d. Mol. Biol. (1990) 215, 477-482
COMMUNICATIONS N e w Foldback Transposable Element TFBI F o u n d in Histone Genes of the Midge Chironomus thummi Th. Hankeln Institut fiir Genetilc Ruhr- Universitdt P.O. Box 10 21 48, D-4630 Bochum 1, F . R . G
and E. R. Schmidt~f Institut fi~r Genetilc Johannes Gulenberg- U niver sit~it Becherweg 32, D-6500 Mainz, F.R.G. (Received 10 October 1989; accepted 2 J u l y 1990) A new Foldback transposable element (TFB1) has been found in the histone H1-H3 intergenic region in the midge Chironomus thummi thummi. TFB1 has long terminal inverted repeats, composed of shSrt, degenerate subrepeats and is flanked by nine or ten base-pair "target site" duplications. TFB1 is present in at least two adjacent histone gene units in Ch. th. thummi, indicating a homogenization of histone gene repeats. The copy number and chromosomal distribution of TFB1 are different in the closely related subspecies Ch. th. thummi and Ch. th. piger, showing that amplification, elimination and transposition of TFB1 have occurred recently during evolution.
Transposable DNA elements constitute a ubiquitous component of the eukaryotic genome (for a review, see Berg & Howe, 1989). Four major classes of transposable elements have been identified, which can be defined by their structural characteristics (Finnegan & Fawcett, 1986). These are the viral-like and non-viral-like retrotransposons, the elements with short terminal inverted repeats (e.g. P elements), and the less well investigated Foldback (FB:~) transposable elements, which are characterized by long, modularly constructed terminal inverted repeats (IVRs: Potter et al., 1980; Truett et al., 1981; Potter, 1982a,b). FB elements are involved in mutational rearrangements within the Drosophila genome (Collins & Rubin, 1983, 1984; Levis et al., 1982), but the mechanism of their transposition is still unclear (Smyth Templeton & Potter, 1989). Here, we report the finding of a new Foldback transposable element (TFB1) from the midge Chironomus thummi. Our results indicate that TFB1 t Author to whom correspondence should be addressed. :~Abbreviations used: FB, foldback; IVR(s), inverted repeat(s); bp, base-pair(s); kb, l03 base-pairs; nt, nucleotide. 0022-2836/90/200477-06 $03.00/0
477
has been involved in major genomic alterations that happened during the divergent evolution of the closely related subspecies Ch. th. thummi and Ch. th. piger. (a) Sequence characteristics of TFB1 The TFBI (Thummi Foldback-like) element was found during the analysis of the Ch. th. thummi histone gene clone A Ctt500-7, which contains at least one complete histone gene repeating unit (Fig. 1; Th. Hankeln & E. R. Schmidt, unpublished results). Sequencing of the H1-H3 intergenic region (subclone pKH2A-1 l, Fig. 1) revealed the presence of large IVRs separated by 746 bp ("loop"). The IVRs are flanked by short direct repeats of 9 bp and l0 bp (Fig. 2(a)). This element (1048 bp long) shares the structural features of Drosophila FB transposable elements (Potter et al., 1980; Truett et al., 1981) and sea urchin TU elements (Lieberman et al., 1983; Hoffman-Liebermann et al., 1985). The left and right IVRs of TFB1 have a length of 162 bp and 140 bp, respectively. This length difference is due mainly to the (imperfect) duplication of a 2 0 b p sequence in the left IVR (Fig. 2(a): nt no. 45-64 is a duplication of nt no. 65-84). © 1990 Academic Press Limited
478
Th. Hankeln and E. R. Schmidt 10
I
S
kCtt
I
I
500-7
I
i
i
E
X
I
i
H4 I
15
I
I
I
t
I
kb
l
i
B Sac
K
P E
X
B Sac S
II
I
II,
!
II I--
H2A H2B
H1
I . . . . I H3
I
H2AH2B
I
PKH2A-1 1
Figure I. Partial restriction map of Ch. th. thummi histone gene clone )~ Ctt500-7. The clone was isolated by plaque filter hybridization (Benton & Davis, 1977) from a Ch. th. thummi genomic library prepared in 2EMBL3 (Frischauf et al.. 1983), using as probe the radioactively labelled (Feinberg & Vogelstein, 1983) Drosophila histone gene repeat cDM500 (Lifton et al., 1977). Low stringency hybridization conditions (55°C, 0"9 M-NaCI, 0'09 M-trisodium citrate, pH 7) were used. At least 2 histone gene repeating units (defined by EcoRI restriction sites) are present in tandem orientation in )~CttS00-7. The position of the histone genes and their transcriptional orientation is indicated. The TFBl element (boxed) is found at exactly the same site in the H1/H3-intergenic region in 2 neighbouring histone gene repeating units. The TFBl-containing region was cloned into pUCl8 (Yanisch-Perron et al., 1985) to give subclone pKH2A-I 1. This subclone was dideoxy-sequenced (Sanger el al., 1977), using double-stranded supereoit plasmid DNA as template (Chen & Seeburg, 1985). Template preparation was according to Holmes & Quigley (1981), sequencing reactions were performed with the Pharmacia T7 polymerase sequencing kit.
