scholarly journals Identification of a calicivirus isolate of unknown origin

2003 ◽  
Vol 84 (10) ◽  
pp. 2837-2845 ◽  
Author(s):  
Angelika Oehmig ◽  
Mathias Büttner ◽  
Frank Weiland ◽  
William Werz ◽  
Klaus Bergemann ◽  
...  
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Unknown Origin ◽  
Open Reading Frames ◽  
Coding Region ◽  
Electron Microscopic ◽  
Significant Similarity ◽  
Western Blots ◽  
Reading Frame ◽  
A Minor

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3′ non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus. In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

Discovery and Characterization of a Novel Large Non-Coding RNA Transcript, the Mouse Alpha Gene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4279-4279
Author(s):  
Rui Lin ◽  
Cheng Liu ◽  
Thomas S. Edgington
Keyword(s):  
Rna Polymerase Ii ◽  
Chromosomal Abnormalities ◽  
Full Length ◽  
Open Reading Frames ◽  
Gene Profiling ◽  
Significant Similarity ◽  
Reading Frame ◽  
Non Coding Rna ◽  
Alpha Gene ◽  
Rna Transcript

Abstract Recently, a new class of RNAs has been identified that, like mRNAs, are usually transcribed by RNA polymerase II, but lack significant translated open reading frames. They are non-coding RNAs (ncRNAs) that exert roles directly as RNAs, and function as genetic regulators, or riboregulators. In the present study, we discovered a novel very large ncRNA. From differential gene profiling of CD31 positive tumor microvascular endothelial cells from a murine colon carcinoma, we amplified an ~380 bp nucleotide sequence, DP100. This RNA was present in tumor cells as well as tumor vascular endothelium. The full-length DP100 transcript is an ~7 Kb RNA lacking an open reading frame set for more than 66 amino acids. Also the hypothetically predicted proteins lacked significant similarity to known proteins, consistent with identity of DP100 as an unusually large non-coding RNA transcript. Further bioinformatic search demonstrated that the full-length DP100 sequence is highly homologous to the human alpha gene, which encodes an 8.5 Kb, non-coding RNA transcript, and is located in a region implicated in chromosomal abnormalities of human tumors. The full-length DP100, here designated the mouse alpha gene, is evolutionally highly conserved among vertebrates, suggesting its functional significance. The role for this ncRNA in cellular behavior is under investigation.

scholarly journals Characterization of a novel intracellular heparanase that has a FERM domain

2002 ◽  
Vol 364 (1) ◽  
pp. 265-274 ◽  
Author(s):  
Karen J. BAME ◽  
Indumati VENKATESAN ◽  
Jean DEHDASHTI ◽  
Jeffrey McFARLANE ◽  
Rebecca BURFEIND
Keyword(s):  
Cho Cells ◽  
Heparan Sulphate ◽  
Chinese Hamster ◽  
Protein Activity ◽  
Western Blots ◽  
Ferm Domain ◽  
Erm Proteins ◽  
Cytoplasmic Proteins ◽  
Core Proteins ◽  
Major Species

The catabolism of cell-surface heparan sulphate proteoglycans is initiated by endosomal heparanases, which are endoglycosidases that cleave the glycosaminoglycans off core proteins and degrade them to shorter oligosaccharides. We have purified previously four intracellular heparanase activities from Chinese hamster ovary (CHO) cells [Bame, Hassall, Sanderson, Venkatesan and Sun (1998) Biochem. J. 336, 191–200], and in the present study we characterize further the most abundant activity (C1A heparanase). This enzyme purifies as a family of 37–48kDa proteins from both CHO cells and the rat liver, with the major species being 37 and 40kDa. Amino acid sequence analysis shows the purified C1A heparanase protein is highly homologous with the N-terminal domain, or FERM domain, of the ≈80kDa proteins ezrin, radixin and moesin (ERM proteins, after ezrin-radixin-moesin). This domain, which is also found in erythrocyte protein 4.1, links cytoplasmic proteins to membranes. Antibodies against the FERM domain recognize all the C1A heparanase proteins on Western blots, suggesting that the smaller species are derived from a larger protein. Activity binds to, and is affected by, molecules known to interact with FERM domains, supporting the hypothesis that the intracellular C1A heparanase is the purified FERM domain protein. Since bacterially expressed FERM domains of radixin and moesin lack heparanase activity, and some tryptic peptides generated from the enzyme do not have a match in any ERM protein, it appears that, rather than being derived from ezrin, radixin or moesin, C1A heparanase may be a new member of the FERM domain family.

scholarly journals Translational efficiency is regulated by the length of the 3' untranslated region.

