P transposable elements and their use as genetic tools in drosophila

TINS-June

231

1985

Reviews Transposan tagging

P transposable elements and their use as genetic tools in Drosopl;ila Gerald M. Rubin P e l e m e n t s h a v e p r o v e n to b e u s e f u l to Drosophila m o l e c u l a r geneticists as tools f o r ' t r a n s p o s o n t a g g i n g ' a n d D N A - m e d l a t e d g e n e transfer. T h e o b l e c t z v e o f this article ts to d e s c r : b e these t w o m e t h o d s a n d to p o i n t o u t their s t r e n g t h s a n d hmttatlons

Transposable elements Transposable elements are segments of D N A that move as discrete umts from place to place in the genome. They have been found in bacteria, fungi, plants and animals The frequency at which these elements move depends on a vanety of poorly understood factors including element structure, genetic background, and environmental influences. Transposable elements can comprise a substantial fraction of an organism's genome For example, approximately five percent of the D r o s o p h i l a m e l a n o g a s t e r genome consists of transposable elements More than 20 different transposable e l e m e n t families have been identified in D m e l a n o g a s t e r and these fall into four structural classes 1. The insertion of a transposable element into a new genomlc site often leads to the reactivation of a gene located at that site Indeed, a large fraction of spontaneous mutations m D r o s o p h i l a appear to be due to transposable element insertions P-transposable elements are of particular interest because their mobdlty has been shown to be under genetic control and can be manipulated experimentally 2_ Naturally occurnng strains that contain P elements (P strains) generally have between 30 and 50 P elements in their genomes A b o u t one-third of these elements are 3 kb in length, the remainder consists of a heterogeneous set of smaller elements that appear to have been derived from the 3 kb element by internal deletion 3 Within P strains the elements do not transpose at high rates and files with such stable P elements are said to have the P cytotype P cytotype ~s apparently determined by the P elements themselves In contrast, files lacking P elements are called M-strain files, and are said to possess the M cytotype. W h e n P-strain males are crossed to M-

strain females, P elements are introduced into the M cytotype The offspring of such a cross show a set of genetic aberrations, collectively known as hybrid dysgenesls, all of which are confined to the germ line of the dysgenic h y b n d and all of which appear to result from destabihzation and transposition of P elements These may include chromosomal rearrangements, gonadal sterility and, most important in the present context, the reduction of new visible and lethal mutations by P element insertion However, In the cross between an Mstrain male and P-strain female, or in a P x P cross, the P elements are malntamed in the P cytotype, and no hybrid dysgenesls is observed

a.

dysgenie

Transposon tagging refers to the use of transposable elements as mobde units of D N A sequence homology to blochemlcally 'tag" a gene as an aid to its molecular cloning'*. Many interestmg genetic loci m D r o s o p h d a have been identified by their phenotypLc effects on development, physiology, or behavior For a large fraction of these, however, neither the product of the gene nor its exact cytogenet~c location is known For such genes, transposon tagging may be the cloning method of choice A typical protocol would be as follows P-strain males and M-strata females are mated, leading to the induction of P-element transpositions in the germ hne of their progeny These progeny are bred and their offspnng are screened or selected for new mutations in the gene of interest In most cases, the new mutations recovered will have resulted from Pelement insertions The gene ~s now 'tagged' with a P element and D N A corresponding to the mutant allele can be retrieved from a genomlc D N A hbrary, made m a bacteriophage ~.vector using D N A isolated from the mutant strata, by virtue of ~ts sequence

cross

P(~

×

M(~

sperm DNA c o n t a i n s P elements

P element t r a n s p o s i t i o n s occur in developing germ line

b.

P-element

-mediated

gene

transfer

microinjection cloned P element

M embryo

Fig. l. The analogy between the behavior of P elements during a dysgemc cross (a) and during Pelement-mediated gene transfer (b) Is dlustrated In a dvsgemc cross the P elements are introduced into an M cytotype egg via the paternal chromosomes In P-element-mediated gene transfer P elements are also introduced Into an M-slram environment, but m this case by mtcrom]ecllon into an embryo whose parents were both from M strains In both cases P elements transpose at h:gh rates m the developing germ hne ~ ) 1985, E l s e v a e r S~aenee P u b h s h e r s [$ V , A m s t e r d a m