Apart from this difference, the two inverted sequences show nearly perfect sequence identity. The IVRs of TFB1 are composed of short, degenerate tandem subrepeats of variable lengths. Drosophila FB elements and sea urchin TU elements also have modularly organized IVRs. In the case of the left IVR of TFB1, the subrepeats obviously extend into sequences that, by comparison to the right IVR, do not belong to the stem but constitute the loop. Thus, the left IVR is longer than the right one. Varying copy numbers of subrepeats in the left and right IVR have been described for Drosophila melanogaster FB3 and FB4 (Truett et al., 1981; Potter, 1982a,b) and might be explained by unequal interchromosomal crossing over between subrepeats or by slippage of the nascent DNA strand during replication. The TFB1 element is flanked by short direct repeats (9 or 10 bp at the left and the right end, respectively). These duplications were probably generated during the integration of TFB1 into the Ch. th. thummi histone gene unit, because the sequence has been found unduplicated in a sequenced histone gene repeat from the closely related subspecies Ch. th, piger. A comparison of the "filled" and "empty" target sites (Fig. 2(b)) shows that the l0 bp sequence flanking the right end of T F B l corresponds to the empty site. The 9 bp sequence flanking the left end obviously is the result of a 1 bp deletion, which occurred during or after the integration event. Nothing is known to date about the mechanisms of FB element transposition. Smyth Templeton & Potter (1989) have described a novel protein encoded by the loop of a Drosophila FB element, which could function in FB transposition. Within TFB1, we have found several short open reading frames, which may be joined via possible splice sites.
However, the significance of these open reading frames remains to be evaluated. (b) Genomic organization of T F B 1 "Zoo" blot hybridization with TFBI subcione pKH2A-ll shows that the repetitive element is present in a wide variety of Chironomus species (Fig. 3(a)). Thus, TFB1 clearly appeared early during evolution of the genus Chironomus. Drosophila FB elements (Silber et al., 1989) and sea urchin TU elements (Hoffman-Liebermann et al., 1985) are also evolutionarily conserved in many species. TFB1 was hybridized to genomic Southern blots of DNA from various populations of Ch. th. thummi and Ch. th. piger (Fig. 3(b)). The results show that TFB1 is present in higher copy number in the genome of Ch. th. thummi than in Ch. th. piger (see also in situ hybridization, Fig. 4). DNA from different populations of the same subspecies does not display much difference in the hybridization pattern, which indicates a predominant stability of TFBl-related sequences in the genome. However, during the time of divergent evolution of Ch. th. thummi and Ch. th. piger TFB1 elements must have had periods of high transpositional activity. This can be concluded from the chromosomal distribution of T F B elements. The chromosomal localization of TFB1, determined by in situ hybridization to larval polytene chromosomes of a Ch. th. thummi x Ch. th. piger F l hybrid (Fig. 4), is largely different in the two subspecies. In Ch, th. thummi, TFBl-homologous sequences are scattered over the genome, including the pericentromeric and telomerle regions. In Ch. th. piger, however, T F B sequences are mainly confined to the pericentromeric region of all chromosomes, with significantly less and weaker signals detectable on the chromo-
Communications
AACGAATTCA CCTCAATCTA AAATTC~AA
479 VSO
AATGCAGCTA CCCT]~]~
VlO0 C A G A T A ~ T A C T G C C A A G A T G C A G A T ~ ' T A C T G C T A A A G T G CAGATGAAAA vz50
.TTAI~I-~-~+CCC . T A A A A T G C A G A T G A A A T G C T GTTG~-~-z-z-i~ AAAAAGGT]LT
v200
A A T G C A G A T G A~-A-A-A-A'.~CGT A T A A A G T G c A G A T A A A A T G CTGCCCALAAT
v250
GTAC,A T ~ ' T A T A A T A A A A T T A A A A T G C A T C A T A T T T G A T A TTATAAAAAT v300 T A T A G T A A A C "~-~-~.-x~l~i-z-z'A A A A A A T T C G T A A G A A A G G A A AG~,-.L-.L-.L-z-x~IC v350 G C A A C G A C T A T T C C T A C C T T G C C C A A A A A C TATACAGCTA v400
T T C T A T A G A G C A G C A C G G A T C A A A T G T A A A A A A C T A G A A G TCTCTACAAT
v450
GTC'~CGTTAT T G A G A T T T C A T G C T G A T T A A ~.-~-~-xCATGCTTGTTrG~ETA
v500
C A G A A A G C G T AI-~-~-I'GCGAT T T T C T C A A T T T G A A A T C T G T TGI~I-xGAAAA
v550
T G A T G T A A A A "~.-~-~-~'AGGCAC A A A I I - ~ - I ' A C T
TCTGAGA~/T TTTAAAACCA v600 ATTTTG ~I~TC G-~-~-~-~-CTCACA A A A T G C G A A A A A C T G A T G T TTGCATCTGA v650
A C A A A A C C A A T A G A T T C A C A C G T G A A A A C G G T G T G A A T T T AAAAAATACA v700 T T T G T A G A T A T A T C A A T G A A A G C T G A C A A A ~,.~-J.-~.-x~,CAA AATTC~C,M~. v750 A A A T A T T C G T G A G T G A C G C T T A A T T A A A T T GAf~-z-I-CTGA CATAACGCCT vSO0
TAA~:~-~-xAGG A A G T A A G T T C T A T G T C A A A A G A A A G C A A T T TTl~-~-z-~-z-i"
v850
A C A A G A T A T A ATAA~.-i=~-xGA A A T T T G T C A c T A T T G T G G T T TCGGAGATAT vg00 T T A A G A A A C C A A A T C A T C A C T C A C G A A T A A "~.~..~ .~ ,~ ~.-~/~-~.-~: CGTAGGGGCT V950
C A A A A A A T T A ATATCCAkACG T T A A A T T A T C T G C A T T A T A C ~-zz-z-~T~'AAA
vlO00
