TGA Codon Research Articles

Method for Preparing Recombinant Galectin-2 Protein without Escherichia coli-Specific Post-translational Modifications.

Galectin-2 (Gal-2) is an animal lectin with specificity for β-galactosides. It is predominantly expressed and suggested to play a protective function in the gastrointestinal tract; therefore, it can be used as a protein drug. Recombinant proteins have been expressed using Escherichia coli and used to study the function of Gal-2. The recombinant human Gal-2 (hGal-2) protein purified via affinity chromatography after being expressed in E. coli was not completely homogeneous. Mass spectrometry confirmed that some recombinant Gal-2 were phosphogluconoylated. In contrast, the recombinant mouse Gal-2 (mGal-2) protein purified using affinity chromatography after being expressed in E. coli contained a different form of Gal-2 with a larger molecular weight. This was due to mistranslating the original mGal-2 stop codon TGA to tryptophan (TGG). In this report, to obtain a homogeneous Gal-2 protein for further studies, we attempted the following methods: for hGal-2, 1) replacement of the lysine (Lys) residues, which was easily phosphogluconoylated with arginine (Arg) residues, and 2) addition of histidine (His)-tag on the N-terminus of the recombinant protein and cleavage with protease after expression; for mGal-2, 3) changing the stop codon from TGA to TAA, which is commonly used in E. coli. We obtained an almost homogeneous recombinant Gal-2 protein (human and mouse). These results have important implications for using Gal-2 as a protein drug.

Evolution of termination codons of proteins and the TAG-TGA paradox

In most eukaryotes and prokaryotes TGA is used at a significantly higher frequency than TAG as termination codon of protein-coding genes. Although this phenomenon has been recognized several years ago, there is no generally accepted explanation for the TAG-TGA paradox. Our analyses of human mutation data revealed that out of the eighteen sense codons that can give rise to a nonsense codon by single base substitution, the CGA codon is exceptional: it gives rise to the TGA stop codon at an order of magnitude higher rate than the other codons. Here we propose that the TAG-TGA paradox is due to methylation and hypermutabilty of CpG dinucleotides. In harmony with this explanation, we show that the coding genomes of organisms with strong CpG methylation have a significant bias for TGA whereas those from organisms that lack CpG methylation use TGA and TAG termination codons with similar probability.

An Efficient Opal-Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells.

Site-specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA-suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual-ncAA incorporation. Using these platforms, we site-specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site-specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

An Efficient Opal‐Suppressor Tryptophanyl Pair Creates New Routes for Simultaneously Incorporating up to Three Distinct Noncanonical Amino Acids into Proteins in Mammalian Cells**

AbstractSite‐specific incorporation of multiple distinct noncanonical amino acids (ncAAs) into proteins in mammalian cells is a promising technology, where each ncAA must be assigned to a different orthogonal aminoacyl‐tRNA synthetase (aaRS)/tRNA pair that reads a distinct nonsense codon. Available pairs suppress TGA or TAA codons at a considerably lower efficiency than TAG, limiting the scope of this technology. Here we show that the E. coli tryptophanyl (EcTrp) pair is an excellent TGA‐suppressor in mammalian cells, which can be combined with the three other established pairs to develop three new routes for dual‐ncAA incorporation. Using these platforms, we site‐specifically incorporated two different bioconjugation handles into an antibody with excellent efficiency, and subsequently labeled it with two distinct cytotoxic payloads. Additionally, we combined the EcTrp pair with other pairs to site‐specifically incorporate three distinct ncAAs into a reporter protein in mammalian cells.

The Phylogenetic Position of the Enigmatic, Polypodium hydriforme (Cnidaria, Polypodiozoa): Insights from Mitochondrial Genomes.

Polypodium hydriforme is an enigmatic parasite that belongs to the phylum Cnidaria. Its taxonomic position has been debated: whereas it was previously suggested to be part of Medusozoa, recent phylogenomic analyses based on nuclear genes support the view that P. hydriforme and Myxozoa form a clade called Endocnidozoa. Medusozoans have linear mitochondrial (mt) chromosomes, whereas myxozoans, as most metazoan species, have circular chromosomes. In this work, we determined the structure of the mt genome of P. hydriforme, using Illumina and Oxford Nanopore Technologies reads, and showed that it is circular. This suggests that P. hydriforme is not nested within Medusozoa, as this would entail linearization followed by recirculation. Instead, our results support the view that P. hydriforme is a sister clade to Myxozoa, and mt linearization in the lineage leading to medusozoans occurred after the divergence of Myxozoa + P. hydriforme. Detailed analyses of the assembled P. hydriforme mt genome show that: (1) it is encoded on a single circular chromosome with an estimated size of ∼93,000 base pairs, making it one of the largest metazoan mt genomes; (2) around 78% of the genome encompasses a noncoding region composed of several repeat types; (3) similar to Myxozoa, no mt tRNAs were identified; (4) the codon TGA is a stop codon and does not encode for tryptophan as in other cnidarians; (5) similar to myxozoan mt genomes, it is extremely fast evolving.

