the transposable genetic element tc1 in the nematode caenorhabditis elegans

4
iews At the most recent Caenorhab- ditis elegans meeting (held at Cold Spring Harbor Labora- tory, May 6-10, 1987), nearly 30% of the 188 meeting abs- tracts referred to a C. elegans transposable genetic element called Tcl. One reason for the great interest in Tcl has to do with the important questions raised by transposable ele- ments in general, such as how they transpose, how they af- fect gene expression, and what kinds of mutations they pro- mote. Most C. elegans work- TIG--August 1987, Vol. 3, no. 8 ¢ The transposable genetic element Tcl in the nematode Caenorhabditis elegans Robert K. Herman and Jocelyn E. Shaw Tcl is a 1.6 kbp DATA sequence present in about 30 copies in some strains of C. elegans and 300 or more copies in other strains. Tcl elements excise much more frequently tn somaffc cells than in the germ line. Germ.line transposition of Tcl has been detected and is under genetic control. Tcl has become very useful as a tool for cloning C. elegans genes zdentified solely by mutation. ers, however, are interested in Tcl for a second reason: to use it as a tool for cloning genes that have been identified by mutation and for which no gene products are known. Study of the developmental genetics of C. elegans, which was pioneered by Sydney Brenner ~, is benefiting from the exceptionally detailed description now available of wild-type C. elegans development. The lineages of all cells generated during development are knownz-4, and the structure and connectivity of the complete net ,,)us system have been determineds-s. The developmental abnormalities of many mutants affected in cell lineage, nervous system development, muscle development or sex determination have been precisely described with reference to wild-type development. Hundreds of genes affecting these aspects of developmen t have been identified by analysis of mutants, but for most of them, gene products are unknown. There is thus great interest in cloning these genes to examine their gene products; in addition, transformation can now be used to see how cloned genes and variants of them function in living animals~. Tcl is contributing to the cloning effort in two ways. First, there are C. elegans strains with hundreds more copies of Tcl distributed throughout their genomes than are found in the wild-type strain N2, on which most genetic and developmental work has been done. Each Tcl dimorphism (nresence or absence of Tcl at a specific site) can be mapped genetically, and the genomic sequence flanking the Tcl can be isolated. Each mapped Tcl site is thus a signpost from which chromosomal walks can be initiated. Such walks are greatly aided by the availability of many groups of clones known to overlap in nucleotide sequence (contigs), which have been generated in a project designed to construct a complete physical map of the C. elegans genome l°. The second use for Tcl is in the direct tagging by insertional mutagenesis of genes to be cloned, as has been done with other transposable elements in other organisms (e.g. Refs 11 and 12). Identification of Tc 1 Tcl was first identified as a consequence of Southern blot comparisons made between two C. elegans strains, Bristol N2 (English strain) and Bergerac BO (French strain). Unique sequence probes identified restriction fragments that were 1.6 kbp larger in BO than in N2, and it was shown that the extra 1.6 kbp was due to the presence of a repetitive element (Tcl) present in about 30 copies in N2 and 300 or more copies in BO (Refs 13 and 14). There was early evidence for frequent Tcl excision in somatic cells, but definitive evidence for germ-line transposition came somewhat later. Moerman and Waterston discovered that the frequency of spontan- eous mutation of the gene uric-22 was much higher in BO than in N2 and suggested that the higher mutability of BO might be due to Tcl transposition 15. Eide and Anderson made use of a special screen developed by Park and Horvitz16 to identify a large number of spontaneous mutations in the myosin heavy chain gene uric-54, which had already been cloned17; they found that a large fraction of spontaneous mutations in BO (but virtually none in N2) were insertions of Tcl (Refs 18 and 19). The genes unc-22 (Ref. 20) and lin-12 (Ref. 21) were the ~st of the genes previously described only genetically to be tagged and cloned by Tcl transposition. Structures of Tc 1 elements and target sites for Tcl insertion Rosenzweig, Liao and Hirsh22 compared the nnc- leotide sequence of a Tcl-filled site in BO with the sequence of the empty (and presumably never occu- pied) site in N2. In place of the 2 bp sequence TA in N2, BO had 1614 bp: TA followed by 1610 bp followed by TA (Fig. 1). One interpretation of this finding is that there was a target duplication of TA upon insertion of Tcl, in which case Tcl is 1610 bp long. Target site duplication ha~ been found for other transposable elements and is thought to arise as a consequence of a staggered cut at the target site during transposition2a. An alternative possibility in the case of Tcl is that the element is 1612 bp long and is inserted between the T and A of the target site without target site duplication 24 (Fig. 1). The sequencing of an additional 16 target sites has not resolved this ambiguity because they all have TA at the insertion site. The 1610 bp sequence has perfect 54 bp inverted repeats at its ends. It also contains two long open reading frames on the same DNA strand. The larger open reading frame corresponds to a basic polypeptide cf 273 amino acids. TATA and CAAT box sequences for transcriptional initiation are present at appropriate spacings 5' to the putative coding region, and a possible polyadenylation signal was identified. The second open reading frame, which potentially encodes a ll2-amino acid polypeptide, is entirely within the first but in a different translational reading frame. No © 1987, Else'aer Pubhmlaons. Cambridge 0168 - 9525/87/$02.00

