Genetic Structure of the Mating-Type Locus of Chlamydomonas reinhardtii

Corresponding author: Patrick J. Ferris, Campus Box 1229, 1 Brookings Dr., Washington University, St. Louis, MO 63130., ferris{at}biosgi.wustl.edu (E-mail)

Communicating editor: S. L. ALLEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Portions of the cloned mating-type (MT) loci (mt⁺ and mt^-) of Chlamydomonas reinhardtii, defined as the 1-Mb domains of linkage group VI that are under recombinational suppression, were subjected to Northern analysis to elucidate their coding capacity. The four central rearranged segments of the loci were found to contain both housekeeping genes (expressed during several life-cycle stages) and mating-related genes, while the sequences unique to mt⁺ or mt^- carried genes expressed only in the gametic or zygotic phases of the life cycle. One of these genes, Mtd1, is a candidate participant in gametic cell fusion; two others, Mta1 and Ezy2, are candidate participants in the uniparental inheritance of chloroplast DNA. The identified housekeeping genes include Pdk, encoding pyruvate dehydrogenase kinase, and GdcH, encoding glycine decarboxylase complex subunit H. Unusual genetic configurations include three genes whose sequences overlap, one gene that has inserted into the coding region of another, several genes that have been inactivated by rearrangements in the region, and genes that have undergone tandem duplication. This report extends our original conclusion that the MT locus has incurred high levels of mutational change.

THE mating-type (MT) locus of the haploid green alga Chlamydomonas reinhardtii, located 30 cM from the centromere of linkage group (chromosome) VI, is involved in generating mating-type plus or minus gametic phenotypes in response to nitrogen starvation (GOODENOUGH et al. 1995 ). The mt⁺ and mt^- versions of this locus segregate 2:2 at meiosis, but early genetic analysis documented that numerous genetic markers that map to the region fail to recombine with one another, suggesting that recombinational suppression is responsible for the observed segregation patterns (GILLHAM 1969 ). This inference was confirmed with the cloning of both the mt⁺ and mt^- loci (FERRIS and GOODENOUGH 1994 ). The locus (Fig 1) consists of an 1-Mb region of recombinational suppression, in the center of which is an 200-kb domain [the rearranged (R) domain] that has undergone numerous translocations and inversions involving four large segments of the domain (Fig 1). These rearrangements presumably suppress meiotic crossing over in the flanking telomere-proximal (T) and centromere-proximal (C) domains of the locus.

View larger version (23K): In this window In a new window Download PPT slide   Figure 1. Transcript map of the MT locus. The top half diagrams the mt- locus and the bottom half the mt+ locus. The T (telomere-proximal), R (rearranged), and C (centromere-proximal) domains are indicated. The four segments of homology within the two R domains are drawn as shaded boxes, with the shape indicating their orientation. The segments are numbered 1–4. The letters a–f indicate regions within the R domain that are unique to mt- or mt+. The genes/transcripts identified in mt- are shown above the line, those for mt+ below the line. Squares represent messages found in vegetative cells. Circles represent messages first turned on in gametes. A + within the circle indicates a message seen only in mt+ gametes; a -, only in mt- gametes; +/-, present in gametes of both mating types. Triangles represent messages limited to the zygote stage. Arrows indicate the direction of transcription when known (or the orientation of the gene in the case of pseudogenes). The scale (in kilobases) is indicated for mt-. Further descriptions of the genes are found in Table 1; data are given on the GENETICS website at http://www.genetics.org/supplemental.

Of the genetic markers under recombinational suppression, three define genes that are selectively transcribed in response to nitrogen starvation and are directly involved with generating either the plus or the minus gametic phenotypes.

1. The Fus1 gene, originally marked by the imp1 mutation, encodes an 810-amino-acid glycoprotein that is necessary for gametic cell fusion. It is located in region c of the mt⁺ R domain (Fig 1) and has no homolog in the mt^- locus (FERRIS et al. 1996 ).

2. The Mid gene, originally marked by the imp11 mutation (the mutant allele and mutant strain are henceforth designated mid-1), encodes a 147-amino-acid regulatory protein, related to a family of nitrogen-sensitive transcriptional regulators (SCHAUSER et al. 1999 ), that induces cells to differentiate as minus gametes. It is located in region f of the mt^- locus and has no homolog in the mt⁺ locus (FERRIS and GOODENOUGH 1997 ).

3. The Sad1 gene, marked by the imp10/imp12 mutations (HWANG et al. 1981 ) and the agl mutation (MATSUDA et al. 1988 ), encodes a 3875-amino-acid protein that serves as the flagellar sexual agglutinin of minus gametes. It is located just centromere-proximal to the mt^- R domain (Fig 1), with an allele located in the homologous position in the mt⁺ locus, an allele that is ordinarily not expressed because its expression is Mid-dependent and plus cells lack the Mid gene. A full report on the characterization of the Sad1 gene is in preparation.

Although several genes involved with mating map to the MT locus, including several new genes that are described in this report, many other gamete-specific genes are not linked to MT and are designated as "autosomal" (GOODENOUGH et al. 1995 ; KURVARI et al. 1998 ). Thus, although the mid-1 strain carries the mt^- chromosome VI and hence lacks an mt⁺ locus, it nonetheless differentiates as a plus gamete and requires only a Fus1 transgene to achieve mating competency (FERRIS and GOODENOUGH 1997 ), indicating that most genes necessary for plus gametogenesis, including the agglutinin gene, are autosomal. To ask why some mating-related genes reside in the MT locus and others in autosomes under Mid regulation is a way of phrasing the unanswered question as to the "purpose" of the MT locus.

The MT locus is also involved in mediating uniparental transmission of organelle genomes during the zygotic phase of the C. reinhardtii life cycle. All four meiotic products of zygote germination ordinarily inherit chloroplast DNA (cpDNA) from the plus parent only and mitochondrial DNA from the minus parent only, the nontransmitted organellar DNAs having been selectively degraded during zygote maturation (ARMBRUST 1998 ; REMACLE and MATAGNE 1998 ). The EZY1 locus comprises seven to eight tandem iterations of a gene that is transcribed immediately after zygote formation and encodes a 414-amino-acid protein that associates with cpDNA and presumably plays some role in its selective transmission patterns (ARMBRUST et al. 1993 ). The Ezy1 gene cluster is located centromere-proximal to the Sad1 gene in both the mt⁺ and mt^- loci (Fig 1).

Mutant alleles in the MT locus that fail to recombine also mark several genes that are expressed in vegetative (mitotic) cells and play no known specific role in gametogenesis or zygote development. Five of these "housekeeping" genes have been cloned and at least partially characterized; all lie outside the R domain at positions designated in Fig 1. The Nic7 (nicotinamide-requiring), Ac29 (acetate-requiring), and Thi10 (thiamine-requiring) gene sequences have been identified by their ability to complement mutant alleles (FERRIS 1995 ). The Ac29 gene has been shown to encode a 495-amino-acid protein homologous to the Arabidopsis protein ALBINO3 (SUNDBERG et al. 1997 ), which is involved in the biogenesis of the chloroplast light-harvesting complex (NAVER et al. 2000 ), and the Thi10 gene encodes hydroxyethylthiazole kinase (K. SHIMOGAWARA, personal communication), an enzyme in the biosynthetic pathway of thiamine. The Mat3 gene encodes a 1209-amino-acid homolog of the retinoblastoma (Rb) protein and is involved in regulation of the cell cycle (ARMBRUST et al. 1995 ; UMEN and GOODENOUGH 2001B ), and the Fa1 gene encodes a 1787-amino-acid protein involved in flagellar morphogenesis (FINST et al. 2000 ).

The finding that such housekeeping genes are intermixed with life-cycle-specific genes suggests that the MT locus arose in an "ordinary" chromosome, in much the same way that the sex chromosomes of mammals were once ordinary chromosomes and continue to encode non-sex-related proteins (LAHN and PAGE 1999 ). However, all of the previously known housekeeping genes mapped outside the rearranged R domain, leaving open the possibility that the DNA within the R domain itself might be either restricted to sex-related functions or largely noncoding—like most of the mammalian Y chromosomes. Northern analysis of the region, reported in this article, documents that this is not the case: Genes prove to be abundant within the R domain, and many of them are expressed in vegetative cells. Therefore, the R domain of chromosome VI has been subjected to numerous local rearrangements while continuing to maintain (most of) its prior genetic activities.

