Regulation of white locus expression: The structure of mutant alleles at the white locus of Drosophila melanogaster

Cell, Vol. 30, 529-541,

September

1982,

Copyright

0 1982

by MIT

Regulation of white Locus Expression: The Structure of Mutant Alleles at the white Locus of Drosophila melanogaster Zuzana Zachar* and Paul M. Bingham* Laboratory of Genetics National Institute of Environmental Health Sciences Research Triangle Park, North Carolina 27709

gion DNA sequences of a selected sample of white mutant alleles. Our results reveal several properties of the organization of the white locus DNA sequences, Results

Summary We have analyzed the structures of 19 mutant alleles at the white locus of Drosophila melanogaster. Thirteen of the mutant alleles in our selected sample arose spontaneously, and of these, seven are associated with insertions of non-white-region DNA sequence elements. Several lines of evidence strongly suggest that these insertions are responsible for their associated mutant alleles, and further suggest that most or all of these insertions are transposons. Moreover, the white locus DNA sequences can be divided into two nonoverlapping domains on the basis of the properties of the two domains as mutational targets. One of these domains behaves, in this regard, in the manner expected of functional coding sequences, whereas the other does not. We propose a model for the nature and function of the presumptive noncoding white locus genetic elements. The two domains of the white locus defined by our studies are approximately coextensive with the functionally distinct subintervals of the locus defined by previous genetic analysis. Lastly, our results strongly suggest that the dominant, mutable wDzL allele results from the insertion of a transposon outside of, but near, the white locus. This putative transposon apparently carries genetic elements that act at a distance to repress expression of the white locus. Introduction Mutant alleles at the X-linked white locus (1-l 5) of Drosophila melanogaster prevent or reduce the deposition of the two chemically distinct screening pigments in the adult eye (for review see Philips and Forrest, 1980). The first mutant allele to be isolated in Drosophila was the w’ allele at white (Morgan, 1910; see Figure 21, and the locus has been subjected to extensive genetic analysis since that time by a large number of investigators. Though the biochemical basis of the phenotypic effects of white mutant alleles is not understood (Philips and Forrest, 19801, several phenomena revealed by genetic analysis suggest that a molecular analysis of the mechanisms of regulation of expression of the locus would be fruitful (Muller, 1932; Green, 1959a, 1959b; Jack and Judd, 1979; Bingham, 1980a). We describe the physical analysis of the white re* Present address: New York at Stony

Department of Biochemistry, State University Brook. Stony Brook. New York 11794.

of

Structures of Wild-Type white Alleles The DNA sequences from the white locus region of the wa’ and Canton-S wc alleles have been cloned previously, and the positions of two chromosomal breakpoints, separated by approximately 14 kb and apparently bracketing the white locus, have been placed on the cloned interval (Bingham et al., 1981; Levis et al., 1982). We have placed additional restriction endonuclease cleavage sites on the map of the Canton-S w+ allele, and have characterized a number of other white alleles by Southern gel analysis and by retrieving and analyzing selected portions of some of these alleles in cloned form (see below). On the basis of these studies, we define two w+ alleles, designated the Canton-S and Oregon-R w+ alleles; the white mutant alleles whose structures we report can be conveniently described as being derived from one of of these w+ these two w+ alleles. The structures alleles and the coordinate system we use to describe the structures of white alleles are diagrammed in Figure 1. The origins of, and eye-color phenotypes produced by, our sample of white mutant alleles are listed in Table 1. Structure of the w ‘lE4 Mutant Allele The mutant allele w’lE4 is a deficiency allele whose deleted segment begins on the interval from the Sac I cleavage site at coordinate -8.9 to the Hind Ill cleavage site at coordinate -6.4 and extends rightward beyond the end of the cloned white region interval at coordinate +25 (Figure 11, as assessed by Southern gel analysis (results not shown). The remaining white region sequences in w”~~ (from coordinates -23 to -8.9) are indistinguishable from the Canton-S w+ allele, with the exception that w”~” lacks the Eco RI cleavage site at coordinate - 15.5 (Figure 1). Structures of Some white Mutant Alleles Not Associated with Large Aberrations A subset of the white mutant alleles that we have analyzed are not associated with large aberrations, as assessed by Southern gel analysis (results not shown). We define large aberrations to be those changes in DNA sequence routinely detectable by restriction mapping, and estimate the sensitivity of this technique to be such that we can detect deletions or insertions involving white region sequences of approximately 50 bp or larger. The wco’ mutant allele has a restriction map indistinguishable from the Canton-S w+ allele on the white region cloned interval. The wEwx mutant allele has a

Cell 530

opll,3lw~

Figure

1. Restriction

Maps

of the Oregon-R

breokporni I and Canton-S

DfUwDzLM

Wild-Type

breakpoint-

white Alleles

(Top) Scale (calibrated in kilobases), showing the coordinate system used to describe the structures of white alleles. This coordinate system has been used previously (Levis et al., 1982); coordinate 0 corresponds to the position of the copia insertion associated with the w” mutant allele (Bingham et al., 1981; Bingham and Judd, 1981). On the Canton-S w+ allele, Hint II cleavage sites have been mapped on the interval from -1.3 to +9.1; Pst I cleavage sites have been mapped on the interval from -20 to +6.7; all other cleavage sites have been previously described (Levis et al., 1982) on the entire interval shown. On the Oregon-R w+ allele, Hint II cleavage sites have been mapped on the interval from -1.3 to +lO; Pst I cleavage sites on the interval from -20 to +9.9; Barn HI cleavages sites on the interval from -10 to +15.6; Eco RI cleavage sites on the interval from - 17 to + 18; Hind Ill cleavage sites on the interval from - 19.5 to +24; Bgl II, Sal I and Sac I cleavage sites on the entire interval shown. In addition to cleavage-site differences between the Canton-S and Oregon-R w+ alleles, Oregon-R lacks 300 bp thatch marks) of sequence length on the interval from the Hint II cleavage site at +7.5 to the Hind Ill cleavage site at +9.0. Also shown is the extent of the deleted segment of the w’lE4 mutant allele: the uncertainty in the placement of the endpoint of this deficiency is indicated by hatch marks. Lastly, the positions of the Df(l)wD”f4 and Dp(l.3)~‘~ chromosomal breakpoints are shown, and the uncertainty in placement of these is indicated by the size of the corresponding boxes (see Figures 6 and 7; Levis et al., 1982). These breakpoints apparently bracket the white locus (Bingham et al., 1981).

