J. Embryol. exp. Morph. 83, Supplement, 137-146 (1984)
Printed in Great Britain © The Company of Biologists Limited 1984
137
Analysis of transcriptional regulation of the s38
chorion gene of Drosophila by P element-mediated
transformation
By LAURA KALFAYAN, BARBARA WAKIMOTO AND
ALLAN SPRADLING
Dept of Embryology, Carnegie Institution of Washington,
115 W. University Parkway, Baltimore, Maryland 21210, U.S.A.
TABLE OF CONTENTS
Summary
Introduction
The chorion gene system
P element-mediated transformation
Expression and regulation of the s38 chorion gene
Discussion
References
SUMMARY
Transcriptional regulation of the s38 chorion gene was studied using P elementmediated germline transformation. A 5-27 kb DNA fragment containing the s38
gene and 5'- and 3'-flanking sequences, was tested for its ability to be transcribed
with correct developmental specificity. Five single-insert transformed lines were
generated by microinjection of this DNA fragment cloned into a marked P
element transformation vector. In each line, the transformed gene was transcribed according to the precise developmental pattern followed by the native
s38 gene. The 1-3 kb at the 5' end of this tested fragment was fused to the E. coli
lac z gene. This fragment was also capable of initiating transcription of E. coli
lac z RNA with the developmental profile of the native s38 gene. In vitro deletion
studies are underway to determine which sequences in the 1-3 kb fragment are
necessary for regulating the developmental expression of the gene.
INTRODUCTION
The regulation of gene expression in eucaryotes has been the focus of much
study in the field of developmental biology. Inducible gene systems, such as those
138
L. KALFAYAN, B. WAKIMOTO AND A. SPRADLING
regulated by heavy metals or hormones, are attractive because they lend themselves to simple experimental manipulation, and the differences in RNA levels
between induced and uninduced states can be dramatic. Genes exhibiting major
changes in the level of expression during development in response to less-wellcharacterized signals are equally interesting to study, and provide the opportunity to teach us about timing or tissue-specific factors or signals controlling their
expression. The approaches to understanding gene regulation range from defining hormonal parameters to classical genetics to in vitro mutagenesis and DNA
sequencing. In the latter approach, the cloned gene can be altered specifically,
and then re-introduced by transfection or injection into an environment where
the effect of the mutation can be studied (McKnight, Gavis, Kingsbury & Axel,
1981; McKnight & Kingsbury, 1982; Brinster etal. 1981; Palmiter etal. 1982a;
Palmiter, Chen & Brinster, 19826).
Our laboratory has concentrated its efforts on understanding the regulation of
gene expression of a set of genes encoding the chorion or eggshell of Drosophila.
The chorion genes exhibit dramatic time- and tissue-specific developmental
regulation (Spradling etal. 1981; Griffin-Shea, Thireos & Kafatos, 1982). The
P element transformation techniques recently developed in this laboratory have
enabled us to ask direct questions about the specific function of the sequences
surrounding chorion genes and the importance of the organization of these genes
for their proper developmental regulation. In this paper we describe our studies
on the controls governing expression of the s38 chorion gene which encodes a
major polypeptide of relative molecular mass 38 X 103.
THE CHORION GENE SYSTEM
The chorion of Drosophila is an architecturally complex structure composed
of three layers and approximately 20 polypeptides (Waring & Mahowald, 1979).
Eggshell protein synthesis and construction take place during the late stages of
oocyte development and closely follow the synthesis of the major chorion RNAs
(Waring & Mahowald, 1979; Spradling & Mahowald, 1979).
Transcription of the chorion genes occurs in the ovarian follicle cells according
to a precisely timed developmental programme. All of the major chorion RNAs
are detected in the last stages (11-14) of oogenesis (Parks & Spradling, 1981;
Griffin-Shea et al. 1982). Within these stages, however, RNA synthesis from
each gene proceeds with an independently timed pattern. The s38 RNA is detected maximally at stage 12, which lasts 1-5 h, and is also present during stages 11
(a 20-min time span) and stage 13 (a 2-5 h period). The RNA from the s38 gene
is highly abundant, accounting for approximately 10 % of the poly A+ RNA in
the stage-12 egg chambers (unpublished observations).