We also report the characterization of several genes that are found in one MT locus but not the other, expanding our understanding of the coding capacity of MT and providing additional evidence for high mutational change in the region (FERRIS et al. 1997 ).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Northern analysis:
The C. reinhardtii strains used to prepare RNA for Northern analysis were wild-type strains CC-620 (mt⁺) and CC-621 (mt^-) all strains are available from the Chlamydomonas Genetics Center, Duke University (Durham, North Carolina). Cultures were maintained in continuous light on Tris-acetate-phosphate (TAP) medium (HARRIS 1989 ) solidified with 1.5% agar. Vegetative RNA was prepared from cells in logarithmic growth in flasks of TAP medium. Gametes were obtained by transferring cells maintained on plates for at least 7 days (MARTIN and GOODENOUGH 1975 ) to nitrogen-free high salt minimal media (HARRIS 1989 ) for 1–2 hr. Zygotes were produced by mating equal numbers of plus and minus gametes and harvesting after 30 min or 3 hr. Preparation of Northern blots was as described (FERRIS et al. 2001 ). Northern blots were stripped and reused several times during the course of these experiments. Most of the probes were prepared using restriction fragments purified from the EMBL3 genomic phage clones that comprise the chromosome walk through the MT loci (FERRIS and GOODENOUGH 1994 ), radiolabeled with [-³²P]dCTP (DuPont/New England Nuclear Research Products) by random priming.

Isolation of cDNA clones:
The cDNA clones for pr6(+), pr6(-), Mta1, Mta2, and Ezy2 were identified by screening plaque lifts of a cDNA expression library in Uni-ZAPXR (Stratagene, La Jolla, CA) prepared from 1-hr zygotic poly(A)+ RNA (ARMBRUST et al. 1993 ) by hybridization with appropriate radiolabeled genomic probes. Inserts from positive clones were excised as Bluescript SK plasmids with R408 helper phage according to the manufacturer's instructions. The cDNA that was eventually used as probe 6 was first cloned fortuitously as a consequence of its cross-hybridization to a probe derived from DNA flanking the Mid gene; subsequent analysis then identified the location of the corresponding gene in the T domain.

DNA sequencing and analysis:
The strategy for DNA sequencing included subcloning, gene-specific primers, nested deletions using the double strand nested deletion kit (Pharmacia, Piscataway, NJ) and use of the GPS-1 genome priming system (New England Biolabs, Beverly, MA). Some sequence data were obtained by making single-stranded DNA according to AUSUBEL et al. 1989 , which was used for dideoxy sequencing with the sequenase kit (United States Biochemical, Cleveland). The bulk of the sequencing was performed with the ABI PRISM dye terminator cycle sequencing ready reaction kit using double-stranded plasmid DNA and subsequent analysis on an ABI DNA sequencer. Sequence data were compiled and analyzed using the Genetics Computer Group sequence analysis software package for VAX/VMS computers (DEVEREUX et al. 1984 ). Sequences were further investigated using the NCBI BLAST program, the TMpred, and the COILS program (LUPAS 1996 ).

The sequences described in this article have the following GenBank accession numbers: Nic7 partial genomic, AY032929; pr6(-) cDNA, AY032930; pr6(+) cDNA, AY032931; Mtd1 cDNA, AF417574; Pr46 genomic, AF387366; Pdk genomic, AF387365; Ezy2 genomic (mt⁺), AF399653; -Ezy2 genomic (mt^-), AF399654; autosomal a region, left border of the duplication, AF417573; autosomal a region, right border of the duplication (Mta2 and Mta3 genes), AF309495; mt⁺ a region, left border of the duplication, AF417572; mt⁺ a region, right border of the duplication (Mta1, -Mta2, and -Mta3 genes), AF417571.

RNase protection analysis:
Total RNA was isolated essentially as described by KIRK and KIRK 1985 . Poly(A)+ RNA was isolated with the BioMag mRNA purification kit (Perseptive Diagnostics). The generation and use of the Ezy1 antisense probe was described previously (ARMBRUST et al. 1993 ). The Ezy2 probe was generated by subcloning into Bluescript II SK a 600-bp BamHI/XhoI fragment from the coding region of the Ezy2 cDNA. The resulting plasmid was linearized with SmaI, and T7 RNA polymerase was used to transcribe an antisense probe of 197 nucleotides. The protected Ezy2 probe is 172 nucleotides. The Ambion (Austin, TX) RPA II kit was used for all RNase protection assays. Ten micrograms of total RNA was used for each RNase protection assay.

Uniparental inheritance crosses:
Genetic crosses were performed using standard protocols (HARRIS 1989 ). The strains used in the control cross were CC-118 (mt⁺ sr-u-2-60) and CC-124 (wild-type mt^-). A mid-1 mt^- (Fus1) cross to CC-421 (nic7 ac29a mt^- spr-u-1-27-3), described previously (FERRIS and GOODENOUGH 1997 ), generated a progeny clone (B32) mid-1 mt^- (Fus1) spr-u-1-27-3 that was crossed to CC-1952 (wild-type mt^-). The following were added to the media as necessary: 4 µg/ml nicotinamide, 100 µg/ml spectinomycin, and 100 µg/ml streptomycin.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Transcriptional patterns in the MT locus: methodology:
Northern blots containing poly(A)+ RNA from vegetative cells of both mating types, gametes of both mating types, and zygotes 30 min and 3 hr into development were prepared, and these were screened with 128 probes from the MT locus. The data are presented on the GENETICS website at http://www.genetics.org/supplemental. The probes were chosen to give near total coverage of the R domain (90% covered, with six gaps of 2–3 kb, and most <1 kb). The C and T domains were covered less extensively (except near the R domain borders), primarily using probes known to give single-copy bands on Southern blots (FERRIS and GOODENOUGH 1994 ). The T domain had 35% coverage from probe 1 to the T/R border; the C domain had 75% coverage from the R/C border to the swamp (Fig 1; cf. FERRIS and GOODENOUGH 1994 ).

From these primary data we attempted to identify all the bona fide genes within the R domains of the mt⁺ and mt^- loci, an analysis complicated by false negatives and false positives.

False negatives (a gene failing to be identified by the Northern analysis) could result for several reasons.

1. The message is of low abundance and the blots are not sensitive enough. For example, probe 54, known to contain part of the Mid gene, and probe 93, known to contain part of the Mat3 gene, did not generate Northern-blot signals under the conditions used.

2. Signals produced by cross-hybridizing repetitive sequences in the probe may obscure gene-specific signals. For example, one cannot discern the Nic7 mRNA against the smeared hybridization signals produced by probe 5 (Fig 2A).

   
   

   View larger version (71K): In this window In a new window Download PPT slide   Figure 2. Northern blots hybridized to selected MT locus probes. (A–D) Poly(A)+ RNA was isolated from mt+ vegetative cells (veg+), mt- vegetative cells (veg-), mt+ gametes (gam+), mt- gametes (gam-), zygotes 30 min after mating (zyg 30'), or zygotes 3 hr after mating (zyg 3 hr). The size of the RNA is indicated on the right (in kilobases). (A) Blot hybridized with probe 5 (Nic7 gene). (B) Blot hybridized with probe 6. (C) Blot hybridized with probe 61 (Mtd1 gene). (D) Blot hybridized with probes derived from the Mta1 and Mta2 cDNAs. (E) Blot hybridized with an Mta1 cDNA probe. Total RNA was isolated from veg+ and gam+, from zygotes 2.5 hr after mating (zyg 2.5 hr), and from gametes of an mt+/mt- diploid (diploid gamete). (F) Poly(A)+ RNA from the designated stages hybridized with the 6.5-kb XhoI fragment from the 16-kb repeat of the Ezy2 locus of mt+(Fig 7); the 3.9-kb Ezy2 signal is visible only in the 1-hr zygote sample (the minor band beneath it is assumed to be artifactual since it is not always present; cf. Fig 9).

