Localization of a gene for a minor chorion protein in Drosophila melanogaster: A new chorion structural locus

DEVELOPMENTAL

BIOLOGY

102.504-508

(1984)

BRIEF NOTES Localization of a Gene for a Minor Chorion Protein in Drosophila melanogastec A New Chorion Structural Locus CLAUDINE ZIGHERA YANNONI’ AND WILLIAM H. PETRI Department of Biology, Boston College, Chestnut Hill, Massachusetts 02167 Received January 4, 1983;accepted in revised form October 24, 1988 A minor chorion protein (called ~‘70)with an approximate molecular weight of ‘70,066D has been characterized in Drosophila melanogaster. The Staket geographic strain was found to carry an electophoretic variant of this eggshell component and was used to determine the chromosomal location of the ~70 gene. Our results establish a new locus for a chorion gene near yellow on the X chromosome and represent the first mapping of a quantitatively minor eggshell protein. INTRODUCTION

The Drosophila melanogaster eggshell system offers an excellent opportunity to study and understand the temporal and functional organization of a complex developmental program. Elucidation of the control mechanisms in the eggshell system requires knowledge of chromosomal locations of as many eggshell structural genes as possible. Over 15 putative chorion proteins have been described (Petri et aL, 1976; Waring and Mahowald, 1979; Yannoni and Petri, 1980; Mindrinos et al, 1980). Genes for the six quantitatively major protein species have been located either biochemically by in situ hybridization of molecular probes to salivary gland chromosomes or genetically by utilizing electrophoretic variants of chorion proteins (Spradling et aL,l979,1980; Yannoni and Petri, 1980,1981, and unpublished results; Griffin-Shea et uZ., 1980, 1982; Spradling, 1981). Both techniques now agree that the genes for the 936 and ~38 proteins are clustered near the ocel&ss locus at 7Fl2 on the X chromosome and that genes for the ~15, ~16, ~18, and s19 proteins are grouped near the sepia locus at 66D on the third chromosome. In this report we characterize an approximately 70,000 D minor chorion protein on one-dimensional SDS-urea gels and describe its nonequilibrium pH gradient electrophoresis (NEPHGE) position relative to previously described one-dimensional NEPHGE components. We describe an electrophoretic molecular weight and NEPHGE variant of this protein found among our collection of over 300 geographic strains, and we present evidence that the structural gene for this ~70 chorion component maps close to the yellow locus on the tip of the first chromosome. We thus locate the ’ Present address: Dept. Molec. Genetics and Microbiology, U. Mass. Medical Ctr., Worcester, MA 01665. 6012-1666/64 $2.06 Copyright All rights

@ 1984 by Academic Press, Inc. of reproduction in any form reserved.

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first structural gene for a minor chorion protein and identify a new locus involved in eggshell development. MATERIALS

AND METHODS

The ~70 variant strain (Staket) was obtained from the Umea, Sweden, collection via Steve Beckendorf (University of California, Berkeley). Sources and detailed genotypes of our standard wild-type strain (Oregon R); the multiply marked and inverted chromosomal localizing strain (BLT), and multiply marked first chromosome strain (CBl) are given in our previous paper, Yannoni and Petri (1980). Marked and balanced stocks containing the deletions shown in Fig. 4 were utilized for deletion mapping studies. All marked strains used were monomorphic for the standard (Oregon-R) form of the ~70 protein. Procedures for conditioning of flies, dissection, and in vitro labeling of whole ovaries and staged follicles, SDS-urea electrophoresis, NEPHGE (previously described as IF), and autofluorography were followed exactly as previously described (Yannoni and Petri, 1980). For correlation of urea-SDS gel and NEPHGE components, standard and variant chorion protein samples were run in lanes adjacent to one another on a preparative NEPHGE slab gel. This was fractionated by cutting out 34 approximately 2-mm wide segments each including both standard and variant lanes. The standard edge of each segment was marked with india ink, and the slice equilibrated and electrophoresed as previously described (Yannoni and Petri, 1980). RESULTS

I. Characterization of Minor Chon’on Protein on UreaSDS Gels In scanning our geographic strain collection on ureaSDS gels we found in the Staket strain an electrophoretic

BRIEF NOTES

505

variant of an approximately 70,000-D protein (~‘70)which we believed to be a minor chorion component. A number of criteria were used for confirmation of the minor band as a bona fide chorion protein. (1) This protein copurifies with shell along with the six major chorion components already identified (Fig. la). (2) In whole-ovary culture of monomorphic standard (Ore-R, BLT, Xple) or variant Staket strains and in staged egg chamber culture of various standard/variant (Std/Var) heterozygote combinations, this protein labels relatively well with tritiated proline in concert with other chorion proteins (Figs. lb and c) as predicted from the amino acid analysis of chorion (Petri et aL, 1976). (3) Production of ~70 has the developmental stage specificity expected of a chorion protein (King, 1970), in particular, it shows a developmental labeling pattern similar to ~36 and ~38 with synthesis occurring primarily in stages 11 through

