 transcription. Although used rarely in normal development, this novel exon could be spliced into the Nkx2-5 coding region in several ways, thereby potentially creating novel Nkx2-5 protein isoforms, whose transcriptional activity is greatly diminished as compared to wild-type Nkx2-5. An enhancer that directs expression in pharynx, spleen, thyroid and stomach was identified within 3.5 kb of exon 1 between the coding exon 1 and the novel upstream exon 1a. Two or more enhancers upstream of exon 1a were capable of driving expression in the cardiac crescent, throughout the myocardium of the early heart tube, then in the outflow tract and right ventricle of the looped heart tube. A negative element was also located upstream of exon1a, which interacted in complex ways with enhancers to direct correct spatial expression. In addition, potential autoregulatory elements can be cooperatively stimulated by Nkx2-5 and GATA-4. Our results demonstrate that a complex suite of interacting regulatory domains regulate Nkx2-5 transcription. Dissection of these elements should reveal essential features of cardiac induction and positive and negative signaling within the cardiac field.

scholarly journals The TRACE-Seq method tracks recombination alleles and identifies clonal reconstitution dynamics of gene targeted human hematopoietic stem cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Rajiv Sharma ◽  
Daniel P. Dever ◽  
Ciaran M. Lee ◽  
Armon Azizi ◽  
Yidan Pan ◽  
...  
Keyword(s):  
Genetic Diseases ◽  
Basic Research ◽  
Gene Correction ◽  
Coding Region ◽  
Hematopoietic Stem ◽  
Hematopoietic Reconstitution ◽  
Exon 1 ◽  
Template Library ◽  
Donor Template

AbstractTargeted DNA correction of disease-causing mutations in hematopoietic stem and progenitor cells (HSPCs) may enable the treatment of genetic diseases of the blood and immune system. It is now possible to correct mutations at high frequencies in HSPCs by combining CRISPR/Cas9 with homologous DNA donors. Because of the precision of gene correction, these approaches preclude clonal tracking of gene-targeted HSPCs. Here, we describe Tracking Recombination Alleles in Clonal Engraftment using sequencing (TRACE-Seq), a methodology that utilizes barcoded AAV6 donor template libraries, carrying in-frame silent mutations or semi-randomized nucleotides outside the coding region, to track the in vivo lineage contribution of gene-targeted HSPC clones. By targeting the HBB gene with an AAV6 donor template library consisting of ~20,000 possible unique exon 1 in-frame silent mutations, we track the hematopoietic reconstitution of HBB targeted myeloid-skewed, lymphoid-skewed, and balanced multi-lineage repopulating human HSPC clones in mice. We anticipate this methodology could potentially be used for HSPC clonal tracking of Cas9 RNP and AAV6-mediated gene targeting outcomes in translational and basic research settings.

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.

Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants

Development ◽  
1993 ◽  
Vol 119 (2) ◽  
pp. 419-431 ◽  
Author(s):  
T.J. Lints ◽  
L.M. Parsons ◽  
L. Hartley ◽  
I. Lyons ◽  
R.P. Harvey
Keyword(s):  
Progenitor Cells ◽  
Myogenic Differentiation ◽  
Homeobox Genes ◽  
Homeobox Gene ◽  
Heart Tube ◽  
New Members ◽  
Mesoderm Development ◽  
Early Entry ◽  
Developing Heart ◽  
Valuable Marker

We have isolated two murine homeobox genes, Nkx-2.5 and Nkx-2.6, that are new members of a sp sub-family of homeobox genes related to Drosophila NK2, NK3 and NK4/msh-2. In this paper, we focus on the Nkx-2.5 gene and its expression pattern during post-implantation development. Nkx-2.5 transcripts are first detected at early headfold stages in myocardiogenic progenitor cells. Expression preceeds the onset of myogenic differentiation, and continues in cardiomyocytes of embryonic, foetal and adult hearts. Transcripts are also detected in future pharyngeal endoderm, the tissue believed to produce the heart inducer. Expression in endoderm is only found laterally, where it is in direct apposition to promyocardium, suggesting an interaction between the two tissues. After foregut closure, Nkx-2.5 expression in endoderm is limited to the pharyngeal floor, dorsal to the developing heart tube. The thyroid primordium, a derivative of the pharyngeal floor, continues to express Nkx-2.5 after transcript levels diminish in the rest of the pharynx. Nkx-2.5 transcripts are also detected in lingual muscle, spleen and stomach. The expression data implicate Nkx-2.5 in commitment to and/or differentiation of the myocardial lineage. The data further demonstrate that cardiogenic progenitors can be distinguished at a molecular level by late gastrulation. Nkx-2.5 expression will therefore be a valuable marker in the analysis of mesoderm development and an early entry point for dissection of the molecular basis of myogenesis in the heart.