Algorithm Model Determination of DNA Primer Design For The Success PCR Process

Deoxyribonucleic Acid Primer Design is an important factor in the success of the Polymerase Chain Reaction process because it will determine the value of Guanine and Cytosine content and Melting temperature for the genome amplification process in the adequacy of research data. To get a good primer design, a simulation process is needed to get the desired results, namely Guanine and Cytosine content between 50%-60%, Melting temperature 50o -65oC, a minimum length of 70 base pair sequences and the temperature difference between the forward primer and reverse primer less than 5oC. Usually researchers use the Primer3plus or NCBI Primer-BLAST software to get the Deoxyribonucleic Acid Primer Design results. Because there are limitations in the use of the software, the researchers, researchers want to use software that are suitable for current and future research needs. For this reason, a new algorithm model is needed to make software to support the needs of researchers. The method used in the algorithm model is to use Start Codon ATG and Stop Codon TAA, TAG and TGA with sequence lengths of 18, 21 and 24 base pairs. The raw data used is the Deoxyribonucleic Acid of the Typhonium Flagelliforme plant which has potential anti-cancer compounds. The new algorithm model resulted in 22 (twenty two) Deoxyribonucleic Acid Primer Design options, all of which met the data constraints and research requirements.

Premature Termination Codon of 1Dy12 Gene Improves Cookie Quality in Ningmai9 Wheat.

The area between middle and lower reaches of the Yangtze River is the largest region for soft wheat production in China. In soft wheat breeding, the lack of germplasm with desirable quality for end-use products is a barrier. Ningmai9 is the main variety of soft wheat planted in this area. To create germplasm with better quality and yield potential than Ningmai9, mutants of HMW-GSs in Ningmai9 induced by ethylmethanesulfonate (EMS) were obtained. SDS-PAGE showed that two mutants, md10 and md11, were HMW-GS 1Dy deletions. DNA sequencing confirmed that one mutation was caused by a C/T substitution, resulting in the change of CAA encoding glutamine into the termination codon TAA, and another mutation was due to a G/A substitution in the central repetitive domain of the coding region, causing TGG encoding tryptophan to become the termination codon TGA. The premature termination codon of the 1Dy12 gene affected the expression of 1Dy12 and kept the mRNA at a lower transcription level during the kernel development stage in comparison with the wild type. HMW-GS 1Dy12 deletion mutants decreased the content of HMW-GSs and glutenin macropolymers, mixograph envelope peak time and TIMEX width, water solvent retention capacity (WSRC), and lactic acid solvent retention capacity (LASRC). In the HMW-GS 1Dy12 deletion lines, the sugar-snap cookie diameter was 8.70–8.74 cm, which was significantly larger than that in the wild type of 8.0 cm. There were no significant differences in spike number, kernel number, thousand kernel weight, and yield between the deletion lines and wild type. Overall, the study indicated that the knockout of the HMW-GS gene induced by EMS is an effective way to improve wheat quality, and deletion mutants of HMW-GS 1Dy12 decrease gluten strength and increase sugar snap cookie diameter without yield penalty in Ningmai9 wheat.

T-G-A Deficiency Pattern in Protein-Coding Genes and Its Potential Reason

If a stop codon appears within one gene, then its translation will be terminated earlier than expected. False folding of premature protein will be adverse to the host; hence, all functional genes would tend to avoid the intragenic stop codons. Therefore, we hypothesize that there will be less frequency of nucleotides corresponding to stop codons at each codon position of genes. Here, we validate this inference by investigating the nucleotide frequency at a large scale and results from 19,911 prokaryote genomes revealed that nucleotides coinciding with stop codons indeed have the lowest frequency in most genomes. Interestingly, genes with three types of stop codons all tend to follow a T-G-A deficiency pattern, suggesting that the property of avoiding intragenic termination pressure is the same and the major stop codon TGA plays a dominant role in this effect. Finally, a positive correlation between the TGA deficiency extent and the base length was observed in start-experimentally verified genes of Escherichia coli (E. coli). This strengthens the proof of our hypothesis. The T-G-A deficiency pattern observed would help to understand the evolution of codon usage tactics in extant organisms.

