scholarly journals Cloning and Expression Analysis of Genes Encoding Lytic Endopeptidases L1 and L5 from Lysobacter sp. Strain XL1

2012 ◽  
Vol 78 (19) ◽  
pp. 7082-7089 ◽  
Author(s):  
Y. S. Lapteva ◽  
O. E. Zolova ◽  
M. G. Shlyapnikov ◽  
I. M. Tsfasman ◽  
T. A. Muranova ◽  
...  
Keyword(s):  
Stop Codon ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Cloning And Expression ◽  
Start Codon ◽  
Lytic Enzymes ◽  
Homologous Proteins ◽  
Logarithmic Growth Phase ◽  
Transcription Terminator ◽  
Content Type

ABSTRACTLytic enzymes are the group of hydrolases that break down structural polymers of the cell walls of various microorganisms. In this work, we determined the nucleotide sequences of theLysobactersp. strain XL1alpAandalpBgenes, which code for, respectively, secreted lytic endopeptidases L1 (AlpA) and L5 (AlpB).In silicoanalysis of their amino acid sequences showed these endopeptidases to be homologous proteins synthesized as precursors similar in structural organization: the mature enzyme sequence is preceded by an N-terminal signal peptide and a pro region. On the basis of phylogenetic analysis, endopeptidases AlpA and AlpB were assigned to the S1E family [clan PA(S)] of serine peptidases. Expression of thealpAandalpBopen reading frames (ORFs) inEscherichia coliconfirmed that they code for functionally active lytic enzymes. Each ORF was predicted to have the Shine-Dalgarno sequence located at a canonical distance from the start codon and a potential Rho-independent transcription terminator immediately after the stop codon. ThealpAandalpBmRNAs were experimentally found to be monocistronic; transcription start points were determined for both mRNAs. The synthesis of thealpAandalpBmRNAs was shown to occur predominantly in the late logarithmic growth phase. The amount ofalpAmRNA in cells ofLysobactersp. strain XL1 was much higher, which correlates with greater production of endopeptidase L1 than of L5.

scholarly journals utR.annotation: a tool for annotating genomic variants that could influence post-transcriptional regulation

2021 ◽  
Author(s):  
Yating Liu ◽  
Joseph Dougherty
Keyword(s):  
Stop Codon ◽  
R Package ◽  
Initiation Site ◽  
Open Reading Frames ◽  
Start Codon ◽  
Translation Initiation Site ◽  
Mouse Species ◽  
Translational Regulators ◽  
Post Transcriptional Regulation ◽  
Annotated Translation

Whole genome sequencing of patient populations is identifying thousands of new variants in UnTranslated Regions(UTRs). While the consequences of UTR mutations are not as easily predicted from primary sequence as coding mutations are, there are some known features of UTRs modulate their function. utR.annotation is an R package that can be used to annotate potential deleterious variants in the UTR regions for both human and mouse species. Given a CSV or VCF format variant file, utR.annotation provides information of each variant on whether and how it alters known translational regulators including:upstream Open Reading Frames (uORFs), upstream Kozak sequences, polyA signals, the Kozak sequence at the annotated translation initiation site, start codon, and stop codon, conservation scores in the variant position, and whether and how it changes ribosome loading based on a model from empirical data.

scholarly journals A Victorivirus and Two Novel Mitoviruses Co-Infected the Plant Pathogen Nigrospora oryzae

Viruses ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 83 ◽  
Author(s):  
Hong Liu ◽  
Rui Liu ◽  
Chang Li ◽  
Hui Wang ◽  
Hong Zhu ◽  
...  
Keyword(s):  
Stop Codon ◽  
Pathogenic Fungus ◽  
Genomic Analysis ◽  
Open Reading Frames ◽  
Start Codon ◽  
Phenotypic Change ◽  
Virus Like Particle ◽  
Viral Particles ◽  
Overlapping Open Reading Frames ◽  
Nigrospora Oryzae

