Cell, vol.

I 8,1303-l

316.

December

1979,

Copyright

0 1979

by MIT

Use of a cDNA Library for Studies on Evolution and Developmental Expression of the Chorion Multigene Families G. K. Sim, F. C. Kafatos, C. W. Jones M. D. Koehler Cellular and Developmental Biology Harvard University Cambridge, Massachusetts 02138 A. Efstratiadis Department of Biological Chemistry Harvard Medical School Boston, Massachusetts 02115 T. Maniatis Division of Biology California Institute of Technology Pasadena, California 91125

and

A cDNA library has been constructed from an RNA preparation highly enriched in silkmoth chorion mRNAs. Many distinct clones have been identified from this library using a stepwise procedure: scoring for infrequent hexanucleotide restriction enzyme recognition sequences; detailed characterization with restriction enzymes that recognize relatively frequent tetranucleotide sequences; probing the arrangement of the corresponding sequences in chromosomal DNA by the Southern procedure; and detailed cross-hybridization analysis. Unique clones, as well as two classes of distinct but related clones, were revealed by hybridization. The crosshybridlzation analysis was greatly facilitated by a newly developed, semiquantitative dot hybridization procedure. The same procedure made it feasible to conveniently estimate the relative abundance of several different sequences in an mRNA mixture. Cloned sequences which scored as relatively abundant in total chorion mRNA were tested with stagespecific chorion mRNA at a very stringent criterion of hybridization. They were thus characterized as early, middle or late sequences with respect to development. The characterized cDNA clones can now be used as probes for studying the evolution, chromosomal organization and regulated developmental expression of the chorion multigene families. Introduction The chorion (eggshell) of the silkmoth Antheraea polyphemus consists of more than 100 different proteins. Each protein is synthesized and deposited by the follicular epithelial cells surrounding the oocyte at a specific time during the 2 day period of choriogenesis, which occurs at the end of oogenesis. In the ovaries of a single animal, eight strings of follicles (ovarioles) are present, each constituting a developmental series of progressively more mature follicles.

The protein-synthetic profile of the follicular epithelial cells at a particular stage of choriogenesis is characteristic for that stage both in vivo and in organ Culture. Thus the expression of the chorion Structural genes at the level of protein synthesis occurs according to a strict developmental program (for review see Kafatos et al., 1977a). Partial protein sequencing data indicate that chorion proteins are encoded by multigene families (Regier et al., 1978a; see also Rodakis, 1978). Within each of the two major size classes of proteins, A and B, individual components show extensive sequence homology. They are clearly distinct, however, as iS evident from internal amino acid substitutions, deletions or insertions. We consider the A and B classes as products of different multigene families, because in the regions sequenced thus far A and B proteins differ substantially from each other. The chorion multigene families are apparently linked (Goldsmith and Basehoar, 1978; Goldsmith and Clermont-Rattner, 1979): genetic analysis in the related silkmoth, Bombyx mori, has permitted mapping of multiple protein variants in three neighboring clusters on a single chromosome (n = 28). We report in this paper the formation of a library of probes for individual chorion mRNAs by molecular cloning of double-stranded cDNA (ds-cDNA). Distinct cloned sequences are selected by restriction endonuclease analysis and DNA blotting-hybridization procedures. These clones are characterized with respect to sequence homologies and expression during development using a newly developed “dot hybridization” procedure. Results Preparation of the Library Our general strategy is summarized in Figure 1. Nineteen distinct clones which were selected and classified according to that scheme are summarized in Table 1. The sequential steps of this scheme are further described below. Poly(A)+ chorion mRNA was prepared from pooled follicles representing all stages of choriogenesis, as described by Efstratiadis and Kafatos (1976) and Kafatos et al. (1977a). In this tissue, approximately 95% of total protein synthesis is chorion-specific; size selection of the mRNA provides additional significant enrichment, since chorion proteins are unusually small. The G+C content of the mRNA preparation is high, as expected for sequences encoding the glytine- and alanine-rich chorion proteins. Its cell-free translation yields almost exclusively identifiable chorion proteins (Kafatos et al., 1977a, 1978; G. Thireoz and F. C. Kafatos, manuscript in preparation). Chorion mRNA was used as template for double stranded cDNA synthesis (Efstratiadis et al., 1976)

Cell 1304

Products having an average size of 550 bp (400-700 bp) were purified by polyacrylamide gel electrophoresis and inserted into the unique Eco RI site of the kanamycin-resistant plasmid pML-21 by the poly(dA)poly(dT) tailing procedure (Jackson, Symons and Berg, 1972; Lobban and Kaiser, 1973; Maniatis et al., 1976). The recombinant molecules were used to transform E. coli strain HBl 01, and a portion of the kanamycin-resistant clones were screened by colony hybridization (Grunstein and Hogness, 1975) using 32P-chorion cDNA as probe. A total of 550 independent clones which scored as positive were preserved as the chorion cDNA library. Individual clones were designated pApC-c (for pML-21 Antheraea polyphemus chorion cDNA) and numbered. For convenience, we will refer to these plasmids as PC. Selection and Characterization of Putatively Distinct Clones by Restriction Endonuclease Analysis Comparison of the size distributions of chorion proteins and chorion mRNAs indicates that most if not all mature mRNAs are monocistronic (Gelinas and Kafatos, 1973; Kafatos et al., 1977a). Since more than 100 different chorion proteins exist, we expect more than 100 different species of chorion sequences among the clones, assuming that the ds-cDNA synthesis and the cloning procedure are nonselective. Our objective was to identify in a manageable way a large number of clones carrying distinct sequences, from a population of several hundred independent transformants, many of which will carry the same sequence.