Case Report: Successful Management of a 29-Day-Old Infant With Severe Hyperlipidemia From a Novel Homozygous Variant of GPIHBP1 Gene

BackgroundSevere hyperlipidemia is characterized by markedly elevated blood triglyceride levels and severe early-onset cardiovascular diseases, pancreatitis, pancreatic necrosis or persistent multiple organ failure if left untreated. It is a rare autosomal recessive metabolic disorder originated from the variants of lipoprotein lipase gene, and previous studies have demonstrated that most cases with severe hyperlipidemia are closely related to the variants of some key genes for lipolysis, such as LPL, APOC2, APOA5, LMF1, and GPIHBP1. Meanwhile, other unidentified causes also exist and are equally worthy of attention.MethodsThe 29-day-old infant was diagnosed with severe hyperlipidemia, registering a plasma triglyceride level as high as 25.46 mmol/L. Whole exome sequencing was conducted to explore the possible pathogenic gene variants for this patient.ResultsThe infant was put on a low-fat diet combined with pharmacological therapy, which was successful in restraining the level of serum triglyceride and total cholesterol to a low to medium range during the follow-ups. The patient was found to be a rare novel homozygous duplication variant-c.45_48dupGCGG (Pro17Alafs*22) in GPIHBP1 gene-leading to a frameshift which failed to form the canonical termination codon TGA. The mutant messenger RNA should presumably produce a peptide consisting of 16 amino acids at the N-terminus, with 21 novel amino acids on the heels of the wild-type protein.ConclusionsOur study expands on the spectrum of GPIHBP1 variants and contributes to a more comprehensive understanding of the genetic diagnosis, genetic counseling, and multimodality therapy of families with severe hyperlipidemia. Our experience gained in this study is also contributory to a deeper insight into severe hyperlipidemia and highlights the importance of molecular genetic tests.

Plastomes in the holoparasitic family Balanophoraceae: Extremely high AT content, severe gene content reduction, and two independent genetic code changes

The transition to a heterotrophic lifestyle in angiosperms is characterized by convergent evolutionary changes. Plastid genome remodeling includes dramatic functional and physical reductions with the highest degrees observed in fully heterotrophic plants. Genes related to photosynthesis are generally absent or pseudogenized, while a few genes related to other metabolic processes that take place within the plastid are almost invariably maintained. The family Balanophoraceae consists of root holoparasites that present reduced plastid genomes with an extraordinarily elevated AT content and the single genetic code change ever documented in land plant plastomes (the stop codon TAG now codes for tryptophan). Here, we studied the plastomes of Lophophytum leandri and Ombrophytum subterraneum (Balanophoraceae) that showed the remarkable absence of the gene trnE, a highly biased nucleotide composition, and an independent genetic code change (the standard stop codon TGA codes for tryptophan). This is the second genetic code change identified in land plant plastomes. Analysis of the transcriptome of Lophophytum indicated that the entire C5 pathway typical of plants is conserved despite the lack of trnE in its plastome. A hypothetical model of plastome evolution in the Balanophoraceae is presented.

The nonsense mutation stop+4 model correlates with motor changes in Duchenne muscular dystrophy

The aim was to assess 3-year longitudinal data using 6MWT in 26 ambulant boys affected by DMD carrying nonsense mutations and to compare their results to other small mutations. We also wished to establish, within the nonsense mutations group, patterns of change according to several variables. Patients with nonsense mutations were categorized according to the stop codon type newly created by the mutation and also including the adjacent 5′ (upstream) and 3′ (downstream) nucleotides. No significant difference was found between nonsense mutations and other small mutations (p > 0.05) on the 6MWT. Within the nonsense mutations group, there was no difference in 6MWT when the patients were subdivided according to: Type of stop codon, frame status of exons involved, protein domain affected. In contrast, there was a difference when the stop codon together with the 3′ adjacent nucleotide (“stop+4 model”) was considered (p < 0.05) with patients with stop codon TGA and 3′ adjacent nucleotide G (TGAG) having a more rapid decline. Our finding suggest that the stop+4 model may help in predicting functional changes. This data will be useful at the time of interpreting the long term follow up of patients treated with Ataluren that are becoming increasingly available.

