A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA

Abstract

Approximately 6000 specific DNA deletion events occur during development of the somatic macro-nucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis -acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetra-hymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis -acting sequences outside the deleted DNA.

Figure 1

Restriction map of Tlr1. The arrows represent the long inverted repeat. Within the inverted repeat are the 19mer tandem repeats: *, 19A (ATTATTTCTTTTTACATTT) and #, 19B (TTTCTCATTTTATGAAAAG)_. The bold lines represent macronucleus-destined DNA and the thin lines micronucleus-limited DNA. The lines above the map indicate the fragments used in cloning and for making probes. B, BglII; C, ClaI; E, EcoRI; H, HindIII; R, RsaI; S, Sau3A.

Figure 2

Rearrangement of a construct with the Tlr1 inverted repeat and flanking sequences. (A) The WT.cam construct and the major rearrangement product. Bold lines represent macronuclear DNA and the thin line is the micronucleus-limited DNA. The arrows show the inverted repeat. *, 19A tandem repeats; #, 19B tandem repeats; hatched bar, 346 bp of pBR322; cm^R, chloramphenicol resistance gene; B, BglII; C, ClaI; E, EcoRI; H, HindIII; M, BamHI; N, NotI; R, RsaI. The NotI or BamHI fragment of the unrearranged construct is 7.9 kb and the rearranged product utilizing the major in vivo junction is expected to be 2.2 kb. (B) Southern analysis of pDWT.cam transformants. Whole cell DNA was digested with BamHI and probed with the probe indicated by the open bar in (A). Lane 1 contains pDWT.cam plasmid DNA digested with BamHI to release the 7.9 kb unrearranged construct fragment. Lanes 2–7 have DNA from pDWT.cam transformants C10, F5, A8, H3, F2 and G3 that show a 2.2 kb band corresponding to accurately rearranged construct. Lane 8 contains DNA from a cell line transformed with pD5H8 vector.

Figure 3

Rearrangement of a construct without the HindIII fragment containing flanking sequences to the right of the element. (A) Restriction map of the IR.cam construct and the predominant rearrangement product, ap1.7. The bold lines represent macronuclear DNA and the thin line micronuclear-limited DNA for the major chromosomal rearrangement. Arrows, inverted repeat; *, 19A tandem repeats; #, 19B tandem repeats; hatched bar, 346 bp of pBR322; cm^R, chloramphenicol resistance gene; B, BglII; C, ClaI; E, EcoRI; H, HindIII; N, NotI; S, Sau3A. The open bar indicates the probe for the blots in (B), (C) and (D). The NotI fragment of the unrearranged construct is 7.1 kb and the rearranged product utilizing the junctions used in vivo is expected to be 1.5 kb. (B) Southern analysis of pDIR.cam transformants. Whole cell DNA was digested with NotI to release the construct from the rDNA. Lane 1, negative control lane containing DNA from a pD5H8 transformant. Lanes 2–10, DNA from independent pDIR.cam transformants. Seven of the nine transformants show an aberrant rearrangement that produced a 1.7 kb NotI fragment. (C) Whole cell DNA from pDIR.cam transformant A8 digested with NotI in lane1, NotI and ClaI in lane 2, and NotI and BglII in lane 3. (D) Whole cell IR.cam transformant A8 DNA digested with HindIII in lane 1, and HindIII and Sau3A in lane 2.

Figure 4

PCR mapping of ap1.7. (A) Diagrammatic representation of the pDIR.cam construct showing the location of the oligonucleotide primers. Vertical arrows indicate the boundaries of the deleted region for ap1.7 (B and C) PCR amplification of whole cell DNA from the IR.cam A8 transformant. M, marker lanes. (B) Left end: lanes 1–3 were primed with Tet 4, lanes 4–6 with Tet 3 and lanes 7–9 with Tet 2. The first of each set was against Tetrahymena DNA specific oligonucleotide Tet 5; the second and third sets with construct-specific oligonucleotides C1 and C2, respectively. (C) Right end: lane 1 was primed with Tet 3 and construct-specific oligonucleotide C3. Lanes 2, 3 and 4 were primed with Tet 3, Tet 2 and Tet 1, respectively, against construct-specific oligonucleotide C4.

Figure 5

Rearrangement of a construct with deletion of the 19mer repeats from the right inverted repeat. (A) Restriction map of the pDΔ.cam construct and the rearrangement. The bold lines represent macronuclear DNA and the thin line micronuclear-limited DNA. Arrows, inverted repeat; *, 19A tandem repeats; #, 19B tandem repeats; hatched bar, 346 bp of pBR322; cm^R, chloramphenicol resistance gene; B, BglII; C, ClaI; E, EcoRI; H, HindIII; M, BamHI; N, NotI; R, RsaI. The open bar indicates the probe for the blot in (B). The unrearranged construct is within a 7.4 kb BamHI fragment and for the accurately rearranged product the fragment is expected to be 2.2 kb. (B) Southern blot of whole cell DNA digested with BamHI to release the construct from the rDNA and probed with sequences from the left side of the element. Lane 1 contains the pDΔ.cam plasmid DNA restricted with BamHI to release the 7.44 kb unrearranged construct. Lane 2 contains DNA from a pD5H8 transformant as a negative control. Lanes 3–9 contain DNA from pDΔ.cam transformants, and show a 2.2 kb band corresponding to an accurately rearranged construct.