As a preliminary selection step, we digested cDNA clones with restriction enzymes that recognize hexanucleotide sequences. Such enzymes cleave the parent plasmid only once or a few times. Hence by comparing the cleavage pattern of each hybrid plasmid with that of pML-21, it was possible to determine unambiguously whether a particular cloned insertion carried the hexanucleotide sequence in question (Figure 2a). This analysis was based on the presence or absence of a particular recognition sequence rather than on its frequency of occurrence, since multiple closely spaced sites within the insertion would generate fragments which might not be detectable on the 1% agarose gel used in this experiment (because of the small amount of DNA used and the porosity of the gel). By scoring 78 clones with a number of different enzymes, we classified their insertions into 14 groups, each characterized by the presence of a different set of restriction sites. Although it is obvious that each group may contain a number of different sequences, we selected only one or two members of each group, and thus limited further analysis to a small but presumptively diverse sample of nineteen clones. As a second step, we analyzed the patterns of restriction fragments generated by digestion of the selected clones with enzymes that recognize tetranucleotide sequences and hence cleave at higher frequencies. The enzyme Hha I proved particularly useful for our purposes because it cleaves the cloned cDNA sequences only moderately frequently, and because of the relatively large size of the fragments flanking the Eco RI site into which ds-cDNA was inserted (Figure 2b). In a blot hybridization pattern (Southern, 1975) of Hha l-digested hybrid plasmid DNA hybridized with 32P-chorion cDNA, we were able to identify all but the smallest fragments bearing chorion mRNA sequences (Figure 3b). The “junction fragments” carrying both insertion and pML-21 sequences, linked by poly(dA)-poly(dT) bridges, were identified by size directly from the restriction pattern (Figures 2b and 3a); the identification was confirmed by Southern hybridization with 32P-poly(rA) (Figure 3~). Identification of both junction and internal fragments permitted sizing of the insertions and their classification according to the absence or presence and distribution of Hha I sites. Corroborative experiments were performed with Alu I, Hinf I and Hae Ill (data not shown). Insertions which differed in terms of hexanucleotide and/or tetranucleotide recognition sites, and which were also comparable in size to the chorion mRNA population, were considered distinct (Table 1). Although it is theoretically possible that two apparently unique sequences in fact correspond to overlapping partial segments of the same sequence, truncated during ds-cDNA synthesis, this possibility did not materialize among these clones (see below). It is important that the estimated total sizes of the insertions (Table 1) are comparable to the sizes of chorion

cDNA 1305

Library

of the Chorion

Table

1. Summary

Multigene

of Identification

Families

of 19 Distinct

Clone

W

Infrequent

I

pc8

680

Sst I

PC7

640

Sst I

~~406

600

Sst I

PC10

560

Sst I

PC9

550

Km

I

PC401

630

Kw

I

pc602

480

PC405

760

Sst I

pc809

780

Sst I

pc607

730

Sst I

Kpn I

PC403

730

Sst I

Kpn I

~~601

450

pc402

650

PC409

575

Kpn I

pcl8

740

Kpn I

PC11

650

Unique

Clones Number of Hha I Sites”

Size

Hybridization Class

II

cDNA

Restriction

Sites

Kpn I

Main Distinguishing Features

2 2

Kpn I

Bgll

2 b

1 Bgl1

0 Hind Ill

1

Kpn I

Hind Ill

1

Kpn I

Hind Ill

i

1 Hind II

Barn HI

1

Bgl I

c

1 Hind II

Bgl I

Hind II

Eco RI

Hind Ill

Pst I Xho I

1

I

0

’

0 0 1

Hind II

No CrossHybridization

0

~~605

475

PC4

630

Eco RI

1

PC404

670

1

0

I

a When 1 or 2 is listed as the number of Hha I sites, this represents a minimum estimate; very closely spaced sites (cl 00 bp) are scored as single sites under our conditions. b Within this class, all clones can be easily distinguished from each other by the extent of cross-hybridizations, except for two pairs, pc8/pc408 and pc401 /pc602 (Figure 7). In addition, pc8 and pc7 are distinguished from each other by a Kpn I site. pc408 differs from both of the above in the size of its internal Hha I fragment (280 bp rather than 220 bp). and in having one or two additional infrequent restriction sites. pcl0 differs from all three of the above by the lack of a detectable internal Hha I fragment; it also differs from pc8 and pc408 by lacking one or two infrequent restriction sites. pc9 differs from all four of the above by the lack of Hha I and Sst I sites, and from all but pc408 by its combination of Bgl I and Kpn I sites. pc401 differs from all five of the above by its Hind Ill site, and from the first four by its lack of an Sst I site. pc602 is very similar to pc401 according to all criteria; it differs from it in the blot-hybridization pattern of chromosomal DNA (Figure 4) and in lacking an Hpa II site located between restriction sites which are represented in both ~~401 and pc602 (A. Ephrussi. unpublished observations). c Within the cross-hybridization Class II, pc405 and pc609 are distinguished from each other by a total of four infrequent restriction sites, and by a low degree of cross-hybridization (Table 2). pc807 is distinguished from the above by the fact that it cross-hybridizes extensively with both; it also differs from pc405 by a total of two infrequent restriction sites, and from pc809 by a total of three infrequent restriction sites. pc403 differs from the other members of the class by two infrequent restriction sites in each case: it is distinct from pc405 and pc609 on the basis of low crosshybridization, and from pc607 since pc607 but not pc403 cross-hybridizes extensively with the other two members of the class.

