Exon Mutant

4.1.2 Genomic Deletion of Exon 3 in the Growth Hormone Receptor (d3-GHR)

Exon 3 of the GHR is flanked by two almost identical retroelements comprised of 251 bp, which are situated at 577 and 1821 bp from the exon, respectively. Some individuals exhibit a homologous recombination of these two retroelements, and produce 2.7-kb deletion, giving rise to two different isoforms of the GHRGHR: the full-length GHR, which retains the exon 3 (fl-GHR or fl/fl), and the exon-3-deleted GHR (d3-GHR), which is either homozygous (d3/d3) or heterozygous (fl/d3). Deletion of exon 3 produces a lack of amino acid residues 7–28 and the amino acid substitution A6D close to the N-terminus.73 This induces subtle conformational changes in the extracellular domain, which do not affect the affinity, binding capacity, or internalization of both GHR isoforms, but which could facilitate ligand-induced activation of GHR (Fig. 1).73,80 Around 50% of the Western population is homozygous for the fl-GHR, 30–40% is heterozygous (fl-GHR/d3GHR) and 10–20% is homozygous for the d3 variant.73,80

Preliminary studies did not consider this deletion to have a significant impact on the GHRs function.81,82 However, subsequent reports demonstrated its association with a differential GH transduction.80

In vitro studies have demonstrated how transcriptional activity of homo- and heterodimers of the d3-GHR are around 30% higher than that of the fl-GHR homodimers.80 In this regard, clinical studies in GH-deficient children carrying the d3-GHR variant have reported a better response to treatment with recombinant human GH.80,83 In GH-deficient adults, however, some studies,84,85 but not all,86 have demonstrated this greater sensitivity to GH treatment in d3-GHR carriers.

Several studies, mostly multicenter, retrospective, and with a relatively small number of patients, have addressed the impact of the d3-GHR allele in acromegaly.87–96 Patients carrying the d3-GHR variant exhibited a close relationship between serum GH and IGF-I concentrations; specifically, for a given IGF-I serum concentration, GH levels were lower in d3-GHR carriers.87 Similarly, the presence of d3-GHR was associated to a higher frequency of biochemical discrepant results, with elevated IGF-I levels, but normal GH, both during SSA treatment, and after surgical intervention. In this regard, in a series of 84 patients with acromegaly, 70% of those who presented with biochemical discrepant results were d3-GHR allele carriers.90 In addition, in one of the largest series of acromegalic patients from a single center,89 patients with heterodimers d3/fl-GHR, and especially those with homodimers d3/d3-GHR, presented a more active disease, higher rate of diabetes mellitus, and lower probability of achieving adequate biochemical control with either surgery, SSA and/or radiotherapy. Moreover, IGF-I levels were higher in homo- and heterozygous patients for d3-GHR, in comparison to patients with the fl-GHR allele. Furthermore, insufficient biochemical control was observed in 54% of patients who were fl/fl-GHR, 55% of those with d3/fl-GHR, and 77% of patients who were homozygous for the d3-GHR allele. This study suggested, in fact, that the best predictor of persistent elevated IGF-I levels during or after treatment was the presence of the d3-GHR allele, with even better predictive values than basal GH/IGF-I levels, age, or tumor size.89

The potential role of the d3-GHR in acromegaly and its outcome has been specifically addressed in several well-known studies.89–91,94,97,98 None of them found a significant association between the presence of this allele and basal or biochemical characteristics, although subtle differences were found regarding weight, body mass index, and the percentage of macroadenomas in two of them.91,94 Turning to the potential influence of this exon deletion in the development of comorbidities during long-term follow-up, some studies have described a greater prevalence of osteoarthropathy, vertebral fractures, dolicocolon, and colonic polyps, as well as a greater probability of maintaining normal tolerance to glucose, in carriers of the d3-GHR variant.92,93,97

Regarding response to treatment with PEG, in two studies,91,94 where patients received PEG in monotherapy, carriers of the d3-GHR allele exhibited a better response: IGF-I normalization occurred earlier and the required PEG dose was 21–27% lower. Meanwhile, in the multicenter study performed by our group,94 a multivariate analysis showed that presence of the d3-GHR allele and male sex were the only two relevant predictors of PEG dose necessary to achieve IGF-I normalization (Table 1).30,94 Another multicenter study performed in Italy with 127 acromegalic patients (49% of whom were on combination therapy with SSA + PEG), found no association between the presence of the d3-GHR allele and response to treatment with PEG, either when used as monotherapy or in combination schemes with SSA.95 However, given the observed deviation from Hardy–Weinberg equilibrium of the d3-GHR genotype distribution in their study, as well as in previous ones, they suggested an association of the d3-GHR variant with a phenotype characterized by resistance to traditional treatments and difficulties in disease control.95

Table 1. Factors Influencing Doses of Pegvisomant.

FactorEffectGH/IGF-I levelDirect correlation with PEG dose requiredSexHigher doses required in men

Higher GH and GHBP levels with lower IGF-I in womenRadiotherapyReduces PEG dose requiredWeightIncreases PEG dose requiredType 2 diabetes mellitusIncreased insulin promotes increased hepatic GHR and therefore requires higher PEG dosesCombination therapy with SSADecreased insulin levels, and subsequent reduced GHR, so less PEG dose requiredd3-GHRLower doses of PEG and fewer months to normalize IGF-IPolymorphism promoter region IGF-IControversial reports

Abbreviations: GH, growth hormone; IGF-I, insulin-like growth factor; PEG, pegvisomant; d3-GHR, exon-3-deleted GH receptor; GHBP, growth hormone binding protein; SSA, somatostatin analogs.

Adapted from Ref. [30].

