Genetic and biochemical analysis of the TLA1 gene in Chlamydomonas reinhardtii

Abstract

The Chlamydomonas reinhardtii genomic DNA database contains a predicted open reading frame (ORF-P) without an apparent stop-codon and unknown coding sequence, located in close proximity and immediately upstream of the TLA1 gene (GenBank Accession No. AF534570). The latter was implicated in the regulation of the light-harvesting chlorophyll antenna size of photosynthesis (Tetali et al. Planta 225:813-829, 2007). To provide currently lacking information on ORF-P and its potential participation in TLA1 gene expression, thus in the regulation of the chlorophyll antenna size, genetic and biochemical analyses were undertaken. The coding and UTR regions of the ORF-P were defined and delineated from those of the adjacent TLA1 gene. ORF-P is shown to encode a protein with a distinct RING-like zinc finger domain that is present in numerous eukaryotic proteins, believed to play a role in cellular ubiquitination, leading to regulation of cellular processes like signaling, growth, transcription, and DNA repair. It is further shown that the two genes share a 74-bp overlap between the 3' UTR region of ORF-P and the 5' UTR region of TLA1. However, they possess distinct start and stop codons and separate coding sequences, and transcribed as separate mRNAs without any trans-splicing between them. Complementation experiments showed that the TLA1 gene alone is sufficient to rescue the truncated chlorophyll antenna size phenotype of the tla1 mutant. Protein sequence alignments in C. reinhardtii and the colorless microalga Polytomella parva suggested that TLA1 defines the relationship between nucleus and organelle in microalgae, indirectly affecting the development of the chlorophyll antenna size.

Fig. 1

Identification of an incomplete predicted open reading frame (ORF-P) in the Chlamydomonas reinhardtii database. a Genomic map of the ORF-P showing predicted exons (thick black arrow) and introns (thin black lines in-between exons). The uncharacterized genomic DNA region between ORF-P and TLA1 is depicted by a dotted line. The small black arrows (6, 7; 2, 3) denote primers used for genomic DNA PCR and RT-PCR. The known start codon (ATG) and the stop codon (TAA) of the TLA1 gene are denoted. Numbers refer to the base pairs of the putative exons, introns and distance between the start codon of ORF-P and start codon of TLA1. b Lanes 1 and 2 show the genomic DNA PCR product (772 bp) obtained with ORF-P-specific primer 6 and primer 7 (Exon-1/Exon-3 of ORF-P; Table 1); lanes 3 and 4 show the RT-PCR product (296 bp) obtained with the same primer set. (Genomic DNA contamination in the RNA preparation gave rise to the 772 bp product in the RT-PCR reaction in lanes 3 and 4.). Lane S refers to 1 kb plus DNA ladder. c RT-PCR using TLA1 gene-specific Exon-1 “primer 2” and Exon-2 “reverse primer 3R” (359 bp product; lane 1, Table 1) and ORF-P gene-specific Exon-1 “primer 6” and Exon-3 “primer 7” (296 bp product; lane 2, Table 1). d Complete genomic DNA map of the C. reinhardtii ORF-P and TLA1 genes. The positional alignment of the TLA1 gene with respect to the ORF-P is shown. Thick black arrows denote exons, while thin black lines denote introns. Untranslated regions of the two genes are denoted by dotted lines. Note that the 3′ UTR of ORF-P overlaps the 5′ UTR of TLA1 by 74 bp. The start and stop codons of the ORF-P and TLA1 genes are labeled. Numbers denote the base pairs of the respective DNA regions, i.e., 5′ UTR, exons, introns and 3′ UTR of the ORF-P

Fig. 2

cDNA sequence of the ORF-P 3′ and TLA1 5′ ends. a Overlap between the cDNA sequence of ORF-P and TLA1 is highlighted in gray. Gene-specific primers used for RT-PCR are color coded and underlined. Shown in uppercase bold and underlined fonts are “primer 12F” (blue), “primer 9F” (red), “primer 11F” (green), and “primer 3F” (black). The stop codon TGA of ORF-P and start codon ATG of TLA1 are shown in uppercase bold magenta font. b RT-PCR results using combinations of ORF-P- and TLA1-specific primers. Lane 1 shows 403-bp RT-PCR product obtained with TLA1 forward “primer 11F” and second exon-specific reverse “primer 3R”. Lane 2 shows no RT-PCR result with ORF-P 3′ UTR-specific forward “primer 12F” and TLA1 5′ UTR-specific reverse “primer 11R” (reverse sequence of “primer 11F”). Lane 3 shows 496-bp RT-PCR product obtained with TLA1 5′ UTR-specific forward “primer 9F” and second exon-specific reverse “primer 3R”. Lane 4 shows no RT-PCR result with ORF-P 3′ UTR-specific forward “primer 12F” and TLA1 second exon-specific reverse “primer 3R”. Lane 5 shows 143 bp RT-PCR product obtained with ORF-P 3′ UTR-specific forward “primer 12F” and TLA1 5′ UTR-specific reverse “primer 9R” (reverse sequence of “primer 9F”). F and R stand for forward primer and reverse primer, respectively

