scholarly journals Insertion of Exogenous Genes within the ORF1a Coding Region of Porcine Astrovirus

Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2119
Author(s):  
Yanjie Du ◽  
Teng Liu ◽  
Yifeng Qin ◽  
Qinting Dong ◽  
Ying Chen ◽  
...  
Keyword(s):  
Hypervariable Region ◽  
Coiled Coil ◽  
Genetically Engineered ◽  
Rapid Screening ◽  
Insertion Mutagenesis ◽  
Coding Region ◽  
Reading Frame ◽  
Promising Tool ◽  
Porcine Astrovirus ◽  
Insertion Sites

A tagged or reporter astrovirus can be a valuable tool for the analysis of various aspects of the virus life cycle, and to aid in the development of genetically engineered astroviruses as vectors. Here, transposon-mediated insertion mutagenesis was used to insert a 15-nucleotide (nt) sequence into random sites of open reading frame 1a (ORF1a) based on an infectious full-length cDNA clone of porcine astrovirus (PAstV). Five sites in the predicted coiled-coil structures (CC), genome-linked protein (VPg), and hypervariable region (HVR) in ORF1a of the PAstV genome were identified that could tolerate random 15 nt insertions. Incorporation of the commonly used epitope tags, His, Flag, and HA, into four of the five insertion sites permitted the production of infectious viruses and allowed recognition by specifically tagged monoclonal antibodies. The results of immuno-fluorescent assays showed that Flag-tagged ORF1a protein overlapped partially with capsid and ORF2b proteins in the cytoplasm. Improved light-oxygen-voltage (iLOV) gene was also introduced at the insertion sites of CC, VPg, and HVR. Only one viable recombinant reporter PAstV expressing iLOV inserted in HVR was recovered. Biological analysis of the reporter virus showed that it displayed similar growth characteristics, and yet produced less infectious virus particles, when compared with the parental virus. The recombinant virus carrying the iLOV fused with the HVR of ORF1a protein maintained its stability and showed green fluorescence after 15 passages in cell cultures. The resultant fluorescently tagged virus could provide a promising tool for the rapid screening of antiviral drugs as well as allowing the visualization of PAstV infection and replication in living cells.

scholarly journals Personalized Development of Antisense Oligonucleotides for Exon Skipping Restores Type XVII Collagen Expression in Junctional Epidermolysis Bullosa

2021 ◽  
Vol 22 (7) ◽  
pp. 3326
Author(s):  
Michael Ablinger ◽  
Thomas Lettner ◽  
Nicole Friedl ◽  
Hannah Potocki ◽  
Theresa Palmetzhofer ◽  
...  
Keyword(s):  
Epidermolysis Bullosa ◽  
Antisense Oligonucleotides ◽  
Exon Skipping ◽  
Junctional Epidermolysis Bullosa ◽  
Reading Frame ◽  
Collagen Expression ◽  
Promising Tool ◽  
Mucous Membranes ◽  
Personalized Approach ◽  
Xvii Collagen

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal–epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

The destabilizing elements in the coding region of c-fos mRNA are recognized as RNA

1993 ◽  
Vol 13 (8) ◽  
pp. 5034-5042
Author(s):  
C L Wellington ◽  
M E Greenberg ◽  
J G Belasco
Keyword(s):  
Codon Usage ◽  
Mammalian Cells ◽  
Mrna Decay ◽  
Polypeptide Chain ◽  
Coding Region ◽  
Protein Coding ◽  
Present Evidence ◽  
Reading Frame ◽  
Nascent Polypeptide

The protein-coding region of the c-fos proto-oncogene transcript contains elements that direct the rapid deadenylation and decay of this mRNA in mammalian cells. The function of these coding region instability determinants requires movement of ribosomes across mRNAs containing them. Three types of mechanisms could account for this translational requirement. Two of these possibilities, (i) that rapid mRNA decay might be mediated by the nascent polypeptide chain and (ii) that it might result from an unusual codon usage, have experimental precedent. Here, we present evidence that the destabilizing elements in the c-fos coding region are not recognized in either of these two ways. Instead, the ability of the c-fos coding region to function as a potent mRNA destabilizer when translated in the +1 reading frame indicates that the signals for rapid deadenylation and decay reside in the sequence or structure of the RNA comprising this c-fos domain.

Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases

1988 ◽  
Vol 8 (8) ◽  
pp. 3439-3447 ◽  
Author(s):  
W Bajwa ◽  
T E Torchia ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Regulatory Gene ◽  
Data Bank ◽  
Noncoding Region ◽  
Striking Similarity ◽  
Coding Region ◽  
Reading Frame ◽  
Carbon Regulation ◽  
Sequence Similarities

GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GAL1, GAL7, GAL10, and MEL1 genes, the GAL3 gene is severely glucose repressed. Like the MEL1 gene, but in contrast to the GAL1, GAL7, and GAL10 genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGal elements of the other galactose-melibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GAL1-specified galactokinase of Saccharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.

IDENTIFICATION, SEQUENCE AND EXPRESSION OF A CRUSTACEAN CARDIOACTIVE PEPTIDE (CCAP) GENE IN THE MOTHMANDUCA SEXTA

2001 ◽  
Vol 204 (16) ◽  
pp. 2803-2816 ◽  
Author(s):  
P. K. LOI ◽  
S. A. EMMAL ◽  
Y. PARK ◽  
N. J. TUBLITZ
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Gene Structure ◽  
Expression Patterns ◽  
Amino Acid Residues ◽  
Coding Region ◽  
Genome Database ◽  
Reading Frame ◽  
Crustacean Cardioactive Peptide ◽  
Embryonic Life

SUMMARYThe crustacean cardioactive peptide (CCAP) gene was isolated from the tobacco hawkmoth Manduca sexta. The gene has an open reading frame of 125 amino acid residues containing a single, complete copy of CCAP. Analysis of the gene structure revealed three introns interrupting the coding region. A comparison of the M. sexta CCAP gene with the Drosophila melanogaster genome database reveals significant similarities in sequence and gene structure.The spatial and temporal expression patterns of the CCAP gene in the M. sexta central nervous system were determined in all major post-embryonic stages using in situ hybridization techniques. The CCAP gene is expressed in a total of 116 neurons in the post-embryonic M. sextacentral nervous system. Nine pairs of cells are observed in the brain, 4.5 pairs in the subesophageal ganglion, three pairs in each thoracic ganglion(T1-T3), three pairs in the first abdominal ganglion (A1), five pairs each in the second to sixth abdominal ganglia (A2-A6) and 7.5 pairs in the terminal ganglion. The CCAP gene is expressed in every ganglion in each post-embryonic stage, except in the thoracic ganglia of first- and second-instar larvae. The number of cells expressing the CCAP gene varies during post-embryonic life,starting at 52 cells in the first instar and reaching a maximum of 116 shortly after pupation. One set of thoracic neurons expressing CCAP mRNA shows unusual variability in expression levels immediately prior to larval ecdysis. Using previously published CCAP immunocytochemical data, it was determined that 91 of 95 CCAP-immunopositive neurons in the M. sexta central nervous system also express the M. sexta CCAP gene, indicating that there is likely to be only a single CCAP gene in M. sexta.

KIF3B gene silent variant leading to sperm morphology and motility defects and male infertility

2021 ◽  
Author(s):  
Raheleh Heydari ◽  
Mehrshad Seresht-Ahmadi ◽  
Shahab Mirshahvaladi ◽  
Marjan Sabbaghian ◽  
Anahita Mohseni-Meybodi
Keyword(s):  
Male Infertility ◽  
Protein Expression ◽  
Binding Site ◽  
Sperm Count ◽  
Critical Role ◽  
Coiled Coil ◽  
Control Group ◽  
Coding Region ◽  
Exon 2 ◽  
Coiled Coil Domain

