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Global warming may impact biodiversity by disrupting biological interactions, including long-term insect-microbe mutualistic associations. Symbiont-mediated insect tolerance to high temperatures is an ecologically important trait that significantly influences an insect's life history. Disruption of microbial symbionts that are required by insects would substantially impact their pest status. Diaphorina citri , a worldwide citrus pest, is associated with the mutualistic symbionts Candidatus Carsonella ruddii and Candidatus Profftella armatura. Wolbachia is also associated with D. citri , but its contribution to the host is unknown. Symbiont density is dependent on a range of factors, including the thermosensitivity of the host and/or symbiont to heat stress. Here, we predicted that short-term heat stress of D. citri would disrupt the host-symbiont phenological synchrony and differentially affect the growth and density of symbionts. We investigated the effects of exposing D. citri eggs to different temperatures for different periods of time on the growth dynamics of symbionts during the nymphal development of D. citri (first instar to fifth instar) by real-time polymerase chain reaction. Symbiont densities were assessed as the number of gene copies, using specific molecular markers: 16S rRNA for Carsonella and Profftella and ftsZ for Wolbachia. Statistical modeling of the copy numbers of symbionts revealed differences in their growth patterns, particularly in the early instars of heat-shocked insects. Wolbachia was the only symbiont to benefit from heat-shock treatment. Although the symbionts responded differently to heat stress, the lack of differences in symbiont densities between treated and control late nymphs suggests the existence of an adaptive genetic process to restore phenological synchrony during the development of immatures in preparation for adult life. Our findings contribute to the understanding of the potential deleterious effects of high temperatures on host-symbiont interactions. Our data also suggest that the effects of host exposure to high temperatures in symbiont growth are highly variable and dependent on the interactions among members of the community of symbionts harbored by a host. Such dependence points to unpredictable consequences for agroecosystems worldwide due to climate change-related effects on the ecological traits of symbiont-dependent insect pests. [ABSTRACT FROM AUTHOR]

Abstract: Naturally selected nickel (Ni) tolerance in Alyssum lesbiacum has been proposed to involve constitutively high levels of endogenous free histidine. Transgenic Arabidopsis thaliana expressing a Salmonella typhimurium ATP phosphoribosyl transferase enzyme (StHisG) resistant to feedback inhibition by histidine contained approximately 2-fold higher histidine concentrations than wild type plants. Under exposure to a toxic Ni concentration, biomass production in StHisG expressing lines was between 14- and 40-fold higher than in wild-type plants. This suggested that enhancing the first step in the histidine biosynthesis pathway is sufficient to increase the endogenous free histidine pool and Ni tolerance in A. thaliana. [Copyright &y& Elsevier]

Plasmodium knowlesi variant antigens are expressed at the surface of infected erythrocytes and are encoded by the Schizont Infected Cell Agglutination variant antigen (SICAvar) multigene family. The 3′ region of the SICAvar gene locus encoding the 205kDa variant antigen expressed in the Pk1(B+)1+ parasites was found to be altered compared to the Pk1(A+) parental clone. Here we report that this alteration is the result of a DNA rearrangement and that the original and altered 205 SICAvar alleles appear to encode bona fide variant antigens. Importantly, 205A and 205B SICAvar RNA sequences are detectable in similar apparent quantities as determined by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) amplification experiments. However, expression of the 205kDa SICA protein at the surface of the infected erythrocyte is not characteristic of the Pk1(A+) parasites and the 205 SICAvar transcript has not been detected in Pk1(A+) parasites by northern blot analysis. Furthermore, we report that many distinct SICAvar transcripts were detected in P. knowlesi Pk1(B+)1+ cDNA library hybridization screens. Of special interest, in light of these data, distinctive differences at the 3′ end of the 205A and 205B alleles are observed, which may be of functional importance. An analysis of the 3′ untranslated region (UTR) of SICAvar genes in more than 100 sequences revealed a surprising common sequence pattern characterized by blocks of imperfect, GT-rich, heptad repeated motifs (Block I), followed by A and T rich homopolymers (Block II) and in a large number of genes, GC-rich segments (Block III). We show that this region undergoes extensive recombination and that the preferential stability of the 205 SICAvar transcript in Pk1(B+)1+ parasites may be associated with the presence of its specific Block III sequences. We speculate that the conserved yet polymorphic SICAvar 3′UTR sequences, and comparable regions in P. falciparum var genes, function in the stage-specific and developmentally regulated post-transcriptional gene silencing (PTGS) of variant antigen transcripts. [Copyright &y& Elsevier]

