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Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Excision of the Drosophila transposable element mariner: identification and characterization of... The EMBO Journal vol.7 no.7 1988 pp.2185-2189, Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor capable of coding for a 346-amino-acid polypeptide chain Ann Meetha M.Medhora, H.MacPeek and (Jacobson et al., 1986). The element is present in 20-30 Daniel L.Hartl copies at dispersed locations in the genome of D. mauritiana Department of Genetics, Washington University School of Medicine, but is less abundant in other members of the melanogaster St Louis, MO 63110, USA species For two in group. example, copies occur typical Communicated by M.Ashburner strains of D. one or two in D. simulans, copies sechellia, and the mariner element is apparently absent in all strains of Genetic and molecular evidence presented in this paper D. melanogaster examined so far (K. Maruyama and demonstrate that the Mos factor for inherited mosaicism D.L.Hartl, unpublished). Most copies of mariner present is a of special copy the transposable element mariner. in the genome of D. mauritiana are apparently free of gross Mosaicism observed in the presence of the Mos (Mosaic) deletions because most copies are approximately the expected factor results from a high frequency of excision of the 1.3 kb in length. mariner element from an insertion site near the white- The excision factor, denoted Mos (Mosaic eyes), was in eye gene Drosophila mauritiana. The Mos factor originally recovered from rare mosaic flies occurring in a promotes the excision of mariner elements from genomic cross involving the wpch allele of D. mauritiana. Genetic insertion sites other than the site in wpchI, and it also studies were carried out primarily using strains of D.simulans promotes its own loss from the genome. Putative trans- into which both the wph allele and Mos had been introduced of positions Mos to new genomic sites have also been by repeated backcrossing. Initial genetic and molecular observed. A of copy mariner present at a particular site characterization of Mos revealed it to be a dominant in a Mos strain has been shown to be missing in derived autosomal factor present in chromosome number 3, which in strains which the Mos factor has been lost, and in acted by means of somatic excision of mariner from its strains with putative transpositions. We propose that this location in Wpch (Bryan et al., 1987). of is identical to the copy mariner Mos factor. Additional characterization of the excision factor Mos is Key words: ele- Drosophila/mariner/mosaicism/transposable presented in this paper. We show: (i) Action of the Mos ment/white gene factor results in excision of mariner elements from sites in the genome in in addition to the site Wpch. (ii) Action of the Mos factor can result in its own excision, detected pheno- typically by the occurrence of infrequent non-mosaic wph Introduction flies in the originally Mos-bearing mosaic strain. The mariner excision factor Mos is a genetic element (iii) Exceptional mosaic segregants from Mos-bearing identified by its ability to promote a high frequency of parents contain inherited mosaic factors in the X chromo- somatic excision of the transposable element mariner from some, chromosome 2 or alternative sites in chromosome 3, an insertion site near the white in maurtiana gene Drosophila suggesting that Mos is itself capable of transposition. (iv) The (Bryan et al., 1987). The mariner insertion occurs in the original third-chromosome Mos activity is associated with white-peach (Wpch) allele, interrupting the region of the 5' a particular chromosomal copy of mariner which is deleted untranslated leader upstream from the first exon, and results in non-Mos excision products. These results suggest that the in a peach-like color et Excision eye (Jacobson al., 1986). mariner excision factor Mos is itself a special copy of of mariner from this location during eye development mariner. restores pigment production in some cells and is identified phenotypically by the occurrence of somatic mosaicism in Results which the have in an otherwise eyes pigmented patches peach-colored et Excision Evidence that the Mos factor results in excision of mariner background (Bryan al., 1987). strain Wpch events occur infrequently in the original white-peach elements from other than that in shown in positions iS and give rise to occasional flies with a few facets Figure 1. The Southern blot was with a pigmented probed single copy from a (Jacobson and and genomic isolated Hartl, 1985; Haymer Marsh, 1986). sequence sequences flanking from a D.simulans clone However, in strains containing the inherited excision factor mariner element selected at random of is which contains Mos, somatic excision the mzariner element so frequent from a Mos library prepared WPch; strain, in both D.simulans mariner elements and additional that is mosaic with endogenous every fly multiple pigmented patches also increases introduced from D. mauritiana in the course of both eyes. The excision factor elements dramatically the allele rise to When with this the rate at which backcrossing. probed anonymous (unknown white-peach gives pheno- derivatives in line cells et the BamHI-Hindm typically wild-type germ (Bryan genomic location) probe, genomic of Mos flies are to a 4.8-kb al., 1987). digests wpch; expected give is 1286 in to the The mariner element transposable bp length, hybridization band, corresponding mariner-containing four in the a 3.5-kb band flanked terminal inverted fragment present library, plus (smaller by 28-bp repeats containing frame which results from the somatic excision of and includes a by 1.3 kb), mismatches, single, long open reading ©IRL Press Limited, Oxford, England M.M.Medhora, A.H.MacPeek and D.L.Hartl pch pch + + Mos nt 0C pch nt ' e + 2 + +Mos Select Gic -3 5 _ nt e + w pch $X; + Mos +;, pch nt' e + .. flies for ebony colored, mosaic-eyed Screen (1) non-mosaic flies (2) non-ebony, or loss of Mos activity. Fig. 2. Mating scheme to detect transposition of DNA with Fig. 1. Filter hybridization of digest genomic probed males that are for autosomal First mating produces heterozygous a mariner element at an site labeled fragment adjacent to anonymous as well as the third-chromosomal Mos. In the next markers net and e, Each lane contains DNA from a in the genome. single fly: 1, ebony-body, non-mosaic flies are candidates for Mos generation, wPch D. simulansID. mauritiana hybrid, non-mosaic; 2, transposition, and non-ebony, non-mosaic flies are candidates for Mos wOch; Mos D.mauritiana D.simulanslD.mauritiana hybrid, mosaic; 3, excision. insertion at the site. The lower wild-type, without mariner anonymous from somatic excision of mariner in the Mos band in