To summarize, deletion of exon 3 of the GHR in patients with acromegaly seems to influence the clinical picture of the disease, its severity, and maybe the response to PEG. In this regard, blockage of the d3-GHR, which is functionally more active, may determine a greater effect than blockage of the native, fl/fl-GHR, which exhibits a lower functional activity. Possible explanations for the inconsistencies observed in results from different studies may concern the fact that the exon 3 deletion of the GHR gene still has a limited impact in clinical endocrinology,99 or because studies have included a small and heterogeneous group of acromegalic patients. In any case, studies that specifically address the significance and clinical relevance of the d3-GHR in acromegaly deem necessary to open a new gate for targeted therapies based on pharmacogenomics, in order to personalize and individualize management of this rare disease.

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The Immune-Related Roles and the Evolutionary History of Dscam in Arthropods

Sophie A.O. Armitage, Daniela Brites, in The Evolution of the Immune System, 2016

3.4 Dscam Clusters of Duplicated Exons

The three alternative exon clusters in Dscam-hv have arisen by reiterative exon duplication and deletion.88 For the sake of simplicity, we will use the exon nomenclature of the D. melanogaster Dscam-hv, that is, exons 4, exons 6, and exons 9. Generally, the exon duplications of Dscam-hv are believed to be the outcome of homologous recombination among neighboring exons with similar sequence composition.88 A feature common to the exons of the three clusters is that exons resulting from duplication (paralogs) diverged extensively, whereas exons resulting from speciation (orthologs) are conserved. As an example, the Drosophila species or Daphnia species that have been examined have their own "set" of alternative exons, indicating that alternative exons evolved independently in different pancrustacean groups.8,77,88 When looking within each taxonomic genus, duplicated exons (paralogs) are quite diverse, but the vast majority of paralogs have an orthologous exon in the other con-generic species (with some exceptions for clusters of exons 6 and 9).8,77,88 This suggests that most exons duplicated and diverged by accumulating mutations in the ancestors of each genus. The high conservation of amino-acid sequences of orthologous exons indicates that they had not diverged much since the split of the extant species from their most recent common ancestor, indicating that selection acts to preserve the ancient diversity that had been created. An exception to this is amino-acid sequence variation in the orthologous regions of exons 4 and 6 that encode epitope II, as discussed previously. Despite this general pattern of sequence evolution, the three clusters of exons seem to have undergone different patterns of exon radiation.8,69,77,88 (Fig. 10.3). The number of exons in cluster 4 tends to be more conserved among species of the same genus, whereas exons of clusters 6 and 9 seem to have higher duplication rates.88 This could reflect that the different clusters specialized in different functions prior to species divergence, as suggested by Crayton and coworkers,69 which is an attractive hypothesis, given the dual role of Dscam-hv in the nervous and immune systems.31 Alternatively, it could also simply reflect protein structural constraints.77

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Expression Arrays: Discovery and Validation

Neal M. Poulin, Torsten O. Nielsen, in Cell and Tissue Based Molecular Pathology, 2009

EXON ARRAYS

Exon arrays represent the next generation of expression microarrays and are still in exploratory stages, with no large-scale studies reported as of this writing. With exon arrays, oligonucleotide probes are designed that query specific exons in target mixtures. Up to 80% of human transcripts are alternately spliced, and this represents a fundamental mechanism for tissue-specific gene function. In this manner, different protein domains may be included in specific isoforms, depending on splicing, and a single gene can give rise to numerous diverse biologic activities. If exon-specific assays are rigorously developed, they have the potential to shed light on this critical level of gene regulation.

The importance of publicly available and nonproprietary probe sequences should not be underestimated. Stanford University has recently contributed in this area with the release of two nonproprietary sets of 70-mer exon-specific probes, termed human and mouse exonic evidence-based oligonucleotides. These sets are designed to provide isothermal probes for constitutive and alternate human and mouse exons, and they include probes spanning known splice junctions from curation of published studies.

Modification to the labeling protocol is required for exon arrays, because reverse transcription is 3′ biased when primed with oligo-dT primers. The use of random hexamer primers avoids this problem but results in extensive labeling of ribosomal RNA (rRNA) and other non-mRNA species, which consumes reagents and increases background. Methods for removal of the major rRNA species have been developed, including subtractive hybridization for 28S and 18S species.

Affymetrix has recently offered a whole genome exon array and introduced modifications to its protocol, including the preparation of labeled cDNA targets from amplified complementary RNA (cRNA). Agilent provides a completely customizable array, which can be configured for any set of user-specified probes. However, in terms of catalog arrays, Agilent produces alternate splicing arrays through licensing from third-party bioinformatics enterprises.

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RNA Turnover in Eukaryotes: Analysis of Specialized and Quality Control RNA Decay Pathways

Daiki Matsuda, ... Lynne E. Maquat, in Methods in Enzymology, 2008

2.1 Rule for which nonsense codons trigger NMD

An exon–exon junction complex (EJC) of proteins deposited ≈20- to 24- nucleotides upstream of exon–exon junctions during pre-mRNA splicing is considered to be a primary determinant of NMD in mammalian cells (Le Hir et al., 2000, 2001). EJC function is one characteristic that distinguishes NMD in mammals from NMD in, for example, S. cerevisiae or C. elegans (reviewed in Isken and Maquat, 2007). As a rule, translation termination at a nonsense codon located more than ≈50- to 55-nucleotides upstream of an exon–exon junction generally triggers NMD. It is proposed that translation termination events that lead to NMD involve the SURF complex, which is composed of Smg1, Upf1, and translation termination factors eRF1 and eRF3 (Kashima et al., 2006). When translation terminates sufficiently upstream of an EJC, Upf1 interacts with Upf2 that is bound to the EJC together with Upf3 or Upf3X and becomes phosphorylated by Smg1. Upf1 phosphorylation may lead to translational repression of the targeted mRNA and, consequently, an increased accessibility of the mRNA to degrading complexes (Isken et al., 2008). However, ribosomes that terminate translation either less than ≈50- to 55-nucleotides upstream of the 3′-most exon–exon junction or downstream of this junction are thought to remove all EJCs so as to preclude NMD (Dostie and Dreyfuss, 2002). It follows that normal termination codons, which are usually situated within the last exon, typically lack a downstream EJC and therefore do not generally lead to NMD.