The most abundant organic compound produced by plants is cellulose; however, it has long been accepted that most animals do not produce endogenous enzymes required for its degradation, but rely instead on symbiotic relationships with microbes that produce the necessary enzymes. Here, we present the genomic organisation of an endogenous glycosyl hydrolase family (GHF) 9 gene in redclaw crayfish (Cherax quadricarinatus), consolidated from a cDNA sequence determined by Byrne et al. [Gene 239 (1999) 317–324.]. Comparison with several other invertebrate GHF9 genes reveals the conservation of both intron position/phase and splice sequence, which adds support to an argument for an ancestral animal cellulase gene. Furthermore, two introns in plant GHF9 genes are also identical in position, implying a more ancient origin for this class of animal cellulase.Protein purification from redclaw gastric fluid via fast performance liquid chromatography (FPLC) indicated the presence of two endoglucanase enzymes. The molecular weights of these components were determined by matrix-assisted laser desorption/ionisation—time-of-flight (MALDI-TOF) to be 47,887 Da (Cel1) and 50,295 Da (Cel2). Cel1 is possibly the functional product of the described cellulase gene, with N-terminal amino acid residues identical to the translated amino acid sequence from the corresponding gene region. Cel2 was identical to Cel1 for 7 of 11 N-terminal residues and likely to be the product of a paralogous endoglucanase gene. These results suggest that redclaw crayfish possess at least one and possibly two functional, endoglucanase enzymes, although further work is required to confirm their origin and attributes. [Copyright &y& Elsevier]

Because of its short life span in blood, its low RNA content and its highly condensed nucleus, the granulocyte was initially considered as a terminally differentiated cell unable to express novel genes. However, mature granulocytes still contain a variety of mRNAs and may respond to external stimuli by rapid and complex changes in gene expression. The present work was undertaken to provide a wider view of the gene expression profile in unstimulated circulating PMNs. We used serial analysis of gene expression (SAGE) adapted to downsized extracts (SADE) to cope with their small mRNA content. As cell samples of the highest degree of purity were crucial for this project, we adapted a magnetic cell sorting method to reach the required high levels of purity (99.55±0.19%) together with low activation rates (1.37±0.28). We analyzed 20787 tags identifying 8547 different transcripts, of which 47.8% were unknown, 34.6% were transcripts of known genes, and 13.8% matched with expressed sequence tags (EST). Highly expressed genes were those involved in cell mobility, diapedesis, cell signaling and destruction of micro-organisms. In addition, this method led to the identification of genes which had not previously been reported in granulocytes. These results could provide new molecular markers and a reliable reference for the investigation of pathologies involving alteration of the granulocyte gene expression profile. [Copyright &y& Elsevier]

Toxoplasma gondii (T. gondii)-derived heat shock protein 70 (T.g.HSP70) has been identified as a virulent molecule expressing only in T. gondii tachyzoites during lethal acute infection. Therefore, it is of importance to determine the expression of T.g.HSP70 mRNA in a quantitative manner for analysis of virulence of T. gondii in tissues. We have constructed a competitor T.g.HSP70 and have successfully established a quantitative competitive-reverse transcriptase–polymerase chain reaction (QC-RT-PCR) targeting T.g.HSP70 gene. By using the established QC-RT-PCR method, we have demonstrated that the copy number of T.g.HSP70 mRNA per T. gondii tachyzoite was highest in the lung among the organs examined in interferon-γ knockout (GKO) mice. [Copyright &y& Elsevier]

Two copies of a new mariner-like element (MLE) presenting unusual inverted terminal repeats (ITRs), Mcmar1-1 and Mcmar1-2, were cloned and sequenced in the genome of the phytoparasitic nematode Meloidogyne chitwoodi. Although the sequence features of these Mcmar1 transposons are commonplace and link them to the mariner family, at their extremities they have large 355-pb long inverted terminal repeats that are perfectly conserved. This characteristic distinguishes them from all the other MLEs so far described that have imperfectly conserved ITRs of about 26–30 bp. In consequence, the sequenced full-length Mcmar1-1 element is 2000 bp long, and comprises an uninterrupted open reading frame (ORF) that encodes a putatively active transposase with 340 amino acid residues. The Mcmar1-2 element is a deleted form of Mcmar1-1 that contains a deletion overlapping most of the internal region of the 5′ITR and the 5′ region of the transposase ORF. The presence of large ITRs in different transposons related to the Tc1-mariner super-family is discussed. [Copyright &y& Elsevier]