3. The gene may not be expressed under the growth conditions used or during the life cycle stages tested.

4. The gene may not have been represented in any of our probes, although this is unlikely for the R domain. Given these considerations, the gene density displayed in Fig 1 is very likely to be an underestimate.

False positives result if the probe cross-hybridizes to messages derived from elsewhere in the genome. This could result from repetitive-sequence elements in the probe that are present in unrelated messages, most likely in the 3' untranslated region (UTR), or a probe that detects a transposon or a duplicated gene. In the case of duplicates, the copies in the MT locus might be functional (although we have no documented examples of this), or, like the genes in the a region of mt⁺ (see below), they might be pseudogenes. In constructing Fig 1 and Table 1 we endeavored to eliminate false positives, but this is necessarily subjective. In general, multiple bands or smears were considered false positives, as were cases in which a DNA fragment known to be present in only the mt⁺ or mt^- locus generated signals in RNA blots derived from both mating types.

Several regions where messages were identified by Northern blots were analyzed in more detail by DNA sequencing to confirm that the Northern analysis accurately predicts genes. This was considered particularly important to verify the existence of vegetatively expressed genes within the R domain and to identify new genes involved in the mating process. The Chlamydomonas expressed sequence tag (EST) data from the Kazusa Institute (ASAMIZU et al. 1999 , ASAMIZU et al. 2000 ) and from the Chlamydomonas Genome Project (http://www.biology.duke.edu/chlamy_genome) have been very useful for the first of these purposes. Not surprisingly, however, since these libraries were derived from vegetative cells, no ESTs were identified for any of the gamete- or zygote-specific genes reported here.

Transcriptional patterns in the mt locus: observations:
Fig 1 shows the location of major transcriptional units in the mt⁺ and mt^- loci, with additional information on the various transcripts provided in Table 1. Genes designated by boxes are expressed during the vegetative phase of the life cycle; most of these were also expressed in gametes and early zygotes (Table 1). These presumably represent genes whose products function throughout the life cycle, and they are henceforth referred to as housekeeping genes. Genes designated by circles are expressed only in gametes, with (+) transcripts found only in plus gametes, (-) transcripts found only in minus gametes, and (+/-) found in both; the expression of these genes is presumably regulated directly or indirectly by nitrogen starvation. Genes designated by triangles are expressed only in early zygotes (whether their expression continues into the late stages of zygote development/germination has not been investigated); the expression of these genes is presumably regulated directly or indirectly by gametic cell fusion (MINAMI and GOODENOUGH 1978 ; FERRIS and GOODENOUGH 1987 ).

The following sections describe genes or MT regions that were subjected to in-depth analysis.

Nic7:
Probe (Pr) 5 is a 2.1-kb genomic fragment from the center of the Nic7 gene in the T domain, as defined by rescue of the nic7 mutation using transformation (FERRIS 1995 ). Probe 5 hybridizes to several bands in Northern blots (Fig 2A), precluding identification of the Nic7 transcript. The 2079-bp DNA sequence of this probe was determined (GenBank no. AY032929). A segment of the (GT)_n repeat (KANG and FAWLEY 1997 ) in the fragment may be responsible for the cross-hybridization. (GT repeats are commonly encountered in the EST libraries.)

Database searches with this partial Nic7 sequence yielded no matches to C. reinhardtii ESTs (which is not surprising given that Nic7 is probably a low-abundance message, and the sequence is not near the 5' or 3' ends). After excluding six putative introns from the Chlamydomonas sequence, a significant homology (63% identity) was found to an Arabidopsis protein predicted from genomic sequencing (GenBank no. BAB09392). The function of the Arabidopsis gene is unknown. However, both sequences display a weak homology to prokaryotic quinolinate synthetase A genes (e.g., 24% identity to the Escherichia coli nadA sequence). Since quinolinate synthetase participates in one pathway of NAD biosynthesis (MAGNI et al. 1999 ), and since nic7 mutants require nicotinamide, the Nic7 gene (and its Arabidopsis counterpart) may code for this enzyme.

Pr6:
The Pr6 gene, detected by a cDNA called probe 6, is not expressed in vegetative cells but is transcribed at low levels in gametes, abundantly in 30-min zygotes, and somewhat less abundantly in 3-hr zygotes (Fig 2B). The gene is located in the T domain (Fig 1).

Two distinct classes of Pr6 cDNA clones, with slightly different sequences, are present in 1-hr-zygote cDNA libraries, indicating that both the mt⁺ (CC-620 parent) and mt^- (CC-621 parent) alleles are expressed. Restriction-site polymorphisms allowed us to assign the two cDNA types to their respective alleles (FERRIS and GOODENOUGH 1994 ), hereafter called pr6(+) and pr6(-). The pr6(-) cDNA encodes a 721-amino-acid protein (GenBank no. AY032930), whereas the pr6(+) cDNA (GenBank no. AY032931) contains an extra 8 bp in its coding region, generating a stop codon-producing frameshift that would result in a truncated 455-amino-acid protein. Synonymous and nonsynonymous codon differences also differentiate the two alleles (Table 2).

A single recombinant between nic7 and ac29 has been isolated (SMYTH et al. 1975 ), and this nic7 ac29a mt^- strain (CC-350) and its derivatives (including CC-421) contain the pr6(+) allele in an mt^- strain (confirmed by PCR amplification and sequencing). Zygotes produced in crosses between CC-350 derivatives and mt⁺ strains are viable despite the fact that they carry two copies of the Pr6(+) frameshift allele and no copies of the Pr6(-) allele, indicating that the Pr6 protein either is nonessential for zygote maturation (at least in the laboratory) or remains functional in its truncated form. Uniparental inheritance of chloroplast markers occurs normally in crosses using the Pr6(+)-carrying strains (our unpublished results).

The sequence of Pr6p is 40% identical, over 190 amino acids, to E. coli Endopeptidase IV (P08395), the signal peptide peptidase (ICHIHARA et al. 1986 ); a comparable level of similarity is found to an Arabidopsis EST (GenBank no. AAF24059). The homology resides in the C-terminal portion of the Pr6p protein that is presumed to be missing from the Pr6(+) frameshifted version.

Pyruvate dehydrogenase kinase:
The results using probes 37–39 highlight the problems of false positives and negatives. Probe 38 hybridizes to a 1.6- and a 1.9-kb message; probe 39 hybridizes to a 3.2- and a 1.1-kb message; probe 37, which partly overlaps probe 38, gives a negative result. The four signals all appear to be false positives; in fact, the message for the protein encoded in this region is not visualized.

We sequenced a 5813-bp region from segment 2 of the mt^- R domain (Fig 1) that covers the region represented by these three probes plus a few hundred flanking nucleotides. A BLAST search identified three C. reinhardtii ESTs to this sequence—two from the Chlamydomonas Genome Project mt⁺ set (AW758420 and AW758419 are the 5' and 3' ends, respectively, of the same clone) and one from the Kazusa mt^- set (AV643090)—which all correspond to the same mRNA. We sequenced the AV643090 clone completely to identify intron borders and the 3' end of the gene. This region contains a 4974-bp gene (GenBank no. AF387365) predicted to produce a message of 2.6 kb, which does not correspond to any of the bands seen on Northerns.

The predicted protein product is homologous to both pyruvate dehydrogenase kinase (Pdk) and the closely related branched chain -keto acid dehydrogenase kinase (Bckdk), containing all the conserved motifs (THELEN et al. 1998 ). Since Pdk has been characterized in plant mitochondria whereas Bckdk has not yet been identified in plants, we have opted to call the gene Pdk.

The Pdk gene resides near one end of segment 2 (Fig 1) such that its 3' UTR extends beyond the sequence discontinuity that marks the end of segment 2. This means that the final 146 bp of the mt^- 3' UTR and the final 125 bp of the mt⁺ 3' UTR are unrelated.