mRNAs (Kafatos et al., 1977a). As a consequence, although the insertions may be missing short sequences corresponding to one or both mRNA ends, it is improbable that these missing sequences would contain an infrequent site. The probability is even lower when two insertions differ by two or more infrequent sites. By similar arguments, we may consider different from each other the following groups of sequences: (1) those which are not susceptible to Hha I; (2) those which have only one, often centrally located Hha I site [or multiple but closely spaced (Cl 00 bp) sites which are not revealed by this analysis]; and (3) those with two or more dispersed Hha I sites which are revealed by the present analysis. Thus clones pc7, pc8 and pc408 differ from the remaining 16 clones in the characterized set by having a detectable

internal Hha I fragment (Figure 3b). Among the three, pc408 is distinguished by an internal fragment which is larger than those of pc7 and pc8. Comparison of cDNA Clones with Respect to Cross-Hybridization and Blot-Hybridization Patterns of Chromosomal DNA In conjunction with the results of restriction analysis, cross-hybridization experiments provided unambiguous evidence for the distinctness of the nineteen selected clones. Moreover, the experiments permitted characterization of the clones according to their sequence homology. To detect sequence homology and localize it to particular regions of the sequences, the insertions of cDNA clones were excised by Sl treatment (Hofstetter

Cell 1306

pML 21 Hind Ill

pML 21 Hind Ill

pc IO Hind Ill

pc 401 Hind Ill

pML 21 Hha I

pML 21 Hha I

pc IO Hhal

PC 9 Hha I

1850 O-

70 O60 O-

Figure

2. Restriction

Cleavage

Analysis

of cDNA

Clones

(a) Scoring of cDNA clones for the presence or absence of an infrequent restriction endonuclease site within the cDNA insertion (Hind ill, hexanucleotide recognition sequence). The cloning vector pML-21 is cleaved once by the enzyme Hind Ill to generate a single band-the linearized form of the plasmid. A single band is also generated from hybrid plasmid clones if the chorion insertion lacks a Hind Ill site, as in pcl0. If the chorion insertion is susceptible to Hind Ill, two fragments of lower molecular weights are generated, as in ~~401, These fragments are comparable in size to those generated by combined treatment of pML-21 with Hind Ill and Eco RI. as expected from the fact that the small dscDNA was inserted in the Eco RI site of the parent plasmid. The bands were visualized by staining with ethidium bromide and photographed under short wavelength ultraviolet light. (b) Characterization of the fragments generated by Hha I, an enzyme that recognizes a tetranucleotide sequence. The fragments were resolved on a 1.4% agarose gel and stained with ethidium bromide. The largest (1300 bp) Hha I fragment of PML-21 is shown by combined Hha I-Eco RI digestion to contain the Eco RI site used for insertion of the ds-cDNA. In the absence of an Hha I site in the ds-cDNA insertion, as in pc9. a fragment larger than 1300 bp is observed. If the insertion carries Hha I site(s). as in pcl0, two new fragments, larger than 600 bp and 700 bp, respectively, are present (arrows). These features make Hha I a particularly useful enzyme for analysis of pML-21 hybrid clones.

et al., 19761, labeled with 32P by nick translation and used as hybridization probes against restricted and blotted DNA from all 19 clones. The results are summarized in Table 2; some of the autoradiograms have been presented previously (Maniatis et al., 1977; Sim et al., 1978). Insertions from eight of the nineteen clones failed to cross-hybridize detectably with any other clone, even at low criterion, and were thus clearly unique (Table 1). Insertions from seven other clones (Class I) showed varying degrees of crosshybridization. Finally, another four clones (Class II) cross-hybridized with each other, but not with any of the other clones. In the accompanying paper (Jones et al., 1979) we show that Classes I and II encode B and A chorion proteins, respectively. As Table 2 shows, the pattern of cross-hybridization at two criteria indicated that various clones within each class differ in the extent of their relatedness. Moreover, localized sequence homologies were suggested by the observation that not all fragments of each cDNA insert cross-hybridize equally well to other clones. Interpretation of this observation is, however,

complicated by the possibility that some hybrids are unstable at a highly stringent criterion because of short length rather than because of sequence mismatching; that is, the apparent sequence homologies may be influenced by the exact locations of Hha I sites within the insertions. Similarly, the distribution of sites may affect the relative prominence of specific hybridization, due to the moth DNA probes, above the inevitable background due to pML-21 sequences contaminating the probe (it is difficult to obtain completely pure probes by Sl excision, presumably because of the existence of AT-rich regions within the vector). Distinctness and differences in relatedness were also documented by hybridization of the clones to restricted and blotted moth chromosomal DNA. A particularly informative experiment, using six cross-hybridizing Class I clones, is shown in Figure 4. A restriction enzyme which cuts some but not all of the cloned sequences was used, and hybridization was performed at a criterion low enough to permit crosshybridization. As expected, a number of chromosomal DNA bands hybridized with all the probes. Clear dif-