1996 ◽  
Vol 16 (1) ◽  
pp. 146-156 ◽  
Author(s):  
R L Tanguay ◽  
D R Gallie
Keyword(s):  
Cho Cells ◽  
Posttranscriptional Regulation ◽  
Untranslated Region ◽  
Stop Codon ◽  
Chinese Hamster ◽  
Firefly Luciferase ◽  
Translational Efficiency ◽  
Coding Region ◽  
The Stability ◽  
Reporter Mrna

All polyadenylated mRNAs contain sequence of variable length between the coding region and the poly(A) tail. Little has been done to establish what role the length of the 3' untranslated region (3'UTR) plays in posttranscriptional regulation. Using firefly luciferase (luc) reporter mRNA in transiently transfected Chinese hamster ovary (CHO) cells, we observed that the addition of a poly(A) tail increased expression 97-fold when the length of the 3'UTR was 19 bases but that its stimulatory effect was only 2.3-fold when the length of the 3'UTR was increased to 156 bases. The effect of the luc 3'UTR on poly(A) tail function was orientation independent, suggesting that its length and not its primary sequence was the important factor. Increasing the length of the 3'UTR increased expression from poly(A)- mRNA but had little effect on poly(A)+ mRNA. To examine the effect of length on translational efficiency and mRNA stability, a 20-base sequence was introduced and reiterated downstream of the luc stop codon to generate a nested set of constructs in which the length of the 3'UTR increased from 4 to 104 bases. For poly(A)- reporter mRNA, translational efficiency in CHO cells increased 38-fold as the length of the 3'UTR increased from 4 to 104 bases. Increasing the length of the 3'UTR beyond 104 bases increased expression even further. Increasing the length of the 3'UTR also resulted in a 2.5-fold stabilization of the reporter mRNA. For poly(A)+ mRNA, the translational efficiency and mRNA half-life increased only marginally as the length of the 3'UTR increased from 27 to 161 bases. However, positioning the poly(A) tail only 7 bases downstream of the stop codon resulted in a 39-fold reduction in the rate of translation relative to a construct with a 27-base 3'UTR, which may be a consequence of the poly(A) tail-poly(A)-binding protein complex functioning as a steric block to translocating ribosomes as they approached the termination codon. The optimal length of the 3' noncoding region for maximal poly(A) tail-mediated stimulation of translation is approximately 27 bases. These data suggest that the length of the 3'UTR plays an important role in determining both the translational efficiency and the stability of an mRNA.

scholarly journals The PET54 gene of Saccharomyces cerevisiae: characterization of a nuclear gene encoding a mitochondrial translational activator and subcellular localization of its product.

Genetics ◽  
1989 ◽  
Vol 122 (2) ◽  
pp. 297-305 ◽  
Author(s):  
M C Costanzo ◽  
E C Seaver ◽  
T D Fox
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Gene ◽  
Open Reading Frames ◽  
Gene Encoding ◽  
Reading Frame ◽  
Acid Protein ◽  
Translational Activator ◽  
The Right ◽  
A Minor

Abstract The product of the nuclear Saccharomyces cerevisiae gene PET54 is specifically required, along with at least two other nuclear gene products, for translation of the mitochondrial mRNA encoding subunit III of cytochrome c oxidase (coxIII). We have genetically mapped PET54 (to the right arm of chromosome VII, 4.8 cM centromere-distal to SUF15), and have biochemically characterized the gene and its product. We determined the nucleotide sequence of a 1.6-kb DNA fragment carrying PET54 and identified the PET54 reading frame by determining the sequence of an ochre mutant allele as well as frameshift and frameshift-revertant alleles of the gene. The wild-type PET54 gene encodes a slightly basic 293-amino acid protein. PET54 is expressed from two mRNAs, both with unusual features: a major transcript with an extremely short 5'-untranslated leader, and a minor transcript with a relatively long 5'-leader carrying three short open reading frames. Antiserum raised against a trpE-PET54 fusion protein was used to probe subcellular fractions. These experiments showed that the PET54 protein is specifically associated with mitochondria, suggesting that it is likely to act directly in coxIII translation.

scholarly journals Germ line and embryonic expression of Fex, a member of the Drosophila F-element retrotransposon family, is mediated by an internal cis-regulatory control region.