G C A A C A G C A T T T C A T C T G C A ~-z-~AGGGAA A A T A A A T T T c ATCTGCACTT v1050 TAGCAGTACC ATCTGCATAT TAGGGTAGCT GCATTTTAGG AATTTCTA~ vll00 TTGGGGTAAC GAAT~TGT G T T A T A A T A G A A A T G A C G C C GTTTAAAATG vllS0
C A A T G A T G G C GTCA~I~I-xGA G A G A G T T T A C A G G T T A G C T T TATAGAATGA V1200 -FI-FFgATTGG A C T A G A A T G T T T T A A G C T A C A A G A A A A G T A TTGTTGAGAT V1250 C A T A A A A T T G A A T A A A A A A T *Fl'~Z'T~-~=i-~-i C A T G T T A A T A ~i-~I-~GGTGG TCTTGAAAAA GACCGTTCTA ATGAAATATG AAAGCTT
~(a)
TH
5'-
TTAAAATCATCAACGAA~TTC f
PI
-
TFB
1 -
AACGAATTTCTGTGTTATAATA- 3'
t
5'-
6
TTAAAATCATCAACGAATTT~TGTGCTAAAATA -
3'
(b)
Figure 2. (a) Nucleotide sequence of subclone pKH2A-I 1 containing the TFB1 element. The IVRs are underlined by arrows. The flanking target site duplications are marked by double line arrows. The 1st 3 nucleotides (AAC) of the sequence do not belong to subclone pKH2A-I1, but are shown since they are part of the 5' target site duplication. (b) Comparison of the empty target site from a histone gene repeating unit of Ch. th. piger (clone 6.2, PI) and the filled target site (Ch. th. thummi clone 500-7, TH) of TFBl. The comparison of the empty with the filled target site indicates a deletion of a T in the left duplication (marked by an asterisk (*)). Further sequence differences are marked by dots.
some arms. This corresponds to the result of Southern hybridization. Obviously, there has been amplification/elimination and transposition of TFB1 during the divergent evolution of the subspecies. The higher copy n u m b e r of TFB1 in Ch. th. t h u m ~ i is correlated to the larger genome size of this subspecies. The same phenomenon has been observed for several families of satellite-like,
tandem-repetitive DNA in Chironomus thummi (Schmidt, 1981, 1984; Th. Hankeln & E . R . Schmidt, unpublished results). The structural simi~ larity between F B elements and satellite DNA has been recognized by T r u e t t ¢t al. (1981). Our results suggest common evolutionary mechanisms acting on T F B elements and tandem-repetitive DNA sequences.
480
Th. Hankeln and E. R. Schmidt
I
ct
23@
2
3
4
: ' " ~" • .%
4@
20
I@
0"6@
. . . . .
,/
9.
~.~ :. - ~ - : . ~ , , r
I
-~
'i "
{a)
:
" 2.
(b)
Figure 3. (a) Southern (1975) blot of Hind III-digested DNA from various species of the genus Chironomus (Zoo blot). hybridized with radiolabelled (Feinberg & Vogelstein, 1983) TFBl-containing subclone pKH2A-1 I. plum, Ch. plumosus; pal, Ch.. pallidivittatus; ten, Ch. tenta~a< halo, Ch. halophilus; th, Ch. th. thummi; pi, Ch. th. piger; lur, Ch. luridus; pseu, Ch. pseudothummi; cin, Ch. cingulatus; me]. Ch. melanotus. Molecular sizes or marker bands are indicated in kb. (b) Southern blot of HindIII-digested DNA from different populations of Ch. th. thummi and Ch.. th. piger, hybridized as in (a). l and 2, different Ch. th. thummi populations; 3 and 4, different Ch. th. piger populations. Molecular sizes of marker bands (marked by dots) are the same as in (a).
(c) T F B 1 and histone gene evolution The integration of transposable elements into histone genes or integenic spacers is not unusual. The foldback-like transposon TU has been found within a histone H2B pseudogene of the sea urchin Stronqylocentrotus purpuratu8 (Lieberman et al., 1983). Insertions of transposable elements have been found in a subgroup of D. melanogaster histone gene repeating units (Lifton et al., 1977; Saigo et al., 1981; Matsuo & Yamazaki, 1989). These latter elements are also inserted into an A +T-rich region between the histone HI and H3 genes. The transposable copia-like element 297 has been found inserted within the TATA box of a D. melanogaster H3 gene (Saigo et al., 1981; Ikenaga & Saigo, 1982). These examples indicate that the histone gene family is a preferential site for the evolutionary fixation of mobile elements. The redundancy of these genes probably allows for insertions without this having severe effects on the organism's fitness. The mutations might even be spread among the members of the multigene family and among the population by homogenization mechanisms
("Molecular drive"; Dover, 1982, 1986). We suggest that the multiple occurrence of TFB1 in at least two, but probably more, adjacent histone gene repeating units in Ch. th. thummi (see Fig. l ) is the consequence of homogenization processes acting on histone gene repeats. In that respect, it is interesting to note that both the integrated TFB] and the 1 bp deletion in the left target site duplication have been homogenized in adjacent repeating units (data not shown). Such homogenization events have been postulated (and shown) for tandem-repetitive DNA families (Coen et al., 1982; Strachan et al., 1985). I n situ hybridization of TFB1 shows an especially intense signal at band D3c on chromosome II of Ch. th. thummi (H~igele, 1970). This band contains one of the five histone gene clusters on the midge (Th. Hankeln & E . R . Schmidt, unpublished results). The bright signal {Fig• 4) is probably due to multiple copies of the TFB1 element, present in a number of tandemly arranged histone gene repeating units. Thus, the TFB1 histone gene containing clone 2 Ctt500-7 has probably be~n derived from locus D3c on chromosome II.
