Natural history of transposition in the green alga Chlamydomonas reinhardtii: use of the AMT4 locus as an experimental system

Abstract

The AMT4 locus of the green alga Chlamydomonas reinhardtii, which we mapped to the long arm of chromosome 8, provides a good experimental system for the study of transposition. Most mutations that confer resistance to the toxic ammonium analog methylammonium are in AMT4 and a high proportion of spontaneous mutations are caused by transposon-related events. Among the 15 such events that we have characterized at the molecular level, 9 were associated with insertions of the retrotransposon TOC1, 2 with a small Gulliver-related transposon, and 1 with the Tcr1 transposon. We found that Tcr1 is apparently a foldback transposon with terminal inverted repeats that are much longer and more complex than previously realized. A duplication of Tcr1 yielded a configuration thought to be important for chromosomal evolution. Other mutations in AMT4 were caused by two mobile elements that have not been described before. The sequence of one, which we propose to call the Bill element, indicates that it probably transposes by way of a DNA intermediate and requires functions that it does not encode. The sequence of the other and bioinformatic analysis indicates that it derives from a miniature retrotransposon or TRIM, which we propose to call MRC1 (miniature retrotransposon of Chlamydomonas).

Figure 1.—

Location of transposon-related mutations in AMT4. (A) Diagram of the AMT4 gene showing the relative locations of macrolesions in various strains above the line and locations of primers used to amplify the four segments of the gene (see text) below the line. Each strain name is followed by a lowercase italic letter to indicate the transposon responsible for the lesion: t for TOC1, c for Tcr1, b for Bill, g for Gulliver-like, and m for MRC1. The unknown upstream lesion of CR07 is indicated as CR07u. The seven exons are shown as numbered blocks with the 3′ noncoding tail of exon 7 shaded. (B) Use of PCR to identify macrolesions in the 48 strains from the second selection for methylammonium resistance (CR132–CR180; see text). The four primer sets were those indicated in A, which yielded products of 1.6, 1.1, 1.2, and 1.6 kb, respectively, for regions I–IV. In spite of the short PCR extension time, the TOC1 insert in CR148 is visible. CR136 has a deletion of 276 bp in exon 5 of the AMT4 gene.

Figure 2.—

Sequence surrounding the seven separate sites of the TOC1 insertion in AMT4. The TOC1 inserts, which occurred at the position of the open space, are omitted. They were aligned left to right and their orientation with respect to AMT4 (F, forward; R, reverse) is given after the strain numbers. The sites of TOC1 insertion in CR159 and CR50-6 are the same as in CR49 and each transposon is oriented in the same direction. Conserved adenine nucleotides 2 bp from the insert site are in boldface type and the CA and TG dinucleotides near the insert sites are underlined (see text).

Figure 3.—

Diagram of TOC1 and TOC1-related inserts in AMT4. (A) Diagram of TOC1 and the inserts in CR40 (intact TOC1; 5662 bp), CR 42 (right half; 577 bp), and CR49 (left half; 5085 bp). LTRs are indicated in black and with cross-hatching. The direction of transcription of TOC1 is from left to right, as is the numbering of nucleotides, which corresponds to the standard numbering. The numbers of base pairs in each region are given above the line for TOC1. Intact TOC1 was also found in strains CR135, CR138, CR148, CR153, and CR50-6. (B) Diagram of modifications to AMT4 in CR159 and sequence near the left boundaries of the intron 4/exon 5 duplications that bracket the TOC1 insert (red). The diagram is oriented with respect to the AMT4 gene and therefore the TOC1 insert has the opposite orientation to that in A and the numbering is reversed. The start of exon 5 is indicated by boldface nucleotides and the normal splice site is indicated with a “^” in both upstream and downstream copies of the duplication. The boxed green “T/A” in the downstream copy is the common nt 161 of TOC1 and 1340 of the AMT4 gene. The boxed “T/A” in the upstream copy is nt 1340 of AMT4. Red sequence is from TOC1. Repeated CTGCC sequences (see text) are underlined. Exon numbers are indicated below the line for the AMT4 gene.