Three dsRNAs, in sizes of approximately 2.5–5 kbp, were detected in the plant pathogenic fungus Nigrospora oryzae strain CS-7.5-4. Genomic analysis showed that the 5.0 kb dsRNA was a victorivirus named as Nigrospora oryzae victorivirus 2 (NoRV2). The genome of NoRV2 was 5166 bp in length containing two overlapping open reading frames (ORFs), ORF1 and ORF2. ORF1 was deduced to encode a coat protein (CP) showing homology to the CPs of viruses belonging to the Totiviridae family. The stop codon of ORF1 and the start codon of ORF2 were overlapped by the tetranucleotide sequence AUGA. ORF2 was predicted to encode an RNA-dependent RNA polymerase (RdRp), which was highly similar to the RdRps of victoriviruses. Virus-like particle examination demonstrated that the genome of NoRV2 was solely encapsidated by viral particles with a diameter of approximately 35 nm. The other two dsRNAs that were less than 3.0 kb were predicted to be the genomes of two mitoviruses, named as Nigrospora oryzae mitovirus 1 (NoMV1) and Nigrospora oryzae mitovirus 2 (NoMV2). Both NoMV1 and NoMV2 were A-U rich and with lengths of 2865 and 2507 bp, respectively. Mitochondrial codon usage inferred that each of the two mitoviruses contains a major large ORF encoding a mitoviral RdRp. Horizontal transfer experiments showed that the NoMV1 and NoMV2 could be cotransmitted horizontally via hyphal contact to other virus-free N. oryzae strains and causes phenotypic change to the recipient, such as an increase in growth rate. This is the first report of mitoviruses in N. oryzae.

scholarly journals Bul Proteins, a Nonredundant, Antagonistic Family of Ubiquitin Ligase Regulatory Proteins

2012 ◽  
Vol 11 (4) ◽  
pp. 463-470 ◽  
Author(s):  
Tatiana V. Novoselova ◽  
Kiran Zahira ◽  
Ruth-Sarah Rose ◽  
James A. Sullivan
Keyword(s):  
Stop Codon ◽  
Critical Role ◽  
Adaptor Proteins ◽  
Antagonistic Activity ◽  
Open Reading Frames ◽  
Gene Deletions ◽  
Content Type ◽  
Ww Domains ◽  
Cellular Pathways

ABSTRACT Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.

scholarly journals The Complete Mitochondrial Genome Sequences of the Philomycus bilineatus (Stylommatophora: Philomycidae) and Phylogenetic Analysis

Genes ◽  
2019 ◽  
Vol 10 (3) ◽  
pp. 198 ◽  
Author(s):  
Tiezhu Yang ◽  
Guolyu Xu ◽  
Bingning Gu ◽  
Yanmei Shi ◽  
Hellen Lucas Mzuka ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Mitochondrial Genome ◽  
Stop Codon ◽  
Phylogenetic Analyses ◽  
Complete Mitochondrial Genome ◽  
Transfer Rna ◽  
Amino Acid Sequences ◽  
Start Codon ◽  
Trna Genes ◽  
Rna Genes

The mitochondrial genome (mitogenome) can provide information for phylogenetic analyses and evolutionary biology. We first sequenced, annotated, and characterized the mitogenome of Philomycus bilineatus in this study. The complete mitogenome was 14,347 bp in length, containing 13 protein-coding genes (PCGs), 23 transfer RNA genes, two ribosomal RNA genes, and two non-coding regions (A + T-rich region). There were 15 overlap locations and 18 intergenic spacer regions found throughout the mitogenome of P. bilineatus. The A + T content in the mitogenome was 72.11%. All PCGs used a standard ATN as a start codon, with the exception of cytochrome c oxidase 1 (cox1) and ATP synthase F0 subunit 8 (atp8) with TTG and GTG. Additionally, TAA or TAG was identified as the typical stop codon. All transfer RNA (tRNA) genes had a typical clover-leaf structure, except for trnS1 (AGC), trnS2 (TCA), and trnK (TTT). A phylogenetic analysis with another 37 species of gastropods was performed using Bayesian inference, based on the amino acid sequences of 13 mitochondrial PCGs. The results indicated that P. bilineatus shares a close ancestry with Meghimatium bilineatum. It seems more appropriate to reclassify it as Arionoidea rather than Limacoidea, as previously thought. Our research may provide a new meaningful insight into the evolution of P. bilineatus.