restriction map indistinguishable from Canton-S, ex150 bp of secept that wBwx lacks approximately quence length on the interval between the Sac I cleavage sites at coordinates - 18.0 and - 19.5. (Available observations do not allow us to decide whether this deficiency is likely to be responsible for the wBwX mutation; however, the position of this deletion with respect to other white locus elements to be described below does not exclude such a causal relationship.) The wcrr and w65a25 mutant alleles have restriction maps indistinguishable from the Oregon-R w+ allele, as does wcf, with the exception that wc’ lacks the Hind Ill cleavage site at +S.O. Our stock of the wSat mutant allele carries white region sequences indistinguishable from the CantonS w+ allele on the interval from -23 through the Barn HI cleavage site at +4.7, and indistinguishable from the Oregon-R w+ allele on the interval between the Pst I cleavage site at +4.8 and the Barn HI cleavage site at + 15.6. Each of the wBwx, wcf, wco’, wcrr, wSar and w65s25 mutant alleles produces a mutant eye-color phenotype distinguishable from that produced by any other member of this set (Table 1). All of these mutations map to the left (toward the telomere) of the w” mutation, as defined by fine-scale genetic mapping studies (see Figure 8; Bingham, 1980a; Bingham and Judd, 1981). Structure of the w b” Mutant Allele wbf’ maps genetically to the left of the war allele (see Figure 81, and the structure of wbfr is described in Figure 2. The unique white region sequences of wbr’ have a restriction map indistinguishable from the Or-

egon-R w+ allele, as defined by Southern gel analysis (results not shown). In addition, wbf’ contains an insertion of approximately 9.5 kb of non-white-region sequences (the wb” element) into the interval between the Sal I cleavage sites at -0.4 and -1.3. DNA sequences in the white region are unique (Bingham et al., 1981; Levis et al., 1982); however, a cloned segment (Xwb”22; Figure 2) carrying a portion of the wbf’ element insertion is homologous to repeated sequences in the Canton-S and Oregon-R,, strains of D. melanogaster and in the y w strain of D. simulans (Figure 3). This result demonstrates that the wbf’ element is homologous to repeated sequences in these strains. Previously characterized Drosophila transposable elements are likewise homologous to repeated Drosophila DNA sequences (for review see Spradling and Rubin, 1981; Bingham et al., 19821, and we propose that the wbf’ element is a transposable element. The wbf’ element labels a 4.5 kb Barn HI-Sal I genomic fragment that has autoradiographic intensity of apparently greater than one copy per haploid genome (Figures 2 and 3). The presence of this highstoichiometry fragment suggests that the wb” element may be a member of a relatively homogeneous, copialike element family (Spradling and Rubin, 1981; Bingham et al., 1982). The wb” allele has been observed to be slightly unstable in one stock carrying the allele (Redfield, 1952). Structure of Mutant Alleles at The wa’ mutant allele has been ized physically (Bingham et al., (Bingham and Judd, 19811, and

the wa Site previously character1981) and genetically these results strongly

Regulation 531

of white Locus

Table 1. Origin Described

Expression

and Eye-Color

Phenotype

of white Mutant

Allele

Eye-Color

Phenotype

Origin

wI

Bleached

white

X-ray

1E4

Brick

w”“’ (colored)

red

Alleles

X-ray Spontaneous

Wewx (Brownex) WC’ (coffee)

Reddish

brown

w”” (carrot)

Orange

da’ (satsuma)

Deep ruby

WS5a25

Bleached

brown

X-ray Spontaneous Spontaneous

white

X-ray

W”’ (buff one)

Light yellow

Spontaneous

w”’ (apricot

one)

Orange

brown

Spontaneous

wS2 (apricot

two)

Orange

brown

Spontaneous

t+@ (apricot

three)

Orange

brown

Spontaneous

ti4 (apricot

four)

Orange

brown

Spontaneous

W’

Bleached

we (eosin)

Light yellow-pink

Spontaneousa

wh (honey)

Light yellow

Spontaneous”

wCh (cherry)

Light yellow-pink

Spontaneous

Mottled red on yellow background in z’ti” males

Soontaneous

Spontaneous?

wZm (zeste

mottled)

white

Spontaneous

tips5 (spotted

55)

Dark yellow in males; light yellow in females

w’p’ (spotted

one)

Dark, fine mottling on yellow background; males darker

n+”

two)

Similar to tip’

Spontaneous

w’p3 (spotted

three)

Similar

to tip’

X-ray

tilp4 (spotted

four)

Similar

to tip’

X-ray

wDzL

(spotted

Light yellow in females; nearly wild-type in males

Spontaneous

The wild-type eye color in D. melanogaster is a deep bright Results and Experimental Procedures for further details. a Spontaneous derivative of w’.

red. See

suggest that the war mutant allele results from the insertion of the copia transposon into white region sequences. We have analyzed the structures of the a2 W , wa3 and wa4 mutant alleles, all of which map genetically at the w* site (Green, 1959a) and produce very similar mutant eye-color phenotypes. The wa2 and wa3 alleles are not associated with aberration in white region sequences, as assessed by Southern gel analysis (results not shown), and are indistinguishable in restriction map from the Canton-S and Oregon-R w + alleles, respectively. The unique white region sequences of the wa4 mutant allele are indistinguishable from the Oregon-R wt allele. In addition, wa4 contains an insertion of approximately 10 kb of non-white-region sequences (the we4 element) on the interval from the Sal I cleavage site at

-0.4 to the Hint and 4).

II cleavage

site at +0.5

(Figures

2

Structures of Mutant Alleles at the wsp Site The wsp mutant alleles map genetically to the right of the wal allele (see Figure 8), and the structures of four independently derived wsp alleles are shown in Figures 2 and 5. The residual, unique white region sequences of the wsp’ and wsp” alleles have restriction maps indistinguishable from the Canton-S w+ allele, and the residual, unique white region sequences of the wsp2 and wsp4 alleles have restriction maps indistinguishable from the Oregon-R w+ allele, as defined by Southern gel analysis (results not shown). The wsp’ mutant allele contains an insertion of approximately 8.6 kb (the wsp’ element) on the interval from the Bgl II cleavage site at +5.0 to the Hint II cleavage site at +5.9 (Figure 2). A cloned segment !hwSp’6; Figure 2) containing the wsp’ element insertion is homologous to repeated DNA sequences in the Oregon-Rap and Canton-S D. melanogaster strains and in the y w D. simulans strain (Figure 3). We propose that the wsp’ element is a transposon. The wsp’ element is homologous to a 4.8 kb Barn HI-Sal I genomic fragment that has an autoradiographic stoichiometry of apparently greater than one copy per haploid genome (Figures 2 and 3), suggesting that the of a relatively homoW sp’ element may be a member geneous, cop&like element family (for review see Spradling and Rubin, 1981). Derivatives of wsp’ that may be partial revertants have been reported to occur spontaneously (Lewis, 1956). The wsp3 allele (Figure 5) is associated with a deletion of white region sequences (the wsp3 deletion) on the interval beginning between the Bgl II cleavage site at +5.0 and the Hint II cleavage site at +5.9 and extending rightward to a point on the interval between the Sac I cleavage sites at +21.9 and +22.1, as defined by Southern gel analysis (results not shown). The wsp4 allele (Figure 5) contains a deletion of 1.2 kb of white region sequences (the wsp4 deletion) on the interval between the Bgl II cleavage sites at +5.0 and +6.4. We have retrieved a portion of the wsp4 allele in cloned form (Figure 5). The 0.2 kb Bgl II fragment (corresponding to the 1.4 kb Bgl II fragment from +5.0 to +6.4) from the cloned segment (Xwsp41) is homologous to cloned white region fragments, including the 3.5 kb Eco RI-Hind Ill fragment from +3.3 to +6.8, and to the 1.4 kb Bgl II fragment from +5.0 to +6.4 (results not shown). Furthermore, when this fragment is used to probe genomic Southern gels, it displays homology to a single 3.5 kb Eco RI-Hind Ill fragment and a single 1.4 kb Bgl II fragment from the Canton-S w+ strain comigrating with the corresponding cloned white region fragments (results not shown). These results demonstrate that as expected on the basis of Southern gel analysis, the wsp4 0.2 kb Bgl II fragment is derived from the Canton-S w+ 1.4 kb Bgl

