Hypothetical Open Reading Frames Research Articles

Hypothetical chloroplast reading frame 51 encodes a photosystem I assembly factor in cyanobacteria.

Hypothetical chloroplast open reading frames (ycfs) are putative genes in the plastid genomes of photosynthetic eukaryotes. Many ycfs are also conserved in the genomes of cyanobacteria, the presumptive ancestors of present-day chloroplasts. The functions of many ycfs are still unknown. Here, we generated knock-out mutants for ycf51 (sll1702) in the cyanobacterium Synechocystis sp. PCC 6803. The mutants showed reduced photoautotrophic growth due to impaired electron transport between photosystem II (PSII) and PSI. This phenotype results from greatly reduced PSI content in the ycf51 mutant. The ycf51 disruption had little effect on the transcription of genes encoding photosynthetic complex components and the stabilization of the PSI complex. In vitro and in vivo analyses demonstrated that Ycf51 cooperates with PSI assembly factor Ycf3 to mediate PSI assembly. Furthermore, Ycf51 interacts with the PSI subunit PsaC. Together with its specific localization in the thylakoid membrane and the stromal exposure of its hydrophilic region, our data suggest that Ycf51 is involved in PSI complex assembly. Ycf51 is conserved in all sequenced cyanobacteria, including the earliest branching cyanobacteria of the Gloeobacter genus, and is also present in the plastid genomes of glaucophytes. However, Ycf51 has been lost from other photosynthetic eukaryotic lineages. Thus, Ycf51 is a PSI assembly factor that has been functionally replaced during the evolution of oxygenic photosynthetic eukaryotes.

Genome Sequence of a Microvirus Recovered from Wastewater in Arizona, USA, in October 2020, Encodes a Previously Undescribed DNA-Binding Protein.

ABSTRACTWe describe the genome of Microvirus-AZ-2020, which was identified from wastewater in Arizona, USA, in October 2020. Microvirus-AZ-2020 belongs to subfamily Gokushovirinae and contains six (five known and one hypothetical) open reading frames (ORFs), each with >40 codons. HHPred analysis and Colabfold structure prediction suggest that the hypothetical ORF encodes a previously undescribed putative DNA-binding protein.

Genome Analysis of an Alphabaculovirus Isolated from the Larch Looper, Erannis ankeraria

The larch looper, Erannis ankeraria Staudinger (Lepidoptera: Geometridae), is one of the major insect pests of larch forests, widely distributed from southeastern Europe to East Asia. A naturally occurring baculovirus, Erannis ankeraria nucleopolyhedrovirus (EranNPV), was isolated from E. ankeraria larvae. This virus was characterized by electron microscopy and by sequencing the whole viral genome. The occlusion bodies (OBs) of EranNPV exhibited irregular polyhedral shapes containing multiple enveloped rod-shaped virions with a single nucleocapsid per virion. The EranNPV genome was 125,247 bp in length with a nucleotide distribution of 34.9% G+C. A total of 131 hypothetical open reading frames (ORFs) were identified, including the 38 baculovirus core genes and five multi-copy genes. Five homologous regions (hrs) were found in the EranNPV genome. Phylogeny and pairwise kimura 2-parameter analysis indicated that EranNPV was a novel group II alphabaculovirus and was most closely related to Apocheima cinerarium NPV (ApciNPV). Field trials showed that EranNPV was effective in controlling E. ankeraria in larch forests. The above results will be relevant to the functional research on EranNPV and promote the use of this virus as a biocontrol agent.

Revisiting the Neurospora crassa mitochondrial genome.

