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Because DNA metabarcoding typically employs sequence diversity among mitochondrial amplicons to estimate species composition, nuclear mitochondrial pseudogenes (NUMTs) can inflate diversity. This study quantifies the incidence and attributes of NUMTs derived from the 658-bp barcode region of cytochrome c oxidase I (COI) in 156 marine animal genomes. NUMTs were examined to ascertain if they could be recognized by their possession of indels or stop codons. In total, 309 NUMTs ≥150 bp were detected, with an average of 1.98 per species (range = 0-33) and a mean length of 391 ± 200 bp. Among this total, 75 (24.3%) lacked indels or stop codons. NUMTs appear to pose the greatest interpretational risk when short (313 bp) amplicons are used, such as in environmental DNA studies, dietary analyses or processed fish identification. Employing the standard amplicon length (313 bp) for marine metabarcoding, NUMTs could potentially inflate the operational taxonomic unit (OTU) count by 21% above the true species count while also raising intraspecific variation at COI by 15%. However, when both amplicon length and position are considered, inflation in OTU counts and in barcode variation were just 9% and 10%, respectively, suggesting NUMTs will not seriously distort biodiversity assessments. There was a weak positive correlation between genome size and NUMT count but no variation among phyla or trophic groups. Until bioinformatic advances improve NUMT detection, the best defence involves targeting long amplicons and developing reference databases that include both mitochondrial sequences and their NUMT derivatives.

Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn

Short ORFs (sORFs), that is, occurrences of a start and stop codon within 100 codons or less, can be found in organisms of all domains of life, outnumbering annotated protein-coding ORFs by orders of magnitude. Even though functional proteins smaller than 100 amino acids are known, the coding potential of sORFs has often been overlooked, as it is not trivial to predict and test for functionality within the large number of sORFs. Recent advances in ribosome profiling and mass spectrometry approaches, together with refined bioinformatic predictions, have enabled a huge leap forward in this field and identified thousands of likely coding sORFs. A relatively low number of small proteins or microproteins produced from these sORFs have been characterized so far on the molecular, structural, and/or mechanistic level. These however display versatile and, in some cases, essential cellular functions, allowing for the exciting possibility that many more, previously unknown small proteins might be encoded in the genome, waiting to be discovered. This review will give an overview of the steadily growing microprotein field, focusing on eukaryotic small proteins. We will discuss emerging themes in the molecular action of microproteins, as well as advances and challenges in microprotein identification and characterization.

As a large number of cancers are caused by nonsense mutations in key genes, read-through of these mutations to restore full-length protein expression is a potential therapeutic strategy. Mutations in the adenomatous polyposis coli (APC) gene initiate the majority of both sporadic and hereditary colorectal cancers (CRC) and around 30% of these mutations are nonsense mutations. Our goal was to test the feasibility and effectiveness of APC nonsense mutation read-through as a potential chemo-preventive therapy in Familial Adenomatous Polyposis (FAP), an inherited CRC syndrome patients. Ten FAP patients harboring APC nonsense mutations were treated with the read-through inducing antibiotic erythromycin for 4 months. Endoscopic assessment of the adenomas was performed at baseline, after 4 and after 12 months. Adenoma burden was documented in terms of adenoma number, maximal polyp size and cumulative polyp size per procedure. Tissue samples were collected and subjected to molecular and genetic analyses. Our results show that in the majority of patients the treatment led to a decrease in cumulative adenoma burden, median reduction in cumulative adenoma size and median reduction in adenoma number. Molecular and genetic analyses of the adenomas revealed that the treatment led to a reduced number of somatic APC mutations, reduced cellular proliferation and restoration of APC tumor-suppressing activity. Together, our findings show that induced read-through of APC nonsense mutations leads to promising clinical results and should be further investigated to establish its therapeutic potential in FAP and sporadic CRCs harboring nonsense APC mutations.

Single nucleotide polymorphisms in CCR6 (C-C chemokine receptor type 6) gene have been found to be the possible cause of many diseases like rheumatoid arthritis, psoriasis, lupus nephritis and systemic sclerosis and other autoimmune diseases. Therefore, identification of structurally and functionally important polymorphisms in CCR6 is important in order to study its potential malfunctioning and discovering therapeutic targets. Several bioinformatics tools were used to identify most damaging nsSNPs that might be vital for CCR6 structure and function. The in silico tools included PROVEAN, SIFT, SNP&GO and PolyPhen2 followed by I-Mutant MutPred and ConSurf. Phyre2 and I-TASSER were used for protein 3-D Modelling while gene-gene interaction was predicted by STRING and GeneMANIA. Our study suggested that three nsSNPs rs1376162684, rs751102128 and rs1185426631 are the most damaging in CCR6 gene while 7 missense SNPs rs1438637216, rs139697820, rs768420505, rs1282264186, rs1394647982, rs769360638 and rs1263402382 are found to revert into stop codons. Prediction of post-transcriptional modifications highlighted the significance of rs1376162684 because it effected potential phosphorylation site. Gene-gene interactions showed relation of CCR6 with other genes depicting its importance in several pathways and co-expressions. In future, studying diseases related to CCR6 should include investigation of these 10 nsSNPs. Being the first of its type, this study also proposes future perspectives that will help in precision medicines. For such purposes, CCR6 proteins from patients of autoimmune diseases should be explored. Animal models can also be of significance find out the effects of CCR6 in diseases.

The CGA arginine codon is a rare codon in Saccharomyces cerevisiae. Thus, full‐length mature protein synthesis from reporter genes with internal CGA codon repeats are markedly reduced, and the reporters, instead, produce short‐sized polypeptides via an unknown mechanism. Considering the product size and similar properties between CGA sense and UGA stop codons, we hypothesized that eukaryote polypeptide‐chain release factor complex eRF1/eRF3 catalyses polypeptide release at CGA repeats. Herein, we performed a series of analyses and report that the CGA codon can be, to a certain extent, decoded as a stop codon in yeast. This also raises an intriguing possibility that translation termination factors eRF1/eRF3 rescue ribosomes stalled at CGA codons, releasing premature polypeptides, and competing with canonical tRNAICG to the CGA codon. Our results suggest an alternative ribosomal rescue pathway in eukaryotes. The present results suggest that misdecoding of low efficient codons may play a novel role in global translation regulation in S. cerevisiae., One of the rare codons in Saccharomyces cerevisiae, CGA, was found to be misdecoded as stop‐codon to a certain extent by the eRF1/eRF3 translation termination complex. Our genetic study of eRF1, a protein tRNA mimic canonically recognizing all three stop codons, suggests a novel class of ribosome rescue pathway for the inefficient translation.