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Page 1: The transposable genetic element Tc1 in the nematode Caenorhabditis elegans

i e w s At the most recent Caenorhab- ditis elegans meeting (held at Cold Spring Harbor Labora- tory, May 6-10, 1987), nearly 30% of the 188 meeting abs- tracts referred to a C. elegans transposable genetic element called Tcl. One reason for the great interest in Tcl has to do with the important questions raised by transposable ele- ments in general, such as how they transpose, how they af- fect gene expression, and what kinds of mutations they pro- mote. Most C. elegans work-

T I G - - A u g u s t 1987, Vol. 3, no. 8

¢

The transposable genetic element Tcl in the nematode

Caenorhabditis elegans Robert K. Herman and Jocelyn E. Shaw

Tcl is a 1.6 kbp DATA sequence present in about 30 copies in some strains of C. elegans and 300 or more copies in other strains. Tcl elements excise much more frequently tn somaffc cells than in the germ line. Germ.line transposition of Tcl has been detected and is under genetic control. Tcl has become very useful as a tool for

cloning C. elegans genes zdentified solely by mutation.

ers, however, are interested in Tc l for a second reason: to use it as a tool for cloning genes that have been identified by mutation and for which no gene products are known.

Study of the developmental genetics of C. elegans, which was pioneered by Sydney Brenner ~, is benefiting from the exceptionally detailed description now available of wild-type C. elegans development. The lineages of all cells generated during development are known z-4, and the structure and connectivity of the complete net ,,)us system have been determined s-s. The developmental abnormalities of many mutants affected in cell lineage, nervous system development, muscle development or sex determination have been precisely described with reference to wild-type development. Hundreds of genes affecting these aspects of developmen t have been identified by analysis of mutants, but for most of them, gene products are unknown. There is thus great interest in cloning these genes to examine their gene products; in addition, transformation can now be used to see how cloned genes and variants of them function in living animals ~.

Tcl is contributing to the cloning effort in two ways. First, there are C. elegans strains with hundreds more copies of Tcl distributed throughout their genomes than are found in the wild-type strain N2, on which most genetic and developmental work has been done. Each Tcl dimorphism (nresence or absence of Tcl at a specific site) can be mapped genetically, and the genomic sequence flanking the Tcl can be isolated. Each mapped Tcl site is thus a signpost from which chromosomal walks can be initiated. Such walks are greatly aided by the availability of many groups of clones known to overlap in nucleotide sequence (contigs), which have been generated in a project designed to construct a complete physical map of the C. elegans genome l°. The second use for Tc l is in the direct tagging by insertional mutagenesis of genes to be cloned, as has been done with other transposable elements in other organisms (e.g. Refs 11 and 12).

