Identification of the minus-dominance gene ortholog in the mating-type locus of Gonium pectorale

Abstract

The evolution of anisogamy/oogamy in the colonial Volvocales might have occurred in an ancestral isogamous colonial organism like Gonium pectorale. The unicellular, close relative Chlamydomonas reinhardtii has a mating-type (MT) locus harboring several mating-type-specific genes, including one involved in mating-type determination and another involved in the function of the tubular mating structure in only one of the two isogametes. In this study, as the first step in identifying the G. pectorale MT locus, we isolated from G. pectorale the ortholog of the C. reinhardtii mating-type-determining minus-dominance (CrMID) gene, which is localized only in the MT- locus. 3'- and 5'-RACE RT-PCR using degenerate primers identified a CrMID-orthologous 164-amino-acid coding gene (GpMID) containing a leucine-zipper RWP-RK domain near the C-terminal, as is the case with CrMID. Genomic Southern blot analysis showed that GpMID was coded only in the minus strain of G. pectorale. RT-PCR revealed that GpMID expression increased during nitrogen starvation. Analysis of F1 progeny suggested that GpMID and isopropylmalate dehydratase LEU1S are tightly linked, suggesting that they are harbored in a chromosomal region under recombinational suppression that is comparable to the C. reinhardtii MT locus. However, two other genes present in the C. reinhardtii MT locus are not linked to the G. pectorale LEU1S/MID, suggesting that the gene content of the volvocalean MT loci is not static over time. Inheritance of chloroplast and mitochondria genomes in G. pectorale is uniparental from the plus and minus parents, respectively, as is also the case in C. reinhardtii.

Figure 1.—

Alignment of four MID proteins from G. pectorale (GpMID), P. starrii (PlestMID), C. reinhardtii (CrMID), and C. incerta (CiMID). Solid and shaded backgrounds indicate identity in 100% or in 75% of the sequences aligned, respectively. Five amino acids composing a leucine zipper are marked with asterisks. A line marks the RWP-RK domain of 47 amino acids used for the phylogenetic analyses (Figure 5).

Figure 2.—

Exon–intron structure of MID genes from G. pectorale (GpMID), P. starrii (PlestMID), C. reinhardtii (CrMID), and C. incerta (CiMID). Corresponding parts of the coding regions (CDS) are interconnected by dotted lines. The line displayed above GpMID indicates the region used as a probe for the DNA gel blot analysis.

Figure 3.—

GpMID is present only in MT⁻. Genomic DNA isolated from a minus strain, Kaneko3 (labeled “−” below the gel) and a plus strain, Kaneko4 (labeled “+”), was digested with either SacII or PstI and hybridized with the GpMID probe whose location is shown in Figure 2. Size standards are shown on the left. GpMID is detected as a single band only in the minus strain.

Figure 4.—

Semiquantitative RT–PCR analysis of GpMID. The Kaneko3 strain was cultured either in nitrogen-free SVM (N-free SVM) for 18 hr or in ordinary SVM. Poly(A)⁺ RNA from these cultures was reverse transcribed and PCR amplified using primers for GpMID and for the EF1-like gene, which served as an internal control.

Figure 5.—

Maximum-likelihood (ML) tree (based on WAG model) of four MID proteins and 25 RWP-RK domains from C. reinhardtii (Cr) and V. carteri (Vc) genome databases. Branch lengths are proportional to the estimated amino acid substitutions, which are indicated by the scale bar above the tree. Numbers to the left of branch points indicate bootstrap values of the ML, neighbor-joining (based on the JTT model), and maximum parsimonious analyses, respectively, of the same data matrix and are included only if the value is ≥50%. Only CrNIT2 and the MID genes are characterized.

Figure 6.—

Scoring the nuclear markers in the F₁ progeny. A sampling of the DNA gel blot and PCR–RFLP analyses on G. pectorale progeny strains for the six nuclear markers. Diploid strains are marked with asterisks. Parental strains were Mongolia 1 (M1) and Mongolia 4 (M4). Size markers (in base pairs) are indicated to the right. The panels do not display the same sets of progeny. The presence/absence of MID (top band) is shown with ACT as an internal control (bottom band).

Figure 7.—

A complete representation of which alleles were inherited in the set of G. pectorale F₁ progeny examined. Shaded boxes indicate inheritance of the phenotype (mating type: MT) or genotype (chlp, chloroplast; mito, mitochondria) of the plus parent [M1 (MT⁺)]; open boxes indicate the minus parent [M4 (MT⁻)]. A circle within a square indicates that the mating phenotype was not observed, or there were no data. A half-shaded/half-open square indicates biparental transmission of mitochondrial markers.

Figure 8.—

Uniparental inheritance of organellar genomes in progeny of G. pectorale crosses. (A) Genomic DNA of the two parent strains (Mongolia 1 and 4, M1 and M4, respectively) and 11 F₁ progeny were digested with PstI, and the resulting Southern blot was hybridized with a portion of the G. pectorale psaA gene to visualize chloroplast DNA. (B) (Top) Genomic DNA of the two parent strains (M1 and M4) and 11 progeny were digested with PstI, and the resulting Southern blot was hybridized with a portion of the C. reinhardtii mitochondrial genome to visualize mitochondrial DNA. One of the parents (P2) used in a second cross is indicated. (Bottom) Parents and progeny of an F₂ cross using F₁ progeny P1 and P2 as parents. Positions of size markers (in kilobases) are indicated at the right.