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PubMed2026

Whole-genome sequencing of adenovirus 41 directly from wastewater using nested overlapping PCR and MinION.

Human adenovirus F41 (HAdV-F41) is one of the leading causes of children's acute gastroenteritis and was recently linked to an outbreak of severe acute hepatitis of unknown etiology among children during 2021 to 2022. While most evidence is based on clinical data, wastewater-based epidemiology offers a community-level approach to monitoring circulating strains and enhancing outbreak preparedness. In this study, we developed an overlapping amplicon-based whole-genome sequencing approach to directly detect HAdV-F41 from archived wastewater samples, using nested PCR with 13 primer sets. Archived wastewater samples were collected between 2021 and 2022 from three treatment plants in Seattle, USA. The viral load ranged from 1.2 × 103 to 8.4 × 103 genome copies per liter. The Oxford Nanopore platform was used for whole-genome sequencing. Complete or partial (>84%) HAdV-F41 genomes were recovered from wastewater samples, with mean coverage depths ranging from 10³ to 10⁵. The consensus sequences showed more than 99% similarity to reference genomes in the NCBI database. The phylogenetic analysis revealed that 2 sequences clustered within lineage 2a and 11 within lineage 2b, reflecting that at least two sub-lineages were circulating in the community at that time. Our results demonstrate that the overlapping amplicon-based whole-genome sequencing approach using the Oxford Nanopore platform reliably recovers HAdV-F41 genomes from wastewater. This method offers high-resolution genomic surveillance of circulating, clinically relevant HAdV-F41, supporting wastewater-based epidemiology as a valuable tool for detecting emerging variants and strengthening the early warning system for future disease outbreaks.IMPORTANCEHuman adenovirus F41 is a primary cause of childhood gastroenteritis and has been linked to recent outbreaks of severe acute hepatitis in children, yet community-level genomic surveillance of this virus remains limited. This study shows that wastewater can be used to recover nearly complete HAdV-F41 genomes through a targeted overlapping-amplicon sequencing strategy on the Oxford Nanopore platform. By applying this method to archived wastewater samples, we detected the simultaneous circulation of multiple viral lineages in a large city. These findings extend wastewater-based epidemiology beyond SARS-CoV-2 and emphasize its importance for monitoring clinically significant enteric viruses. The method described here offers a scalable tool for tracking viral evolution in communities and enhancing early warning systems for future outbreaks.

Wastewater↗

PubMed2026

Complete genome characterization, phylogenetic analysis, and capsid P2 variation of a goose astrovirus genotype 2 isolate from Guizhou, China.

Goose astrovirus genotype 2 (GAstV-2) is associated with gout and renal disease in goslings, but its occurrence in Guizhou Province remains poorly documented. We isolated a GAstV-2 strain, designated GZJP2024, from goslings with visceral gout on a farm in Jinping County, Guizhou Province, China. PCR detected GAstV-2 but not goose parvovirus, goose reovirus, Tembusu virus, fowl adenovirus, or goose astrovirus genotype 1. Serial passage in goose embryos produced mortality and hemorrhagic lesions during the third passage. Whole-genome sequencing yielded a 7,251-nt genome containing three overlapping open reading frames (ORF1a, ORF1b, and ORF2). Sequence identity and phylogenetic analyses assigned GZJP2024 to the GAstV-2 lineage. ORF1b was the most conserved coding region, whereas ORF2 was more variable. Comparison with consensus sequences from representative GAstV-2 strains identified five amino acid substitutions in ORF1a and ten in ORF2. Four ORF2 substitutions (E456D, L540Q, S608T, and A614T) occurred in the capsid P2 domain and overlapped or neighbored predicted B-cell epitope-rich regions. Template-based mapping placed E456D, S608T, and A614T on exposed regions of a spike-like capsid structure. These findings document a GAstV-2 isolate from a gout-affected goose farm in Guizhou and provide sequence data for future regional surveillance.

