The TOGp protein is a new human microtubule-associated protein homologous to the Xenopus XMAP215

1998 ◽  
Vol 111 (10) ◽  
pp. 1371-1383 ◽  
Author(s):  
S. Charrasse ◽  
M. Schroeder ◽  
C. Gauthier-Rouviere ◽  
F. Ango ◽  
L. Cassimeris ◽  
...  
Keyword(s):  
Sequence Comparison ◽  
Immunofluorescence Microscopy ◽  
Open Reading Frame ◽  
Microtubule Assembly ◽  
Human Cell Lines ◽  
Reading Frame ◽  
Protein Map ◽  
Dividing Cells ◽  
Perinuclear Cytoplasm

We have recently identified a 6,449 bp cDNA, termed colonic, hepatic tumor over-expressed gene (ch-TOG), that is highly expressed in human tumors and brain. Its single open reading frame encodes a putative 218,000 Da polypeptide, TOGp. Antibodies generated against a bacterially expressed TOGp fragment specifically recognize a 218, 000 Da polypeptide in two human cell lines and in brain. Immunofluorescence microscopy using affinity-purified TOGp antibodies revealed that the distribution of TOGp was dependent upon the cell cycle. During interphase, TOGp was found concentrated in the perinuclear cytoplasm, where it co-localized with ER markers. In contrast anti-TOGp antibodies stained centrosomes and spindles in mitotic cells. TOGp co-sedimented with taxol-stabilized microtubules in vitro. Moreover, a TOGp enriched fraction promotes microtubule assembly both in solution and from nucleation centers. Finally, sequence comparison and immunologic cross-reaction suggest that TOGp is homologous to XMAP215, a previously described microtubule associated protein (MAP) from Xenopus eggs. These results suggest that TOGp is a MAP and that TOGp/XMAP215 may be necessary for microtubules rearrangements and spindle assembly in rapidly dividing cells.

scholarly journals Characterization of proteins immunologically related to brain microtubule-associated protein MAP-1B in non-neural cells

1989 ◽  
Vol 92 (4) ◽  
pp. 607-620
Author(s):  
J. Diaz-Nido ◽  
J. Avila
Keyword(s):  
Mitotic Spindle ◽  
Polyclonal Antibodies ◽  
Microtubule Assembly ◽  
Neural Cells ◽  
Microtubule Network ◽  
Cytoplasmic Microtubule ◽  
Protein Map ◽  
Dividing Cells

Brain microtubule-associated protein MAP-1 is composed of at least two polypeptides, MAP-1A and MAP-1B, which are among the main components of the neural cytoskeleton. Specific monoclonal and polyclonal antibodies against MAP-1B stain nuclei, mitotic spindles, centrosomes and the cytoplasmic microtubule network of different non-neural cells studied by immunofluorescence microscopy. It appears that these cells contain two proteins of 325K and 220K (K = 10(3) Mr), which are immunologically related to brain MAP-1B. The 325K protein, which is localized to the cytoplasmic microtubule network, the centrosome and the mitotic spindle, seems to be structurally related to the neural MAP-1B, as judged from their similar peptide maps and phosphorylation patterns. The 220K protein, which is localized to the nuclear matrix in interphase cells and to the mitotic spindle in dividing cells, has a proteolytic profile different from that of neural MAP-1B and is phosphorylated to a much lesser extent than the 325K protein. Both proteins bind tubulin in vitro, which suggests that they may participate in microtubule assembly in vivo; the 325K protein could perform such a role during the entire cell cycle, while the 220K protein could be implicated in the formation of the mitotic spindle.

The Drosophila suppressor of sable gene encodes a polypeptide with regions similar to those of RNA-binding proteins

1991 ◽  
Vol 11 (2) ◽  
pp. 894-905
Author(s):  
R A Voelker ◽  
W Gibson ◽  
J P Graves ◽  
J F Sterling ◽  
M T Eisenberg
Keyword(s):  
Rna Processing ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Open Reading Frame ◽  
In Vitro Translation ◽  
Rna Recognition Motif ◽  
Reading Frame ◽  
Cdna Sequences ◽  
Suppressor Of Sable