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Developmental Disabilities and Metabolic Disorders

Mary Lee Gregory, ... Bruce K. Shapiro, in Neurobiology of Brain Disorders, 2015

Whole Exome and Whole Genome Sequencing

Exons are the regions of genes that code for proteins. The exome refers collectively to all of the exons in an individual. The genome represents all chromosomal genetic data including coding and non-coding (intron) regions, approximately 3 billion DNA letters. Exons represent only 1.5% of the genome but result in around 85% of monogenic diseases. Whole exome sequencing (WES) therefore focuses on the areas of the chromosome most likely to result in disease and significantly decreases the amount of data that must be examined. WES will not detect mutations in the introns. WES is now available for clinical use, but it is expensive.

Whole genome sequencing (WGS) examines both exons and introns. It necessarily involves large amounts of information and therefore is likely to result in many findings of unknown significance, particularly as so much of the intron data is poorly understood. WGS is not in clinical use currently, but is likely to be available soon. Of note, WES and WGS analyze gene sequence and are therefore very useful for detecting small sequence changes but do not look at copy variants. A large deletion or duplication of genetic material may not be detected on WES and WGS. Therefore, it is always recommended to perform a standard microarray before WES (and WGS when it is available). As arrays and gene sequencing use different technologies and gather different information, they should be considered complementary tests.48

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Alternative Splicing

Scott A. Akker, ... Shern L. Chew, in Encyclopedia of Endocrine Diseases, 2004

Splice Sites

The splice sites that flank exons are necessary but not sufficient to explain exon recognition. Splice sites have a varying degree of similarity to highly degenerate consensus sequences. In fact, splice site-like sequences that also match the consensus occur with great frequency throughout the genome and define a set of pseudosites and pseudoexons that never undergo splicing. Such pseudoexons may outnumber true exons 10 to 1. Thus, there is a spectrum of exons: those that are always spliced (constitutive exons), those that are spliced only under certain conditions (alternatively spliced exons), and those that never undergo splicing (pseudoexons). As a rule, alternatively spliced exons tend to have splice sites with a weaker match to the consensus than constitutive exons, and this allows them to be more amenable to the effects of other sequence elements and factors. Experimentally enhancing the splice site strength of alternatively spliced exons usually causes them to be included constitutively. The splice site strength of the exons neighboring an alternatively spliced exon is also important. Strengthening the 5′ splice site of the upstream exon or the 3′ splice site of the downstream exon can lead to an increased degree of skipping of the middle exon.

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Alternative Splicing

K. Lynch, L. Motta-Mena, in Encyclopedia of Biological Chemistry (Second Edition), 2013

Types of Alternative Splicing Patterns

Although some exons in an RNA messenger transcript are constitutively spliced, that is, they are always included in the final message, the splicing of other exons is regulated (Figure 2). The most common type of regulated splicing event is what is called a cassette exon, which is an exon that is sometimes included and sometimes excluded from the mature mRNA. In some cases, cassette exons are mutually exclusive in which case only one exon from a group of two or more variants is selected. There are also instances where alternate 5′ or 3′ splice sites, located in tandem, can be used to produce different mRNA isoforms that differ in the length of a particular exon. By combining the use of alternative promoters and alternative splicing, the 5′-end of a transcript can be altered. Similarly, coordinate regulation of polyadenylation and splicing can alter the 3′-end of a transcript. Finally, in some cases the failure to remove a particular intron (i.e., intron retention event) in an mRNA can occur.

Figure 2. Patterns of pre-mRNA alternative splicing. The most common patterns of alternative splicing are shown along with alternate products. Cases in which alternative promoter sites or alternative polyA sites are coupled with alternative splicing are indicated. Exons are depicted as boxes and introns as lines; dashed lines indicate the splice sites used in the splicing reaction. Constitutive exons are shown in gray and regulated exons are shown as light gray or black boxes.

As shown in Figure 2, each of these altered splicing patterns has the potential to alter the coding sequence of the mRNA to give rise to a protein of distinct sequence and function. Frequently, individual protein domains are encoded by discrete exons, such that functionality can be determined by modular inclusion or exclusion of a particular domain-encoding exon. Alternatively, differential use of an exon, or inclusion of an intron may result in the introduction of a premature stop codon. In such cases, the mRNA or protein may be destabilized, such that alternative splicing becomes a mechanism to regulate the absolute level of protein expression. Finally, alternative splicing that changes the 5′- or 3′-UTR (UTR, untranslated region) of a message can determine the presence or absence of sequences that regulate message stability or translation. Thus, even changes in noncoding regions of an mRNA can ultimately control the expression or function of the resulting protein.

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Gene Trapping

K. Asakawa, K. Kawakami, in Brenner's Encyclopedia of Genetics (Second Edition), 2013

Exon Trapping

An exon trap construct contains a reporter gene placed downstream of a DNA fragment with a splice acceptor. When the construct is integrated in an intron of a gene, splicing between the splice acceptor site in the trap construct and the splice donor site of the 5′ upstream exon produces a chimeric mRNA for the trapped gene and the reporter gene, resulting in the reporter expression similar to that of the trapped gene. Exon trapping is widely used to identify a gene based on the expression of the reporter gene and/or to disrupt the gene function. The term 'gene trapping' is often used to refer to exon trapping in a narrow sense.

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A worldwide yearly survey of new data in adverse drug reactions

Michael T. Bowen, in Side Effects of Drugs Annual, 2014

Growth Hormone Receptor Antagonists [SEDA-33, 911; SEDA-34, 708; SEDA-35, 792]

Exon 3-deleted GHR A cross-sectional, multicenter study of 127 acromegalic patients did not find any association between exon 3-deleted GHR (d3GHR) and efficacy or adverse reactions to pegvisomant treatment [46C]. d3GHR patients did deviate from the Hardy–Weinburg equilibrium which the authors conclude required further investigation to determine whether this may be associated with poor response to pegvisomant.

