scholarly journals Thermal Instability of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from a Temperature-Conditional Chloroplast Mutant of Chlamydomonas reinhardtii

1993 ◽  
Vol 101 (4) ◽  
pp. 1189-1194 ◽  
Author(s):  
Z. Chen ◽  
S. Hong ◽  
R. J. Spreitzer
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Thermal Instability ◽  
Bisphosphate Carboxylase ◽  
Chloroplast Mutant

The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation

1994 ◽  
Vol 14 (6) ◽  
pp. 4011-4019
Author(s):  
J A Nelson ◽  
P B Savereide ◽  
P A Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Marker Gene ◽  
Small Subunit ◽  
Direct Selection ◽  
Nuclear Transformation ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Dominant Selectable Marker ◽  
Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

scholarly journals The Intracellular Localization of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase in Chlamydomonas reinhardtii

1998 ◽  
Vol 116 (4) ◽  
pp. 1585-1591 ◽  
Author(s):  
Olga N. Borkhsenious ◽  
Catherine B. Mason ◽  
James V. Moroney
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Intracellular Localization ◽  
Bisphosphate Carboxylase

scholarly journals The CRY1 gene in Chlamydomonas reinhardtii: structure and use as a dominant selectable marker for nuclear transformation.

1994 ◽  
Vol 14 (6) ◽  
pp. 4011-4019 ◽  
Author(s):  
J A Nelson ◽  
P B Savereide ◽  
P A Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Selectable Marker ◽  
Marker Gene ◽  
Small Subunit ◽  
Direct Selection ◽  
Nuclear Transformation ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Dominant Selectable Marker ◽  
Cry1 Gene

We have cloned and sequenced the CRY1 gene, encoding ribosomal protein S14 in Chlamydomonas reinhardtii, and found that it is highly similar to S14/rp59 proteins from other organisms, including mammals, Drosophila melanogaster, and Saccharomyces cerevisiae. We isolated a mutant strain resistant to the eukaryotic translational inhibitors cryptopleurine and emetine in which the resistance was due to a missense mutation (CRY1-1) in the CRY1 gene; resistance was dominant in heterozygous stable diploids. Cotransformation experiments using the CRY1-1 gene and the gene for nitrate reductase (NIT1) produced a low level of resistance to cryptopleurine and emetine. Resistance levels were increased when the CRY1-1 gene was placed under the control of a constitutive promoter from the ribulose bisphosphate carboxylase/oxygenase small subunit 2 (RBCS2) gene. We also found that the 5' untranslated region of the CRY1 gene was required for expression of the CRY1-1 transgene. Direct selection of emetine-resistant transformants was possible when transformed cells were first induced to differentiate into gametes by nitrogen starvation and then allowed to dedifferentiate back to vegetative cells before emetine selection was applied. With this transformation protocol, the RBCS2/CRY1-1 dominant selectable marker gene is a powerful tool for many molecular genetic applications in C. reinhardtii.

scholarly journals Effects of mutations at the two processing sites of the precursor for the small subunit of ribulose-bisphosphate carboxylase in Chlamydomonas reinhardtii

2002 ◽  
Vol 366 (3) ◽  
pp. 989-998 ◽  
Author(s):  
Cédric INVERNIZZI ◽  
Jonathan IMHOF ◽  
Gabriela BURKARD ◽  
Katharina SCHMID ◽  
Arminio BOSCHETTI
Keyword(s):  
Amino Acids ◽  
Chlamydomonas Reinhardtii ◽  
Small Subunit ◽  
Ribulose Bisphosphate Carboxylase ◽  
Cleavage Sites ◽  
Bisphosphate Carboxylase ◽  
Molecular Masses ◽  
Processing Site

The role of the two processing sites in the precursor of the small subunit (SS) of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSS) of Chlamydomonas reinhardtii was studied by introducing mutations at the cleavage sites for the stromal processing peptidases SPP-1 and SPP-2, which hydrolyse wild-type pSS (20.6kDa) to an intermediate-sized product iSS (18.3kDa) and to the mature SS (16.3kDa), respectively. The mutations introduced into cDNA resulted in exchange of (a) two amino acids flanking processing site 1, or (b) one or (c) both amino acids flanking processing site 2. Mutation (a) prevented pSS from being processed at site 1 but not from cleavage at site 2. Mutation (c) abolished the action of SPP-2 but not SPP-1. When pSS with mutation (c) was imported into isolated chloroplasts, iSS accumulated while SS formation was abolished. However, mature SS was produced even in the absence of iSS synthesis (mutation a). Import of pSS bearing mutation (b), which only partially inhibited processing at the SPP-2 site, slowed the rate of SS formation down whereas iSS and some slightly smaller derivatives accumulated. These experiments suggested that in Chlamydomonas processing of pSS can occur in two steps, whereby the first step is facultative. The same three mutations were studied in vivo after transformation of SS-deficient C. reinhardtii T60-3 with mutated genomic DNA. Growth and photosynthesis was as in control transformants, except for the slower-growing transformants (mutation c) where no mature SS was immuno-detected. However, pSS fragments with molecular masses between those of iSS and SS were present even in the ribulose-1,5-bisphosphate carboxylase/oxygenase holoenzyme.

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