Translation of the genetic code occurs in a cycle where ribosomes engage mRNAs, synthesize protein, and then disengage in order to repeat the process again. The final part of this process – ribosome recycling, where ribosomes dissociate from mRNAs – involves a complex molecular choreography of specific protein factors to remove the large and small subunits of the ribosome in a coordinated fashion. Errors in this process can lead to the accumulation of ribosomes at stop codons or translation of downstream open reading frames (ORFs). Ribosome recycling is also critical when a ribosome stalls during the elongation phase of translation and must be rescued to allow continued translation of the mRNA. Here we discuss the molecular interactions that drive ribosome recycling, and their regulation in the cell. We also examine the consequences of inefficient recycling with regards to disease, and its functional roles in synthesis of novel peptides, regulation of gene expression, and control of mRNA-associated proteins. Alterations in ribosome recycling efficiency have the potential to impact many cellular functions but additional work is needed to understand how this regulatory power is utilized.

The infectious salmon anaemia virus (ISAV) is capable of causing a significant disease in Atlantic salmon, which has resulted in considerable financial losses for salmon farmers around the world. Since the first detection of ISAV in Canada in 1996, it has been a high priority for aquatic animal health management and surveillance programmes have led to the identification of many genetically distinct ISAV isolates of variable virulence. In this study, we evaluated the virulence of three ISAV isolates detected in Atlantic Canada in 2012 by doing in vivo-controlled disease challenges with two sources of Atlantic salmon. We measured viral loads in fish tissues during the course of infection. Sequences of the full viral RNA genomes of these three ISAV isolates were obtained and compared to a high-virulence and previously characterized isolate detected in the Bay of Fundy in 2004, as well as a newly identified ISAV NA-HPR0 isolate. All three ISAV isolates studied were shown to be of low to mid-virulence with fish from source A having a lower mortality rate than fish from source B. Viral load estimation using an RT-qPCR assay targeting viral segment 8 showed a high degree of similarity between tissues. Through genomic comparison, we identified various amino acid substitutions unique to some isolates, including a stop codon in the segment 8 ORF2 not previously reported in ISAV, present in the isolate with the lowest observed virulence.

Although the human neurotropic polyomavirus, JC virus (JCV), was isolated almost a half century ago, understanding the molecular mechanisms governing its biology remains highly elusive. JCV infects oligodendrocytes and astrocytes in the central nervous system (CNS) and causes a rare fatal brain disease known as progressive multifocal leukoencephalopathy (PML) in immunocompromised individuals including AIDS. It has a small circular DNA genome (∼5 kb) and generates two primary transcripts from its early and late coding regions, producing several predicted alternatively spliced products mainly by cis-splicing. Here, we report the discovery and characterization of two novel open reading frames (ORF1 and ORF2) associated with JCV late transcripts, generated by an unusual splicing process called trans-splicing. These ORFs result from (i) the trans-splicing of two different lengths of the 5'-short coding region of VP1 between the coding regions of agnoprotein and VP2 after replacing the intron located between these two coding regions and (ii) frame-shifts occurring within the VP2 coding sequences terminated by a stop codon. ORF1 and ORF2 are capable of encoding 58 and 72 aa long proteins respectively and are expressed in infected cells and PML patients. Each ORF protein shares a common coding region with VP1 and has a unique coding sequence of their own. When the expression of the unique coding regions of ORFs is blocked by a stop codon insertion in the viral background, the mutant virus replicates less efficiently when compared to wild-type, suggesting that the newly discovered ORFs play critical roles in the JCV life cycle.

Summary A previous study revealed a strong association between the DMRT3:Ser301STOP mutation in horses and alternate gaits as well as performance in harness racing. Several follow-up studies have confirmed a high frequency of the mutation in gaited horse breeds and an effect on gait quality. The aim of this study was to determine when and where the mutation arose, to identify additional potential causal mutations and to determine the coalescence time for contemporary haplotypes carrying the stop mutation. We utilized sequences from 89 horses representing 26 breeds to identify 102 SNPs encompassing the DMRT3 gene that are in strong linkage disequilibrium with the stop mutation. These 102 SNPs were genotyped in an additional 382 horses representing 72 breeds, and we identified 14 unique haplotypes. The results provided conclusive evidence that DMRT3:Ser301STOP is causal, as no other sequence polymorphisms showed an equally strong association to locomotion traits. The low sequence diversity among mutant chromosomes demonstrated that they must have diverged from a common ancestral sequence within the last 10 000 years. Thus, the mutation occurred either just before domestication or more likely some time after domestication and then spread across the world as a result of selection on locomotion traits.

A spontaneous medaka ro mutant shows abnormal wobbling and rolling swimming behaviors. By positional cloning, we mapped the ro locus to a region containing the gene encoding Contactin1b (Cntn1b), which is an immunoglobulin (Ig)-superfamily domain-containing membrane-anchored protein. The ro mutant had a deletion in the cntn1b gene that introduced a premature stop codon. Furthermore, cntn1b mutants generated by the CRISPR/Cas9 system and trans-heterozygotes of the CRISPR mutant allele and ro had abnormal swimming behavior, indicating that the cntn1b gene was responsible for the ro-mutant phenotype. We also established zebrafish cntn1a and cntn1b mutants by transcription activator-like effector nucleases (TALENs). Zebrafish cntn1b but not cntn1a mutants showed abnormal swimming behaviors similar to those in the ro mutant, suggesting that Cntn1b plays a conserved role in the formation or function of the neural circuits that control swimming in teleosts. Although Cntn1-deficient mice have abnormal cerebellar neural circuitry, there was no apparent histological abnormality in the cerebellum of medaka or zebrafish cntn1b mutants. The medaka cntn1b mutants had defective optokinetic response (OKR) adaptation and abnormal rheotaxis (body positioning relative to water flow). Medaka and zebrafish cntn1b mutants are effective models for studying the neural circuits involved in motor learning and motor coordination.