The mitochondrial genome of Neurospora crassa has been less studied than its nuclear counterpart, yet it holds great potential for understanding the diversity and evolution of this important fungus. Here we describe a new mitochondrial DNA (mtDNA) complete sequence of a N. crassa wild type strain. The genome with 64839bp revealed 21 protein-coding genes and several hypothetical open reading frames with no significant homology to any described gene. Five large repetitive regions were identified across the genome, including partial or complete genes. The largest repeated region holds a partial nd2 section that was also detected in Neurospora intermedia, suggesting a rearrangement that occurred before the N. crassa speciation. Interestingly, N. crassa has a palindrome adjacent to the partial nd2 repeated region possibly related to the genomic rearrangement, which is absent in N. intermedia. Finally, we compared the sequences of the three available N. crassa complete mtDNAs and found low levels of intraspecific variability. Most differences among strains were due to small indels in noncoding regions. The revisiting of the N. crassa mtDNA forms the basis for future studies on mitochondrial genome organization and variability.

A workflow to identify novel proteins based on the direct mapping of peptide-spectrum-matches to genomic locations

BackgroundSmall Proteins have received increasing attention in recent years. They have in particular been implicated as signals contributing to the coordination of bacterial communities. In genome annotations they are often missing or hidden among large numbers of hypothetical proteins because genome annotation pipelines often exclude short open reading frames or over-predict hypothetical proteins based on simple models. The validation of novel proteins, and in particular of small proteins (sProteins), therefore requires additional evidence. Proteogenomics is considered the gold standard for this purpose. It extends beyond established annotations and includes all possible open reading frames (ORFs) as potential sources of peptides, thus allowing the discovery of novel, unannotated proteins. Typically this results in large numbers of putative novel small proteins fraught with large fractions of false-positive predictions.ResultsWe observe that number and quality of the peptide-spectrum matches (PSMs) that map to a candidate ORF can be highly informative for the purpose of distinguishing proteins from spurious ORF annotations. We report here on a workflow that aggregates PSM quality information and local context into simple descriptors and reliably separates likely proteins from the large pool of false-positive, i.e., most likely untranslated ORFs. We investigated the artificial gut microbiome model SIHUMIx, comprising eight different species, for which we validate 5114 proteins that have previously been annotated only as hypothetical ORFs. In addition, we identified 37 non-annotated protein candidates for which we found evidence at the proteomic and transcriptomic level. Half (19) of these candidates have close functional homologs in other species. Another 12 candidates have homologs designated as hypothetical proteins in other species. The remaining six candidates are short (< 100 AA) and are most likely bona fide novel proteins.ConclusionsThe aggregation of PSM quality information for predicted ORFs provides a robust and efficient method to identify novel proteins in proteomics data. The workflow is in particular capable of identifying small proteins and frameshift variants. Since PSMs are explicitly mapped to genomic locations, it furthermore facilitates the integration of transcriptomics data and other sources of genome-level information.

Complete mitogenome assemblies from a panel of 13 diverse potato taxa

Mitochondrial DNA is maternally inherited and is shown to affect nuclear–cytoplasmic interactions in potato. Analyzing the mitogenome helps understand the evolutionary relationships and improve breeding programs in potato. We report complete mitogenome sequences from a panel of 13 potato accessions of various taxa. Each mitogenome has three independent circular molecules, except one of the S. bukasovii sample BUK2, which has a single circular molecule. Each mitogenome code for 37 non-redundant protein-coding genes, three rRNAs, 20 tRNAs, and 19 hypothetical open reading frames. Phylogenetic analysis reveals congruency between plastome and mitogenome phylogeny.

Novel modulators of p53-signaling encoded by unknown genes of emerging viruses.

The p53 transcription factor plays a key role both in cancer and in the cell-intrinsic response to infections. The ORFEOME project hypothesized that novel p53-virus interactions reside in hitherto uncharacterized, unknown, or hypothetical open reading frames (orfs) of human viruses. Hence, 172 orfs of unknown function from the emerging viruses SARS-Coronavirus, MERS-Coronavirus, influenza, Ebola, Zika (ZIKV), Chikungunya and Kaposi Sarcoma-associated herpesvirus (KSHV) were de novo synthesized, validated and tested in a functional screen of p53 signaling. This screen revealed novel mechanisms of p53 virus interactions and two viral proteins KSHV orf10 and ZIKV NS2A binding to p53. Originally identified as the target of small DNA tumor viruses, these experiments reinforce the notion that all viruses, including RNA viruses, interfere with p53 functions. These results validate this resource for analogous systems biology approaches to identify functional properties of uncharacterized viral proteins, long non-coding RNAs and micro RNAs.

