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At least 19 recordsLinked to original sources

PubMed2026

Evaluating detection of Histophilus somni immunoglobulin-binding protein A DR2 Fic: A species-specific gene target for recombinase polymerase amplification relative to long-read sequencing of respiratory samples from feedlot calves.

Histophilosis is an important cause of morbidity and mortality as well as antimicrobial use in feedlot cattle across North America. Detection of Histophilus somni by culture is challenging, and there is no standardized tool for distinguishing isolates that carry virulence factors most likely to contribute to disease. The DR2 repeat of H. somni-associated virulence factor 'immunoglobulin-binding protein A' (ibpA DR2) harbors a Fic domain that mediates host cell cytotoxicity and is essential for histophilosis. For rapid detection of ibpA DR2 in extracted DNA, we developed a real-time recombinase polymerase amplification (RPA) assay with a runtime of 24 min at 39 °C. DNA from H. somni-RPA-positive respiratory swabs (n = 73) was screened for ibpA DR2 using the novel RPA assay and long-read metagenomic sequencing, as well as nanopore whole-genome sequencing (WGS) of H. somni isolated from the same samples. IbpA DR2 was identified in 71% and 70% of tested samples using RPA and WGS, respectively, and in ≤41% of samples using metagenomic sequencing. The likelihood of detection by RPA did not differ (OR 1.1, 95% CI (0.42, 2.9), P > 0.99) from WGS; however, agreement between these assays was only fair (κ = 0.31). Conversely, RPA (OR 3.4, 95% CI (1.6, 8.2)) and WGS (OR 8.0, 95% CI (2.4, 42)) were more likely (P < 0.001) to detect ibpA DR2 than metagenomic sequencing, likely reflecting limited coverage of H. somni by metagenomics. This study demonstrated that RPA and long-read WGS detected ibpA DR2 with similar frequencies in extracted DNA and H. somni isolates, respectively. Further testing of non-target isolates confirmed the analytical specificity of ibpA DR2 to H. somni. Further investigation of the diagnostic validity for RPA-based ibpA DR2 detection is required in a larger cohort of field samples, as a rapid screening tool for H. somni most likely to contribute to disease.

Animals↗

PubMed2026

Molecular diagnostics and integrated management challenges of tobacco streak virus: Current status and future perspectives.

Tobacco streak virus (TSV) is an economically important viral pathogen causing severe yield and quality losses in several agricultural, horticultural and medicinal crops worldwide. Its complex epidemiology involving sap transmission, infected pollen and pollen-feeding thrips, together with symptom similarity to other necrosis-inducing pathogens, frequently results in misdiagnosis and delayed disease management. This review critically evaluates recent advances in TSV diagnostics and integrated disease management strategies. Particular emphasis is placed on the transition from conventional biological and serological assays to advanced molecular diagnostics including reverse transcription polymerase chain reaction (RT-PCR), quantitative real-time PCR, multiplex PCR and emerging isothermal amplification technologies such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The review also highlights emerging innovations including CRISPR/Cas-based diagnostics in addition, integrated management approaches involving phytosanitation, weed reservoir management, vector ecology-based, host resistance breeding, RNA interference (RNAi) and genome editing technologies are critically analysed. Major challenges including inadequate field validation, limited multiplex capability, poor assay standardization and scarcity of resistant cultivars are discussed. Future objectives to develop quick, field-adaptable and durable TSV detection and management methods are additionally discussed.

CRISPR/Cas diagnostics↗

PubMed2006

DNA detection using recombination proteins.