lane 2 results DNA was obtained a genotype. The flanking by screening partial Table I. Excision and apparent transposition of Mos factor from strain Z8 Materials and Genomic genomic library (see methods). and The mol. wt markers are DNA digested with BamHI HindlIl. Strain Number of Excision Excision Transposition double of X DNA. from EcoRI-HindIII digests bacteriophage from wpch of Mos of Mos offspring (class 1) (class 2) (class 3) from this location. The somatic excision mariner anonymous Z3 14320 111 (1.55%) 10 (0.14%) 6 (0.08%) band is in the Mos strain but not in the non- readily apparent Z8 14 057 127 (1.81%) 78 (1.11%) 14 (0.20%) that excision of this mariner Mos control (Figure 1), implying Z8 10 982 62 (1.18%) 46 (0.84%) 10 (0.18%) occur in the Mos strain. A similar element does experiment using a different anonymous probe isolated at random from number of because each using half the total progeny counted, a D. mauritiana indicated somatic excision in Mos library of are detectable of the three exceptional types progeny only strains from this unrelated location also (G.Bryan, unpub- of the that have the in the 50% offspring appropriate conclude that excision of the mariner element lished). We background genotype. strains is not restricted to the mariner element present in Mos the rate of reversion was somewhat greater Although wlch wpch includes most or all mariner in the allele, but probably in the first with Z8 than in the second (P < experiment elements present in the genome. the absolute amount of the difference was not large. 0.01), Possible excision and/or of the Mos element transposition Mos excision and were not Rates of putative transposition was by means of the matings outlined in itself investigated different in the two Z8 However, significantly experiments. D.simulans males of genotype wpch; Figure 2. Mosaic Z3 with the rate of Mos excision was / cross of non- in comparing Z8, netl e + + Mos were obtained from the +; -7-fold greater in the Z8 strain than in Z3 (P < 0.0001), males. mosaic e females with wpch; Mos mosaic wPch; net; pch while the rates of w reversion and putative Mos trans- in net veins and The recessive allele net and e result wing The for the were not significantly different. reason the order is position ebony body color respectively. (Although gene between Z3 and Z8 is differences in Mos excision rates if e were to the left of Mos could be on the written as Mos, unclear. males were crossed other side.) The doubly heterozygous of obtained from A sample of exceptional progeny class 1, en masse with wpch; net; e females. Exceptional progeny was discussed in a a somewhat different mating scheme, were of three classes. et restriction previous report (Bryan al., 1987). Diagnostic Females with which result from (1) wild-type eye color, Wpch fragments from the putative revertant alleles from were of the mariner element in wpch. germ line excisions - wpch found to be 1.3 kb smaller than those in itself, Non-mosaics with wild-type (non-ebony) body color, (2) mariner element. It has not been implying excision of the which the evidence below indicates as resulting from self- whether the excision events are determined primarily precise excision of the Mos factor. or most of the revertant alleles result imprecise. However, (3) Mosaics with ebony body color, which contain in wild-type phenotypes. heritable mosaic factors in the X chromosome, in chromo- apparently A of nine of class 2 from the some 2 or in the chromosome 3. sample exceptional progeny ebony-bearing Z8 was mated with non- Two Mos strains of D.simulans, Z3 and Z8, were exam- second experiment individually Wpch Z8 in order to confirm that the exceptional ined using the mating scheme in Figure 2. The Z3 and mosaic flies strains were independently by repeated back- non-mosaic parents did not produce mosaic progeny in future produced No in of the D. mauritiana mosaic strain E25H to generations. mosaicism was detected these progenies, crossing of the Mos factor. D.simulans. Since there was no statistically significant indicating apparent permanent loss Filter DNA from the Mos revertants, tendency for exceptional types of progeny to occur in clusters hybridizations using when probed with mariner DNA, indicated the consistent (indicated by homogeneity in frequency of exceptions found loss of a particular mariner-containing fragment. An example among culture bottles), the data have been pooled and are in Table I. Strain Z8 was studied in two indepen- is shown in Figure 3, in which a BamHI-HindIll digest presented dent experiments. The frequencies given were calculated of single Mos revertant flies from the Z8 strain was com- 2186 Mos excision factor R R R R R M M M M M M. wprF wpc T T T kb Zr kb Nv, 9.46- 6.75- -6.75 -5.05 * I ^. -4.26 Fig. 3. Filter hybridization showing mariner elements in individual Z8 D.simulans obtained from the mating scheme in Figure 2. Lanes as follows: M, mosaic flies; R, non-mosaic putative Mos excisions Fig. 4. Filter hybridization showing manriner elements in exceptional (exceptional progeny of class ii). Arrow indicates a band hybridizing progeny of class 3, in which the Mos factor maps to a new with mariner that is present in all mosaics and absent in all putative chromosomal location. Each lane contains DNA from an Mos individual excisions. Genomic DNA digested with BamHI-HindIII, and fly, as follows: M, individual Z8 mosaics; @pch, white-peach with of non- probed internal Sall-SphI fragment mariner. Molecular weight mosaic controls; T, descendants of independently obtained exceptional markers are from a HindIll digest of bacteriophage DNA. progeny of class 3 in which the Mos factor had been mapped to the X, 2 and 3 chromosomes (left to right respectively). The 5. 1-kb band pared with non-revertant single flies from the same strain, (arrow) corresponding to the presence of Mos in Z8 is missing in the using a Sail -SphI internal fragment of mariner as the probe putative transpositions. Restriction digests, probe and mol. wt markers (Jacobson et al., 1986). The mosaics and ('M' lanes) clearly as in Figure 3. consistently show the presence of a DNA hybridizing frag- ment of - 5.1 kb (arrow), which is absent in the A total of 11 exceptional progeny invariably were of this type. Unless revertants ('R' lanes). A total of 30 independent revertants these represent de novo occurrences of heritable mosaic were examined in this manner, and all were missing the factors, the exceptions represent either transpostion of Mos 5.1-kb band. The hybridizing band in has been into the homologous chromosome question 3 or possible recombina- cloned and similar blots carried out using a tion in the male. sequence flanking the mariner element as the probe. With this non- (ii) Mosaic progeny approximately equal numbers probe, of ebony