Genome Analysis of a Novel Clade b Betabaculovirus Isolated from the Legume Pest Matsumuraeses phaseoli (Lepidoptera: Tortricidae).

Matsumuraeses phaseoli is a Lepidopteran pest that primarily feeds on numerous species of cultivated legumes, such as Glycine and Phaseolus. It is widely distributed in northeast Asia. A novel granulovirus, designated as Matsumuraeses phaseoli granulovirus (MaphGV), was isolated from pathogenic M. phaseoli larvae that dwell in rolled leaves of Astragalus membranaceus, a Chinese medicinal herb. In this study, using next-generation sequencing, we report the complete genome of MaphGV. MaphGV genome comprises a double-stranded DNA of 116,875 bp, with 37.18% GC content. It has 128 hypothetical open reading frames (ORFs). Among them, 38 are baculovirus core genes, 18 are lepidopteran baculovirus conserved genes, and 5 are unique to Baculoviridae. MaphGV has one baculovirus repeat ORF (bro) and three inhibitors of apoptosis proteins (iap), including a newfound iap-6. We found two atypical baculoviral homologous regions (hrs) and four direct repeats (drs) in the MaphGV genome. Based on phylogenetic analysis, MaphGV belongs to Clade b of Betabaculovirus and is closely related to Cydia pomonella granulovirus (CpGV) and Cryptophlebia leucotreta granulovirus (CrleGV). This novel baculovirus discovery and sequencing are invaluable in understanding the evolution of baculovirus and MaphGV may be a potential biocontrol agent against the bean ravaging pest.

Genome Analysis of a Novel Clade II.b Alphabaculovirus Obtained from Artaxa digramma.

Artaxa digramma is a lepidopteran pest distributed throughout southern China, Myanmar, Indonesia, and India. Artaxa digramma nucleopolyhedrovirus (ArdiNPV) is a specific viral pathogen of A. digramma and deemed as a promising biocontrol agent against the pest. In this study, the complete genome sequence of ArdiNPV was determined by deep sequencing. The genome of ArdiNPV contains a double-stranded DNA (dsDNA) of 161,734 bp in length and 39.1% G+C content. Further, 149 hypothetical open reading frames (ORFs) were predicted to encode proteins >50 amino acids in length, covering 83% of the whole genome. Among these ORFs, 38 were baculovirus core genes, 22 were lepidopteran baculovirus conserved genes, and seven were unique to ArdiNPV, respectively. No typical baculoviral homologous regions (hrs) were identified in the genome. ArdiNPV had five multi-copy genes including baculovirus repeated ORFs (bros), calcium/sodium antiporter B (chaB), DNA binding protein (dbp), inhibitor of apoptosis protein (iap), and p26. Interestingly, phylogenetic analyses showed that ArdiNPV belonged to Clade II.b of Group II Alphabaculoviruses, which all contain a second copy of dbp. The genome of ArdiNPV was the closest to Euproctis pseudoconspersa nucleopolyhedrovirus, with 57.4% whole-genome similarity. Therefore, these results suggest that ArdiNPV is a novel baculovirus belonging to a newly identified cluster of Clade II.b Alphabaculoviruses.

Genomic analysis of a novel isolate Heliothis virescens ascovirus 3i (HvAV-3i) and identification of ascoviral repeat ORFs (aros).

Ascoviruses are circular double-stranded DNA viruses that infect insects. Herein we sequenced and analyzed the genome of the previously unrecorded ascovirus isolate Heliothis virescens ascovirus 3i (HvAV-3i). The genome size is 185,650bp with 181 hypothetical open reading frames (ORFs). Additionally, definition based on ascovirus repeated ORFs (aros) is proposed; whereby the 29 aros from all sequenced Ascoviridae genomes are divided into six distinct groups. The topological relationship among the isolates of Heliothis virescens ascovirus 3a is (HvAV-3f, {HvAV-3h, [HvAV-3e, (HvAV-3g, HvAV-3i)]}) with every clade well supported by a Bayesian posterior probability of 1.00 and a Bootstrap value of 100%.