The nucleotide sequence of the Drosophila melanogaster suppressor of sable [su(s)] gene has been determined. Comparison of genomic and cDNA sequences indicates that an approximately 7,860-nucleotide primary transcript is processed into an approximately 5-kb message, expressed during all stages of the life cycle, that contains an open reading frame capable of encoding a 1,322-amino-acid protein of approximately 150 kDa. The putative protein contains an RNA recognition motif-like region and a highly charged arginine-, lysine-, serine-, aspartic or glutamic acid-rich region that is similar to a region contained in several RNA-processing proteins. In vitro translation of in vitro-transcribed RNA from a complete cDNA yields a product whose size agrees with the size predicted by the open reading frame. Antisera against su(s) fusion proteins recognize the in vitro-translated protein and detect a protein of identical size in the nuclear fractions from tissue culture cells and embryos. The protein is also present in smaller amounts in cytoplasmic fractions of embryos. That the su(s) protein has regions similar in structure to RNA-processing protein is consistent with its known role in affecting the transcript levels of those alleles that it suppresses.

scholarly journals Ovine herpesvirus 2 replicates initially in the lung of experimentally infected sheep

2008 ◽  
Vol 89 (7) ◽  
pp. 1699-1708 ◽  
Author(s):  
Hong Li ◽  
Cristina W. Cunha ◽  
Christopher J. Davies ◽  
Katherine L. Gailbreath ◽  
Donald P. Knowles ◽  
...  
Keyword(s):  
Lymphoproliferative Disease ◽  
Infected Sheep ◽  
Open Reading Frame ◽  
Malignant Catarrhal Fever ◽  
Defence Mechanism ◽  
Reading Frame ◽  
Initial Infection ◽  
Nasal Secretions ◽  
Replication Site

Ovine herpesvirus 2 (OvHV-2), a rhadinovirus in the subfamily Gammaherpesvirinae, is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a frequently fatal lymphoproliferative disease primarily of ruminants worldwide. Inability to propagate the virus in vitro has made it difficult to study OvHV-2 replication. Aerosol inoculation of sheep with OvHV-2 from nasal secretions collected from naturally infected sheep during shedding episodes results in infection of naive sheep, providing an excellent system to study OvHV-2 initial replication in the natural host. In this study, we showed that OvHV-2 delivered through the nasal route by nebulization resulted in infection in all lambs, but no infection was established in any lambs after intravenous or intraperitoneal injection. In nebulized lambs, while it was not detected initially in any other tissues, OvHV-2 DNA became detectable in the lung at 3 days post-infection (p.i.), increased to about 900 copies per 50 ng DNA at 5 days p.i., reached peak levels (∼7500 copies) at 7 days p.i., and then declined to an average of 800 copies at 9 days p.i. Transcripts of OvHV-2 open reading frame 25 (coding for the capsid protein), an indicator of virus replication, were only detected in lung tissues, and were positively correlated with OvHV-2 DNA levels in the lungs. In addition, selected immune response genes were also highly expressed in the lung at 5 and 7 days p.i. The data indicate that lung is the primary replication site for OvHV-2 during initial infection in sheep and suggest that viral replication is promptly controlled by a host defence mechanism.

scholarly journals Design of in vitro Transcribed mRNA Vectors for Research and Therapy

2019 ◽  
Vol 73 (5) ◽  
pp. 391-394
Author(s):  
Marina Tusup ◽  
Lars E. French ◽  
Mara De Matos ◽  
David Gatfield ◽  
Thomas Kundig ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Messenger Rna ◽  
Untranslated Region ◽  
Open Reading Frame ◽  
Purification Method ◽  
Reading Frame ◽  
Mrna Purification ◽  
Functional Regions ◽  
In Cells

The use of in vitro transcribed messenger RNA (ivt mRNA) for vaccination, gene therapy and cell reprograming has become increasingly popular in research and medicine. This method can be used in vitro (transfected in cells) or administered naked or formulated (lipoplexes, polyplexes, and lipopolyplexes that deliver the RNA to specific organs, such as immune structures, the lung or liver) and is designed to be an immunostimulatory or immunosilent agent. This vector contains several functional regions (Cap, 5' untranslated region, open reading frame, 3' untranslated region and poly-A tail) that can all be optimised to generate a highly efficacious ivt mRNA. In this study, we review these aspects and report on the effect of the ivt mRNA purification method on the functionality of this synthetic transient genetic vector.

scholarly journals Murine cytomegalovirus open reading frame m29.1 augments virus replication both in vitro and in vivo

2007 ◽  
Vol 88 (11) ◽  
pp. 2941-2951 ◽  
Author(s):  
Mohammad M. Ahasan ◽  
Clive Sweet
Keyword(s):  
Virus Replication ◽  
Post Infection ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Open Reading Frame ◽  
Murine Cytomegalovirus ◽  
Reading Frame ◽  
Codon Mutation