Cell 532

:

-5

-IyLyy

I doti

g$$ 4

d 0

-5

,

II fragment, and furthermore, suggest that there are few, if any, non-white-region sequences at the wsp4 breakpoint. Thus the wsp4 deletion may be simply derived from the Oregon-R w+ allele by elimination of white region sequences without the mediation of nonwhite-region sequences (for example, transposableelement sequences). Our stock of the wsp2 allele (Figure 5) contains a deletion of approximately 200 bp of white region sequences (the w Jp2 deletion) on the interval from the Bgl II cleavage site at +5.0 to the Hint II cleavage site at +5.9. The wsp2 allele is associated as well with additional DNA sequences (apparently non-white-region sequences; the w sp2 insertion) on the interval defined by the Hint II cleavage site at +1.2 and the Barn HI cleavage site at + 1.7, as assessed by Southern gel analysis (results not shown). We have not characterized the w sp2 insertion in detail. Notice that the wsp2 deletion occurs on the same small interval on which the wsp’ element insertion occurs, and that is affected by the wsp3 and wsp4 deletions (Figures 2 and 5). We emphasize that each of these four wsp alleles produces a very similar mutant eye-color phenotype idiosyncratic to these four alleles (for references see Lindsley and Grell, 1968) and propose that the wsp2 deletion, but not the wsp2 insertion, is responsible for the wsp2 mutant allele.

W’ .,‘s

0 --

Figure 2. Structures of Some white Mutant Alleles Associated Insertion of Non-white-Region DNA Sequence Elements

with

Major horizontal lines (with restriction endonuclease cleavage sites indicated): structure of a portion of the white region DNA sequences in the case of the indicated allele. Scales below these lines (calibrated in kilobases) indicate the relationship of these sequences to cloned unique white region sequences (Figure 1). Boxes: DNA-sequenceelement insertions associated with the indicated white mutant alleles. In (a). (c) and Cd) the dimensions of a cloned segment of the corresponding white allele is indicated; the uncertainty in positioning of the termini of these cloned segments is indicated by hatch marks. (d) Structure of a portion of the w’ allele and its w” and wB derivatives. wh iS indistinguishable from w’ except for the loss of 1.1 kb of

Structures of Mutant Alleles at the we Site We have characterized three independently derived mutant alleles that apparently affect genetic elements near the site of the we mutation (see Figure 8). In contrast with the set of wsp mutant alleles, members of this set produce mutant eye-color phenotypes quite distinct from one another (Table 1). The w’ mutant allele resides between wa’ and wsp’, as defined by fine-scale genetic mapping experiments (Lewis, 1952; Green, 1959a). As defined by Southern gel analysis (results not shown), the restriction map of the unique white region sequences in this allele is indistinguishable from that of the Canton-S w+ allele on the interval from -5.8 to +4.7, and is indistinguishable from that of the Oregon-R w+ allele on the interval from +4.7 to + 13, with the exception that w’ lacks the Hind Ill cleavage site at +9.0. In addition, the w’ allele contains an insertion of 5.7 kb of non-whiteregion sequences (the w’ element) on the interval from the Hind Ill cleavage site at +3.3 to the Barn HI cleavage site at +4.7 (Figure 2). The w’ allele is slightly unstable and has given rise spontaneously to the we and wh derivatives, both of sequence length internal to the w’ element on an interval bounded by the two element-internal Sal I cleavage sites and including the Hind Ill cleavage site between these two Sal I cleavage sites. we is indistinguishable from w’ except for the loss of the w’ element-internal Eco RI cleavage site, the acquisition of a new element-internal Xho I cleavage site and an increase of 0.2 kb in the distance between white region cleavage sites to the left of the element insertion and conserved, element-internal cleavage sites.

Regulation 533

of white Locus

Expression

Figure 3. Each of the Distinct DNA-Sequence-Element Insertions Associated with Four white Mutant Alleles Is Homologous to Reiterated Drosophila Sequences I2

56

34

b-4

ui% which produce more darkly pigmented eyes than the parental w’ allele (for references see Lindsley and Grell, 1968). Both we and wh are indistinguishable from w ’ except for changes largely or entirely internal to the w’ element insertion (see Figure 2 for details). A cloned portion of the wh element (w’ element derivative) is homologous to repeated sequences in the Canton-S and Oregon-& D. melanogaster strains and in the y w strain of D. simulans (Figures 2 and 3). We propose that the w’ element and its we and wh derivatives are transposons. We have examined the structure of the wch allele in our wCh wy stock, and find it to be indistinguishable from our stock of the wB allele (Figures 21, in spite of its reportedly independent origin (Safir, 1913). The we and wch alleles, as originally described, are very similar or identical in all respects analyzed, including finescale genetic map position (for references see Lindsley and Grell, 1968). We have not analyzed other to identify a wCh allele distinW ch stocks to attempt guishable from the we allele. The w’“’ mutant allele (Becker, 1960; Kalisch and Becker, 1970; B. H. Judd, personal communication) maps genetically between the wa and wsp sites. The W Zm allele produces a wild-type eye-color phenotype in an otherwise wild-type genetic background, but produces an obviously mutant eye-color phenotype in the presence of the z’ mutant allele at the zeste locus (Becker, 1960). As defined by Southern gel analysis (results not shown), the unique white region sequences in the wZm allele are indistinguishable from the Canton-S w+ allele on the interval from the Hind III cleavage site at -5.8 to the Barn HI cleavage site at +4.7, and are indistinguishable from the Oregon-R wc allele on the interval from the Pst I cleavage site at +4.8 to the Bgl II cleavage site at + 13, with the exception that wZm lacks the Hind Ill cleavage site at +9.0. In addition, wZm contains an insertion of approximately 7.5 kb of non-white-region sequences (the wZm element) on the interval from the Hind Ill cleavage site at +3.3 to the

78

;

(A-C) Results of probing Southern filters of Barn HI-Sal I digests of Oregon-&. CantonS and D. simulans (y w) DNAs with the indicated cloned segment. These three cloned segments are described in Figure 2. (D) Result of probing a Southern filter of the following digests with the Xw”‘2 cloned segment (see Figure 6 and text): Eco RI-Sal I digest of Oregon-R?> (lane 1). w”’ (lane 2), Harwich (lane 3) or Canton-S (lane 4) DNA; or Hind Ill-Barn HI digest of Oregon-Rpp (lane 51, wc” (lane 6), Harwich (lane 7) or Canton-S (lane 8) DNA.

