An ancestral genomic sequence that serves as a nucleation site for de novo gene birth

A short non-coding sequence present between the gamma-glutamyltransferase 1 (GGT1) and gamma-glutamyltransferase 5 (GGT5) genes, termed a spacer sequence has been detected in the genomes of Mus musculus, the house mouse and in Philippine tarsier, a primitive ancestral primate. It is highly conserved during primate evolution with certain sequences being totally invariant from mouse to humans. Evidence is presented to show this intergenic sequence serves as a nucleation site for the initiation of diverse genes. We also outline the birth of the human lincRNA gene BCRP3 (BCR activator of RhoGEF and GTPase 3 pseudogene) during primate evolution. The gene developmental process involves sequence initiation, addition of a complex of tandem transposable elements and addition of a segment of another gene. The sequence, initially formed in the Old World Monkeys such as the Rhesus monkey (Macaca mulatta) and the baboon (Papio anubis), develops into different primate genes before evolving into the human BCRP3 gene; it appears to also include trial and error during sequence/gene formation. The protein gene, GGT5 may have also formed by spacer sequence initiation in an ancient ancestor such as zebrafish, but spacer and GGT5 gene sequence drift during evolution produced a divergence that precludes further assessment.

Variation in Plastome Sizes Accompanied by Evolutionary History in Monogenomic Triticeae (Poaceae: Triticeae)

To investigate the pattern of chloroplast genome variation in Triticeae, we comprehensively analyzed the indels in protein-coding genes and intergenic sequence, gene loss/pseudonization, intron variation, expansion/contraction in inverted repeat regions, and the relationship between sequence characteristics and chloroplast genome size in 34 monogenomic Triticeae plants. Ancestral genome reconstruction suggests that major length variations occurred in four-stem branches of monogenomic Triticeae followed by independent changes in each genus. It was shown that the chloroplast genome sizes of monogenomic Triticeae were highly variable. The chloroplast genome of Pseudoroegneria, Dasypyrum, Lophopyrum, Thinopyrum, Eremopyrum, Agropyron, Australopyrum, and Henradia in Triticeae had evolved toward size reduction largely because of pseudogenes elimination events and length deletion fragments in intergenic. The Aegilops/Triticum complex, Taeniatherum, Secale, Crithopsis, Herteranthelium, and Hordeum in Triticeae had a larger chloroplast genome size. The large size variation in major lineages and their subclades are most likely consequences of adaptive processes since these variations were significantly correlated with divergence time and historical climatic changes. We also found that several intergenic regions, such as petN–trnC and psbE–petL containing unique genetic information, which can be used as important tools to identify the maternal relationship among Triticeae species. Our results contribute to the novel knowledge of plastid genome evolution in Triticeae.

DNA methylation affects pre-mRNA transcriptional initiation and processing in Arabidopsis

Background: DNA methylation may regulate pre-mRNA transcriptional initiation and processing, thus affecting gene expression. Unlike animal cells, plants, especially Arabidopsis thaliana, have relatively low DNA methylation levels, limiting our ability to observe any correlation between DNA methylation and pre-mRNA processing using typical short-read sequencing. However, with newly developed long-read sequencing technologies, such as Oxford Nanopore Technology Direct RNA sequencing (ONT DRS), combined with whole-genome bisulfite sequencing, we were able to precisely analyze the relationship between DNA methylation and pre-mRNA transcriptional initiation and processing using DNA methylation-related mutants. Results: Using ONT DRS, we generated more than 2 million high-quality full-length long reads of native mRNA for each of the wild type Col-0 and mutants defective in DNA methylation, identifying a total of 117,474 isoforms. We found that low DNA methylation levels around splicing sites tended to prevent splicing events from occurring. The lengths of the poly(A) tail of mRNAs were positively correlated with DNA methylation. DNA methylation before transcription start sites or around transcription termination sites tended to result in gene-silencing or read-through events. Furthermore, using ONT DRS, we identified novel transcripts that we could not have otherwise, since transcripts with intron retention and fusion transcripts containing the uncut intergenic sequence tend not to be exported to the cytoplasm. Using the met1-3 mutant with activated constitutive heterochromatin regions, we confirmed the effects of DNA methylation on pre-mRNA processing. Conclusion: The combination of ONT DRS with whole-genome bisulfite sequencing was a powerful tool for studying the effects of DNA methylation on splicing site selection and pre-mRNA processing, and therefore regulation of gene expression.

