Changes In Coding Sequence Research Articles

The epigenome as a target for cancer chemoprevention.

Epigenetic events, a key driving force in the development of cancer, are alterations in gene expression without changes in the DNA coding sequence that are heritable through cell division. Such changes occur throughout all stages of tumorigenesis, including the early phases, and are increasingly recognized as major mechanisms involved in silencing tumor suppressor genes. Epigenetic changes can be reversed by the use of small molecules and, thus, such changes are promising targets for cancer chemopreventive drug development. This review examines the basis for targeting the epigenome as a prevention strategy, focusing on understanding the epigenetic changes that occur before the development of frank malignancy, when chemopreventive intervention will have the maximal impact.

Molecular analysis of echovirus 13 isolates and aseptic meningitis, Spain.

Echovirus 13 (EV13), considered rare, was reported worldwide in 2000, mostly related to aseptic meningitis outbreaks. In Spain, 135 EV13 isolates were identified. The genetic relationships between 64 representative strains from Spain and other reported isolates from the United States, Germany, Italy, Japan, and Sweden were described by analyzing the partial sequence of the major capsid protein (VP1) gene. The strains from Spain were clearly identified as EV13 (79.5% similarity with the EV13 reference strain) and were grouped phylogenetically into two different clusters (by origination on either the Iberian Peninsula or Canary Islands). Isolates from Germany from 2000 clustered with the Canary Islands group. The isolates from other countries obtained before 2000 were genetically distant. Changes in EV13 coding sequence involved several differences in the C-terminal extreme of the VP1 protein. Part of the neutralizing antigenic site III has been described in this genome region in poliovirus and swine vesicular disease virus.

Evolution of a pigmentation gene, the melanocortin-1 receptor, in primates.

The melanocortin-1 receptor (MC1R) forms a critical switch in the production of orange/red pheomelanin and black/brown eumelanin pigments during hair development in mammals. The molecular evolution of the melanocortin-1 receptor gene was investigated in a broad range of primate species, including several groups with large differences in distribution of orange/red and black hairs. Primate MC1R has been subject to purifying selection throughout most of its evolution, with small changes in selective constraint being detected early in primate evolution. In contrast to the situation in humans and domestic mammals, many intraspecific and intrageneric differences in primate coat color cannot be attributed to changes in the MC1R coding sequence. Nevertheless, important changes in the biochemical function of MC1R are suggested by mutations in sites of known functional importance, particularly in New World monkeys and lemurs. The evolution of the MC1R in lion tamarins is anomalous, with a combination of a high nonsynonymous to synonymous substitution rate (dN/dS) ratio, deletions, and substitutions.

A common polymorphism in the oxygen-dependent degradation (ODD) domain of hypoxia inducible factor-1α (HIF-1α) does not impair Pro-564 hydroxylation

BackgroundThe hypoxia-inducible factor (HIF) transcription complex, which is activated by low oxygen tension, controls a diverse range of cellular processes including angiogenesis and erythropoiesis. Under normoxic conditions, the α subunit of HIF is rapidly degraded in a manner dependent on hydroxylation of two conserved proline residues at positions 402 and 564 in HIF-1α in the oxygen-dependent degradation (ODD) domain. This allows subsequent recognition by the von Hippel-Lindau (VHL) tumor suppressor protein, which targets HIF for degradation by the ubiquitin-proteasome pathway. Under hypoxic conditions, prolyl hydroxylation of HIF is inhibited, allowing it to escape VHL-mediated degradation. The transcriptional regulation of the erythropoietin gene by HIF raises the possibility that HIF may play a role in disorders of erythropoiesis, such as idiopathic erythrocytosis (IE).ResultsPatients with IE were screened for changes in the HIF-1α coding sequence, and a change in the ODD domain that converts Pro-582 to Ser was identified in several patients. This same change, however, was also detected at a significant frequency, 0.073, in unaffected controls compared to 0.109 in the IE patient group. In vitro hydroxylation assays examining this amino acid change failed to reveal a discernible effect on HIF hydroxylation at Pro-564.ConclusionThe Pro582Ser change represents a common polymorphism of HIF-1α that does not impair HIF-1α prolyl hydroxylation. Although the Pro582Ser polymorphism is located in the ODD domain of HIF-1α it does not diminish the association of HIF-1α with VHL. Thus, it is unlikely that this polymorphism accounts for the erythrocytosis in the group of IE patients studied.