UP2

- 4.8kb

- 2.3kb - l7kb 1”

k

- 0.9kb - 0.7kb - 0.8kb

Figure

4. Southern

Gel Analysis

of the wa4 Mutant

Allele

(A) Result of probing Southern filters of Hint II digests of Oregon-R (w’) and w” DNAs with the white region segment extending from the Sal I cleavage site at -0.4 to the Hind Ill cleavage site at +3.3 (Figure 1). (B) Result of probing a Southern filter of these same two digested DNAs with the white region fragment between the Sal I cleavage sites at - 1.3 and -0.4. Sal I cleavage sites are Hint II cleavage sites and, collectively, these results and those in which the same DNAs are analyzed after digestion with other restriction endonucleases demonstrate that the wt and wB4 alleles are indistinguishable on the intervals from the Hint II cleavage sites at - 1.3 to -0.4, +0.5 to +1.2 and +I .2 to +6.0. and that the wa4 allele has (considering only the results shown) at least 3.1 kb of additional DNA sequences on the interval defined by the Hint II cleavage sites at -0.4 and +0.5. (See also Figure 2.)

Barn HI cleavage site at +4.7 (Figure 2). We have not attempted to retrieve the wZ”’ allele in cloned form; however, the genetic instability of w’“’ (for review see

Cell 534

0

Figure

‘5

5. Structures

‘IO

115

l 20

of the wEP2, wsp3 and wsP’ Mutant

L

Alleles

Central horizontal line: unique white region sequences. Scale below this line (calibrated in kilobases): relationship of these sequences to the cloned white region interval (Figure 1). Each of these wsp alleles corresponds to a deletion of white region sequences, including a portion of the interval from the Sgl II cleavage site at +5.0 to the Hint II cleavage site at +5.9. Solid bars: size of the deleted segment. Open bars: the uncertainty in placement of the deleted segment. The w”’ allele we have analyzed is associated, in addition, with an apparent insertion of undetermined size on an interval defined by the Hint II cleavage site at +1.2 and the Sam HI cleavage site at +I .7 (see text). (Top) extent of a cloned segment of the w“” allele (hwEO’ 1 ), the uncertainty in placement of the termini of this segment is indicated by hatch marks.

Green, 1976) suggests that the wZm element is a transposon. It has been suggested, on the basis of its interaction with z’, that wZm is a local, tandem duplication of white region sequences (for references see Lindsley and Grell, 1968). Our analysis of this allele reveals no such duplication, and we suggest that the ~‘“‘element insertion produces a mutation that partially mimics local duplication of white region sequences. However, we note that a small, tandem duplication (less than -500 bp) of white region sequences immediately contiguous to the wZm element insertion (for example, a target-site duplication generated by insertion of the wLm element transposon) would escape detection by the techniques we have used here. Moreover, a large duplication (larger than the white region cloned interval: Figure 1) including the w’“’ element insertion in both copies might also escape detection. The wsps5 mutant allele may have arisen spontaneously (R. Grell and E. Lewis, personal communication), and produces a dark yellow eye color in hemizygous males and a light yellow eye color in homozygous females. Though ii produces an eye-color phenotype superficially similar to the wsp alleles, its eye-color phenotype is nonetheless readily distinguishable from that of the wsp alleles described above. We have subjected this allele to additional genetic analysis, and find it to be, formally, an enhancer of z’ in hemizygous males; z+ w”~~/Y males have dark yellow eyes, whereas z’ w@~/Y males have nearly white eyes. On the other hand, wsp55 does not interact strongly with either wDZL or z’ in heterozygous females, in contrast with we; z+ w’~~~/z’ wrdpdp+females have orange eyes similar to those of z+ wa/z’ wrdp+

females, and wsp55/wDzL females have yellow-orange eyes similar to those of w+/wDzL females (for discussion of these genetic tests see Green, 1959a; Jack and Judd, 1979; Bingham, 1980a). The unique white ‘region sequences of wsp5= are indistinguishable from the Canton-S w+ allele (Figure l), with the exception that wsp55 lacks the Hind Ill cleavage site at +9.0, as assessed by Southern gel analysis (results not shown). In addition, ws’55 contains an insertion of 5.8 kb of non-white-region sequences (the wsp55 element) on the interval from the Hind Ill cleavage site at +3.3 to the Barn HI cleavage site at +4.7 (Figure 2). Repeated attempts to retrieve the wsp55 element in cloned form have been unsuccessful (see Experimental Procedures); however, the restriction map of the W sp55 element (Figure 2) suggests that it has direct terminal repeats similar to the long terminal repeats of some characterized Drosophila transposons (for review see Spradling and Rubin, 1981) and we propose that the wsp55 element is a transposon. Structure of Crossovers between we and w Op’ The crossovers analyzed were isolated in the course of an unrelated genetic study (B. H. Judd and P. M. Bingham, unpublished observations) among the progeny of we/y z’ wsp’ spl females. (The we stock used was nominally wCh; however, as discussed above, this stock carries a white allele indistinguishable from we, and we will designate it here as w”.) From among approximately 250,000 progeny of such females examined, four presumptive we wsp’ spl recombinants and two presumptive y z’ w+ recombinants were recovered. We have analyzed two examples of each class. As assessed by Southern gel analysis (results not shown), the two yz’ w+ recombinants analyzed carry wild-type white alleles indistinguishable from the parental wsp’ (Figure 2) on the interval from the Hind Ill cleavage site at -5.8 through the Bgl II cleavage site at +5.0, and indistinguishable from the parental we allele (Figure 2) on an interval from the Barn HI cleavage site at +4.7 to the Hind Ill cleavage site at + 10. These results demonstrate that the crossover events giving rise to these w+ recombinants occurred between the we element insertion and the wsp’ element insertion, and that these wild-type recombinants carry neither of these insertions. As defined by Southern gel analysis (results not shown), the two we wsp’ spl recombinants analyzed were indistinguishable from the parental we allele (Figure 2) on the interval from the Hind Ill cleavage site at -5.8 to the Bgl II cleavage site at +5.0, and were indistinguishable from the parental wsp’ allele (Figure 2) on the interval from the Barn HI cleavage site at +4.7 through the Hind Ill cleavage site at +9.0. These results demonstrate that the crossover events giving rise to these double-mutant recombinants occurred