Identification of Tc 1 Tcl was first identified as a consequence of

Southern blot comparisons made between two C. elegans strains, Bristol N2 (English strain) and Bergerac BO (French strain). Unique sequence probes identified restriction fragments that were 1.6 kbp larger in BO than in N2, and it was shown that the extra 1.6 kbp was due to the presence of a repetitive

element (Tcl) present in about 30 copies in N2 and 300 or more copies in BO (Refs 13 and 14).

There was early evidence for frequent Tcl excision in somatic cells, but definitive evidence for germ-line transposition came somewhat later. Moerman and Waterston discovered that the frequency of spontan- eous mutation of the gene uric-22 was much higher in BO than in N2 and suggested that the higher mutability of BO might be due to Tcl transposition 15. Eide and Anderson made use of a special screen developed by Park and Horvitz 16 to identify a large number of spontaneous mutations in the myosin heavy chain gene uric-54, which had already been cloned17; they found that a large fraction of spontaneous mutations in BO (but virtually none in N2) were insertions of Tcl (Refs 18 and 19). The genes unc-22 (Ref. 20) and lin-12 (Ref. 21) were the ~ s t of the genes previously described only genetically to be tagged and cloned by Tcl transposition.

Structures of Tc 1 e lements and target s ites for Tcl inser t ion

Rosenzweig, Liao and Hirsh 22 compared the nnc- leotide sequence of a Tcl-filled site in BO with the sequence of the empty (and presumably never occu- pied) site in N2. In place of the 2 bp sequence TA in N2, BO had 1614 bp: TA followed by 1610 bp followed by TA (Fig. 1). One interpretation of this finding is that there was a target duplication of TA upon insertion of Tcl, in which case Tcl is 1610 bp long. Target site duplication ha~ been found for other transposable elements and is thought to arise as a consequence of a staggered cut at the target site during transposition 2a. An alternative possibility in the case of Tcl is that the element is 1612 bp long and is inserted between the T and A of the target site without target site duplication 24 (Fig. 1). The sequencing of an additional 16 target sites has not resolved this ambiguity because they all have TA at the insertion site.

The 1610 bp sequence has perfect 54 bp inverted repeats at its ends. It also contains two long open reading frames on the same DNA strand. The larger open reading frame corresponds to a basic polypeptide cf 273 amino acids. TATA and CAAT box sequences for transcriptional initiation are present at appropriate spacings 5' to the putative coding region, and a possible polyadenylation signal was identified. The second open reading frame, which potentially encodes a ll2-amino acid polypeptide, is entirely within the first but in a different translational reading frame. No

© 1987, Else'aer Pubhmlaons. Cambridge 0168 - 9525/87/$02.00

Page 2: The transposable genetic element Tc1 in the nematode Caenorhabditis elegans

TIC, - - August 1987, Vol, 3, n,~. 8 review

1,6A12 bp 1'6A10bp ! I, I

IR ORF IR FiE. 1. Mejor features of ~'cl and its i~etti~n target site, based on n=cl~eti~ seq~ndr_~ "z'z4 and showing the terminal invert~ repeats (IE) and the largest open r e ~ i ~ ~a~r,~ (ORF).

TATA or CAAT box sequences with appropriate spacings were found for the latter sequence. Possibly the same message is translated in two ways.

Structurally, Tcl resembles the P elements of Drosophila, the Ae-Ds elements of maize, and the bacterial IS elements; and as is the case for these elements, Tcl transcripts are not abtmdant. Unlike P elements and Ac-Ds elements, for which abbreviated variants are common, nearly all the Tc l copies seem to be the same size 13"t4. Slight heterogeneities in restriction enzyme sites have been noted within the Tcl family, however t°'~s.