Communications
481
Figure 4. In situ hybridization of TFBl-containing subclone pKH2A-I 1 to larval polytene chromosome of Ch. th. thvmmi x Ch. th. pitier F 1 hybrids. The DNA probe was biotin-labelled (Langer-Safer et al., 1982) according to the Feinberg & Vogelstein (1983) method. The FITC fluorescence immunodetection system (DetekIf, Enzo Biochem.) was used as described (Hankeln & Schmi'dt, 1987; Schmidt et al., 1988). The partially paired Ch. th. thummi and Ch. th. piger chromosomes II are indicated by th and pi, respectively. Centromere regions are marked by C. The brightly hybridizing histone gene locus D3c (according to the map presented by H~gele (1970)) is marked by an arrow. The corresponding phase contrast micrograph is shown.
The other four histone gene loci do not show any hybridization with T F B 1 , which means t h a t the integration of T F B l and its spreading to other histone gene repeating units by homogenization mechanisms is specific for the histone gene locus II, D3c. Such a locus-specific evolution of histone genes seems not probable in D. melanogaster, where all histone genes are known to be clustered a t one chromosomal site only (Pardue et al., 1977). In Ch. th. piger, however, only a very weak signal is found at histone locus D3c after hybridization with T F B I , indicating t h a t only one or a few copies of T F B l are present. We thank Mrs Bettina Welch for expert technical assistance and Mrs Heidi Sommerfeld for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Schm 523/3-6).
References Benton, W. D. & Davis, R. W. (1977). Science, 196, 180-182. Berg, D. E. & Howe, M. M. (1989). Editors of Mobile DNA, Amer. Soc. Mierobiol., Washington, DC. Chen, E. ¥. & Seeburg, P. H. (1985). DNA, 4, 165-170. Coen, E., Straehan, T. & Dover, G. (1982). J. Mol. Biol. 158, 17-35.
Collins, M. & Rubin, G. M. (1983). Nature (London), 303, 259-260. Collins, M. & Rubin, G. M. (1984). Nature (London), 308, 323-327. Dover, G. A. (1982). Nature (London), 299, l l l - l l 7 . Dover, G. A. (1986). Trends Genct. 2, 159-165. Feinberg, A. P. & Vogelstein, B. (1983). Anal Bioehem. 132, 6-13. Finnegan, D. J. & Fawcett, D. H. (1986). In Oxford Surveys on Eucaryotic Genes (Maclean, N., ed.), vol. 3, pp. 1-62, Oxford University Press, Oxford. Frischauf, A.-M., Lehraeh, H., Poustka, A. & Murray, N. (1983). J. Moi. Biol. 170, 827-842. H~gele, K. (1970). Chromosoma (Berlin), 31, 91-138. Hankeln, T. & Schmidt, E. R. (1987). J. Mol. Evol. 26, 311-319. Hoffman-Liebermann, B., Liebermann, C., Kedes, L. H. & Cohen, S. N. (1985). Mol. Cell. Biol. 5, 991-1001. Holmes, D. S. & Quigley, M. (1981). Anal. Biochem. 114, 193-197. Ikenaga, H. & Saigo, K. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 4143-4147. Langer-Safer, P. R., Levine, M. & Ward, D.C. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 4381-4385. Levis, R., Collins, M. & Rubin, G. M. (1982). Cell, 30, 551-565. Liebermann, D., Hoffman-Liebermann, B., Weinthal, J., Childs, G., Maxson, R., Mauron, A., Cohen, S. N. & Kedes, L. (1983). Nature (London), 306, 342-347. Lifton, R. P., Goldberg, M. L., Karp, R. W. & Hogness,
482
Th. Hankeln and E. R. Schmidt
D. S. (1977). Cold Spring Harbor Syrup. Quant. Biol. 42, 1047-1051. Matsuo, Y. & Yamazaki, T. (1989}. Nuel. Acids Res. 17, 225-238. Pardue, M. L., Kedes, L. H., Weinberg, E. S. & Birnstiel, M. L. (1977). Chromosoma (Berlin), 63, 135-151. Potter, S. S. (1982a). Nature (London), 297, 201-204. Potter, S. S. (1982b). Mol. Gen, Genet. 188, 107-110. Potter, S. S, Truett, M., Phillips, M. & Maher, A. (1980). Cell, 20, 639-647. Saigo, K., Millstein, L. & Thomas, C. A., Jr (1981). Cold Spring Harbor Syrup. Quant. Biol. 45, 815-827. Sanger, F., Nieklen, S. & Coulson, A. R. (1977). Proc. Nat. Aead. Sci., U.S.A. 74, 5463-5468. Schmidt, E. R. (1981). F E B S Letters, 129, 21-24.
Sehmidt, E. R. (1984). J. Mol. Biol. 178, 1-15. Schmidt, E. R., Keyl, H.-G. & Hankeln, Th. (1988). Chromosoma (Berlin), 96, 353-359. Silber, J., Bazin, C., Lemeunier, F., Aulard, S. & Voloviteh, M. (1989). J. Mol. Evol. 28, 220-224. Southern, E. M. (1975). J. Mol. Biol. 98, 503-519. Smyth Templeton, N. & Potter, S. S. (1989). EMBO J. 8, 1887-1894. Straehan, T., Webb, D. & Dover, G. A. (1985). EMBO J. 4, 1701-1708. Truett, M. A., Jones, R. S. & Potter, S. S. (198]). Cell, 24, 753-763. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Gene, 33, 103-119.