scholarly journals Variability of PSPAL1 (phenylalanine ammonia-lyase gene-1) proximal promoter sequence and expression in pea challenged with Mycosphaerella pinodes

2014 ◽  
Vol 50 (No. 2) ◽  
pp. 163-170 ◽  
Author(s):  
S. Okorska ◽  
D. Michalczyk ◽  
A. Okorski ◽  
A. Piotrowicz-Cieślak ◽  
P. Pupel ◽  
...  
Keyword(s):  
Signal Transmission ◽  
Infected Plant ◽  
Promoter Sequence ◽  
Proximal Promoter ◽  
Mycosphaerella Pinodes ◽  
Post Inoculation ◽  
Relative Level ◽  
Exon 1 ◽  
Weak Negative Correlation ◽  
High Level

Part of the PSPAL1 gene (corresponding to the proximal promoter, exon 1 and intron) from eight pea varieties was sequenced and compared to the published sequence of PSPAL1 gene from Midoriusui cultivar (GenBank: D10002.1). The sequences showed a very high level of identity (96–99%), except in five varieties there occurred a motif TTATTACAAAATATTA close to the Goldberg-Hogness (TATA) box, and it was not detected in the other four varieties, including Midoriusui. Plants of eight pea varieties were subjected to controlled infection with Mycosphaerella pinodes and the disease index was determined (it ranged from 5.2 to 42.3%). The PSPAL1 gene of the most resistant cultivar (Walor) contained the above-mentioned motif and that of the most susceptible (Polar) did not. However, the relationship was not clear in varieties with intermediate levels of resistance. In four varieties (Walor, Ezop, Ramrod and Polar) the expression level of PSPAL1 gene in leaves was analysed (1, 3, 6, 9, 12 and 15 h post inoculation) and it showed a weak negative correlation with disease severity (R= – 0.53). The activation of PSPAL1 gene occurred not only in infected pea leaves but also in stems and – to a much lower degree – in roots (with the relative level of PSPAL1 transcripts amounting to 0.15 in roots and 38.75 in leaves), indicating some kind of signal transmission beyond the infected plant tissues.

Identifying Novel Blood Proteins and Protein Isoforms Using Tandem Mass Spectra.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1616-1616
Author(s):  
Damian Fermin ◽  
Baxter B. Allen ◽  
Thomas W. Blackwell ◽  
Rajasree Menon ◽  
Marcin Adamski ◽  
...  
Keyword(s):  
Human Genome ◽  
Genomic Sequence ◽  
Complete Sequence ◽  
Open Reading Frames ◽  
Protein Isoforms ◽  
Blood Protein ◽  
Tandem Mass ◽  
Blood Proteins ◽  
Coding Region ◽  
Significant Alignment

Abstract Blood is a complex fluid that samples all tissues in the human body. Despite complete sequence determination of the human genome, defining genes and gene products remains a challenge. Here, we apply tandem mass spectroscopy as new source of unbiased data to interrogate genomic sequence and identify novel protein coding sequences. A six-frame translation of the Human genome was used as the query database to search for novel blood proteins in the data from the HUPO PPP. Significance is assessed using a Poisson statistical model incorporating the length of the matching sequence and the frequency of spectrum matches observed in searching the database [Nat Biotech 2006 24(3):333–8]. Matches are binned by X!Tandem hyperscore, and statistics for each score class are considered independently. The overall probability that the matches to an ORF occurred at random is calculated as the product of the probability that the matches in each score category occurred at random. The expected number of random matches, E, is calculated as the product of the probability that an ORF match occurred at random multiplied by the number of ORFs searched. The confidence in an ORF identification is 1/(1+E). An open reading frame is considered significant if confidence is greater than 95%. Expanding recently published work [Genome Biol2006; 7(4):R35], we have identified 837 significant open reading frames coding for 18852 peptides falling within 914 exons of 413 genes. Out of 8856 candidate ORFs outside the boundaries of known genes, 3246 of them achieved a confidence >= 0.95. We also required the XG ORFs to be supported by at least 3 distinct ESTs. Twenty four of the XG ORFs were found to have a significant alignment to the mouse genome. Of these, 13 of the alignments encompassed a coding region for one of the diagnostic peptides associated with the ORF. Gene models for the XG ORFS were derived from the GENSCAN prediction made for their coding regions. This analysis suggests that alternative splicing of blood protein genes is common and that much remains to be learned about the protein constituents of blood.