Murine cytomegalovirus mutant Rc29, with a premature stop codon mutation in the m29 open reading frame (ORF), produced no apparent phenotype in cell culture or following infection of BALB/c mice. In contrast, a similar mutant virus, Rc29.1, with a premature stop codon mutation in its m29.1 ORF, showed reduced virus yields (2–3 log10 p.f.u. ml−1) in tissue culture. Mutant virus yields in BALB/c mice were delayed, reduced (∼1 log10 p.f.u. per tissue) and persisted less well in salivary glands compared with wild-type (wt) and revertant (Rv29.1) virus. In severe combined immunodeficiency mice, Rc29.1 virus showed delayed and reduced replication initially in all tissues (liver, spleen, kidneys, heart, lung and salivary glands). This delayed death until 31 days post-infection (p.i.) compared with wt (23 days p.i.) but at death virus yields were similar to wt. m29 gene transcription was initiated at early times post-infection, while production of a transcript from ORF m29.1 in the presence of cycloheximide indicated that it was an immediate-early gene. ORFs m29.1 and M28 are expressed from a bicistronic message, which is spliced infrequently. However, it is likely that each ORF expresses its own protein, as antiserum derived in rabbits to the m29.1 protein expressed in bacteria from the m29.1 ORF detected only one protein in Western blot analysis of the size predicted for the m29.1 protein. Our results suggest that neither ORF is essential for virus replication but m29.1 is important for optimal viral growth in vitro and in vivo.

scholarly journals In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication

Virology ◽  
2005 ◽  
Vol 332 (1) ◽  
pp. 206-215 ◽  
Author(s):  
Soonjeon Youn ◽  
Julian L. Leibowitz ◽  
Ellen W. Collisson
Keyword(s):  
Open Reading Frame ◽  
Reading Frame ◽  
Infectious Bronchitis

scholarly journals Mouse Hepatitis Virus 3C-Like Protease Cleaves a 22-Kilodalton Protein from the Open Reading Frame 1a Polyprotein in Virus-Infected Cells and In Vitro

1998 ◽  
Vol 72 (3) ◽  
pp. 2265-2271 ◽  
Author(s):  
Xiao Tao Lu ◽  
Amy C. Sims ◽  
Mark R. Denison
Keyword(s):  
Hepatitis Virus ◽  
Mouse Hepatitis Virus ◽  
Open Reading Frame ◽  
Cleavage Sites ◽  
Reading Frame ◽  
Amino Terminal ◽  
Infected Cells ◽  
Carboxy Terminal ◽  
A Site

ABSTRACT The 3C-like proteinase (3CLpro) of mouse hepatitis virus (MHV) is predicted to cleave at least 11 sites in the 803-kDa gene 1 polyprotein, resulting in maturation of proteinase, polymerase, and helicase proteins. However, most of these cleavage sites have not been experimentally confirmed and the proteins have not been identified in vitro or in virus-infected cells. We used specific antibodies to identify and characterize a 22-kDa protein (p1a-22) expressed from gene 1 in MHV A59-infected DBT cells. Processing of p1a-22 from the polyprotein began immediately after translation, but some processing continued for several hours. Amino-terminal sequencing of p1a-22 purified from MHV-infected cells showed that it was cleaved at a putative 3CLpro cleavage site, Gln_Ser4014 (where the underscore indicates the site of cleavage), that is located between the 3CLpro domain and the end of open reading frame (ORF) 1a. Subclones of this region of gene 1 were used to express polypeptides in vitro that contained one or more 3CLpro cleavage sites, and cleavage of these substrates by recombinant 3CLpro in vitro confirmed that amino-terminal cleavage of p1a-22 occurred at Gln_Ser4014. We demonstrated that the carboxy-terminal cleavage of the p1a-22 protein occurred at Gln_Asn4208, a sequence that had not been predicted as a site for cleavage by MHV 3CLpro. Our results demonstrate the usefulness of recombinant MHV 3CLpro in identifying and confirming cleavage sites within the gene 1 polyprotein. Based on our results, we predict that at least seven mature proteins are processed from the ORF 1a polyprotein by 3CLpro and suggest that additional noncanonical cleavage sites may be used by 3CLpro during processing of the gene 1 polyprotein.

scholarly journals The Major Open Reading Frame of the β2.7 Transcript of Human Cytomegalovirus: In Vitro Expression of a Protein Posttranscriptionally Regulated by the 5′ Region

