From the
The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is
essential for phototransduction. Besides being found abundantly in
retina, norpA gene products are expressed in a variety of
tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to
phototransduction. We have identified a second subtype of norpA protein
that is generated by alternative splicing of norpA RNA. The
alternative splicing occurs at a single exon that is excluded from
mature norpA transcripts when a substitute exon of equal size
is retained. The net difference between the two subtypes of norpA
protein is 14 amino acid substitutions occurring between amino acid
positions 130 and 155 of the enzyme. Results from Northern analyses
suggest that norpA subtype I transcripts are most abundantly
expressed in adult retina, while subtype II transcripts are most
abundant in adult body. Moreover, norpA subtype I RNA can be
detected by the reverse transcription-polymerase chain reaction in
extracts of adult head tissue but not adult body nor at earlier stages
of Drosophila development. Conversely, norpA subtype
II RNA can be detected by reverse transcription-polymerase chain
reaction throughout development as well as in heads and bodies of
adults. Furthermore, norpA subtype I RNA is easily detected in
retina using tissue in situ hybridization analysis, while
subtype II RNA is not detectable in retina but is found in brain. Since
only norpA subtype I RNA is found in retina, we conclude that
subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a
variety of tissues not known to contain phototransduction machinery,
subtype II protein is likely to be utilized in signaling pathways other
than phototransduction. The amino acid differences between the two
subtypes of norpA protein may reflect the need for each subtype to
interact with signaling components of different signal-generating
pathways.
Phosphatidylinositol-specific phospholipase C (PLC)
PLC is known to be a
diverse family of enzymes whose members differ in structure and tissue
distribution (Rhee et al., 1989; Meldrum et al.,
1991). PLC enzymes have been grouped into four major classes
(PLC-
The major types of PLC appear to be activated by
different receptor classes (Meldrum et al., 1991; Jones and
Carpenter, 1992; Rhee and Choi, 1992a, 1992b). PLC-
A PLC for which
a function in vivo has been identified is encoded by the norpA gene of Drosophila melanogaster. Strong
mutations in the norpA gene have long been known to abolish
the photoreceptor potential, rendering the fly blind (Hotta and Benzer,
1970; Pak et al., 1970). The identification of the norpA gene product as a PLC emerged from findings that norpA mutations severely reduce PLC activity in eye (Yoshioka et
al., 1985) and that the deduced protein is similar in structure
and amino acid sequence to mammalian PLC (Bloomquist et al.,
1988). These findings, along with a large body of evidence indicating
that invertebrate phototransduction is mediated by PLC, led to the
conclusion that the norpA gene encodes a PLC that is utilized
in phototransduction in Drosophila (Bloomquist et
al., 1988; Pak and Shortridge, 1991).
However, norpA is also known to be a complex gene whose expression is not limited
to the retina nor utilized exclusively in phototransduction. Zhu et
al.(1993) demonstrated that the norpA gene is expressed
in embryo, adult body (thorax and abdomen), leg, and brain, as well as
retina. Since body, leg, and brain are not known to contain visual
machinery, it was proposed that the norpA-encoded PLC is
utilized in signaling pathways in addition to phototransduction. Zhu et al.(1993) also observed different sizes of norpA transcripts on Northern analyses, raising questions whether norpA transcripts are alternatively spliced and if this leads
to yet unidentified subtypes of norpA protein. If so, this would be in
parallel with three other PLC-
An
advantage of using the norpA mutant of Drosophila as
a model to study PLC function is that it is much easier to deduce the
identity of the signaling pathways involved by examining the mutant
phenotype. Here, we demonstrate that the norpA gene encodes at
least two subtypes of PLC by alternative splicing of the nascent RNA
and that each is likely to be utilized in completely different
signaling pathways.
To map the location of exons of norpA (3, 4,
4A, and 5) within ge-nomic DNA, a cosmid clone (COSM1) that contains
the norpA gene (Bloomquist et al., 1988) was used as
template for PCR reactions. PCR was carried out on COSM1 DNA using
oligonucleotide primers made to sites within exons. Location of the
exons within the genomic DNA and approximate size of the intervening
sequences was deduced by identifying and sizing the amplified products
on agarose gels. Nucleotide sequences at intron/exon boundaries, as
well as within the exons, was determined by carrying out DNA sequencing
reactions on COSM1 DNA using oligonucleotide primers made against sites
within the exons.
Probes were
generated by terminal labeling of 80-bp DNA fragments that correspond
to norpA exon 4 or exon 4A using terminal transferase in the
presence of digoxygenin 11-dUTP (Boehringer Mannheim) according to
manufacturer's instructions. The treated tissue sections were
hybridized overnight at 37 °C in hybridization solution (35%
deionized formamide, 5% dextran sulfate, 1
Amplification of norpA cDNA was carried out using PCR primer sets (see ``Materials
and Methods'') that are complimentary to the previously reported norpA cDNA (Bloomquist et al., 1988). A variety of
primer set combinations were tested in the RT-PCR reactions, and only
single amplification products were observed (data not shown).