cDNA

Library

of the Chorion

Multigene

Families

1307

ferences were, however, evident in the intensity of a number of bands. Thus band F was particularly prominent with probes pc9, pc401 and ~~602, which were distinguished from each other by the intensities of bands A and H (high in pc9 and ~~401) and band G (high in pc401 and ~~602). Clones pc8, pc408 and pcl0 proved to be a separate subgroup of closely related sequences, characterized by the relative prominence of bands B-E. Homology Relationships Analyzed by Dot Hybridization We used a convenient and semi-quantitative dot-hybridization procedure (Kafatos et al., 1978; Kafatos, Jones and Efstratiadis, 1979) for evaluating in detail the extent of sequence homology between clones belonging to Class I. In these experiments the problems of variable length were reduced, since the cDNA insert was maintained intact. Briefly, multiple samples of cloned DNAs, identical in amount, were spotted adjacent to each other on a filter, in dots of uniform diameter. The filter was then hybridized with 32PcRNA, synthesized by E. coli RNA polymerase using as template the Sl -excised insertion of a single cDNA clone. Consistent results were also obtained using excised and nick-translated individual DNA inserts directly as probes (data not shown). The extent of similarity was evaluated either by hybridization of a single filter followed by stepwise melting and autoradiography or by hybridization of a series of such filters at various temperatures. Typical results of the two methods are presented in Figures 5 and 7. Because of our primary interest in RNA-DNA hybridizations at high criterion (see below), we chose to perform all reactions in the presence of formamide. Our standard hybridization buffer contained 50% formamide and 0.6 M NaCI; the melting buffer contained 50% formamide and 0.3 M NaCl. Figure 5 presents the results of a hybridization-andmelting experiment using pc408 cRNA as the probe. At 50°C the cross-hybrids with pcl0 and pc401 DNA were less intense than the pc408 self-hybrid, but significantly above the background control (pML-21 vector DNA). Subsequent washing at 63°C resulted in disappearance of the pc401 hybrid and partial melting of the pcl0 hybrid. Even the pcl0 hybrid disappeared after melting at 71 “C, leaving only the self-hybrid detectable. Clearly, pcl0 is more related to pc408 than is ~~401. In Figure 6, a similar experiment was analyzed quantitatively by excising the dots following hybridization, melting the hybrids individually at progressively higher temperatures, and determining the proportion of cRNA that melted off at each step using liquid scintillation counting. Reproducible results were obtained in three such experiments. The melting profiles were steep (Kafatos et al., 1979). After correction for length and conversion to 0.6 M NaCI, the T, of

pc408 cRNA hybridized with pc408 DNA was calculated as approximately 72’C. Relative to this pc408 self-hybrid, the AT, of the cross-hybrids was 1.21.5’C for pc8, 6-7°C for pcl0 and 10°C for ~~401. Under the conditions of these experiments, which did not saturate the DNA dots (see legend to Figure 61, the extent of hybridization also differed for the various clones: considering the extent of self-hybridization of pc408 as 1 OO%, cross-hybridization was 66-88% for pc8, 21-32% for pcl0 and 13% for ~~401. Thus according to both extent of hybridization and T,, the degree of similarity to pc408 is pc8>pclO>pc401. This conclusion is in full agreement with partial sequence comparisons of these clones (Jones et al., 1979); it is also consistent with Figure 4. Figure 7a illustrates results of the alternative type of experiment: replicate hybridizations under conditions ranging from very permissive to very stringent. In this case, filters carrying dots of DNA from twelve chorion cDNA clones, and pML-21 DNA as control (Figure 7b), were hybridized with cRNAs at various temperatures, from 40’ to 64°C (approximately 32” to 8°C below the T, of the self-hybrid). The existence of sequence homology between all seven members of Class I (Table 1) was apparent in the 40°C experiment using pc401 cRNA. With this probe, it was clear that pc602 is the clone most closely related to ~~401, both according to the intensity of the cross-hybrid and by its formation even at high criterion (58” and 64°C hybridizations); it was also evident that the pc401/ pc602 pair shares greater sequence homology with pcl0 than with the remaining Class I clones (50°C hybridization). On the other hand, pcl0 is more closely related to pc408 and pc8 than to pc401 and pc602 (40’ to 58°C hybridizations using pcl0 cRNA). Finally, the hybridizations using pc408 cRNA confirmed that pc408 is very similar to pc8, and more similar to pcl0 than to pc401 and ~~602. These relationships are schematized in Figure 7c. Of the remaining five clones in Figure 7, four (~~405, ~~403, pc607 and pc609) are members of the separate cross-hybridizing Class II. As expected from Table 2, none of these clones detectably hybridized with Class I cRNAs. The last clone, pc18, is a member of the “unique” class (Table 1): it does not cross-hybridize with any other clone. This is particularly interesting since, like Class II, pcl8 encodes an A-type chorion protein (Jones et al., 1979). From these experiments, it is clear that many distinct chorion sequences can be assayed individually by dot hybridization, provided that a sufficiently high criterion is used. Some pairs of sequences (~~401 and ~~602; pc408 and 1~81, however, are so similar (AT, of lo to 2°C) that they must be assayed together. Developmental Characterization of cDNA Clones Reconstitution experiments have demonstrated that the relative concentrations of multiple RNA sequences