revertants show a hybridizing band of 5.1 kb, but in the and non-ebony and approximately equal numbers of females revertants the band is 1.3 kb smaller, which is consistent and males. This result indicates presence of the mosaic factor with of deletion a mariner element from the in either chromosome 2 or chromosome 4. A total of 10 fragment detected by the probe (data not These results putative transpositions were of this type. shown). suggest that in the mosaic phenotype the Z8 Mos strain is associated (iii) Mosaic progeny all female, indicating presence of the with a mariner in element at a particular site the genome, mosaic factor in the X chromosome. A total of two putative which is lost to upon reversion the non-mosaic state. transpositions were of this type. sample of 23 exceptional progeny of class 3 from the Progeny tests of type (i) and (iii) provide unambiguous Z8 strain was also studied. As demonstrated below, these evidence of the presence of heritable mosaic factors in individuals contain heritable mosaic factors in new locations chromosome 2 and the X chromosome. Subsequent crosses in X have shown that the genome, including the chromosome, chromosome the mosaic strains containing these factors 2 and the chromosome 3. are as stable as ebony-bearing The relatively those containing the original Mos simplest to the occurrence of these factor located in hypothesis explain exceptions is chromosome 3 (data not shown). that the Mos factor in the chromosome 3 origial wild-type Strains containing heritable mosaic factors in the X is capable of and the result 2 transposition, exceptional progeny chromosome, chromosome and the ebony-bearing chromo- from such events. some 3 were tested for the of transposition presence the characteristic Exceptional class 3 were mated to the mosaic 5.1-kb restriction associated progeny map mariner-containing fragment factor. Mosaic males were crossed with mariner D. simulans with the original Mos factor in chromosome 3. As expected, females in order to isolate the new mosaic factor in this restriction was absent from the as fragment strains, F1 mosaic males were shown in 4. Each lane DNA heterozygous form. The Figure contains from a resulting single with mariner. The restriction then crossed with e and the individual probed enzymes and Wpch; net; females, F2 progeny Three of were found the were the same as used for 3. The lanes classified and counted. probe Figure types progeny in the labeled 'M' are individual Z8 and the characteristic F2 generation: mosaics, 5. 1-kb band is indicated with an arrow. DNA from (i) All mosaic progeny phenotypically ebony, indicating in the chromosome. individuals three class 3 presence of the mosaic factor representing exceptional progeny e-bearing 2187 M.M.Medhora, A.H.MacPeek and D.L.Hartl in the lanes labeled 'T'. The mosaic factors in elements Ac and P are 4.5 and 2.9 kb respectively, and are shown to chromosome X, 2 and 3 deletion derivatives are common in the genome. In contrast, these strains were mapped respectively (left to right). Although each newly derived mariner elements longer than 1.3 kb have not been observed, mosaic 'T' strain contains a heritable mosaic factor, all lack even in strains containing the Mos element, and deletion mariner element associated with mosaicism derivatives are not common. Moreover, the 1.3-kb mariner the particular in the Z8 strain. element contains a single open reading frame. At least five copies of mariner from anonymous positions in the genomes of D.maruitiana, D.simulans and D.yakuba have been Discussion sequenced, and all preserve the same open reading frame and are not bp (K.Maruyama and mariner element from longer than 1286 Heritable excision of the transposable D.L.Hartl, unpublished). The mariner element shares a from other sites in the results the WPCh allele and genome number of similarities with the transposable element Tc 1 in from factor initially mapped a trans-acting designated Mos, the nematode Caenorhabditis elegans. For example, et The basis Tcl to chromosome 3 (Bryan al., 1987). genetic contains a single open reading frame, excises in both germ to be either a mutation in a host of Mos was postulated gene line and somatic cells, and maintains the full 1.6-kb length of mariner that acts in trans. The trans action or another copy among most copies examined (Rosenzweig et al., 1983; mobile elements is observed both in of genetic commonly D.E.Moerman and R.H.Waterston, personal com- in and For I.Mori, prokaryotes and eukaryotes (Berg Howe, 1988). elements in munication). example, the first reported transposable maize, Several models for the molecular basis of Mos may and Enhancer-Inhibitor Activator-Dissociation(Ac-Ds) (En- for its to excision and account ability promote transposition of autonomous and non- 1), both involve two-part systems that Mos of mariner. One class of models assumes activity autonomous elements (McClintock, 1947, 1948, 1949; results from nucleotide sequence alterations within the Mos The autonomous elements Ac and Peterson, 1953, 1960). for Mos is based element. A second class of models activity En code for that direct not their own proteins only transposi- effects mediated on position effects, as through position by but also mobilize the non-autonomous Ds and I tion, help or enhancers to chromatin structure or promoters external The non-autonomous elements often contain elements. the element. mutations, such as large deletions in their protein-coding Mos factor a The occurrence of the suggests possible regions. for the increase or decrease evolutionary mechanism rapid The possibility that Mos was a special copy of mariner of elements in the in the number of copies transposable was tested using genetically marked strains of D.simulans of the rate transposition, presence genome. By increasing 3-10 of mariner. The data indicate containing copies genetic in the number of the Mos factor may lead to an increase at a due that Mos itself is lost spontaneously high frequency Mos of mariner elements. At the same time, promotes also occur that contain to excision. Exceptional segregants well as in somatic excision of mariner in the line as germ mosaic factors in the X chromosome heritable chromosome, number of cells, therefore tending to decrease the copies. marked chromosome 3. The 2 or a genetically simplest of Mos is observed in its A third important characteristic is that these arise from transpositions hypothesis exceptions and dysgenic effects (D.Garza, G.Bryan D.L.Hartl, of the Mos but we cannot exclude the that factor, possibility that Mos is harmful to fitness unpublished), which suggest of de novo the strain a Mos-bearing generates high frequency will to be eliminated natural selection. The and tend by from Mos itself. In heritable mosaic factors different any forces on the