Conserved regions (human accelerated regions)

2018 ◽  
pp. 1-3
Author(s):  
Evelyn Jagoda ◽  
Terence D. Capellini
Keyword(s):  
Conserved Regions ◽  
Human Accelerated Regions

scholarly journals Development of Bacteriophage Virus-Like Particle Vaccines Displaying Conserved Epitopes of Dengue Virus Non-Structural Protein 1

Vaccines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 726
Author(s):  
Nikole L. Warner ◽  
Kathryn M. Frietze
Keyword(s):  
Dengue Virus ◽  
Population Based ◽  
Ns1 Protein ◽  
Structural Protein ◽  
Denv Serotypes ◽  
Conserved Regions ◽  
Virus Like Particle ◽  
Dependent Cell ◽  
Infected Cells ◽  
Plasma Leakage

Dengue virus (DENV) is a major global health problem, with over half of the world’s population at risk of infection. Despite over 60 years of efforts, no licensed vaccine suitable for population-based immunization against DENV is available. Here, we describe efforts to engineer epitope-based vaccines against DENV non-structural protein 1 (NS1). NS1 is present in DENV-infected cells as well as secreted into the blood of infected individuals. NS1 causes disruption of endothelial cell barriers, resulting in plasma leakage and hemorrhage. Immunizing against NS1 could elicit antibodies that block NS1 function and also target NS1-infected cells for antibody-dependent cell cytotoxicity. We identified highly conserved regions of NS1 from all four DENV serotypes. We generated synthetic peptides to these regions and chemically conjugated them to bacteriophage Qβ virus-like particles (VLPs). Mice were immunized two times with the candidate vaccines and sera were tested for the presence of antibodies that bound to the cognate peptide, recombinant NS1 from all four DENV serotypes, and DENV-2-infected cells. We found that two of the candidate vaccines elicited antibodies that bound to recombinant NS1, and one candidate vaccine elicited antibodies that bound to DENV-infected cells. These results show that an epitope-specific vaccine against conserved regions of NS1 could be a promising approach for DENV vaccines or therapeutics to bind circulating NS1 protein.

Demonstration of the indolepyruvate decarboxylase gene homologue in different auxin-producing species of the Enterobacteriaceae

1994 ◽  
Vol 40 (12) ◽  
pp. 1072-1076 ◽  
Author(s):  
Wolfgang Zimmer ◽  
Barbara Hundeshagen ◽  
Edith Niederau
Keyword(s):  
Polymerase Chain Reaction ◽  
Klebsiella Oxytoca ◽  
Pyruvate Decarboxylase ◽  
Enterobacter Agglomerans ◽  
Chain Reaction ◽  
Acetic Acid Production ◽  
Conserved Regions ◽  
Polymerase Chain ◽  
Gene Homologue ◽  
Indole 3 Acetic Acid

Different Enterobacteriaceae were assayed for their ability to produce the plant hormone indole-3-acetate with the aim to study the distribution of the indole-3-pyruvate pathway, which is known to be involved in the production of indole-3-acetate in a root-associated Enterobacter cloacae strain. Other E. cloacae strains, and also Enterobacter agglomerans strains, Pantoea agglomerans, Klebsiella aerogenes, and Klebsiella oxytoca were found to convert tryptophan into indole-3-acetate. As it was also intended to identify the conserved regions of the indole-3-pyruvate decarboxylase, which is involved in producing indole-3-acetate in the E. cloacae strain, oligonucleotide primers were synthesized for different regions of the corresponding gene. One pair of these primers allowed us to amplify a segment of the predicted size by the polymerase chain reaction with DNA of the seven different Enterobacteriaceae that produce indole-3-acetate. Segments of five strains were cloned and sequenced. All sequences showed significant homology to the indole-3-pyruvate decarboxylase gene. As in addition a positive DNA–DNA hybridization signal was detected in the seven strains using the E. cloacae or E. agglomerans segments as a probe, indole-3-acetate biosynthesis is suggested to be catalyzed via the indole-3-pyruvate pathway not only in E. cloacae but also in the other soil-living Enterobacteriaceae. Conserved regions were detected in the indole-3-decarboxylase by alignment of the now-available five different partial sequences. These regions should enable identification of the gene in other bacterial families or even in plants.Key words: indole-3-pyruvate decarboxylase, indole-3-acetic acid production, auxin, polymerase chain reaction, Enterobacteriaceae.

Strategy for the enrichment of protein biomarkers from diverse bacterial select agents

2021 ◽  
Vol 28 ◽  
Author(s):  
Sasikumar Sabna ◽  
Dev Vrat Kamboj ◽  
Ravi Bhushan Kumar ◽  
Prabhakar Babele ◽  
Sakshi Rajoria ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Species Identification ◽  
Immune Serum ◽  
Tandem Ms ◽  
Environmental Matrices ◽  
Bacterial Proteins ◽  
Conserved Regions ◽  
Groel Protein ◽  
Ms Analysis ◽  
Enrichment Strategy

Background: Some pathogenic bacteria can be potentially used for nefarious applications in the event of bioterrorism or biowarfare. Accurate identification of biological agent from clinical and diverse environmental matrices is of paramount importance for implementation of medical countermeasures and biothreat mitigation. Objective: A novel methodology is reported here for the development of a novel enrichment strategy for the generally conserved abundant bacterial proteins for an accurate downstream species identification using tandem MS analysis in biothreat scenario. Methods: Conserved regions in the common bacterial protein markers were analyzed using bioinformatic tools and stitched for a possible generic immuno-capture for an intended downstream MS/MS analysis. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of 60 kDa chaperonin GroEL. Hyper-immune serum was raised against recombinant synthetic GroEL protein. Results: The conserved regions of common bacterial proteins were stitched for a possible generic immuno-capture and subsequent specific identification by tandem MS using variable regions of the molecule. Phylogenetic analysis of selected proteins was carried out and synthetic constructs were generated for the expression of conserved stitched regions of GroEL. In a proof-of-concept study, hyper-immune serum raised against recombinant synthetic GroEL protein exhibited reactivity with ~60 KDa proteins from the cell lysates of three bacterial species tested. Conclusion: The envisaged methodology can lead to the development of a novel enrichment strategy for the abundant bacterial proteins from complex environmental matrices for the downstream species identification with increased sensitivity and substantially reduce the time-to-result.

scholarly journals Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction. Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA

1992 ◽  
Vol 287 (1) ◽  
pp. 195-200 ◽  
Author(s):  
J S Miles
Keyword(s):  
Polymerase Chain Reaction ◽  
Schizosaccharomyces Pombe ◽  
Binding Domain ◽  
Amino Acid Sequences ◽  
Chain Reaction ◽  
Conserved Regions ◽  
Reductase Gene ◽  
Degenerate Oligonucleotide ◽  
Polymerase Chain ◽  
Cytochrome P 450

1. Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance. 2. Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe. 3. A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774. This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium. 4. Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions. 5. The Sc. pombe cytochrome P-450 reductase gene was shown to contain no introns.

scholarly journals Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Wenhua Kuang ◽  
Huanyu Zhang ◽  
Manli Wang ◽  
Ning-Yi Zhou ◽  
Fei Deng ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Active Site ◽  
Salt Bridge ◽  
Sulfhydryl Oxidase ◽  
Dimer Interface ◽  
Conserved Regions ◽  
Occlusion Bodies ◽  
Budded Virus ◽  
And Function ◽  
Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

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