Glycine decarboxylase complex subunit H, Pr46a, and Pr46b:
Probe 46, a 5.4-kb Sal fragment from segment 3 of the mt⁺ R domain, hybridizes to a single 1.1-kb transcript seen only in vegetative cells; however, the sequence of probe 46 matched ESTs representing three different transcripts, two of 1.1 kb and one of 1.4 kb (Fig 3). In mt⁺ and mt^- genomic Southern blots, probe 46 hybridized to a single band, indicating that these three genes are present only in the MT locus.

View larger version (7K): In this window In a new window Download PPT slide   Figure 3. Genomic structure of the Pr46/GdcH region of segment 3. Open boxes represent untranslated regions; solid boxes represent coding sequences; thin lines represent introns. Only the 3' half of the GdcH gene is within the sequenced region. Arrows indicate direction of transcription. Key to the restriction sites used here and in Fig 5 and Fig 7: B, BamHI; E, EcoRI; H, HindIII; S, SmaI; Sa, SalI; Sc, SacI; X, XhoI; and Xb, XbaI.

The sequence of the leftmost message (Fig 3) encodes the mitochondrial enzyme glycine decarboxylase complex subunit H (GdcH), which participates in photorespiration (OLIVER 1994 ). The 3' half of the GdcH gene is included in probe 46, and it is well represented in the C. reinhardtii EST database. Several alternative poly(A) addition sites are represented in the EST collections, the most common (shown in Fig 3) located within the first intron of the adjacent gene (Pr46a). That is, there is partial overlap between the GdcH and Pr46a transcripts (Fig 3).

Gene Pr46a is represented by four ESTs, all from the Kazusa collection and hence derived from the mt^- allele of the gene. One of these (AV390703) was sequenced to determine the intron locations and the 3' end. The predicted Pr46a protein of 96 amino acids is highly conserved (80% identity to an Arabidopsis protein, 75% identity to a Caenorhabditis elegans protein) but of unknown function. The sequence does not appear in the yeast genome. A number of polymorphisms exist between the mt⁺ and mt^- alleles, only one of which is in the coding region, resulting in an Ile in mt⁺ and a Thr in mt^- at position 69, a poorly conserved region of the protein.

Gene Pr46b is represented by a single EST in the Kazusa collection (AV626473); this was sequenced to determine the positions of the two introns and the 3' end. One EST from the Chlamydomonas Genome Project collection confirmed the 3' end, and a second includes additional 5' sequence. The predicted Pr46b protein of 267 amino acids shows 30% identity to a human cDNA (GenBank no. AK023156) and its mouse homolog (GenBank no. AK006639), of unidentified function. Again there are polymorphisms between the mt⁺ genomic sequence and the mt^- cDNA: The six changes in the sequenced coding regions are all synonymous, suggesting that the gene is under selection.

Remarkably, the Pr46a and Pr46b mRNAs also overlap, in this case by 1005 bp: The 3' end of one message is within the last intron of the other gene and vice versa (Fig 3). The 3' UTR of each message overlaps part of the 3' UTR and part of the coding region of the other, but there is no overlap of their coding regions.

Region f:
In a previous publication we documented that the Mid gene, marked by the mid-1 mutation, resides in region f, which is flanked by segments 3 and 4 and unique to the mt^- R domain (FERRIS and GOODENOUGH 1997 ; Fig 1). Subsequently, Christoph Beck and colleagues generated a strain (CC-3712) with a deletion that covers all of region f plus 8–9 kb of segment 3 and 10–12 kb of segment 4 (our unpublished data). The deletion mutant (mid-2) has the expected pseudo-plus sterile phenotype of a mid mutant (GOODENOUGH et al. 1982 ) but undergoes apparently normal vegetative growth under laboratory conditions. Since no transcripts other than Mid hybridize to the regions deleted in mid-2, these regions, corresponding to probes 51–59 and 103, may be free of other genes.

Gene Mtd1:
Region d is a single-copy sequence found only within segment 4 of the mt^- locus (FERRIS and GOODENOUGH 1994 ; Fig 1). Probe 61, a restriction fragment from region d, hybridizes to a 2.2-kb mRNA, found in minus but not plus gametes and barely visible in the 30-min zygote sample (Fig 2C). The cognate gene for this message is called Mtd1. Several Mtd1 cDNA clones were isolated, one of which was sequenced. The 2274-bp message codes for the 625-amino-acid protein shown in Fig 4. No homologs of this protein are in current databases.

View larger version (59K): In this window In a new window Download PPT slide   Figure 4. Predicted sequence of the Mtd1 protein. Predicted transmembrane domains are in shaded boxes; canonical N-glycosylation sites are underlined.

Whatever function the Mtd1 protein provides to mt^- gametes, it cannot be essential in the laboratory, since mt⁺ gametes transformed with the Mid gene can mate as minus and produce meiotic progeny with a mt⁺ partner even though the Mtd1 gene is absent from both parents (FERRIS and GOODENOUGH 1997 ). Fusion between the gametes in such crosses is very slow, however, which suggests a role for Mtd1 in efficient cell fusion, perhaps as a component of the membrane overlying the mt^- mating structure (WEISS et al. 1977 ; GOODENOUGH et al. 1982 ). The predicted Mtd1 protein (Fig 4) has five NXT/S N-glycosylation consensus motifs and three predicted transmembrane segments, which, if threaded sequentially, would place the NXTs in an exterior orientation.

Genes Mta1, Mta2, Mta3, -Mta2, and -Mta3:
Region a was originally defined (FERRIS and GOODENOUGH 1994 ) as a 20-kb sequence between segments 1 and 3 in the mt⁺ R domain that is not present in the mt^- R domain. However, a homologous sequence is present in an autosome, meaning that plus cells carry two copies of the sequence and minus cells carry one. The extent of the duplicated sequence has been defined by comparing restriction maps, cross-hybridizing probes, and sequencing selected sections (Fig 5). Sequences that join the duplicated mt⁺ region to segments 1 and 3 (Fig 5) are absent from both the mt^- locus and from the autosomal domain.

View larger version (24K): In this window In a new window Download PPT slide   Figure 5. Maps of the autosomal (top) and mt+ (bottom) genomic regions carrying the duplicated a regions (bracketed by arrows; see Fig 3 for a key to restriction enzyme abbreviations). The two solid lines just under each map indicate the DNA that has been sequenced. The autosomal map shows the exon-intron structures of the Mta2 and Mta3 genes and the location of a crp2 repetitive element (DAY and ROCHAIX 1989 ). The mt+ map shows the region between segments 1 and 3 (cf. Fig 1), beneath which are indicated the regions corresponding to probes 111–121. The structures of the Mta1 gene and the -Mta2 and -Mta3 pseudogenes are depicted, as well as the locations of three insertions in the intergenic region between the -Mta2 and -Mta3 promoters.

Eleven probes (111–121 in Fig 5) were used in the Northern analysis. Of those flanking the duplicated a region, probes 111 and 112 detect the same 6.5-kb RNA, probe 119 detects a 3.0-kb RNA, and probe 120 detects a 0.9-kb RNA. However, since these signals are present in mt^- lanes as well, we interpret them to be false positives.

Probe 118, which lies within the duplicated a region, detects a 1.8-kb message at all life-cycle stages analyzed. The entire probe has been sequenced, and guided by EST matches we found that this message derives from a gene we call Mta3. The gene (GenBank no. AF309495) has one intron and encodes a predicted gene product of 166 amino acids, with a molecular weight of 18.5 kD, an isoelectric point (pI) of 11.3, and no homologs in the database. Since the Mta3 sequence lies within the duplicated region, the Mta3 ESTs from the vegetative plus library could have originated either from the autosomal copy or from the copy in the mt⁺ locus. However, the three ESTs analyzed all contain sequence polymorphisms specific to the autosomal copy, suggesting that the mt⁺ copy may not be transcribed. This inference is supported by the finding that the mt⁺ copy carries a mutation that deletes the intron 5' splice site so that an alternative splice junction would have to be used for the mt⁺ Mta3 gene to be functional. Our working assumption, therefore, is that the mt⁺ copy of Mta3 is a pseudogene, -Mta3, and that the expressed Mta3 gene is autosomal.