Background The assignment of human leukocyte antigen (HLA) null alleles is clinically relevant in the setting of stem cell transplantation. Cell surface expression profiling and mRNA processing analysis of the HLA-B allele previously designated as B*07:44, have been performed. Materials and Methods Cell surface expression of HLA-B*07:44 was determined using flow cytometry. Genomic full-length and HLA-B*07-specific cDNA sequencing were carried out by Sanger procedure. Results Flow cytometric analysis confirmed previous serologic results and demonstrated a lack of cell membrane expression of the HLA-B protein. The mRNA processing, studied using direct HLA-B*07-specific cDNA sequencing, revealed the presence of a unique, aberrantly spliced mRNA, with a deletion of the last 43 bp on the 5’-end of exon 4. The substitution from T to G at genomic position 1799 compared to B*07:02:01 introduced a new and stronger splice donor site at exon 4. This alternative splicing produced an mRNA containing a premature stop codon at position 280, explaining the absence of mature HLA-B7 protein on the cell surface. Conclusion These findings led us to consider this HLA-B variant as a HLA null allele. The World Health Organization (WHO) Nomenclature Committee has since renamed this variant B*07:44N .

Background Coffin–Siris syndrome is an extremely rare syndrome associated with developmental and congenital anomalies. It is caused by heterozygous pathogenic variants of ARID1A, ARID1B, SMARCA4, SMARCB1, SMARCE1, and SOX11. Methods This case study presents the whole exome sequencing of a patient with characteristic clinical features of Coffin–Siris syndrome. Analysis included Sanger sequencing of complementary DNA and bioinformatic analysis of the variant. Results Analysis of cDNA Sanger sequencing data revealed that the donor splice site variant led to skipping of exon 19. Further, bioinformatic analysis predicted abnormal splicing in a translational frameshift of 11 amino acids and the creation of a premature termination codon. Results found a novel de novo splice site variant c.5025+2T>C in the ARID1B and truncated 1 633 amino acid protein NP_065783.3:p. (Thr1633Valfs*11). Conclusion Truncated ARID1B resulted in loss of the BAF250 domain, which is part of SWI/SNF‐like ATP‐dependent chromatin remodeling complex. The severe clinical manifestation presented by the proband was attributed to the disappearance of the BAF250 domain in the ARID1B protein. Our finding provides strong evidence that this pathogenic variant of exon 19 caused a frameshift mutation in the ARID1B at the terminal exon, resulting in the expression of a severe phenotype of CSS., This case study examined the whole exome sequencing of a patient with characteristic clinical features of Coffin–Siris syndrome and found a novel de novo splice site variant c.5025+2T>C in the ARID1B. Analysis of cDNA Sanger sequencing data obtained from this patient revealed that the donor splice site variant led to skipping of exon 19. The severe clinical manifestation that our proband presented with; cumulated in disappearance of the BAF250 domain in the ARID1B protein. Thus, providing strong evidence that this pathogenic variant of exon 19 caused frameshift mutation in the ARID1B at the terminal exon resulting in the expression of a severe phenotype of CSS.

Translational readthrough generates proteins with extended C‐termini, which often possess distinct properties. Here, we have used various reporter assays to demonstrate translational readthrough of AGO1 mRNA. Analysis of ribosome profiling data and mass spectrometry data provided additional evidence for translational readthrough of AGO1. The endogenous readthrough product, Ago1x, could be detected by a specific antibody both in vitro and in vivo. This readthrough process is directed by a cis sequence downstream of the canonical AGO1 stop codon, which is sufficient to drive readthrough even in a heterologous context. This cis sequence has a let‐7a miRNA‐binding site, and readthrough is promoted by let‐7a miRNA. Interestingly, Ago1x can load miRNAs on target mRNAs without causing post‐transcriptional gene silencing, due to its inability to interact with GW182. Because of these properties, Ago1x can serve as a competitive inhibitor of miRNA pathway. In support of this, we observed increased global translation in cells overexpressing Ago1x. Overall, our results reveal a negative feedback loop in the miRNA pathway mediated by the translational readthrough product of AGO1.

Dom34-Hbs1 plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). Here, we report a conserved function of human Pelota (hPelota) in mRNA quality control of nonstop mRNA. hPelota facilitated the expression of the nonstop products from GFP-Rz mRNA, which lacks a termination codon and a poly(A) tail, in exosome-defective mutant cells. hPelota promoted the dissociation of stalled ribosomes at the 3' end of GFP-Rz mRNA, and mutations in domain A diminished this activity. The hPelota-R45A mutant associated with ribosomes but was defective in peptide release. Finally, hPelota promoted the degradation of GFP-Rz mRNA and suppressed the sequential endonucleolytic cleavages caused by stalled ribosomes at the 3' end of mRNA in dom34∆ mutant cells.

The novel allele HLA-C*16:123N has a single nucleotide difference with HLA-C*01:02:01:01 in exon 2.

Summary Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects life span and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments.

Summary Classical genetic studies in European rabbits (Oryctolagus cuniculus) suggested the presence of two alleles at the brown coat colour locus: a wild-type B allele that gives dense black pigment throughout the coat and a recessive b allele that in the homozygous condition (b/b genotype) produces brown rabbits that are unable to develop black pigmentation. In several other species, this locus is determined by mutations in the tyrosinase-related protein 1 (TYRP1) gene, encoding a melanocyte enzyme needed for the production of dark eumelanin. In this study, we investigated the rabbit TYRP1 gene as a strong candidate for the rabbit brown coat colour locus. A total of 3846 bp of the TYRP1 gene were sequenced in eight rabbits of different breeds and identified 23 single nucleotide polymorphisms (SNPs; 12 in intronic regions, five in exons and six in the 3′-untranslated region) and an insertion/deletion of 13 bp, in the 3′-untranslated region, organised in a few haplotypes. A mutation in exon 2 (g.41360196G>A) leads to a premature stop codon at position 190 of the deduced amino acid sequence (p.Trp190ter). Therefore, translation predicts a truncated TYRP1 protein lacking almost completely the tyrosinase domain. Genotyping 203 rabbits of 32 different breeds identified this mutation only in brown Havana rabbits. Its potential functional relevance in disrupting the TYRP1 protein and its presence only in brown animals strongly argue for this non-sense mutation being a causative mutation for the recessive b allele at the brown locus in Oryctolagus cuniculus.