rates outcome of these depends quantitatively that Mos is a these results the view transposable case, support and excision of mariner elements of transposition (including element. excision of the Mos factor balanced transposition and itself), of mariner Molecular analysis identified a particular copy selective elimination of Mos due to its against the dysgenic or of the Mos that correlated with the presence absence An theoretical is in some effects. intriguing possibility that, factor are this factor. Strains that have lost the Mos missing cases, a Mos-like derivative of a transposable element might Putative with heritable copy of mariner. transcriptions arise and proliferate, and, even as it is being eliminated by in the also lack this mosaic factors in new positions genome selection, result in a rapid increase in the number of copies but contain one or more additional copy of mariner, they of its sister elements. The increased copy number may persist mariner elements. even after the Mos-like elements have been completely be drawn between the Mos-mariner An analogy may eliminated by selection, and in the absence of Mos-like and the derived P element because system designated PA2-3, elements, the genome with the increased number of copies the PA2-3 behaves like the Mos factor in the promoting contains no evidence of the mechanism that produced the P in and deletion and of elements somatic transposition germ result. the breaks down cells (Laski et al., 1986). However, analogy construct in which the 2-3 because PA2-3 is an engineered intron has been the need for the deleted, eliminating splicing Materials and methods that normally confines P-element activity to the germ line. strains Drosophila of mariner Naturally occurring copies exhibit both germinal Strains D.simulans wPh and wPch; Mos were constructed as D.simulans and somatic instability (Jacobson and Hartl, 1985; Haymer described et introducing the Vlch and Mos genes from (Bryan al., 1987) by and Marsh, 1986). D. mauritiana into D.simulans through repeated backcrossing. Ten series of independent backcrosses were carried out to generate independent lines The molecular structure of mariner exhibits one poten- of D.simulans wxh; Mos, called Zi-Z1O, which were individually difference with similar tially significant superficially maintained. The strain Ach; net; e strain of D.simulans was constructed transposable elements in eukaryotes. Autonomous elements, by crossing D.sinulans wPch females with D.simulans net; e males obtained such as Ac in maize and the P element in D. melanogaster, from sib F1 J.Coyne, mating the and selecting individual wPch; net; e virgin females and males from contain multiple open reading frames. The autonomous the F2 to establish the strain. Flies were maintained 2188 Mos excision factor on standard cornmeal medium or Formula 4-24 instant medium (Carolina Biological, Burlington, NC). Filter hybridizations Isolation of DNA, restriction digests, gel electrophoresis and Southern transfers were carried out as described (Jacobson et al., 1986). DNA probes were obtained from isolated restriction fragments of DNA labeled with 32p by the method of Feinberg and Vogelstein (1983) using random hexamers obtained from Pharmacia, Inc. Specific activities of the probes were >6 x 108 DNA. The DNA used as probe to study the excision of c.p.m./1g mariner elements from anonymous positions in the genome was isolated as described below. The mariner Sall - SphI fragment was obtained as described in Jacobson et al. (1986). Construction of partial library from strain Z8 A partial library of genomic DNA was made from the strain Z8 using the commercially available vector Bluescribe M 13 + (Vector Cloning Systems, San Diego, CA). Genomic DNA was isolated as described (Lis et al., 1983; Kune et al., 1985), and 20 tg was digested to completion with BamHI and HindIII. The restriction digest was fractionated by electrophoresis in a 0.8% agarose gel. A slice of gel containing DNA of -4.5 -5.5 kb was cut out and the DNA extracted as described (Vogelstein and Gillespie, 1979). The extracted DNA wasmixed in a ligation reaction with vector DNA that had been digested to completion with the same two enzymes, using -0.5 Ag of fragment and -0.1Itg of vector in a 10-1l reaction volume. The enzymatic reactions were carried out as described (Maniatis et al., 1982). The ligation mixture was used to transform Escherichia coli strain DH5a [F- X- hsdR17 recAl A(lacZYA-argF)]. The resulting library was screened by colony filter hybridization using a32P-labeled Sall-SphI internal fragment ofmariner as probe. Positively hybridizing colonies were purified, the cloned DNA isolated, digested with PstI, transferred to filters and hybridized with the probe. A 1.7-kb fragment of DNA flanking the mariner element in the genome was isolated from the clone pMl 1O. This DNA did not hybridize with mariner, and was used to study excision of the mariner element in the strain Z8, as described in the legend of Figure 1. Acknowledgements We are grateful for the generous support of our laboratory colleagues, especially Danny Garza for helpful discussion in planning the genetic crosses, Kyoko Maruyama for providing plasmids and continuous support, Glenn Bryan for fly strains, and Suhas Phadnis and Bob DuBose for their comments on the manuscript. This work was supported by grant number GM33741 from the US National Institutes of Health. References Berg,D.E. and Howe,M.M. (eds) (1988) Mobile DNA. American Society for Microbiology, Washington, DC, in press. Bryan,G.J., Jacobson,J.W. and Hartl,D.L. (1987) Science, 235, 1636- 1638. Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem., 132, 6-13. Haymer,D.S. and Marsh,J.L. (1986) Dev. Genet., 6, 281 -291. Jacobson,J.W. and Hartl,D.L. (1985) Genetics, 111, 57-65. Jacobson,J.W., Medhora,M.M. and Hartl,D.L. (1986) Proc. Natl. Acad. Sci. USA, 83, 8684-8688. Kuner,J.M., Nakanishi,M., Ali,Z., Drees,B., Gustavson,E., Theis,J., Kauvar,L., Kornberg,T. and O'Farrell,P.H. (1985) Cell, 42, 309-316. Laski,F.A., Rio,D.C. and Rubin,G.M. (1986) Cell, 44, 7-19. Lis,J.T., Simon,J.A. and Sutton,C.A. (1983) Cell, 35, 403-410. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. McClintock,B. (1947) Carnegie Inst. Washington Year Book, 46, 146-152. McClintock,B. (1948) Carnegie Inst. Washington Year Book, 47, 155-169. McClintock,B. (1949) Carnegie Inst. Washington Year Book, 48, 142-154. Peterson,P.A. (1953) Genetics, 38, 682-683. Peterson,P.A. (1960) Genetics, 45, 115-133. Rosenzweig,B., Liao,L.W. and Hirsh,D. (1983) Nucleic Acids Res., 11, 4201 -4209. Vogelstein,B. and Gillespie,D. (1979) Proc. Natl. Acad. Sci. USA, 76, 615-619. Received on March 14, 1988; revised on April 25, 1988 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor.

Medhora, M. M.; MacPeek, A. H.; Hartl, D. L.