DNA amplification is essential to most nucleic acid testing strategies, but established techniques require sophisticated equipment or complex experimental procedures, and their uptake outside specialised laboratories has been limited. Our novel approach, recombinase polymerase amplification (RPA), couples isothermal recombinase-driven primer targeting of template material with strand-displacement DNA synthesis. It achieves exponential amplification with no need for pretreatment of sample DNA. Reactions are sensitive, specific, and rapid and operate at constant low temperature. We have also developed a probe-based detection system. Key aspects of the combined RPA amplification/detection process are illustrated by a test for the pathogen methicillin-resistant Staphylococcus aureus. The technology proves to be sensitive to fewer than ten copies of genomic DNA. Furthermore, products can be detected in a simple sandwich assay, thereby establishing an instrument-free DNA testing system. This unique combination of properties is a significant advance in the development of portable and widely accessible nucleic acid-based tests.

Bacillus subtilis↗

PubMed2026

An RPA-assisted homogeneous electrochemical DNA sensor for on-site eDNA detection toward early warning of crown-of-thorns starfish outbreaks.

Crown-of-thorns starfish (COTS) outbreaks seriously threaten coral reef ecosystems, while conventional monitoring approaches are time-consuming and often lack sufficient sensitivity for early warning. Existing electrochemical DNA sensors usually require complex electrode-surface immobilization procedures, which can lead to uneven probe distribution, significant steric hindrance, and poor stability. Meanwhile, the low concentration of environmental DNA (eDNA) in marine environments further complicates detection. To overcome these challenges, this study developed a homogeneous electrochemical DNA sensor assisted by recombinase polymerase amplification (RPA) for COTS eDNA detection. Target DNA was first amplified by RPA, and the amplification products were then hybridized in solution with capture probe (CP)-modified magnetic beads (MB) and biotin-labeled signal probe (SP) to form sandwich-structured MB complexes. These complexes were subsequently magnetically enriched and immobilized on the electrode surface for electrochemical signal readout. Under optimized conditions, the sensor displayed a linear response to COTS genomic DNA from 3.77 fg/μL to 1 ng/μL, with an LOD of 2.02 fg/μL and an LOQ of 3.77 fg/μL. The sensor was applied to Xisha Islands samples, and the results agreed with droplet digital PCR (ddPCR) (P > 0.05), demonstrating its potential for sensitive and reliable on-site COTS eDNA detection.

Animals↗

PubMed2026

A CRISPR-Based Rapid Detection Assay for Crayfish Plague (Aphanomyces astaci) From Environmental Samples.

Crayfish plague, caused by Aphanomyces astaci (Aa), is an infectious disease invasive in Europe, where its rapid spread has resulted in sharp declines of native crayfish species. Monitoring currently relies on a highly sensitive, but costly and time-consuming qPCR approach. Here, we designed a simplified, rapid and cost-efficient molecular assay for on-site detection of Aa. The novel rapid assay employs a combination of isothermal recombinase polymerase amplification and CRISPR-Cas12a-based detection that can be coupled with fluorescence or lateral flow visualisation. We demonstrate that the novel assay can detect A. astaci from tissue and environmental DNA with higher sensitivity than the available qPCR assay and readily distinguishes Aa from its non-pathogenic sister taxon A. fennicus. We tested two genomic marker sites for Aa that discriminate closely related oomycetes and incorporate field-deployable lateral flow and fluorescence readouts. Our work will make crayfish plague monitoring broadly accessible to practitioners and non-academic stakeholders as a tool to curb further Aa-driven loss of Europe's imperilled freshwater crustaceans and strengthen preparedness against future incursions of the pathogen in other regions.

Cas12a↗

PubMed2024

Phase Transition of Wax Enabling CRISPR Diagnostics for Automatic At-Home Testing of Multiple Sexually Transmitted Infection Pathogens.