Calcium regulated protein, CarP, responds to multiple host signals and mediates calcium regulation of Pseudomonas aeruginosa virulence

Pseudomonas aeruginosa is an opportunistic pathogen causing life-threatening infections. Previously, we showed that elevated calcium (Ca2+) levels increase the production of virulence factors in P. aeruginosa. In the effort to characterize the Ca2+ regulatory network, we identified a Ca2+–regulated β-propeller protein, CarP, and showed that expression of the encoding gene is controlled by the Ca2+-regulated two-component system CarSR. Here, by using a Galleria melonella model, we showed that CarP plays a role in regulating P. aeruginosa virulence. By using RNA-Seq, RT-PCR, RT-qPCR, and promoter fusions, we determined that carP is transcribed into at least two transcripts and regulated by several bacterial and host factors. The transcription of carP is elevated in response to Ca2+ in P. aeruginosa cystic fibrosis isolates and PAO1 laboratory strain. Elevated Fe2+ also induces carP. Simultaneous addition of Ca2+ and Fe2+ increased the carP promoter activity synergistically, which requires the presence of CarR. In silico analysis of the intergenic sequence upstream of carP predicted recognition sites of RhlR/LasR, OxyR, and LexA, suggesting regulation by quorum sensing (QS) and oxidative stress. In agreement, the carP promoter was activated in response to stationary-phase PAO1 supernatant and required the presence of elevated Ca2+ and CarR, but remained silent in the triple mutant lacking rhlI, lasI, and pqsA synthases. We also showed that carP transcription is regulated by oxidative stress and that CarP contributes to P. aeruginosa Ca2+-dependent H2O2 tolerance. The multifactorial regulation of carP suggests that CarP plays an important role in P. aeruginosa adaptations to host environments. IMPORTANCE P. aeruginosa is a human pathogen causing life-threatening infections. It is particularly notorious for its ability to adapt to diverse environments within the host. Understanding the signals and the signaling pathways enabling P. aeruginosa adaptation is imperative for developing effective therapies to treat infections caused by this organism. One host signal of particular importance is calcium. Previously, we identified a component of P. aeruginosa calcium-signaling network, CarP, whose expression is induced by elevated levels of calcium. Here we show that carP plays an important role in P. aeruginosa virulence and is up-regulated in P. aeruginosa strains isolated from sputa of patients with cystic fibrosis. We also identified several bacterial and host factors that regulate the transcription of carP. Such multifactorial regulation highlights the interconnectedness between regulatory circuits and together with the pleotropic effect of CarP on virulence suggests the importance of this protein in P. aeruginosa adaptations to the host.

Histone modifications associated with gene expression and genome accessibility are dynamically enriched at Plasmodium falciparum regulatory sequences