1996 ◽  
Vol 16 (6) ◽  
pp. 2998-3007 ◽  
Author(s):  
B Kerber ◽  
S Fellert ◽  
H Taubert ◽  
M Hoch
Keyword(s):  
Germ Line ◽  
Expression Patterns ◽  
Regulatory Region ◽  
Open Reading Frames ◽  
Regulatory Control ◽  
Nucleic Acid Binding ◽  
Coding Region ◽  
Reading Frame ◽  
Cis Acting ◽  
Retrotransposon Family

The F elements of Drosophila melanogaster belong to the superfamily of long interspersed nucleotide element retrotransposons. To date, F-element transcription has not been detected in flies. Here we describe the isolation of a member of the F-element family, termed Fex, which is transcribed in specific cells of the female and male germ lines and in various tissues during embryogenesis of D. melanogaster. Sequence analysis revealed that this element contains two complete open reading frames coding for a putative nucleic acid-binding protein and a putative reverse transcriptase. Functional analysis of the 5' region, using germ line transformation of Fex-lacZ reporter gene constructs, demonstrates that major aspects of tissue-specific Fex expression are controlled by internal cis-acting elements that lie in the putative coding region of open reading frame 1. These sequences mediate dynamic gene expression in eight expression domains during embryonic and germ line development. The capacity of the cis-regulatory region of the Fex element to mediate such complex expression patterns is unique among members of the long interspersed nucleotide element superfamily of retrotransposons and is reminiscent of regulatory regions of developmental control genes.

scholarly journals The Immunogenic Glycoprotein gp35-37 of Human Herpesvirus 8 Is Encoded by Open Reading Frame K8.1

1998 ◽  
Vol 72 (8) ◽  
pp. 6725-6731 ◽  
Author(s):  
Marc-Steffen Raab ◽  
Jens-Christian Albrecht ◽  
Alexander Birkmann ◽  
Svenja Yağuboğlu ◽  
Dieter Lang ◽  
...  
Keyword(s):  
Phorbol Esters ◽  
Genomic Sequence ◽  
Human Herpesvirus ◽  
Body Cavity ◽  
Lytic Replication ◽  
Open Reading Frames ◽  
Human Herpesvirus 8 ◽  
Western Blots ◽  
Reading Frame ◽  
Herpesvirus 8

ABSTRACT Human herpesvirus 8 (HHV-8) is likely to be involved in the pathogenesis of Kaposi’s sarcoma (KS) and body cavity-based lymphomas (BCBLs). The HHV-8 genome is primarily in a latent state in BCBL-derived cell lines like BCBL-1, but lytic replication can be induced by phorbol esters (R. Renne, W. Zhang, B. Herndier, M. McGrath, N. Abbey, D. Kedes, and D. E. Ganem, Nat. Med. 2:342–346, 1996). A 35- to 37-kDa glycoprotein (gp35-37) is the polypeptide most frequently and intensively recognized by KS patient sera on Western blots with induced BCBL-1 cells. Its apparent molecular mass is reduced to 30 kDa by digestion with peptide-N-glycosidase F. By searching the known HHV-8 genomic sequence for open reading frames (ORF) with the potential to encode such a glycoprotein, an additional, HHV-8-specific reading frame was identified adjacently to ORF K8. This ORF, termed K8.1, was found to be transcribed primarily into a spliced mRNA encoding a glycoprotein of 228 amino acids. Recombinant K8.1 was regularly recognized by KS patient sera in Western blots, and immunoaffinity-purified antibodies to recombinant K8.1 reacted with gp35-37. This shows that the immunogenic gp35-37 is encoded by HHV-8 reading frame K8.1, which will be a useful tool for studies of HHV-8 epidemiology and pathogenesis.

scholarly journals Felis domesticus papillomavirus, isolated from a skin lesion, is related to canine oral papillomavirus and contains a 1·3 kb non-coding region between the E2 and L2 open reading frames

2002 ◽  
Vol 83 (9) ◽  
pp. 2303-2307 ◽  
Author(s):  
Masanori Terai ◽  
Robert D. Burk
Keyword(s):  
Phylogenetic Analysis ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Domestic Cat ◽  
Homology Search ◽  
Coding Region ◽  
Reading Frame ◽  
Blast Homology Search ◽  
Reading Frames ◽  
Felis Domesticus

We have characterized the complete genome (8300 bp) of an isolate of Felis domesticus papillomavirus (FdPV) from a domestic cat with cutaneous papillomatosis. A BLAST homology search using the nucleotide sequence of the L1 open reading frame demonstrated that the FdPV genome was most closely related to canine oral papillomavirus (COPV). A 384 bp non-coding region (NCR) was found between the end of L1 and the beginning of E6, and a 1·3 kbp NCR was located between the end of E2 and the beginning of L2. Phylogenetic analysis placed FdPV in the E3 clade with COPV. Both viruses contain the atypical second NCR, which has no homology with sequences in existing databases.

scholarly journals Exocytosis of pinocytic contents by Chinese hamster ovary cells.