Probe 117, which also lies within the duplicated a region, detects two messages—one 2.2 kb and one 0.8 kb—both of which are absent from vegetative cells, present in gametes and 30-min zygotes, and at reduced levels in 3-hr zygotes (Fig 2D). The 2.2-kb species is present in gametes of both mating types, whereas the 0.8-kb species is present in plus gametes only (Fig 2D). cDNA clones that correspond to each have been isolated.

The smaller 0.8-kb message derives from a gene we call Mta1, which is present in the mt⁺ copy of the a region but absent from the autosomal copy. The Mta1 gene is expressed in mt⁺/mt^- diploid gametes (Fig 2E), indicating that its expression is not repressed by the Mid protein (diploids differentiate as minus gametes; EBERSOLD 1967 ). The mt⁺-unique gene Fus1 is also expressed in diploid gametes (FERRIS et al. 1996 ), suggesting that gamete-specific genes unique to the mt⁺ locus have lost, or never acquired, Mid repressibility.

The Mta1 gene encodes a predicted 126-amino-acid protein, Mta1 (Fig 6), of 14.6 kD, pI 7. Its C terminus is predicted to adopt a coiled-coil motif, generating BLAST matches to proteins such as lamin B. Amino acids 48–102, the main components of the coiled-coil domain, comprise five imperfect repeats of an 11-amino-acid sequence (Fig 6). A strikingly similar 11-amino-acid repeat domain is found in the ROPE protein of Plasmodium chabaudi (WERNER et al. 1998 ), where the motif is proposed to form a leucine histidine-zipper that interacts with other proteins.

View larger version (11K): In this window In a new window Download PPT slide   Figure 6. Predicted sequence of the Mta1 protein, with the five 11-amino-acid long repeats aligned. The H and L residues conserved in the homologous protein from Plasmodium (WERNER et al. 1998 ) are shown in boldface type.

The larger message detected by probe 117 derives from a gene that is expressed from the autosomal a region in gametes of both mating types but not vegetative cells. As reported elsewhere (FERRIS et al. 2001 ), this gene encodes a 386-amino-acid hydroxyproline-rich glycoprotein of unknown function. In our previous publication (FERRIS et al. 2001 ) we called the gene a2 and the protein A2; using the nomenclature adopted for the present article, we call the gene Mta2 and the protein Mta2.

When the autosomal and mt⁺ genomic sequences are compared, it becomes clear that the Mta1 coding region has been inserted into the Mta2 gene in the mt⁺ locus (Fig 5): The promoter region, the 5' UTR (and its intron), and the first nine codons of Mta1 correspond to the Mta2 sequences in the autosome, after which the two sequences diverge completely, with the rest of the Mta1 sequence being totally unrelated to the autosomal Mta2 sequence. Downstream of the 3' end of the Mta1 gene, Mta2 sequences pick up again: Although most of the second Mta2 exon and a portion of its second intron are missing, the remainder of the gene is present. Since these Mta2 sequences are not included in the Mta1 transcript, this means that transcriptional termination signals downstream of the Mta1 gene prevent expression of the adjacent Mta2 sequences. We therefore designate this region as -Mta2.

Table 2 shows the level of homology between Mta2 and -Mta2 and between Mta3 and -Mta3. The density of codon and noncodon differences is comparable in the two gene pairs, consistent with the possibility that the two pseudogenes were created at a similar time during C. reinhardtii evolution.

Insertions in the a region:
As detailed in the DISCUSSION, the configuration of the a sequences in the mt⁺ locus is most readily explained by proposing that the Mta1 gene transposed into the region, thereby inactivating the resident Mta2 gene and creating -Mta2. The presence of three insertions between the -Mta2 and -Mta3 sequences (Fig 5), which may have participated in Mta3 inactivation, offers additional evidence of transpositional activity in the region.

The first insertion is a 1278-bp sequence related to the TOC2 element described by DAY 1995 . The insertion has a perfect 14-bp inverted repeat at the two ends that is identical to one of the 14-bp TOC2 inverted repeats. The 60 bp at the left end of the insertion is an 83% match to one end of TOC2, and the 26 bp at the right end is a 92% match to the other end of TOC2 and, like TOC2, the insertion has created a 7-bp target-site duplication. However, the bulk of the insertion otherwise bears little resemblance to TOC2 or to any other sequence in the database.

The second insertion is a 249-bp sequence that resembles the 12-kb Gulliver transposon (FERRIS 1989 ): It has perfect 15-bp inverted repeats at the two ends that are a 14/15 match for the Gulliver right-end inverted repeat and creates an 8-bp target-site duplication like Gulliver. However, the sequence between the inverted repeats bears no resemblance to the limited sequences available for full-length Gulliver elements (FERRIS 1989 ).

The third insertion is a 361-bp sequence with a direct repeat of 34 bp at each end (1-bp mismatch). There is no unambiguous target-site duplication and no homology to previously characterized Chlamydomonas transposons.

The Ezy2 gene cluster:
An obvious structural difference between the mt⁺ and mt^- locus is a 16-kb DNA sequence tandemly repeated six to eight times in segment 3 of the mt⁺ R domain (Fig 1). This sequence is found in the mt^- locus as a single copy, split in two, a portion resident at the end of segment 3 and the remainder resident in the C domain (FERRIS and GOODENOUGH 1994 ).

To determine whether gene(s) are located within the 16-kb element, an mt⁺ genomic clone of the repeat unit was used to probe Northern blots. No signals were detected using vegetative or gametic samples, whereas a single 3.9-kb mRNA was detected in the 1-hr zygote sample (Fig 2F). A cDNA library generated from 1-hr zygotes was also screened with the probe, and one full-length cDNA was recovered and sequenced. An open reading frame of 3078 bp defines the unit gene, hereafter called Ezy2 (Early zygote 2; Fig 7). A genomic copy was also sequenced, which showed polymorphisms in its 3' UTR sequence to the full-length cDNA. Additional partial cDNAs were also characterized, some displaying polymorphisms to the full-length clone, suggesting that several, and perhaps all, of the Ezy2 repeats are transcribed.

View larger version (15K): In this window In a new window Download PPT slide   Figure 7. Structure of the Ezy2 region and comparison of Ezy2 and -Ezy2. (Top) The organization of 1.5 repeats of the 16-kb repeat unit (see Fig 3 for a key to restriction enzyme abbreviations), where a single repeat unit is indicated by the arrow with a solid circle on one end and an arrowhead on the other end. The locations of the 6.5- and 9.5-kb XhoI fragments used as probes are indicated by the double-headed arrows. Indicated at the top are the portion of the 16-kb repeat that resides in segment 3 of mt- and the portion that resides in the C domain of mt- (cf. Fig 1 and Fig 10). (Bottom) Comparison of the Ezy2 and -Ezy2 gene structures. The direction of transcription is indicated; a pair of double-headed arrows shows the location and extent of the exon-intron duplication. The structure shown for the -Ezy2 gene is hypothetical in the sense that it is no longer transcriptionally active.

The predicted Ezy2 polypeptide is shown in Fig 8. It displays a putative 42-amino-acid chloroplast transit peptide (Fig 8, boxed): An alanine follows the initiator methionine and the N-terminal region displays a high content of valine, alanine, and serine, albeit there are fewer arginines than expected for a transit peptide (VON HEIJNE et al. 1989 ). The VXA predicted cleavage site (FRANZEN et al. 1990 ) follows position 42, generating a mature polypeptide of 983 amino acids. The predicted size of Ezy2, minus the transit peptide, is 104 kD, and the predicted pI is 9.9, meaning that it might interact with DNA or with an acidic protein such as Ezy1 (ARMBRUST et al. 1993 ). However, no obvious DNA-binding or protein-binding motifs are present within the sequence, and no informative matches have been identified in the database. An intriguing feature of the sequence is that it displays a perfect internal direct repeat of 214 amino acids (Fig 8, boldface type followed by italics).