Nucleotide sequences of the amplified norpA cDNA matched the norpA sequence reported by Bloomquist et al.(1988)
except within a small region of 80 nucleotides occurring between
nucleotides 1038 and 1119 (Fig. 1). This difference was found in
both body and leg cDNA and corresponds to a region of the norpA cDNA that Masai and Hotta(1991) described as exon number 4.
However, the exon 4 domain of the Bloomquist sequence is missing from
these amplified cDNAs. The region that replaces exon 4 in these body
and leg tissues was designated as exon 4A. No other sequence
differences were found between the Bloomquist sequence and the norpA cDNA obtained from body or leg.
As shown in Fig. 4, norpA subtype I (exon 4) transcripts can easily be detected by RT-PCR in
wild-type head and to a lesser extent in eya head and norpA mutant head, but not in body or at earlier developmental
times. Conversely, norpA subtype II transcripts are detectable
in all lanes tested but most easily detected in adult body (thorax and
abdomen) (Fig. 4). These data are in agreement with the Northern
blot results that show norpA subtype I RNA is most abundantly
found in head, more specifically in compound eye, and subtype II RNA to
be most abundant in body. However, the ability of RT-PCR assays to
detect lower abundance transcripts refine and expand the Northern blots
by identifying other tissues and developing times that express norpA.
Mutations in the norpA gene have long been known to
drastically affect the light-evoked responses of photoreceptor cells,
and it is well accepted that the norpA gene encodes
phospholipase C that is essential for phototransduction (Bloomquist et al., 1988; Pak and Shortridge, 1991; Ranaganathan et
al., 1991). However, norpA gene products have also been
proposed to operate in other signaling pathways in addition to
phototransduction (Zhu et al., 1993). Here, we have identified
a second subtype of norpA (designated subtype II) and find
that it is not detectable in retina but is easily detected in other
tissues not known to contain phototransduction machinery. Conversely, norpA subtype I transcripts are easily detectable in retina
but not easily detected in other tissues. Since norpA subtype
I transcripts are found in retina, but subtype II transcripts are not,
we conclude that norpA subtype I protein is utilized in
phototransduction. Moreover, norpA subtype II transcripts are
found in tissues not known to contain phototransduction machinery,
indicating that subtype II protein is utilized in signaling pathways
other than phototransduction.
The results of the RT-PCR analyses
showing that norpA subtype I transcripts are present in head
of eya mutant (Fig. 4) do not contradict results from
the Northern blot analyses, which fail to detect subtype I expression
in eya head (Fig. 3). RT-PCR is known to be much more
sensitive than Northern blots in detecting very low amounts of RNA
products (Fuqua et al., 1990). Even though eya mutants lack compound eyes, the ocellar visual organs on the top
of the head are present (Bonini et al., 1993). Moreover, the
light-evoked responses in ocelli are known to be abolished by strong norpA mutations (Hu et al., 1978). Inasmuch as norpA
subtype I protein is utilized in the phototransduction pathway in
compound eyes, it is logical to expect that the ocellar
phototransduction pathway utilizes subtype I protein as well. Thus, the norpA subtype I transcripts detected in eya mutant
head by RT-PCR may be derived from the ocellar visual organs and are
too low in abundance in eya head RNA pools to be detected by
Northern blot analysis.
Moreover, the capacity of RT-PCR analyses to
detect low abundance transcripts is likely the reason that RT-PCR
detects norpA subtype II transcripts in wild-type head (Fig. 4), but these are not observed in the Northern analysis (Fig. 3). Zhu et al. (1993) reported that norpA protein
is expressed in brain as well as compound eyes and ocelli. This
localization in brain is in complete agreement with the tissue in
situ hybridization analyses shown in the present work that
localizes norpA subtype II RNA in brain (Fig. 5).
However, these signals are only weakly visible in brain whenever the
tissue in situ hybridization reactions are carried out on head
(data not shown). Thus, it is likely that norpA subtype II
transcripts are not expressed in sufficient amounts in head to be
detected by Northern blot analysis. Furthermore, the ability of RT-PCR
to detect low abundance transcripts is why norpA RNA is
detected in norpA mutant (Fig. 4).