Abstract Sperm structural and functional defects are leading causes of male infertility. Patients with immotile sperm disorders suffer from axoneme failure and show a significant reduction in sperm count. The kinesin family member 3B (KIF3B) is one of the genes involved in the proper formation of sperm with a critical role in intraflagellar and intramanchette transport. A part of exon 2 and exons 3–5 of the KIF3B encodes a protein coiled-coil domain that interacts with IFT20 from the IFT protein complex. In the present study, the coding region of KIF3B coiled-coil domain was assessed in 88 oligoasthenoteratozoospermic patients, and the protein expression was evaluated in the mature spermatozoa of the case and control groups using immunocytochemistry and western blotting. According to the results, there was no genetic variation in the exons 3–5 of the KIF3B, but a new A > T variant was identified within the exon 2 in 30 patients, where nothing was detected in the control group. In contrast to healthy individuals, significantly reduced protein expression was observable in oligoasthenoteratozoospermic (OAT) patients carrying variation where protein organization was disarranged, especially in the principal piece and midpiece of the sperm tail. Besides, the protein expression level was lower in the patients’ samples compared to that of the control group. According to the results of the present study the NM_004798.3:c.1032A > T, p.Pro344 = variant; which has been recently submitted to the Clinvar database; although synonymous, causes alterations in the transcription factor binding site, exon skipping, and also exonic splicing enhancer-binding site. Therefore, KIF3B can play an important role in spermatogenesis and the related protein reduction can cause male infertility.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

Cloning of the Nocardia corallina polyhydroxyalkanoate synthase gene and production of poly-(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly-(3-hydroxyvalerate-co-3-hydroxyheptanoate)

1998 ◽  
Vol 44 (7) ◽  
pp. 687-691 ◽  
Author(s):  
Brian Hall ◽  
Jennifer Baldwin ◽  
Ho Gun Rhie ◽  
Douglas Dennis
Keyword(s):  
Fatty Acids ◽  
Ralstonia Eutropha ◽  
Carbon Sources ◽  
Pha Synthase ◽  
Broad Host Range ◽  
Coding Region ◽  
Reading Frame ◽  
Nocardia Corallina ◽  
Odd Chain Fatty Acids ◽  
Chain Fatty Acids

The polyhydroxyalkanoate (PHA) synthase gene (phaCNc) from Nocardia corallina was identified in a lambda library on a 6-kb BamHI fragment. A 2.8-kb XhoII subfragment was found to contain the ntact PHA synthase. This 2.8-kb fragment was subjected to DNA sequencing and was found to contain the coding region for the PHA synthase and a small downstream open reading frame of unknown function. On the basis of DNA sequence, phaCNc is closest in homology to the PHA synthases (phaCPaI and phaCPaII) of Pseudomonas aeruginosa (approximately 41% identity and 55% similarity). The 2.8-kb XhoII fragment containing phaCNc was subcloned into broad host range mobilizable plasmids and transferred into Escherichia coli, Klebsiella aerogenes (both containing a plasmid bearing phaA and phaB from Ralstonia eutropha), and PHA-negative strains of R. eutropha and Pseudomonas putida. The recombinant strains were grown on various carbon sources and the resulting polymers were analyzed. In these strains, the PHA synthase from N. corallina was able to mediate the production of poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) containing high levels of 3-hydroxyhexanoate when grown on hexanoate and larger even-chain fatty acids and poly(3-hydroxyvalerate-co-3-hydroxyheptanoate) containing high levels of 3-hydroxyheptanoate when grown on heptanoate or larger odd-chain fatty acids. Key words: polyhydroxyalkanoates (PHAs), Nocardia corallina, biodegradable, polyester.

scholarly journals Novel TRIM5 Isoforms Expressed by Macaca nemestrina

2007 ◽  
Vol 81 (22) ◽  
pp. 12210-12217 ◽  
Author(s):  
Greg Brennan ◽  
Yury Kozyrev ◽  
Toshiaki Kodama ◽  
Shiu-Lok Hu
Keyword(s):  
Coiled Coil ◽  
Macaca Nemestrina ◽  
Cdna Clones ◽  
Reading Frame ◽  
Spry Domain ◽  
Immunodeficiency Virus ◽  
Hiv Type 1 ◽  
Hiv 1

