Article Figures & Data
Figures
- Fig. 1.
Maps of the macronuclear A51 gene (51Amac) and of the inserts of phages λ51Amic, λ51Gmic, and λ51Gmac. Coding sequences are represented by thick arrows in the macronuclear sequences. IESs are shown as black boxes in the micronuclear sequences. Double-headed arrows indicate paralogous IES pairs (IESs occurring at homologous positions in the two genes). Open boxes at the ends of the phage inserts symbolize the vector arms. The positions and lengths of probes a, b, c, d, and e are shown. Restriction sites: M, MnlI; P, PstI (only sites relevant to Fig. 2 and 3 are shown).
- Fig. 2.
Southern blot analysis of the macronuclear genome of sexual progeny of clones transformed with λ51Gmac, λ51Gmic, and λ51Amic. Total DNA from mass-autogamy samples was digested withMnlI. Because the mic/mac ploidy ratio is ∼1/250, only macronuclear DNA is visible. For each sample, the copy number of the phage insert in the maternal macronucleus is indicated by a vertical bar above the lane. Lane W1 is control DNA from uninjected cells. Lanes W2 and W3 contain the same DNA as in lane W1, mixed with a small amount of λ51Amic and λ51Gmic DNA, respectively, prior to digestion withMnlI. Symbols on the right indicate the positions of IES-retaining and wild-type macronuclear fragments. The same blot was hybridized successively with probes a, b, c, and d (Fig. 1).
- Fig. 3.
Southern blot analysis of the excision of IESs 51G4404 and 51G2832 in sexual progeny of clones transformed with λ51Gmic. Samples G1 to G6 and W1 to W3 are the same as in Fig. 2. Each of the mass-autogamy samples G2 to G6 is followed by a series of karyonidal samples representing individual clones isolated from the same mass autogamy. Vertical bars above the lanes indicate the copy numbers of phage inserts in maternal macronuclei. W0 is a control mass autogamy from an uninjected cell; 0a and 0b are karyonidal clones from this mass autogamy. All samples were digested with PstI. The same blot was hybridized successively with probes d and e (see Fig. 1). In addition to the PstI fragment containing IES 51G2832, probe e cross-hybridizes with the central PstI fragment from theG gene (upper band in lower panel), due to the repeated structure of this region of the coding sequence.
- Fig. 4.
Southern blot analysis of the excision of IESs 51A-712, 51A6649, and 51A2591 in sexual progeny of clones transformed with plasmids p51A-712mac, p51A-712mic, and p29A6649mic (see map in Fig. 5). Plasmid copy numbers in the maternal macronucleus are indicated by the vertical bars above the lanes. Mass-autogamy samples were digested withMnlI and BstBI. The same blot was successively hybridized with probes a, b (Fig. 1), and f (see map; M,MnlI; B, BstBI).
- Fig. 5.
Summary of the results showing the sequence specificity of the inhibition of IES excision by maternal sequences. The maps show the phage and plasmid inserts used. IESs are shown as black boxes. Paralogous IES pairs are indicated by double-headed arrows. The names of the five IESs showing inhibition are boxed. In the lower panel, the plus sign indicates that excision inhibition was observed. nd, not determined.
- Fig. 6.
Quantitative analysis of the excision of different IESs as a function of the copy number of specific sequences in the maternal macronucleus. “% excision in new macronucleus” is the fraction of rearranged copies in the new macronuclear genome, as measured in mass-autogamy samples. (A) Excision of IESs 51G4404 and 51G2832 after transformation of the maternal macronucleus with λ51Gmac or λ51Gmic. (B) Excision of IESs 51A2591, 51A6649, and 51A-712 after transformation of the maternal macronucleus with λ51Amic. (C and D) Filled symbols show the quantification of the maternal effects of IESs present on the endogenous macronuclear chromosomes and are superimposed on the same graphs as in panels A and B, respectively, to compare the inhibition efficiencies of IES-retaining chromosomes with those of the phage inserts.
- Fig. 7.
Internal IES-like segments excised from the macronuclear maintained copies of 51A2591 (A) and 51A6649 (B). Only the sequences of internal segments are shown. Boldface TAs are the excision boundaries of internal segments. Boxed TAs are the excision boundaries of the whole IESs. The sequences of homologous segments of theA29 allele are also shown; differences with theA51 allele are underlined. The 40-bp segment in 29A2591 is not excised in the macronuclear maintained version.
- Fig. 8.
Sequences of the ends of tested IESs. The left (L) and right (R) end sequences of each IES are aligned with the degenerate consensus established from the general IES sample (18, 19). All sequences are written 5′ to 3′. Subscripts in the general consensus indicate the fraction of sequences in the general sample showing the preferred nucleotide at each position. Sequences marked with asterisks are the ends of internal IES-like segments within 51A2591 (51A28) and 51A6649 (51A29).