Background Danon disease is an X‐linked dominant hereditary condition caused by mutations in the gene encoding lysosomal‐associated membrane protein 2 (LAMP2), leading to failure of lysosome binding to autophagosomes, accumulation of glycogen in the heart, and abnormal cardiac function. Methods We describe identification of a mutation in LAMP2, c.741+1G>T, in a family with Danon disease by whole exome sequencing. Results Pathology examination of patient skeletal muscle biopsy showed myogenic damage and autophagic vacuoles with sarcolemmal features (AVSF). Numerous autophagic vacuoles accumulated in muscle cells were detected by electron microscopy, indicating abnormal autophagy function. Conclusion The mutation did not result in loss of mRNA exons; rather, a 6‐nucleotide (two‐codon) insertion, where the latter was a stop codon, leading to early termination of LAMP2 protein translation. The resulting truncated protein lacks an important transmembrane domain, which will impair lysosome/autophagosome fusion, damage autophagy function, and result in the clinical manifestations of Danon disease.

The addition of new and versatile chemical and biological properties to proteins pursued through incorporation of non-canonical amino acids is at present primarily achieved by stop codon suppression. However, it is critical to find new “blank” codons to increase the variety and efficiency of such insertions, thereby taking ‘sense codon recoding’ to center stage in the field of genetic code expansion. Current thought optimistically suggests the use of the pyrrolysine system coupled with re-synthesis of genomic information towards achieving sense codon reassignment. Upon review of the serious experimental challenges reported in recent studies, we propose that success in this area will depend on the re-synthesis of genomes, but also on ‘rewiring’ the mechanism of protein synthesis and of its quality control.

Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.

Aminoglycoside-mediated read-through of stop codons was recently demonstrated for a variety of diseases in vitro and in vivo. About 30 percent of human genetic diseases are the consequence of nonsense mutations. Nonsense mutations in obesity-associated genes like the melanocortin 4 receptor (MC4R), expressed in the hypothalamus, show the impact of premature stop codons on energy homeostasis. Therefore, the MC4R could be a potential pharmaceutical target for obesity treatment and targeting MC4R stop mutations could serve as proof of principle for nonsense mutations in genes expressed in the brain. We investigated four naturally occurring nonsense mutations in the MC4R (W16X, Y35X, E61X, Q307X) located at different positions in the receptor for aminoglycoside-mediated functional rescue in vitro. We determined localization and amount of full-length protein before and after aminoglycoside treatment by fluorescence microscopy, cell surface and total enzyme linked immunosorbent assay (ELISA). Signal transduction properties were analyzed by cyclic adenosine monophosphate (cAMP) assays after transient transfection of MC4R wild type and mutant receptors into COS-7 cells. Functional rescue of stop mutations in the MC4R is dependent on: (i) triplet sequence of the stop codon, (ii) surrounding sequence, (iii) location within the receptor, (iv) applied aminoglycoside and ligand. Functional rescue was possible for W16X, Y35X (N-terminus), less successful for Q307X (C-terminus) and barely feasible for E61X (first transmembrane domain). Restoration of full-length proteins by PTC124 could not be confirmed. Future pharmaceutical applications must consider the potency of aminoglycosides to restore receptor function as well as the ability to pass the blood-brain barrier.

For simple retroviruses, such as the Rous sarcoma virus (RSV), post-transcriptional control elements regulate viral RNA splicing, export, stability, and packaging into virions. These RNA sequences interact with cellular host proteins to regulate and facilitate productive viral infections. One such element, known as the RSV stability element (RSE), is required for maintaining stability of the full-length unspliced RNA. This viral RNA serves as the mRNA for the Gag and Pol proteins and also as the genome packaged in progeny virions. When the RSE is deleted from the viral RNA, the unspliced RNA becomes unstable and is degraded in a Upf1-dependent manner. Current evidence suggests that the RSE inhibits recognition of the viral gag termination codon by the nonsense-mediated mRNA decay (NMD) pathway. We believe that the RSE acts as an insulator to NMD, thereby preventing at least one of the required functional steps that target an mRNA for degradation. Here, we discuss the history of the RSE and the current model of how the RSE is interacting with cellular NMD factors.

The investigation of protein–protein interactions is an essential part of biological research. To obtain a deeper insight into regulatory protein networks, the identification of the components, domains and especially single residues that are involved in these interactions is helpful. A widespread and attractive genetic tool for investigation of protein–protein interactions is the yeast two-hybrid system. This method enables large-scale screens and its application is cheap and relatively simple. For identification of the amino acids in a protein sequence that are essential for interaction with a specific partner, yeast two-hybrid assays can be combined with random mutagenesis of the sequence of interest. A common problem with such an experiment is the generation of stop codons within the mutagenized fragments, leading to the isolation of many false positives when screening for loss of interaction using the two-hybrid method. To overcome this problem, we modified the yeast two-hybrid system to allow selection for sequences without stop codons. To achieve this, we fused the ScURA3 marker-gene in frame to the mutagenized fragments. We show here that this marker is fully functional when fused to a two-hybrid construct with a nuclear localization signal, such as a Gal4 activation domain and a prey protein, thus allowing selection of stop-free sequences on media without uracil. Using the Rho-binding domain from a Bni1-like formin and different Rho-type GTPases from Ashbya gossypii as examples, we further show that our system can be used to screen large numbers of transformants for loss of protein–protein interactions in combination with random mutagenesis. Copyright © 2011 John Wiley & Sons, Ltd.