Electrophysiologic and phenotypic features of an autosomal cone-rod dystrophy caused by a novel CRX mutation

Purpose To reexamine a large Albertan family previously reported with a progressive cone dystrophy with variable phenotype and to map the disorder using molecular genetic techniques. Design Observational case series. Participants Twenty-nine subjects (10 affected) from four generations of a large kindred were clinically examined. Twenty-three of these individuals, as well as two unaffected spouses, were included in the molecular genetic study. Subject ages ranged from 17 to 91 years of age. Methods Disease status and associated ocular abnormalities were assessed primarily by measurement of visual acuity, color vision, fundus photography, and both full-field and multifocal electroretinography (ERG and mfERG). Linkage of the disorder to the rhodopsin gene was studied using microsatellites. A mutational screen of the CRX gene was performed to identify coding sequence changes. Main outcome measures Visual acuity and color discrimination were reduced in clinically affected individuals; full-field flash ERG was used to measure function of both cones and rods. mfERG and fundus photography allowed documentation of the observed macular changes. Results We noted a variable, adult-onset macular dystrophy, progressing in some cases to a retinitis pigmentosa-like phenotype. Both photopic and scotopic full-field ERG amplitudes were reduced by approximately 50%, demonstrating involvement of both photoreceptor systems. A reduced b-wave amplitude with a relatively preserved a-wave was observed at both cone and rod levels. Macular involvement was confirmed by mfERG. The rhodopsin locus was excluded by haplotype analysis. A novel frameshift mutation was detected in exon III of the CRX retinal homeobox gene. ERG and molecular genetic findings were consistent with the reclassification of this disease as an autosomal dominant cone-rod dystrophy (CRD) Conclusions We report a novel CRX mutation causing autosomal dominant CRD. Observed ERG changes suggest that this mutation primarily impairs inner retinal function. Because retinal expression of CRX is limited to photoreceptors, this dysfunction may be the result of faulty photoreceptor communication with second-order retinal neurons. We propose misexpression of gated cation channels caused by altered CRX activity as one putative mechanism by which a sole photoreceptor defect may selectively impair neurotransmission without disrupting the upstream events of phototransduction.

Competition between SsrA tagging and translational termination at weak stop codons in Escherichia coli.

SsrA is a tmRNA involved in tagging polypeptides on stalled ribosomes. The resulting fusion proteins are then degraded. We purified endogenous SsrA-tagged proteins by means of a genetically engineered SsrA and identified some of them. Analysis of the proteins suggested that they are tagged at their C-terminal extremities. One of them, ribokinase, is expressed from a messenger with a poorly efficient stop codon, leading to translational recoding events. A change in the ribokinase coding sequence from a weak to a strong translational stop sequence (UGAc to UAAu) annihilated SsrA tagging. Translational termination by UGA recruits the translational release factor (RF) 2. We observed that SsrA tagging of ribokinase was inversely correlated with RF2 activity, revealing a dynamic competition between translational termination and SsrA tagging.

Evaluation of FOXP2 as an autism susceptibility gene.

A mutation in the gene FOXP2 was recently identified as being responsible for a complicated speech and language phenotype in a single large extended pedigree. This gene is of interest to autism because it lies in one of the most consistently linked autism chromosomal regions of interest. We therefore tested this gene for its involvement in autism in a large sample of autism families. We completely sequenced the exon containing the mutation, screened the remaining coding sequence using SSCP technology, and identified and genotyped two novel intronic tetranucleotide repeat polymorphisms that were then analyzed for evidence of linkage and linkage disequilibrium (LD). We identified two families in which heterozygous deletions of a small number of glutamines in a long poly-glutamine stretch were found in one parent and the autistic probands; no other non-conservative coding sequence changes were identified. Linkage and LD analyses were performed in 75 affected sibling pair families and in two subgroups of this sample defined by the presence/absence of severe language impairment. One allele appeared to have an opposite pattern of transmission in the language based subgroups, but otherwise the linkage and LD analyses were negative. We conclude that FOXP2 is unlikely to contribute significantly to autism susceptibility.