New formulation In an open-label, randomised, single-dose, two-way crossover, phase I study of healthy male and female subjects (N = 28), the safety and tolerability of a new 1 × 30 mg/ml subcutaneous injection formulation of pegvisomant did not differ from the marketed 2 × 15 mg/ml subcutaneous formulation [47c]. The new formulation may serve to minimise injection-site reactions by decreasing the number of injections required for effective treatment, although larger studies are required to properly assess this possibility.

Elevated intrahepatic lipid A randomised, controlled trial (N = 18) of pegvisomant combined with somatostatin analogue treatment versus somatostatin analogue monotherapy found that co-treatment with pegvisomant resulted in elevation of intrahepatic lipid which could account for the transient elevation in liver enzymes observed in up to 25% of patients undergoing pegvisomant treatment [48c].

Lipohypertrophy Case studies of two Caucasian women (aged 51 and 71 years) with acromegaly with pegvisomant-related lipohypertrophy at the abdominal injection site found that switching the injection site to the two thighs resulted in reversal of the lipohypertrophy at the abdominal injection site but re-emergence at the new injections sites [49A]. These cases indicate that injection-site lipohypertrophy appears to be reversible and could be minimised through careful monitoring by physical and radiological examination and regular rotation of injection site.

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Animal Models of Osteogenesis Imperfecta

Charlotte L. Phillips, ... Bettina A. Gentry, in Osteogenesis Imperfecta, 2014

P3H1−/− Mouse

Exons 1–3 of the P3H1 or leprecan 1 (Lepre1) gene were deleted by homologous recombination to create the P3H1−/− null mouse.106 Heterozygous P3H1/+ mice do not exhibit any observable phenotype. P3H1−/− mice exhibit delayed postnatal growth with shortening of their long bone segments (rhizomelia) and are smaller with decreased BMD relative to wild-type and P3H1/+ littermates.106P3H1−/− mice develop kyphoscoliosis which progressively worsens with age. Femoral stiffness and force to failure is significantly lower in P3H1−/− mice compared to P3H1/+ and wild-type mice.106 Type I collagen-rich tissues, tendon and skin, are also impacted by the mutation; P3H1−/− mouse tail tendons have abnormal morphology and demonstrate an increase in small diameter fibrils,106 with an altered distribution of fibril sizes ranging from 20 to 100 nm compared to a range of 50 to 400 nm in wild-type mice. P3H1−/− skin is thinner than that of wild-type mice, and the reticular dermis has relatively normal looking collagen fibrils interspersed with clumped areas of fibrils and open spaces not seen in wild-type skin.

Exon Structure

Related terms:

Exon

Intron

Peptide

C-Terminus

Peptide Sequence

Nested Gene

Alpha Chain

Mammal

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Marapsin

Kavita Raman, George H. Caughey, in Handbook of Proteolytic Enzymes (Third Edition), 2013

Structural Chemistry

The intron/exon structure (intron number, phase and placement) of marapsin gene PRSS27 is distinct from that of trypsin and of most S1A family proteases, but is not unique. It is similar to the structure of genes encoding a subgroup of type I transmembrane serine peptidases, notably γ-tryptase/TPSG, prostasin/PRSS8 and testisin/PRSS21 [1]. This supports evidence from amino acid sequence alignments that human marapsin, although not itself a transmembrane peptidase, is related to the aforementioned subgroup of type I transmembrane peptidases and evolved from a membrane-anchored ancestor by dropping its hydrophobic C-terminal tail. The division of the prepro-segment into three separate exons is especially idiosyncratic. The predicted 12-residue human marapsin propeptide, after cleavage-activation of pro-marapsin at Arg34, likely remains attached to the 256-residue catalytic domain via disulfide linkage involving Cys26 of the propeptide and Cys144 of the catalytic domain, by analogy to similar arrangements in chymotrypsin and many other serine peptidases. There are four additional predicted disulfide pairs, all present in classical locations for mammalian serine peptidases. The catalytic domain begins at residue 35 with Met, which is very rare for serine proteases, but is highly conserved among marapsins. A rendering of a model of marapsin based on a crystal-derived structure of prostasin is shown in Figure 595.1. Human and mouse marapsin contain predicted sites of N-glycosylation and both enzymes are modified post-translationally by N-glycosylation when expressed as recombinant proteins in mammalian cells, as indicated by increased electrophoretic mobility of the proteases incubated with glycosidases [1,5]. The 291-residue catalytic domain of mouse marapsin includes a 35-residue hydrophobic C-terminal extension (absent in the human enzyme), which hydropathy analysis reveals to possess the length and features typical of a transmembrane segment with a short cytoplasmic extension [1,5]. When mouse marapsin containing this segment is expressed in mammalian cells as an epitope-tagged pseudozymogen, soluble marapsin is released into medium by cells incubated with bacterial phosphatidylinositol-specific phospholipase C [3], suggesting that the peptide anchor is swapped for a lipid – namely glycophosphatidylinositol. In accord with predictions that human marapsin lacks a peptide or lipid anchor, native human marapsin is secreted from the human esophageal cell line, Het-1A [4].

Figure 595.1. Richardson diagram of a model of human marapsin based on crystal-derived structure 3gym of human prostasin. The marapsin homology model was created with SWISS-MODEL and rendered using the Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California at San Francisco. Side chains of 'catalytic triad' residues (His57, Asp102 and Ser195 by chymotrypsinogen numbering) are shown in ball-and-stick format.

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Peptidyl-dipeptidase A/angiotensin I-converting enzyme

Pierre Corvol, ... Florent Soubrier, in Handbook of Proteolytic Enzymes (Second Edition), Volume 1, 2004

Gene Structure

The complete intron–exon structure of the human ACE gene was determined from the restriction mapping of genomic clones and by sequencing the intron–exon boundaries (Hubert et al., 1991). The human ACE gene contains 26 exons: the somatic ACE mRNA is transcribed from exon 1 to 26, but exon 13 is removed by splicing from the primary RNA transcript; the germinal ACE mRNA is transcribed from exon 13 to 26 (Figure 82.4). The structure of the human ACE gene provides further support for the hypothesis of the duplication of an ancestral gene. Exons 4–11 and 17–24, which encode the two homologous domains of the enzyme, are highly similar in size and in sequence. In contrast, the intron sizes are not conserved.