We have previously generated a transgenic mouse strain (LSL-TβRICA) containing a Cre-inducible constitutively active TGFβ type I receptor (Bartholin L. et al. 2008. Generation of mice with conditionally activated transforming growth factor beta signaling through the TbetaRI/ALK5 receptor, GENESIS). Transgene expression depends on the excision of a floxed-transcriptional STOP (LSL, Lox-STOP-Lox) located upstream the TβRICA coding sequence. In order to evaluate the correct excision of the STOP signal in the presence of Cre-recombinase, we developed a rapid screening based on an original PCR genotyping strategy. More precisely, we designed a set of primers flanking the LSL containing region. The size of the amplified products will differ according to recombination status of the LSL-TβRICA allele. Indeed, the size of the STOP containing PCR product is 1.93 kb, but is reduced to 0.35 kb when the STOP signal is removed after Cre-mediated recombination. We validated excision in several compartments, including pancreas, liver, T lymphocytes and embryos using different Cre expressing transgenic mouse strains. This represents a simple and efficient way of monitoring the tissue specific recombination of the LSL-TβRICA allele.

Sequence analysis of 168 cDNA clones encoding 12 nicotinic acetylcholine receptor subunits, Tcasalpha1-Tcasalpha11 and Tcasbeta1, from the red flour beetle, Tribolium castaneum, revealed extensive post-transcriptional modification and multiple alleles. The greatest diversity was found for Tcasa6, where 18 unique transcripts, as a result of alternative and optional exon usage, were seen. A novel alternative exon 8d was found in one Tcasalpha6 transcript. Tcasalpha5 transcripts did not contain previously reported exons 8-10. Six subunits had transcripts that contained unspliced introns, which introduced premature stop codons. Intron 3' splice site variants were seen at six intron boundaries across five subunits. A-to-I RNA editing was seen only in Tcasalpha6. Alleles were found for all subunit genes, except Tcasalpha1 and Tcasalpha10. Transcriptional and allelic diversity are discussed with respect to receptor function and potential interactions with insecticides.

Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.

Benign hereditary chorea (BHC) is a rare autosomal dominant nonprogressive movement disorder. In some cases the phenotype includes, besides choreoathetosis, thyroid dysfunction and pulmonary infections in infancy, as expressed by the name "Brain-Thyroid-Lung syndrome". Mutations in the thyroid transcription factor-1 (TITF-1) gene have been identified in some BHC families. We present the phenotypic features of a family with chorea, hypothyroidism, and lung dysfunction. All affected individuals suffered from a nonprogressive chorea with infancy onset. All showed short stature and some webbed neck. One patient suffered from psychosis at the age of 27 years another from lung carcinoma. In all affected individuals, a novel mutation consisting of heterozygous C to A substitution at position 650 of the coding sequence of the TITF-1 gene, exon 3 was detected, leading to a premature stop at codon 217 (S217X). We describe the unique phenotypic features and intrafamilial variability expressing this novel mutation.

Microsporidia are fungal-like unicellular eukaryotes which develop as obligate intracellular parasites. They differentiate into resistant spores that are protected by a thick spore wall composed of a glycoprotein-rich outer layer or exospore and a chitin-rich inner layer or endospore. In this study performed on the silkworm pathogen Nosema bombycis, we analyzed the spore wall proteins (SWPs) by proteomic-based approaches, MALDI-TOF MS and LC-MS/MS, and 14 hypothetical spore wall proteins (HSWPs) or peptides were obtained in total. Furthermore, we have examined the SWPs by SDS-PAGE and three main spore wall peptides were detected with molecular weights of 32.7 kDa (SWP32), 30.4 kDa (SWP30), and 25.3 kDa (SWP25), respectively. By N-terminal amino acid residue sequencing, and searching the genomic DNA shotgun database of N. bombycis, the complete ORFs of SWP30 and SWP32 were obtained, which encode for a 278- and a 316-amino acid peptide, respectively. Mouse polyclonal antibodies were raised against SWP30 and SWP32 recombinant proteins produced in Escherichia coli, and the results of indirect immunofluorescence assay (IFA) and immunoelectron microscopy (IEM) analyses indicated SWP30 to be an endosporal protein while SWP32 was shown to be an exosporal protein. Both SWP30 and SWP32 are included in the 14 HSWPs identified by MS, confirming the results of the proteomic-based approaches.

While all essential mammalian mitochondrial factors involved in the initiation and elongation phases of translation have been cloned and well characterized, little is known about the factors involved in the termination process. In the present work, we report the functional analysis of human mitochondrial translation release factors (RF). Here, we show that HMRF1, which had been previously denoted as a human mitochondrial RF, was inactive in in vitro translation system, although it is a mitochondrial protein. Instead, we identified another human mitochondrial RF candidate, HMRF1L, and demonstrated that HMRF1L is indeed a mitochondrial protein that functions specifically as an RF for the decoding of mitochondrial UAA and UAG termination codons in vitro. The identification of the functional mitochondrial RF brings us much closer to a detailed understanding of the translational termination process in mammalian mitochondria as well as to the unraveling of the molecular mechanism of diseases caused by the dys-regulation of translational termination in human mitochondria.

Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co-segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6-320A-->T). Its effect on mRNA processing was evaluated in-vitro: the in-frame inclusion of a 75-bp pseudo-exon carrying a premature stop was found, representing the first report of pseudo-exon activation as a mechanism leading to afibrinogenaemia.

The human tissue kallikreins, 15 secreted serine proteases, may play diverse roles in pathophysiology. The National Center for Biotechnology Information's dbSNP was mined for polymorphisms located within the kallikrein ( KLK ) locus using custom-designed “ParSNPs” and “LocusAnnotator” software tools. Using “ParSNPs”, a filterable catalogue of 1856 KLK polymorphisms (1023 validated) was generated. “LocusAnnotator” was used to annotate the KLK locus sequence with gene and polymorphism features. A second locus was examined to validate the use of both programs on a non-kallikrein locus. This report may assist in the informed selection of KLK polymorphisms for future association and biochemical studies in relation to disease. Furthermore, “ParSNPs” and “LocusAnnotator” are available at no cost from our website ( www.acdcLab.org/annotations ) to examine other loci.