Epigenetics in colorectal cancer

Malignant transformation is now known to require a series of molecular alterations that disrupt a limited number of pathways including autocrine and paracrine responses to growth factors, cell-cycle control, senescence, motility, and invasion. Studies on hereditary cancers have established genetic changes as the primary driving force for these molecular alterations. Recently, however, it has been recognized that epigenetic changes, defined as clonal changes in gene expression without accompanying changes in primary DNA coding sequence, can also be a driving force in neoplastic transformation, for selected genes, and in specific tumors. DNA methylation within gene promoters and associated alterations in histone acetylation appear primary mediators of epigenetic inheritance in cancer cells. In the large intestine, aberrant DNA methylation arises very early, initially in normal-appearing mucosa, and may be part of the age-related field defect observed in sporadic colorectal neoplasia. Aberrant methylation also contributes to later stages of colon cancer formation and progression through a hypermethylator phenotype termed cytosine phosphoguanosine (CpG) island methylator phenotype (CIMP), which appears to be a defining event in approximately half of all sporadic tumors. In sporadic colon cancer, CIMP has distinct epidemiologic and clinical features and is responsible for most cases of microsatellite instability related to hMLH1 inactivation. The recognition of epigenetic changes as a driving force in colorectal neoplasia opens new areas of research in disease epidemiology, risk assessment, screening, and treatment.

Consequences of Hox gene duplication in the vertebrates: an investigation of the zebrafish Hox paralogue group 1 genes.

As a result of a whole genome duplication event in the lineage leading to teleosts, the zebrafish has seven clusters of Hox patterning genes, rather than four, as described for tetrapod vertebrates. To investigate the consequences of this genome duplication, we have carried out a detailed comparison of genes from a single Hox paralogue group, paralogue group (PG) 1. We have analyzed the sequences, expression patterns and potential functions of all four of the zebrafish PG1 Hox genes, and compared our data with that available for the three mouse genes. As the basic functions of Hox genes appear to be tightly constrained, comparison with mouse data has allowed us to identify specific changes in the developmental roles of Hox genes that have occurred during vertebrate evolution. We have found variation in expression patterns, amino acid sequences within functional domains, and potential gene functions both within the PG1 genes of zebrafish, and in comparison to mouse PG1 genes. We observed novel expression patterns in the midbrain, such that zebrafish hoxa1a and hoxc1a are expressed anterior to the domain traditionally thought to be under Hox patterning control. The hoxc1a gene shows significant coding sequence changes in known functional domains, which correlate with a reduced capacity to cause posteriorizing transformations. Moreover, the hoxb1 duplicate genes have differing functional capacities, suggesting divergence after duplication. We also find that an intriguing function 'shuffling' between paralogues has occurred, such that one of the zebrafish hoxb1 duplicates, hoxb1b, performs the role in hindbrain patterning played in mouse by the non-orthologous Hoxa1 gene.

Protein stabilization: a common consequence of mutations in independently derived v-Myc alleles

Myc is overexpressed in many cancers as a result of gene rearrangement or amplification, but coding sequence changes which cluster in the N-terminal transactivation domain also appear to play a role in tumour progression. The prototypic v-Myc gene of MC29 virus differs from avian c-Myc by a series of mutations, including a change at a regulatory phosphorylation site within the mutational hotspot (thr-61) which is known to potentiate transformation in vitro. We now show that the mutation at thr-61 stabilizes the v-Myc protein (turnover difference) and that this single mutation is both necessary and sufficient for the phenotype. A major involvement of the proteasome in Myc degradation was confirmed, but surprisingly, a dilysine motif adjacent to thr-61 proved not to be the ubiquitin target. Two other v-Myc genes which carry a mutation at thr-61 (avian MH2) or a large deletion encompassing this domain (feline T17) were found to be stabilized to a similar extent as MC29, showing that stabilization is a common feature of independently derived Myc oncogenes. These results suggest a common selective process in the genesis of these three viral oncoproteins and a mechanistic link with Jun, Fos and Myb oncoproteins which are also stabilized relative to their cellular counterparts.