Capsid P2↗

PubMed1992

A novel calcium-binding protein from Euglena gracilis. Characterisation of a cDNA encoding a 74-kDa acidic-repeat protein targeted across the endoplasmic reticulum.

We have isolated a novel cDNA from Euglena gracilis that encodes a protein composed of 24.9% aspartate with an estimated pI of 3.56, and a deduced molecular mass of 73,542 Da. The first 20 or so amino acids are hydrophobic and resemble a signal sequence. The rest of the polypeptide is composed of a 23-amino-acid repeat. There are 30 repeats, of which 23 are full length. Part of the consensus sequence derived from the repeats has some similarity to the loop of the EF-hand type calcium-binding motif. Evidence is presented that a fusion protein of this novel protein with beta-galactosidase can bind calcium. Northern blotting indicates a single transcript of 2.3 kb (the same size as the cDNA). In-vitro translation of the cDNA gives a protein that migrates on SDS/PAGE with an apparent molecular mass of 120-125 kDa. The protein is processed into a smaller, protease-protected form (110-120 kDa) when translated in the presence of canine pancreatic microsomal vesicles. This suggests that the protein is targeted across the endoplasmic reticulum membrane in vivo, and is the first report of a signal sequence from E. gracilis. We propose that the cDNA obtained encodes a novel calcium-binding protein that is either secreted or resident in the endomembrane system of E. gracilis, and call it the acidic-repeat protein.

Amino Acid Sequence↗

PubMed1992

cis-acting sequences involved in exon selection in the chicken beta-tropomyosin gene.

The chicken beta-tropomyosin gene contains an internal pair of mutually exclusive exons (6A and 6B) that are selected in a tissue-specific manner. Exon 6A is incorporated in fibroblasts and smooth muscle cells, whereas exon 6B is skeletal muscle specific. In this study we show that two different regions in the intron between the two mutually exclusive exons are important for this specific selection in nonmuscle cells. Sequences in the 3' end of the intron have a negative effect in the recognition of the 3' splice site, while sequences in the 5' end of the intron have a positive effect in the recognition of the 5' splice site. First, sequences in exon 6B as well as in the intron upstream of exon 6B are both able to inhibit splicing when placed in a heterologous gene. The sequences in the polypyrimidine stretch region contribute to splicing inhibition of exons 5 or 6A to 6B through a mechanism independent of their implication in the previously described secondary structure around exon 6B. Second, we have identified a sequence of 30 nucleotides in the intron just downstream of exon 6A that is essential for the recognition of the 5' splice site of exon 6A. This is so even after introduction of a consensus sequence into the 5' splice site of this exon. Deletion of this sequence blocks splicing of exon 6A to 6B after formation of the presplicing complex. Taken together, these results suggest that both the mutually exclusive behavior and the choice between exons 6A and 6B of the chicken beta-tropomyosin gene are trans regulated.

Animals↗

PubMed1992

Expression and characterization of human FKBP52, an immunophilin that associates with the 90-kDa heat shock protein and is a component of steroid receptor complexes.

Using an FK506 affinity column to identify mammalian immunosuppressant-binding proteins, we identified an immunophilin with an apparent M(r) approximately 55,000, which we have named FKBP52. We used chemically determined peptide sequence and a computerized algorithm to search GenPept, the translated GenBank data base, and identified two cDNAs likely to encode the murine FKBP52 homolog. We amplified a murine cDNA fragment, used it to select a human FKBP52 (hFKBP52) cDNA clone, and then used the clone to deduce the hFKBP52 sequence (calculated M(r) 51,810) and to express hFKBP52 in Escherichia coli. Recombinant hFKBP52 has peptidyl-prolyl cis-trans isomerase activity that is inhibited by FK506 and rapamycin and an FKBP12-like consensus sequence that probably defines the immunosuppressant-binding site. FKBP52 is apparently common to several vertebrate species and associates with the 90-kDa heat shock protein (hsp90) in untransformed mammalian steroid receptor complexes. The putative immunosuppressant-binding site is probably distinct from the hsp90-binding site, and we predict that FKBP52 has different structural domains to accommodate these functions. hFKBP52 contains 12 protein kinase phosphorylation-site motifs and a potential calmodulin-binding site, implying that posttranslational phosphorylation could generate multiple isoforms of the protein and that calmodulin and intracellular Ca2+ levels could affect FKBP52 function. FKBP52 transcripts are present in a variety of human tissues and could vary in abundance and/or stability.