The insertion of Tcl shows a bias for specific target sequences. Eleven independent Tcl insertions in uric- 54 were found to have occurred at just four sites (at the nucleotide level), all in exons (D. Eide and P. Anderson*). [By contrast, in uric-22, which is clearly a favored gene for Tcl insertion, 12 Tcl insertioas occurred in at least ten separate sites, spanning about 30 kbp (Ref. 20)]. Inspection of 17 target sequences that have been analysed shows that the only absolutely conserved sequence is the TA insertion site. The sequences flanking the TA targets are not random, however, and a 9 bp consensus target sequence has been proposed (I. Mori, G. Benian, D. Moerman, and R. Waterston, pets. commun.).

Tcl excision: somatic versus germ-line High frequency excision of the Tcl element from a

particular genomic site can be recognized on a Southern blot. Unique sequence flanking several Tcl elements has been cloned and used to probe Southern blots of genomic DNA. In each case the probe hybridizes to two bands: a heavy band corresponding to the Tcl-bearing fragment and a weak band, 1.6 kbp smaller, corresponding to the Tcl empty site. Such empty sites seem to be generated by most, if not all, Tcl elements in BO (Refs 26, 19, 20). The fact that the empty site is 1.6 kbp smaller suggests that excision is often precise or nearly precise. The empty sites are produced almost exclusively by somatic cells, since they are passed on to progeny at relatively low frequency 2~. As a consequence of the low frequency germ-line excision, the fraction of sites that axe empty

* Names cited without a reference refer to presentations made at the Cold Spring Harbor C. elegans meeting.

is low iv_ embryos, increases during larval develop- ment, and then drops as the germ line becomes more prominent in the adult zT. The much higher excision of Tc l in somatic cells than in the germ line is just the opposite of the behavior of P elements in Drosophila during hybrid dysgenesis: the excision and transposi- tion of P elements ;~ germ-line specific (owing to a germ-line specificity in the splicing of the P element transcript) ~.

Emmons and colleagues have measured the rate of formation of empty sites at five different genomic sites in BO worms arrested as first stage larvae by starvation in buffer 26. At all five sites excision continued for the two weeks that the worms remained alive, by which time more than 10% of sites had been vacated. Thus excision does not seem to require DNA replication. At two of the five sites, N2 also has Tc l elements; both showed somatic excision but at a lower rate than in BO.

Somatic excision of Tc l from within uric-,54 has been followed by the method just described and also by the identification of somatic mosaics, uric-54 mutants are both paralysed and unable to lay eggs because their muscles are defective. Eide and Anderson found that a BO strain carrying a Tc l element in uric-54 gave about 1% egg laying-proficient animals TM. These animals usually also showed improved ability to move, and when their longitudinal strips of body muscle cells were inspected by polarized light microscopy, they were often found to have patches of birefringence characteristic of wild-type body muscle (although not fully wild-type). The frequency of germ-line reversion of the same Tcl element was about 4 x 10-6; by contrast, empty sites reached a level of 1-5%.

K-S. Ruan and S. Emmons (Ref. 29 and pers. commun.) and D. Eide and P. Anderson have cloned and sequenced several vacated sites generated by somatic excision of Tcl elements at four different locations; most of the excisions were imprecise, usually leaving behind 4 bp insertions, but all of the six excisions examined at one particular site were precise, suggesting that flanking sequence may influence the accuracy of excision. Germ-line Tc l excisions from two sites in unc-54 (D. Eide and P. Anderson) and one site in uric-22 (.1. Kiff, D. Moerman and R. Waterson) have been studied by sequencing revertants. Despite the selection imposed for

Page 3: The transposable genetic element Tc1 in the nematode Caenorhabditis elegans