1998 ◽  
Vol 72 (10) ◽  
pp. 8425-8429 ◽  
Author(s):  
Giovanna Bergamini ◽  
Marko Reschke ◽  
Maria Concetta Battista ◽  
Maria Cristina Boccuni ◽  
Fabio Campanini ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Cytomegalovirus Infection ◽  
Intracellular Localization ◽  
Protein Product ◽  
Transient Transfection ◽  
Open Reading Frame ◽  
Major Open Reading Frame ◽  
Reading Frame ◽  
Human Cytomegalovirus Infection

ABSTRACT β2.7 is the major early transcript produced during human cytomegalovirus infection. This abundantly expressed RNA is polysome associated, but no protein product has ever been detected. In this study, a stable peptide of 24 kDa was produced in vitro from the major open reading frame (ORF), TRL4. Following transient transfection, the intracellular localization was nucleolar and the expression was posttranscriptionally inhibited by the 5′ sequence of the transcript, which harbors two short upstream ORFs.

scholarly journals Ilarviruses Encode a Cucumovirus-Like 2b Gene That Is Absent in Other Genera within the Bromoviridae

1998 ◽  
Vol 72 (8) ◽  
pp. 6956-6959 ◽  
Author(s):  
Hong-Wu Xin ◽  
Liang-Hui Ji ◽  
Simon W. Scott ◽  
Robert H. Symons ◽  
Shou-Wei Ding
Keyword(s):  
Open Reading Frame ◽  
Latent Virus ◽  
Evolutionary Origin ◽  
Protein Species ◽  
Subgenomic Rna ◽  
Reading Frame ◽  
2B Protein ◽  
Sequence Similarities

ABSTRACT We found that RNA 2 of the four ilarviruses sequenced to date encodes an additional conserved open reading frame (ORF), 2b, that overlaps the 3′ end of the previously known ORF, 2a. A novel RNA species of 851 nucleotides was found to accumulate to high levels in plants infected with spinach latent virus (SpLV). Further analysis showed that RNA 4A is a subgenomic RNA of RNA 2 and encodes all of ORF 2b. Moreover, a protein species of the size expected for SpLV ORF 2b was translated in vitro from the RNA 4A-containing virion RNAs. The data support the suggestion that the SpLV 2b protein is translated in vivo. The 2b gene of ilarviruses, which is not encoded by alfamoviruses and bromoviruses, shares several features with the previously reported cucumovirus 2b gene; however, their encoded proteins share no detectable sequence similarities. The evolutionary origin of the 2b gene is discussed.

scholarly journals A highly conserved mouse gene with a propensity to form pseudogenes in mammals.

1988 ◽  
Vol 8 (7) ◽  
pp. 2797-2803 ◽  
Author(s):  
D L Heller ◽  
K M Gianola ◽  
L A Leinwand
Keyword(s):  
Dna Sequence ◽  
Cdna Clone ◽  
Restriction Analysis ◽  
In Vitro Transcription ◽  
Open Reading Frame ◽  
Cdna Clones ◽  
Reading Frame ◽  
Mouse Cdna ◽  
Processed Pseudogenes

A mouse cDNA clone corresponding to an abundantly transcribed poly(A)+ mRNA was found to be represented by 200 copies in mammalian genomes. To understand the origin and nature of this sequence family, we studied two genomic members and two cDNA clones from mouse liver. The DNA sequence of the coding strand of a full-length cDNA clone was shown to have an open reading frame capable of encoding a 25-kilodalton polypeptide that has not been previously described. In vitro transcription-translation experiments verified the presence of an open reading frame encoding a protein of the predicted size. Restriction analysis of genomic DNA and DNA sequence analysis of genomic clones indicated that many of the 200 members of this family represent processed pseudogenes, with one or a small number of active structural genes. The vast majority of the genomic copies are heterogeneous in length, truncated at their 5' ends with respect to the mRNA, and do not appear to have intervening sequences. Two distinct genomic members of this family were sequenced and found to represent incomplete copies of the mRNA. Both are 5' truncated at slightly different points with respect to the mRNA. Both pseudogenes have multiple base changes, insertions, and deletions relative to the mRNA, and one of them encodes the poly(A) tail of the mRNA. The expression of this gene family is highest in rapidly dividing cells such as early mouse embryos and testis, but was seen in all tissues tested. This gene shows extremely high sequence conservation, extending to chicken, amphibian, and nematode genomes. Surprisingly, the gene appears to exist in only one copy in these organisms.

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