FIG. 2. Two-dimensional (2D) gel analysis of ~‘70.(a) Autofluorograph of a 2D gel showing the relative position of the 8’70to the other major eggshell components. Whole ovaries from a homozygous standard strain were incubated in vitro in the presence of [aHlproline for 1 hr, dissolved in toto and subjected to electrophoresis. (b) Autofluorograph of an enlarged portion of a 2D gel prepared similar to (a) except that a variant/standard heterozygote ovary was used. (c) Coomassie brilliant blue staining pattern in an enlarged portion of a 2D gel using purified eggshell from a variant/standard heterozygote.

13 (Fig. lc). (4) In addition, the ~70 protein displays the premature hydrogen peroxide-induced crosslinking typical of chorion proteins (data not shown, for example see Mindrinos et aA, 1980).

FIG. 1. Characterization of 870 protein on SDS-urea gels. (a). Coomassie brilliant blue (CBB)-stained, purified eggshell components from a standard strain (Ore-R) displayed on a 7-15% gradient gel. (b) Autofluorograph of 7-15% gradient gel containing in each track approximately seven stage 12 egg chambers from a homozygous variant or standard strain labeled in vitro for 1 hr with [Qlproline. (c) Autofluorograph of a 7.5% gel containing in each track approximately seven staged egg chambers from variant/standard heterozygotes (Staket/CBl) which were labeled in vitro for 1 hr in the presence of [‘Hlproline.

II. Localization of the s70 Protein on 1-D and 2-D NEPHGE Gels We extended our general electrophoretic characterization of the ~70 protein to one- and two-dimensional (l- and 2-D) NEPHGE gels in the hope of improving resolution between the standard and variant forms. In addition, 1D NEPHGE gels allow the simultaneous comparison of many more samples on a single gel than is easily accomplished on SDS gels and 2D gels can reveal the presence of multiple isoelectric forms of the 970 if they exist. Initial analysis of eggshell protein migration distances in the second dimension of NEPHGE gels suggested that the ~70 component of interest occupied a relatively neutral position in the IF dimension (Fig. 2a). However, since additional compo-

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DEVELOPMENTALBIOLOGY

nents on the acid side of this gel also occupy positions close to the predicted 70 kD neighborhood (Fig. Ba), we felt it necessary to take additional steps to assure ourselves of the correct NEPHGE location of the ~70protein for which we had discovered a variant. To accomplish this, both the standard and variant radioactively labeled proteins were applied in immediately adjacent positions on an isoelectric focusing gel. At the end of the electrophoretic separation, the slab was sliced laterally into 34 sections so that each slice contained similarly migrating components of both the standard and variant strains. All 34 slices were individually rerun on SDS-urea gels and selected examples are displayed in Fig. 2b. Generally, alignment of standard and variant components is quite precise, for example, notice the ~36 proteins in slices 21-2’7 and the ~16 in slices 5-10. Because the stage distribution of the egg chambers in standard and variant ovaries may have differed somewhat, comparisons of labeling intensity between similar components of the two strains are not meaningful. Only slice 21 contained both a slow standard form and a fast variant form of an approximately 70,000 D component suggesting that this slice alone and not slice 9 or 10 (which contains another, more acidic, roughly 70 to 73 kD component) revealed the NEPHGE position of the ~70 of interest. This result indicates that on one-dimensional NEPHGE, the ~70 should migrate

VOLUME102,1984

just to the acid side of the ~36. In fact, using hindsight, careful observation reveals a minor NEPHGE component possessing a stage-specific labeling pattern identical to the ~70 migrating just above the ~36 (Fig. 3a). Examination of 2-D gels of Std/Var heteroxygotes (Figs. 2b and c) shows that the Staket ~70 varies slightly in charge as well as molecular weight. However, this charge variation is not resolved in our one-dimensional NEPHGE gels; and hence, our genetic studies utilize the urea-SDS gel system. In addition, examination in two dimensions of stained, purified eggshell prepar:i tions from Std/Var heterozygotes (Fig. 3~) shows that both standard and variant forms of the ~70 copurify with shell confirming that the variant protein is also an eggshell protein. Two-dimensional gel analysis of [3H]proline incorporation patterns in staged egg chambers confirmed our earlier finding (Fig. 1) that production of the ~70 occurs primarily in stages 11 through 13 (data not shown). III. Linkage Analysis All F1 progeny resulting from crosses of the monomorphic ~70 standard and variant strains show. approximately equal labeling intensities in variant.1 and standard forms of ~70 and show identical stage specific labeling patterns for both (Figs. lc and 2b). Using the

FIG. 3. Correlation of one-dimensional (1D) NEPHGE and SDS-urea gel patterns of ~70 protein. (a) Autofluorograph from a 1D NEPHGE gel of a developmental series from a homozygous standard strain. Approximately seven egg chambers of each indicated stage were labeled in vitro for 1 hr with [‘Hlproline and analyzed in adjacent gel tracks. The indicated position of the ~70 was deduced from information provided by (b). (b) Autofluorographic results from SDS electrophoresis of selected fractions from the 34 slices prepared as described in the text from a 1D NEPHGE gel where homozygous standard and variant samples were run in tandem. Slice 1 represents the acid end of the NEPHGE gel and slice 33 the basic.