Amino Acid Isomerases↗

PubMed2025

Detecting SARS-CoV-2 cryptic lineages using publicly available whole genome wastewater sequencing data.

Beginning in early 2021, unique and highly divergent lineages of SARS-CoV-2 were sporadically found in wastewater sewersheds using a sequencing strategy focused on amplifying the most rapidly evolving region of SARS-CoV-2, the receptor binding domain (RBD). Because these RBD sequences did not match known circulating strains and their source was not known, we termed them "cryptic lineages". To date, more than 20 cryptic lineages have been identified using the RBD-focused sequencing strategy. Here, we identified and characterized additional cryptic lineages from SARS-CoV-2 wastewater sequences submitted to NCBI's Sequence Read Archives (SRA). Wastewater sequence datasets were screened for individual sequence reads that contained combinations of mutations frequently found in cryptic lineages but not contemporary circulating lineages. Using this method, we identified 18 cryptic lineages that appeared in multiple (2-81) samples from the same sewershed, including 12 that were not previously reported. Partial consensus sequences were generated for each cryptic lineage by extracting and mapping sequences containing cryptic-specific mutations. Surprisingly, seven of the mutations that appeared convergently in cryptic lineages were reversions to sequences that were highly conserved in SARS-CoV-2-related enteric bat Sarbecoviruses. The apparent reversion to bat Sarbecovirus sequences is consistent with the notion that SARS-CoV-2 adaptation to replicate efficiently in respiratory tissues preceded the COVID-19 pandemic.

SARS-CoV-2↗

PubMed1992

Characterization of an intron 12 splice donor mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis, the most common lethal genetic disease in the white population, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Analysis of DNA from a pancreatic insufficient patient by chemical mismatch cleavage and subsequent DNA sequencing led to the identification of a potential splice mutation in the CFTR gene. A transition of the invariant guanosine to adenosine (1898 + 1G > A) was found at the splice donor site of intron 12. To determine the effect of this mutation on the patient's CFTR transcripts, RNA from the nasal epithelium was reverse transcribed and amplified by the polymerase chain reaction (RT-PCR). Direct sequencing of the PCR products revealed that the transcript from the chromosome with the 1898 + 1G > A mutation had skipped exon 12 entirely, resulting in a joining of exons 11 and 13. Deletion of exon 12 results in the removal of a highly conserved region which encodes the Walker B consensus sequence of the first nucleotide-binding fold of CFTR.

Adult↗

PubMed2026

Performance comparison of rapid and native barcoding methods for Oxford Nanopore sequencing of Poliovirus Viral Protein 1 (VP1) amplicons.

Accurate and timely sequencing of poliovirus is critical for global eradication efforts, particularly for molecular epidemiology based on the typing region of the genome, viral protein 1 (VP1). While Oxford Nanopore Technologies (ONT) sequencing has expanded capabilities for poliovirus surveillance, the relative performance of different ONT library preparation methods, including ligation-based (Native Barcoding) and transposase-based (Rapid Barcoding) approaches, has not been systematically evaluated. In this study, we compared rapid barcoding and native barcoding workflows for sequencing VP1 amplicons from 17 type 2 poliovirus-positive samples, each processed in triplicate. Native barcoding generated significantly more sequencing output, producing approximately 2.3-fold greater total read yield than rapid barcoding, and demonstrated higher run-to-run reproducibility (R2 = 0.979-0.998 vs. 0.847-0.929, respectively; p < 0.001). In addition, native barcoding generated 80% of the total yield achieved by rapid barcoding within approximately 7 h, whereas rapid barcoding required approximately 40 h to reach the same output. Despite these differences, both methods produced identical VP1 consensus sequences across all samples, with comparable read quality (median per-base Q-scores of approximately Q17-Q18). Rapid barcoding provided substantial practical advantages, reducing hands-on library preparation time (55 vs. 200 min) and per-sample cost ($12.82 vs. $16.54), while simplifying workflow and reducing technical complexity. These findings indicate that sequencing yield may not be a determinant of downstream analytical outcomes for poliovirus VP1 ONT sequencing. Rapid barcoding therefore represents a cost-effective and efficient approach for routine poliovirus surveillance, whereas native barcoding remains advantageous in applications requiring rapid data generation or maximal sequencing depth.