Fig. 3

Deduced amino acid sequence of the ORF-P-encoded, RDP1 protein. a The RDP1 gene encodes a protein of 410 amino acids. The C3HC4 RING zinc finger domain is shown in bold. b Comparison of the conventional C3HC4 RING zinc finger motif (upper) with that present in RDP1 (lower). C and H denote conserved cysteine and histidine residues, respectively, and X represents generic amino acids. The non-canonical residues are highlighted in red. Note that the spacing between the fourth and fifth cysteine is one amino acid in RDP1 in contrast to two amino acids in the conventional C3HC4 RING zinc finger

Fig. 4

Phenotype of wild type, tla1 mutant and tla1-complemented strains of C. reinhardtii. Minimal media (TBP) liquid cultures of wild type (a), tla1 mutant (b) and two complemented strains, tla1-comp1 (c) and tla1-comp2 (d) are shown. Cell densities of the four cultures were approximately, 6 × 10⁶ cells/mL. The tla1 mutant strain was complemented with a single copy of the wild-type TLA1 gene. The tla1 mutant showed a pale green coloration phenotype (b), indicative of the low-level chlorophyll concentration in these cells, whereas the wild type (a) and putative complemented strains tla1-comp1 (c) and tla1-comp2 (d) had a dark green coloration similar to that of the wild type

Fig. 5

PCR and RT-PCR analysis of wild type, tla1 and two tla1-complemented strains. Lane 1 tla1-comp1, lane 2 tla1-comp2, lane 3 tla1 mutant, lane 4 wild type. a Genomic DNA PCR product of 781 bp was obtained with forward PsaD 5′ UTR “primer 5” and reverse TLA1 Exon-2 “primer 4” (Fig. 1S and Table 1S; supplementary material). No products were obtained with the tla1 mutant (lane 3) or wild type samples (lane 4). b RT-PCR products of 665 bp were obtained with forward PsaD 5′ UTR “primer 5” and reverse (TLA1 Exon-2 “primer 4” (Fig. 1S and Table 1S; supplementary material) from the cDNA of the tla1-complements (lanes 1 and 2). No products were obtained from the cDNA of the tla1 mutant (lane 3) or wild type samples (lane 4). c Genomic DNA PCR product of 519 bp was obtained with forward TLA1 5′ UTR “primer 1” and reverse Exon-2 “primer 3” from the wild type sample only (lane 4). d RT-PCR product of 403 bp was obtained with forward TLA1 5′ UTR “primer 1” and reverse Exon-2 “primer 3” with the wild type cDNA only lane 4). e RT-PCR products of 642 bp were obtained with all samples when using forward Exon-1 “primer 2” and reverse Exon-2 “primer 4”. Genomic DNA and RNA for PCR and RT-PCR were extracted from cells grown photosynthetically in minimal TBP liquid media

Fig. 6

SDS-PAGE and western blot analysis of Chlamydomonas reinhardtii proteins. Lane 1 tla1-comp1, lane 2 tla1-comp2, lane 3 tla1 mutant, lane 4 wild type. a SDS-PAGE of total cell protein extracts. Lanes were loaded on an equal Chl basis (1.5 nmol Chl per lane). Lane S shows the pre-stained low molecular weight markers. b, c Western blot analysis of C. reinhardtii total cell protein extracts. Lanes, loaded as in a, were probed with TLA1-specific (b) and Lhcb-specific polyclonal antibodies (c). Note the substantially lower levels of the TLA1 and Lhcb proteins in lane 3 (tla1 mutant) relative to that in the other samples

Fig. 7

ClustalW generated amino acid sequence alignment of the TLA1 protein from Chlamydomonas reinhardtii and Polytomella parva. The sequence alignment revealed a 50% identity and 67% homology between the two proteins