The algal selenoproteomes

BackgroundSelenium is an essential trace element, and selenocysteine (Sec, U) is its predominant form in vivo. Proteins that contain Sec are selenoproteins, whose special structural features include not only the TGA codon encoding Sec but also the SECIS element in mRNA and the conservation of the Sec-flanking region. These unique features have led to the development of a series of bioinformatics methods to predict and research selenoprotein genes. There have been some studies and reports on the evolution and distribution of selenoprotein genes in prokaryotes and multicellular eukaryotes, but the systematic analysis of single-cell eukaryotes, especially algae, has been very limited.ResultsIn this study, we predicted selenoprotein genes in 137 species of algae by using a program we previously developed. More than 1000 selenoprotein genes were obtained. A database website was built to record these algae selenoprotein genes (www.selenoprotein.com). These genes belong to 42 selenoprotein families, including three novel selenoprotein gene families.ConclusionsThis study reveals the primordial state of the eukaryotic selenoproteome. It is an important clue to explore the significance of selenium for primordial eukaryotes and to determine the complete evolutionary spectrum of selenoproteins in all life forms.

Identification of a male sterile gene Ms in Brassica rapa L.

Male sterile lines are important for hybrid seed production in Brassica rapa. In our previous study, we obtained a multiple-allele inherited male sterile line of Chinese cabbage. To identify the male sterile gene Ms in B. rapa, we used B. rapa L. var. chinensis (‘AB03’; MsMs) and B. rapa var. purpuraria (‘AB04’; MsfMsf) as parents to construct an F2 segregating population, and mapped the Ms gene on chromosome A07 between single sequence repeat (SSR) markers SSRY17 and SSRY22, which flank the gene at genetic distances of 0.11 cM and 0.31 cM, respectively. The interval between the two markers was 231.3 kb and comprised 21 genes. Whole-genome resequencing and sequence comparison revealed that only one gene, Bra015018, harbored an insertion–deletion (InDel) in an exon in the mapped interval. This InDel led to premature termination of translation due to generation of the stop codon TGA. Bra015018 was homologous to AT1G34355, which encoded a forkhead-associated (FHA) domain-containing protein that was related to plant growth and floral development. Genotyping revealed that male sterility was fully co-segregated with the InDel. qRT-PCR analysis showed that Bra015018 was specifically expressed in the stamen. Consequently, we predicted that Bra015018 was a candidate gene of Ms.

Widespread use of the “ascidian” mitochondrial genetic code in tunicates

Background: Ascidians, a tunicate class, use a mitochondrial genetic code that is distinct from vertebrates and other invertebrates. Though it has been used to translate the coding sequences from other tunicate species on a case-by-case basis, it is has not been investigated whether this can be done systematically. This is an important because a) some tunicate mitochondrial sequences are currently translated with the invertebrate code by repositories such as NCBI GenBank, and b) uncertainties about the genetic code to use can complicate or introduce errors in phylogenetic studies based on translated mitochondrial protein sequences. Methods: We collected publicly available nucleotide sequences for non-ascidian tunicates including appendicularians such as Oikopleura dioica, translated them using the ascidian mitochondrial code, and built multiple sequence alignments covering all tunicate classes. Results: All tunicates studied here appear to translate AGR codons to glycine instead of serine (invertebrates) or as a stop codon (vertebrates), as initially described in ascidians. Among Oikopleuridae, we suggest further possible changes in the use of the ATA (Ile → Met) and TGA (Trp → Arg) codons. Conclusions: We recommend using the ascidian mitochondrial code in automatic translation pipelines of mitochondrial sequences for all tunicates. Further investigation is required for additional species-specific differences.

Stop codons and the +4 nucleotide may influence the efficiency of G418 in rescuing nonsense mutations of the HERG gene.