Poliovirus↗

PubMed2025

Long-Read Sequencing of the MUC1 VNTR: Genomic Variation, Mutational Landscape, and Its Impact on ADTKD Diagnosis and Progression.

BACKGROUND: ADTKD-MUC1 is caused by frameshift mutations in MUC1 gene that produce a frameshifted protein (MUC1fs) toxic to kidney cells. The gene's variable number of tandem repeats (VNTR), with high GC content, makes it largely inaccessible to standard sequencing. As a result, both the reference sequence and natural variation in this region remain poorly defined, complicating mutation detection and data interpretation. Standard methods also fail to pinpoint the exact VNTR unit affected, limiting insight into mutation mechanisms and genotype-phenotype correlations. METHODS: We employed Single Molecule, Real-Time (SMRT) sequencing and characterized the genomic sequence of MUC1 in 300 individuals including 279 individuals from 143 families suspected of having ADTKD-MUC1. We compared these results to those obtained using the CLIA-approved mass spectrometry-based probe extension (PE) assay, which specifically detect the most prevalent 59dupC mutation. We correlated the structural features of the MUC1 VNTR with the rate of kidney function decline in affected individuals. RESULTS: We identified MUC1 consensus sequences for 205 unique VNTR alleles, with 9 distinct types of frameshift mutations present on 52 distinct mutated VNTR alleles. MUC1 frameshift mutations were identified in 71 of 143 families (50%) with suspected ADTKD, comprising 135 genetically affected individuals (48%). The SMRT assay exhibited complete concordance and revealed that the PE assay is capable of detecting frameshift mutations in approximately 85% of affected families. The constellation of VNTR structures supports a genotype-progression model, in which fast progressors exhibit a significantly lower number of repeat units on the wild-type allele and a higher number of repeats on the mutation-bearing allele, including an increased number of frameshifted repeat units. CONCLUSIONS: SMRT sequencing outperforms current diagnostic methods for ADTKD-MUC1 and reveals the prognostic value of VNTR structures. Although their contribution to disease progression is modest (~6% variance explained), it remains biologically and clinically meaningful.

Autosomal Dominant Tubulointerstitial Kidney Disea↗

PubMed2025

Misdetection of frameshifts in SARS-CoV-2 genomes: need for additional harmonisation and efficient monitoring of data workflows.

Five years after the outbreak of the SARS-CoV-2 pandemic in 2020, diagnostic laboratories have moved from massive sequencing of thousands of samples to routine surveillance of SARS-CoV-2 cases, as with all other respiratory viruses. Surveillance remains of paramount importance to prevent a further SARS-CoV-2 surge, as the virus has been shown to mutate rapidly and can render available drugs and vaccines ineffective. During the pandemic, several bioinformatics pipelines and workflows have been developed to streamline analysis, shorten turnaround time and ensure reproducibility. As the number of samples decreases, laboratories are moving towards more flexible sequencing strategies and optimizing the cost per sample. However, workflow redesigns, even if individual steps have proven successful time and time again, can lead to challenges when changes in a bioinformatics pipeline are introduced (e.g. version updates, implementation of new features, etc.), a new combination of viral mutations emerge or a change in wet-lab procedures leads to unpredictable results. Here, we present a report of misidentified frameshift mutations in the consensus sequence of SARS-CoV-2, which led to an incorrect assumption of mutations in the spike and nucleocapsid viral proteins with the potential to affect PCR detection or even antigen testing. This investigation exemplifies the need for better awareness of the challenges that can occur even when using routinely applied protocols and analytical workflows and highlights the need for cooperation between experts of NGS, bioinformaticians and decision-makers towards more harmonized data workflows.