Sexually transmitted infections (STIs) significantly impact women's reproductive health. Rapid, sensitive, and affordable detection of these pathogens is essential, especially for home-based self-testing, which is crucial for individuals who prioritize privacy or live in areas with limited access to healthcare services. Herein, an automated diagnostic system called Wax-CRISPR has been designed specifically for at-home testing of multiple STIs. This system employs a unique strategy by using the solid-to-liquid phase transition of wax to sequentially isolate and mix recombinase polymerase amplification (RPA) and CRISPR assays in a microfluidic chip. By incorporating a home-built controlling system, Wax-CRISPR achieves true one-pot multiplexed detection. The system can simultaneously detect six common critical gynecological pathogens (CT, MG, UU, NG, HPV 16, and HPV 18) within 30 min, with a detection limit reaching 10-18 M. Clinical evaluation demonstrates that the system achieves a sensitivity of 96.8% and a specificity of 97.3% across 100 clinical samples. Importantly, eight randomly recruited untrained operators performe a double-blinded test and successfully identified the STI targets in 33 clinical samples. This wax-transition-based one-pot CRISPR assay offers advantages such as low-cost, high-stability, and user-friendliness, making it a useful platform for at-home or field-based testing of multiple pathogen infections.

Sexually Transmitted Diseases↗

PubMed2025

Application of engineered CRISPR/Cas12a variants with altered protospacer adjacent motif specificities for the detection of isoniazid resistance mutations in Mycobacterium tuberculosis.

UNLABELLED: Drug-resistant tuberculosis (TB) is a major global public health concern. Although isoniazid is currently considered one of the most effective first-line drugs for TB treatment, its efficacy is limited by the emergence of resistance. Therefore, it is imperative to develop new methods for detecting drug-resistant TB. In this study, we developed a nucleic acid detection system based on the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a_RR protein. The system combines recombinase polymerase amplification with an engineered CRISPR/Cas12a_RR protein to enable rapid and specific detection of the katG G944C mutation in isoniazid-resistant Mycobacterium tuberculosis (Mtb). It could detect the target DNA at concentrations as low as 1% in a mixed sample. Compared with TaqMan quantitative polymerase chain reaction and DNA sequencing, the CRISPR/Cas12a_RR system demonstrated superior detection performance in terms of sensitivity, specificity, and cost-effectiveness. Furthermore, it effectively differentiated between drug-resistant Mtb strains from wild-type Mtb strains in clinically isolated samples, with the entire detection process completed in 60 min. In conclusion, the CRISPR/Cas12a_RR detection system offers a novel, rapid, simple, sensitive, and specific approach for identifying isoniazid-resistant Mtb, with significant potential for clinical application, particularly in resource-limited settings. IMPORTANCE: This study presents a novel method for detecting isoniazid-resistant Mycobacterium tuberculosis (Mtb) using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a mutants, offering rapid detection, cost-effectiveness, and high specificity, and thereby providing a promising new avenue for detecting isoniazid-resistant Mtb.

Isoniazid↗

PubMed2025

Whole genome study and construction of SHERLOCK detection method for endemic strains of Burkholderia pseudomallei in Hainan based on third-generation sequencing.

UNLABELLED: Burkholderia pseudomallei (Bp) is a gram-negative bacterium found in soil and surface water. It is also the pathogen that causes melioidosis disease in humans and animals. This study aimed to obtain the whole genome sequence of the endemic strain of Bp in Hainan, using third-generation sequencing (TGS) technology, and elucidate the genome structure, function, and genetic evolution. Additionally, the study aimed to achieve rapid and specific identification of these endemic strains using specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) detection technology, providing a new strategy for the early diagnosis of melioidosis. Utilizing the PacBio platform for TGS technology, we completed whole genome sequencing of 16 Bp strains from Hainan. High-precision and complete genome sequences were obtained through quality control and genome assembly of the sequencing data. Additionally, we established a nucleic acid detection technology platform based on SHERLOCK, which could be completed from nucleic acid extraction to result reading within 1-2 hours, demonstrating good sensitivity and specificity (both are 100%). The lateral chromatography strip method does not require special equipment and holds promise as an immediate screening method for the early diagnosis of melioidosis. IMPORTANCE: Melioidosis is a highly pathogenic infectious disease caused by a gram-negative bacterium of Burkholderia pseudomallei (Bp). The traditional gold standard for diagnosing melioidosis is still isolation and culture from clinical samples. Although this method has high specificity, it has low sensitivity and is time-consuming, which often leads to misdiagnosis or missed diagnosis of melioidosis, affecting subsequent treatment. In this study, recombinase polymerase amplification technology and clustered regularly interspaced short palindromic repeats/Cas13a technology were combined to establish the Specific High-sensitivity Enzymatic Reporter Unlocking detection technology, which can achieve rapid and accurate identification of Bp, providing a new method for the early diagnosis of melioidosis.