Passiflora plastome sequencing reveals widespread genomic rearrangements

AbstractAlthough past studies have included Passiflora among angiosperm lineages with highly rearranged plastid genomes (plastomes), knowledge about plastome organization in the genus is limited. So far only one draft and one complete plastome have been published. Expanded sampling of Passiflora plastomes is needed to understand the extent of the genomic rearrangement in the genus, which is also unusual in having biparental plastid inheritance and plastome‐genome incompatibility. We sequenced 15 Passiflora plastomes using either Illumina paired‐end or shotgun cloning and Sanger sequencing approaches. Assembled plastomes were annotated using Dual Organellar GenoMe Annotator (DOGMA) and tRNAscan‐SE. The Populus trichocarpa plastome was used as a reference to estimate genomic rearrangements in Passiflora by performing whole genome alignment in progressiveMauve. The phylogenetic distribution of rearrangements was plotted on the maximum likelihood tree generated from 64 plastid encoded protein genes. Inverted repeat (IR) expansion/contraction and loss of the two largest hypothetical open reading frames, ycf1 and ycf2, account for most plastome size variation, which ranges from 139 262 base pairs (bp) in P. biflora to 161 494 bp in P. pittieri. Passiflora plastomes have experienced numerous inversions, gene and intron losses along with multiple independent IR expansions and contractions resulting in a distinct organization in each of the three subgenera examined. Each Passiflora subgenus has a unique plastome structure in terms of gene content, order and size. The phylogenetic distribution of rearrangements shows that Passiflora has experienced widespread genomic changes, suggesting that such events may not be reliable phylogenetic markers.

Mitochondrial genome sequence of tuber-bearing wild potato, Solanum commersonii Dunal

We report two complete mitochondrial genome sequences of a tuber-bearing wild potato species (Solanum commersonii). The genomes are circular DNA molecules with lengths of 213,676 bp and 338,427 bp containing 80 nonredundant genes totally, including 34 protein-coding genes, 25 hypothetical open reading frames, 18 tRNA genes, and 3 rRNA genes. Phylogenetic analysis using common protein-coding sequences confirmed that S. commersonii belongs to the Solanoideae subfamily in the Solanaceae family.

The complete mitochondrial genome sequences of potato (Solanum tuberosum L., Solanaceae)

Potato (Solanum tuberosum) from the Solanaceae is the fourth most important food crop worldwide. In this study, five complete mitochondrial genome sequences of S. tuberosum were characterized through de novo assembly of whole genome sequencing data. The resulting circular mitochondrial DNA molecules ranged from 49,171 bp to 297,014 bp in size and contained a total of 80 non-redundant genes, comprising 34 protein-coding genes, 24 hypothetical open reading frames, 19 tRNA genes, and 3 rRNA genes. Phylogenetic analysis using common protein-coding sequences confirmed that S. tuberosum belongs to the Solanoideae subfamily in the Solanaceae family.

The complete mitochondrial genome of Hericium coralloides (Hericiaceae, Basidiomycota)

In this study, the complete mitochondrial genome of the medicinal mushroom Hericium coralloides (Hericiaceae, Basidiomycota) was sequenced. This mitochondrial genome is 72,961 bp in length and consisted of 14 protein-coding genes, 21 hypothetical open reading frames, 2 ribosomal RNA subunits and 27 transfer RNAs. The overall nucleotide composition of is 41.33% A, 40.71% T, 9.06% C and 8.90% G, with GC content of 17.96%. A phylogenetic tree with the complete mitochondrial genome sequences of Hericium coralloides together with 9 other affinis mushrooms was constructed. The newly achieved mitochondrial genome sequence seem to be useful for addressing taxonomic issues and studying related evolution events, which would contribute to enrich the fungal mitochondrial genome resource and promote the biological research.

Genome analysis of Heliothis virescens ascovirus 3h isolated from China.

