The Drosophila melanogaster actin 5C gene uses two transcription initiation sites and three polyadenylation sites to express multiple mRNA species

At least six mRNAs are made from the Drosophila melanogaster act5C gene. We investigated the structures of these RNAs in detail and determined that they are heterogeneous at both their 5' and 3' ends. At the 5' end there were two nonhomologous leader exons which were alternately spliced to the remainder of the gene. These leader exons mapped to 1.7 and 0.7 kilobases, respectively, upstream of a common splice acceptor site which was eight base pairs 5' to the translation initiator AUG. Exon 1 is 147 bases in length, while exon 2 is 111 bases. A consensus TATA sequence was found roughly 30 base pairs upstream from exon 1, but none was found in the analogous position upstream of exon 2. The transcript length diversity arose principally from the use of three polyadenylation sites. This gave rise to RNA molecules with 3'-untranslated regions of roughly 375, 655, and 945 base pairs. With two start sites and three termination sites, this gene has the potential to produce six different transcripts. All six possible transcripts were present in whole fly mRNA. Transcripts containing the two different leader exons were found in roughly the same relative quantities through development. In contrast, the various 3' ends were differentially represented through development.

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The mouse carbonic anhydrase I gene contains two tissue-specific promoters

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3308-3313.1989 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3308-3313

Author(s):

P Fraser ◽

P Cummings ◽

P Curtis

Keyword(s):

Carbonic Anhydrase ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Exon 2 ◽

Tissue Specific ◽

Isolation And Characterization ◽

Carbonic Anhydrase I ◽

Erythroleukemia Cells ◽

Exon 1

We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.

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The mouse carbonic anhydrase I gene contains two tissue-specific promoters.

Molecular and Cellular Biology ◽

10.1128/mcb.9.8.3308 ◽

1989 ◽

Vol 9 (8) ◽

pp. 3308-3313 ◽

Cited By ~ 28

Author(s):

P Fraser ◽

P Cummings ◽

P Curtis

Keyword(s):

Carbonic Anhydrase ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Exon 2 ◽

Tissue Specific ◽

Isolation And Characterization ◽

Carbonic Anhydrase I ◽

Erythroleukemia Cells ◽

Exon 1

We report the isolation and characterization of the mouse carbonic anhydrase I (CAI) gene. Direct RNA sequence analysis of the 5' nontranslated regions of CAI mRNA from mouse colon and mouse erythroleukemia cells demonstrated tissue specificity in the lengths and sequences of CAI transcripts. Analysis of several mouse CAI genomic clones showed that the transcripts arose from a single CAI gene with two tissue-specific promoters and eight exons. CAI transcripts in the colon were found to initiate just upstream of the erythroid exon 2 of the CAI gene region sequence. Erythroid transcripts originated from a novel promoter upstream of exon 1, which was located more than 10 but less than 250 kilobases upstream of exon 2. Erythroid exon 1 contained only a nontranslated sequence, which was spliced to exon 2 via a cryptic splice acceptor site located in the region that encoded the colon mRNA 5' nontranslated sequence. The remaining exon-intron junctions were conserved in comparison with those of the CAII and CAIII genes.

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Sequence requirements for transcriptional arrest in exon 1 of the murine adenosine deaminase gene

Molecular and Cellular Biology ◽

10.1128/mcb.10.4.1484-1491.1990 ◽

1990 ◽

Vol 10 (4) ◽

pp. 1484-1491

Author(s):

V Ramamurthy ◽

M C Maa ◽

M L Harless ◽

D A Wright ◽

R E Kellems

Keyword(s):

Adenosine Deaminase ◽

Transcription Initiation ◽

Substantial Reduction ◽

Initiation Site ◽

Xenopus Laevis Oocytes ◽

Functional Assay ◽

Base Pairs ◽

Base Pair Fragment ◽

Exon 1 ◽

Translation Initiation Codon

We have previously shown that a transcription arrest site near the 5' end of the murine adenosine deaminase (ADA) gene is significantly involved in the regulation of ADA gene expression. To facilitate the analysis of this transcription arrest site, we have analyzed the transcription products from cloned ADA gene fragments injected into Xenopus laevis oocytes. When genomic fragments spanning the 5' end of the ADA gene were injected into oocytes, a 96-nucleotide (nt) ADA RNA was the major transcription product. The 5' end of this RNA mapped to the transcription initiation site for the ADA gene, and its 3' terminus mapped 7 nt downstream of the translation initiation codon within exon 1. A 300-base-pair fragment of genomic DNA spanning the 5' end of the ADA gene was sufficient to generate the 96-nt transcript which accounted for approximately one-half of the transcription products from injected templates. Deletion of a segment of approximately 65 base pairs, located immediately downstream of the 3' terminus of the 96-nt transcript, resulted in a substantial reduction in the synthesis of the 96-nt transcript and a corresponding increase in the production of larger transcripts. These studies show that the transcriptional apparatus of X. laevis oocytes responds to the transcription arrest site associated with exon 1 of the murine ADA gene and that oocyte injections provide a convenient functional assay for additional mechanistic studies.