Regulation 535

of white Locus

Expression

between the we element insertion and the wsp’ element insertion, and that these recombinants carry both of these insertions. Structures of the w OzL Mutant Allele and Several of Its Spontaneous Derivatives The wDzL allele arose in the inbred Oregon-R,, stock (Bingham, 1980a); and the restriction maps of unique white region sequences of the wDzL and Oregon-F& w+ alleles are indistinguishable, as assessed by Southern gel analysis (results not shown). The Oregon-f&, w+ allele, in turn, is indistinguishable from the Oregon-R w+ allele (Figure l), with the exception of one or more apparently complex differences on the interval from the Hind Ill cleavage site at -6.4 to the Sac I cleavage site at - 19.5. We have not characterized the restriction map of the Oregon-R** w+ allele on this interval of divergence with the Oregon-R w+ allele. The wDzL allele (Figure 6) contains, in addition, 13-14 kb of non-white-region sequences (the wDzL element) inserted on the interval from the Eco RI cleavage site at +9.5 to the Hind Ill cleavage site at + 10, as assessed by Southern gel analysis (results not shown). The wDzL allele is unstable and reverts spontaneously at a relatively high rate (Bingham, 1980b). The two revertants of wDzL that we have analyzed, W rD’ and wrD5, have structures indistinguishable from that of wDzL, except for the loss of a substantial portion of the wDzL element with the loss of few, if any, contiguous white region sequences (Figure 6). In par-

Figure 6. Structure of the Spontaneous Derivatives

w “‘

Mutant

Allele

and

Several

of Its

Central horizontal line: the structure of a portion of the wD” allele. Scale (calibrated in kilobases): relationship of these sequences to cloned white region sequences (see Figures 1 and 6). The wDz‘ allele is associated with the insertion of 13-14 kb of non-white-region sequences (the wDzL element; solid bar) on the interval shown. Also shown are the structures of two wDzL revertants (w’~’ and V/O’). Both are associated with deletion of wDz‘ element-internal sequences, including all mapped, element-internal cleavage sites. Solid bars: size of the presumptive deleted segments. Open bars: the uncertainty in placement of these deleted segments. The dimensions of cloned are segments of these revertants (hw’O’ 2 and hwrD54, respectively) indicated. (Top) Structures of two spontaneous deletion derivatives of wDz‘ (for genetic and cytogenetic analysis of these derivatives see Bingham, 1960b). In both cases, Df(l)w’“2 and Df(7)wDzL14, unique white region sequences beginning at or very near the wDzL element insertion site and extending rightward beyond the end of the cloned white region interval are deleted (see Figure 7 and text). Solid bars: minimal extent of the deleted segments. Open bars: maximal extent.

titular, both wrD and wrD5 are indistinguishable from DZLon the intervals from the Eco RI cleavage site at +9.5 leftward and from the Hind Ill cleavage site at + 10.0 rightward, but lack all mapped wDzL elementinternal cleavage sites (Figure 6), as assessed by Southern gel analysis (results not shown). Moreover, both revertants retain non-white-region sequences (presumably wDzL element residues) on the interval containing the parental wDzL element (Figure 6; see below). Repeated attempts to retrieve the presumptive wDzL element residues associated with wrD’ and wrD5 have produced only cloned segments containing residues of non-white-region sequences at the w” element insertion site smaller than those present in these alleles in the fly. In particular, we have characterized in detail one cloned segment containing the wDzL element insertion site retrieved from wrD’, hwrD’2 (Figure 6). This cloned segment contains additional sequences at the w” element insertion site of approximately 1.5 kb (results not shown), whereas the wrD’ allele itself carries additional sequences on this interval of approximately 4 kb (Figure 6). Similarly, hwrD54 carries approximately 1.8 kb of additional sequences at the wDzL element insertion site (results not shown), whereas wrD5 itself carries approximately 3.3 kb (Figure 6). These results suggest that the presumptive wDzL element residues in wrD’ and wrD5 may be unstable when cloned in bacteriophage lambda vectors. The cloned segments AtiD’2 and XwrD54 are homologous to D. melanogaster repeated sequences, as assessed by Southern gel analysis (Figure 3; other results not shown). If it is assumed that the additional sequences at the wDzL element insertion site in these cloned segments are residues of the sequences at this site in wrD’ and wrD5, respectively, it follows that all or a portion of these additional sequences in wrD’ and wrD5 are homologous to repeated Drosophila sequences. This inference, in turn, strongly suggests that all or a portion of the wDzL element is homologous to repeated Drosophila sequences. In addition to revertants, wDZL gives rise spontaneously to other mutations, among them chromosomal rearrangements (Bingham, 1980b). Df(7)wDzL2 and Df(l)wD” 74 are cytologically visible deficiency mutations arising spontaneously in w” stocks that delete large chromosomal segments beginning at or very near white and extending rightward (Bingham, 1980b). Both of these deficiencies delete white region segments beginning on the interval containing the wDzL element insertion and extending rightward (Figure 6). In particular, Southern gel analysis and in situ hybridization (Figure 7; other results not shown) demonstrate that white region sequences on the interval extending leftward from the Eco RI cleavage site at +9.5 are intact in these deficiency chromosomes, whereas sequences extending rightward from the Hind Ill cleavage site at + 10 are deleted (Figure 6). W

Cell 536

Df(l)wDZL 14 is associated with a w+ allele at white, whereas Df(7)wDzL2 retains a parental wDzL white allele (Bingham, 1980b). We have analyzed the structures of the wDzL element residues at these deficiency breakpoints by parallel Southern gel analysis with control w+ and wDZL DNAs, using the Eco RI-Hind Ill fragment between +9.0 and +9.5 as sequence probe (results not shown). These experiments demonstrate that the lefthand portion of the wDZL element is apparently intact through at least the Pst I cleavage site near the center of the element in the Df(l)wDzL2 case (Figure 6). However, the wDzL element residue, if any, at the Df(7)wDzL 74 breakpoint lacks at least the leftmost cleavage sites for Hint II, Hind Ill, Bgl II and Pst I, which are present in the parental wDzL element (Figure 6). Discussion Evidence That Large Aberrations in white Region DNA Sequences Are Responsible for Their Associated white Mutant Alleles We propose that the large aberrations associated with a subset of analyzed white mutations cause those mutations. Several lines of evidence support this hypothesis. First, if it is assumed that the wsp2 deletion, and not the wsp2 insertion, is responsible for the wsp2 mutation (see Results), the 11 analyzed white mutant alleles associated with large aberrations form a physical map precisely colinear with the fine-scale genetic map (Figure 8). Second, previous analysis of the w” allele (Bingham et al., 1981; Bingham and Judd, 1981) and our analysis of crossovers between we and wsp’ (see Results) suggest that the we, wsp’ and w*’ mutant alleles are indissolubly linked to their respective large aberrations. Third, secondary mutation of two white mutant alleles, w’ (Figure 2) and wDzL (Figure 61, is associated with physical changes inter-

nal to the DNA-sequence-element insertions to be responsible for these mutations.