Changes in the Pattern of Twisted Gastrulation Gene Expression Among Drosophila Species

A long-standing hypothesis posits that morphological changes may be more likely to result from changes in regulation of gene expression than from changes in the protein coding sequences of genes. We have compared the expression pattern of the twisted gastrulation (tsg) gene among five Drosophila species: D. melanogaster, D. simulans, D. subobscura, D. mojavensis, and D. virilis. The tsg gene encodes a secreted protein that is required for the specification of dorsal midline fates in the Drosophila early embryo. TSG is unlike other secreted growth and differentiation factors in Drosophila in that its expression pattern can be experimentally varied and still result in normal development. Because of this, its regulatory region may be freer to diverge than that of other developmental genes whose misexpression may lead to lethal defects. Thus, the tsg gene may be a good indicator of the frequency and nature of evolutionary changes affecting patterns of gene expression. Over approximately 60 million years (Myr), the tsg gene has retained a dorsal-on/ventral-off pattern and a middorsal region of expression; but there have been marked changes in the middorsal domain of expression as well as the appearance/loss of other domains of expression along the anterior/posterior axis. Changes between closely related species (approximately 2-5 Myr since divergence) that are not reflected among more distantly related species suggest frequent changes in gene expression over evolutionary time. These changes in gene expression may serve as the raw material for eventual evolutionary changes in morphology.

An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells

The green fluorescent protein (GFP) from the jellyfishAequorea victoriahas become an important marker of gene expression. However, the sensitivity of wild-type GFP has been below that of standard reporter proteins, such as β-galactosidase, which utilize enzymatic amplification. To improve the detection of GFP in transfected mammalian cells, we have constructed a unique GFP variant which contains chromophore mutations that make the protein 35 times brighter than wild-type GFP, and is codon-optimized for higher expression in mammalian cells. These changes in the GFP coding sequence provide an enhanced GFP (EGFP) that greatly increases the sensitivity of the reporter protein. We show that the EGFP expression vector delivered into mammalian cells gives rise to bright fluorescence that is readily detectable following a 16-24 hr transfection interval. Visual detection of transfected cells with EGFP appears to be more sensitive than equivalent measurements with β-galactosidase catalyzed conversion of the X-gal substrate. We conclude that EGFP allows sensitive and convenient detection of gene transfer in mammalian cells.

Molecular Analysis of the Presenilin 1 (S182) Gene in “Sporadic” Cases of Alzheimer's Disease: Identification and Characterisation of Unusual Splice Variants

Mutations of the presenilin 1 (PS-1) gene at the Alzheimer's disease (AD) FAD3 locus on chromosome 14q24.3 are responsible for the majority of familial early-onset AD. As genes responsible for familial forms of AD are obvious candidates for further investigation in "sporadic" disease, we performed a molecular analysis of PS-1 transcripts extracted from brain tissues of a series of histologically confirmed cases of "sporadic" AD (n=10) and also from histologically "normal" (non-Alzheimer) age-matched brain controls (n=5). No sequence changes in the PS-1 coding sequence were detected after analysis by reverse transcription-PCR. This suggests that the frequency of mutations in the PS-1 (S182) coding region in "sporadic" Alzheimer's disease in very low. However, we demonstrated that the PS-1 gene is highly variably spliced. One splice variant involves the 5' untranslated region of the PS-1 gene only and hence encodes for normal PS-1. Six further splice variants involve coding regions of the PS-1 gene and result in truncated proteins lacking specific transmembrane domains. Most of these variants do not coincide with recognized sites of introns in the PS-1 gene. One of these variants, resulting in the loss of transmembrane domain TM-VII, was found only in an AD patient.

Two alternatively spliced forms of the human insulin-like growth factor I receptor have distinct biological activities and internalization kinetics.

Two alternatively spliced human insulin-like growth factor I (IGF I) receptor mRNA transcripts have been described that differ by three nucleotides (CAG) starting at position 2829. This results in an amino acid coding sequence change from Thr-Gly to Arg in the extracellular portion of the receptor beta subunit. To investigate the functional significance of this sequence difference, we obtained full-length cDNAs for the CAG+ and CAG- receptor forms, transfected CHO cells, and isolated multiple clones expressing approximately equal numbers of receptors (0.57-0.73 x 10(6) receptors/cell). The two receptors bound IGF I with similar affinity (Kd approximately 1.7 nM), but the CAG- form exhibited an approximately 2-fold increase in IGF I stimulation of receptor autophosphorylation, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, thymidine incorporation, and IRS-1-associated phosphatidylinositol 3-kinase activity. In contrast to its higher signaling activity, the rate of receptor-mediated internalization of IGF I by the CAG-receptor was decreased by 50% in comparison with the CAG+ receptor. We conclude that proteins corresponding to the two alternatively spliced human IGF I receptor transcripts are biologically active, but have distinct signaling properties. These experiments further indicate a previously unrecognized role for the extramembranous portion of the beta subunit in receptor signaling and internalization.