Genes encoding the most abundant chorion proteins are located in two genomic clusters (Spradling et al. 1980). This organization facilitates an unusual kind
of regulation in Drosophila. Prior to transcription, the DNA in each cluster
Transcriptional regulation of the s38 gene
139
selectively amplifies in the ovarian follicle cells (Spradling & Mahowald, 1980).
The chorion genes are then abundantly transcribed, producing large quantities
of RNA and protein within the last 5 h of oogenesis.
The s38 gene, located in the X-chromosome chorion gene cluster (position
7F1) is centrally located within a region that amplifies 16-fold (Spradling, 1981).
It is surrounded by at least nine other transcription units that are active during
the late stages of oogenesis (Parks, Kalfayan & Spradling, 1982 and unpublished
observations). We were interested in learning whether the clustering and the
amplification of these genes was actually required for properly regulated initiation of transcription. If not, we wished to define the minimum DNA sequences
necessary for regulation of both timing and tissue-specific expression of the
genes. We found that at most, 1-3 kilobases (kb) of the DNA at and including
some of the 5'- portion of the s38 gene regulates the timed expression of this
gene.
P ELEMENT-MEDIATED
TRANSFORMATION
The regulation of chorion and other genes' expression can be studied with
relative ease in Drosophila now that the technique of P element-mediated germline transformation is possible (Spradling & Rubin, 1982; Rubin & Spradling,
1982). Genetic studies led to the discovery of the transposable P elements and
defined two types of elements, complete and defective (Engels, 1981). More
recently, both classes of P elements have been cloned and sequenced (O'Hare
& Rubin, 1983). The complete elements, capable of catalysing their own transposition in a permissive environment (an M strain), are 2-9 kilobases and are
terminated by inverted 31 base pair repeats. The defective elements, ranging in
size from 0-5-1-6 kb lack sequences from the middle portion of the DNA, but
have conserved the 31 base repeat at their ends. They are capable of transposing
only in the presence of complete P elements. Thus, the middle portion of the
element is thought to encode a trans-acting transposase-like product while the
ends are required for the integration.
In our transformation system, cloned P elements are injected directly into the
pole plasm of preblastoderm, M-strain embryos. Intact P elements are used to
catalyse the transposition of defective element vectors that carry between their
ends the cloned gene (such as the s38 gene) to be studied. The transposon
integrates into random chromosomal sites in the developing germline cells, and
is subsequently stably inherited. An additional marker gene, such as the rosy+
gene is also incorporated into the transposon so that transformation events into
rosy— strains can be assayed by examination of the eye colour.
EXPRESSION AND REGULATION OF THE
s38 chorion gene
A marked transposon (pi A25) containing the s38 gene was constructed for use
in this study (Wakimoto, Kalfayan & Spradling, 1983; DeCicco et al. 1983) and
140
L. KALFAYAN, B. WAKIMOTO AND A. SPRADLING
is shown in Fig. 1. The essential features of this transposon are the P element
ends, a cloned restriction fragment containing the wild-type rosy gene, and an
altered s38 gene. The s38 gene produces a l-4kb RNA that is wholly contained
within a 4-7 kb Eco Rl restriction fragment (Spradling, 1981; Parks et al. 1982).
This fragment has approximately 700 bases flanking the 5' end of the gene and
2-2 kb past the 3' end. Since no mutations in the s38 gene have been discovered,
the gene was altered by the insertion of a heterologous DNA fragment (derived
from the bacteriophage ml3) into the transcribed portion of the gene. This
change was designed to produce a 2-0 kb RNA that could be distinguished from
the s38 transcript by size and by hybridization to the heterologous inserted DNA
sequence.
Co-injection of the transposon, plA25, with the complete cloned P element,
p7r25-l, into rosy- M embryos resulted in a number of transformants which were
used to establish five different single insert lines. The genetic and cytological data
will be presented in detail in a later publication. In addition, two multiple insert
lines containing transposons at different sets of sites were obtained.