We are reporting a putative multifunctional Type I polyketide synthase (PKS) gene from the apicomplexan Cryptosporidium parvum (CpPKS1). The 40 kb intronless open reading frame (ORF) predicts a single polypeptide of 13,414 amino acids with a molecular mass of 1516.5 kDa. Sequence analysis identified at least 29 enzymatic domains within this protein. These domains are organized into an N-terminal loading unit, seven polyketide chain elongation modules, and a carboxy terminator unit. The loading domain consists of an acyl-CoA ligase (AL) and an acyl carrier protein (ACP). All seven elongation modules contain between two and five of the six domains required for the elongation of two-carbon (C2) acyl units, i.e. ketoacyl synthase, acyl transferase, dehydrase, enoyl reductase, ketoreductase and/or ACP. The carboxy terminator is homologous to various reductases, suggesting that the final elongated product is not hydrolytically released by thioesterases as observed in most Type I PKS and all fatty acid synthetase (FAS) systems, but by a reducing reaction, which has been demonstrated in some non-ribosomal peptide synthase systems. The protein sequence and domain organization of CpPKS1 protein resembles a previously reported C. parvum fatty acid synthase (CpFAS1), which is encoded by a 25 kb ORF. Maximum likelihood phylogenetic analysis of acyl transferases within PKS/FAS from C. parvum and other organisms clearly differentiates acetate-extending clades from those incorporating propionate. All acyl transferase domains from CpPKS1, and a previously reported CpFAS1, clustered within the acetate-extending group, suggesting the likelihood that only non-methylated C2 units are incorporated by C. parvum polyketide and fatty acid synthases. The expression of CpPKS1 was confirmed by reverse transcription-polymerase chain reaction and immunofluorescence microscopy. Many polyketides are medically significant antibiotics, anticancer agents, toxins, or signaling molecules. Therefore, it is interesting to speculate what role CpPKS1 might play in this apicomplexan and the disease caused by this opportunistic infection of AIDS patients. [Copyright &y& Elsevier]

Protein casein kinase 2 (CK2) is a heterotetramer composed of two catalytic (α) and two regulatory (β) subunits. In Drosophila melanogaster, four genes encoding for one CK2α (DmCK2α) and three CK2β (DmCK2β, DmCK2β′ and DmCK2βtes) subunits have been identified. Here, we have focused on the role of DmCK2β. Evaluation of DNA complementary to RNA sequences and Western blot analysis revealed a complex pattern of DmCK2β transcription and the existence of at least three distinct DmCK2β isoforms in adult flies. The phenotype of the viable DmCK2β mutation mushroom bodies undersizedP1 (DmCK2βmbuP1) described in this study implicates a role for DmCK2β in cell proliferation or cell survival during brain development. The isolation of a complete loss of function allele of DmCK2β allowed us to assay the importance of various structural domains for the in vivo function of DmCK2β. Expression of in vitro mutagenised DmCK2β transgenes in a DmCK2β null mutant background demonstrated an absolute requirement of the β/β dimerisation motif for CK2β function, whereas removal of all N-terminal phosphorylation sites of DmCK2β resulted in reduced viability. In contrast, a mutation in the predicted destruction box motif did not interfere with DmCK2β function. [Copyright &y& Elsevier]

