Deletion screening at the hypoxanthine-guanine phosphoribosyltransferase locus in chinese hamster cells using the polymerase chain reaction

1989 ◽  
Vol 9 (3) ◽  
pp. 177-187 ◽  
Author(s):  
Zhidong Xu ◽  
Yongjia Yu ◽  
Abraham W. Hsie ◽  
C. Thomas Caskey ◽  
Belinda Rossiter ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Chinese Hamster ◽  
Chain Reaction ◽  
Chinese Hamster Cells ◽  
Polymerase Chain ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

Analysis of fibrinogen γ-chain truncations shows the C-terminus, particularly γIle387, is essential for assembly and secretion of this multichain protein

Blood ◽  
2002 ◽  
Vol 99 (10) ◽  
pp. 3654-3660 ◽  
Author(s):  
Nobuo Okumura ◽  
Fumiko Terasawa ◽  
Hitoshi Tanaka ◽  
Masako Hirota ◽  
Hiroyoshi Ota ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Messenger Rna ◽  
Culture Media ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Chain Reaction ◽  
Ovary Cells ◽  
C Terminus ◽  
Polymerase Chain

To examine the role of the fibrinogen γ chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated γ chains, terminating between residues γ379 and the C-terminus, γ411. The variant fibrinogens were synthesized from altered γ-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with γ chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the γ chain, from 1.4 μg/mL for normal fibrinogen to 0.39 μg/mL for γ 387 fibrinogen. Immunoassays of cell lysates showed that all variant γ chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription–polymerase chain reaction demonstrated that the γ-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the γ chain are essential for fibrinogen assembly, and more specifically, that γ387 is critical. We propose that the loss of residue γ387 destabilized the structure of γ chain, preventing assembly of αγ and βγ dimers, essential intermediates in the assembly of normal fibrinogen.

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