SARS-CoV-2↗

PubMed1992

Clustering of fibrillin (FBN1) missense mutations in Marfan syndrome patients at cysteine residues in EGF-like domains.

The Marfan syndrome is an autosomal dominant heritable disorder of connective tissue with prominent involvement of the ocular, skeletal, and cardiovascular systems. The gene on chromosome 15 encoding fibrillin (FBN1), a 350-kDa glycoprotein component of the extracellular microfibril, is the site of defect in most, if not all cases. Complementary DNA sequence reveals a gene composed largely of epidermal growth factor-like repeats, each containing six predictably spaced cysteine residues. To date, two FBN1 gene missense mutations have been reported. Here we describe the identification of three new missense mutations in the FBN1 gene in patients with the Marfan syndrome. All of the 5 characterized missense mutations occur within the epidermal growth factor-like repeats of the FBN1 gene. In addition, 4 of 5 involve the substitution of cysteine residues and 3 of 5 substitute the third cysteine in the epidermal growth factor-like motif consensus sequence. These data suggest that defined residues within EGF-like domains of FBN1 have particular significance and, when altered, play a pivotal role in expression of the Marfan phenotype.

Amino Acid Sequence↗

PubMed2025

Determining genotype and antimicrobial resistance of Salmonella Typhi in environmental samples by amplicon sequencing.

BACKGROUND: Estimates of the burden of typhoid fever due to Salmonella enterica serovar Typhi (S. Typhi) rely on data from clinical surveillance, which is rarely done in low income settings and is also limited by the poor sensitivity of the assays used and the reliance on health seeking by patients. Environmental surveillance for S. Typhi shed by symptomatic and asymptomatic individuals in wastewater offers a sensitive surveillance tool that could help to inform burden estimates. Sequencing S. Typhi direct from wastewater concentrates has the potential to identify circulating genotypes and associated antimicrobial resistance (AMR) genes, supporting public health interventions such as vaccination and antimicrobial usage. METHODOLOGY AND PRINCIPAL FINDINGS: We designed a multiplex targeted amplicon sequencing protocol for genotyping and determining AMR in S. Typhi from wastewater samples, targeting SNPs that identify genotypes of interest and both chromosomal and plasmid-borne AMR. PCR products were sequenced using the Oxford Nanopore Technologies (ONT) MinION, and genotypes and AMR identified using the GenoTyphi program. We tested this approach on samples from south India from both hospital outflow and wastewater collected from the community. All samples tested were suspected to be positive for S. Typhi following quantitative PCR for ttr, tviB, and staG gene targets. Out of 110 samples tested we were able to determine a genotype and/or AMR for 8. All samples that gave a genotype call suggested a genotype consistent with those found in clinical cases in India during the same time period and produced consensus sequences that clustered with S. Typhi when included in a phylogenetic tree. CONCLUSIONS: In this study, we provide proof of concept data for amplicon sequencing of S. Typhi in wastewater which with further optimisation could be used to complement clinical surveillance data or provide data on S. Typhi presence in the absence of clinical surveillance. This information can inform public health interventions, and the concept could be applied to other pathogens of interest for genotyping from environmental surveillance samples.

Salmonella typhi↗

PubMed1992

The angiotensin I-converting enzyme (kininase II): molecular organization and regulation of its expression in humans.