Burkholderia pseudomallei↗

PubMed2026

Rapid CRISPR-based bovine embryo sexing to streamline genotype-informed cattle breeding.

Cattle in vitro fertilisation and embryo transfer programmes increasingly rely on embryo-level selection to accelerate genetic gain, but current sexing and genotyping workflows can be costly, slow and logistically demanding. This study developed an efficient, low-resource workflow for bovine embryo sexing that combines whole genome amplification (WGA) with recombinase polymerase amplification-CRISPR-Cas12a (RPA-Cas12a). It also assessed whether the same WGA biopsy products could be used for downstream single nucleotide polymorphism (SNP) microarray genotyping. A one-tube RPA-Cas12a assay targeting the bovine Y-chromosome S4 repeat was developed for fluorescence and lateral flow assay (LFA) readouts. Analytical sensitivity was assessed using serially diluted bovine genomic DNA (gDNA), and breed robustness was tested using male and female gDNA from five major beef breeds and Holstein cattle. The workflow was then applied to WGA products from 22 bovine blastocyst biopsies, with sex calls validated against an established real-time PCR melt curve assay and 100K SNP microarray genotyping. The assay detected male bovine gDNA down to 100 pg using both fluorescence and LFA readouts, with no signal from female gDNA. Male-specific detection was consistent across all breeds tested. All WGA-RPA-Cas12a sex calls from blastocyst biopsies were concordant with real-time PCR and SNP microarray sex calls, and WGA biopsy products produced genome-wide SNP call rates above 85%. This workflow provides a practical approach for rapid bovine embryo sex triage and could reduce unnecessary cryopreservation and genotyping while improving the efficiency of genotype-informed cattle breeding programmes.

Bovine embryo↗

PubMed2002

DNA sequence analysis of interlocus recombination between the human T-cell receptor gamma variable (GV) and beta diversity-joining (BD/BJ) sequences on chromosome 7 (inversion 7).

V(D)J recombinase-mediated recombination between the T-cell receptor (TCR) gamma variable (GV) genes at chromosome 7p15 and the TCR beta joining (BJ) genes at 7q35 leads to the formation of a hybrid TCR gene. These TCR gamma/beta interlocus rearrangements occur at classic V(D)J recombination signal sequences (RSS) and, because the loci are in an inverted orientation, result in inversion events that are detectable in the chromosome structure as inv(7)(p15;q35). Similar rearrangements involving oncogenes and either TCR or immunoglobulin genes mediated by the V(D)J recombinase are found in lymphoid malignancies. Oligonucleotide primers that allow polymerase chain reaction (PCR) amplification across the inv(7) genomic recombination junction sequence have been described. Southern blot analysis has been primarily used to confirm the GV/BJ hybrid nature of the product, with limited information on the DNA sequence of these recombinations. We have modified this PCR method using total genomic DNA from the mononuclear cells in peripheral blood samples to increase specificity and to allow direct sequencing of the translocation junction that results from the recombination between the GV1 and BJ1 families of TCR genes in 25 examples from 11 individuals (three adults, one child, six newborns, and one ataxia telangiectasia (AT) patient). We focused on samples from newborns based on previous studies indicating that the predominant hypoxanthine-guanine phosphoribosyl transferase (HPRT) mutations in newborns are V(D)J recombinase-mediated deletion events and that the frequency of these mutations decreases with increasing age. Although the dilution series-based PCR assay utilized does not yield sharply defined quantitative endpoints, results of this study strongly suggest that inv(7) recombinations in newborns occur at equal or lower frequencies than those seen in adults. Consistent with the PCR primer pairs, all sequenced products contain a GV1 and a BJ1 segment and most also contain a BD1 segment. GV1s2 and 1s4 were the most frequently found GV1 genes (8 and 9 examples, respectively) and BJ1s5 and 1s6 were the most frequently found BJ1 genes (9 and 10 examples, respectively). These results demonstrate the effectiveness of this methodology for assessing GV/BJ interlocus rearrangements mediated by V(D)J recombinase.