0 3 7 8 - 5 9 1 2 / 8 5 F ~ 2 00

232 homology to the P element Since P strains contain about 50 P elements in their genomes, many extraneous P elements will be present in addition to the one responsible for the mutation Thus only 1 of 50 recombinant phages which show P element homology will contain the gene of interest Only that phage, however, will contain D N A sequences that will label the cytogenetic location of the mutant when hybridized m s i t u to polythene chromosomes Several tactics can be employed to avoid having to isolate and screen large numbers of phages by hybridization in s t t u First, the mutant strain can be back-crossed for several generations to M-strain flies to remove most of the unwanted P elements Second, the chromosomal region containing the mutation can be mlcrodissected and a D N A library made from just this small fraction of the genome s Finally, strains containing one or only a few P elements can be constructed by Pelement-mediated transformation (see below) and such strains can be used instead of naturally occurring P strains as the source of P elements in the initial cross This last approach has the added advantage that P elements can be used that have been modified to contain genetic markers or other features to permit their easy identification and cloning The m a j o r strength of transposon tagging as a cloning method is that only the approximate cytogenetmc location of the mutation need be known Thus it can be readily applied to most of the D r o s o p h d a genes already identified_ Moreover, screens can be lmtiated for new mutations with a particular phenotype using P element insertion as the mutagen, thereby facditatlng the subsequent cloning of those genes A major weakness m the method results from target site preferences for insertion inherent to the P element. Thus not all genomlc sites are mutated with equal frequency and perhaps only one half of all genes will be mutated at high enough rates to make their isolation by this approach practical P-element-mediated gene transfer The strategy for using P elements as vectors for gene transfer is based on mimicking the events that take place during a dysgemc cross between P and M strains (see Fig 1) In such a cross, P elements on paternally contributed chromosomes enter the M cytotype egg and are induced to transpose at

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high rates A n analogous situation occurs if D N A containing a 3 kb P element is microlnjected into an M cytotype embryo shortly after fertilization This element can transpose from the rejected D N A to the germline chromosomes of the host embryo in a reaction that is catalysed by a protein encoded by the P element 6,7 Smaller P elements that lack the D N A sequences encoding this protean can also transpose if co-InJected with the 3 kb element. O t h e r D N A segments of interest can be transferred into the germhne if they are inserted within such internally deleted P elements and then co-inJected with the 3 kb element s. Fig 2 shows a typical protocol for such a gene transfer e x p e n m e n t Since genes transferred using P element vectors are incorporated into the germhnes of their hosts their function can be assayed in all cell types and developmental stages in subsequent generations A l t h o u g h the transferred genes are not inserted at

rosy

their normal chromosomal locations, they appear to be regulated properly and, In nearly all cases, exhibit correct tissue and temporal ~pectflotv ol expression~-t i Uses of gene transfer The experimental power ot gene transfer methods is evident at two levels The most straightforward and routine apphcation is to determine whether a cloned segment of D N A encodes the same product that is nusslng or altered in a particular mutant 0 e genetic complementatlon) As an example of this type of apphcation, suppose you wish to clone a gene wluch when mutated is known to result m abnormal morphology in a region of the brain. You have no idea what the biochemical function of this gene is, but you know the location of the mutation on the cytogenetic map and have b e e n able to isolate D N A from this region of the chromosome from wild-type flies either by trans-

÷

3 kb P element helper plasmld

Q

transposon plasmld

rosy

+

select ry -/- offspring

Y

G 1" P [ ry "/- "1, ry - / r y single or multiple P [ r y -/- ] insertions

Fig, 2. Typtcal protocol for a gene transfer experiment. D N A of the plusmut p~25_1, winch carries a 3 kb P elemenl, and D N A o f the rosy tran,~poson plasmut, which cames a wdd-type rosy gene/,nserted into a small P dement (to generate a rosy transposon), are co.mlected into an embryo that ~s homozygoas for a mutat~n m the rosy gene. The inclusion of the plasmut p~25 1 ts necessary, since the rosy transposon can only transpose with the aut of protein factors encoded by the 3 k b 'hdlm" P element_ Approximately 10% o f the inlected embryos wdl survive to become fertile adults (GO adults ) and, m about one-thxrd of these, transposmon of the rosy tran~poson from the mlected plasmld D N A to germlme chromosomes will have occurred Since transposuton only occurs tn the germ line, the exprexgion o f the introduced rosy gene is not evutent m the somatic tissues of the GO adult~, but If they are mated to ry- mdivMuals the expression o f the rosy gene can be assayed m the next (G1) generaaon In those off~pnng that show expressmn of the rosy gene (ry+ off~pnng) single or mul~ple cop~es o f the rosy transposon (P[ry+]) are found m~erted m the chromosomes and these are stably inherited m future gmlerations The rosy gene (the structural gene for the enzyme xanthme dehydrogenase) affects eye color and thus is easily scored Genes whose f'uncaone are doCfieuh to assay can be tran~erred by construcnng a transposon that contains both the gene of interest and the rosy gene Success[~_l transfer of the ennre transposon can be detected by scoring for rosy gene fimcnon and then the ry + progeny can be assayed for the function o f the second gene