scholarly journals Blackcurrant Leaf Chlorosis Associated Virus: Next-Generation Sequencing Reveals an Extraordinary Virus with Multiple Genomic Components Including Evidence of Circular RNA

2018 ◽  
Author(s):  
Delano James ◽  
James Phelan ◽  
Daniel Sanderson
Keyword(s):  
Next Generation Sequencing ◽  
Stop Codon ◽  
Distinct Species ◽  
Circular Rna ◽  
Open Reading Frames ◽  
Start Codon ◽  
Next Generation ◽  
Leaf Chlorosis ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Blackcurrant leaf chlorosis associated virus (BCLCaV) was detected recently by next-generation sequencing (NGS) and proposed as a new and distinct species in the genus Idaeovirus. Genomic components of BCLCaV that were detected and confirmed include: 1) RNA-1 that is monocistronic and encodes the replicase complex; 2) a bicistronic RNA-2 that encodes a movement protein (MP) and the coat protein (CP) of the virus, with open reading frames (ORF) that overlap by a single adenine (A) nucleotide (nt) representing the third position of an opal stop codon of the MP ORF2a and the first position of the start codon of the CP ORF2b; 3) a subgenomic form of RNA-2 (RNA-3) that contains ORF2b; and 4) a concatenated form of RNA-2 that consists of a complementary and inverted RNA-3 conjoined to the full-length RNA-2. Analysis of NGS-derived paired-end reads revealed the existence of bridge reads encompassing the 3’-terminus and 5’-terminus of RNA-2 or RNA-3 of BCLCaV. The full RNA-2 or RNA-3 could be amplified using outward facing or abutting primers; also RNA-2/RNA-3 could be detected even after three consecutive RNase R enzyme treatments with denaturation at 95 oC preceding each digestion. Evidence was obtained indicating that there are circular forms of BCLCaV RNA-2 and RNA-3.

scholarly journals Transcriptional and Translational Landscape of Equine Torovirus

2018 ◽  
Vol 92 (17) ◽  
Author(s):  
Hazel Stewart ◽  
Katherine Brown ◽  
Adam M. Dinan ◽  
Nerea Irigoyen ◽  
Eric J. Snijder ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Rna Synthesis ◽  
Gastrointestinal Disease ◽  
Ribosome Profiling ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Comparative Genomic ◽  
Human Pathogens ◽  
Content Type ◽  
Reading Frames

ABSTRACT The genus Torovirus (subfamily Torovirinae, family Coronaviridae, order Nidovirales) encompasses a range of species that infect domestic ungulates, including cattle, sheep, goats, pigs, and horses, causing an acute self-limiting gastroenteritis. Using the prototype species equine torovirus (EToV), we performed parallel RNA sequencing (RNA-seq) and ribosome profiling (Ribo-seq) to analyze the relative expression levels of the known torovirus proteins and transcripts, chimeric sequences produced via discontinuous RNA synthesis (a characteristic of the nidovirus replication cycle), and changes in host transcription and translation as a result of EToV infection. RNA sequencing confirmed that EToV utilizes a unique combination of discontinuous and nondiscontinuous RNA synthesis to produce its subgenomic RNAs (sgRNAs); indeed, we identified transcripts arising from both mechanisms that would result in sgRNAs encoding the nucleocapsid. Our ribosome profiling analysis revealed that ribosomes efficiently translate two novel CUG-initiated open reading frames (ORFs), located within the so-called 5′ untranslated region. We have termed the resulting proteins U1 and U2. Comparative genomic analysis confirmed that these ORFs are conserved across all available torovirus sequences, and the inferred amino acid sequences are subject to purifying selection, indicating that U1 and U2 are functionally relevant. This study provides the first high-resolution analysis of transcription and translation in this neglected group of livestock pathogens. IMPORTANCE Toroviruses infect cattle, goats, pigs, and horses worldwide and can cause gastrointestinal disease. There is no treatment or vaccine, and their ability to spill over into humans has not been assessed. These viruses are related to important human pathogens, including severe acute respiratory syndrome (SARS) coronavirus, and they share some common features; however, the mechanism that they use to produce sgRNA molecules differs. Here, we performed deep sequencing to determine how equine torovirus produces sgRNAs. In doing so, we also identified two previously unknown open reading frames “hidden” within the genome. Together these results highlight the similarities and differences between this domestic animal virus and related pathogens of humans and livestock.

scholarly journals Defective RNA of a Novel Mycovirus with High Transmissibility Detrimental to Biocontrol Properties of Trichoderma spp.