Edited by S. Brenner
COMMUNICATIONS N e w Foldback Transposable Element TFBI F o u n d in Histone Genes of the Midge Chironomus thummi Th. Hankeln Institut fiir Genetilc Ruhr- Universitdt P.O. Box 10 21 48, D-4630 Bochum 1, F . R . G
and E. R. Schmidt~f Institut fi~r Genetilc Johannes Gulenberg- U niver sit~it Becherweg 32, D-6500 Mainz, F.R.G. (Received 10 October 1989; accepted 2 J u l y 1990) A new Foldback transposable element (TFB1) has been found in the histone H1-H3 intergenic region in the midge Chironomus thummi thummi. TFB1 has long terminal inverted repeats, composed of shSrt, degenerate subrepeats and is flanked by nine or ten base-pair "target site" duplications. TFB1 is present in at least two adjacent histone gene units in Ch. th. thummi, indicating a homogenization of histone gene repeats. The copy number and chromosomal distribution of TFB1 are different in the closely related subspecies Ch. th. thummi and Ch. th. piger, showing that amplification, elimination and transposition of TFB1 have occurred recently during evolution.
Transposable DNA elements constitute a ubiquitous component of the eukaryotic genome (for a review, see Berg & Howe, 1989). Four major classes of transposable elements have been identified, which can be defined by their structural characteristics (Finnegan & Fawcett, 1986). These are the viral-like and non-viral-like retrotransposons, the elements with short terminal inverted repeats (e.g. P elements), and the less well investigated Foldback (FB:~) transposable elements, which are characterized by long, modularly constructed terminal inverted repeats (IVRs: Potter et al., 1980; Truett et al., 1981; Potter, 1982a,b). FB elements are involved in mutational rearrangements within the Drosophila genome (Collins & Rubin, 1983, 1984; Levis et al., 1982), but the mechanism of their transposition is still unclear (Smyth Templeton & Potter, 1989). Here, we report the finding of a new Foldback transposable element (TFB1) from the midge Chironomus thummi. Our results indicate that TFB1 t Author to whom correspondence should be addressed. :~Abbreviations used: FB, foldback; IVR(s), inverted repeat(s); bp, base-pair(s); kb, l03 base-pairs; nt, nucleotide. 0022-2836/90/200477-06 $03.00/0
477
has been involved in major genomic alterations that happened during the divergent evolution of the closely related subspecies Ch. th. thummi and Ch. th. piger. (a) Sequence characteristics of TFB1 The TFBI (Thummi Foldback-like) element was found during the analysis of the Ch. th. thummi histone gene clone A Ctt500-7, which contains at least one complete histone gene repeating unit (Fig. 1; Th. Hankeln & E. R. Schmidt, unpublished results). Sequencing of the H1-H3 intergenic region (subclone pKH2A-1 l, Fig. 1) revealed the presence of large IVRs separated by 746 bp ("loop"). The IVRs are flanked by short direct repeats of 9 bp and l0 bp (Fig. 2(a)). This element (1048 bp long) shares the structural features of Drosophila FB transposable elements (Potter et al., 1980; Truett et al., 1981) and sea urchin TU elements (Lieberman et al., 1983; Hoffman-Liebermann et al., 1985). The left and right IVRs of TFB1 have a length of 162 bp and 140 bp, respectively. This length difference is due mainly to the (imperfect) duplication of a 2 0 b p sequence in the left IVR (Fig. 2(a): nt no. 45-64 is a duplication of nt no. 65-84). © 1990 Academic Press Limited
478
Th. Hankeln and E. R. Schmidt 10
I
S
kCtt
I
I
500-7
I
i
i
E
X
I
i
H4 I
15
I
I
I
t
I
kb
l
i
B Sac
K
P E
X
B Sac S
II
I
II,
!
II I--
H2A H2B
H1
I . . . . I H3
I
H2AH2B
I
PKH2A-1 1
Figure I. Partial restriction map of Ch. th. thummi histone gene clone )~ Ctt500-7. The clone was isolated by plaque filter hybridization (Benton & Davis, 1977) from a Ch. th. thummi genomic library prepared in 2EMBL3 (Frischauf et al.. 1983), using as probe the radioactively labelled (Feinberg & Vogelstein, 1983) Drosophila histone gene repeat cDM500 (Lifton et al., 1977). Low stringency hybridization conditions (55°C, 0"9 M-NaCI, 0'09 M-trisodium citrate, pH 7) were used. At least 2 histone gene repeating units (defined by EcoRI restriction sites) are present in tandem orientation in )~CttS00-7. The position of the histone genes and their transcriptional orientation is indicated. The TFBl element (boxed) is found at exactly the same site in the H1/H3-intergenic region in 2 neighbouring histone gene repeating units. The TFBl-containing region was cloned into pUCl8 (Yanisch-Perron et al., 1985) to give subclone pKH2A-I 1. This subclone was dideoxy-sequenced (Sanger el al., 1977), using double-stranded supereoit plasmid DNA as template (Chen & Seeburg, 1985). Template preparation was according to Holmes & Quigley (1981), sequencing reactions were performed with the Pharmacia T7 polymerase sequencing kit.