The EMBO Journal , Volume 7 (7) – Jul 1, 1988
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Springer Journals
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Copyright © European Molecular Biology Organization 1988
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0261-4189
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10.1002/j.1460-2075.1988.tb03057.x
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Abstract

The EMBO Journal vol.7 no.7 1988 pp.2185-2189, Excision of the Drosophila transposable element mariner: identification and characterization of the Mos factor capable of coding for a 346-amino-acid polypeptide chain Ann Meetha M.Medhora, H.MacPeek and (Jacobson et al., 1986). The element is present in 20-30 Daniel L.Hartl copies at dispersed locations in the genome of D. mauritiana Department of Genetics, Washington University School of Medicine, but is less abundant in other members of the melanogaster St Louis, MO 63110, USA species For two in group. example, copies occur typical Communicated by M.Ashburner strains of D. one or two in D. simulans, copies sechellia, and the mariner element is apparently absent in all strains of Genetic and molecular evidence presented in this paper D. melanogaster examined so far (K. Maruyama and demonstrate that the Mos factor for inherited mosaicism D.L.Hartl, unpublished). Most copies of mariner present is a of special copy the transposable element mariner. in the genome of D. mauritiana are apparently free of gross Mosaicism observed in the presence of the Mos (Mosaic) deletions because most copies are approximately the expected factor results from a high frequency of excision of the 1.3 kb in length. mariner element from an insertion site near the white- The excision factor, denoted Mos (Mosaic eyes), was in eye gene Drosophila mauritiana. The Mos factor originally recovered from rare mosaic flies occurring in a promotes the excision of mariner elements from genomic cross involving the wpch allele of D. mauritiana. Genetic insertion sites other than the site in wpchI, and it also studies were carried out primarily using strains of D.simulans promotes its own loss from the genome. Putative trans- into which both the wph allele and Mos had been introduced of positions Mos to new genomic sites have also been by repeated backcrossing. Initial genetic and molecular observed. A of copy mariner present at a particular site characterization of Mos revealed it to be a dominant in a Mos strain has been shown to be missing in derived autosomal factor present in chromosome number 3, which in strains which the Mos factor has been lost, and in acted by means of somatic excision of mariner from its strains with putative transpositions. We propose that this location in Wpch (Bryan et al., 1987). of is identical to the copy mariner Mos factor. Additional characterization of the excision factor Mos is Key words: ele- Drosophila/mariner/mosaicism/transposable presented in this paper. We show: (i) Action of the Mos ment/white gene factor results in excision of mariner elements from sites in the genome in in addition to the site Wpch. (ii) Action of the Mos factor can result in its own excision, detected pheno- typically by the occurrence of infrequent non-mosaic wph Introduction flies in the originally Mos-bearing mosaic strain. The mariner excision factor Mos is a genetic element (iii) Exceptional mosaic segregants from Mos-bearing identified by its ability to promote a high frequency of parents contain inherited mosaic factors in the X chromo- somatic excision of the transposable element mariner from some, chromosome 2 or alternative sites in chromosome 3, an insertion site near the white in maurtiana gene Drosophila suggesting that Mos is itself capable of transposition. (iv) The (Bryan et al., 1987). The mariner insertion occurs in the original third-chromosome Mos activity is associated with white-peach (Wpch) allele, interrupting the region of the 5' a particular chromosomal copy of mariner which is deleted untranslated leader upstream from the first exon, and results in non-Mos excision products. These results suggest that the in a peach-like color et Excision eye (Jacobson al., 1986). mariner excision factor Mos is itself a special copy of of mariner from this location during eye development mariner. restores pigment production in some cells and is identified phenotypically by the occurrence of somatic mosaicism in Results which the have in an otherwise eyes pigmented patches peach-colored et Excision Evidence that the Mos factor results in excision of mariner background (Bryan al., 1987). strain Wpch events occur infrequently in the original white-peach elements from other than that in shown in positions iS and give rise to occasional flies with a few facets Figure 1. The Southern blot was with a pigmented probed single copy from a (Jacobson and and genomic isolated Hartl, 1985; Haymer Marsh, 1986). sequence sequences flanking from a D.simulans clone However, in strains containing the inherited excision factor mariner element selected at random of is which contains Mos, somatic excision the mzariner element so frequent from a Mos library prepared WPch; strain, in both D.simulans mariner elements and additional that is mosaic with endogenous every fly multiple pigmented patches also increases introduced from D. mauritiana in the course of both eyes. The excision factor elements dramatically the allele rise to When with this the rate at which backcrossing. probed anonymous (unknown white-peach gives pheno- derivatives in line cells et the BamHI-Hindm typically wild-type germ (Bryan genomic location) probe, genomic of Mos flies are to a 4.8-kb al., 1987). digests wpch; expected give is 1286 in to the The mariner element transposable bp length, hybridization band, corresponding mariner-containing four in the a 3.5-kb band flanked terminal inverted fragment present library, plus (smaller by 28-bp repeats containing frame which results from the somatic excision of and includes a by 1.3 kb), mismatches, single, long open reading ©IRL Press Limited, Oxford, England M.M.Medhora, A.H.MacPeek and D.L.Hartl pch pch + + Mos nt 0C pch nt ' e + 2 + +Mos Select Gic -3 5 _ nt e + w pch $X; + Mos +;, pch nt' e + .. flies for ebony colored, mosaic-eyed Screen (1) non-mosaic flies (2) non-ebony, or loss of Mos activity. Fig. 2. Mating scheme to detect transposition of DNA with Fig. 1. Filter hybridization of digest genomic probed males that are for autosomal First mating produces heterozygous a mariner element at an site labeled fragment adjacent to anonymous as well as the third-chromosomal Mos. In the next markers net and e, Each lane contains DNA from a in the genome. single fly: 1, ebony-body, non-mosaic flies are candidates for Mos generation, wPch D. simulansID. mauritiana hybrid, non-mosaic; 2, transposition, and non-ebony, non-mosaic flies are candidates for