Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii.

Abstract

The nitrate reductase structural gene of Chlamydomonas reinhadt has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-l. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binig domains of tobacco and Arabidopsis thauliana nitrate reductases and human NADH cytochrome bs reductase. The identity ofthe cloned gene as nitrate reductase was confirmed by its ability to complement a nit-i mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, sign tly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.

Characterization of the alternative oxidase of Chlamydomonas reinhardtii in response to oxidative stress and a shift in nitrogen source

“…reinhardtii CC-2454 was transformed with pAOX1 as well as pMN24 which contains the complete nitrate reductase (nit-1) gene (Fernandez et al 1989). Nuclear cotransformation was carried out using the 'glass-bead' method described by Kindle (1990).…”

“…Pelleted cells were resuspended in 20 ml gamete autolysin (Harris 1989) to aid in the removal of cell wall components. Following a 45-min incubation at 28 C, cells were harvested, washed once in SGII NO3 medium (Fernandez et al 1989) and resuspended in SGII NO3 to a cell concentration of approximately 8-9 · 10 7 cells ml À1 . In a sterile 15-ml polypropylene tube, 0.6 ml of cells were vortexed for 30 s in the presence of 0.2 ml of 20% (w/v) polyethylene glycol 8000, 0.6 g of acid-washed 0.5-mm glass beads, 6 mg pAOX01 and 3 mg pMN24.…”

“…This construct was introduced into Chlamydomonas strain CC-2454 (nit1-305; cw15 mt þ ) together with pMN24 (Fernandez et al 1989) which contains the gene encoding nitrate reductase (nit). Cells were plated on TAP NO3 medium and after about 10-14 days approximately 1% of the nit þ colonies exhibited a blue staining indicating the presence of the Aox1/Ars reporter gene and Ars activity.…”

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

“…Plasmids pMN24 and pMN56 contain the complete C. reinhardtii gene for nitrate reductase (NIT1) (7); the 9-kb XbaI-EcoRI fragment of pMN24 was subcloned into pUC119 to create pMN56. pl.O3 is an RBCS2 subclone generously provided by M. Goldschmidt-Clermont (University of Geneva, Geneva, Switzerland) (8).…”

“…A few reports of selection using heterologous genes have appeared (11,13,38), although no heterologous gene has allowed efficient recovery of transformants. Several C. reinhardtii genes corresponding to auxotrophic mutants, including the genes for nitrate reductase (NITJ) (7,19), argininosuccinate lyase (ARG7) (4), and oxygen-evolving enhancer protein 1 (OEEI) (29), have been cloned and characterized for use as efficient transformation markers. Dominant C. reinhardtii mutations that confer resistance to herbicides and other drugs are promising candidates for providing selectable marker genes.…”