Apart from this difference, the two inverted sequences show nearly perfect sequence identity. The IVRs of TFB1 are composed of short, degenerate tandem subrepeats of variable lengths. Drosophila FB elements and sea urchin TU elements also have modularly organized IVRs. In the case of the left IVR of TFB1, the subrepeats obviously extend into sequences that, by comparison to the right IVR, do not belong to the stem but constitute the loop. Thus, the left IVR is longer than the right one. Varying copy numbers of subrepeats in the left and right IVR have been described for Drosophila melanogaster FB3 and FB4 (Truett et al., 1981; Potter, 1982a,b) and might be explained by unequal interchromosomal crossing over between subrepeats or by slippage of the nascent DNA strand during replication. The TFB1 element is flanked by short direct repeats (9 or 10 bp at the left and the right end, respectively). These duplications were probably generated during the integration of TFB1 into the Ch. th. thummi histone gene unit, because the sequence has been found unduplicated in a sequenced histone gene repeat from the closely related subspecies Ch. th, piger. A comparison of the "filled" and "empty" target sites (Fig. 2(b)) shows that the l0 bp sequence flanking the right end of T F B l corresponds to the empty site. The 9 bp sequence flanking the left end obviously is the result of a 1 bp deletion, which occurred during or after the integration event. Nothing is known to date about the mechanisms of FB element transposition. Smyth Templeton & Potter (1989) have described a novel protein encoded by the loop of a Drosophila FB element, which could function in FB transposition. Within TFB1, we have found several short open reading frames, which may be joined via possible splice sites.
However, the significance of these open reading frames remains to be evaluated. (b) Genomic organization of T F B 1 "Zoo" blot hybridization with TFBI subcione pKH2A-ll shows that the repetitive element is present in a wide variety of Chironomus species (Fig. 3(a)). Thus, TFB1 clearly appeared early during evolution of the genus Chironomus. Drosophila FB elements (Silber et al., 1989) and sea urchin TU elements (Hoffman-Liebermann et al., 1985) are also evolutionarily conserved in many species. TFB1 was hybridized to genomic Southern blots of DNA from various populations of Ch. th. thummi and Ch. th. piger (Fig. 3(b)). The results show that TFB1 is present in higher copy number in the genome of Ch. th. thummi than in Ch. th. piger (see also in situ hybridization, Fig. 4). DNA from different populations of the same subspecies does not display much difference in the hybridization pattern, which indicates a predominant stability of TFBl-related sequences in the genome. However, during the time of divergent evolution of Ch. th. thummi and Ch. th. piger TFB1 elements must have had periods of high transpositional activity. This can be concluded from the chromosomal distribution of T F B elements. The chromosomal localization of TFB1, determined by in situ hybridization to larval polytene chromosomes of a Ch. th. thummi x Ch. th. piger F l hybrid (Fig. 4), is largely different in the two subspecies. In Ch, th. thummi, TFBl-homologous sequences are scattered over the genome, including the pericentromeric and telomerle regions. In Ch. th. piger, however, T F B sequences are mainly confined to the pericentromeric region of all chromosomes, with significantly less and weaker signals detectable on the chromo-
Communications
AACGAATTCA CCTCAATCTA AAATTC~AA
479 VSO
AATGCAGCTA CCCT]~]~
VlO0 C A G A T A ~ T A C T G C C A A G A T G C A G A T ~ ' T A C T G C T A A A G T G CAGATGAAAA vz50
.TTAI~I-~-~+CCC . T A A A A T G C A G A T G A A A T G C T GTTG~-~-z-z-i~ AAAAAGGT]LT
v200
A A T G C A G A T G A~-A-A-A-A'.~CGT A T A A A G T G c A G A T A A A A T G CTGCCCALAAT
v250
GTAC,A T ~ ' T A T A A T A A A A T T A A A A T G C A T C A T A T T T G A T A TTATAAAAAT v300 T A T A G T A A A C "~-~-~.-x~l~i-z-z'A A A A A A T T C G T A A G A A A G G A A AG~,-.L-.L-.L-z-x~IC v350 G C A A C G A C T A T T C C T A C C T T G C C C A A A A A C TATACAGCTA v400
T T C T A T A G A G C A G C A C G G A T C A A A T G T A A A A A A C T A G A A G TCTCTACAAT
v450
GTC'~CGTTAT T G A G A T T T C A T G C T G A T T A A ~.-~-~-xCATGCTTGTTrG~ETA
v500
C A G A A A G C G T AI-~-~-I'GCGAT T T T C T C A A T T T G A A A T C T G T TGI~I-xGAAAA
v550
T G A T G T A A A A "~.-~-~-~'AGGCAC A A A I I - ~ - I ' A C T
TCTGAGA~/T TTTAAAACCA v600 ATTTTG ~I~TC G-~-~-~-~-CTCACA A A A T G C G A A A A A C T G A T G T TTGCATCTGA v650
A C A A A A C C A A T A G A T T C A C A C G T G A A A A C G G T G T G A A T T T AAAAAATACA v700 T T T G T A G A T A T A T C A A T G A A A G C T G A C A A A ~,.~-J.-~.-x~,CAA AATTC~C,M~. v750 A A A T A T T C G T G A G T G A C G C T T A A T T A A A T T GAf~-z-I-CTGA CATAACGCCT vSO0
TAA~:~-~-xAGG A A G T A A G T T C T A T G T C A A A A G A A A G C A A T T TTl~-~-z-~-z-i"
v850
A C A A G A T A T A ATAA~.-i=~-xGA A A T T T G T C A c T A T T G T G G T T TCGGAGATAT vg00 T T A A G A A A C C A A A T C A T C A C T C A C G A A T A A "~.~..~ .~ ,~ ~.-~/~-~.-~: CGTAGGGGCT V950
C A A A A A A T T A ATATCCAkACG T T A A A T T A T C T G C A T T A T A C ~-zz-z-~T~'AAA
vlO00