Noval Mutation in Four Saudi Families with Glanzmann Thrombasthenia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1136-1136
Author(s):  
Tarek Owaidah ◽  
Hala Abalkhail ◽  
Abdulrahman Al Musa ◽  
Hasan Mosmali ◽  
Albanyan Abdulmajeed ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Stop Codon ◽  
Direct Sequencing ◽  
Family Members ◽  
Premature Stop Codon ◽  
Type I ◽  
Bleeding Tendency ◽  
Coding Region ◽  
Glanzmann Thrombasthenia ◽  
Exon 1

Abstract Abstract 1136 Introduction: Glanzmann thrombasthenia (GT) is a rare autosomal recessive inherited bleeding disorder characterized by an impaired platelet aggregation and variable bleeding tendency. Inherited genetic mutations in integrin alpha IIb and beta3 (ITGA2B, ITGB3) result in a heterogeneity of the thrombasthenia phenotypes. It is phenotypically expressed in homozygotes or compound heterozygotes, given that 50% of normal aIIbb3 is sufficient to guarantee unimpaired platelet function that result in asymptomatic carriers. Defects in ITGB3 result in failure of binding of B3 and alpha IIb. These defects had been reported in Arabs (Iraqi Jews). We are reporting some results of Saudi GT genotype project. Materials & Methods: In this study, we analyzed the entire coding region ITGB3 gene using polymerase chain reaction (PCR) and direct sequencing with primers specifically designed to amplify the coding region of exon 1–15 and exon /Intron boundaries in a cohort of 51 GT patients diagnosed and treated in our institute. Results: Out of 51 cases from 20 families had mutational screening of the ITGB3 gene with the aim to detect the causative pathogenic mutations to enable the pre-symptomatic diagnosis in at risk family members. In this study we detect 1 novel germline mutation c.2190delC (p.Ser703fs) in exon 13. The mutation is predicted to result in premature stop codon and protein truncation. The mutation was detected in 6 patients in homozygous stat (3 males and 3 females). Three tested samples from the patients family members detected the mutation in heterozygous state and all of them were asymptomatic with normal PFA and Intact expression of Platelet Glycoprotiens CD41(Gpllb), CD42a(GPIX), CD42b(GPlb), and CD61(Gpllla). All the GT patients with this mutation were type I GT with Prolonged PFA and complete absence of CD41(Gpllb) and CD61(Gpllla) glycoprotein. Conclusion: The result of this study represents the first Molecular analysis of ITGB3 gene in Saudi Arabia and displays the existence of novel pathogenic and possibly a founder effect in Saudi families. Disclosures: No relevant conflicts of interest to declare.

Characterization Of a Weakly Expressed KIR2DL1 Allele

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4847-4847
Author(s):  
Hongchuan Li ◽  
Andrew R Huehn ◽  
Postbaccalaureate Fellow ◽  
Paul W Wright ◽  
Research Biologist ◽  
...  
Keyword(s):  
Single Copy ◽  
Surface Expression ◽  
Genetic Alterations ◽  
Proximal Promoter ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Class I Mhc ◽  
Complete Sequencing ◽  
Weak Expression ◽  
Distal Promoter

Abstract The variegated expression of the human KIR family of class I MHC receptors provides an interesting model system for the study of stochastic activation of gene transcription. Previous studies have linked distal KIR promoter transcription to the initiation of KIR expression from the proximal promoter. In order to identify novel genetic alterations associated with decreased KIR expression, a group of 182 donors was characterized for KIR gene content, KIR transcripts, and FACS analysis of KIR surface expression. An individual was discovered that possessed a single copy of the KIR2DL1 gene but had a low level of gene expression by either FACS or Q-PCR.  Complete sequencing of the KIR2DL1 gene confirmed the presence of an intact coding region. Analysis of promoter elements revealed a cluster of three single nucleotide polymorphisms (SNPs) in the distal promoter approximately 1 kb upstream from the start of KIR2DL1 translation. These SNPs are also found in the distal promoter region of the non-transcribed KIR2DL5*002 allele as well as the KIR3DP1 pseudogene. One of these SNPs creates a functional binding site for the ZEB1 transcription factor.  Individuals possessing the ZEB1 site in their KIR2DL1 promoter produce high levels of a non-translatable distal KIR2DL1transcript that inhibits transcription from the proximal promoter, resulting in weak expression of this allele. This research was supported in part by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. Funded by NCI Contract HHSN261200800001E. Disclosures: Miller: Coronado Biosciences: Scientific Advisory Board Other.