No ascovirus isolated from China has been sequenced so far. Therefore, in this study, we aimed to sequence the genome of Heliothis virescens ascovirus 3h (HvAV-3h) using the 454 pyrosequencing technology. The genome was found to be 190,519-bp long with a G+C content of 45.5%. We also found that it encodes 185 hypothetical open reading frames (ORFs) along with at least 50 amino acids, including 181 ORFs found in other ascoviruses and 4 unique ORFs. Gene-parity plots and phylogenetic analysis revealed a close relationship between HvAV-3h and three other HvAV-3a strains and a distant relationship with Spodoptera frugiperda ascovirus 1a (SfAV-1a), Trichoplusia ni ascovirus 6a (TnAV-6a), and Diadromus pulchellus ascovirus 4a (DpAV-4a). Among the 185 potential genes encoded by the genome, 44 core genes were found in all the sequenced ascoviruses. In addition, 25 genes were found to be conserved in all ascoviruses except DpAV-4a. In the HvAV-3h genome, 24 baculovirus repeat ORFs (bros) were present, and the typical homologous repeat regions (hrs) were absent. This study supplies information important for understanding the conservation and functions of ascovirus genes as well as the variety of ascoviral genomes.

Transcriptome-based analysis of the saprophytic fungus Abortiporus biennis – response to oxalic acid

In this study, the transcriptomic-based response of the white rot fungus Abortiporus biennis to oxalic acid induction was reported. The whole transcriptome of A. biennis was analysed using the RNA-based sequencing technology and Solid 5500 platform. De novo assembly of reads generated 37,719 contigs. A molecular function for 26,280 unique transcripts was assigned. The analysis of the A. biennis transcriptome predicted 635 hypothetical open reading frames encoding carbohydrate active enzymes distributed in 122 families. 82 genes were identified, whose expression level was significantly changed after oxalic acid addition. Among them, 18 genes were up-regulated and 64 genes were down-regulated. Genes coding for putative cellulose and hemicellulose degrading enzymes were predominantly up-regulated in the mycelium induced with oxalic acid; it was in the case of cellulases and xylanases (hemicellulases), in particular, β-glucosidase and endo-1,4-β-xylanases. On the contrary, several genes coding for lignolytic enzymes were down-regulated, with the significant repression level in the case of versatile peroxidase.Finally, we identified putative genes involved in oxalate metabolism. Among the transcripts detected in the A. biennis transcriptome, one was annotated as coding for putative oxalate decarboxylase (ODC) and nine transcripts were annotated as formate dehydrogenases (FDH). The addition of oxalic acid to the culture caused upregulation of the gene coding for ODC and three genes for FDH. Amongst the transcripts of putative FDH genes, one designated as NODE_36057, demonstrated the highest induction level recorded in this study after the oxalic acid addition.

Genome Sequencing and Analysis of Catopsilia pomona nucleopolyhedrovirus: A Distinct Species in Group I Alphabaculovirus.

The genome sequence of Catopsilia pomona nucleopolyhedrovirus (CapoNPV) was determined by the Roche 454 sequencing system. The genome consisted of 128,058 bp and had an overall G+C content of 40%. There were 130 hypothetical open reading frames (ORFs) potentially encoding proteins of more than 50 amino acids and covering 92% of the genome. Among all the hypothetical ORFs, 37 baculovirus core genes, 23 lepidopteran baculovirus conserved genes and 10 genes conserved in Group I alphabaculoviruses were identified. In addition, the genome included regions of 8 typical baculoviral homologous repeat sequences (hrs). Phylogenic analysis showed that CapoNPV was in a distinct branch of clade “a” in Group I alphabaculoviruses. Gene parity plot analysis and overall similarity of ORFs indicated that CapoNPV is more closely related to the Group I alphabaculoviruses than to other baculoviruses. Interesting, CapoNPV lacks the genes encoding the fibroblast growth factor (fgf) and ac30, which are conserved in most lepidopteran and Group I baculoviruses, respectively. Sequence analysis of the F-like protein of CapoNPV showed that some amino acids were inserted into the fusion peptide region and the pre-transmembrane region of the protein. All these unique features imply that CapoNPV represents a member of a new baculovirus species.