Plasmodium vivax merozoite surface protein-9 (Pvmsp-9) is characterized here along with orthologues from the related simian malarias Plasmodium cynomolgi and Plasmodium knowlesi. We show that although the corresponding MSP-9 proteins do not have acidic–basic repeated amino acid (aa) motifs, they are related to the Plasmodium falciparum acidic–basic repeat antigen (ABRA) also known as p101. Recognition of this new interspecies Plasmodium MSP family stems from the prior identification of related MSP termed PvMSP-185, PcyMSP-150, and PkMSP-110 on the surface of P. vivax, P. cynomolgi and P. knowlesi merozoites. A clone containing the nearly complete P. knowlesi gene encoding PkMSP-110/MSP-9 provided a hybridization probe and initial sequence information for the design of primers to obtain the P. vivax and P. cynomolgi orthologues using polymerase chain reaction (PCR) amplification strategies. The P. vivax, P. cynomolgi and P. knowlesi msp-9 genes encode proteins that range in calculated molecular mass from 80 to 107 kDa, have typical eukaryotic signal peptides and diverse repeated motifs present immediately upstream of their termination codon. Another feature conserved among these proteins, including the P. falciparum ABRA protein, is the positions of four cysteine residues near the N-terminus, suggesting this conservation maintains structural and perhaps functional characteristics in the MSP-9 family. Rabbit polyclonal antisera raised against recombinantly expressed N-termini of P. knowlesi and P. vivax MSP-9 cross-react with the counterpart proteins in immunofluorescence and immunoblot assays. Comparative interspecies investigations of the potential role(s) of Plasmodium MSP-9 in merozoite invasion of erythrocytes and as a malaria vaccine candidate can now be pursued. [Copyright &y& Elsevier]

The enteric protozoan Cryptosporidium parvum infects intestinal epithelial cells in a wide range of hosts, causing severe gastrointestinal disease. The invasive sporozoite stage most likely attaches to and invades host cells through multiple host receptor/parasite ligand interactions. Preliminary evidence suggests that the glycoprotein products of the Cpgp40/15 gene, gp40 and gp15, are involved in these interactions. In addition, the Cpgp40/15 gene that encodes these glycopeptides is highly polymorphic in genotype I isolates, suggesting that the gene products may be subject to immune selection. In this study, we characterized the Cpgp40/15 gene in a genotype I isolate and compared expression of the Cpgp40/15 gene in isolates of both genotype. Cpgp40/15 is a single copy gene in both TU502 (genotype I) and GCH1 (genotype II) isolates. However, Northern blot analysis revealed the presence of two transcripts, 2.3 and 1.5 kb in size, in mRNA from GCH1 as well as TU502-infected Caco-2A cells. Accumulation of the two Cpgp40/15 mRNAs peaked 12–24 h post-infection. Using 3′RACE analysis, three polyadenylation sites were identified 371, 978 and 1002 bp downstream of the GCH1 Cpgp40/15 stop codon. Two of these polyadenylation sites were also used in TU502. The sequences of the GCH1 Cpgp40/15 3′untranslated regions (3′UTRs) were identical to genomic sequence and shared 96.7% homology with TU502 3′UTRs. Actinomycin D treatment of GCH1-infected Caco-2A cells followed by Northern blot analysis, revealed that the stability of the 1.5 kb message was considerably greater than that of the 2.3 kb transcript. [Copyright &y& Elsevier]

Background & Aims: HBV infects over 257 million people worldwide and is associated with the development of hepatocellular carcinoma (HCC). Integration of HBV DNA into the host genome is likely a key driver of HCC oncogenesis. Here, we utilise targeted long-read sequencing to determine the structure of HBV DNA integrations as well as full isoform information of HBV mRNA with more accurate quantification than traditional next generation sequencing platforms., Methods: DNA and RNA were isolated from fresh frozen liver biopsies collected within the GS-US-174-0149 clinical trial. A pan-genotypic panel of biotinylated oligos was developed to enrich for HBV sequences from sheared genomic DNA (∼7 kb) and full-length cDNA libraries from poly-adenylated RNA. Samples were sequenced on the PacBio long-read platform and analysed using a custom bioinformatic pipeline., Results: HBV-targeted long-read DNA sequencing generated high coverage data spanning entire integrations. Strikingly, in 13 of 42 samples (31%) we were able to detect HBV sequences flanked by 2 different chromosomes, indicating a chromosomal translocation associated with HBV integration. Chromosomal translocations were unique to each biopsy sample, suggesting that each originated randomly, and in some cases had evidence of clonal expansion. Using targeted long-read RNA sequencing, we determined that upwards of 95% of all HBV transcripts in patients who are HBeAg-positive originate from cccDNA. In contrast, patients who are HBeAg-negative expressed mostly HBsAg from integrations., Conclusions: Targeted lso-Seq allowed for accurate quantitation of the HBV transcriptome and assignment of transcripts to either cccDNA or integration origins. The existence of multiple unique HBV-associated inter-chromosomal translocations in non-HCC CHB patient liver biopsies suggests a novel mechanism with mutagenic potential that may contribute to progression to HCC., Lay Summary: Fresh frozen liver biopsies from patients infected with HBV were subjected to targeted long-read RNA and DNA sequencing. Long-read RNA sequencing captures entire HBV transcripts in a single read, allowing for resolution of overlapping transcripts from the HBV genome. This resolution allowed us to quantify the burden of transcription from integrations vs. cccDNA origin in individual patients. Patients who were HBeAg-positive had a significantly larger fraction of the HBV transcriptome originating from cccDNA compared with those who were HBeAg-negative. Long-read DNA sequencing captured entire integrated HBV sequences including multiple kilobases of flanking host sequence within single reads. This resolution allowed us to describe integration events flanked by 2 different host chromosomes, indicating that integrated HBV DNA are associated with inter-chromosomal translocations. This may lead to significant transcriptional dysregulation and drive progression to HCC., Competing Interests: All authors are employed by Gilead Sciences Inc. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2022 The Authors.)