F1

m

I ~ x N2 4. m . ~

~V~w

X ~

SOUTHERN BLOT N 2 PROBED WITH Tc 1

T Ill ,

X _ _

Self and r ::> select m/m

F~

m

4

0 Repeal prev=ous

two steps many tumes

Y

m

m

Fig. 2. Scheme for Tel-tagging a C. elegans gene to be cloned z°'zl. The BO strain contains about300 or more copies of Tcl, and N2 contains about 30 stable copies (the illustrated Southern blot showing five bands for N2 is ~ ,aplified for diagrammatic purposes). A modification of this scheme is to substitute a low-copy number Tcl-transposing strain for BO (see text). Caenorhabditis elegans hermaphrodites have five pairs of autosomes (l-V) and a pair of X chromosomes.

restored gene function, the great majority of sequenced germ-line excisions were imprecise. Most revertants at one unc-54 site left behind the same 4 bp insertion often seen after somatic excision; the Tcl insertion site was in an exon but very near an exon- intron border, and the extra 4 bp left by the excision appears to have shifted the splice site by 4 bp, with the consequence that a wild-type polypeptide was generated. Reversions at the other sites included short in-frame insertions, duplications of the se- quences flanking the insertion site, and 1-2 kbp deletions. Thus, although Tcl excision in BO is much less frequent in the germ line than in the somatic cells, the spectra of vacated sites in the two cell classes appear to be similar if not identical. In conjunction with the investigations into Tcl excision in vivo, an in vitro system desig~led to monitor Tcl excision (R. Plasterk) should help to elucidate the mechanisms of Tcl movement.

Extraehromosomal copies of Tc 1 Extrachromosomal copies of Tcl have been

identified in BO (Refs 30 and 31), at a level between 0.1 and 1.0 copy per cell. The predominant form was a 1.6 kbp linear molecule with ends corresponding to the ends of the integrated Tcl. Also identified were relaxed and supercniled circular copies of the element. It has been suggested that the extrachromosomal copies of Tcl are the product of somatic excision and may be intermediates in the transposition process~ However, the concentration of extrachromosomal copies did not increase in starved BO larvae 2s, in

TIG m August 1987, VoL 3, no. 8

which the fraction of empty Tcl sites did increase; thus, if the extrachromosomal copies are the product of somatic excision then they must he unstable.

Mutator st ra ins Although no C. elegans strain devoid of Tc l

elements has been found, different strains exhibit very different frequencies of germ-line Tcl transposition, and it is important to understand what is responsible for the variation. Tcl copy number is dearly not solely responsible. Transposition, whether measured by insertion in uric-54 or uric-22, is much greater for BO and TR679 (see below), high-copy number strains, than for N2, a low-copy number strain; but DH424 (isolated in California) is a high-copy number strain and shows little if any Tcl transposition ]s'xs. Moennan and Waterston also found that two other Bergerac lines (FR and BL1), both high-copy number strains, failed to show the high frequency unc-22 mutation (later shown to be largely due to Tcl insertion) characteristic of the Bergerac BO strain is. Con- versely, Tcl-transposing strains having only about 45-60 copies of Tcl have been generated from mutator/N2 hybrids (M. Finney and R. Horvitz; I. Mori, D. Moerman and R. Waterston), and an N2 strain with mutator activity for uric-22 and increased Tc l copy number has been discovered (C. Trent andJ. Hodgldn, pers. commun.).

The uric-22 mutator activity in BO was shown to depend on genetic background, not on the uric-22 region itself: an uric-22 region from N2 became mutable when put in a BO background, and a BO unc- 22 region was stabilized in an N2 background ~~. Early attempts to localize genetically mutators responsible for Tcl transposition in BO indicated .that they were polygenlc. More recently, mutators in different derivatives of BO/N2 hybrids have been mapped to three linkage groups, and it has been suggested that the mutators may themselves transpose (I. Mori, D. Moerman and R. Waterston).

P. Anderson and colleagues have identified strains following EMS mutagenesis that show enhanced frequencies of spontaneous Tcl excision and trans- position in the germ line. One of these strains, called TR679, and some of its derivatives have been used for transposon tagging (see below). Portions of the mutator activity of TR679 have been mapped to different linkage groups; the enhanced mutator activity of TR679 may be polygenic or transposable (or both).