proposed

The Nature of Spontaneous and X-Ray-Induced Mutation in Drosophila The choice of white mutant alleles that had been previously analyzed genetically imposes upon us an implicit selection for mutant alleles that produce some degree of eye pigmentation (Lewis, 1956; Green, 1959a; Bingham, 1980a). (We define such mutant alleles, operationally, to be leaky.) In addition to white deficiency alleles (Figure 1; our unpublished results), only w’ and w65a25 among our sample are tight (that is, they produce bleached white eyes). Moreover, previous genetic analysis (Green, 1959a) demonstrates that most spontaneous and x-ray-induced white mutations mapping in the lefthand portion of the locus (to the left of w”) are tight. Thus our choice of leaky mutant alleles in the lefthand portion of the locus represents a selection against the majority phenotypic class of such alleles and, therefore, possibly a selection against the majority class of mutational lesions. In contrast, available observations (Green, 1959a; LeFever, 1973; Bingham, 1980a, 1980b) do not exclude the possibility that our sample of mutations mapping in the righthand portion of the locus is representative. If our sample of mutations is representative with respect to mutational lesion, it follows that the majority of spontaneous white mutations arising in laboratory stocks and mapping in the righthand portion of the locus result from the insertion of (apparently transposable) DNA sequence elements (see Results; Figure 8). Moreover, the restriction maps of the element insertions associated with seven of the mutant alleles analyzed (Figures 2, 3, 6 and 8) suggest that each is a member of a different transposable-element family. Lastly, all three possibly representative, x-rayinduced white mutant alleles examined (w’lE4, wsp3

B op//,zyw*5fb7 white, region

white \region

I

pob.2: +9 to +9.5 Figure 7. In Situ Hybridization of the wDz‘ Element Insertion

to the Polytene Site

pbe: Salivary

Dp(7.2)~~‘~~ is the insertion of an X-chromosome our unpublished observations). (A) The probe is the from Am1.l (Levis et al., 1982). containing white Df(7)wD”2 deficiency, whereas sequences from +

Gland

Chromosomes

40 lo *I6

of the Df(l)wDz‘2;

Dp(l,2)~*‘~~

Strain

with Probes

from Either Side

segment carrying the entire white region into the second chromosome (Bingham et al.. 1981; Eco RI-Hind Ill fragment between +9 and +gS. (6) The probe is the Eco RI-Hind Ill fragment region sequences from +lO to +16. Sequences from +Q to +9.5 are not deleted in the 10 to + 16 are deleted. (See also Figures 1 and 6.)

Regulation 537

of white Locus

Expression

wayx Figure

8. Summary

of Structures

of Leaky

white Mutant

wm l@’ , ( f/

we fl;

fzL

Alleles

Open horizontal bar: physical mapof the white region. Scale (calibrated in kilobases; Figure 1): positions of white region elements. Mutant alleles above bar: those associated with large aberrations. Alleles below bar: those not associated with large aberrations. Solid bars: approximate sizes and positions of deletion aberrations. Solid triangles: approximate positions of DNA-sequence-element insertions. The origins of, and eye-color phenotypes produced by. these white mutant alleles are listed in Table 1. (Bottom) Genetic map of selected white mutant alleles. (For discussion of the fine-scale genetic analysis of white see Green, 1959a; LeFever. 1973; Singham, 1980a; Singham and Judd, 1981 .I Mutant alleles not associated with large aberrations are positioned on the physical map with respect to mutations that are associated with large aberrations on the basis of their relative genetic map positions and under the assumption that all the lesions in question (with the possible exception of the wBWX lesion; see Results) reside between the Df(l)d” 14 and Dr~f1.3)~~~ breakpoints, at approximately +9.8 and -2.5, respectively (Figure 1). Approximate minimal dimensions of the lefthand (stippled box) and righthand (hatched box) domains of white locus DNA sequences are given as we define them (see Discussion).

and wsp4; Figures tions.

1 and 5) are associated

with dele-

The Molecular Basis of the wDzL Mutation It has been proposed, on the basis of genetic analysis (Bingham, 1980a, 1980b), that the dominant, mutable W “’ allele results from the insertion of a transposable element into or to the right of the rightmost genetic elements of the white locus. Several of our observations strongly support this hypothesis and lead us to refine it. First, w “’ is associated with a 13-14 kb DNA sequence element (the wDzL element) insertion into white region sequences, and reversion of wDZL is associated with the deletion of sequences largely or entirely internal to the wDzL element (Figure 6). Second, cloned portions of presumptive wDzL element residues are homologous to repeated Drosophila DNA sequences, as are previously characterized Drosophila transposons (for review see Spradling and Rubin, 1981; Bingham et al., 1982). Third, the wDzL element insertion is at the extreme righthand end of the array of known white locus mutations (Figure 8), and deficiency mutations stimulated by wDzL whose large deleted segments begin very near and presumably at the wDzL element insertion site and extend rightward (away from white) can be associated with either w+ or wDzL white alleles (Figure 6). (The capacity of transposons to catalyze rearrangement of contiguous sequences is well established in bacterial systems; for review see Kleckner, 1981.)

Moreover, one such deficiency, retaining a wDzL white allele, retains much and possibly all of the wDzL element at the deletion breakpoint (contiguous to white), whereas a second such deficiency, associated with reversion of wDzL to w+, retains little, if any, of the wDzL element at the deletion breakpoint (Figure 6). Genetic analysis of wDzL strongly suggests that this mutation causes the repression of white expression (Bingham, 1980a), and we propose the following mechanistic model for the effects of the wDzL element insertion on white expression. The wDzL element is inserted outside of the white locus, and the wDzL mutant allele does not result from the disruption of white region sequences at the insertion point of the element. The wDzL element carries genetic elements that act at a distance (at least in cis) on the white locus to repress its expression. Available observations do not allow us to attempt to decide whether the effects of wDzL in trans (Bingham, 1980a) result from direct action in trans of wDZL-element-borne genetic elements or are mediated indirectly by white locus genetic elements. A Two-Domain Model for the Organization of white Locus DNA Sequences Some of the results of our analysis are summarized in Figure 8; on the basis of these results, the locus can be divided into two nonoverlapping domains. We define these domains as follows. The lefthand domain is contained between, but does not include, the Dp(7,3)wzh breakpoint (Figure 1; Levis et al., 1982) on the left (approximate coordinate - 1.5) and the we