1982 ◽  
Vol 93 (3) ◽  
pp. 632-637 ◽  
Author(s):  
C J Adams ◽  
K M Maurey ◽  
B Storrie
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Fluid Phase ◽  
Cell Fractionation ◽  
General Phenomenon ◽  
Electron Microscopic ◽  
Cultured Fibroblasts ◽  
Ovary Cells

The extent of exocytosis of pinocytic vesicle contents was studied in suspension-cultured Chinese hamster ovary (CHO) cells using horseradish peroxidase (HRP) as a pinocytic content marker. HRP was shown to be internalized via fluid-phase pinocytosis in CHO cells. After an HRP pulse of 2.5-10 min a rapid decrease of 30-50% in cell-associated HRP activity was observed within 10-20 min at 37 degrees C. During this time the loss of cell-associated HRP was accompanied by an equivalent increase in extracellular HRP. After this rapid exocytosis of HRP, the remaining peroxidase activity decreased with a t1/2 of 6-8 h, the known lysosomal half-life of HRP. In pulse-chase experiments HRP was chased into a nonexocytic compartment. Based on cell fractionation and electron microscopic experiments, this nonexocytic compartment was identified as a lysosome and the compartment from which exocytosis occurs as a pinosome. The occurrence of pinocytic content exocytosis in cultured fibroblasts suggests that exocytosis of pinocytic vesicle contents is a general phenomenon.

Heterogeneity and microtubule interaction of the CHO1 antigen, a mitosis-specific kinesin-like protein. Analysis of subdomains expressed in insect sf9 cells

1994 ◽  
Vol 107 (12) ◽  
pp. 3485-3499 ◽  
Author(s):  
R. Kuriyama ◽  
S. Dragas-Granoic ◽  
T. Maekawa ◽  
A. Vassilev ◽  
A. Khodjakov ◽  
...  
Keyword(s):  
Cho Cells ◽  
Sequence Similarity ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sf9 Cells ◽  
Coding Region ◽  
Expression Library ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Calculated Molecular Mass

The CHO1 antigen is a mitosis-specific kinesin-like motor located at the interzonal region of the spindle. The human cDNA coding for the antigen contains a domain with sequence similarity to the motor domain of kinesin-like protein (Nislow et al., Nature 359, 543, 1992). Here we cloned cDNAs encoding the CHO1 antigen by immunoscreening of a CHO Uni-Zap expression library, the same species in which the original monoclonal antibody was raised. cDNAs of CHO cells encode a 953 amino acid polypeptide with a calculated molecular mass of 109 kDa. The N-terminal 73% of the antigen was 87% identical to the human clone, whereas the remaining 27% of the coding region showed only 48% homology. Insect Sf9 cells infected with baculovirus containing the full-length insert produced 105 and 95 kDa polypeptides, the same doublet identified as the original antigen in CHO cells. Truncated polypeptides corresponding to the N-terminal motor and C-terminal tail produced a 56 and 54 kDa polypeptide in Sf9 cells, respectively. Full and N-terminal proteins co-sedimented with, and caused bundling of, brain microtubules in vitro, whereas the C-terminal polypeptide did not. Cells expressing the N terminus formed one or more cytoplasmic processes. Immunofluorescence as well as electron microscopic observations revealed the presence of thick bundles of microtubules, which were closely packed, forming a marginal ring just beneath the cell membrane and a core in the processes. The diffusion coefficient and sedimentation coefficient were determined for the native CHO1 antigen by gel filtration and sucrose density gradient centrifugation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The CHO Cell Clustering Response to Pertussis Toxin: History of Its Discovery and Recent Developments in Its Use

Toxins ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 815
Author(s):  
Mary C. Gray ◽  
Richard L. Guerrant ◽  
Erik L. Hewlett
Keyword(s):  
Pertussis Toxin ◽  
Cho Cells ◽  
Cellular Response ◽  
Molecular Mechanisms ◽  
Chinese Hamster ◽  
Biologically Active ◽  
In Vitro Assays ◽  
Cho Cell ◽  
Electron Microscopic

Chinese hamster ovary (CHO) cells respond to pertussis toxin (PT) with a novel clustering pattern, which is dependent on biologically active PT. Since its description in 1983, this cellular response has been refined and used extensively for detection and quantification of PT activity, as well as anti-PT antibodies. There are limitations, however, in the use of this phenomenon as originally described. They are: (1) a subjective, observer-dependent scoring system; (2) the requirement for 16–24 h incubation in order for the response to be clearly detectable; and (3) apparent interference from non-toxin materials. To overcome these limitations, a number of alternative in vitro assays for PT, using CHO cells or other cell types, have been developed and are described elsewhere in this publication. In addressing the challenges associated with the CHO cell assay, we discovered that changes in the electrical impedance-based “normalized cell index” of PT-treated CHO cells obtained with the ACEA xCELLigence instrument enable objective detection/quantification of the PT-induced effect in as little as 3–4 h. To the best of our knowledge, the molecular basis for this intriguing response remains unknown. We present here electron microscopic (EM) images of control and PT-treated cells, which suggest some potential molecular mechanisms.

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