Hind III
EcoRl
M13
\
Inject into ry 506 embryos
with p;r 25-1
Fig. 1. Construction of the s38-M13 transposon. A 4-7 kb Eco-Rl fragment containing the s38 gene was modified by the insertion of a 572 bp fragment from M13 into
the transcribed portion of the s38 gene. The arrow indicates the approximate position
of the insert (open box) near the 5' end of the message and the direction of transcription. The resulting 5-27 kb Eco Rl fragment was cloned into the Eco Rl site of the
transformation vector PV11 so that the region containing chorion and rosy+ gene
sequences wasflankedby P element ends (solid blocks). This plasmid was co-injected
with the complete P element, p25.1 into rosy- M embryos.
Transcriptional regulation of the s38 gene
141
Examination of restricted genomic DNA from each single insert transformed
line showed that the transposons had integrated, unrearranged, into the genome.
An example of this work, showing that the transformed Eco Rl fragment containing the s38-ml3 gene is of the expected size, is shown in Fig. 2.
Transcripts from the transformed genes were detected in each transformed
line during the proper developmental time period. Staged poly A+ RNA from
each of the late stages of oogenesis as well as from pooled early stages was
prepared and screened for homology to the inserted ml3 sequence. The same
RNA preparations were then rescreened with DNA homologous to the s38
message. The results in all cases were the same, and a typical example taken from
X ry~ 4 5
4.3-
~_
pP
X ry~ 4
^ttk.
m^»
5
pP
-»^i —527
3-4-
201-613-
pUC 8
S38
Fig. 2. Analysis of transformed DNA in single insert lines. Two to three micrograms
of genomic DNA from males (who do not amplify their chorion genes) from rosy—
flies-and from'transformed lines No. 4 and 5 was restricted with Eco Rl, separated
on 1 % agarose gels and transferred to nitrocellulose filters. The DNA blots were
hybridized with radiolabelled plasmid DNAs from either pUC 8 (which contains
sequences homologous to the inserted M13 DNA and should hybridize specifically
to the transformed gene) or plO3.47 (which contains the 4-7kb Eco Rl fragment
containing the s38 chorion gene and should hybridize to both the endogenous and the
transformed DNA). DNA was digested with Hindlll and Eco Rl. Lanes marked pP
are Eco Rl digested cloned plA25 fragments.
142
L. KALFAYAN, B. WAKIMOTO AND A. SPRADLING
P[s38-ry]4
p
2-9-
1-9 10 11 12 13 14 1-9 10 11 12 13 14
«te
1-4-
pUC8
s38
Fig. 3. Developmental profile of the s38-M13 transcript. Poly A + RNA was isolated
from staged egg chambers of the transformed line No. 4 and assayed by Northern
blotting for the presence of the s38-M13 transcript. The RNA was size fractionated
on 1 % agarose-formaldehyde gels. Lanes labelled 1-9 contain RNA from the early
stages from 5 ovaries. Other lanes contain RNAs from 100 egg chambers at the stage
indicated, except for the stage 11 lane, which has only 50 egg chambers of RNA. The
same blot was hybridizedfirstwith the radiolabelled pUC 8 probe (see legend for Fig.
2), washed free of hybridized material and rehybridized to labelled plO3.47.
one of the single insert lines is shown in Fig. 3. Clearly, transcription of the
transformed gene initiates in parallel with that of the endogenous gene. Differences in the relative amounts of the transformed versus the endogenous RNAs
in stages 11-13 are apparent, however, and can be explained in part by the fact
that this transformed gene does not amplify, while the endogenous gene is undergoing amplification between stages 11 and 13. In addition the altered message
may be less stable than its normal counterpart. No transformed transcripts were
detected in different stages of development (data not shown).
A second transposon, pACSn, was constructed to begin to test the limits of the
DNA sequences required for developmental regulation of the s38 gene. In this
plasmid 1-3 kb at the 5' end of the 4-7 kb fragment containing the s38 gene was
fused to the lac z gene of E. coli (Fig. 4). The DNA was injected as described
above and RNA from the transformed lines was analysed for hybridization to the
cloned lac z gene. As witnessed with the larger fragment, the transposed gene
was regulated with the proper developmental timing for the s38 gene (Fig. 5).