View larger version (75K): In this window In a new window Download PPT slide   Figure 8. Predicted sequence of the Ezy2 protein. The putative chloroplast transit peptide is boxed. The first internal 214-amino-acid repeat is in boldface type, and the second is in italics.

Fig 9A shows the pattern of Ezy2 expression during zygote development as monitored by RNase protection assays. The message appears almost immediately after zygote formation, peaks at 30 min, is greatly reduced by 2 hr, and is undetectable by 4 hr into zygote development. By comparison, Ezy1 expression peaks later (Fig 9A), as does the expression of most other zygote-specific genes (FERRIS and GOODENOUGH 1987 ; UCHIDA et al. 1993 ; KURIYAMA et al. 1999 ; SUZUKI et al. 2000 ).

View larger version (30K): In this window In a new window Download PPT slide   Figure 9. RNase protection analysis of Ezy2 gene expression. (A) Total RNA was extracted from gametes and from zygotes 5, 10, 30, 60, 120, or 240 min after zygote formation and was hybridized with Ezy1 or Ezy2 antisense RNA probes. The Ezy1 probe is 176 nucleotides (nt), and the protected fragment is 118 nt. The Ezy2 probe is 197 nt and the protected fragment is 172 nt. (B) RNase protection analysis of Ezy1 and Ezy2 message levels in either wild-type zygotes or zygotes resulting from a cross between mt- and mid-1 (Fus1) gametes. Total RNA was isolated from wild-type mt+/mt- zygotes 1 hr after the gametes were mixed or from the mt- mid-1 (Fus1) zygotes at the indicated intervals. The lower expression of the Ezy1 message in the mutant zygotes is presumably due to the fact that cell fusion is not efficient in these matings.

To determine whether the bisected copy of Ezy2 in the mt^- locus is expressed, a mating was performed between a normal minus strain and a mid-1 mt^- strain transformed with the Fus1 gene [mid-1 mt^- (Fus1)]. The mid-1 mutant, lacking a functional Mid gene, differentiates as plus and, when transformed with Fus1, is able to mate with minus gametes and form apparently normal zygotes (FERRIS et al. 1996 ) that carry two copies of the bisected Ezy2 gene in their mt^- chromosomes but no copies of the full-length Ezy2 sequences because they lack mt⁺ chromosomes. When these zygotes were subjected to RNase protection assays, no Ezy2 expression was detected (Fig 9B), indicating that the bisected sequence is a nonfunctional gene that we henceforth designate -Ezy2. As a control, RNase protection was also performed using the Ezy1 sequence, a gene tandemly repeated in both the mt⁺ and mt^- loci (Fig 1 and ARMBRUST et al. 1993 ), and expression was detected (Fig 9B), demonstrating that transcription of mt-linked genes is not generally impaired in these unusual zygotes.

The mt⁺ Ezy2 gene is 6 kb, with a contiguous "spacer" of 10 kb, meaning that the repeats in mt⁺ segment 3 span 100–140 kb. The gene has one intron in the 5' UTR and seven introns in the coding region (Fig 7). The 214-amino-acid internal repeat is encoded by exons 3 and 4 (Fig 7). The first internal repeat is 829 bp and the second is 842 bp, the length differences created by three insertions/deletions (indels) in the intervening intron. The introns are otherwise identical, and one synonymous codon difference is found between the duplicated exons. Restriction analysis indicates that the internal repeat is present in all the mt⁺ Ezy2 copies.

The genomic sequence of -Ezy2 was also determined. Whereas the restriction maps of the mt⁺ versions of Ezy2 are very similar, the restriction maps of Ezy2 and -Ezy2 share few common sites (FERRIS and GOODENOUGH 1994 ). However, the overall sequence homology between them is sufficiently high, and the intron/exon structure sufficiently well preserved, to allow an unambiguous alignment (Fig 7). In the -Ezy2 sequence, the spacer domain has been truncated at a downstream position, the missing portion now being located in the C domain (Fig 7). The most obvious difference between the coding regions of Ezy2 and -Ezy2 is that -Ezy2 lacks exon 4 and hence the internal direct repeat (Fig 7). In addition, a frameshift at the 5' end of -Ezy2 shifts the location of the first candidate initiator methionine to a more downstream position (Fig 7), and numerous nucleotide differences and indels have accumulated throughout the two versions of the gene (Table 2).

Zygote development in the absence of a mt⁺ locus:
As noted earlier, because the mid-1 mt^- mutant lacks a functional Mid gene, it expresses plus gametic traits; moreover, if it has been transformed with the Fus1 gene from the mt⁺ locus, it is able to fuse with mt^- gametes, generating zygotes that have two copies of the mt^- version of chromosome VI. These zygotes are apparently able to mature and germinate normally, indicating that the program for zygote development does not require genes such as Mta1 or Ezy2 that are exclusively encoded in the mt⁺ locus.

We went on to ask whether the uniparental transmission of plus cpDNA is affected in these zygotes. Table 3 compares the transmission patterns of chloroplast markers in control crosses and crosses in which a mid-1 mt^- (Fus1) strain served as the plus parent. Inheritance of chloroplast traits is seen to be biparental in the crosses involving the strain lacking an mt⁺ chromosome.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Coding capacity of the MT locus:
The rearranged R domain of the C. reinhardtii MT locus as well as the flanking T and C domain sequences that are under recombinational suppression are shown to contain genes that are expressed throughout the life cycle of the organism as well as genes expressed exclusively during the gametic or the zygotic phases of the life cycle. Although a comparable transcription map has not yet been generated for other regions of the C. reinhardtii genome, this distribution of genes is what one would expect if an ordinary chromosome had undergone large-scale rearrangements and had also gained a few gene sequences in one homolog but not the other. Large-scale rearrangements are found in the mouse T locus, which includes genes affecting male fertility (SILVER 1985 ; LYON et al. 2000 ), and in the self-incompatibility loci of Brassica plants (CASSELMAN et al. 2000 ; KUSABA et al. 2001 ), and genes-without-homologs characterize mating-type loci in the fungi (KRONSTAD and STABEN 1997 ; BADRANE and MAY 1999 ) and XY chromosome pairs in mammals (LAHN and PAGE 1999 ). Unusual architecture therefore appears to be a common feature of sex-related chromosomal domains.

A particular goal of this study was to ascertain whether the four large segments of rearranged DNA in the R domain contain active genes or are instead noncoding structural elements, as is the case, for example, for most of the mammalian Y chromosome (LAHN and PAGE 1997 ). Numerous active genes were in fact identified throughout the R domain (Fig 1 and Table 1). One of the genes (Pdk) encodes pyruvate dehydrogenase kinase, an enzyme that plays a key role in controlling pyruvate dehydrogenase activity and hence the TCA cycle and cellular respiration (ZOU et al. 1999 ). A second (GdcH) encodes glycine decarboxylase complex subunit H, an enzyme of the photorespiration pathway (OLIVER 1994 ; SRINIVASAN and OLIVER 1995 ). Two other sequenced genes (Pr46a and Pr46b), while of unknown function, have well-conserved homologs in several multicellular eukaryotes. Therefore, the R domain of chromosome VI appears to have maintained many, and perhaps all, of its prior genetic activities while having been subjected to numerous local rearrangements and insertion/deletion events.

It is widely assumed that one of the functions of meiotic recombination is to promote genomic integrity, and it has been demonstrated that chromosomes prevented from engaging in meiotic recombination are subject to deterioration, a model for the ontogeny of XY differentiation (CHARLESWORTH 1991 ; RICE 1994 ). One would therefore not expect important enzymes such as quinolinate synthetase, glycine decarboxylase, hydroxyethylthiazole kinase, and pyruvate dehydrogenase kinase, and important transcriptional regulators such as Rb, to be encoded in genomic regions that are under heavy recombinational suppression. Presumably any costs incurred by this suppression are offset by the advantage it confers, but the nature of the advantage has yet to be determined.