Figure 82.4. Organization of the human ACE gene. The relative locations of the 26 exons (vertical bars) are shown. Exon 13 (hatched bar) is specific to the testicular ACE mRNA. The two promoters are indicated by vertical arrows.

The human somatic and germinal forms of ACE mRNA are transcribed from a single gene (Hubert et al., 1991) and the two species of ACE mRNA are generated by the initiation of transcription from alternative start sites under the control of separate promoters. There are two functional promoters in the ACE gene. The somatic ACE promoter is located on the 5′ side of the first exon of the gene. Positive regulatory elements are found inside the 132 bp region upstream from the transcription start site and there are negative regulatory elements between positions −132 and −343 and between −472 and −754 (Testut et al., 1993).

The germinal ACE promoter is located on the 5′ side of the 5′ end of the germinal ACE mRNA. Primer extension and RNase protection assays on mouse, rabbit and human germinal RNA demonstrated that the transcription initiation site was located inside the ACE gene (Howard et al., 1990; Hubert et al., 1991; Kumar et al., 1991). Thus intron 12, which corresponds to the genomic sequence 5′ to the germinal specific exon 13, as deduced from the complete analysis of the ACE gene in humans, was proposed as the putative germinal ACE promoter. Unequivocal evidence for the promoter-function of this sequence was obtained by using intron 12, in transgenic mice, to drive the transcription of a reporter gene in a germinal-specific fashion, in elongating spermatozoa (Langford et al., 1991). In another series of transgenic mice, a 91 bp fragment of intron 12 of the ACE gene was used as the promoter and was sufficient to confer a germinal cell-restricted pattern of transcription to the transgene (Howard et al., 1993). Further mapping of the elements controlling transcription was achieved by DNAse footprinting and gel mobility shift assays which demonstrated that a sequence between positions −42 and −62 specifically binds nuclear factors from testes extracts and contains a consensus cAMP-responsive element (Howard et al., 1993). This element is able to bind transcription factors of the cAMP-responsive element binding protein (CREB) family. CREMτ, (cAMP-response element modulator), one of the CREM isoforms, belongs to this family and was shown to bind and to transactivate the germinal ACE promoter (Zhou et al., 1996). In addition, mice homozygous for the knockout of the gene coding for CREM, do not express germinal ACE (Kessler et al., 1998). Taken together, these results show that CREMτ expression in elongating spermatids and binding on the germinal ACE promoter, allows the cell- and stage-specific expression of this isoform.

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Pharmacology

D.M. Soderlund, in Comprehensive Molecular Insect Science, 2005

5.1.3.3 Functional Characterization of Sodium Channel Splice Variants

The conservation of alternative exon structure and the developmental and anatomical regulation of alternative exon usage in the para sodium channel gene of D. melanogaster and its orthologs in other insect species imply that alternative splicing may generate a family of sodium channel proteins with differing functional properties, as has been found for other ion channels and receptors (Harris-Warwick, 2000). Most of the optional exons identified in para orthologs to date (see Figure 2) are located in intracellular domains of the sodium channel protein. These exons may therefore be involved in the regulation of sodium channel expression or function as the result of interactions with protein kinases or G proteins (Cukierman, 1996; Cantrell and Catterall, 2001). This interpretation is consistent with the existence in exons a and i of consensus protein kinase A phosphorylation sites (O'Dowd et al., 1995; Ingles et al., 1996). Similarly, exons 2 and 4 of the VmNa sodium channel are located in intracellular domains, and exon 2 contains contains a consensus protein kinase C phosphorylation site (Wang et al., 2003). The sole exception so far to the intracellular location of optional exons is exon 1 of the VmNa sodium channel sequence, which is located in the transmembrane region of domain II (Wang et al., 2003).

So far, there is little direct evidence bearing on the functional significance of the alternative splicing of optional exons. In embryonic D. melanogaster neurons, functional sodium channels were detected only in those cells having para transcripts containing exon a (O'Dowd et al., 1995). This study also documented enhanced sodium current expression in cells expressing channels that contain both exons a and i. Whereas these findings imply a critical role for exon a alone and the combination of exons a and i together in sodium channel regulation, the direct comparison in functional expression assays using X. laevis oocytes of variants of para that differ only by the presence or absence of exon a did not find any effects of exon a on sodium current expression or properties in this system (Warmke et al., 1997).

In contrast to the optional exons, the mutually exclusive exons in para orthologs occur within the transmembrane regions of homology domains II and III (Figure 2). There is no information on the functional role of the alternative splicing of exons c and d. In D. melanogaster, these exons differ by only two of 55 amino acid residues (Loughney et al., 1989). In M. domestica, all functional channels apparently contain only exon d because exon c contains an in-frame stop codon (Lee et al., 2002). These observations suggest that alternative splicing at the c/d site may play a role in posttranscriptional regulation rather than in the generation of functionally distinct channel variants.

The most significant functional effects of alternative exon usage have been documented for splice variants at the exon k/l site. Unlike exons c and d, exons k and l (corresponding to exons G2 and G1 in B. germanica) differ substantially in amino acid sequence (Lee et al., 2002; Tan et al., 2002a). Expression of paraCSMA variants containing either exon G1 or G2 in oocytes documents differences in the voltage dependence of both activation and inactivation of these channels (Tan et al., 2002a). Unexpectedly, this study also found substantial differences between these variants in their sensitivity to the pyrethroid insecticide deltamethrin. These results provide the first experimental evidence for functional differences between splice variants of insect sodium channels. Alternative splicing at the k/l site in B. germanica and V. destructor also appears to be involved in the expression of inactive channel variants, in that exon G3 in the paraCSMA sequence encodes a truncated channel (Tan et al., 2002a) and the absence of exon 3 in the VmNa sequence encodes a channel lacking one of the four voltage sensor regions (Wang et al., 2003).