Cell 1308

cDNA

Library

of the Chorion

Multigene

Families

1309

Table

2. Cross-Hybridization

of Chorion

cDNA

401

Clones

9

IO 8 602 406

& D

PCW DNAs from the nineteen selected hybrid clones and the parent plasmid were restricted with Hha I. fractionated in parallel on a 1.4% agarose gel (compare Figure 3a) and transferred to nitrocellulose filter (Southern, 1975). A series of identical filters were then hybridized with sixteen different 32P-labeled insertions, excised (Hofstetter et al., 1976) from the respective clones; three insertions (pc6. pc403 and ~~607) could not be easily excised and used as probes, presumably because of short poly(dA&oly(dT) tails. Hybridizations were carried out in 4 x SSC at 65°C (low criterion, L). After the first set of autoradiograms were developed, the filters were washed in 2 x SSC, 50% formamide, 65’C (high criterion, H) and new autoradiograms were prepared. Dashes in the first column represent the moth DNAcontaining fragments generated by Hha I digestion of various cDNA clones: corresponding dashes in other columns indicate which of these fragments detectably cross-hybridize with each probe at each criterion (pc7 to pc609. L and H columns). pc403 has two Hha I fragments of comparable length, which were not resolved in this experiment. Probes derived from eight clones (~~601, ~~402. pc409. pcl8, pcl I, ~~605. pc4 and ~~404) failed to cross-hybridize with any other clone and are not shown in this table. Self-hybridization is indicated by slanted dark lines. The pattern of cross-hybridization defines the classes of clones, I and II.

in a mixture can be estimated by dot hybridization (Kafatos et al., 1979). This approach was used to determine the developmental stages during which various cloned sequences are represented in cytoplasmic mRNA. Choriogenic follicles are arranged in a developmental progression in each of eight ovarioles in the moth (Kafatos et al., 1977a) and can be obtained sequentially by dissection. For absolute staging of the follicles, however, it is necessary to evaluate their Figure

3. Identification

and Sizing

of Chorion

DNA-Containing

Fragments

E

Figure

4. Southern

Images

of Chorion

Genes

in the Moth Genome

DNA from whole pupae was restricted with Hind Ill. fractionated in a wide slot of a 1% agarose gel and transferred to a nitrocellulose filter. Strips of 6 mm width, each containing 10 pg DNA, were cut from the filter and hybridized to nick-translated “P-DNA from each of six Class I clones (spec. act. IO’ cpm/pg). Hybridizations were carried out in 30% formamide at 50°C in 4 X SSC (0.6 M NaCI, 0.06 M NaCitrate) for 36 hr and the filters were washed in 2 x SSC, 50% formamide at 55°C before exposure. A-H identify bands which hybridize to different extents with different clones.

protein-synthetic profiles, since different different numbers of choriogenic follicles (Paul and Kafatos, 1975). From each animals, the follicles of one ovariole were in pApC-c

animals have per ovariole of fourteen labeled with

Clones

(a) Ethidium bromide-stained pattern of 19 different pApC-c clones cleaved by the restriction enzyme Hha I and electrophoresed in parallel on a 1.4% agarose gel. Each lane contained -1 fig of DNA. (b) An autoradiogram of a duplicate of (a). transferred onto nitrocelulose filter and hybridized to 32P-labeled chorion cDNA in 4 x SSC at 65’C for 16 hr. All the junction chorion/pML-21 fragments hybridize; the only exception is the lower junction fragment of pc401 (arrow in Figure 3a). which is known by sequence analysis to contain only 30 bp of chorion sequence (Jones et al., 1979). In addition, arrows indicate internal Hha I fragments (220-260 bp) derived from chorion DNA insertions. Some of the junction fragments are present as diffuse rather than sharp bands as a consequence of heterogeneity in the length of the poly(dA)-poly(dT) linkers that arose with many generations of propagation. (c) An autoradiogram of an experiment similar to (b). except that 32P-labeled poly(rA) was used as probe and hybridization was carried out at low criterion (6 X SSC at 42°C) for 24 hr. The junction fragments hybridize; dots identify pairs of fragments with similar mobility, as well as the weakly hybridizing lower fragment of pc401 which is known to contain a poly(dA)-poly(dT) joint only approximately 30 bp long (C. W. Jones, unpublished observations).

Cell 1310

Figure 5. Discrimination between Cross-Hybridizing Dot Hybridization and Stepwise Melting

Sequences

by

DNAs from linearized clones ~~401, pc406 and pcl0 and pML-21 DNA as control were spotted on filters. The probe was cRNA synthesized on Sl-excised chorion DNA from clone ~~406. Afler 12 hr of hybridization at 50°C (in the standard hybridization mixture of 50% formamide. 0.6 M NaCI; Kafatos et al., 1979) replicate filters were washed and autoradiographed (top, left and right). One filter was then melted at 63°C (50% formamide, 0.3 M NaCI) and autoradiographed. melted again at 71 “C and autoradiographed once again (bottom, left and right, respectively). Note that the pc401 cross-hybrid disappears first, and then the pcl0 cross-hybrid.

30

20

IO

50

55

60

65

Temperature 3H-leucine for 1 hr, while those of the other seven were pooled by rank position and frozen. The newly synthesized proteins of the labeled follicles were analyzed by polyacrylamide slab gel electrophoresis followed by fluorography: each resulting “synthetic profile” revealed the absolute developmental stage of that follicle position in that particular animal (Paul and Kafatos, 1975). Figure 8 shows the synthetic profiles of a series of follicles, corresponding to all the developmental stages of choriogenesis. Follicles from the various animals were then combined by absolute developmental stage and homogenized; mRNA was prepared from the cytoplasmic fraction by Mg’+ precipitation (Palmiter, 1974) followed by phenol-chloroform extraction and oligo(dT)-cellulose chromatography (Efstratiadis and Kafatos, 1976). The mRNA was fragmented with alkali and end-labeled with Y-~‘P-ATP. Seventeen radioactive RNA probes corresponding to the seventeen protein synthetic stages were thus generated. Identical filters were prepared, each bearing DNA dots from twelve distinct clones (including two not yet described, pcl4 and ~~271) which were judged to be relatively “abundant” by a dot-hybridization experiment using mRNA from pooled stages of development (Kafatos et al., 1979). Each filter was then hybridized with a different developmental stage-specific mRNA probe and the hybridization profiles were determined by autoradiography. The experiment was performed twice, at a moder-