Background: Efficient differentiation of stem cells into three-dimensional (3D) osteogenic construct is still an unmet challenge. These constructs can be crucial for patients with bone defects due to congenital or traumatic reasons. The modulation of cell fate and function as a consequence of interaction with the physical and chemical properties of materials is well known., Methods: The current study has examined the osteogenic differentiation potential of human skeletal populations following culture on glass surfaces, as a monolayer, or in glass tubes as a pellet culture. The 3D prosperities were assessed morphometrically and the differentiation was evaluated through molecular characterization as well as matrix formation., Results: Early temporal expression of alkaline phosphatase expression of skeletal populations was observed following culture on glass surfaces. Skeletal populations seeded on glass tubes, adhered as a monolayer to the tube base and subsequently formed 3D pellets at the air -media interface. The pellets cultured on glass displayed 4.9 ± 1.3 times the weight and 2.9 ± 0.1 the diameter of their counterpart cultured in plastic tubes and displayed enhanced production of osteogenic matrix proteins, such a collagen I and osteonectin. The size and weight of the pellets correlated with surface area in contrast to cell numbers seeded. Global DNA methylation level was decreased in pellets cultured on glass. In contrast, gene expression analysis confirmed upregulation extracellular matrix proteins and osteogenesis-related growth factors., Conclusion: This simple approach to the culture of skeletal cells on glass tubes provides a scaffold-free, 3D construct platform for generating pellets enabling analysis and evaluation of tissue development and integration of multiple constructs with implications for tissue repair and regenerative application on scale-up., Competing Interests: None., (© 2021 The Authors. Published by Elsevier Inc.)

Testicular torsion and detorsion (TTD) is a serious urological condition affecting young males that is underlined by an ischemia reperfusion injury (tIRI) to the testis as the pathophysiological mechanism. During tIRI, uncontrolled production of oxygen reactive species (ROS) causes DNA damage leading to germ cell apoptosis (GCA). The aim of the study is to explore whether inhibition of NADPH oxidase (NOX), a major source of intracellular ROS, will prevent tIRI-induced GCA and its association with endoplasmic reticulum (ER) stress. Sprague-Dawley rats (n = 36) were divided into three groups: sham, tIRI only and tIRI treated with apocynin (a NOX inhibitor). Rats undergoing tIRI endured an ischemic injury for 1 h followed by 4 h of reperfusion. Spermatogenic damage was evaluated histologically, while cellular damages were assessed using real time PCR, immunofluorescence staining, Western blot and biochemical assays. Disrupted spermatogenesis was associated with increased lipid and protein peroxidation and decreased antioxidant activity of the enzyme superoxide dismutase (SOD) as a result of tIRI. In addition, increased DNA double strand breaks and formation of 8-OHdG adducts associated with increased phosphorylation of the DNA damage response (DDR) protein H2AX. The ASK1/JNK apoptosis signaling pathway was also activated in response to tIRI. Finally, increased immuno-expression of the unfolded protein response (UPR) downstream targets: GRP78, eIF2-α1, CHOP and caspase 12 supported the presence of ER stress. Inhibition of NOX by apocynin protected against tIRI-induced GCA and ER stress. In conclusion, NOX inhibition minimized tIRI-induced intracellular oxidative damages leading to GCA and ER stress., (© 2020 The Author(s).)