G C A A C A G C A T T T C A T C T G C A ~-z-~AGGGAA A A T A A A T T T c ATCTGCACTT v1050 TAGCAGTACC ATCTGCATAT TAGGGTAGCT GCATTTTAGG AATTTCTA~ vll00 TTGGGGTAAC GAAT~TGT G T T A T A A T A G A A A T G A C G C C GTTTAAAATG vllS0
C A A T G A T G G C GTCA~I~I-xGA G A G A G T T T A C A G G T T A G C T T TATAGAATGA V1200 -FI-FFgATTGG A C T A G A A T G T T T T A A G C T A C A A G A A A A G T A TTGTTGAGAT V1250 C A T A A A A T T G A A T A A A A A A T *Fl'~Z'T~-~=i-~-i C A T G T T A A T A ~i-~I-~GGTGG TCTTGAAAAA GACCGTTCTA ATGAAATATG AAAGCTT
~(a)
TH
5'-
TTAAAATCATCAACGAA~TTC f
PI
-
TFB
1 -
AACGAATTTCTGTGTTATAATA- 3'
t
5'-
6
TTAAAATCATCAACGAATTT~TGTGCTAAAATA -
3'
(b)
Figure 2. (a) Nucleotide sequence of subclone pKH2A-I 1 containing the TFB1 element. The IVRs are underlined by arrows. The flanking target site duplications are marked by double line arrows. The 1st 3 nucleotides (AAC) of the sequence do not belong to subclone pKH2A-I1, but are shown since they are part of the 5' target site duplication. (b) Comparison of the empty target site from a histone gene repeating unit of Ch. th. piger (clone 6.2, PI) and the filled target site (Ch. th. thummi clone 500-7, TH) of TFBl. The comparison of the empty with the filled target site indicates a deletion of a T in the left duplication (marked by an asterisk (*)). Further sequence differences are marked by dots.
some arms. This corresponds to the result of Southern hybridization. Obviously, there has been amplification/elimination and transposition of TFB1 during the divergent evolution of the subspecies. The higher copy n u m b e r of TFB1 in Ch. th. t h u m ~ i is correlated to the larger genome size of this subspecies. The same phenomenon has been observed for several families of satellite-like,
tandem-repetitive DNA in Chironomus thummi (Schmidt, 1981, 1984; Th. Hankeln & E . R . Schmidt, unpublished results). The structural simi~ larity between F B elements and satellite DNA has been recognized by T r u e t t ¢t al. (1981). Our results suggest common evolutionary mechanisms acting on T F B elements and tandem-repetitive DNA sequences.
480
Th. Hankeln and E. R. Schmidt
I
ct
23@
2
3
4
: ' " ~" • .%
4@
20
I@
0"6@
. . . . .
,/
9.
~.~ :. - ~ - : . ~ , , r
I
-~
'i "
{a)
:
" 2.
(b)
Figure 3. (a) Southern (1975) blot of Hind III-digested DNA from various species of the genus Chironomus (Zoo blot). hybridized with radiolabelled (Feinberg & Vogelstein, 1983) TFBl-containing subclone pKH2A-1 I. plum, Ch. plumosus; pal, Ch.. pallidivittatus; ten, Ch. tenta~a< halo, Ch. halophilus; th, Ch. th. thummi; pi, Ch. th. piger; lur, Ch. luridus; pseu, Ch. pseudothummi; cin, Ch. cingulatus; me]. Ch. melanotus. Molecular sizes or marker bands are indicated in kb. (b) Southern blot of HindIII-digested DNA from different populations of Ch. th. thummi and Ch.. th. piger, hybridized as in (a). l and 2, different Ch. th. thummi populations; 3 and 4, different Ch. th. piger populations. Molecular sizes of marker bands (marked by dots) are the same as in (a).
(c) T F B 1 and histone gene evolution The integration of transposable elements into histone genes or integenic spacers is not unusual. The foldback-like transposon TU has been found within a histone H2B pseudogene of the sea urchin Stronqylocentrotus purpuratu8 (Lieberman et al., 1983). Insertions of transposable elements have been found in a subgroup of D. melanogaster histone gene repeating units (Lifton et al., 1977; Saigo et al., 1981; Matsuo & Yamazaki, 1989). These latter elements are also inserted into an A +T-rich region between the histone HI and H3 genes. The transposable copia-like element 297 has been found inserted within the TATA box of a D. melanogaster H3 gene (Saigo et al., 1981; Ikenaga & Saigo, 1982). These examples indicate that the histone gene family is a preferential site for the evolutionary fixation of mobile elements. The redundancy of these genes probably allows for insertions without this having severe effects on the organism's fitness. The mutations might even be spread among the members of the multigene family and among the population by homogenization mechanisms
("Molecular drive"; Dover, 1982, 1986). We suggest that the multiple occurrence of TFB1 in at least two, but probably more, adjacent histone gene repeating units in Ch. th. thummi (see Fig. l ) is the consequence of homogenization processes acting on histone gene repeats. In that respect, it is interesting to note that both the integrated TFB] and the 1 bp deletion in the left target site duplication have been homogenized in adjacent repeating units (data not shown). Such homogenization events have been postulated (and shown) for tandem-repetitive DNA families (Coen et al., 1982; Strachan et al., 1985). I n situ hybridization of TFB1 shows an especially intense signal at band D3c on chromosome II of Ch. th. thummi (H~igele, 1970). This band contains one of the five histone gene clusters on the midge (Th. Hankeln & E . R . Schmidt, unpublished results). The bright signal {Fig• 4) is probably due to multiple copies of the TFB1 element, present in a number of tandemly arranged histone gene repeating units. Thus, the TFB1 histone gene containing clone 2 Ctt500-7 has probably be~n derived from locus D3c on chromosome II.