MT-unique sequences:
We also examined closely six regions of the MT locus that are found in one chromosome but not the other; these are hereafter referred to as MT-unique sequences. We were unable to detect any genes in two of these—region b in mt⁺ and region e in mt^- (see data at http://www.genetics.org/supplemental)—albeit it is of interest that region b is duplicated, in inverted orientation, at a site 1 cM telomere-proximal to the mt⁺ locus (FERRIS and GOODENOUGH 1994 ), yet another example of autosome/MT duplication. The remaining four MT-unique sequences appear to contain one active gene apiece. Each is restricted in expression to the gametic phase of the life cycle; two are plus specific and two are minus specific.

1. Region a in mt⁺ contains the gene Mta1 that is expressed in plus gametes only. The Mta1 protein is predicted to contain a leucine-histidine zipper and is of unknown function.

2. Region c in mt⁺ contains the Fus1 gene, encoding the Fus1 protein, that is expressed in plus gametes only and is necessary for plus-mediated gametic cell fusion (FERRIS et al. 1996 ).

3. Region d in mt^- contains the Mtd1 gene that is expressed in minus gametes only. The predicted Mtd1 gene product is a putative triple-span membrane protein with putative extracellular N-glycosylation sites. When mt⁺ gametes are transformed with the Mid gene, which causes them to differentiate as minus, their flagellar agglutination is strong but their cell fusion is very slow and erratic. Since such gametes lack the Mtd1 gene, there is a pleasing symmetry to the possibility that regions c and d might contain genes Fus1 and Mtd1 that code for plus and minus cell-fusion proteins, respectively. The Mtd1 sequence shows no homology to known membrane-fusion motifs, so if it proves to participate in membrane fusion it may do so by a novel mechanism.

4. Region f in mt^- contains the Mid gene that is expressed in minus gametes only. The Mid protein is necessary for minus gametic differentiation (FERRIS and GOODENOUGH 1997 ). Whereas there would be a pleasing symmetry in the postulate that the Mta1 protein is necessary for plus gametic differentiation, this is ruled out by the ability of mid-1 and mid-2 mutants to differentiate as plus gametes in the absence of an Mta1 gene.

In addition to these four genes, the Ezy2 gene is MT-unique as well, being expressed from the mt⁺ locus only. It differs from the four genes above in three respects: It is present in multiple tandem copies; its expression is initiated in the zygote rather than in the gamete; and it is not strictly unique to the mt⁺ locus in that a nonexpressed Ezy2 pseudogene is located in the mt^- locus.

Codon bias:
The first two genes to be sequenced from the C. reinhardtii MT locus were Fus1 (FERRIS et al. 1996 ) and Mid (FERRIS and GOODENOUGH 1997 ), and both had the surprising property of lacking the codon bias found in all other C. reinhardtii genes, generating the suggestion that bias might be relaxed because these genes both reside in the R domain and/or because both lack homologs. Table 1 documents that neither suggestion is generally applicable: The R-domain genes identified in this study all show moderate to strong codon bias (B value) and a high percentage of GC, including Mta1 and Mtd1, which have no homologs, and Ezy2, which no longer has a functional homolog. Therefore, the absence of bias in Fus1 and Mid remains unexplained, although it may indicate that they have been without homologs longer than the other genes (KLIMAN and HEY 1993 ).

Chloroplast DNA inheritance:
During the first 2 hr of zygote maturation in C. reinhardtii, cpDNA derived from the mt^- parent is normally degraded by nuclease digestion whereas cpDNA from the mt⁺ parent is preserved and later selectively replicated (UMEN and GOODENOUGH 2001A ), resulting in the uniparental-plus pattern of inheritance of chloroplast-encoded traits (ARMBRUST 1998 ). It has been postulated that this system is analogous to modification/restriction systems in bacteria, with the plus cpDNA being selectively "protected" by methylation so that it resists cutting by methylation-sensitive restriction enzymes in the zygote (SAGER and KITCHIN 1975 ). However, recent studies do not support such a model (UMEN and GOODENOUGH 2001A ), and the molecular basis for uniparental-plus inheritance awaits elucidation.

MATAGNE and MATHIEU 1983 observed that when heterozygous diploid (mt⁺/mt^-) minus strains were crossed with either haploid plus or homozygous diploid (mt⁺/mt⁺) plus strains, cpDNA transmission was biparental. These findings were interpreted to indicate that "protection" of plus cpDNA in the mt⁺/mt^- parent is dependent on the presence of the mt⁺ locus and is not subject to "minus dominance" (i.e., is not Mid-repressible). We show here that the Mta1 gene is restricted in expression to mt⁺ gametes and is not Mid-repressible (Fig 2E). However, our results would seemingly argue against a role for Mta1, or any other gene in the mt⁺ locus, in cpDNA protection since, in the absence of a mt⁺ chromosome (e.g., in mid-1 mt^- (Fus1) x mt^- crosses), zygotes give rise to viable meiotic progeny. If both plus and minus cpDNA were unprotected and hence destroyed in the early zygote, the cross would presumably be lethal, as is indeed the case in a related system (VANWINKLE-SWIFT et al. 1994 ).

The mid-1 mt^- (Fus1) x mt^- cross is not lethal, but neither is it normal: cpDNA is inherited biparentally (Table 3), suggesting that the missing mt⁺ chromosome is somehow necessary for the selective destruction of minus cpDNA in the zygote. For example, if the mt⁺-encoded Ezy2 protein participates in cpDNA destruction and is selectively targeted to minus chloroplasts in the zygote (perhaps because minus chloroplasts carry specific receptors for Ezy2 translocation; cf. BAUER et al. 2000 ), then biparental inheritance would be expected to occur in the absence of Ezy2.

Taken together, the results available at present are best explained by proposing that the mt⁺ locus encodes both a protection function and a destruction function, with Mta1 being a candidate participant in protection and Ezy2 in destruction. Both of these functions would be operative in the heterozygous-diploid crosses of MATAGNE and MATHIEU 1983 , generating two sets of protected genomes and hence biparental inheritance. By contrast, neither set of functions would be operative in our crosses, which would also result in biparental inheritance because neither set of unprotected genomes would be destroyed.

Evolutionary history of the MT locus:
A common way to model the evolution of separate sexes (heterothallism, dioecy) is to start with a self-fertile (homothallic, monoecious) ancestor and propose steps that would lead to self-sterility (e.g., CHARLESWORTH 1991 ). A homothallic lineage ancestral to C. reinhardtii can most simply be thought of as having a Mid gene in chromosome VI that switched "on" in some cells and "off" in others, the former cells expressing minus-specific genes and hence differentiating as minus gametes, and the latter cells expressing plus-specific genes and differentiating as plus gametes. Indeed, this is the inferred pattern of gene expression in the distantly related homothallic species C. monoica (VANWINKLE-SWIFT et al. 1998 ). The loss of Mid from a copy of chromosome VI would then generate a self-sterile plus-only clone carrying a proto-mt⁺ locus, while the loss of the off switch from the Mid gene in another copy of chromosome VI would generate a self-sterile minus-only clone carrying a proto-mt^- locus.

To model the subsequent "invasion" of the homothallic population by these two chromosomes, one can invoke the benefits of outcrossing as driving the process. Alternatively, or in addition, one can invoke positive selection for advantageous genes linked to the proto-mt loci and propose that the linkage would come to be buttressed by recombinational suppression (CHARLESWORTH 1991 ; RICE 1994 ; TRICKETT and BUTLIN 1994 ; but see FILATOV et al. 2000 ). One suggestion along these lines has been that linkage disequilibrium came to preserve an adaptive association between mating type and genes involved in organelle DNA inheritance (HURST 1992 ; HURST and HAMILTON 1992 ).

We can now consider possible origins of the MT-unique genes, using Fus1 as an example. There are two possibilities: Either Fus1 originally happened to reside in the proto-MT region of chromosome VI and was subsequently lost from the proto-mt^- chromosome or it was originally autosomal and then moved into the proto-mt⁺ locus, subsequently losing its autosomal representation and its Mid-repressibility. In either case, once any mating-related gene like Fus1 became MT-unique, it would become dependent on its MT-linkage for correct expression in plus or minus gametes. Thus, the acquisition of one or more MT-unique gametogenesis genes would lead to a selective advantage for chromosomal rearrangements or other changes that (further) suppress recombination in the region, thereby assuring that a gene like Fus1 is expressed in mt⁺ gametes and not expressed in mt^- gametes.