Figure 6. Melting Profiles Chorion Sequences

70

75

(‘C)

of Self- and Cross-Hybrids

between

Cloned

Dots of DNA from linearized chorion cDNA clones were hybridized with “P-cRNA synthesized from the Sl -excised chorion sequence of pc406 (-250 NT average length). Hybridization (24 hr at 50°C) and melting of the hybrids was performed as in Figure 5 (Kafatos et al., 1979). The percentage of radioactivity released at each melting step was evaluated by liquid scintillation. Similar results were obtained in three experiments (50” and 55’C hybridizations). In the experiment shown here, the extent of hybridization with ~406 DNA was 754 cpm (Cerenkov; -0.09 ng. or 0.4% of saturation, since 3% of the pc406 DNA is chorion anti-message strand); hybridization with pc6 DNA was 500 cpm. with pcl0 DNA 156 cpm and with pc401 101 cpm. The hybridization mixture (2.5 ml) contained -50 ng “P-cRNA. and the hybridizable filter-bound DNA (anti-message strand of all four clones) was -100 ng.

ately high and a very high criterion (59’ and 64’C, or approximately 13’ and 8°C below the T, of selfhybrids). Figure 9 presents the results. Clearly, it is possible to categorize chorion sequences according to when during development they are represented in mRNA. Although temporal specificity can be seen at both temperatures, it is most evident at the higher criterion. pc271 is an early, only moderately abundant sequence. It appears as early as Stage la, and is most prominent at Stages lb to II. A second group, ~~403, ~~607, pc609, pcl0, pc8 and ~~408. are middle sequences, being most prominent at Stages Ic to IV, or even later (~~403, ~~10); within this group, pc8 and ~408 appear to be slightly earlier. A third group,

cDNA

Library

of the Chorion

Multigene

Families

1311

Figure 7. Discrimination between Sequences of Varying Similarity by Dot Hybridization at Different Criteria Sixteen replicate filters, each bearing DNA dots from twelve hybrid clones plus pML-21, arranged as shown in (b). were hybridized for 38 hr (in 50% formamide, 0.6 M NaCI). For each filter, a unique combination of temperature and probe was used, as indicated: all probes were cRNAs synthesized on Sl-excised chorion sequence from cDNA clones. The autoradicgrams are shown in (a). pc18, which encodes an A-type protein (Jones et al.. 19791, does not cross-hybridize at any criterion, even with the Class II clones ~~405, ~~403, pc807 and pc609 (Table l), which also encode A-type proteins (Jones et al., 1979). The experiments with cRNAs of ~~401, pcl0 and ~408 show the varying degrees of cross-hybridization between the seven clones of Class I (Table 1). which encode B-type chorion proteins (Jones et al., 1979). The inferred degrees of sequence homology between these clones are diagrammed in (c) in the form of a dendrogram; the lengths of the lines are arbitrary, but their relative positions indicate the degrees of relatedness. Dotted lines indicate that the sequence homology between pc7 and pc9 is unknown, since neither one was used as a source of cRNA; it is clear, however, that both of these clones are relatively distant from the other five (see the 40’ and 50°C hybridizations). The results are also consistent with Figure 4.

~~401, ~~602, pc9 and pcl6, are late sequences: they first become prominent at about Stage le to II, peak at approximately Stages VI to VIII and remain relatively abundant until nearly the end of choriogenesis, while the middle sequences become undetectable. Within this group, pc9 may be slightly later than the rest. pcl4 hybridizes throughout most of choriogenesis. Subsequent experiments showed, however, that it is a double transformant, containing two different recombinant plasmids, which are distinguished by the presence or absence of Hha I sites in their respective inserted sequences. One of these is a middle and the other a late sequence (data not shown). At least 200 fold changes in relative abundance can be observed across development. For example, relative to ~~401, the intensity of pc271 is approximately 4 fold higher at Stage lb, but at least 50 fold lower at Stage VI. Some of the developmental changes in mRNA concentration are notably rapid. Most of the early and middle sequences appear within the 3 hr separating Stages la and lb (Paul and Kafatos, 1975). Conversely, the late sequences disappear within the 3 hr separating Stages Xc and Xd.

Discussion General Strategies for the Construction and Screening of a cDNA Library In the 4 years since its introduction, cloning of dscDNA has become a widely used technique for obtaining DNA corresponding to individual mRNA species. The utility of this procedure was initially demonstrated by the purification of a few very abundant sequences, such as the mRNAs for globins or ovalbumin (Rougeon, Kourilsky and Mach, 1975; Higuchi et al., 1976; Maniatis et al., 1976). This report confirms the expectation (Kafatos et al., 1977b; Maniatis et al., 19771, based on the very nature of the procedure, that cDNA cloning can be used to generate an entire library of clones, each representing in homogeneity individual sequences, even homologous or relatively rare ones, from an initially very complex mRNA mixture. Clearly, generation and characterization of a cDNA library is an appropriate first step in the analysis of developmentally regulated sets of genes, including multigene families. The small family of vitellogenin genes has been recently studied by procedures similar to those used here (Wahli et al., 1979).