The importance of the local sequence context in determining how efficiently aminoglycosides rescue nonsense mutations has been established previously in disease models. Different stop codons appear to facilitate the termination process with differing efficiencies. Furthermore, the efficiency with which termination is suppressed may also be influenced by the local sequence context surrounding the stop codon. The strongest bias has usually been identified with the nucleotide base that immediately follows the stop codon in the majority of experiments. However, how the sequence context influences the efficiency of aminoglycosides in rescuing the human ether-a-go-go-related (HERG) protein in mammalian cells remains to be fully elucidated. Therefore, the present study was devised to examine the susceptibility of different termination codons on the HERG gene and the +4 nucleotide immediately following them to be suppressed by aminoglyco-sides in 293 cells. The 293 cells were transiently transfected with the wild-type or mutant genes. The read-through effect was subsequently examined by adding aminoglycoside G418 into the culture medium, followed by incubation of the cells for 24 h. An immunofluorescence method was then used to observe the protein expression of HERG prior to and following drug treatment. Patch clamping was performed to evaluate the function of the HERG protein. These experiments revealed that stop codons TGA and TAA in the R1014X mutant were more susceptible to treatment with the drug G418. Similar results were observed with the W927X-TGA and W927X-TAA mutants. Subsequently, R1014X-TGAC, R1014X-TGAG and R1014X-TGAA mutants were constructed based on the R1014X-TGAT mutant. The level of red fluorescence was observed prior to and following the administration of G418 using antibodies targeting the N- or C-terminus of the HERG protein. However, the tail current density was found only to increase with the R1014X-TGAT mutant following G418 treatment. Taken together, the results of the present study suggest that the type of premature stop codon and the context of the nucleotide immediately following at the +4 position, may determine the pharmacological rescue efficiency of the HERG gene.

Response of a novel selenium-dependent glutathione peroxidase from thick shell mussel Mytilus coruscus exposed to lipopolysaccharide, copper and benzo[α]pyrene

Glutathione peroxidase (GPx) plays an important antioxidant role in cellular defense against environmental stress. In the present study, a novel selenium-dependent glutathione peroxidase termed McSeGPx firstly identified in thick shell mussel Mytilus coruscus. McSeGPx consists of 197 amino acid residues, characterized with one selenocysteine residue encoded by an opal stop codon TGA, one selenocysteine insertion sequence (SECIS) in the 3ʹ untranslated region (UTR), two active site motifs and one signature sequence motif. McSeGPx transcripts were constitutively expressed in all examined tissues, and were significantly induced in gills and digestive glands with the stimulations of lipopolysaccharide (LPS), copper (Cu) and benzo[α]pyrene (B[α]P). Additionally, rough increases in McSeGPx activity were detected in both tissues under the challenge of LPS, Cu and B[α]P. Collectively, these results suggested that McSeGPx affiliate to selenocysteine dependent GPx (SeGPx) family and might play an important role in mediating the environmental stressors and antioxidant response in M. coruscus.

CITRIC: cold-inducible translational readthrough in the chloroplast of Chlamydomonas reinhardtii using a novel temperature-sensitive transfer RNA

BackgroundThe chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnWUCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA.ResultsA mutated version of our trnWUCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene.ConclusionsCITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.

Post-transcriptional regulation of chicken PPARγ transcript variant 3 by upstream open reading frame

Peroxisome proliferator-activated receptor gamma (PPARγ) is a critical regulator of adipogenesis. Our previous study showed that unlike human and mouse PPARγ transcripts, several chicken PPARγ transcript variants contain upstream open reading frames (uORFs) in their 5'untranslated region (5'TR). To decipher the role of uORFs in post-transcriptional regulation of chicken PPARγ gene, we constructed wild-type (psiCHECK2-cPPARγ3-5'UTR-WT) and a uORF mutant (the upstream ATG (uATG) was mutated to stop codon TGA) 5'UTR reporters (psiCHECK2-cPPARγ3- 5'UTR-Mut) of chicken PPARγ transcript variant 3 (cPPARγ3). These two reporters were individually transfected into immortalized chicken pre-adipocytes (ICPA) and DF1 cells, and the renilla luciferase (hRluc) activity and mRNA expression level were detected by reporter assay and qRT-PCR. The results showed that the hRluc activity of the mutated 5'UTR was significantly higher than that of the wild-type 5'UTR in ICPA cells (P<0.01), and the hRluc activity of the mutated 5'UTR tended to be higher than that of the wild-type 5'UTR in DF1 cells, but this difference did not reach statistical significance (P>0.05). The qRT-PCR analysis showed, in ICPA cells, the hRluc mRNA expression was significantly lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct (P<0.01). In DF1 cells, the hRluc mRNA expression tended to be lower in the cells transfected with the mutated 5'UTR construct than in the cells transfected with the wild-type 5'UTR construct, but this difference did not reach statistical significance (P>0.05). To further gain insight into the post-transcriptional regulation of cPPARγ3 by the uORF, we constructed the expression plasmids bearing the full-length coding region of chicken PPARγ gene plus either wild-type or mutant uORF 5'UTR (pcDNA3.1-cPPARγ3-WT and pcDNA3.1-cPPARγ3-Mut). These two constructed PPARγ expression plasmids were individually transiently transfected into both ICPA and DF1 cells, and PPARγ mRNA and protein levels were assayed by qRT-PCR and western blotting. The result showed that in both cell lines, PPARγ mRNA expression was significantly lower in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.05). In contrast, western blot analysis showed that PPARγ protein level was significantly higher in the cells transfected with pcDNA3.1-cPPARγ3-Mut than in the cells transfected with pcDNA3.1-cPPARγ3-WT (P<0.001). Taken together, our results demonstrate that the uORF in 5'UTR of the cPPARγ3 inhibits its translation.