A mating-related gene could originate in or move into an MT locus by chance, and the loss of additional gene representation and of Mid regulation could also occur by chance. Alternatively, there may be some selective advantage to a cis-configuration of gametogenesis genes, as opposed to regulating their expression in trans. Since our results indicate that such "gene acquisition" events have occurred several times during the evolutionary history of the MT locus in C. reinhardtii, a selective advantage is suggested, but its nature remains to be identified.

Mutational profile of the MT locus:
The most striking feature of the MT locus is its unusual chromosomal organization (FERRIS and GOODENOUGH 1994 ). The present study provides additional examples of unconventional configurations.

As summarized in Fig 5, the a region, present in the mt⁺ locus and absent from the mt^- locus, is a sequence that is duplicated in an autosome and flanked by DNA that carries no identified genes. The autosomal copies of the a-region genes (the gamete-specific Mta2 and the housekeeping Mta3) are functional, whereas their mt⁺ counterparts are pseudogenes (-Mta2 and -Mta3). Of particular interest is the gamete-specific Mta1 gene in the mt⁺ locus, which co-opts the upstream regulatory elements and the first nine codons of an Mta2 sequence and then diverges into a unique open reading frame (ORF), the resulting gene being a chimera (Fig 5).

The most likely scenario for the generation of this chimera is to propose that the Mta1 sequence inserted into a preexisting Mta2 gene, thereby capturing a gamete-specific promoter, which is analogous to the acquisition of a testis-specific promoter by the Cdic gene in Drosophila melanogaster (NURMINSKY et al. 1998 ). This scenario leaves open many questions: Where did the Mta1 sequence come from? Was it autosomal or MT-linked and what were its original upstream sequences? Did an intact Mta2 gene originally exist in the mt⁺ locus, which was then disrupted by the Mta1 transposition event? Or was the chimeric sequence constructed in an autosome, perhaps in a duplicated copy of Mta2, and then transposed to the mt⁺ locus?

The GdcH, Pr46a, and Pr46b genes in segment 3 illustrate a different kind of unusual gene overlap. As summarized in Fig 3, each of these genes overlaps one of the others at its 3' end, but none of these overlaps have led to gene inactivation since all three are transcribed. Nothing is known about how these relationships were established, but, given the high density of rearrangements in the MT locus, it is possible that the three genes were once separated and were subsequently brought together. This is, to our knowledge, the first report of nuclear gene overlap in C. reinhardtii.

The major rearrangements involving segments 1–4 have generated two mutations characterized in this study. First, the distal portion of the 3' UTR of the Pdk gene, located at one edge of segment 2, contains region-b sequences in the mt⁺ locus and completely different segment 1 sequences in the mt^- locus (Fig 1). Presumably one of these sequences represents the original 3' UTR and the other was created by rearrangement; it is not known whether these differences affect the properties of the two gene transcripts. Second, rearrangements involving segment 3 of the mt^- locus have disrupted the Ezy2 gene. The large number of differences between Ezy2 and -Ezy2 compared to other gene/pseudogene pairs in the MT locus (Table 2) suggests that this event occurred in the more distant past.

The Ezy2 configurations are particularly intriguing in that they entail four different kinds of alterations: (1) rearrangement of gene order; (2) inactivation of the gene in the mt^- locus; (3) endoduplication of an exon in the mt⁺ locus gene; and (4) tandem duplication of the endoduplicated gene to generate six to eight copies. Fig 10 presents a possible scenario for the sequence of these three events, with details given in the legend.

View larger version (27K): In this window In a new window Download PPT slide   Figure 10. Postulated sequence for the evolution of Ezy2 genes. The original Ezy2, present in both mating types, is depicted as a single-copy gene flanked by an untranscribed "spacer" and containing one copy of the now duplicated exon 3. A double-stranded break occurred within the spacer region of the mt- copy, allocating a portion of the spacer to what is now the mt- C domain (boxed arrowhead) and the rest of the gene/spacer to what is now the mt- segment 3. [Segment 3 is presently inverted and is also separated from the C domain by segment 4 (Fig 1); the timing of these rearrangements vis-à-vis the evolution of the Ezy2 sequences is unknown.] The loss of the C-domain spacer sequences may have participated in rendering the mt- copy of the gene transcriptionally inactive and hence a pseudogene, or transcriptional inactivation may have occurred for other reasons: For a zygote-specific gene linked to mating type, loss of gene activity in one of the two loci may be difficult to select against. The mt+ copy, meanwhile, underwent a duplication of exon 3 and then subsequently underwent an expansion in copy number, perhaps in part to make up for the loss of the mt- copy. An alternative scenario would propose that Ezy2 was originally a multigene family in both mating types, each gene containing an unduplicated exon. In mt-, the subsequent chromosomal rearrangements deleted all but one copy, which subsequently became a pseudogene. In mt+, the exon duplication occurred in one copy and then spread by concerted evolution (SWANSON and VAQUIER 1998 ).

Several highly expressed autosomal zygote-specific genes have previously been found to exist as near-neighbor duplicates, including two cases in which both copies are functional (UCHIDA et al. 1999 ; SUZUKI et al. 2000 ) and one case in which one copy is now a pseudogene (MATTERS and GOODENOUGH 1992 ). Two additional examples of apparent near-neighbor duplicates of zygote-specific genes—detected by Pr 72/74 and by Pr 100 (Table 1)—have been found in this study. However, the long tandem iteration of Ezy2 genes and the nearby tandem cluster of zygote-specific Ezy1 genes in both chromosomes (Fig 1) clearly represent a distinctive phenomenon and one that appears to be a recurring theme in sexual evolution. In D. melanogaster, for example, a recent 10-fold tandem iteration of a sperm-specific gene is found in the X chromosome (NURMINSKY et al. 1998 ), and tandemly repeated genes are the rule in the human Y chromosome (reviewed in LAHN and PAGE 1997 ). Indeed, features of the AZF region of the human Y (SAXENA et al. 1996 ) offer striking parallels to the MT locus. AZF contains multiple copies, >99% identical in sequence, of a gene called DAZ (Deleted in Azoospermia), an RNA-binding protein essential for male fertility. The DAZ sequence is found as well in human chromosome 3, where expression is restricted to the germ cells of both sexes. During primate evolution, a copy of this autosomal gene transposed to the Y, where one of its exons underwent internal amplification, after which the modified gene itself underwent amplification.

The data reported here, combined with previous studies, reveal the MT locus to be an unusual and dynamic region of the C. reinhardtii genome, harboring translocations, inversions, large indels, genes without homologs, genes that transpose (FERRIS and GOODENOUGH 1997 ), tandem gene duplications, gene inactivation events, and, in two genes, unusual codon bias. Moreover, the sex-related genes in the locus have been shown to be undergoing rapid evolution between species (FERRIS et al. 1997 ). And yet, despite these anomalies, the locus continues to encode large numbers of housekeeping genes that presumably occupied this region of chromosome VI long before it took on its modern configuration and novel functions. Since most of the C. reinhardtii life cycle is carried out in the haploid state, the presence of these presumably essential genes may keep a selective brake on what would, in a predominantly diploid organism, be a far more extensive, Y chromosome-like reconfiguration of the region. If so, then the MT locus may offer a unique opportunity to observe sex-chromosome evolution in progress.

	FOOTNOTES

² Present address: Marine Molecular Biotechnology Laboratory, School of Oceanography, University of Washington, Seattle, WA 98195.

	ACKNOWLEDGMENTS

We thank Christoph Beck for providing the mid-2 deletion strain, the Kazusa Institute for sending several cDNA clones, and Kosuke Shimogawara for providing the sequence of the Thi10 gene. We also thank Chunsheng Luo, Eileen Westphale, and Linda Small for excellent technical assistance. This study was supported by grants from the National Science Foundation (MCB-9904667) and the U.S. Public Health Services (GM-26150).

Manuscript received June 15, 2001; Accepted for publication October 9, 2001.

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