Mos wOch; Mos D.mauritiana D.simulanslD.mauritiana hybrid, mosaic; 3, excision. insertion at the site. The lower wild-type, without mariner anonymous from somatic excision of mariner in the Mos band in lane 2 results DNA was obtained a genotype. The flanking by screening partial Table I. Excision and apparent transposition of Mos factor from strain Z8 Materials and Genomic genomic library (see methods). and The mol. wt markers are DNA digested with BamHI HindlIl. Strain Number of Excision Excision Transposition double of X DNA. from EcoRI-HindIII digests bacteriophage from wpch of Mos of Mos offspring (class 1) (class 2) (class 3) from this location. The somatic excision mariner anonymous Z3 14320 111 (1.55%) 10 (0.14%) 6 (0.08%) band is in the Mos strain but not in the non- readily apparent Z8 14 057 127 (1.81%) 78 (1.11%) 14 (0.20%) that excision of this mariner Mos control (Figure 1), implying Z8 10 982 62 (1.18%) 46 (0.84%) 10 (0.18%) occur in the Mos strain. A similar element does experiment using a different anonymous probe isolated at random from number of because each using half the total progeny counted, a D. mauritiana indicated somatic excision in Mos library of are detectable of the three exceptional types progeny only strains from this unrelated location also (G.Bryan, unpub- of the that have the in the 50% offspring appropriate conclude that excision of the mariner element lished). We background genotype. strains is not restricted to the mariner element present in Mos the rate of reversion was somewhat greater Although wlch wpch includes most or all mariner in the allele, but probably in the first with Z8 than in the second (P < experiment elements present in the genome. the absolute amount of the difference was not large. 0.01), Possible excision and/or of the Mos element transposition Mos excision and were not Rates of putative transposition was by means of the matings outlined in itself investigated different in the two Z8 However, significantly experiments. D.simulans males of genotype wpch; Figure 2. Mosaic Z3 with the rate of Mos excision was / cross of non- in comparing Z8, netl e + + Mos were obtained from the +; -7-fold greater in the Z8 strain than in Z3 (P < 0.0001), males. mosaic e females with wpch; Mos mosaic wPch; net; pch while the rates of w reversion and putative Mos trans- in net veins and The recessive allele net and e result wing The for the were not significantly different. reason the order is position ebony body color respectively. (Although gene between Z3 and Z8 is differences in Mos excision rates if e were to the left of Mos could be on the written as Mos, unclear. males were crossed other side.) The doubly heterozygous of obtained from A sample of exceptional progeny class 1, en masse with wpch; net; e females. Exceptional progeny was discussed in a a somewhat different mating scheme, were of three classes. et restriction previous report (Bryan al., 1987). Diagnostic Females with which result from (1) wild-type eye color, Wpch fragments from the putative revertant alleles from were of the mariner element in wpch. germ line excisions - wpch found to be 1.3 kb smaller than those in itself, Non-mosaics with wild-type (non-ebony) body color, (2) mariner element. It has not been implying excision of the which the evidence below indicates as resulting from self- whether the excision events are determined primarily precise excision of the Mos factor. or most of the revertant alleles result imprecise. However, (3) Mosaics with ebony body color, which contain in wild-type phenotypes. heritable mosaic factors in the X chromosome, in chromo- apparently A of nine of class 2 from the some 2 or in the chromosome 3. sample exceptional progeny ebony-bearing Z8 was mated with non- Two Mos strains of D.simulans, Z3 and Z8, were exam- second experiment individually Wpch Z8 in order to confirm that the exceptional ined using the mating scheme in Figure 2. The Z3 and mosaic flies strains were independently by repeated back- non-mosaic parents did not produce mosaic progeny in future produced No in of the D. mauritiana mosaic strain E25H to generations. mosaicism was detected these progenies, crossing of the Mos factor. D.simulans. Since there was no statistically significant indicating apparent permanent loss Filter DNA from the Mos revertants, tendency for exceptional types of progeny to occur in clusters hybridizations using when probed with mariner DNA, indicated the consistent (indicated by homogeneity in frequency of exceptions found loss of a particular mariner-containing fragment. An example among culture bottles), the data have been pooled and are in Table I. Strain Z8 was studied in two indepen- is shown in Figure 3, in which a BamHI-HindIll digest presented dent experiments. The frequencies given were calculated of single Mos revertant flies from the Z8 strain was com- 2186 Mos excision factor R R R R R M M M M M M. wprF wpc T T T kb Zr kb Nv, 9.46- 6.75- -6.75 -5.05 * I ^. -4.26 Fig. 3. Filter hybridization showing mariner elements in individual Z8 D.simulans obtained from the mating scheme in Figure 2. Lanes as follows: M, mosaic flies; R, non-mosaic putative Mos excisions Fig. 4. Filter hybridization showing manriner elements in exceptional (exceptional progeny of class ii). Arrow indicates a band hybridizing progeny of class 3, in which the Mos factor maps to a new with mariner that is present in all mosaics and absent in all putative chromosomal location. Each lane contains DNA from an Mos individual excisions. Genomic DNA digested with BamHI-HindIII, and fly, as follows: M, individual Z8 mosaics; @pch, white-peach with of non- probed internal Sall-SphI fragment mariner. Molecular weight mosaic controls; T, descendants of independently obtained exceptional markers are from a HindIll digest of bacteriophage DNA. progeny of class 3 in which the Mos factor had been mapped to the X, 2 and 3 chromosomes (left to right respectively). The 5. 