Extensive mitochondrial gene rearrangements in Ctenophora: insights from benthic Platyctenida

BackgroundComplete mitochondrial (mt) genomes have been sequenced for thousands of animals and represent a molecule of choice for many evolutionary studies. Nevertheless, some animal groups have remained under-sampled. Ctenophora (comb jellies) is one such example, with only two complete mt sequences determined hitherto for this phylum, which encompasses ca. 150–200 described species. This lack of data derives from the extremely fast mt evolutionary rate in this lineage, complicating primer design and DNA amplification. Indeed, in the two ctenophore mt genomes sequenced to date, i.e. those of Mnemiopsis leidyi (order Lobata) and Pleurobrachia bachei (order Cydippida), both rRNA and protein coding genes exhibit an extraordinary size reduction and have highly derived sequences. Additionally, all tRNAs, and the atp6 and atp8 genes are absent. In order to determine whether these characteristics are shared by other ctenophores, we obtained the complete mt genomes of three benthic ctenophores belonging to the so far unsampled order of Platyctenida: Coeloplana loyai, Coeloplana yulianicorum and Vallicula multiformis.ResultsThe mt genomes of benthic ctenophores reveal the same peculiarities found in Mnemiopsis and Pleurobrachia, demonstrating that the fast evolutionary rate is a general trait of the ctenophore mt genomes. Our results also indicate that this high evolutionary rate not only affects the nucleotide substitution but also gene rearrangements. Indeed, gene order was highly rearranged among representatives of the different taxonomic orders in which it was close to random, but also quite variable within Platyctenida, in which the genera Coeloplana and Vallicula share only four conserved synteny blocks. However, the two congeneric Coeloplana species display exactly the same gene order. Because of the extreme evolutionary rate, our phylogenetic analyses were unable to resolve the phylogenetic position of ctenophores within metazoans or the relationships among the different Ctenophora orders. Comparative sequence-analyses allowed us to correct the annotation of the Pleurobrachia mt genome, confirming the absence of tRNAs, the presence of both rRNA genes, and the existence of a reassignment of codon TGA from tryptophan to serine for this species.ConclusionsSince Platyctenida is an early diverging lineage among Ctenophora, our findings suggest that the mt traits described above are ancestral characteristics of this phylum.

First report of an atypical new Aspergillus parasiticus isolates with nucleotide insertion in aflR gene resembling to A. sojae.

Aflatoxins are toxic and carcinogenic secondary metabolites produced primarily by the filamentous fungi Aspergillus flavus and Aspergillus parasiticus and cause toxin contamination in food chain worldwide. Aspergillus oryzae and Aspergillus sojae are highly valued as koji molds in the traditional preparation of fermented foods, such as miso, sake, and shoyu. Koji mold species are generally perceived of as being nontoxigenic and are generally recognized as safe (GRAS). Fungal isolates were collected from a California orchard and a few were initially identified to be A. sojae using β-tubulin gene sequences blasted against NCBI data base. These new isolates all produced aflatoxins B1, B2, G1, and G2 and were named as Pistachio Winter Experiment (PWE) strains. Thus, it is very important to further characterize these strains for food safety purposes. The full length of aflR gene of these new isolates was sequenced. Comparison of aflR DNA sequences of PWE, A. parasiticus and A. sojae, showed that the aflatoxigenic PWE strains had the six base insertion (CTCATG) similar to domesticated A. sojae, but a pre-termination codon TGA at nucleotide positions 1153-1155 was absent due to a nucleotide codon change from T to C. Colony morphology and scanning microscopic imaging of spore surfaces showed similarity of PWE strains to both A. parasiticus and A. sojae. Concordance analysis of multi locus DNA sequences indicated that PWE strains were closely linked between A. parasiticus and A. sojae. The finding documented the first report that such unique strains have been found in North America and in the world.