5'-flanking region responsible for endosperm-specific expression of rice prolamine chimeric gene in transgenic tobacco.

The 5' upstream region (-680-(+)40) containing the complete signal peptide coding sequence of the rice seed storage prolamine gene was amplified in vitro with polymerase chain reaction from the genome of Chinese super rice cultivar Zhonghua 8. Physical map and DNA sequence analysis showed strong homology with the 5'-flanking region of rice prolamine gene reported by Kim in 1988. No changes in the signal peptide coding sequence and a long leader sequence with several small ORFs were found. Chimeric gene containing 5'-flanking region of the prolamine gene has been transcriptionally fused with the beta-glucuronidase reporter gene. The fusion junction was confirmed by both physical map and DNA sequence analysis. The resultant chimeric gene was used to transform the tobacco explants by Ti binary system of Agrobacterium tumefaciens LBA4404. With dot and Southern blotting hybridization, three transgenic tobacco plants with the copies of chimeric GUS genes as many as 20 were obtained. Histochemical analysis revealed that the GUS activity in the endosperm tissues of tobacco seeds at the developmental stage was about 20 DAP. No GUS activity was found in leaves, stems, roots and flowers of the transgenic tobacco plants. Therefore, we concluded that the 5'-upstream cis-elements from -680 to -18 were enough to confer the endosperm-specific and temporal expression of rice prolamine gene.

Rates of amino acid change in the envelope protein correlate with pathogenicity of primate lentiviruses.

A spectrum of pathogenicity has been observed for primate lentiviruses in their natural hosts. For example, human immunodeficiency virus type 1 (HIV-1) is a potent etiologic agent for AIDS in man, whereas there is no evidence to date which indicates that simian immunodeficiency virus from African green monkeys (SIVAGM) causes immunodeficiency in AGM. We measured the relative rates of amino acid change, as the ratio of the number of nonsynonymous to synonymous (silent) nucleotide substitutions, for six primate lentiviruses evolving in their respective hosts. These rates for the external envelope glycoprotein (gp120) and gag coding sequences are 2-3 times higher for pathogenic HIV-1 and SIVmac (macaque) than for minimally pathogenic SIVAGM and SIVsmm (sooty mangabey), and intermediate for HIV-2. We speculate that the increased rates of nonsynonymous changes in gp120 and gag coding sequences are due to viral escape from immune surveillance and are indicative of higher immunogenicity of these proteins in their hosts. Based on these results and available experimental data, we conclude that there is a positive correlation between lentiviral pathogenicity and immunogenicity of the Env and Gag proteins in a given host. This hypothesis is consistent with recent data suggesting that immune system activation or autoimmunity induced by viral antigens may be important in the pathogenesis of AIDS.

DNA base sequence changes in spontaneous and ethyl methanesulfonate-induced mutations of a chromosomally-integrated gene in Chinese hamster ovary cells

A series of spontaneous and ethyl methanesulfonate-induced 6-thioguanine-resistant mutants were isolated in the CHO-10T5 cell line. This cell line was constructed by the introduction of a shuttle vector containing the Escherichia coli gpt gene into a hypoxanthine-guanine phosphoribosyltransferase deficient derivative of the Chinese hamster cell line CHO-K1. Shuttle vector sequences were recovered from many of the mutant cell lines by the COS cell fusion technique and the DNA base sequence of the gpt genes was determined whenever possible. The base sequences were determined for gpt genes recovered from 29 spontaneous mutants. Of these 29 mutants, 9 have single base substitutions, 1 has a small duplication, 17 have simple deletions, 1 has a deletion with additional bases inserted at the deletion site, and 1 has no change in the gpt coding sequence. Many of the deletions were less than 20 basepairs in length and several occurred in a region previously observed to be a hotspot for spontaneous deletions. The generation of the deletion/insertion mutation may have involved a quasi-palindromic intermediate. A total of 59 ethyl methansesulfonate-induced mutants were isolated and vector sequences were recovered from 50 mutants. All 50 mutants sequenced had single base substitutions and most (45) were G:C to A:T transitions. While there were no strong hotspots in this collection of mutations, the site distribution was obviously nonrandom. Many of the G:C to A:T transitions either produced a nonsense codon or occurred at glycine codons.