Cell 1312

Figure

8. Changing

Patterns

of Chorion

Protein

Synthesis

during

Development

Protein-synthetic profiles are presented, corresponding to all seventeen stages of choriogenesis. plus the last prechoriogenic stage (0). A single ovariole (from one of the animals also used in Figure 9) was labeled for 30 min in culture with 3H-leucine (80 Ci/mmole). Sequential follicles were then deyolked. dissolved in sample buffer and analyzed by SDS-polyacrylamide slab gel electrophoresis. The fluorogram shows the newly synthesized proteins at each developmental stage. Stage identifications are consistent with those previously based on gels of lower resolution (Paul and Kafatos. 1975); (+) and (-) represent late and early phases, respectively, for each stage. The A, 6, C and D classes of chorion proteins have been defined previously (Paul and Kafatos, 1975). El and E2 are a newly recognized class of chorion proteins (Regier, Mazur and Kafatos. : 980). Numbers identify specific bands within the A, S and C classes

Many variations are possible for each step of constructing a cDNA library (for a recent review, see Efstratiadis and Villa-Komaroff, 1979). With respect to screening, a scheme such as that summarized in Figure 1 is very convenient for recovery of a large number of distinct clones. In addition to scoring for infrequent restriction sites, other preliminary screening steps may be used-for example, parallel colony hybridizations with probes derived from different developmental stages, if one is interested in sequences which are uniquely expressed at a specific stage. After the initial screening step(s), distinctness can be firmly established by detailed restriction and hybridization analyses. Comparison of blot hybridization patterns of chromosomal DNA is a convenient general method for recognizing uniqueness of cDNA clones, whereas cross-hybridizations are particularly important for analysis of multigene families. All the tests for distinctness are complicated by the possibility that apparently different clones may be partials of the same sequence. This is one important reason why the cloned ds-cDNA must be as comparable to the mRNA length as possible. In addition to optimizing the reactions for full length ds-cDNA syn-

thesis (Efstratiadis and Villa-Komaroff, 19791, it is also possible to size-select the ds-cDNA (as we have done) before tailing, if the desired sequences fall in a known and narrow size range. Sequence Homologies of Cloned Chorion cDNAs A large number of distinct cDNA clones have been characterized in this paper. Eleven of these can be assigned to two classes by cross-hybridization analysis (Table 2). Additional clones are unique-that is, do not detectably cross-hybridize with other clones in this set; the unique category includes the eight clones identified in Table 1, plus pc271 (our preliminary unpublished observations). The accompanying paper (Jones et al., 1979) shows by DNA sequencing that five members of the cross-hybridization classes encode chorion proteins of the two major families, A and 6. Because of their relatedness, we conclude that all eleven clones in the cross-hybridization classes correspond to chorion mRNA sequences. In addition, one of the unique clones (~~18) has also been shown to encode an A protein (Jones et al., 1979). Although we do not have formal proof that the remaining unique clones also represent chorion sequences, this is prob-

cDNA

Library

of the Chorion

Multigene

Families

1313

Figure 9. Changing Abundance Chorion RNA Sequences during

of Specific Development

Abundance of specific sequences in stagespecific RNA was evaluated at two criteria (59” and 64’C hybridizations in 50% formamide, 0.6 M NaCI). Multiple replicate filters were prepared with dots of DNA from twelve clones. The arrangement of DNA dots and the clones from which they were obtained are indicated at bottom right, together with the autoradiogram of such a filter hybridized with pML-21 “P-DNA to visualize the dots. In the 59OC experiment, pML-21 DNA was spotted instead of pc271 DNA. as a control. RNA was prepared from the cytoplasm of staged follicles (Stages la-Xd; compare Figure 6) by Mg2+ precipitation, phenol-chloroform extraction and binding to oligo(dT)-cellulose. and was end-labeled with r-“P-ATP after alkali fragmentation. Individual filters were hybridized with the stage-specific probes for 26 hr (59°C experiment) or 40 hr (64°C experiment), washed and autoradiographed. Exposures were adjusted to compensate for slight differences in the radioactivity of the probes or their contamination with rRNA (Efstratiadis and Kafatos. 1976); the selection of exposures was such as to make the intensities of each dot monotonically variable across development. The results were evaluated by visual comparison to the autoradiogram of a 2 fold dilution series of 32P-DNA directly spotted on nitrocellulose (Standard). Clones can be easily classified as early (pc271), middle (~~403. pc607, pc609, pcl0. pc8. ~~408) or late (pc401, ~602, pc9. pd 8) sequences. ~14 hybridizes throughout most of choriogenesis because it isadouble transformant, containing one middle and one late sequence (data not shown). Note the increased temporal specificity of specific clone hybridization in the 64’C versus the 54“C experiment, and the more extensive differences in intensity between clones which are known to cross-hybridize, such as ~401 and pc9.