Background The malaria parasite Plasmodium falciparum has an unusually euchromatic genome with poorly conserved positioning of nucleosomes in intergenic sequences and poorly understood mechanisms of gene regulation. Variant histones and histone modifications determine nucleosome stability and recruit trans factors, but their combinatorial contribution to gene regulation is unclear. Results Here, we show that the histone H3 acetylations H3K18ac and H3K27ac and the variant histone Pf H2A.Z are enriched together at regulatory sites upstream of genes. H3K18ac and H3K27ac together dynamically mark regulatory regions of genes expressed during the asexual life cycle. In contrast, H3K4me1 is depleted in intergenic sequence and dynamically depleted upstream of expressed genes. The temporal pattern of H3K27ac and H3K18ac enrichment indicates that they accumulate during S phase and mitosis and are retained at regulatory sequences until at least G1 phase and after cessation of expression of the cognate genes. We integrated our ChIPseq data with existing datasets to show that in schizont stages H3K18ac, H3K27ac and Pf H2A.Z colocalise with the transcription factor PfAP2-I and the bromodomain protein PfBDP1 and are enriched at stably positioned nucleosomes within regions of exposed DNA at active transcriptional start sites. Using transient transfections we showed that sequences enriched with colocalised H3K18ac, H3K27ac and Pf H2A.Z possess promoter activity in schizont stages, but no enhancer-like activity. Conclusions The dynamic H3 acetylations define P. falciparum regulatory sequences and contribute to gene activation. These findings expand the knowledge of the chromatin landscape that regulates gene expression in P. falciparum.

An lp17-encoded small non-coding RNA with a potential regulatory role in mammalian host adaptation by the Lyme disease spirochete

The bacterial agent of Lyme disease, Borrelia burgdorferi, relies on an intricate gene regulatory network to transit between the disparate Ixodes tick vector and mammalian host environments. We recently reported that a B. burgdorferi mutant lacking an intergenic region of lp17 displayed attenuated murine tissue colonization and pathogenesis due to altered antigen expression. In this study, a more detailed characterization of the putative regulatory factor encoded by the region was pursued through genetic complementation of the mutant with variants of the intergenic sequence. In cis complemented strains featuring mutations aimed at eliminating potential BBD07 protein translation were capable of full tissue colonization, suggesting that the region encodes an sRNA. In trans complementation resulted in elevated transcription levels and was found to completely abolish infectivity in both immunocompetent and immunodeficient mice. Quantitative analysis of transcription of the putative sRNA by wild type B. burgdorferi showed it to be highly induced during murine infection. Lastly, targeted deletion of this region resulted in significant changes to the transcriptome, including genes with potential roles in transmission and host adaptation. The findings reported herein strongly suggest that this lp17 intergenic region encodes for an sRNA with a critical role in the gene regulation required for adaptation and persistence of the pathogen in the mammalian host.Author SummaryLyme disease continues to emerge as a devastating infection that afflicts hundreds of thousands of people annually in the United States and abroad, highlighting the need for new approaches and targets for intervention. Successful development of these therapies relies heavily on an improved understanding of the biology of the causative agent, Borrelia burgdorferi. This is particularly true for the critical points in the life cycle of the pathogen where it must transition between ticks and mammals. Variation in the levels of bacterial gene expression is the lynchpin of this transition and is known to be driven partly by the activity of regulatory molecules known as small non-coding RNAs (sRNAs). In this work, we characterize one of these sRNAs by providing experimental evidence that the transcribed product does not code for a protein, by testing the effects of its overproduction on infectivity, and by interrogating whether its activity causes changes in expression levels of genes at the level of transcription. The findings of this study provide further evidence that regulatory sRNA activity is critical for transmission and optimal infectivity of B. burgdorferi and contribute to the recently growing effort to attribute specific roles to these important molecules in the context of Lyme disease.

Polymorphic SINEC_Cf Retrotransposons in the Genome of the Dog (Canis familiaris)