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Genetic evidence for promoter competition in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4608 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4608-4615 ◽

Cited By ~ 20

Author(s):

J E Hirschman ◽

K J Durbin ◽

F Winston

Keyword(s):

Transcription Initiation ◽

Initiation Site ◽

Base Change ◽

Wild Type ◽

Base Pairs ◽

Cis Acting ◽

Initiation Of Transcription ◽

In Trans ◽

Tata Sequence ◽

Transcription Signals

The his4-912 delta mutation is an insertion of the long terminal repeat (delta) of the yeast retrotransposon Ty into the HIS4 promoter region, such that the delta is 97 base pairs upstream of the HIS4 transcription initiation site. Strains carrying the his4-912 delta allele are His- at 23 degrees C; this phenotype can be reversed either by growth at 37 degrees C or by mutations in trans-acting SPT genes. Under conditions in which his4-912 delta confers a His- phenotype. HIS4 transcription initiates at the delta initiation site, rather than at the HIS4 initiation site, producing a longer, nonfunctional transcript. Under conditions in which the strain is His+, transcription initiates at the wild-type HIS4 initiation site. To understand how transcription is balanced between the delta and HIS4 promoters, we have selected for cis-acting suppressors of his4-912 delta. Two classes defined by six independent mutations restore synthesis of a functional HIS4 transcript. The first class is an A-to-G base change 1 base upstream of the proposed delta TATA sequence. These mutants do not synthesize the delta-initiated transcript; instead, they synthesize only the wild-type HIS4 transcript. The second class of mutations alters base pairs surrounding the functional HIS4 TATA sequence. The two strongest His+ mutants of this class synthesize the wild-type HIS4 transcript at levels consistent with their His+ phenotype. Surprisingly, these two mutants also have a reduced level of the delta-initiated transcript relative to the his4-912 delta parent. Analysis of these mutants indicates that the level of transcription from one promoter can affect the level of transcription from the other promoter and suggests that delta and HIS4 transcription signals compete for initiation of transcription from each site.

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Sanger sequencing as a first-line approach for molecular diagnosis of Andersen-Tawil syndrome

F1000Research ◽

10.12688/f1000research.11610.1 ◽

2017 ◽

Vol 6 ◽

pp. 1016 ◽

Cited By ~ 6

Author(s):

Armando Totomoch-Serra ◽

Manlio F. Marquez ◽

David E. Cervantes-Barragán

Keyword(s):

Molecular Diagnosis ◽

Sanger Sequencing ◽

Heterozygous Mutation ◽

Coding Region ◽

First Line ◽

Base Pairs ◽

Exon 2 ◽

Clinical Criteria ◽

Exon 1 ◽

Next Generation Sequencing Ngs

In 1977, Frederick Sanger developed a new method for DNA sequencing based on the chain termination method, now known as the Sanger sequencing method (SSM). Recently, massive parallel sequencing, better known as next-generation sequencing (NGS), is replacing the SSM for detecting mutations in cardiovascular diseases with a genetic background. The present opinion article wants to remark that “targeted” SSM is still effective as a first-line approach for the molecular diagnosis of some specific conditions, as is the case for Andersen-Tawil syndrome (ATS). ATS is described as a rare multisystemic autosomal dominant channelopathy syndrome caused mainly by a heterozygous mutation in the KCNJ2 gene. KCJN2 has particular characteristics that make it attractive for “directed” SSM. KCNJ2 has a sequence of 17,510 base pairs (bp), and a short coding region with two exons (exon 1=166 bp and exon 2=5220 bp), half of the mutations are located in the C-terminal cytosolic domain, a mutational hotspot has been described in residue Arg218, and this gene explains the phenotype in 60% of ATS cases that fulfill all the clinical criteria of the disease. In order to increase the diagnosis of ATS we urge cardiologists to search for facial and muscular abnormalities in subjects with frequent ventricular arrhythmias (especially bigeminy) and prominent U waves on the electrocardiogram.