BRIEF NOTES

same rationale that we have previously discussed in detail (Yinnoni and Petri, 1980), this evidence strongly suggests that (1) the Staket geographic strain carries a varia’nt of the ~‘70, and that (2) in mapping the source of the xariation we are mapping the structural gene for this protein. Studies for the linkage group analysis of this chorion gene gore carried out as previously described (Yannoni and Petri, 1980). In the cross utilized, BLT females crossed to males whose autosomes are BLT/Var and whose sex-chromosome constitution is Var/Y, all resulting females (96) showed the presence of both the variant and standard forms of the ~70. This indicates X-ch,romosome linkage, since only the X chromosome will be heterozygous in all cases. Specific chromosomal localization of this gene was carried out following the approach detailed by Yannoni and Petri (1980) using strain CBl which carries a multiply marked X. Heterozygous females (Staket/CBl) were crossed to CBl males. All females phenotypically CBl (y, cv, v,f) carried only the standard form and all females phenotypically wild-type (g+, cv+, v+, f+) carried both the standard and variant forms, confirming that the ~70 gene resides on the X chromosome. Analysis of recombinant females indicates that the ~70 gene is tightly linked to gellow (87/88 cases), which is located at the tip of the first chromosome at 0.0 far from the only previously identified X-chromosome site for chorion structural genes. This result was confirmed by electrophoretic analysis of a group of deletion heterozygotes constructed by crossing Staket flies to various stocks which contain assorted X-chromosome deletions (Fig. 4) and which display only the standard form of the ~70. Only Df(l)lB5; ZB15/Staket flies lacked the standard form of the ~70 as would be expected if the ~70 gene were within the deleted region. All of the other deletion heterozygotes produced both variant and standard types in roughly equal amounts as judged by labeling intensity. Deletions RA2 and KA14 which cover the ocelliless region at 23.1 map units were included in our study as added evidence that the ~70 gene is not within this area where the coordinately synthesized ~36 and 938 components are clustered along with two unidentified transcription units (Spradling, 1981). We crossed strain Df(1) RC40/FM7 carrying a deletion of X-chromosome bands 4Bl to 4Fl to Staket specifically to delete the 4B area which has been shown to hybridize with polyA containing RNAs from stage 12 and 13 egg chambers (Spradling and Mahowald, 1979). DISCUSSION

The many quantitatively minor, putative chorion proteins (Waring and Mahowald, 1979; Margaritis et al,

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FIG. 4. Approximate locations of deletions used in confirmation of ~70 chromosomal location. Modified from Bridges’s map originally published as a supplement to the J. Hered 26,1935.

1980; Mindrinos, 1980) have received less attention than the six major ones, and until now none of their gene loci have been determined. Although proteins are classified as being “minor” components of the chorion based primarily on their relatively low intensity of Coomassie brilliant blue staining and or proline radiolabeling, it remains possible that some of these so called “minor” components may in fact equal or exceed the majors in their quantitative contribution to the eggshell. Low affinity for Coomassie, low amounts of proline, and unusual solubility properties could all contribute to disguising an actually major chorion component as a minor one. It is with this caveat in mind that the current designation of major and minor chorion proteins should be viewed. Biochemical localization of the six major chorion genes proceeded rapidly in part due to two factors: the relatively abundant amounts of mRNA for these proteins, and the clustering of all six genes into two small chromosomal areas. The first factor made it relatively easy to find chorion cDNA clones in libraries made from latestage follicles, and the second factor allowed identification of additional chorion genes by examination of genomic clones obtained using a few initial cDNA probes. However, perhaps because of relatively low amounts of mRNA coding for minor chorion proteins, cDNA clones of these components have not been reported. It is unlikely that genes for most of the minor chorion proteins