Communications
481
Figure 4. In situ hybridization of TFBl-containing subclone pKH2A-I 1 to larval polytene chromosome of Ch. th. thvmmi x Ch. th. pitier F 1 hybrids. The DNA probe was biotin-labelled (Langer-Safer et al., 1982) according to the Feinberg & Vogelstein (1983) method. The FITC fluorescence immunodetection system (DetekIf, Enzo Biochem.) was used as described (Hankeln & Schmi'dt, 1987; Schmidt et al., 1988). The partially paired Ch. th. thummi and Ch. th. piger chromosomes II are indicated by th and pi, respectively. Centromere regions are marked by C. The brightly hybridizing histone gene locus D3c (according to the map presented by H~gele (1970)) is marked by an arrow. The corresponding phase contrast micrograph is shown.
The other four histone gene loci do not show any hybridization with T F B 1 , which means t h a t the integration of T F B l and its spreading to other histone gene repeating units by homogenization mechanisms is specific for the histone gene locus II, D3c. Such a locus-specific evolution of histone genes seems not probable in D. melanogaster, where all histone genes are known to be clustered a t one chromosomal site only (Pardue et al., 1977). In Ch. th. piger, however, only a very weak signal is found at histone locus D3c after hybridization with T F B I , indicating t h a t only one or a few copies of T F B l are present. We thank Mrs Bettina Welch for expert technical assistance and Mrs Heidi Sommerfeld for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (Schm 523/3-6).
References Benton, W. D. & Davis, R. W. (1977). Science, 196, 180-182. Berg, D. E. & Howe, M. M. (1989). Editors of Mobile DNA, Amer. Soc. Mierobiol., Washington, DC. Chen, E. ¥. & Seeburg, P. H. (1985). DNA, 4, 165-170. Coen, E., Straehan, T. & Dover, G. (1982). J. Mol. Biol. 158, 17-35.
Collins, M. & Rubin, G. M. (1983). Nature (London), 303, 259-260. Collins, M. & Rubin, G. M. (1984). Nature (London), 308, 323-327. Dover, G. A. (1982). Nature (London), 299, l l l - l l 7 . Dover, G. A. (1986). Trends Genct. 2, 159-165. Feinberg, A. P. & Vogelstein, B. (1983). Anal Bioehem. 132, 6-13. Finnegan, D. J. & Fawcett, D. H. (1986). In Oxford Surveys on Eucaryotic Genes (Maclean, N., ed.), vol. 3, pp. 1-62, Oxford University Press, Oxford. Frischauf, A.-M., Lehraeh, H., Poustka, A. & Murray, N. (1983). J. Moi. Biol. 170, 827-842. H~gele, K. (1970). Chromosoma (Berlin), 31, 91-138. Hankeln, T. & Schmidt, E. R. (1987). J. Mol. Evol. 26, 311-319. Hoffman-Liebermann, B., Liebermann, C., Kedes, L. H. & Cohen, S. N. (1985). Mol. Cell. Biol. 5, 991-1001. Holmes, D. S. & Quigley, M. (1981). Anal. Biochem. 114, 193-197. Ikenaga, H. & Saigo, K. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 4143-4147. Langer-Safer, P. R., Levine, M. & Ward, D.C. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 4381-4385. Levis, R., Collins, M. & Rubin, G. M. (1982). Cell, 30, 551-565. Liebermann, D., Hoffman-Liebermann, B., Weinthal, J., Childs, G., Maxson, R., Mauron, A., Cohen, S. N. & Kedes, L. (1983). Nature (London), 306, 342-347. Lifton, R. P., Goldberg, M. L., Karp, R. W. & Hogness,
482
Th. Hankeln and E. R. Schmidt
D. S. (1977). Cold Spring Harbor Syrup. Quant. Biol. 42, 1047-1051. Matsuo, Y. & Yamazaki, T. (1989}. Nuel. Acids Res. 17, 225-238. Pardue, M. L., Kedes, L. H., Weinberg, E. S. & Birnstiel, M. L. (1977). Chromosoma (Berlin), 63, 135-151. Potter, S. S. (1982a). Nature (London), 297, 201-204. Potter, S. S. (1982b). Mol. Gen, Genet. 188, 107-110. Potter, S. S, Truett, M., Phillips, M. & Maher, A. (1980). Cell, 20, 639-647. Saigo, K., Millstein, L. & Thomas, C. A., Jr (1981). Cold Spring Harbor Syrup. Quant. Biol. 45, 815-827. Sanger, F., Nieklen, S. & Coulson, A. R. (1977). Proc. Nat. Aead. Sci., U.S.A. 74, 5463-5468. Schmidt, E. R. (1981). F E B S Letters, 129, 21-24.
Sehmidt, E. R. (1984). J. Mol. Biol. 178, 1-15. Schmidt, E. R., Keyl, H.-G. & Hankeln, Th. (1988). Chromosoma (Berlin), 96, 353-359. Silber, J., Bazin, C., Lemeunier, F., Aulard, S. & Voloviteh, M. (1989). J. Mol. Evol. 28, 220-224. Southern, E. M. (1975). J. Mol. Biol. 98, 503-519. Smyth Templeton, N. & Potter, S. S. (1989). EMBO J. 8, 1887-1894. Straehan, T., Webb, D. & Dover, G. A. (1985). EMBO J. 4, 1701-1708. Truett, M. A., Jones, R. S. & Potter, S. S. (198]). Cell, 24, 753-763. Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Gene, 33, 103-119.
Edited by S. Brenner