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Terminal Differentiation: REST

S. Aigner, G.W. Yeo, in Encyclopedia of Neuroscience, 2009

REST/NRSF: Gene Organization, Alternative Splicing, and Protein Structure

The REST/NRSF gene and its exon structure are evolutionarily conserved from human to fugu (pufferfish). The gene is not found in flies or nematodes, suggesting that REST/NRSF is specific to the vertebrate lineage. It consists of three alternative first exons (exons I–III), located in the 5′ untranslated region (5′ UTR); three constitutively spliced exons (IV–VI); and a short (28 base pairs) alternatively spliced internal exon (exon N). The full-length REST/NRSF protein comprises three known functional domains: a DNA-binding domain and two repressor domains, which are located at the N- and C-termini of the protein. The two repressor domains function independently of each other and serve to recruit distinct transcriptional regulation complexes (Figure 1(b)). The protein also has lysine- and proline-rich regions; however, their significance is unknown. REST/NRSF's DNA binding domain encompasses an array of eight highly conserved zinc fingers, small independently folded nucleic acid binding motifs found in many other sequence-specific nucleic acid binding proteins. Thus, with its repressor and DNA binding domains, REST/NRSF shows an architecture typical of most transcription factors.

Figure 1. (a) Structure of the REST/NRSF gene and alternative isoform REST4. Exons are indicated as boxes and introns as dashed lines. Exon numbers in roman characters from I to VI are shown above the respective exons; the neuronal-specific exon located between exons V and VI is indicated as exon N. Exons I–III are alternative 5′ UTRs that are spliced to exon IV, represented by lines connecting exons at the intron–exon boundaries. Lines above the primary transcript represent the REST/NRSF primary isoform, and lines below the primary transcript represent the REST4 alternative isoform. Arrows indicate termination codons for REST/NRSF and REST4. (b) REST/NRSF protein includes eight zinc fingers near the N-terminal repressor domain and one zinc finger in the C-terminal domain. REST/NRSF contains a lysine-rich and a proline-rich region upstream of the C-terminal repressor domain. REST4 includes exon N, leading to a truncated protein encompassing the N-terminal repressor domain and five zinc fingers. (c) The conserved REST/NRSF DNA binding site, termed NRSE/RE1, is approximately 21 bp of long and contains of two highly conserved half-site motifs that are separated by two non conserved nucleotides and are flanked by several poorly conserved nucleotides. In this depiction of conserved mammalian NRSE/RE1 sites, generated with pictogram (http://genes.mit.edu/pictogram), the degree of conservation at each position is represented by the size of the nucleotide letter.

Although little is known about the function of alternative splicing events that affect the 5′ UTR of the REST/NRSF gene, the splice variant produced by inclusion of exon N, located within the region of the gene that encodes the DNA binding domain, has received a great deal of attention because it is primarily found in neurons. Its protein product, termed REST4, is a truncated version of REST/NRSF that terminates after the fifth zinc finger and therefore lacks the C-terminal repressor domain (Figure 1(a)). Thus, REST4 is likely to have a function distinct from full-length REST/NRSF, which will be discussed later.

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Genome-Wide Perspectives on Vitamin D Receptor–Mediated Control of Gene Expression in Target Cells

J. Wesley Pike, ... Nancy A. Benkusky, in Vitamin D (Fourth Edition), 2018

Organization of the VDR Gene

The initial organization of the intron–exon structure of the human VDR chromosomal gene was determined in 1988 and corresponded to the sequence of the human VDR initially reported by Baker et al. [71,75]. Delineation of the mouse and human genome sequences revealed that the VDR was expressed from a single gene in both species with the human homolog located on chromosome 12; these details have been confirmed via total genome sequencing. The precise structural arrangement of the gene, which spanned over 50 kb, emerged as a result of the restriction mapping of several recovered cosmid and lambda clones and determination of nucleotide sequence, completing the structure of the gene shortly thereafter [76] (Fig. 9.9). Eight exons comprise the coding sequence of the VDR protein. The first of these is exon 2, which contains the most proximal 3 bp of the 5′ noncoding sequence, the translation start site, and nucleotide sequence that encodes the first DNA-binding zinc finger module. Exon 3 is located ∼15 kb downstream and encodes the second zinc finger module. Exons 4, 5, and 6 define the hinge region that separates the DNA binding and ligand-binding domains. Exons 6 through 9 encode a final portion of the hinge and the carboxy-terminal ligand-binding E/F domain together with ∼3200 nucleotides of 3′ noncoding sequence. It is clear that the human chromosomal gene for the VDR is not unlike that of other steroid receptor genes in size and exon organization, with the exception that an extra insertion exon is present that is a determinant of the enlarged hinge region in the VDR gene. From a sequence point of view, the 5′ end of the human VDR gene is highly complex, featuring a single major and at least one or more minor promoters responsible for the production of several alternatively spliced RNAs [76]. Although the mouse VDR gene appears to be transcribed from a single promoter [211,212], more recent analyses suggest that this conclusion may be premature [213]. With respect to the human VDR gene, two short exons of 77 and 81 bp located upstream of exon 2 and termed 1a and 1c account for the 5′ untranslated region sequence originally reported by Baker et al. [71]. The primary TATA-containing promoter (P1) is located upstream of exon 1a, whereas a secondary GC-rich promoter (P2) devoid of a TATA sequence is located upstream of exon 1. The variable use of exons 1b and 1c lead to the production of alternatively spliced mRNAs whose nature and function remain unknown. Additional exons upstream of exon 1a have also been suggested, including a potential promoter located upstream of exon 1f [214,215]. Although there is some indication that the use of these promoters is cell type-specific [214], the relative abundance of most of the unusual VDR RNAs that are transcribed from these upstream regions is exceedingly low and thus their contribution to the overall expression of the VDR protein and to an unusual larger form of the receptor that has been suggested is currently unclear [216]. In the postgenomic era, the organization, the annotated sequences comprising this gene, and its location on chromosome 12 are readily available on the UCSC genome browser.