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Structure of the glucocorticoid receptor (NR3C1) gene 5′ untranslated region: identification, and tissue distribution of multiple new human exon 1

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01822 ◽

2005 ◽

Vol 35 (2) ◽

pp. 283-292 ◽

Cited By ~ 176

Author(s):

Jonathan D Turner ◽

Claude P Muller

Keyword(s):

Glucocorticoid Receptor ◽

Untranslated Region ◽

Splice Variants ◽

Splice Acceptor Site ◽

Specific Expression ◽

Exon 2 ◽

Tissue Specific ◽

Tissue Specific Expression ◽

Human Exon ◽

Exon 1

The 5′ untranslated region (UTR) of the glucocorticoid receptor (GR) plays a key role in determining tissue-specific expression and protein isoforms. Analysis of the 5′ UTR of the human GR (hGR) has revealed 11 splice variants of the hGR exon 1, based on seven exon 1s, four of which (1-D to 1-F and 1-H) were previously unknown. All of the exon 1 variants have unique splice donor sites and share a common exon 2 splice acceptor site. Due to an upstream in-frame TGA stop codon the predicted translation from all splice variants is identical. The four new exon 1s show remarkable similarity with their rat homologues. Exon 1-D starts and finishes 17 and 36 bp upstream of the corresponding ends of the rat exon 14. Exon 1-E is only 6 bp longer than its homologue exon 15. Exon 1-F contains two short inserts of 11 and 6 bp when compared with the rat 17. 1-H is 18 bp longer than the corresponding rat 111. In addition to these new exons, we found that the human exon 1-C occurs as three distinct splice variants, covering the region homologous to the rat exons 19 and 110. All of the alternative hGR exons 1s presented here were found to be transcribed in human tissue. The human hippocampus expresses mRNA of all the exon 1 variants, while the expression of the other exon 1s seems to be tissue specific. While exon 1-D is only in the hippocampus, exons 1-E and 1-F are also detected in the immune system, and exon 1-H additionally in the liver, lung and smooth muscle. The 5′ region of the hGR is more complex than previously thought, and we suggest that each of these untranslated first exons have a distinct proximal promoter region, providing additional depth to the mechanisms available for tissue-specific expression of the hGR isoforms.

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

Journal of Endocrinology ◽

10.1677/joe.0.1510215 ◽

1996 ◽

Vol 151 (2) ◽

pp. 215-223 ◽

Cited By ~ 9

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

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A Novel Splice Acceptor Site Mutation which Produces Multiple Splicing Abnormalities Resulting in Protein S Deficiency Type I

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614631 ◽

1999 ◽

Vol 82 (07) ◽

pp. 65-71 ◽

Cited By ~ 14

Author(s):

Hideki Tatewaki ◽

Hiroko Iida ◽

Mutsuko Nakahara ◽

Hiroko Tsuda ◽

Sachiko Kinoshita ◽

...

Keyword(s):

Molecular Mechanisms ◽

Protein S ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Type I ◽

Protein S Deficiency ◽

Site Mutation ◽

Exon 2 ◽

S Gene ◽

S Deficiency

SummaryIn an attempt to explore the molecular mechanisms for protein S deficiency, a patient with such a deficiency was examined at the DNA, RNA and protein levels. Nucleotide analyses revealed that the proband, the mother and the grandmother had a G → C substitution in the invariant AG dinucleotide at the splicing acceptor site of intron A/exon 2. This patient was heterozygous for this substitution and the mutant allele was inherited from the proband’s mother and grandmother. Reverse transcription-polymerase chain reaction analysis demonstrated several kinds of splicing abnormalities such as exon skipping and cryptic splicing, in addition to correct splicing. Semiquantitation of mRNA for the protein S gene revealed that the amount of the proband’s mRNA was reduced to 60% of normal. Thus, this mutation impaired the normal processing of mRNA for the protein S gene, resulting in the subject’s severe protein S deficiency.

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Alternative RNA splicing generates transcripts encoding a thorax-specific isoform of Drosophila melanogaster myosin heavy chain

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2511-2519.1986 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2511-2519

Author(s):

S I Bernstein ◽

C J Hansen ◽

K D Becker ◽

D R Wassenberg ◽

E S Roche ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Rna Splicing ◽

Splice Acceptor Site ◽

Acceptor Site ◽

Sequencing Studies ◽

Alternatively Spliced ◽

Muscle Types ◽

Terminal Amino

Genomic and cDNA sequencing studies show that transcripts from the muscle myosin heavy-chain (MHC) gene of Drosophila melanogaster are alternatively spliced, producing RNAs that encode at least two MHC isoforms with different C termini. Transcripts encoding an MHC isoform with 27 unique C-terminal amino acids accumulate during both larval and adult muscle differentiation. Transcripts for the second isoform encode one unique C-terminal amino acid and accumulate almost exclusively in pupal and adult thoracic segments, the location of the indirect flight muscles. The 3' splice acceptor site preceding the thorax-specific exon is unusually purine rich and thus may serve as a thorax-specific splicing signal. We suggest that the alternative C termini of these two MHC isoforms control myofilament assembly and may play a role in generating the distinctive myofilament organizations of flight muscle and other muscle types.

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