T I N S - June 1985 poson tagging, microdtssection cloning 5, or chromosome walking 12. In order to Identify those DNA sequences corresponding to the gene, lndwidual D N A segments from this chromosomal region can be inserted into P element vectors, reintroduced into the organism, and tested for their ability to rescue the m u t a n t phenotype_ In this way the gene can be cloned and identified without any p n o r knowledge of the nature of its product The full battery of molecular biological methods can then be applied to the isolated gene A more sophisticated application of gene transfer involves the wilful manipulation of a gene and then determining the biological consequences of the changes introduced Specifically, the structure of a cloned gene can be altered by mutagenesis m vitro and then put back into the genome where the function of the altered gene can be assessed Specific changes in the amino acid sequence of the protein can be engineered and the effect(s) of these changes determined in the organism's normal environment, where behavioral or developmental phenotypes caused by the altered gene can be monitored In addition, mutations outside the protein coding region of the gene can be made and assayed tn vtvo to determine which cts-actmg D N A sequences control proper tissue speclfioty and developmental timing of gene expressxon More complex mutations can also be constructed tn vitro, including mutations that cause a gene to be expressed in a cell type where it is not normally active For example, suppose a gene encoding a cell surface protein specific for cell type 'A" has been isolated The protein coding portion of this gene could be joined to the control region of a second gene whose expression IS limited to another cell type, 'B', and the fusion gene introduced into the genome_ Flies carrying the fusion gene should now express the protein on the surface of cell type 'B', directed by the fusion gene, as well as on the surface of cell type ' A ' (due to the unaltered copy of the gene present in the fly's genome) If this cell surface protein ts

233 involved in cell-cell recognition, specific developmental abnormalities might be expected. One obvious problem with this type of approach is that the incorrect expression of many important genes will be lethal to the organism While genetic tools exist for handling recessive lethal mutations in Drosophda, mutations hke the one described above would most hkely be dominant The classical way to overcome this difficulty is to make expression of the mutant gene conditional For example, fusions can be made to transcriptional promoters that are inducible by environmental factors, such as heat shock Unfortunately, even the heatInducible hsp70 promoter, the best currently known for this purpose in Drosophda 13, shows some transcription during the life cycle of the fly even when not intentionally induced Genetic analysis of sex determination in Drosophda predicts the existence of sex-specific promoters 14. Fusions to such promoters should result In expression in only one sex. A n alternative approach would be to make fusions to promoters that are specific for non-essential cell types For example, flies without eyes are viable under laboratory conditions and therefore fusions made to a promoter for a photoreceptor-speofic gene (e g rhodopsln) would be expected to be viable even if the fusion product resulted in death of the photoreceptor cells or otherwise disrupted eye development. Similarly, the flight muscle is not essential and a gene for a flight muscle-specific actln has been isolated 15_ O t h e r potential applications of gene transfer remain to be developed For example, many genes have been cloned by virtue of their differential expression in certain cell types or developmental stages. However, these genes frequently do not correspond to known mutations, and thus the phenotype of an individual lacking the gene function cannot be discerned Although with sufficient effort mutations In such a gene can be induced by classical means, a convenient and reliable method that utilizes the cloned

copy of the gene to inactivate the corresponding chromosomal copy would be particularly useful Methods based on homologous recombination, such as those used in yeast t6, are not likely to be feasible in Drosophila It is possible that methods based on the production of an antisense R N A t7 can be adapted to Drosophda, although the ability of this method to completely inactivate a gene ts unclear Even in their present state of development, transposon tagging and Pelement-mediated gene transfer, when used in combination with classical Drosophda genetics, allow many experimental approaches to molecular neuroblology that are not currently feasible in other orgamsms Selected references 1 Rubm, G M (1983) m Mobde Geneac Elements (James A Shapiro, ed ), pp 329362, Academic Press, New York 2 Engels, W R (1983) Annu Rev Genet 17, 315-344 3 O ' H a r e . K and Rubm G M (1983) Cell34, 25-35 4 Bmgham. P M , Levis, R and Rubm, G M (1981) Cell 25,693-704 5 Scalenghe. F , Tuvco, E . Edstrom, J E , P~rrotta, V and Melh, M (1981) Chromosoma 82, 205-216 6 Spradhng. A C and Rubm, G M (1982) Scwnce 218. 341-347 7 Karess. R E and Rubm, G M (1984) Cell 38, 135-146 8 Rubm, G M a_qd Spradlmg, A C (1982) Science 218, 348-353 9 Scholntck, S B . Morgan. B A and Hirsh, J (1983) Cell 34, 37-45 10 Spradhng, A C and Rubm, G M (1983) Cell 34.47-57 l l Goldberg, D , Posakony, J and Manlatls, T (1983) Cell 34, 59-73 12 Bender, W , Sp~erer, P and Hogness, D S (1983) J Mol Btol 168, 17-33 13 L~s, J T , Simon. J A and Sutton, C A (1983) Cell 35,403-410 14 Baker, B S and Belote, J M (1983) Annu Rev Genet 17. 345-393 15 Fyrberg, E A , Mahaffey, J W , Bond, B J and Davtdson, N (1983) Ce1133. 115123 16 Scherer, S and Davis, R W (1979) Proc Nail Acad Sct USA 76, 4951-4955 17 Izant, J G and Welntraub, H (1984) Cell 36, 1007-1015 Gerald M Rubm ts at the Department o f Biochemistry, Umverstty of Cahforma at Berkeley, Berkeley, CA 04720, USA