Background: Genomic testing is often limited by the exhaustible nature of human tissue and blood samples. Here we describe biotinylated amplicon sequencing (BAmSeq), a method that allows for the creation of PCR amplicon based next-generation sequencing (NGS) libraries while retaining the original source DNA., Design and Methods: Biotinylated primers for different loci were designed to create NGS libraries using human genomic DNA from cell lines, plasma, and formalin-fixed paraffin embedded (FFPE) tissues using the BAmSeq protocol. DNA from the original template used for each BAmSeq library was recovered after separation with streptavidin magnetic beads. The recovered DNA was then used for end-point, quantitative and droplet digital PCR (ddPCR) as well as NGS using a cancer gene panel., Results: Recovered DNA was analyzed and compared to the original DNA after one or two rounds of BAmSeq. Recovered DNA revealed comparable genomic distributions and mutational allelic frequencies when compared to original source DNA. Sufficient quantities of recovered DNA after BAmSeq were obtained, allowing for additional downstream applications., Conclusions: We demonstrate that BAmSeq allows original DNA template to be recovered with comparable quality and quantity to the source DNA. This recovered DNA is suitable for many downstream applications and may prevent sample exhaustion, especially when DNA quantity or source material is limiting.

Digital PCR in droplets (ddPCR) is an emerging method for more and more applications in DNA (and RNA) analysis. Special requirements when establishing ddPCR for analysis of genetically modified organisms (GMO) in a laboratory include the choice between validated official qPCR methods and the optimization of these assays for a ddPCR format. Differentiation between droplets with positive reaction and negative droplets, that is setting of an appropriate threshold, can be crucial for a correct measurement. This holds true in particular when independent transgene and plant-specific reference gene copy numbers have to be combined to determine the content of GM material in a sample. Droplets which show fluorescent units ranging between those of explicit positive and negative droplets are called 'rain'. Signals of such droplets can hinder analysis and the correct setting of a threshold. In this manuscript, a computer-based algorithm has been carefully designed to evaluate assay performance and facilitate objective criteria for assay optimization. Optimized assays in return minimize the impact of rain on ddPCR analysis. We developed an Excel based 'experience matrix' that reflects the assay parameters of GMO ddPCR tests performed in our laboratory. Parameters considered include singleplex/duplex ddPCR, assay volume, thermal cycler, probe manufacturer, oligonucleotide concentration, annealing/elongation temperature, and a droplet separation evaluation. We additionally propose an objective droplet separation value which is based on both absolute fluorescence signal distance of positive and negative droplet populations and the variation within these droplet populations. The proposed performance classification in the experience matrix can be used for a rating of different assays for the same GMO target, thus enabling employment of the best suited assay parameters. Main optimization parameters include annealing/extension temperature and oligonucleotide concentrations. The droplet separation value allows for easy and reproducible assay performance evaluation. The combination of separation value with the experience matrix simplifies the choice of adequate assay parameters for a given GMO event.

A 14 kDa protein homologous to the γ-d-glutamyl-l-diamino acid endopeptidase members of the NlpC/P60 Superfamily has been described in Dermatophagoides pteronyssinus and Dermatophagoides farinae but it is not clear whether other species produce homologues. Bioinformatics revealed homologous genes in other Sarcopteformes mite species (Psoroptes ovis and Blomia tropicalis) but not in Tetranychus urticae and Metaseiulus occidentalis. The degrees of identity (similarity) between the D. pteronyssinus mature protein and those from D. farinae, P. ovis and B. tropicalis were 82% (96%), 77% (93%) and 61% (82%), respectively. Phylogenetic studies showed the mite proteins were monophyletic and shared a common ancestor with both actinomycetes and ascomycetes. The gene encoding the D. pteronyssinus protein was polymorphic and intronless in contrast to that reported for D. farinae. Homology studies suggest that the mite, ascomycete and actinomycete proteins are involved in the catalysis of stem peptide attached to peptidoglycan. The finding of a gene encoding a P60 family member in the D. pteronyssinus genome together with the presence of a bacterial promotor suggests an evolutionary link to one or more prokaryotic endosymbionts.