Quantitative and functional characterization of the hyper-conserved protein of Prochlorococcus and marine Synechococcus.

A large fraction of any bacterial genome consists of hypothetical protein-coding open reading frames (ORFs). While most of these ORFs are present only in one or a few sequenced genomes, a few are conserved, often across large phylogenetic distances. Such conservation provides clues to likely uncharacterized cellular functions that need to be elucidated. Marine cyanobacteria from the Prochlorococcus/marine Synechococcus clade are dominant bacteria in oceanic waters and are significant contributors to global primary production. A Hyper Conserved Protein (PSHCP) of unknown function is 100% conserved at the amino acid level in genomes of Prochlorococcus/marine Synechococcus, but lacks homologs outside of this clade. In this study we investigated Prochlorococcus marinus strains MED4 and MIT 9313 and Synechococcus sp. strain WH 8102 for the transcription of the PSHCP gene using RT-Q-PCR, for the presence of the protein product through quantitative immunoblotting, and for the protein's binding partners in a pull down assay. Significant transcription of the gene was detected in all strains. The PSHCP protein content varied between 8±1 fmol and 26±9 fmol per ug total protein, depending on the strain. The 50 S ribosomal protein L2, the Photosystem I protein PsaD and the Ycf48-like protein were found associated with the PSHCP protein in all strains and not appreciably or at all in control experiments. We hypothesize that PSHCP is a protein associated with the ribosome, and is possibly involved in photosystem assembly.

The chloroplast genome of Hyoscyamus niger and a phylogenetic study of the tribe Hyoscyameae (Solanaceae).

The tribe Hyoscyameae (Solanaceae) is restricted to Eurasia and includes the genera Archihyoscyamus, Anisodus, Atropa, Atropanthe, Hyoscyamus, Physochlaina, Przewalskia and Scopolia. Even though the monophyly of Hyoscyameae is strongly supported, the relationships of the taxa within the tribe remain unclear. Chloroplast markers have been widely used to elucidate plant relationships at low taxonomic levels. Identification of variable chloroplast intergenic regions has been developed based on comparative genomics of chloroplast genomes, but these regions have a narrow phylogenetic utility. In this study, we present the chloroplast genome sequence of Hyoscyamus niger and make comparisons to other solanaceous plastid genomes in terms of gene order, gene and intron content, editing sites, origins of replication, repeats, and hypothetical open reading frames. We developed and sequenced three variable plastid markers from eight species to elucidate relationships within the tribe Hyoscyameae. The presence of a horizontally transferred intron in the mitochondrial cox1 gene of some species of the tribe is considered here a likely synapomorphy uniting five genera of the Hyoscyameae. Alternatively, the cox1 intron could be a homoplasious character acquired twice within the tribe. A homoplasious inversion in the intergenic plastid spacer trnC-psbM was recognized as a source of bias and removed from the data set used in the phylogenetic analyses. Almost 12 kb of plastid sequence data were not sufficient to completely resolve relationships among genera of Hyoscyameae but some clades were identified. Two alternative hypotheses of the evolution of the genera within the tribe are proposed.

Genome sequence and analysis of Buzura suppressaria nucleopolyhedrovirus: a group II Alphabaculovirus.

The genome of Buzura suppressaria nucleopolyhedrovirus (BusuNPV) was sequenced by 454 pyrosequencing technology. The size of the genome is 120,420 bp with 36.8% G+C content. It contains 127 hypothetical open reading frames (ORFs) covering 90.7% of the genome and includes the 37 conserved baculovirus core genes, 84 genes found in other baculoviruses, and 6 unique ORFs. No typical baculoviral homologous repeats (hrs) were present but the genome contained a region of repeated sequences. Gene Parity Plots revealed a 28.8 kb region conserved among the alpha- and beta-baculoviruses. Overall comparisons of BusuNPV to other baculoviruses point to a distinct species in group II Alphabaculovirus.