2019 ◽  
Vol 7 (11) ◽  
pp. 507 ◽  
Author(s):  
Jiaqi You ◽  
Kang Zhou ◽  
Xiaolin Liu ◽  
Mingde Wu ◽  
Long Yang ◽  
...  
Keyword(s):  
Stop Codon ◽  
Terrestrial Ecosystems ◽  
Biological Properties ◽  
Open Reading Frames ◽  
Plant Diseases ◽  
Biocontrol Agents ◽  
Start Codon ◽  
Trichoderma Spp ◽  
Trichoderma Species ◽  
Defective Rna

Trichoderma species are a group of fungi which is widely distributed in major terrestrial ecosystems; they are also commonly used as biocontrol agents for many plant diseases. A virus, namely Trichoderma harzianum hypovirus 1 (ThHV1), was identified in T. harzianum isolate T-70, and also infected isolate T-70D, together with its defective RNA (ThHV1-S). The ThHV1 genome possessed two Open Reading Frames (ORFs), namely ORF1 and ORF2. The start codon of ORF2 overlapped with the stop codon of ORF1 in a 43 nt long region. The polypeptide encoded by ORF2 of ThHV1 shared sequence similarities with those of betahypoviruses, indicating that ThHV1 is a novel member of Hypoviridea. Isolate T-70D, carrying both ThHV1 and ThHV1-S, showed abnormal biological properties, notably a decreased mycoparasitism ability when compared with isolate T-70. Both ThHV1 and ThHV1-S could be vertically transmitted to conidia and horizontally transmitted to T. harzianum isolate T-68 and T. koningiopsis T-51. The derivative strains carrying both ThHV1 and ThHV1-S showed decreased mycoparasitism ability, whereas strains carrying ThHV1 alone were normal, indicating that ThHV1-S is closely associated with the decreased mycoparasitism ability of T. harzianum isolate T-70D. ThHV1 was widely detected in isolates of T. harzianum, T. koningiopsis and T. atroviride originating from soil of China. Therefore, viruses in fungal biocontrol agents may also be a factor associated with the stability of their application.

scholarly journals Gene Cloning and Expression and Secretion ofListeria monocytogenes Bacteriophage-Lytic Enzymes inLactococcus lactis

2000 ◽  
Vol 66 (7) ◽  
pp. 2951-2958 ◽  
Author(s):  
Susanne Gaeng ◽  
Siegfried Scherer ◽  
Horst Neve ◽  
Martin J. Loessner
Keyword(s):  
Stop Codon ◽  
Surrounding Medium ◽  
Virus Multiplication ◽  
Starter Cultures ◽  
Proteolytic Processing ◽  
Lytic Activity ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Lytic Enzymes ◽  
Dairy Starter Cultures

ABSTRACT Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenesencode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa l-alanoyl-d-glutamate peptidase and Ply511 (36.5 kDa) acts asN-acetylmuramoyl-l-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introducedply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SP slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SP slpA–ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3′-end coding sequence ofSP slpA–ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511Δ(S262–K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competentSPSlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511ΔC into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

scholarly journals Distinct Actions by Paenibacillus sp. Strain E18 α-l-Arabinofuranosidases and Xylanase in Xylan Degradation

2013 ◽  
Vol 79 (6) ◽  
pp. 1990-1995 ◽  
Author(s):  
Pengjun Shi ◽  
Xiaoyan Chen ◽  
Kun Meng ◽  
Huoqing Huang ◽  
Yingguo Bai ◽  
...  
Keyword(s):  
High Performance ◽  
Synergistic Effects ◽  
Amino Acid Sequences ◽  
Water Soluble ◽  
Open Reading Frames ◽  
Content Type ◽  
Xylan Degradation ◽  
Wheat Arabinoxylan ◽  
Paenibacillus Sp ◽  
Family 10 Xylanase