We investigated the fate of aberrant mRNAs lacking in-frame termination codons (called nonSTOP mRNA) in mammalian cells. We found that translation of nonSTOP mRNA was considerably repressed although a corresponding reduction of mRNA was not observed. The repression appears to be post-initiation since (i) repressed nonSTOP mRNAs were associated with polysomes, (ii) translation of IRES-initiated and uncapped nonSTOP mRNA were repressed, and (iii) protein production from nonSTOP mRNA associating with polysomes was significantly reduced when used to program an in vitro run-off translation assay. NonSTOP mRNAs distributed into lighter polysome fractions compared to control mRNAs encoding a stop codon, and a significant amount of heterogeneous polypeptides were produced during in vitro translation of nonSTOP RNAs, suggesting premature termination of ribosomes translating nonSTOP mRNA. Moreover, a run-off translation assay using hippuristanol and RNAse protection assays suggested the presence of a ribosome stalled at the 3' end of nonSTOP mRNAs. Taken together, these data indicate that ribosome stalling at the 3' end of nonSTOP mRNAs can block translation by preventing upstream translation events.

The efficiency of stop codons read-through in yeast is controlled by multiple interactions of genetic and epigenetic factors. In this study, we demonstrate the participation of the Hal3-Ppz1 protein complex in regulation of read-through efficiency and manifestation of non-Mendelian anti-suppressor determinant [ISP(+)]. Over-expression of HAL3 in [ISP(+)] strain causes nonsense suppression, whereas its inactivation displays as anti-suppression of sup35 mutation in [isp(-)] strain. [ISP(+)] strains carrying hal3Delta deletion cannot be cured from [ISP(+)] in the presence of GuHCl. Since Hal3p is a negative regulatory subunit of Ppz1 protein phosphatase, consequences of PPZ1 over-expression and deletion are opposite to those of HAL3. The observed effects are mediated by the catalytic function of Ppz1 and are probably related to the participation of Ppz1 in regulation of eEF1Balpha elongation factor activity. Importantly, [ISP(+)] status of yeast strains is determined by fluctuation in Hal3p level, since [ISP(+)] strains have less Hal3p than their [isp(-)] derivatives obtained by GuHCl treatment. A model considering epigenetic (possibly prion) regulation of Hal3p amount as a mechanism underlying [ISP(+)] status of yeast cell is suggested.

Summary Pyrrolysine and selenocysteine have infiltrated natural genetic codes via the translation of canonical stop codons. UGA translation as selenocysteine is absolutely dependent on message context. Here we describe the first experimental examination of contextual requirements for UAG translation as pyrrolysine. A hexahistidine-tagged Methanosarcina barkeri mtmB1 gene, encoding monomethylamine methyltransferase MtmB1, was introduced into Methanosarcina acetivorans. Host mtmB expression was minimized by growth on methanol and recombinant mtmB1 products monitored by anti-MtmB and anti-hexahistidine immunoblotting. UAG translation was not compromised, as recombinant MtmB1 was 1% of cellular protein with only trace UAG-terminated mtmB1 product detectable. Untranslated regions flanking mtmB1 were not required for UAG translation, but loss of a downstream pyrrolysine insertion sequence (PYLIS) significantly increased the UAG-termination product of mtmB1 and decreased the UAG-translation product, which nonetheless contained pyrrolysine. An in-frame UAG within a bacterial uidA transcript was translated in the methanogen as pyrrolysine with 20% efficiency, suggesting UAG translation in the absence of evolved context. However, predominant UAG-directed termination with enhancement of UAG translation by the PYLIS appears analogous to cis-acting elements for UGA translation as selenocysteine, although different mechanisms may underlie these recoding events.

Dystrophic epidermolysis bullosa (DEB) is caused by mutations in the COL7A1 gene encoding collagen, the major component of anchoring fibrils. Premature termination codon (PTC) mutations in both alleles usually lead to the Hallopeau-Siemens variant that shows the most severe phenotype. We experienced a case of the non-Hallopeau-Siemens variant (nHS-RDEB), which had a mild clinical severity although it has PTC mutations in both alleles. Our patient was a compound heterozygote for a nonsense mutation (R669X) in exon 15 and a nonsense mutation (E2857X) in exon 116. But we confirmed the existence of some anchoring fibrils on electron micrograph. This suggested that a PTC close to the 3' end of COL7A1 does not completely abolish the collagen VII mRNA. We hypothesized that the truncated procollagen VII from the mutant allele with a nonsense mutation (E2857X) in exon 116 included two out of eight cysteines needed for disulfide bond formation, and hence a few functional anchoring fibrils could be formed.

International audience; Fatty acid-binding protein type 1 (FABP1), commonly termed liver-type fatty acid-binding protein (L-FABP), is encoded by a single gene in mammals. We cloned and sequenced cDNAs for two distinct FABP1s in zebrafish coded by genes designated fabp1a and fabp1b. The zebrafish proteins, FABP1a and FABP1b, show highest sequence identity and similarity to the human protein FABP1. Zebrafish fabp1a and fabp1b genes were assigned to linkage groups 5 and 8, respectively. Both linkage groups show conserved syntenies to a segment of mouse chromosome 6, rat chromosome 4 and human chromosome 2 harboring the FABP1 locus. Phylogenetic analysis further suggests that zebrafish fabp1a and fabp1b genes are orthologs of mammalian FABP1 and most likely arose by a whole-genome duplication event in the ray-finned fish lineage, estimated to have occurred 200-450 million years ago. The paralogous fabp10 gene encoding basic L-FABP, found to date in only nonmammalian vertebrates, was assigned to zebrafish linkage group 16. RT-PCR amplification of mRNA in adults, and in situ hybridization to whole-mount embryos to fabp1a, fabp1b and fapb10 mRNAs, revealed a distinct and differential pattern of expression for the fabp1a, fabp1b and fabp10 genes in zebrafish, suggesting a division of function for these orthogolous and paralogous gene products following their duplication in the vertebrate genome. The differential and complementary expression patterns of the zebrafish fabp1a, fapb1b and fabp10 genes imply a hierarchical subfunctionalization that may account for the retention of both the duplicated fabp1a and fabp1b genes, and the fabp10 gene in the zebrafish genome.