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exist in the already described genomic clones. Only four transcription units have been detected in the 66D cluster, and all of these are accounted for by major chorion proteins (Griffin-Shea et aL, 1982). Two of the four transcription units in the ‘7F cluster have also been assigned to major chorion genes (Spradling, 1981). Hence, at most, genes for two minor chorion proteins may be within already cloned areas, and we can predict that additional chromosomal areas will also be involved in eggshell synthesis. Using geographic strain variants, we have identified one of these new areas. An advantage to using natural variants and genetic techniques for the mapping of minor shell components is that one works directly with the protein of interest. The genetic locus of any shell protein for which an electrophoretic variant can be identified may be mapped using the basic methods we have described here and elsewhere (Yannoni and Petri, 1980,198l). The quantity of protein synthesized does not significantly influence the method. It is important to note that our method actually maps the source of protein variation which could reside in the structural gene for a modifying enzyme as well as in the structural gene for the shell protein in question. We have adapted a standard criterion for evaluating the behavior of variant and standard proteins in heterozygotes as an indicator of whether or not the source of variation resides in the shell gene (Yannoni and Petri, 1980, 1981). The general validity of our assumptions is borne out by the fact that the results from our mapping of variants in all six major chorion genes agrees (Yannoni and Petri, 1980,1981, and unpublished observations) in all cases with the results derived from in situ hybridization of molecular probes. Hence, we are quite confident that we have in fact localized the structural gene for the s70 protein. Several minor proteins have already been described. Waring (1979) describes several high-molecular-weight components, one of which (C60-50) shows the same developmental stage specificity and coordinate synthesis with s36 and ~38 that we have observed in the case of the ~70.Mindrinos et al. (1980) have also reported several high-molecular-weight minor proteins and identified them as chorion components based on premature crosslinking with peroxide. One of these proteins also shows a developmental stage specificity during stages 11-13. However, because of the approximate nature of these molecular weights and lack of additional comparative data, it is difficult to be certain whether our s70 is the same as one of the proteins already described. Recently,

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Komitopoulou et al. (1983) have described a number of female-sterile mutations affecting the eggshell. Among these a number fall into three complementation groups near the tip of the X chromosome. Although biochemical alterations have not been investigated in any of these, it remains possible that the s70 locus resides within one of these groups. We thank Mr. Hilton Kwan for expert photographic assistance and Mr. Timothy Murphy and Ms. Lynne Ripley for skillful assistance in typing this manuscript. This work has been supported by NIH Grant GM 24185. REFERENCES GRIFFIN-SHEA,R., THIREOS,G., and KAFATOS, F. C. (1980). Chorion cDNA clones of D. melanogaster and their use in studies of sequence homology and chromosomal location of chorion genes. CeU 19,915922. GRIFFIN-SHEA,R., THIREOS,G., and KAFATOS,F. C. (1982).Organization of a cluster of four chorion genes in L?rosophiZaand ita relationship to developmental expression and amplification. Deu. Bid 91, 325336.

KING, R. C. (1970). “Ovarian Development in Drosophila melanogaster.” Academic Press, New York. KOMITOPOULOU, K., GANS,M., MARGARITIS,L. H., KAFATOS,F. C., and MASSON,M. (1983). Isolation and characterization of sex-linked female-sterile mutants in Drosophila melonogoster with special attention to eggshell mutants. Genetics 105,897-920. MARGARITIS,L. H., KAFATOS, F. C., and PETRI, W. H. (1980). The eggshell of Drosophila melanogaster. I. Fine structure of the layers and regions of the wild-type eggshell. J. CeU sci. 43, l-35. MINDRINOS,M., PETRI, W. H., GALANOPOULOS, V. K., LOMBARD,M. F., and MARGARITIS,L. H. (1980).Crosslinking of the Drosophila chorion involves a peroxidase. Wilhelm Roux’s Arch. 189.18’7-196. PETRI,W. H., WYE~AN,A. R., and KAFATOS,F. C. (1976). Specific protein synthesis in cellular differentiation. Deu. Biol 49, 185-199. SPRADLING,A. C., and MAHOWALLI,A. P. (1979). Identification and genetic localization of mRNAs from ovarian follicle cells of Dro sophilu melanogaster. Cell 16,589-598. SPRADLING,A. C., DIGAN, M. E., and MAHOWALD,A. P. (1980). Two clusters of genes for major chorion proteins of Drosophila me&nogaetw. Cell 19, 905-914. SPRADLING,A. C. (1981). The organization and amplification of two cbromosomal domains containing Drosophila chorion genes. Cell 27, 193-201. WARING,G. L., and MAHOWALD,A. P. (1979). Identification and time of synthesis of chorion proteins in Drosophila nwlunogaster. CeU 16, 599-607.

YANNONI,C. Z., and PETRI,W. H. (1980). Characterization by isoelectric focusing of chorion protein variants in Drosophila mekznog&erand their use in developmental and linkage analysis. Wilhelm RQUX’S Arch 189, 17-24. YANNONI, C. Z., and PETRI, W. H. (1981). Drosophila m~lanogaster eggshell development: Localization of the s19 chorion gene. Wilhelm Rwuds Arch. 190,301~303.