ABSTRACTWe cloned aPaenibacillussp. strain E18 5.3-kb xylanolytic gene cluster that contains three open reading frames encoding two family 43 α-l-arabinofuranosidases (Abf43A and Abf43B) and one family 10 xylanase (XynBE18). The deduced amino acid sequences of Abf43A and Abf43B were at most 68% and 63% identical to those of two putative family 43 proteins fromClostridiumsp. strain DL-VIII (EHI98634.1 and EHI98635.1), respectively, but were only 11% identical to each other. Recombinant Abf43A and Abf43B had similar activities at 45°C and pH 6.0 but varied in thermostabilities and substrate specificities. Abf43B was active against only 4-nitrophenyl α-l-arabinofuranoside, whereas Abf43A acted on 4-nitrophenyl α-l-arabinofuranoside, wheat arabinoxylan, 4-nitrophenyl α-d-xylopyranoside, and sugar beet arabinan. The sequential and combined effects on xylan degradation by XynBE18, Abf43A, and Abf43B were characterized. For beechwood, birchwood, and oat spelt xylans as the substrates, synergistic effects were found when XynBE18 and Abf43A or Abf43B were incubated together and when the substrates were first incubated with Abf43A or Abf43B and then with XynBE18. Further high-performance liquid chromatography (HPLC) analysis showed that the amounts of xylobiose and xylose increased sharply in the aforementioned reactions. For water-soluble wheat arabinoxylan as the substrate, Abf43A not only released arabinose but also had a synergistic effect with XynBE18. Synergy may arise as the result of removal of arabinose residues from xylans by α-l-arabinofuranosidases, which eliminates steric hindrance caused by the arabinose side chains and which allows xylanases to then degrade the xylan backbone, producing short xylooligosaccharides.

scholarly journals Functional Identification of Two Novel Genes from Pseudomonas sp. Strain HZN6 Involved in the Catabolism of Nicotine

2012 ◽  
Vol 78 (7) ◽  
pp. 2154-2160 ◽  
Author(s):  
Jiguo Qiu ◽  
Yun Ma ◽  
Yuezhong Wen ◽  
Liansheng Chen ◽  
Lifei Wu ◽  
...  
Keyword(s):  
Amine Oxidase ◽  
Bartonella Henselae ◽  
Insertion Site ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Functional Identification ◽  
Novel Genes ◽  
Content Type ◽  
Transposon Insertion ◽  
Arthrobacter Nicotinovorans

ABSTRACTNicotine is a natural alkaloid produced by tobacco plants, and the mechanisms of its catabolism by microorganisms are diverse. In the present study, we reported the mutation, cloning, and identification of two novel genes involved in nicotine degradation from the newly isolatedPseudomonassp. strain HZN6. Transposon mutagenesis identified a HZN6 mutant in which the nicotine-degrading pathway was blocked at pseudooxynicotine. A 3,874-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR. Two open reading frames (designatedpaoandsap) were analyzed, and the deduced amino acid sequences shared 29% identity with 6-hydroxy-l-nicotine oxidase fromArthrobacter nicotinovoransand 49% identity with an aldehyde dehydrogenase fromBartonella henselae. Bothpaoandsapwere cloned and functionally expressed in recombinantEscherichia coliBL21. Thepaogene encoded a novel pseudooxynicotine amine oxidase with noncovalently bound flavin adenine dinucleotide (FAD) and exhibited substrate specificity removing the methylamine from pseudooxynicotine with the formation of 3-succinoylsemialdehyde-pyridine and hydrogen dioxide. Thesapgene encoded a NADP+-dependent 3-succinoylsemialdehyde-pyridine dehydrogenase that catalyzed the dehydrogenation of 3-succinoylsemialdehyde-pyridine to 3-succinoyl-pyridine. Genetic analyses indicated that thepaogene played an essential role in nicotine or pseudooxynicotine mineralization in strain HZN6, whereas thesapgene did not. This study provides novel insight into the nicotine-degrading mechanism at the genetic level inPseudomonasspp.

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