Mutation of the p53 Gene in Human Acute Myelogenous Leukemia

AbstractHeterogeneity of p53 protein expression is seen in blast cells of patients with acute myelogenous leukemia (AML). p53 protein is detected in the blasts of certain AML patients but not in others. We have identified p53 protein variants with abnormal mobility on gel electrophoresis and/or prolonged half-life (t½). We have sequenced the p53 coding sequence from primary blast cells of five AML patients and from the AML cell line (OCIM2). In OCIM2, a point mutation in codon 274 was identified that changes a valine residue to aspartic acid. A wild type p53 allele was not detected in these cells. Two point mutations (codon 135, cysteine to serine; codon 246, methionine to valine) were identified in cDNA from blasts of one AML patient. Both mutations were present in blast colonies grown from single blast progenitor cells, indicating that individual leukemia cells had sustained mutation of both p53 alleles. The cDNAs sequenced from blast samples of four other patients, including one with prolonged p53 protein t½ and one with no detectable p53 protein, were fully wild type. Thus, the heterogeneity of p53 expression cannot be explained in all cases by genetic change in the p53 coding sequence. The prolonged t½ of p53 protein seen in some AML blasts may therefore reflect changes not inherent to p53. A model is proposed in which mutational inactivation of p53, although not required for the evolution of neoplasia, would confer a selective advantage, favoring clonal outgrowth during disease progression.

Mutation of the p53 gene in human acute myelogenous leukemia

Heterogeneity of p53 protein expression is seen in blast cells of patients with acute myelogenous leukemia (AML). p53 protein is detected in the blasts of certain AML patients but not in others. We have identified p53 protein variants with abnormal mobility on gel electrophoresis and/or prolonged half-life (t 1/2). We have sequenced the p53 coding sequence from primary blast cells of five AML patients and from the AML cell line (OCIM2). In OCIM2, a point mutation in codon 274 was identified that changes a valine residue to aspartic acid. A wild type p53 allele was not detected in these cells. Two point mutations (codon 135, cysteine to serine; codon 246, methionine to valine) were identified in cDNA from blasts of one AML patient. Both mutations were present in blast colonies grown from single blast progenitor cells, indicating that individual leukemia cells had sustained mutation of both p53 alleles. The cDNAs sequenced from blast samples of four other patients, including one with prolonged p53 protein t 1/2 and one with no detectable p53 protein, were fully wild type. Thus, the heterogeneity of p53 expression cannot be explained in all cases by genetic change in the p53 coding sequence. The prolonged t 1/2 of p53 protein seen in some AML blasts may therefore reflect changes not inherent to p53. A model is proposed in which mutational inactivation of p53, although not required for the evolution of neoplasia, would confer a selective advantage, favoring clonal outgrowth during disease progression.

Genomic and cDNA actin sequences from a virulent strain of Entamoeba histolytica.

Invasiveness of Entamoeba histolytica strains that cause acute amoebiasis is characterized by aggressive behavior associated with cell motility and actin function. Analysis of actin genes from E. histolytica was initiated by devising methods for the isolation of biologically active nucleic acids, which allowed the preparation of cDNA and genomic DNA libraries. E. histolytica actin-encoding cDNAs and genomic clones have been isolated from libraries prepared from the virulent HM1:IMSS strain using a heterologous actin probe. Nucleotide sequence analysis of three independent cDNA clones and one genomic clone reveals a highly unusual codon bias and the absence of intervening sequences in E. histolytica actin. The coding sequence of the genomic clone is identical to that of two of the three cDNA clones. These represent at least two distinct mRNAs differing only by five silent changes in the protein coding sequence. Multiple genomic copies of the actin gene can be detected by Southern hybridization. E. histolytica actin exhibits a higher degree of homology to cytoplasmic than to muscle actin. Although the protein has been shown not to bind DNase I, the inferred amino acid sequence indicates conservation of all residues implied to participate in this binding.