Figure 9.9. Organization of the mouse and human vitamin D receptor (VDR) transcription units. Linear depiction of the mouse and human VDR gene where the arrows indicate exons containing either an initiation or a termination codon and vertical bars represent exons. Functional promoters are present upstream of exon 1 in the mouse and exons 1c and 1a in the human. A possible promoter may be present near exons 1f of the human gene. Dotted lines encompass exons 3–10 in the mouse gene and exons 2–9 in the human gene that encode the mouse and human proteins (shown in the top figure). LBD, ligand-binding domain.

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Genes and Genomics

Peijun Zhang, Xiang Jia Min, in Applied Mycology and Biotechnology, 2005

3.2.3.9 FELINES

FELINES, or Finding and Examining Lots of Intron 'N' Exon Structures (http://www.genome.ou.edu/informatics.html) (Drabenstot et al. 2003) is a package including 5 applications developed in Perl. The package provides utilities to automate intron and exon database construction and analysis from EST and genomic sequence data.

The package is divided into three layers. The alignment layer (wiscrs.pl) pairs and aligns EST sequences to their homologous genomic sequences. The script employs NCBI BLAST (blastall) to pair EST and genomic sequences. E-value, the cutoff of which can be defined by users, is used to evaluate the homology of the pairs. Next, alignments are generated by Spidey, a tool for aligning mRNAs to genomic sequences (Wheelan et al. 2001). The extraction layer (gumbie.pl) extracts the intron and exon regions from the Spidey alignment files produced by the alignment layer and parses the resulting datasets into the respective database. The parsing filter criteria are fully customizable in an option file. The analysis layer consists of three applications (icat.pl, findmner.pl, and cattracts.pl). The icat.pl performs two tasks. First, it filters imported intron databases against the defined filter criteria to ensure that the imported intron datasets are comparable to those generated by FELINES. Secondly, icat.pl extracts user defined conserved intron motifs. The findmners.pl identifies all user-defined fixed-length sequences and reports their statistics. The cattracts.pl searches multiple consensus elements simultaneously. The FELINES package is available at the website mentioned above.

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MOLECULAR BIOLOGY OF CARTILAGE MATRIX

SERGIO LINE, ... YOSHIHIKO YAMADA, in Cellular and Molecular Biology of Bone, 1993

B Gene Structure

Rat and human aggrecan contains 15 exons that span approximately 100 kb. The exon structure of this gene corresponds almost exactly to the structural domains predicted in the cDNA sequence. The Gl domain is encoded by exons 3, 4, and 5, which correspond to subdomains A, B, and B', respectively. The IGD domain is coded by exon 6, whereas domains B and B' correspond to exons 7 and 8, respectively. The two glycosaminoglycan attachment domains, the KS and CS domains, are encoded by two distinct exons: exon 9 for KS and the large exon 10 for CS. The exception is domain G3, where the three subdomains are coded by the last five exons. The 372-bp untranslated sequence is mostly contained in exon 1, whereas the small exon 2 contains the sequence for the signal peptide.

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Caerulein

Toshio Sekiguchi, in Handbook of Hormones, 2016

Synthesis and Release

Gene, mRNA, and Precursor

In S. tropicalis, two caerulein genes have been identified in a four-exon structure [3]. The lengths of the caerulein mRNA are 428 and 418 bp, encoding peptides of 98 and 91 aa residues, respectively [3].

Distribution of mRNA

The caerulein gene is expressed in the skin.

Tissue Content

In adult H. caerulea, the content of caerulein is approximately 300–1,000 μg/g of fresh skin [6]. Caerulein content in the dorsal region of the skin is higher compared with that in the ventral region [6]. In contrast, the caerulein level is below 1 μg/g of wet weight in the dorsal region of larval skin [7].

Regulation of Synthesis and Release

Dockray and Hopkins reported that a caerulein-like substance is released by X. laevis in response to treatment with adrenaline [8]. This substance stimulates contraction of the guinea pig gallbladder and pancreatic secretions in rats [8]. Amino acid analysis of the secreted substance has a similar aa composition to that of caerulein [8]. Seasonal changes in caerulein synthesis have been observed [5]. For example, L. splendida synthesizes caerulein that stimulates smooth muscle contraction during the reproductive summer season. In the winter, synthesis of a less active, desulfated form of caerulein increases, and another caerulein peptide subtype (caerulein 1.2), which has relatively low activity, is released [5].

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Biochemistry of Lipids, Lipoproteins and Membranes

William L. Smith, Frank A. Fitzpatrick, in New Comprehensive Biochemistry, 1996

2.7 Regulation of PGHS-1 and PGHS-2 gene expression

PGHS-1 and PGHS-2 are encoded by separate genes, the intron/exon structures of which are illustrated in Fig. 6 [1,8]. Apart from the first two exons, the intron/exon arrangements are similar. However, the PGHS-2 gene (~ 8 kb) is considerably smaller than the PGHS-1 gene (~ 22 kb). The PGHS-1 gene is on human chromosome 9, while the PGHS-2 gene is located on human chromosome l.

Fig. 6. Intron/exon structures of the genes for PGHS-1 and PGHS-2.

The expressions of the PGHS-1 and PGHS-2 genes are regulated in quite different ways. PGHS-1 is expressed more or less constitutively in almost all tissues, whereas PGHS-2 is absent from cells unless induced in response to cytokines, tumor promoters, or growth factors [8,10]. Apparently, cells use PGHS-1 to produce prostaglandins needed to regulate 'housekeeping activities' typically involving rapid responses to circulating hormones (Fig. 2). PGHS-2 apparently produces prostanoids which function during specific stages of cell differentiation or replication. Recent evidence indicating that PGHS-2 is concentrated on the nuclear envelope suggests that at least some of the prostanoids formed via PGHS-2 operate at the level of the nucleus [6].

