Molecular cloning of higher plant homologues of the high-affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergillus nidulans

The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our transformation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

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Differential Expression of Aspergillus nidulans Ammonium Permease Genes Is Regulated by GATA Transcription Factor AreA

Eukaryotic Cell ◽

10.1128/ec.5.2.226-237.2006 ◽

2006 ◽

Vol 5 (2) ◽

pp. 226-237 ◽

Cited By ~ 30

Author(s):

Brendon J. Monahan ◽

Marion C. Askin ◽

Michael J. Hynes ◽

Meryl A. Davis

Keyword(s):

Aspergillus Nidulans ◽

Sufficient Conditions ◽

Optimal Growth ◽

Ammonium Transporter ◽

Ammonium Uptake ◽

Ammonium Transport ◽

Gata Factor ◽

High Affinity ◽

Gata Transcription Factor ◽

Nitrogen Conditions

ABSTRACT The movement of ammonium across biological membranes is mediated in both prokaryotes and eukaryotes by ammonium transport proteins (AMT/MEP) that constitute a family of related sequences. We have previously identified two ammonium permeases in Aspergillus nidulans, encoded by the meaA and mepA genes. Here we show that meaA is expressed in the presence of ammonium, consistent with the function of MeaA as the main ammonium transporter required for optimal growth on ammonium as a nitrogen source. In contrast, mepA, which encodes a high-affinity ammonium permease, is expressed only under nitrogen-limiting or starvation conditions. We have identified two additional AMT/MEP-like genes in A. nidulans, namely, mepB, which encodes a second high-affinity ammonium transporter expressed only in response to complete nitrogen starvation, and mepC, which is expressed at low levels under all nitrogen conditions. The MepC gene product is more divergent than the other A. nidulans AMT/MEP proteins and is not thought to significantly contribute to ammonium uptake under normal conditions. Remarkably, the expression of each AMT/MEP gene under all nitrogen conditions is regulated by the global nitrogen regulatory GATA factor AreA. Therefore, AreA is also active under nitrogen-sufficient conditions, along with its established role as a transcriptional activator in response to nitrogen limitation.

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Integrated genomic and transcriptomic insights into the two-component high-affinity nitrate transporters in allotetraploid rapeseed

Plant and Soil ◽

10.1007/s11104-018-3652-3 ◽

2018 ◽

Vol 427 (1-2) ◽

pp. 245-268 ◽

Cited By ~ 5

Author(s):

Ying-peng Hua ◽

Ting Zhou ◽

Hai-xing Song ◽

Chun-yun Guan ◽

Zhen-hua Zhang

Keyword(s):

High Affinity ◽

Two Component ◽

Nitrate Transporters

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petD mRNA maturation in Chlamydomonas reinhardtii chloroplasts: role of 5' endonucleolytic processing

Molecular and Cellular Biology ◽

10.1128/mcb.14.9.6180-6186.1994 ◽

1994 ◽

Vol 14 (9) ◽

pp. 6180-6186

Author(s):

W Sakamoto ◽

N R Sturm ◽

K L Kindle ◽

D B Stern

Keyword(s):

Chlamydomonas Reinhardtii ◽

Fusion Gene ◽

Deletion Mutants ◽

Chloroplast Genes ◽

Coding Region ◽

Higher Plant ◽

Glucuronidase Activity ◽

Mrna Maturation ◽

Processing Site ◽

And Function

Complex processing of primary transcripts occurs during the expression of higher-plant chloroplast genes. In Chlamydomonas reinhardtii, most chloroplast genes appear to possess their own promoters, rather than being transcribed as part of multicistronic operons. By generating specific deletion mutants, we show that petD, which encodes subunit IV of the cytochrome b6/f complex, has an RNA processing site that is required for accumulation of monocistronic petD mRNA in petD promoter deletion mutants; in such mutants, transcription of petD originates from the upstream petA promoter. The 5' ends of transcripts initiated at the petD promoter are probably also generated by processing, since the 5' end of monocistronic petD mRNA is the same in wild-type strains as it is in the petD promoter mutants. The location and function of the processing site were further examined by inserting petD-uidA fusion genes into the chloroplast genome (uidA is an Escherichia coli gene that encodes beta-glucuronidase). When a promoterless petD-uidA fusion gene was inserted downstream of petA, a monocistronic uidA transcript accumulated, which was apparently initiated at the petA promoter and was processed at a site corresponding precisely to the petD mRNA 5' end. When a construct including only sequences downstream of +25 relative to the mature mRNA 5' end was inserted into the same site, a dicistronic petA-uidA transcript accumulated but no monocistronic uidA transcript could be detected, suggesting that a processing site lies at least partially within the region from -1 to +25. Beta-glucuronidase activity was not detected in transformants that accumulated only the dicistronic petA-uidA transcript, suggesting that the first 25 bp of the 5' untranslated region are required for translation initiation. One explanation for this translational defect is that Chlamydomonas chloroplasts cannot translate the second coding region of some dicistronic messages.

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