1-kb band pared with non-revertant single flies from the same strain, (arrow) corresponding to the presence of Mos in Z8 is missing in the using a Sail -SphI internal fragment of mariner as the probe putative transpositions. Restriction digests, probe and mol. wt markers (Jacobson et al., 1986). The mosaics and ('M' lanes) clearly as in Figure 3. consistently show the presence of a DNA hybridizing frag- ment of - 5.1 kb (arrow), which is absent in the A total of 11 exceptional progeny invariably were of this type. Unless revertants ('R' lanes). A total of 30 independent revertants these represent de novo occurrences of heritable mosaic were examined in this manner, and all were missing the factors, the exceptions represent either transpostion of Mos 5.1-kb band. The hybridizing band in has been into the homologous chromosome question 3 or possible recombina- cloned and similar blots carried out using a tion in the male. sequence flanking the mariner element as the probe. With this non- (ii) Mosaic progeny approximately equal numbers probe, of ebony revertants show a hybridizing band of 5.1 kb, but in the and non-ebony and approximately equal numbers of females revertants the band is 1.3 kb smaller, which is consistent and males. This result indicates presence of the mosaic factor with of deletion a mariner element from the in either chromosome 2 or chromosome 4. A total of 10 fragment detected by the probe (data not These results putative transpositions were of this type. shown). suggest that in the mosaic phenotype the Z8 Mos strain is associated (iii) Mosaic progeny all female, indicating presence of the with a mariner in element at a particular site the genome, mosaic factor in the X chromosome. A total of two putative which is lost to upon reversion the non-mosaic state. transpositions were of this type. sample of 23 exceptional progeny of class 3 from the Progeny tests of type (i) and (iii) provide unambiguous Z8 strain was also studied. As demonstrated below, these evidence of the presence of heritable mosaic factors in individuals contain heritable mosaic factors in new locations chromosome 2 and the X chromosome. Subsequent crosses in X have shown that the genome, including the chromosome, chromosome the mosaic strains containing these factors 2 and the chromosome 3. are as stable as ebony-bearing The relatively those containing the original Mos simplest to the occurrence of these factor located in hypothesis explain exceptions is chromosome 3 (data not shown). that the Mos factor in the chromosome 3 origial wild-type Strains containing heritable mosaic factors in the X is capable of and the result 2 transposition, exceptional progeny chromosome, chromosome and the ebony-bearing chromo- from such events. some 3 were tested for the of transposition presence the characteristic Exceptional class 3 were mated to the mosaic 5.1-kb restriction associated progeny map mariner-containing fragment factor. Mosaic males were crossed with mariner D. simulans with the original Mos factor in chromosome 3. As expected, females in order to isolate the new mosaic factor in this restriction was absent from the as fragment strains, F1 mosaic males were shown in 4. Each lane DNA heterozygous form. The Figure contains from a resulting single with mariner. The restriction then crossed with e and the individual probed enzymes and Wpch; net; females, F2 progeny Three of were found the were the same as used for 3. The lanes classified and counted. probe Figure types progeny in the labeled 'M' are individual Z8 and the characteristic F2 generation: mosaics, 5. 1-kb band is indicated with an arrow. DNA from (i) All mosaic progeny phenotypically ebony, indicating in the chromosome. individuals three class 3 presence of the mosaic factor representing exceptional progeny e-bearing 2187 M.M.Medhora, A.H.MacPeek and D.L.Hartl in the lanes labeled 'T'. The mosaic factors in elements Ac and P are 4.5 and 2.9 kb respectively, and are shown to chromosome X, 2 and 3 deletion derivatives are common in the genome. In contrast, these strains were mapped respectively (left to right). Although each newly derived mariner elements longer than 1.3 kb have not been observed, mosaic 'T' strain contains a heritable mosaic factor, all lack even in strains containing the Mos element, and deletion mariner element associated with mosaicism derivatives are not common. Moreover, the 1.3-kb mariner the particular in the Z8 strain. element contains a single open reading frame. At least five copies of mariner from anonymous positions in the genomes of D.maruitiana, D.simulans and D.yakuba have been Discussion sequenced, and all preserve the same open reading frame and are not bp (K.Maruyama and mariner element from longer than 1286 Heritable excision of the transposable D.L.Hartl, unpublished). The mariner element shares a from other sites in the results the WPCh allele and genome number of similarities with the transposable element Tc 1 in from factor initially mapped a trans-acting designated Mos, the nematode Caenorhabditis elegans. For example, et The basis Tcl to chromosome 3 (Bryan al., 1987). genetic contains a single open reading frame, excises in both germ to be either a mutation in a host of Mos was postulated gene line and somatic cells, and maintains the full 1.6-kb length of mariner that acts in trans. The trans action or another copy among most copies examined (Rosenzweig et al., 1983; mobile elements is observed both in of genetic commonly D.E.Moerman and R.H.Waterston, personal com- in and For I.Mori, prokaryotes and eukaryotes (Berg Howe, 1988). elements in munication). example, the first reported transposable maize, Several models for the molecular basis of Mos may and Enhancer-Inhibitor Activator-Dissociation(Ac-Ds) (En- for its to excision and account ability promote transposition of autonomous and non- 1), both involve two-part systems that Mos of mariner. One class of models assumes activity autonomous elements (McClintock, 1947, 1948, 1949; results from nucleotide sequence alterations within the Mos The autonomous elements Ac and Peterson, 1953, 1960). for Mos is based element. A second class of models activity En code for that direct not their own proteins only transposi- effects mediated on position effects, as through position by but also mobilize the non-autonomous Ds and I tion, help or enhancers to chromatin structure or promoters external The non-autonomous elements often contain elements. the element. mutations, such as large deletions in their protein-coding Mos factor a The occurrence of the suggests possible regions. for the increase or decrease evolutionary mechanism rapid The possibility that Mos was a special copy of mariner of elements in the in the number of copies transposable was tested using genetically marked strains of D.simulans of the rate transposition, presence genome. By increasing 3-10 of mariner. The data indicate containing copies genetic in the number of the Mos factor may lead to an increase at a due that Mos itself is lost spontaneously high frequency Mos of mariner elements. At the same time, promotes also occur that contain to excision. Exceptional segregants well as in somatic excision of mariner in the line as germ mosaic factors in the X chromosome heritable chromosome, number of cells, therefore tending to decrease the copies. marked chromosome 3. The 2 or a genetically simplest of Mos is observed in its A third important characteristic is that these arise from transpositions hypothesis exceptions and dysgenic effects (D.Garza, G.Bryan D.L.Hartl, of the Mos but we cannot exclude the that factor, possibility that Mos is harmful to fitness unpublished), which suggest of de novo the strain a Mos-bearing generates high frequency will to be eliminated natural selection. The and tend by from Mos itself. In heritable mosaic factors