Sequestosome 1/p62 (p62) mutations are associated with PDB; however, there are limited data regarding functional consequences. We report a novel mutation in exon 7 (K378X) in a patient with polyostotic Paget's disease of bone. p62 mutants increased NF-κB activation and significantly potentiated osteoclast formation and bone resorption in human primary cell cultures. Introduction:Sequestosome 1/p62 (p62) mutations are associated with Paget's disease of bone (PDB); however, there are limited data regarding functional consequences. One report has linked the common P392L mutation in the p62 ubiquitin binding associated (UBA) domain with increases in NF-κB activity, a transcription factor essential for osteoclastogenesis. To further clarify the functional impact of p62 mutations associated with PDB, we assessed the effect of p62 mutation (a novel mutation: K378X, and previously reported mutations: P392L and E396X) on RANK-induced NF-κB activation and compared this with the effect of wildtype p62. In addition, we studied the effect of p62 mutation on osteoclast formation and bone resorption. Materials and Methods: We performed co-transfection experiments with expression plasmids for p62 (wildtype or mutated) and RANK and an NF-κB luciferase reporter gene. Luciferase activities were recorded after addition of luciferin to cellular lysates. RAW264.7 cells stably expressing enhanced green fluorescent protein (EGFP)-tagged p62 (wildtype, K378X, or P392L) or EGFP alone were assessed for changes in cell proliferation. Additionally, these cells were stimulated with RANKL to produce osteoclast-like cells (OLCs). Primary human monocytes collected from the K378X-affected patient and a control subject were stimulated to form OLCs and bone resorption data were obtained. Results: The novel mutation introduces a premature stop codon in place of Lys-378 and thereby eliminates the entire p62 UBA domain; this and two additional natural mutations (P392L, E396X) increased NF-κB activation compared with wildtype p62. Wildtype p62 consistently inhibited NF-κB activation compared with empty vector. UBA mutations (K378X and P392L) significantly increased the number of OLCs formed in response to RANKL and also the number of nuclei of the OLCs. K378X-affected human monocytes formed more OLCs with more nuclei and increased bone resorption compared with control monocytes. Conclusions: Our data show that mutation of the p62 UBA domain results in increased activation of NF-κB and osteoclast formation and function compared with wildtype p62. These results may partially explain the mechanism by which p62 mutation contributes to the pathogenesis of PDB.

Summary Bacterial release factors (RFs) 1 and 2 catalyse translation termination at UAG/UAA and UGA/UAA stop codons respectively. It has been shown that limiting the amount of ribosomal protein L11 affects translation termination at UAG and UGA differently. To understand the functional interplay between L11 and RF1/RF2, we isolated 21 distinct mutations in L11 as suppressors of either temperature-sensitive (ts) RF1/RF2 strains or read-through mutants of lacZ nonsense (UAG or UGA) strains. 10 of 21 mutants restored ts lethal growth of RF1 and/or RF2 strains. All the selected L11 mutants, including the RF1ts- and RF2ts-specific suppressors, had the same effect, either enhancing or reducing, on UAG and UGA termination efficiency in vivo. The specific properties of the selected L11 mutations remained unchanged in an RF3 deletion strain. Moreover, ribosomes absent of L11 had equally reduced activity for both RF1- and RF2-mediated peptide release in vitro. These results suggest that, unlike the previous notion, L11 has a common, cooperative role with RF1 and RF2. These L11 mutations were located on the surface of two domains of L11, and interpreted to affect the interaction between L11 and rRNA or the RFs thereby leading to the altered translation termination.

Familial British dementia (FBD) is a disorder characterized by the presence of amyloid deposits in cerebral blood vessels and brain parenchyma coexisting with neurofibrillary tangles in limbic areas. The amyloid subunit (ABri) is a 4 kDa fragment of a 266 amino acid type II single-spanning transmembrane precursor protein encoded by the BRI gene located on chromosome 13. In FBD patients, a single base substitution at the stop codon of this gene generates a larger 277-residue precursor (ABriPP-277). Proteolytic processing by a furin-like enzyme at the C-terminus of the elongated precursor generates the 34 amino acid ABri that undergoes rapid aggregation and fibrillization. ABri is structually unrelated to all known amyloids including A beta, the main component of the amyloid lesions in Alzheimer's disease (AD), indicating that cerebral deposition of amyloid molecules other than A beta can trigger similar neuropathological changes leading to neuronal loss and dementia. These data support the concept that amyloid deposition in the vascular wall and brain parenchyma is of primary importance in the initiation of neurogeneration.

Background and Aims: There is a paucity of population-based epidemiological information regarding hepatitis B virus (HBV) infection in India. The present study was planned to outline the magnitude and pattern of HBV infection, hepatitis B e antigen (HBeAg)-negative infection and the associated viral mutants in India. Methods: A community-based epidemiological study of HBV infection was carried out in West Bengal, India. Serological markers of infection and potential risk factors for HBV transmission were determined. Among the infected individuals, HBV-DNA, genotypes and mutations in the precore (PC) stop codon and basal core promoter (BCP) regions were determined by DNA sequencing and polymerase chain reaction (PCR) restriction fragment length polymorphism methods. Results: Of the 7653 people included in the study, 227 (2.97%) tested positive for hepatitis B surface antigen (HBsAg), of whom 204 (90%) were HBeAg-negative and hepatitis B e antibody (anti-HBe)-positive, and 78% had normal alanine aminotransferase (ALT) levels. HBV-DNA could be detected by PCR in only 32% of people. G1896A PC stop codon mutants were present in 12% of people, BCP mutants in 18% and the remainder (70%) of the HBeAg-negative infections were associated with wild type sequences in these regions. Conclusions: This first general population-based epidemiological study of HBV infection from India suggests that HBV acquisition starts in early childhood and peaks in adulthood. Most infections in the community are e-negative and inactive. The point prevalence of PC stop codon and BCP mutants is low in this primarily inactive and asymptomatic HBV-infected population sample in eastern India.

Gastrointestinal stromal tumors (GISTs) are a specific and rare subset of human gastrointestinal tract tumors. Most GISTs show gain-of-function mutations of KIT, mainly in exon 11, that always maintain the reading frame. We report on data from a 43-year-old Japanese man with recurrent duodenal GIST and a frameshift mutation in KIT exon 13 together with an in-frame deletion in KIT exon 11 detected by genomic DNA sequencing. Deletion of 48 base pairs of KIT exon 11, which preserved the reading frame, was identified in both primary and recurrent tumors, whereas deletion of one nucleotide of codon 642 of KIT exon 13, which changed the reading frame and induced a novel stop codon at amino acid 644, was found only in the recurrent tumor. The predicted protein resulting from the latter would lack part of the kinase domain. To the best of our knowledge, this is the first documentation of a GIST with a frameshift mutation of KIT.