The dog is an exciting genetic system in which many simple and complex traits have now been mapped. For many traits the causal mutation is a polymorphic SINE. To investigate the genome-wide pattern of young SINEC_Cf insertions, we sampled 62 dogs representing 59 breeds and sequenced libraries enriched for SINE flanks. In each dog we detect an average of 10,423 polymorphic loci and all together the libraries identify 81,747 putative polymorphic SINEs. We validated 184 SINEs inserted in protein-coding exons, untranslated regions, introns and intergenic sequence. In dogs both SINEC_Cf and LINEs exhibit a strand bias in introns where antisense copies are more frequent. Antisense polymorphic SINEs also have a higher density in introns. Both SINEs and LINEs drop to very low density near exons. Both sense and antisense polymorphic SINEs also drop to low density upstream of coding exons but not downstream. Antisense polymorphic SINEC_Cfs upstream of coding exons are known to cause narcolepsy, merle, and progressive retinal atrophy in dogs. In other mammals SINE pairs in inverted orientation disrupt gene expression. We find inverted pairs of SINEC_Cf are rare in both introns and intergenic sequence when the two SINEs are separated by less than 100 bp. The lack of inverted pairs is even more pronounced when the SINEs have high sequence identity. Intronic and intergenic LINE pairs show similar patterns. Polymorphic SINEs rarely pair with either SINEC_Cf or SINEC_Cf2. Overall, the high insertion rate of SINEC_Cf provides a natural mutagenesis screen in the dog genome.

Detection of Klebsiella pneumoniae complex in carrots

Background Klebsiella pneumoniae (Kp) is an opportunistic pathogen now acknowledged as an urgent threat to human health because it is multi-drug resistant bacteria. The role of food as reservoir or carrier of Kp is under investigation, at the same time the optimization of the method to detect Kp in food is ongoing. The study was aimed to detect Kp in carrots using conventional method and to screen Real-time PCR performances, targeting intergenic sequence between zur and khe, based on INRA and Pasteur Institute method Methods A total of 60 samples of carrots were tested. The samples (25 g) were added to 225 ml of Buffered peptone water and incubated at 37 °C and at 44 °C for 24h. After incubation broths were subjected to Real-Time PCR, and one loop streaked on SCAI + inositol agar. Plates were then incubated at 37 °C and at 44 °C for 48 h. After incubation a maximum of 5 typical colonies were selected, subcultered on Nutritive agar and identified by MALDI-TOF MS. Results The RT PCR gave 29 positive samples, according to the presence of Kp detected at least in one of the two temperature conditions of broths incubation and 31 negative samples. Conventional culture gave, instead, 13 positive and 47 negative samples. Discordance was highlighted in 16 samples, all positives in RT-PCR and negatives with culture method. Conclusions Real-Time PCR gave not false negative results, then PCR decrease the time needed to perform the detection in negative samples. The discordance of results could be linked to lack of selectivity of the SCAI agar + inositol, not able to distinguish colonies of Klebsiella spp. Vs similar colonies not Klebsiella spp., the presence of DNA and not viable cells. The process of validation of the detection method is still ongoing, trying to improve selectivity using two selective media for plating. It's necessary to do new trials and to select more typical colonies to increase the probability to detect Kp colonies. Key messages Real-Time PCR is able to detect all positive samples, and to shorten the length of the analytical method. To improve sensibility of the analytical method more selective media need to be used.

Conserved novel ORFs in the mitochondrial genome of the ctenophore Beroe forskalii

To date, five ctenophore species’ mitochondrial genomes have been sequenced, and each contains open reading frames (ORFs) that if translated have no identifiable orthologs. ORFs with no identifiable orthologs are called unidentified reading frames (URFs). If truly protein-coding, ctenophore mitochondrial URFs represent a little understood path in early-diverging metazoan mitochondrial evolution and metabolism. We sequenced and annotated the mitochondrial genomes of three individuals of the beroid ctenophore Beroe forskalii and found that in addition to sharing the same canonical mitochondrial genes as other ctenophores, the B. forskalii mitochondrial genome contains two URFs. These URFs are conserved among the three individuals but not found in other sequenced species. We developed computational tools called pauvre and cuttlery to determine the likelihood that URFs are protein coding. There is evidence that the two URFs are under negative selection, and a novel Bayesian hypothesis test of trinucleotide frequency shows that the URFs are more similar to known coding genes than noncoding intergenic sequence. Protein structure and function prediction of all ctenophore URFs suggests that they all code for transmembrane transport proteins. These findings, along with the presence of URFs in other sequenced ctenophore mitochondrial genomes, suggest that ctenophores may have uncharacterized transmembrane proteins present in their mitochondria.