Cell 530

site on the right (approximate coordinate +3.9). (We assume that all white mutations analyzed, with the possible exception of wawx [see Results], reside to the right of the Dp(7,3)wzh breakpoint; Figure 1; Levis et al., 1982.) The lefthand domain of the locus contains the insertion site of the copia transposon associated with the wa’ mutation, the wE4 element insertion site and the wbf’ element insertion site. The lefthand domain contains leaky mutant alleles the majority of which (six of nine) are not associated with large aberrations (Figure 8). The righthand domain of the locus is contained between, but does not include, the wa site (coordinate 0) on the left and the wDzL element insertion site (approximate coordinate +9.8) on the right. The righthand domain contains leaky mutant alleles that are always (seven of seven cases) associated with large aberrations. The probability of generating this distribution of mutational lesions under the hypothesis that the white locus consists of a single, homogeneous domain with respect to its properties as a mutational target is less than 1.5% by the one-tailed form of Fisher’s exact test. We note that the inclusion of the wa site in the lefthand domain of the locus is largely arbitrary. If the four w’ alleles are included instead in the righthand domain of the locus, the probability of generating the observed distribution under the hypothesis that the locus is a single domain is less than 3.6% by this same test. The Behavior of the Two Domains of the white Locus As Mutational Targets Green (1959a) examined 15 independently derived white mutant alleles; seven of these arose spontaneously and eight arose after x-ray mutagenesis. Fourteen of these 15 mutant alleles mapped in a genetic interval corresponding to the lefthand domain of the locus as we have defined it. Moreover, seven of seven transposon insertion mutations that arise in P-M dysgenic hybrids and that occupy at least three separate sites (Rubin et al., 1982) reside in a sequence interval contained within the lefthand domain of the locus as we define it. Lastly, the lesions associated with mutations mapping in the righthand domain of the locus can be interpreted as affecting either of two small regions within that domain (the we site and the wsp site; Figure 8; our unpublished results). On the basis of these observations, we propose that the lefthand domain of the locus is substantially more densely populated with sequences whose alteration or disruption produces a recognizably mutant eyecolor phenotype than is the right hand domain of the locus. We further propose that the apparently rare sequences in the righthand domain of the locus that are effective mutational targets are organized into a small number of physically small genetic elements. We have proposed that the disruption of most of the DNA sequences of the righthand domain of the locus

does not produce a mutant eye-color phenotype under the conditions normally used to screen for white mutations. Consistent with this proposal are the results of genetic analysis of two white alleles that we have been unable to obtain for biochemical analysis: both the wis allele (Rasmuson, 1962) and the w+“ allele (Gethmann, 1971) are unstable, and the instability in each case maps in the genetic interval corresponding to the righthand domain of the locus as we have defined it (Gethmann, 1971). We therefore suggest that both of these alleles result from transposon insertion into the righthand domain of the locus. While w” produces a mutant eye-color phenotype under special circumstances (Rasmuson, 1962), both wis and w* (as well as wZm; Figure 2; see Results) produce wild-type eye-color phenotypes in an otherwise wildtype genetic background (Rasmuson, 1962; Gethmann, 1971). Implications of the Two-Domain Model for the Function of white Locus DNA Sequences The behavior of the lefthand domain of the white locus suggests that it consists largely of functional coding sequences (for example, peptide-coding sequences). First, leaky mutations residing in the lef’thand domain of the locus are usually not associated with large aberrations (see Figure 8; Results). Second, the lefthand domain of the locus is apparently relatively densely populated with DNA sequences whose mutation produces a recognizably mutant eye-color phenotype (see above). Third, in a separate study, seven of seven transposon insertion mutations on an interval contained within the lefthand domain of the locus as we have defined it were associated with tight white mutant alleles, and these mutations reverted to wildtype only in association with apparently precise excision of their respective insertions (Rubin et al., 1982). (The observation that white deficiency alleles are tight [Figure 1; Lefevre and Wilkins, 1966; our unpublished observations] suggests that tight white mutant alleles are, in fact, devoid of white function.) Lastly, genetic analysis reveals no effects in trans of mutations mapping in the lefthand domain of the locus (Green, 1959a; Jack and Judd, 1979; Bingham, 1980a). Consistent with the hypothesis that the lefthand domain of the locus consists of functional coding sequences, P. Bingham and J. Jack (unpublished observations) have recently observed a polyadenylated RNA in w+ pupae (adult eye pigments are deposited during pupal stages) with extensive homology to sequences in the lefthand domain of the locus (coordinates -2.7 to -0.4; Figures 1 and 8) and without detectable homology to the righthand domain of the locus. In contrast, the behavior of the righthand domain of the locus suggests that this domain contains few, if any, functional coding sequences. First, all analyzed mutations mapping in this domain (seven of seven cases), even though leaky, are associated with large

Regulation 539

of white Locus

Expression

aberrations. Second, the behavior of this domain of the locus as a mutational target suggests that it is relatively sparsely populated with DNA sequences whose mutation produces a recognizably mutant eyecolor phenotype and, moreover, suggests that recognizably functional sequences in this domain are grouped into a small number of physically small elements (see above). Third, genetic analysis demonstrates that white mutant alleles mapping in the righthand portion of the locus are functionally distinguishable from those mapping in the lefthand portion (Green, 1959a) and suggests that mutations in the righthand portion of the locus exert effects in trans as well as in cis (Green, 1959a; Jack and Judd, 1979; Bingham, 1980a). On the basis of these genetic studies, it has been suggested that the genetic elements residing in the righthand portion of the locus function to regulate the expression of the locus (Green, 1959a; Jack and Judd, 1979; Bingham, 1980a). Further consistent with this last proposal is the observation of Green (1959b) that the genetic determinants responsible for the different quantities of eye pigment found in different w+ stocks probably map in the righthand portion of the white locus. Recent studies of the papovaviruses suggest that the major early transcription units of both SV40 and polyoma are regulated by physically small genetic elements (gene activators or gene enhancers) capable of acting at distances of greater than one kilobase to stimulate the expression of linked RNA polymerase II transcription units (Banerji et al,, 1981; devilliers and Schaffner, 1981). Other studies suggest that these elements may stimulate expression of linked transcription units in response to tissue-specific signals (Fujimura et al., 1981; Sekikawa and Levine, 1981). On the basis of the properties of the white locus DNA sequences described above, we speculate that the recognizably functional elements of the righthand domain of the locus are two or more gene-activator-like elements that regulate the expression of a primary transcription unit corresponding to the lefthand domain of the locus. If this speculation is correct, it suggests that the white locus gene-activator-like elements exert effects in trans (in the special case of somatically synapsed chromosomes) as well as in c/s (Jack and Judd, 1979; Bingham, 1980a). Some alternatives to our speculation are, of course, not excluded by available experimental observations. We further point out that a model of this general form would account efficiently for the results of genetic analysis of the cuf locus of 0. melanogaster (Johnson and Judd, 1979). Finally, we note that the results of the analysis of white (see above) may have a general technical implication. In particular, if, in the analysis of other metazoan loci, attention is exclusively restricted either to tight mutant alleles or to a relatively small sample (less than approximately 15 in the white case) of mutant

alleles, regions functionally analogous to the righthand domain of white may escape detection. Experimental