Adult↗

PubMed1991

Amplification of snap-back DNA synthesis reactions by the uvsX recombinase of bacteriophage T4.

The uvsX protein of bacteriophage T4 is a recA-type recombinase. This protein has previously been shown to help initiate DNA replication on a double-stranded DNA template by catalyzing synapsis between the template and a homologous DNA single strand that serves as primer. Here, we demonstrate that this replication-initiating activity of the uvsX protein greatly amplifies the snap-back (hairpin-primed) DNA synthesis that is catalyzed by the T4 DNA polymerase holoenzyme on linear, single-stranded DNA templates. Amplification requires the presence of uvsX protein, the DNA polymerase holoenzyme, T4 gene 32 protein, and a T4 DNA helicase, in a reaction that is modulated by the T4 uvsY protein (an accessory protein to the uvsX recombinase). The reaction products consist primarily of large networks of double-stranded and single-stranded DNA. With alkali or heat treatment, these networks resolve into dimer-length single-stranded DNA chains that renature instantaneously to reform a monomer-length double helix. A simple model can explain this uvsX protein-dependent amplification of snap-back DNA synthesis; the mechanism proposed makes several predictions that are confirmed by our experiments.

Adenosine Triphosphatases↗

PubMed1992

Evidence for failure of V(D)J recombination in bone marrow pre-B cells from X-linked agammaglobulinemia.

X-linked agammaglobulinemia (XLA) results from a failure of B lymphoid development. We have previously examined pre-B cell hybrids from three patients with XLA and found them to be limited to production of a novel germ line transcript of the Ig H chain locus composed of a leader sequence (LS) spliced to the constant region of mu chain (C mu) as mRNA and polypeptide. These transcripts result from transcriptional activation of the germ line heavy chain locus from an LS exon upstream of the embryonic JH locus. Germ line LS-C mu transcripts are produced by pre-B cells from normal bone marrow and fetal liver, indicating that they are products of normal pre-B cell development, as part of the process of transcriptional activation to provide access for the recombinase. Bone marrow from three patients with XLA has been examined directly by polymerase chain reaction amplification to determine whether the exclusive production of LS-C mu by XLA pre-B cell hybrids is representative of XLA pre-B cells. I report that LS-C mu is the predominant Ig molecule produced by XLA pre-B cells, with limited production of the D mu product of DJH intermediate stage of V(D)J recombination. Mature VHDJH recombinations were not detected with a variety of primers that amplify VH sequences. I conclude that XLA is associated with a limitation in V(D)J recombination that may cause the failure of pre-B cell development.

Agammaglobulinemia↗

PubMed2002

Factors affecting PCR-mediated recombination.

In the past decade, polymerase chain reaction (PCR) has become an important tool for the identification of previously unknown microorganisms and the analysis of environmental microbial diversity. Several studies published during recent years, however, have demonstrated that products obtained after PCR using Taq or Vent DNA polymerases will contain hybrid molecules when several homologous target sequences such as multigene families, alleles, or RNA viruses are co-amplified. In this report, we examined the recombination frequency and the extent of template switching during PCR using Taq, Pfu and RTth/Vent DNA polymerases. As a test system we constructed a series of plasmids carrying between one and three frame shift mutations in the gene coding for the protease subtilisin or deletions of approximately 100 bp in the lacZ alpha. Highest recombination frequencies were observed when these mutants were co-amplified with Taq followed by RTth/Vent DNA polymerases. Pfu DNA polymerase displayed no discernable recombination activity under normal PCR conditions. Data also suggest that in vivo repair of heteroduplex DNA molecules in Escherichia coli by a RecA-independent mechanism, perhaps the mismatch repair, results in the formation of chimeric molecules. Using Bacillus subtilis as the host, however, can significantly diminish non-PCR RecA-independent in vivo recombination, owing to the fact that transforming DNA molecules enter B. subtilis as single strands. Combined, these results suggest that using Pfu DNA polymerase for amplification and B. subtilis as the host for transformation may significantly reduce chimera formation.