Protein sequencing and molecular cloning of human endothelial angiotensin I-converting enzyme (ACE; kininase II), have led to a description of the structure of the enzyme and to several questions concerning the intracellular maturation of ACE and the mechanisms of enzyme action. With the help of recombinant ACE expression in mammalian cells and site-directed mutagenesis, a model for the maturation of ACE in endothelial cells has been proposed. This model comprises transmembrane anchoring of the membrane-bound ACE near its carboxyterminal extremity, and post-translational cleavage of the anchor in the secreted form. The endothelial ACE displays a high degree of internal homology between two large peptidic domains that each bears a consensus sequence for zinc binding and therefore a putative active site. The testicular ACE, however, encoded from the same gene by a shorter mRNA, contains only the carboxyterminal half of endothelial ACE and therefore a single active site. Expression of ACE mutants with only one intact homologous domain, however, indicates that in endothelial ACE both domains are enzymatically active. Further characterization of these two active sites of endothelial ACE is in progress. In humans, population studies have indicated that the large interindividual variability in plasma ACE levels is partly genetically determined and under the influence of a major gene effect. This was later confirmed and extended by the observation of an insertion-deletion polymorphism of the ACE gene that is associated with the level of ACE in plasma. The clinical implications of these observations are discussed.

Cloning, Molecular↗

PubMed1992

Detection of sequence variants in the gene for human type II procollagen (COL2A1) by direct sequencing of polymerase chain reaction-amplified genomic DNA.

The direct sequencing of the human type II procollagen (COL2A1) gene from polymerase chain reaction (PCR)-amplified genomic DNA is described. Thirty-two regions of the COL2A1 gene were asymmetrically amplified with intron primers which were specifically chosen to amplify a region spanning 500 to 800 bp of sequence encoding one or more exons and their accompanying intervening sequences. Primers for dideoxynucleotide sequencing of the PCR products were then designed to provide complete exon sequence information and to insure that intron:exon splice junction sequence data would be obtained. Amplification and sequencing reactions were performed on an automated workstation to facilitate the handling of multiple DNA templates. The procedure allowed efficient sequencing of over 25,000 bp of each allele of the COL2A1 gene per diploid genome. We used this method for the comparative analyses of COL2A1 sequences in DNA isolated from the blood of 42 unrelated individuals and we identified 21 neutral sequence variants in the gene. The sequence variations were confirmed by independent assays, including restriction enzyme digestion. The sequence variants described here will be important for identifying haplotypes of the type II procollagen gene that will be useful in defining a genetic etiology for diseases of cartilaginous tissues.

Alleles↗

PubMed1992

Structural and functional studies on phi 29 DNA polymerase.

The Bacillus subtilis phage phi 29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr ("K-Y") motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of phi 29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of phi 29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the phi 29 DNA ends, by means of a "sliding-back" mechanism, could also contribute to increase the fidelity of phi 29 DNA replication.

Amino Acid Sequence↗

PubMed1992

Phosphorylatable and epitope-tagged human erythropoietins: utility and purification of native baculovirus-derived forms.

The hematopoietic glycopeptide erythropoietin (EPO) is a prime regulator of red cell production in mammals, yet the precise nature of its interaction with specific cell surface receptors is poorly understood. Towards defining domains of EPO that are involved in receptor activation, we have developed (i) conditions for the expression of recombinant human EPO (rhEPO) at high levels in SF9 cells using modified 2- and 5-liter stirred reactors, (ii) a two-step procedure for the purification of this EPO without denaturation, and (iii) forms of EPO tagged with either a hemagglutinin influenza virus epitope or a consensus sequence for in vitro phosphorylation. Compared to EPO expressed in mammalian cells, rhEPO from SF9 cells in N-glycosylated with simple, neutral oligosaccharides of limited size, yet as purified presently using nondenaturing procedures, possesses exceptionally high in vitro activity (> or = 500,000 U/mg). Thus, this form of EPO should prove advantageous for direct physicochemical analyses. Regarding epitope-tagged and phosphorylatable EPOs, forms modified at the amino terminus (Ala1) fully retained receptor binding and in vitro biological activities. In contrast, forms modified at the carboxy terminus (Cys161) were inactive and did not compete for receptor binding, indicating that integrity of this domain is essential for receptor recognition. For active amino-terminal-modified forms, the specific binding of MAb 12CA5 to native HAI-EPO and the utility of 32P-labeled PHOS-EPO in receptor binding and internalization studies also were demonstrated. The development of these unique, highly active forms of human EPO should advance studies of essential interactions between this cytokine and its cell surface receptor.

