scholarly journals Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines

2001 ◽  
Vol 82 (4) ◽  
pp. 963-969 ◽  
Author(s):  
Anne-Nathalie Volkoff ◽  
Janick Rocher ◽  
Pierre Cérutti ◽  
Marc C. P. Ohresser ◽  
Yves d’Aubenton-Carafa ◽  
...  
Keyword(s):  
Cell Lines ◽  
Spodoptera Littoralis ◽  
Trichoplusia Ni ◽  
Untranslated Region ◽  
Reading Frame ◽  
Short Open Reading Frame ◽  
Hyposoter Didymator ◽  
Insect Cell Lines ◽  
Lepidopteran Cell Lines

An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3′-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.

Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells

1987 ◽  
Vol 7 (7) ◽  
pp. 2435-2443
Author(s):  
I L Andrulis ◽  
J Chen ◽  
P N Ray
Keyword(s):  
Cell Lines ◽  
Untranslated Region ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Coding Region ◽  
Protein Coding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

Expression and characterization of recombinant human α-3/4-fucosyltransferase III from Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn) cells using the baculovirus expression system

2001 ◽  
Vol 353 (3) ◽  
pp. 719-725 ◽  
Author(s):  
Vanessa A. MORAIS ◽  
Jacinta SERPA ◽  
Angelina S. PALMA ◽  
Teresa COSTA ◽  
Luís MARANGA ◽  
...  
Keyword(s):  
Cell Lines ◽  
Trichoplusia Ni ◽  
Secretory Pathway ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Exchange Chromatography ◽  
Sf9 Cells ◽  
Type I ◽  
Baculovirus Expression ◽  
Insect Cell Lines

The human α-3/4-fucosyltransferase III (Fuc-TIII) participates in the synthesis of Lewis determinants. The enzyme from human sources is scarce and heterogeneous. In this paper we describe the expression of a secreted form of Fuc-TIII (SFT3) in two insect cell lines, Spodopterafrugiperda (Sf9) and Trichoplusiani (Tn), using the baculovirus expression system. The Sf9 cells secreted approx. 0.4unit/l (1mg/l) of the enzyme. The Tn cells secreted approx. 3-fold this amount. A large proportion of active protein was accumulated in the two cell lines (50 and 75% respectively for Sf9 and Tn cells, on the fourth day after infection) indicating a possible limitation not only of the folding machinery, but also a saturation of the secretory pathway. SFT3 was purified by cation-exchange chromatography followed by affinity chromatography. The enzyme from the Tn cell line had a lower global charge, possibly due to post-translational modifications, such as phosphorylation or sulphation. The two glycosylation sites from SFT3 were occupied. SFT3 secreted by Sf9 cells was completely deglycosylated by peptide-N-glycanase F, whereas 50% of SFT3 secreted by Tn cells was resistant to deglycosylation by this enzyme. The apparent kinetic parameters determined with the type I acceptor were kcat = 0.4s-1 and Km = 0.87mM for the SFT3 secreted by Tn cells, and kcat = 0.09s-1 and Km = 0.76mM for the SFT3 secreted by Sf9 cells, indicating that the enzymes had substrate affinities within the same order of magnitude as their mammalian counterpart. Furthermore, SFT3 secreted by either cell type showed a clear preference for type 1 carbohydrate acceptors, similarly to human Fuc-TIII.

scholarly journals Isolation of human cDNAs for asparagine synthetase and expression in Jensen rat sarcoma cells.

1987 ◽  
Vol 7 (7) ◽  
pp. 2435-2443 ◽  
Author(s):  
I L Andrulis ◽  
J Chen ◽  
P N Ray
Keyword(s):  
Cell Lines ◽  
Untranslated Region ◽  
Asparagine Synthetase ◽  
Synthetase Activity ◽  
Coding Region ◽  
Protein Coding ◽  
Reading Frame ◽  
Amino Terminal ◽  
Multiple Copies ◽  
Sarcoma Cells

Asparagine synthetase cDNAs containing the complete coding region were isolated from a human fibroblast cDNA library. DNA sequence analysis of the clones showed that the message contained one open reading frame encoding a protein of 64,400 Mr, 184 nucleotides of 5' untranslated region, and 120 nucleotides of 3' noncoding sequence. Plasmids containing the asparagine synthetase cDNAs were used in DNA-mediated transfer of genes into asparagine-requiring Jensen rat sarcoma cells. The cDNAs containing the entire protein-coding sequence expressed asparagine synthetase activity and were capable of conferring asparagine prototrophy on the Jensen rat sarcoma cells. However, cDNAs which lacked sequence for as few as 20 amino acids at the amino terminal could not rescue the cells from auxotrophy. The transferant cell lines contained multiple copies of the human asparagine synthetase cDNAs and produced human asparagine synthetase mRNA and asparagine synthetase protein. Several transferants with numerous copies of the cDNAs exhibited only basal levels of enzyme activity. Treatment of these transferant cell lines with 5-azacytidine greatly increased the expression of asparagine synthetase mRNA, protein, and activity.

Studies on the Specificity of Several Insect Cell Lines to Replication of Baculoviruses

1974 ◽  
Vol 32 ◽  
pp. 260-261
Author(s):  
J. R. Adams ◽  
R. H. Goodwin ◽  
T. A. Wilcox
Keyword(s):  
Tissue Culture ◽  
Cell Lines ◽  
Insect Cell ◽  
Trichoplusia Ni ◽  
Heliothis Zea ◽  
Corn Earworm ◽  
Cabbage Looper ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Insect Cell Lines

Some insect tissue culture cells may be readily infected with inocula of infectious hemolymph from the respective diseased host species or infectious cell culture supernatants from the respective infected tissue culture cells. We were interested in studying the degree of specificity of several insect cell lines to non-host viruses. Viruses investigated were baculoviruses (BV) isolated from corn earworm, Heliothis zea (Boddie); cabbage looper, Trichoplusia ni (Hübner) ; fall armyworm, Spodoptera frugiperda (J. D. Smith); alfalfa looper, Autographa californica (Speyer); gypsy moth, Porthetria dispar (L.) and cotton bollworm, Heliothis armigera (Hubner) on cell lines obtained from T. ni, H. zea, S. frugiperda.

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