Bacillus subtilis↗

PubMed2006

Genomic and functional analysis of the IncP-9 naphthalene-catabolic plasmid NAH7 and its transposon Tn4655 suggests catabolic gene spread by a tyrosine recombinase.

The naphthalene-catabolic (nah) genes on the incompatibility group P-9 (IncP-9) self-transmissible plasmid NAH7 from Pseudomonas putida G7 are some of the most extensively characterized genetic determinants for bacterial aerobic catabolism of aromatic hydrocarbons. In contrast to the detailed studies of its catabolic cascade and enzymatic functions, the biological characteristics of plasmid NAH7 have remained unclear. Our sequence determination in this study together with the previously deposited sequences revealed the entire structure of NAH7 (82,232 bp). Comparison of NAH7 with two other completely sequenced IncP-9 catabolic plasmids, pDTG1 and pWW0, revealed that the three plasmids share very high nucleotide similarities in a 39-kb region encoding the basic plasmid functions (the IncP-9 backbone). The backbone of NAH7 is phylogenetically more related to that of pDTG1 than that of pWW0. These three plasmids carry their catabolic gene clusters at different positions on the IncP-9 backbone. All of the NAH7-specified nah genes are located on a class II transposon, Tn4655. Our analysis of the Tn4655-encoded site-specific recombination system revealed that (i) a novel tyrosine recombinase, TnpI, catalyzed both the intra- and intermolecular recombination between two copies of the attI site, (ii) the functional attI site was located within a 119-bp segment, and (iii) the site-specific strand exchange occurred within a 30-bp segment in the 41-bp CORE site. Our results and the sequence data of other naphthalene-catabolic plasmids, pDTG1 and pND6-1, suggest a potential role of the TnpI-attI recombination system in the establishment of these catabolic plasmids.

Bacterial Proteins↗

PubMed2001

Ectopic transposition of a group II intron in natural bacterial populations.

Self-splicing group II introns are thought to be the evolutionary progenitors of eukaryotic spliceosomal introns. The invasion of novel (ectopic) sites by group II introns is considered to be a key mechanism by which spliceosomal introns may have become widely dispersed. However, the dynamics of these events in populations are unknown. In bacteria, only two group II introns have been shown to splice and to be mobile in vivo. One of these introns, RmInt1 from Sinorhizobium meliloti, which encodes a protein with no endonuclease domain, has been shown to invade the ectopic oxi1 site independently of recombinase. In this study, we analysed ectopic transposition of the RmInt1 intron in a natural population of S. meliloti. We characterized S. meliloti isolates by polymerase chain reaction amplification of a gene, dapB, which is found only on the pRmeGR4b plasmid diagnostic of GR4-type strains. The diversity within this specific field population of bacteria was analysed by restriction fragment length polymorphism using ISRm2011-2 (homing site of RmInt1) and RmInt1 as probes. We found that ectopic transposition of RmInt1 to the oxi1 site occurred in this natural bacterial population. This ectopic transposition was also the most frequent genetic event observed. This work provides further evidence that the ectopic transposition of group II introns is an important mechanism for their spread in natural bacterial populations.

DNA Transposable Elements↗

PubMed1999

Inducible amplification of gene copy number and heterologous protein production in the yeast Kluyveromyces lactis.