Amino Acid Sequence↗

PubMed2025

The Arabidopsis TIRome informs the design of artificial TIR (Toll/interleukin-1 receptor) domain proteins.

The TIR (Toll/interleukin-1 receptor) domain is an ancient protein module that functions in immune and cell death responses across the Tree of Life. TIR domains encoded by plants and prokaryotes function as enzymes to produce diverse small molecule immune signals. Plant genomes can encode hundreds of TIR-domain containing proteins-many of which confer important agricultural disease resistance as TIR-NLR (nucleotide-binding, leucine-rich repeat) immune receptors. Despite their importance, how natural variation influences TIR enzymatic output and immunity-associated cell death is largely unexplored. We assayed a complete collection of the TIR domains of Arabidopsis thaliana Col-0 (the "AtTIRome") to explore variation in TIR metabolite production and cell death signaling. Roughly half of the AtTIRome triggered cell death in transient assays. Artificial TIR proteins designed based on consensus sequences of the AtTIRome's cell death phenotypic classes revealed polymorphisms controlling variation in TIR cell death elicitation and metabolite production. Structure-function analyses of artificial TIRs revealed that natural variation in the "BB-loop", a flexible region overlying the catalytic pocket, determines differences in function across Arabidopsis TIR-containing proteins. We further demonstrate that artificial TIRs are functional on an NLR chassis and that BB-loop variation can tune the activity of a natural TIR-NLR protein. These findings shed light on the diversity of TIR outputs and reveal methods to design and engineer TIR-based immune receptors.

Arabidopsis↗

PubMed1992

Cloned, expressed rat cerebellar nitric oxide synthase contains stoichiometric amounts of heme, which binds carbon monoxide.

The endogenous formation of nitric oxide (NO) has become an area of intense interest as evidence for its biological functions has been obtained in three distinct tissues: circulating macrophages, in which it exerts cytotoxic effects; blood vessels, in which it has been identified as endothelium-derived relaxing factor; and neuronal cells, in which it functions as a neurotransmitter. The formation of NO in brain extracts has been shown to be catalyzed by an enzyme, termed NO synthase, which generates the NO responsible for stimulation of cGMP formation, the highest levels of which occur in the cerebellum. NO synthase catalyzes the formation of citrulline from arginine with the coincident production of NO and has been shown to be a flavoprotein, containing 1 mol each of FAD and FMN, tetrahydrobiopterin, and iron. It is also reported to contain an alpha-helical, calmodulin-binding consensus sequence consistent with its stimulation by calmodulin in the presence of Ca2+. The formation of NO requires incorporation of one of the atoms of molecular oxygen into one of the guanidinium nitrogen atoms of arginine with the coincident formation of citrulline. This communication reports that rat cerebellar NO synthase, cloned and stably expressed in human kidney 293 cells, contains heme in amounts stoichiometric with the flavins FAD and FMN as evidenced by the appearance of a pyridine hemochrome and a reduced CO difference spectrum with an absorbance maximum at approximately 445 nm. The finding of a CO-binding heme moiety explains the presence of iron in the enzyme and suggests a role for prosthetic heme as an oxygenase reaction center. This report also presents evidence for incorporation of delta-[14C]aminolevulinate specifically into immunoprecipitable NO synthase in stably transfected human kidney 293 cells but not in nontransfected cells. Simultaneously, K. A. White and M. A. Marletta [(1992) Biochemistry 31, 6627-6631] have demonstrated a CO-binding heme prosthetic group in purified murine macrophage NO synthase and have suggested the identity of these reaction centers in both the constitutive (cerebellar) and inducible (macrophage) forms of NO synthase.

Amino Acid Oxidoreductases↗