Procedures

Drosophila Strains For references to the isolation and genetic characterization of most of our sample of white mutant alleles. see Lindsley and Grell (1968). The isolation and genetic characterization of the wDZL allele are described by Bingham (1980a. 1980b). The isolation and genetic characterization of the w”~, w65e25 and w”’ alleles are described by LeFever (1973). In Drosophila genetic terminology the superscript 1 is often used only implicitly: however, we have used this superscript explicitly in several cases to avoid confusion between specific and generic allele designations. Thus, though the white spotted one and white apricot one alleles are often designated wsD and wB, respectively, we have designated them wJp’ and wa’. It has been observed previously that the Amherst strain of D. simulans has fewer copies than D. melanogaster of several D. melanogaster transposons (Meselson et al., 1980; Bingham et al.. 1981). In contrast, we find that the y w strain of D. simulans and the Canton-S and Oregon-F& strains of D. melanogaster have similar numbers of copies of the presumptive transposable elements analyzed here (Figure 3) and of the copia transposon (our unpublished observations). In Situ Hybridization In situ hybridization was performed essentially according to the method of Bingham et al. (1981). We have recently experienced some difficulty in staining polytene chromosomes using some commercially available Giemsa powders or stock solutions and conventional neutral procedures. We find that the following alkaline procedure gives good results even with batches of Giemsa stock that give poor results with neutral procedures. We combined 400 volumes of distilled water, 12.5 volumes of absolute methanol, 1 volume of 0.5% (w/v) sodium carbonate and 10 volumes of commercial Giemsa stock solution. Slides were stained overnight in a sealed container. Slides were destained in water until the appropriate level of staining was achieved. Best results were obtained when the sodium carbonate solution was made freshly: sodium carbonate solutions more than a few days old should not be used. Construction and Screening of Phage Clone Libraries Drosophila DNA (100-300 pg/ml) was digested with Mbo I to a massaverage molecular weight of approximately 15 kb. Mbo I digestion was carried out in 10 mM Tris (pH 7.41, 80 mM NaCI. 10 mM MgC12. 1 mM dithiothreitol and 100 pg/ml gelatin. Enzyme concentrations were adjusted to produce the desired level of digestion in 15-30 min. Mbo I digestion was terminated by heat treatment at 70°C for 20 min. Calf intestinal alkaline phosphatase (Boehringer) was added to a concentration of -1 U/ml, and the reaction mixture was incubated for an additional 30 min at 37°C. The reaction mixture was extracted two times with buffered phenol. Drosophila DNA thus treated was mixed with a two to three fold mass excess of phage vector arms (XMG 14). and the mixture was ethanol-precipitated. The precipitated DNA was washed two times with ice-cold 70% ethanol, and the pellet was allowed to air-dry passively. The pellet (lo-30 @9) was resuspended in 25 ~1 of 10 mM Tris. 0.1 mM EDTA (pH 8.0) by allowing the solvent to stand over the pellet overnight at 4°C. The resuspended pellet was brought to concentrations of 10 mM MgC& and 10 mM dithiothreitol, and 6 U T4 ligase was added. The ligation reaction was allowed to proceed for 4-6 hr at 15°C. and the ligated material was packaged immediately according to the procedure of Scalenghe et al. (1981). Yields of lo5 to lo6 pfu/pg of Drosophila DNA were routinely observed. Phage libraries were screened essentially as described by Benton and Davis (1977). In two cases we encountered results suggesting that individual DNA-sequence-element insertions contained sequences rendering phages carrying them incapable of efficient growth. First, recombinant phage clones carrying the entire wb” element were rare in our library

Cell 540

screens. Of 34 cloned segments homologous to sequences immediately flanking the wb” element insertion (the 1.5 kb Sal I fragment between coordinates -1.3 and -2.7 and the 3.6 kb Sal I-Hind Ill fragment between coordinates -0.4 and +3.3), only two were homologous to both probes. During subsequent purification of these two phages on low-plaque-density plates it became clear that neither formed visible plaques. Control experiments demonstrated that the two phages in question were not homologous to either Ml3 or pBR322 but were homologous to hMG14. We were able to retrieve cloned segments carrying a portion of the w”” element, and one of these was analyzed in detail (see Results). Second, nine recombinant clones carrying the entire wsp55 element insertion as defined by their having homology to both the 3.8 kb Hind Ill-Sal I fragment between coordinates -0.4 and +3.3 and the 2.1 kb Eco RI-Barn HI fragment between coordinates +4.7 and +6.8 were isolated. Each of the nine formed pinpoint plaques afler 46-72 hr of incubation. Attempts to grow two of these in liquid culture or on plate stocks produced very poor yields (less than lOa pfu/ml of lysate), and no attempt was made to characterize any of these cloned segments further. Phage Cloning Vector We have used as a cloning vector hMG14 (M. Graham and M. Olson, personal communication). This vector contains the left arm of h7059 (Karn et al., 19801, the right arm of Charon 30 (Rimm et al., 1960) and a dispensable region consisting of a yeast Barn HI fragment containing numerous Eco RI cleavage sites. This last property allows phage arms to be purified in high yields and purity by sucrose gradient velocity sedimentation after digestion of the phage DNA with Barn HI and Eco RI. hMG14 arms accept DNA fragments whose ends are generated by Mbo I, Sau 3A or Barn HI cleavage. Purification of Drosophila DNA Drosophila DNA was isolated from adult flies as described et al. (1961). Southern Gel Analysis Southern gel analysis was carried out essentially method of Southern (1975) as modified by Botchan

by Bingham

according to the et al. (1976).

DNA Sequence Probes Some of the cloned segments used have been described previously (Bingham et al., 1981; Levis et al., 1962). and others are described in the text. Nick translation of sequence probes for Southern gel analysis, screening of phage libraries and in situ hybridization was carried out as described by Bingham et al. (1961). Acknowledgments We are grateful to A. J. Levine, M. M. Green, W. R. Engels and K. B. Marcu for helpful discussions and to M. Olson and M. Graham for making hMG14 available to us before the publication of its description. This work was supported by funds from the National Institute of Environmental Health Sciences-Intramural Research Program to P. M. B. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

May 5. 1982

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