Figure 4.—

Gulliver-related elements in AMT4 and effects of the CR07 insert on splicing (RT–PCR). (A) Sequence of the AMT4 gene of CR07 from the end of intron 3 to the end of exon 4. Target-site duplications are in boldface type and the insert sequence is highlighted in yellow. The splice site leading to the large RT–PCR product (C) is indicated with a “^” and the splice site leading to the small RT–PCR product is indicated with a “^^.” The position of the splice site used in parental strain 4A⁺, which was destroyed by the insertion, is indicated with a “(^).” Potential 3′ splice junction signals are underlined and the splice signal leading to the small RT–PCR product (C) is in green. The complement of the TILL2R primer (see text) is in red. (B) Comparison of the inserts in strains CR165 and CR07 with each other and with Gulliver termini (AF019750 and AF019751). Only Gulliver sequence similar to that of inserts in CR165 or CR07 is shown. The target-site duplications are in boldface type and the terminal inverted repeats are underlined. In addition, CR07 contains an insert of 32 bp with respect to CR165 (indicated by dashes for CR165). It is flanked by a 5-bp target-site duplication in purple. Nucleotides that differ between the three sequences are boxed. (C) RT–PCR products for the beginning of AMT4 from strains 4A⁺ and CR07. Products were amplified from RNA prepared in October 2002 (old RNA) and February 2004 (new RNA) with the ATGF and TILL2R primers (Figure 1). Strains were grown on arginine as nitrogen source. (D) RT–PCR products for 5′ and 3′ regions of AMT4 that bracket the region of the CR07 insertion. Primers were ATGF and TILL1R or Short1 and ENDR, respectively. The TILL1R primer is at the 3′-end of exon 3 and its sequence is given in Kim et al. (2005). (E) RT–PCR of the AMT1 gene from strains 4A⁺ and CR07 grown on arginine or ammonium. “Old RNA” was used for CR07. The PCR product from 4A⁺ genomic DNA (gDNA) is shown for comparison. Primers were AM2 and AM3 (Kim et al. 2005).

Figure 5.—

Tcr1 elements in AMT4. (A) RT–PCR products obtained from derivatives of CR46 carrying small Tcr1-related inserts that remain at the original insertion site (CR46-5, CR46-7, and CR46-8). The primers were −1577F and ATGR (Figure 1). (B) Complete DNA sequence of the small inserts (mini Tcr1) in CR46 derivatives CR46-5, CR46-7, and CR46-8. Terminal inverted repeats of 314 bp characterized in this work are underlined. Direct target-site repeats are in boldface type. The boxed sequences are examples of those that could pair with one another to yield stem-loop structures (1 with 2 or 3, or 2 with 4). The TCTG sequence present in one copy in mini Tcr1 but two copies in Tcr1 (see results) is indicated by letters on a solid background. The “ATG” translational start of AMT4 is indicated by a space just preceding it (bottom line). (C) Diagram of the Tcr1 insert in CR46, smaller inserts in the derivatives characterized in B, and derivatives that carry two Tcr1 inserts (CR46-6, CR46-10, and CR46-11). Arrows indicate the orientation of Tcr1.

Figure 6.—

Sequence of the newly described “Bill” element. The sequence of the insert in CR03 is given with the 8-bp target-site duplication in boldface type and the terminal inverted repeats (TIR) underlined. Internal inverted repeats are double underlined and numbered and internal direct repeats are overlined and numbered. They give a sense of the complexity of the internal structure of the element.

Figure 7.—

Sequences related to the newly described MRC1 element. (A) Sequence comparison for the CR50-1 (94 bp) and CR50-2 (29 bp) inserts. Each insert has the same 5-bp target-site duplication (boldface type) and the first 29 bp of CR50-1 are identical to those of CR50-2. (B) Sequence of the putative retrotransposon MRC1. The LTRs are boxed and the sequence identical to that of CR50-1 is indicated by letters on a shaded background. (C) Diagram of intact MRC1, a single LTR, and portions of the LTR in CR50-1 and CR50-2. Unique sequence in the intact element is indicated with a single line.