different any forces on the rates outcome of these depends quantitatively that Mos is a these results the view transposable case, support and excision of mariner elements of transposition (including element. excision of the Mos factor balanced transposition and itself), of mariner Molecular analysis identified a particular copy selective elimination of Mos due to its against the dysgenic or of the Mos that correlated with the presence absence An theoretical is in some effects. intriguing possibility that, factor are this factor. Strains that have lost the Mos missing cases, a Mos-like derivative of a transposable element might Putative with heritable copy of mariner. transcriptions arise and proliferate, and, even as it is being eliminated by in the also lack this mosaic factors in new positions genome selection, result in a rapid increase in the number of copies but contain one or more additional copy of mariner, they of its sister elements. The increased copy number may persist mariner elements. even after the Mos-like elements have been completely be drawn between the Mos-mariner An analogy may eliminated by selection, and in the absence of Mos-like and the derived P element because system designated PA2-3, elements, the genome with the increased number of copies the PA2-3 behaves like the Mos factor in the promoting contains no evidence of the mechanism that produced the P in and deletion and of elements somatic transposition germ result. the breaks down cells (Laski et al., 1986). However, analogy construct in which the 2-3 because PA2-3 is an engineered intron has been the need for the deleted, eliminating splicing Materials and methods that normally confines P-element activity to the germ line. strains Drosophila of mariner Naturally occurring copies exhibit both germinal Strains D.simulans wPh and wPch; Mos were constructed as D.simulans and somatic instability (Jacobson and Hartl, 1985; Haymer described et introducing the Vlch and Mos genes from (Bryan al., 1987) by and Marsh, 1986). D. mauritiana into D.simulans through repeated backcrossing. Ten series of independent backcrosses were carried out to generate independent lines The molecular structure of mariner exhibits one poten- of D.simulans wxh; Mos, called Zi-Z1O, which were individually difference with similar tially significant superficially maintained. The strain Ach; net; e strain of D.simulans was constructed transposable elements in eukaryotes. Autonomous elements, by crossing D.sinulans wPch females with D.simulans net; e males obtained such as Ac in maize and the P element in D. melanogaster, from sib F1 J.Coyne, mating the and selecting individual wPch; net; e virgin females and males from contain multiple open reading frames. The autonomous the F2 to establish the strain. Flies were maintained 2188 Mos excision factor on standard cornmeal medium or Formula 4-24 instant medium (Carolina Biological, Burlington, NC). Filter hybridizations Isolation of DNA, restriction digests, gel electrophoresis and Southern transfers were carried out as described (Jacobson et al., 1986). DNA probes were obtained from isolated restriction fragments of DNA labeled with 32p by the method of Feinberg and Vogelstein (1983) using random hexamers obtained from Pharmacia, Inc. Specific activities of the probes were >6 x 108 DNA. The DNA used as probe to study the excision of c.p.m./1g mariner elements from anonymous positions in the genome was isolated as described below. The mariner Sall - SphI fragment was obtained as described in Jacobson et al. (1986). Construction of partial library from strain Z8 A partial library of genomic DNA was made from the strain Z8 using the commercially available vector Bluescribe M 13 + (Vector Cloning Systems, San Diego, CA). Genomic DNA was isolated as described (Lis et al., 1983; Kune et al., 1985), and 20 tg was digested to completion with BamHI and HindIII. The restriction digest was fractionated by electrophoresis in a 0.8% agarose gel. A slice of gel containing DNA of -4.5 -5.5 kb was cut out and the DNA extracted as described (Vogelstein and Gillespie, 1979). The extracted DNA wasmixed in a ligation reaction with vector DNA that had been digested to completion with the same two enzymes, using -0.5 Ag of fragment and -0.1Itg of vector in a 10-1l reaction volume. The enzymatic reactions were carried out as described (Maniatis et al., 1982). The ligation mixture was used to transform Escherichia coli strain DH5a [F- X- hsdR17 recAl A(lacZYA-argF)]. The resulting library was screened by colony filter hybridization using a32P-labeled Sall-SphI internal fragment ofmariner as probe. Positively hybridizing colonies were purified, the cloned DNA isolated, digested with PstI, transferred to filters and hybridized with the probe. A 1.7-kb fragment of DNA flanking the mariner element in the genome was isolated from the clone pMl 1O. This DNA did not hybridize with mariner, and was used to study excision of the mariner element in the strain Z8, as described in the legend of Figure 1. Acknowledgements We are grateful for the generous support of our laboratory colleagues, especially Danny Garza for helpful discussion in planning the genetic crosses, Kyoko Maruyama for providing plasmids and continuous support, Glenn Bryan for fly strains, and Suhas Phadnis and Bob DuBose for their comments on the manuscript. This work was supported by grant number GM33741 from the US National Institutes of Health. References Berg,D.E. and Howe,M.M. (eds) (1988) Mobile DNA. American Society for Microbiology, Washington, DC, in press. Bryan,G.J., Jacobson,J.W. and Hartl,D.L. (1987) Science, 235, 1636- 1638. Feinberg,A.P. and Vogelstein,B. (1983) Anal. Biochem., 132, 6-13. Haymer,D.S. and Marsh,J.L. (1986) Dev. Genet., 6, 281 -291. Jacobson,J.W. and Hartl,D.L. (1985) Genetics, 111, 57-65. Jacobson,J.W., Medhora,M.M. and Hartl,D.L. (1986) Proc. Natl. Acad. Sci. USA, 83, 8684-8688. Kuner,J.M., Nakanishi,M., Ali,Z., Drees,B., Gustavson,E., Theis,J., Kauvar,L., Kornberg,T. and O'Farrell,P.H. (1985) Cell, 42, 309-316. Laski,F.A., Rio,D.C. and Rubin,G.M. (1986) Cell, 44, 7-19. Lis,J.T., Simon,J.A. and Sutton,C.A. (1983) Cell, 35, 403-410. Maniatis,T., Fritsch,E.F. and Sambrook,J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. McClintock,B. (1947) Carnegie Inst. Washington Year Book, 46, 146-152. McClintock,B. (1948) Carnegie Inst. Washington Year Book, 47, 155-169. McClintock,B. (1949) Carnegie Inst. Washington Year Book, 48, 142-154. Peterson,P.A. (1953) Genetics, 38, 682-683. Peterson,P.A. (1960) Genetics, 45, 115-133. Rosenzweig,B., Liao,L.W. and Hirsh,D. (1983) Nucleic Acids Res., 11, 4201 -4209. Vogelstein,B. and Gillespie,D. (1979) Proc. Natl. Acad. Sci. USA, 76, 615-619. Received on March 14, 1988; revised on April 25, 1988

Journal

The EMBO Journal – Springer Journals

Published: Jul 1, 1988

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