Relatively little is known concerning the regulation of expression of PGHS-1, although the enzyme is known to be under developmental control. The regulation of expression of PGHS-2 is currently under intensive investigation. Much of what is known about PGHS-2 comes from studies with cultured fibroblast and endothelial cells and purified macrophages [8,10]. Typically, PGHS-2 is induced rapidly (1–3 h) and dramatically (20–80-fold). Growth factors, phorbol esters, and interleukin-1β induce PGHS-2 in fibroblasts and endothelial cells; and bacterial lipopolysaccharide, interleukin-1β, and tumor necrosis factor stimulate PGHS-2 expression ex vivo in monocytes and macrophages [8]. While only a limited number of tissues and cell types have been examined, it is likely that PGHS-2 can be induced in almost any cell or tissue with the appropriate stimuli. Importantly, as noted earlier, PGHS-2 expression, but not PGHS-1 expression, can be completely inhibited by anti-inflammatory glucocorticoids such as dexamethasone [8].

The promoters of the two PGHS genes are indicative of their mode of regulation. PGHS-1 has a TATA-less promoter, a feature common to housekeeping genes. Reporter plasmids constructed with the 5′-upstream region of the PGHS-1 gene have failed to show any significant inducible transcription from this promoter, supporting the concept that regulation of PGHS-1 occurs only developmentally. The PGHS-2 promoter, on the other hand, contains a TATA box, and experiments with reporter plasmids containing the PGHS-2 promoter and upstream 5′-flanking sequence have demonstrated that PGHS-2 is highly regulatable [8,11]. Transcriptional activation of the PGHS-2 gene appears to be one important mechanism for increasing PGHS-2 expression. Transcription of PGHS-2 is unique, in that it can be controlled by multiple signalling pathways, including the cAMP pathway, the protein kinase C pathway (phorbol esters), by viral transformation (src), and by other pleiotropic pathways such as those activated by growth factors, bacterial endotoxin, and inflammatory cytokines.

While the primary structures of the human, mouse, and rat PGHS-2 genes and 5′-flanking regions have been determined, the complex analysis of cis-elements responsible for regulation of this gene are, as yet, in their early stages. The transcriptional control elements necessary for activation of the mouse PGHS-2 gene by phorbol esters and serum are located within the first 371 nucleotides upstream of the mouse PGHS-2 transcription start site. An NF-IL6/C/EBP regulatory element in the rat promoter centered at position − 131 is responsible, at least in part, for increased PGHS-2 gene transcription in rat follicular cells following exposure to cAMP.

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PROTEINASE-ACTIVATED RECEPTORS

R.C. Chambers, in Encyclopedia of Respiratory Medicine, 2006

Structure

The four PAR genes are between 3.5 and 3.7 kb long and share a similar two-exon structure encoding around 400 amino acids (Table 1). Human PAR1, PAR2, and PAR3 cluster together on band q13 on chromosome 5, suggesting that they may have arisen by gene duplication from a single ancestral gene. In contrast, PAR4 is located separately at position p12 on chromosome 19. The sequence homology between human PARs is between 27% and 33%; with the greatest difference noted between PAR4 and the other three PARs in terms of both the N- and C-termini and the cleavage site. Comparison of amino acid sequence alignment between species revealed that the PARs are highly conserved between humans and mice and that homologs of these genes are present in amphibians.

Table 1. Human proteinase-activated receptor expression and pharmacology

No. of amino acidsHigh-affinity-activating proteinasesLow-affinity-activating proteinasesTethered ligand sequenceActivating peptidesNonpeptide antagonistsInactivating proteinasesTissue expressionCell typesPAR1425ThrombinTrypsin, TF/FVIIa/FXa, granzyme A, plasmin, trypsin IV, MMP-1R41↑SFLLRNSFLLRN-NH2, TFLLR-NH2RWJ56110, RWJ58259Cathepsin G, neutrophil proteinase-3, elastase, chymase, Der p1Airways, blood, brain, bone, breast, cardiovascular system, endometrium, immune system, intestine, lung parenchyma, lymph node, nervous system, skinAstrocytes, epithelial cells, endothelial cells, fibroblasts, hematopoietic progenitor cells, keratinocytes, macrophages, mast cells, natural killer cells, neuronal cells, platelets, smooth muscle cells, T cellsPAR2397Trypsin, tryptase, trypsin II, trypsin IVMatriptase/ MT-SP1, TF/FVIIa/FXa, proteinase-3, Der p1, Der p3, Der p9R34↑SLIGKVSLIGKV-NH2, SFLLRN-NH2None to dateElastase, chymaseAirways, blood, brain, cardiovascular system, GI tract, immune system, intestine, nervous system, pancreas, skin, testes, urogenital tract, eyeEndothelial cells, eosinophils, epithelial cells, fibroblasts, keratinocytes, mast cells, macrophages, monocytes, neuronal cells, neutrophils, platelets, smooth muscle cells, T cellsPAR3374ThrombinTrypsin, Factor XaK38↑TFRGAPNone known–Cathepsin GImmune systemMegakaryocytes of the bone marrow , platelets, T cellsPAR4385Thrombin, trypsinCathepsin G,R47↑GYPGQVGYPGQV-NH2, AYPGKF-NH2YD-3UnknownAirway, blood, cardiovascular systemEpithelial cells, smooth muscle cells, endothelial cells, platelets, fibroblasts

Der p1, 3, and 9, house dust mite Dermatophagoides pteronyssinus proteinase 1, 3, and 9.

MMP-1, matrix metalloproteinase-1.

MT-SP1, Membrane-type serine protease 1.

NH2, amide.

Letters denote amino acid sequences in one letter code; arrow denotes cleavage site.

The predicted protein structure of the PARs share several features with classical seven transmembrane G-protein domain-linked receptors with their signature configuration consisting of seven helical hydrophobic transmembrane regions that in turn give rise to three intra- and three extracellular loops, a C-terminal intracellular tail, and a long N-terminal extracellular domain (Figure 1).

Figure 1. Mechanism of activation of proteinase-activated receptor by thrombin. (1) The cleavage of an extracellular fragment of the receptor by thrombin unmasks a tethered ligand that binds to the body of the receptor. (2) The resulting conformational change induces cell signaling via activation of heterotrimeric G-proteins. PAR, proteinase-activated receptor; Gα, β, γ, G-protein subunits.

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