able, considering the purity of the starting mRNA preparation. Thus cDNA clone8 from our library can now be used for detailed studies on evolution (Jones et al., 1979) and genomic organization of the chorion multigene families. The sequence similarities and compositional bias of chorion proteins (which are unusually rich in glycine, alanine and cysteine; Kafatos et al., 1977a) are evident even at the level of restriction analysis. The cloned cDNA sequences appear to be rich in the usually infrequent Kpn I and Sst I recognition sites: of the 76 clones tested, 43 have Kpn I site8 and 33 have Sst I sites; 23 have both Kpn I and Sst I sites. In contrast, none of the insertions are cleaved by Bgl II. Pst I and Xho I cleave only one insertion each, while both Barn HI and Eco RI cleave two. Clones of Class l-that is, those encoding 6 pro-

teins-show a wide range of sequence homologies. Some of them (~~401 vensus ~~602; pc6 versus pc406) are so similar that they can be distinguished only with difficulty, whereas others (for example, pcl0 and PC406 versus pc7 and pc9) show little crosshybridization (Figure 7). Assuming that sequence homology reflects evolutionary relatedness (Jones et al., 1979). it appears that the B family includes both recently and anciently diverged members. Sequence divergence within the A family may be even more extensive (Table 2 and our unpublished observations). Unique clones encoding chorion proteins may represent gene8 which evolved long ago, rapidly or from an origin independent from the rest. Comparisons between different taxa suggest that insect chorion proteins are very diverse (Kafatos et al., 1977a). Alternatively, the homologues of unique clones may

Cell 1314

not have been discovered as yet, either because they belong to small subfamily branches or because they are only expressed at a low level at all stages of choriogenesis combined. The former explanation may be applicable for the abundant pcl8 sequence, homologues to which have been recently found by further screening of the cDNA library (S. G. Tsitilou and J. C. Regier, unpublished observations); the latter explanation may be valid for pcl 1, ~~404, ~~405, pc409, pc601 and ~~605, which have been shown to represent rare sequences (Kafatos et al., 1979). Developmental Expression of Distinct Chorion Sequences The experiments reported in this paper demonstrate the feasibility of discriminating between all but the most closely related sequences by dot hybridization. We have characterized some relatively abundant cloned sequences with respect to timing of accumulation of the corresponding RNA in the cytoplasmic, polyadenylated mRNA fraction during choriogenesis. It should now be feasible to use pulse-labeled nuclear RNA in similar experiments to determine whether the developmental pattern of accumulation is controlled at the level of transcription. Within the accuracy of the experiment shown in Figure 9, three major groups of sequences can be distinguished: early, middle and late. Comparison with the protein-synthetic profiles (Figure 8) reveals correspondingly timed protein groups. A fourth, very late protein group (exemplified by the identified El, E2 and C3 proteins of Figure 8) has no parallel among the “abundant” cDNA clones characterized to date. The proteins encoded by cDNA clones have been determined by cell-free translation of specifically hybridized chorion mRNA (G. Thireos, personal communication). pcl8 has thus been shown to encode an Al protein, pc609 an A4, pcl0 a Bl or 82 and pc401 a B6. Figure 8 shows that the corresponding protein bands are synthesized in vivo within the time periods that would be predicted from the developmental dot hybridizations (Figure 9); the major Al and B6 bands are late, like pcl8 and ~~401, whereas the major A4 and 81-82 bands are characteristic of mid-choriogenesis, like pc609 and pcl0. Although each of the bands in Figure 8 contains multiple components, it would appear from this comparison that polyadenylated chorion mRNAs accumulate in the cytoplasm as they are needed, rather than being nonspecifically accumulated at the outset and then sequentially activated at the translational level. This conclusion is in agreement with an earlier study of much lower resolution (Gelinas and Kafatos, 1977). Experimental

Procedures

Biohazard Containment This work was performed in compliance ments set forth by the NIH Guidelines.

with the PP-EKl

require-

Bacterial Strains The bacterial strains used here are E. coli K12 (hsm-, 1969) gal-. proD. St?‘; Boyer and Roulland-Sussoix. (pML-21: Hershfield et al., 1974).

hrs-. and

recA-, HBlOl

Preparation of Total and Staged Choriogenic Follicles For preparation of stage-specific mRNA. follicles were dissected from developing moths of Antheraea polyphemus and pooled according to their rank position within the ovariole, between the positions corresponding to the end of vitellogenesis (which precedes choriogenesis) and ovulation (which follows the end of choriogenesis; Paul et al., 1972). Seven of the eight ovarioles of each animal were used for preparation of RNA. The eighth was used for determination of absolute stage (Paul and Kafatos, 1975); it was incubated for 30 min in medium containing ‘H-leucine. the follicles were collected individually, cut with scissors to remove the oocyte, dissolved and subjected to gel electrophoresis in a slab gel as described (Regier et al., 1978b). Newly synthesized proteins were detected by fluorography (Banner and Laskey, 1974). Alternatively, for preparation of the total chorion mRNA used in ds-cDNA synthesis, choriogenic follicles were pooled without staging. Construction of Hybrid DNA Molecules Chorion mRNA was purified from the cytoplasmic fraction of follicular cells as described, by Mg’+ precipitation. phenol-chloroform-isoamyl alcohol extraction, binding to oligo(dTI-cellulose and sucrose-gradient centrifugation (Efstratiadis and Kafatos. 1976). Our procedures for synthesizing full-length ds-cDNA and constructing hybrid molecules via the poly(dA>poly(dT) tailing procedure have been published (Efstratiadis et al., 1976; Maniatis et al., 1976). The only modification was that the addition of homopolymers was carried out under the conditions of Roychandry. Jay and Wu (1976). and therefore the X exonuclease treatment was omitted. We aimed at adding an average of 150 nucleotides to each 5’ end. After Sl treatment and prior to tailing, the ds-cDNA was gel-purified to eliminate small (