ABSTRACT The TRIM5 family of proteins contains a RING domain, one or two B boxes, and a coiled-coil domain. The TRIM5α isoform also encodes a C-terminal B30.2(SPRY) domain, differences within which define the breadth and potency of TRIM5α-mediated retroviral restriction. Because Macaca nemestrina animals are susceptible to some human immunodeficiency virus (HIV) isolates, we sought to determine if differences exist in the TRIM5 gene and transcripts of these animals. We identified a two-nucleotide deletion (Δ2) in the transcript at the 5′ terminus of exon 7 in all M. nemestrina TRIM5 cDNA clones examined. This frameshift results in a truncated protein of 300 amino acids lacking the B30.2(SPRY) domain, which we have named TRIM5θ. This deletion is likely due to a single nucleotide polymorphism that alters the 3′ splice site between intron 6 and exon 7. In some clones, a deletion of the entire 27-nucleotide exon 7 (Δexon7) resulted in the restoration of the TRIM5 open reading frame and the generation of another novel isoform, TRIM5η. There are 18 amino acid differences between M. nemestrina TRIM5η and Macaca mulatta TRIM5α, some of which are at or near locations previously shown to affect the breadth and potency of TRIM5α-mediated restriction. Infectivity assays performed on permissive CrFK cells stably transduced with TRIM5η or TRIM5θ show that these isoforms are incapable of restricting either HIV type 1 (HIV-1) or simian immunodeficiency virus infection. The expression of TRIM5 alleles incapable of restricting HIV-1 infection may contribute to the previously reported increased susceptibility of M. nemestrina to HIV-1 infection in vivo.

scholarly journals Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

2002 ◽  
Vol 76 (3) ◽  
pp. 1450-1460 ◽  
Author(s):  
S. Spaderna ◽  
H. Blessing ◽  
E. Bogner ◽  
W. Britt ◽  
M. Mach
Keyword(s):  
Human Cytomegalovirus ◽  
Structural Component ◽  
Laboratory Strain ◽  
Immunoblot Analysis ◽  
Protein Product ◽  
Clinical Isolates ◽  
Coding Region ◽  
Reading Frame ◽  
Infected Cells ◽  
Strain Ad169

ABSTRACT Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.

scholarly journals The Two Drosophila Telomeric Transposable Elements Have Very Different Patterns of Transcription

1999 ◽  
Vol 19 (1) ◽  
pp. 873-881 ◽  
Author(s):  
O. N. Danilevskaya ◽  
K. L. Traverse ◽  
N. C. Hogan ◽  
P. G. DeBaryshe ◽  
M. L. Pardue
Keyword(s):  
Transposable Elements ◽  
Sequence Similarity ◽  
Sequence Divergence ◽  
Full Length ◽  
Coding Region ◽  
Free Standing ◽  
Reading Frame ◽  
Sequence Organization ◽  
Sense Strand ◽  
Cell Fractions

ABSTRACT The transposable elements HeT-A and TARTconstitute the telomeres of Drosophila chromosomes. Both are non-long terminal repeat (LTR) retrotransposons, sharing the remarkable property of transposing only to chromosome ends. In addition, strong sequence similarity of their gag proteins indicates that these coding regions share a common ancestor. These findings led to the assumption that HeT-A andTART are closely related. However, we now find that these elements produce quite different sets of transcripts. HeT-Aproduces only sense-strand transcripts of the full-length element, whereas TART produces both sense and antisense full-length RNAs, with antisense transcripts in more than 10-fold excess over sense RNA. In addition, features of TART sequence organization resemble those of a subclass of non-LTR elements characterized by unequal terminal repeats. Thus, the ancestral gag sequence appears to have become incorporated in two different types of elements, possibly with different functions in the telomere. HeT-Atranscripts are found in both nuclear and cytoplasmic cell fractions, consistent with roles as both mRNA and transposition template. In contrast, both sense and antisense TART transcripts are almost entirely concentrated in nuclear fractions. Also,TART open reading frame 2 probes detect a cytoplasmic mRNA for reverse transcriptase (RT), with no similarity to TARTsequence 5′ or 3′ of the RT coding region. This RNA could be a processed TART transcript or the product of a “free-standing” RT gene. Either origin would be novel. The distinctive transcription patterns of both HeT-A andTART are conserved in Drosophila yakuba, despite significant sequence divergence. The conservation argues that these sets of transcripts are important to the function(s) ofHeT-A and TART.

Sign in / Sign up

Export Citation Format

Share Document