The mutator activities in both BO and TR679 appear to be dominant to inactivity. Mutators could be providing trans-acting factors that promote trans- position of Tcl elements in the genome; if so, they might be analogous to a maize Ac element or a Drosophila P element, each of which is capable of mobilizing non-autonomous elements, or they might be acting in some other way. An apparent distinction between BO and TR679 has been revealed recently by the finding that transposable elements distinct from Tcl are activated in TR679. One such element is called Tc3; it is not homologous to Tcl and has been found in three distinct sites in spontaneous unc-22 mutants of TR679 0. Collins, B. Forbes and P. Anderson) and in two distinct sites in uric-86 mutants recovered in a strain descended from a TR679/N2 hybrid (M. Finney

Page 4: The transposable genetic element Tc1 in the nematode Caenorhabditis elegans

T I G - Auglcst 1987, VoL 3, no. 8

and R. Horvitz). Work on transposable elements in C. elegans other than T c l is in its early stages.

T r a n s p o s o n t a g g i n g a n d g o n e c lon ing In BO, the frequencies of spontaneous Tcl- induced

mutants for uric-54, uric-22 and l in-12 are about 5 x 10 -7, 10 -4 and 5 × 10 - s respectively 19-21. For each of these loci, selective methods for identifying rare mutant individuals were used. For loci for which mutant selection is not possible, the frequency of T c l insertion may be too low to make BO useful for transposon tagging. Enhanced mutator strains such as TR679 have been used successfully by a number of workers to tag genes of interest . In TR679, the frequency of T e l mutation in uric-22 is about 10 -3 (P. Anderson). Other genes have shown lower but still useful frequencies of T e l insertion. Still others have been refractory to T c l insertion, but at least one of these, uric-86, has been a target for other t ransposons (see above).

Of course, finding a spontaneous mutant for a g e n e of in teres t in a Tel- t ransposing strain is only the first s tep in cloning the gene. One must pick out the T c l - tagged sequence from among all the Tcl -bear ing sequences in the mutant. The now-standard method for identifying the relevant T c l is to outcross the mutant to the wild-type strain N2, to pick homozygous mutant self progeny, and then to repeat these two s teps many times 2°'2x (Fig. 2). The chromosomes of the resulting strain should all be N2 except in the vicinity of the spontaneous mutation. Southern blots of DNAs from the mutant strain and from N2 are compared to see if an extra Tcl-containing band can be found. Closely-linked T c l elements may lead to extra bands that are not actually within the gene to be cloned; these may be eliminated by selection for recombination in the intervals on each side of the gene of interest , using genetically marked N2 strains. Once one identifies a Tel-containing fragment that invariably cosegregates with the mutation of interest, fragments of appropriate size can be cloned in E. coli, and a clone containing T c t can be identified. Sequence flanking the T e l can be used for subsequent probing and recovery of more flanking DNA. An alternative to Tel-tagging, as already noted, is to make use of T e l dimorphisms known to be closely linke;! to the gene of interest to initiate a chromosomal walk through the gene (G. Ruvkun, V. Amhros and R. Horvitz). When it is thought that the desired DNA has been isolated, by ei ther method, additional mutant alleles (including rever tants of Tcl-induced muta- tions) that affect pat terns of Sou them blots can provide confirming evidence that the region of interest has in fact been cloned. In addition, transformation with the isolated wild-type genes has been reported to rescue mutants, providing good evidence that the correct DNA sequences were cloned (A. Fire and R. Waterson; J. Way and M. Chaifie). These s trategies have already been successful for several genes. Improvements seem likely as more is learned about T e l and other transposable elements in C. e l e g a ~ .