Activation-induced cytidine deaminase (AID) plays a key role in the induction of somatic hypermutation and class switching at the immunoglobulin loci of B lymphocytes. AID overexpression can induce a mutator phenotype in lymphoid and nonlymphoid cell lines, suggesting that AID by itself is sufficient to trigger hypermutation and class switching. AID expression in vivo is considered to be restricted to germinal center B lymphocytes, yet AID expression is also seen in many B cell lymphomas, hinting at a potential role for the development of these malignancies. We used a GFP-based reversion assay to efficiently evaluate the activation of mutator phenotypes. As expected, AID overexpression in the human Burkitt lymphoma cell line BL70 caused hypermutation. Surprisingly, AID overexpression in the human pre-B cell line Nalm-6 failed to induce a detectable mutator phenotype, indicating that Nalm-6 cells are probably lacking an essential factor(s) to confer AID-induced mutagenesis. This finding supports the concept that AID overexpression by itself must not automatically lead to the onset of a mutator phenotype. In addition, treating Nalm-6 transfectants with thymidine, a potential mutagenic drug, caused profound mutation rates on the GFP transgene. Thus, the GFP-based mutation assay might prove a powerful tool to study protein- and chemical-induced mutator phenotypes in cell lines.

Objective: The precore mutant is detectable in most Israeli patients with persistent hepatitis B virus (HBV) infection. The aim of this study was to determine the prevalence of HBV genotypes, viral load and outcome of precore mutant infection in stable patients and in patients after liver transplantation. Methods: The prevalence of HBV genotype and viral load were investigated in 81 patients with HBV precore mutant infection. Of these, 50 patients (40 males, 10 females; mean age 43.4 ± 11.0 yr) underwent liver transplantation and were serum HBV DNA-negative by hybridization at the time of transplantation. Patients received long-term HBV immunoprophylaxis and immunosuppression, and lamivudine in cases of graft HBV recurrence. The remaining 31 patients were stable, with serum anti-HBe-positivity. Genotypes were tested by restriction fragment length polymorphism of an S gene amplicon. Precore mutations were studied with an INNO-LiPA probe assay. Results: Follow-up was 46.6 ± 37.7 months. Most of the transplanted group was of Middle Eastern origin (53.6%); the remainder were from Eastern Europe (21.4%), Western Europe and the USA (10.8%), Africa (7.1%), and Asia (7.1%). In the transplanted group, the pre-transplant HBV genotype D was the most prevalent (96%), while genotype A was found in only 4%. Eleven patients (22%) developed recurrent HBV infection post-transplantation. There were no differences in genotype distribution between patients with graft reinfection or lamivudine resistance and patients without recurrence. Mean viral load at recurrence was 148.4 × 106 ± 60.4 × 106 copies/mL. The stable group had a similar origin and HBV genotype prevalence, but a lower mean viral load of 12.4 × 106 ± 29.4 × 106 copies/mL (p = 0.007). The prevalence of mutations at the precore region and codon 28 was similar in both groups. Conclusions: The chronic precore mutant HBV-infected patients were characterized as follows: (i) genotype D was the most frequent genotype, (ii) the HBV genotype distribution was similar in patients with stable infection and after liver transplantation, (iii) viral load at recurrence was significantly higher than in stable infection, and (iv) HBV genotype was unrelated to the development of recurrence or lamivudine resistance in the tested population.

The association of precore stop codon mutation (A1896), dinucleotide mutation (T1762/A1764) in the basic core promoter of hepatitis B virus (HBV) genome, and genotype of HBV with fulminant or subfulminant hepatitis remains controversial. We studied HBV genotypes as well as mutations in the precore and basic core promoter regions in 18 hepatitis B carriers with fulminant or subfulminant hepatitis. Genotyping of HBV was performed by polymerase chain reaction-restriction fragment length polymorphism. The presence of A1896 in the precore gene and T1762/A1764 in the basic core promoter gene was determined by the polymerase chain reaction and by direct sequencing. Eighteen age- and sex-matched patients with chronic active hepatitis B served as controls. The HBV was of genotype B in 14, genotype C in 3, and unclassified in 1. Precore A1896 mutation occurred in 12 (67%) of the 18 patients. In contrast, the prevalence of basic core promoter mutation was only 17%. Nevertheless, the distribution of HBV genotype and the prevalence of precore A1896 mutation in the fulminant and subfulminant hepatitis patients were similar to those in 18 control patients. In conclusion, the genomic variability of HBV does not seem to contribute to the fulminant and subfulminant exacerbation of chronic hepatitis B in Taiwanese HBV carriers.

We report the translational readthrough levels induced by the aminoglycosides gentamicin, amikacin, tobramycin, and paromomycin for eight premature stop codon mutations identified in Duchenne's and Becker's muscular dystrophy patients. In a transient transfection reporter assay, aminoglycoside treatment results show that one stop codon mutation is suppressed significantly better (up to 10% stop codon readthrough) than the others; five show lower but statistically significant suppression (

Pyloric atresia-junctional epidermolysis bullosa syndrome (PA-JEB) is an autosomal recessive inherited rare blistering disorder caused by mutations in ITGA6 or ITGB4, genes encoding integrin alpha6 or beta4, respectively. In this study, we have disclosed the mutations in ITGB4 in a Korean patient with PA-JEB. The proband, who showed skin blisters, was diagnosed as having pyloric atresia and died 2 years after birth. Mutational analysis showed a novel 594insC maternal mutation in exon 7, which led to premature termination codon (PTC), and a novel Q425P paternal mutation in exon 11. Q425P mutation was not detected in 200 alleles obtained from a normal healthy Korean control, and was shown to reduce alpha-helix forming ability in integrin beta4 a by Garnier alpha-helicity plot of the protein, indicating that this mutation is pathogenic but not polymorphism. The phenotype in the present case can be explained by (1) the combination of PTC and missense mutation, and (2) amino-acid substitution occurring for the amino acid not preserved in the integrin beta family. Our results contribute to further the accumulation of mutation data for better understanding of the genotype/phenotype correlation in PA-JEB, and may give profound insight into the role of integrins alpha6 and beta4.