Heterologous protein production can be doubled by increasing the copy number of the corresponding heterologous gene. We constructed a host-vector system in the yeast Kluyveromyces lactis that was able to induce copy number amplification of pKD1 plasmid-based vectors upon expression of an integrated copy of the plasmid recombinase gene. We increased the production and secretion of two heterologous proteins, glucoamylase from the yeast Arxula adeninivorans and mammalian interleukin-1beta, following gene dosage amplification when the heterologous genes were carried by pKD1-based vectors. The choice of the promoters for expression of the integrated recombinase gene and of the episomal heterologous genes are critical for the mitotic stability of the host-vector system.

Animals↗

PubMed1992

Convergent and overlapping transcripts of the Chlamydia trachomatis 7.5-kb plasmid.

Transcription of the 7.5-kb cryptic plasmid of Chlamydia trachomatis serovar L2 was investigated. Faint, diffuse transcripts of about 1.6 and 2.2 kb and intense short transcripts of about 250 and 430 bases were identified by Northern blot analysis. The short transcripts were found to have a common 5' end corresponding to bp 501 relative to the unique BamHI site of the plasmid and to terminate at different downstream sites. Putative promoter sequences of TTGCCA and TATATT, which closely resemble the consensus recognition site of Escherichia coli sigma 70, were identified at the -35 and -10 positions upstream from the 5' end of the short transcripts made in chlamydia. Transcripts of similar sizes were also expressed from this promoter in E. coli harboring a recombinant plasmid encoding the short transcripts. The short transcripts encode a common open reading frame (ORF) of 34 codons; however, a strong ribosome binding site was not found in the vicinity of the initiator codon, and it is not known whether the transcripts are translated in vivo. A large ORF of 330 codons, which has been shown to encode a hypothetical protein containing conserved domains of recombinase-like proteins, is antisense to the short transcripts. Transcripts encoding the large ORF could not be detected directly by Northern blot or primer extension analysis. However, transcripts were detected by polymerase chain reaction amplification of the large ORF cDNA and when Southern blots of single-stranded antisense DNA for the large ORF were probed with radiolabeled RNA synthesized by host-free chlamydial reticulate bodies. Thus, both strands of the chlamydial plasmid are transcribed in the region encoding the short transcripts. We propose that the short transcripts play a regulatory role as antisense RNAs.

Amino Acid Sequence↗

PubMed1993

V(D)J recombination generates a high frequency of nonstandard TCR D delta-associated rearrangements in thymocytes.

The standard products of V(D)J recombination are coding junctions, which encode Ag receptor polypeptides, and their commonly excised reciprocal products, signal junctions. Additional nonstandard products also have been detected, mostly in artificial recombination substrate studies. The occurrence of nonstandard products, including pseudonormal, hybrid, and open/shut junctions, indicates significant indeterminacy of the V(D)J recombinase. However, the incidence of nonstandard products for endogenous Ag receptor genes in vivo has not been specifically addressed. The data presented here show that for the TCR-delta locus, D element-associated recombination in mouse thymocytes results in a high incidence of nonstandard recombination products. D delta 1-D delta 2 rearrangements, both chromosome retained and excised episomal products, were studied by polymerase chain reaction amplification, cloning, and sequence analysis. The proximity of D delta 1 and D delta 2 elements, and the fact that both are flanked by 5' and 3' recombination signal sequences with 12-bp and 23-bp spacers, respectively, results in frequent pseudonormal joining. The resulting products are signal junctions retained on the chromosome. Excised episomal products include coding junctions, hybrid junctions formed in apparent violation of the 12/23 spacer rule, and standard signal junctions; some signal junctions show evidence of imprecise cleavage. Evidence for open/shut and/or oligonucleotide capture events was also seen. Similar rearrangements were detectable in thymocytes of mutant scid mice. These findings indicate a high degree of indeterminacy of V(D)J recombinase-mediated D delta 1-D delta 2 rearrangement in both wild-type and scid thymocytes. This indeterminacy affects the productive potential of TCR-delta loci.

Animals↗