R e f e r e n c e s l Brenner, S. (1974) Eenet/cs 77, 71-94 2 Sulstan, J. E. and Horvitz, ]I. R. (1977) Dee. B=oi. 56, 110--

156 3 Kimble, J. and Hirsh, D. (1979) Dev. Bml. 70, 396-417

review 4 Sulston, J. E., Schierenberg, E., White, J. G. and Thomson,

J. N. (1983)D~. Biol. 100, 64-119 5 Ward, S., Thomson, N., White, J. G. and Brenner, S. (1975)

J. Comp. Neurol. 160, 313-,338 6 Ware, R.W., Clark, D., Crossland, K. and Russell, R. L.

(1975) ]. Comp. NeuroL 162, 71-110 7 Albertson, D. G. and Thomson, J. N. (1976) Philos. Trans. R.

Soc. London. Ser. B 275, 299--325 8 White, J. G., Southgate, E., Thomson, J. N. and Brenner, S.

(1986) Philos. Trans. R. S~. Lr~don. Set. B 314, 1-340 9 Fh-e, A. (1986)EMBO ]. 5, 2673--2680

10 Coulson, A., Sulstan, ]., Brannet, S. and Karn, J. (1986)Prec. Nail Acad. Sci. USA 83, 7821-7825

11 Bingham, P. M., Levis, R. and Rubin, G. IVL (1981) Cell 25, 693-704

12 Federoff, N. Y., Furtek, D. B. and Nelson, O. E. (1984)Prec. Nail Acad. Sol. USA 81, 3825-3829

13 Emmnns, S. W., Yesner, L., Ruan, K. and Katzenber8, D. (1983) Cell 32, 55-65

14 Liso, L. W., Rosenzweig, B. and Hirsh, D. (1983) Proc. Nail Acad. Sci. USA 80, 3585-3589

15 Moerman, D. G. and Waterstnn, P,- H. (1984) Genetics 108, 859-877

16 Park, E-C. and Horvitz, H. R. (1986) Ge~e6cs 113, 853-867 17 MacLeod, A. R., Karn, J. and Brenner, S. (1981) Nature 291,

386-390 18 Eide, D. and Anderson, P. (1985) Genetics 109, 67-79 19 E~de, D. and Anderson, P. (1985) Proc. Nail Aead. Sci. USA

82, 1756--1760 20 Moerman, D. G., Benian, G. M. and Waterston, R. H. (1986)

Proc. Nail Aead. Sc~. lISA 83, 2579-2583 21 Greenwa]d, I. (1985) Cell 43, 583-590 22 Rosenzweig, B., Liao, L. W. and Hirsh, D. (1983) Nucleic

Acids Res. 11, 4201-4209 23 Grindley, N. D. F. and Sherratt, D.J. (1983) Cc/d Spying

Harbor Symp. QuanI. Biol. 43, I257-1261 24 Rosergweig, B., Liao, L. W. and Hirsh, D. (1983) Nucleic

Ac/ds Res. 11, 7137-7140 25 Rose, A. M., H~x'is, L.J., Mawji, N. R. and Morris, W. J.

(1985) Can. ]. Bioci~,m. Cell Biol. 63, 752--756 26 Emmons, S. W., Roberts, S. and Ruan, K. (1986) Mol. Gen.

Genet. 202, 410-415 27 Emmons, S. and Yesner, L. (1984) Cell 36, 599-605 28 Lanlu, F. A., Rio, D. C. and Ruhin, G. M. (1986)Ce1144, 7-19 29 Emmorm, S. W., Ruan, K-S., Levitt, A. and Yesner, L. (1985)

Cold Spring Harbor Syr~. Quant. BtoL 50, 313-320 30 Ruan, K-S. and Emmons, S. W. (1984) Proc. Nail Acad. Sci.

USA 81, 4018--4022 31 Rose, A. M. and Snntch, T. P. (1984) Nature 311, 485-486

R. K. Herman and J. E. Shaw are at the DepaTlmeut of Genetics and Cell Biology, Dniversity of Minnesota, S t Paul MN 55108, USA.

Reviews scheduled for forthcoming issues of Trends in Genetics

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