Molecular characterization and SNP identification in HSPB1 gene in Murrah buffalo

2015 ◽  
Author(s):  
Ashwani Arya ◽  
Archana Verma ◽  
I. D. Gupta ◽  
Shahid A. Shergojry ◽  
Ankit Magotra ◽  
...  
Keyword(s):  
Heat Shock ◽  
Marker Assisted Selection ◽  
Bos Taurus ◽  
Nucleotide Position ◽  
Coding Region ◽  
Murrah Buffalo ◽  
Future Improvement ◽  
Pcr Products ◽  
Exon 1 ◽  
Heat Shock Protein Genes

Present study was conducted on 100 lactating Murrah buffaloes maintained at LRC, NDRI Karnal (Haryana) to characterize and to identify genetic polymorphisms in HSPB1 gene. Two sets of primers specific to coding sequence of HSPB1 gene were designed using Primer3 software and PCR products of 631 bp and 670 bp were obtained. Amplicons were custom sequenced and subjected to ClustalW analysis which revealed 8 nucleotide changes, 7 in non coding region and one in coding region in Murrah buffalo sequence as compared to Bos taurus. Only one SNP at nucleotide position G225A in exon 1 of HSPB1 gene was observed in Murrah buffalo, which resulted in two genotypes GG and AG with respective frequency of 0.84 and 0.16 indicating the existence of variability in the sampled population. The study has opened the possibility of identifying and using genetic variations in major heat shock protein genes, for the future improvement of livestock through marker assisted selection.

A pharmacogenetic study of irinotecan: Genetic polymorphisms in the coding region of UGT1A1 gene and SN-38 glucuronidation in the body

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 13078-13078
Author(s):  
K. Araki ◽  
K. Fujita ◽  
Y. Ando ◽  
F. Nagashima ◽  
W. Yamamoto ◽  
...  
Keyword(s):  
Drug Disposition ◽  
Japanese Patients ◽  
The Body ◽  
Wild Type ◽  
Coding Region ◽  
Pharmacogenetic Study ◽  
Time Curve ◽  
Ugt1a1 Gene ◽  
Concentration Time ◽  
Exon 1

13078 Background: Pharmacogenetic testing of UGT1A1*28, a promoter variant of UGT1A1 gene, for estimating a risk of irinotecan toxicity has been introduced into clinical practice. Purpose: To elucidate clinical significance of UGT1A1*6 and UGT1A1*27, the variants in exon 1 that are found mostly in Asians. Methods: Pharmacogenetic relationships were explored between the genotyping results and ratio of area under the concentration-time curve (AUC) of SN-38 and its glucuronide (SN-38G) as a surrogate for a UGT1A1 activity (AUCSN-38/AUCSN-38G) in 36 Japanese patients who received various regimens of irinotecan chemotherapy at doses from 50 to 180 mg/m2. Results: No patients were homozygous for UGT1A1*28 and none had UGT1A1*27. One was heterozygous for UGT1A1*28. Two were homozygous and ten heterozygous for UGT1A1*6, all of whom were wild type with respect to UGT1A1*28. Two patients were simultaneously heterozygous for UGT1A1*28 and UGT1A1*6 that existed on the different chromosomes. The remaining 21 patients did not have any variants studied. The 2 patients who was simultaneously heterozygous for UGT1A1*28 and UGT1A1*6 and the 2 patients who were homozygous for UGT1A1*6 had significantly higher AUCSN-38/AUCSN-38G than the others (P = 0.004). Conclusions: Concurrence of the two UGT1A1 variants, UGT1A1*28 and UGT1A1*6 even in their heterozygous statuses, remarkably altered drug disposition of irinotecan and could enhance susceptibility to the drug. Patients who are homozygous for UGT1A1*6 should also be monitored cautiously. Genotyping of UGT1A1 polymorphisms in the coding region together with UGT1A1*28 is necessary for predicting irinotecan toxicity at least for Asian patient population. [Table: see text] [Table: see text]

Sign in / Sign up

Export Citation Format

Share Document