Transcriptional regulation of the s38 gene
143
Carnegie 1:
Bam/Rl
pACSl:
Sal
or
Bam
lac z
SBB
KxxyvwvwJ
SB
j
Sal or Bam
+
S
Sal
i
\ ;
/
/ / /
/
/
5
/ / / / / / s\
t
Sal
f
pACSn:
s38-lac z
Fig. 4. Construction of the s38-lacz fusion genes in three reading frames. The indicated steps were carried out according to standard procedures. The 1-3 kb Eco RlBam HI fragment at the 5' end of the s38 gene was ligated into a similarly digested
transformation vector, Carnegie 1. A fragment from the lac z gene of E. coli was
fused into the s38 DNA at the Bam HI site in all three orientations, using different
enzymes in a polylinker. An 8-1 kb Sal fragment containing the rosy+ gene was
ligated into the vector between the P element ends. Solid blocks indicate P element
ends. The open box represents the pBR322 backbone sequences necessary to
propagate the plasmid in bacteria. They are lost in the integration into Drosophila
DNA.
DISCUSSION
We have shown that a 1-3 kb DNA sequence, containing approximately 700
nucleotides of sequences flanking the 5' end of the s38 gene is capable of directing
properly timed synthesis of the s38 RNA. We are currently attempting to narrow
this sequence down to a minimum. In addition, the data indicate that the s38 gene
does not need to reside in its normal position within the cluster of DNA that
amplifies. Furthermore, properly timed transcription can occur in the absence of
amplification (data not shown).
144
L. KALFAYAN, B. WAKIMOTO AND A. SPRADLING
P[s38-lacz]
p
1-9 10 11 12 13 14
2-11-5-
0-7-
lac z
Fig. 5. Developmental profile of the s38-lac z transcript. The Northern blot shown
here was prepared essentially as described in the legend for Fig. 3 except that RNA
from 100 egg chambers was used in each of stages 10-13 and from 200 egg chambers
in stage 14. The hybridization probe was a plasmid, pMC1871 containing the lac z
gene.
Several other investigators have used P element-mediated transformation to
study developmental regulation of other Drosophila genes (Scholnick, Morgan
& Hirsch, 1983; Goldberg, Posakony & Maniatis, 1983; Hazelrigg, Levis &
Rubin, 1984; Spradling & Rubin, 1983). The conclusion that emerges from these
studies is that Drosophila genes do not require a great deal of sequence flanking
each gene to direct developmentally regulated expression. Our results address
directly expression at the level of transcription. We have shown that no more
than 700 nucleotides beyond the 5' end of the s38 gene are required for directing
the precisely timed initiation of transcription of this gene.
The chorion genes differ from the other genes that have been studied, however, in their unusual clustered organization and developmentally controlled
amplification. It is therefore interesting to note that neither clustering nor amplification are required for proper developmental regulation of RNA synthesis.
Transcriptional regulation of the s38 gene
145
Recent transformation studies in this laboratory have shown that small fragments of DNA within central region of the amplified cluster of chorion genes
possess the ability to amplify when removed from the cluster, but that the level
of amplification is sensitive to chromosomal position (DeCicco & Spradling,
manuscript in preparation). The sensitivity to chromosomal position can be
buffered in part by surrounding the amplifying sequences with DNA from the
amplified cluster. Interestingly, transcription of the transformed s38 gene is
much lower than that of the endogenous gene. The levels of transformed RNA
vary slightly depending on their chromosomal position, as noted for the rosy+
gene (Spradling & Rubin, 1983), the Ddc+ gene (Scholnick etal. 1983) and the
Adh+ gene (Goldberg et al. 1983). However, the overall level of s38 RNA is
lower than predicted, even after accounting for the absence of amplification.
This is perhaps due to a decreased stability in the altered s38 gene. We are also
currently investigating the possibility that, as for amplification, additional surrounding sequences from the cluster increase the level of synthesis from the
transformed gene due to distant 'enhancer-like' sequences.
Our current strategy for defining the minimum sequences required for timing
and tissue-specific expression of the s38 gene involve the construction of a series
of deletions from the 5' and 3' ends of the 1-3 kb regulatory fragment. We expect
that this approach will tell us whether the timing control is separable from that
for tissue-specific regulation of the gene.
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