Currently, it is
unclear how many signaling pathways utilize the different subtypes of norpA or if there are more splice-variant subtypes yet to be
identified. norpA mutants were originally identified as a
result of their visual system defect (Hotta and Benzer, 1970; Pak et al., 1970). norpA mutants have also been shown to
exhibit altered mating behavior (Markow and Manning, 1980; Tompkins et al., 1982) as well as defective olfactory responses in
maxillary palps (Riesgo-Escovar et al., 1995). There are
likely additional mutant characteristics that remain unidentified,
especially since the presence of norpA expression in brain and
leg point to involvement in signaling pathways other than those
affecting the above mentioned mutant defects. While these data suggest
that upwards of three completely different signaling processes utilize
the norpA-encoded PLC, results of the present work indicate
that the two identified subtypes of norpA comprise the major
splice-variant forms. In the Northern blot analyses, the two
exon-specific probes detect all three of the major transcripts of norpA that are identified when much longer cDNA probes are
used (Fig. 3). However, this does not preclude the possibility
that there are yet unidentified splice-variant forms of norpA that are expressed only in a small subset of cells or in a very
low abundance in tissues.
The significance of the present work lies
in its contribution to defining the role of subtypes of PLC in cellular
signaling. As previously mentioned, phosphatidylinositol-specific PLC
is known to be a family of proteins that have been classified into
major groups (PLC-
(
)cleaves phosphatidylinositol 4,5-bisphosphate to
yield two preeminent second messenger molecules, inositol trisphosphate
and diacylglycerol, in one of the largest classes of cellular signaling
pathways known (Berridge, 1987; Rhee et al., 1989). Signaling
pathways that are mediated by PLC are found in both plants (Drobak,
1992) and animals (Ryu et al., 1987) and have been shown to
modulate a variety of cellular processes such as invertebrate
phototransduction (Pak and Shortridge, 1991), contraction (Volpe et
al., 1986), secretion (Putney, 1988), fertilization (Miyazaki et al., 1993), growth, differentiation (Michell, 1989),
phagocytosis, and chemotaxis (Lew, 1990).
, PLC-
, PLC-
, and PLC-
), based on
immunological and structural differences (Rhee et al., 1989).
The PLC-
, PLC-
, and PLC-
enzymes have been further
classified into subgroups (PLC-
1 through PLC-
4, PLC-
1
through PLC-
3, and PLC-
1 through PLC-
3) based on
differences in amino acid sequence and that each subtype is encoded by
a separate gene (Rhee and Choi, 1992a, 1992b). Emerging evidence
indicates that an additional level of diversity exists beyond the
subgroup classifications because individual genes have been shown to
encode multiple PLC products by alternative splicing of RNA (Shortridge et al., 1991; Ferreira et al., 1993; Bahk et
al., 1994).
activation
appears to be modulated by canonical heptahelical receptors coupled to
G-proteins, while PLC-
enzymes appear to be coupled to growth
factor receptors via tyrosine kinase phosphorylation. It is thought
that individual PLCs within a class are coupled to a single type of
receptor (eg. G-protein-coupled receptors), yet this has not
been proven because so few PLC subtypes have been linked to a
particular receptor or cellular process in vivo. Moreover, it
is unknown whether splice-variant subtypes of PLC are utilized in the
same signaling pathway, a particular type of signaling pathway, or in
completely diverse and separate signaling pathways.
genes that are highly homologous to norpA (Shortridge et al., 1991; Ferreira et
al., 1993; Bahk et al., 1994). It is unknown what role
the cognate splice-variant subtypes play in signaling processes.
Drosophila Strains
D. melanogaster
white (w) mutant was used in all
experiments as a control group because it lacks visual pigment, which
can interfere with some assays, and its genetic background is most
similar to that of norpA mutants used in experiments.
Hereafter, w
is referred to as wild
type because it is wild type for norpA expression. The norpA
/w mutant was used in all
studies as a negative control and is known to exhibit total blindness
(Bloomquist et al., 1988), to have severely reduced amounts of
phospholipase C activity in eye, and to lack detectable amounts of
norpA protein (Zhu et al., 1993). The norpA
mutant is known to express small
amounts of norpA RNA.
(
)The eya (eyes absent) mutant (Bonini et al.,
1993), which completely lacks compound eyes, was used for comparisons
to identify norpA gene products that are expressed in eye.
Cloning and Sequence Analysis of norpA
cDNAs
norpA cDNAs were obtained by reverse
transcribing Drosophila poly(A) RNA followed
by amplifying the norpA cDNA using the polymerase chain
reaction (RT-PCR) (Fuqua et al., 1990). Poly(A)
RNA used as template for RT-PCR was prepared from eya mutant body (thorax and abdomen) and leg according to methods
described by Shortridge et al.(1991). eya mutant was
used as a source for body RNA to obviate the problem of having the
preparations contaminated with tissue from the compound eye. Priming of
the poly(A)
RNA in reverse transcription reactions was
done using oligo-d(T) as well oligonucleotide primers that are
complementary to the norpA cDNA (Bloomquist et al.,
1988). Amplification of norpA cDNA by PCR was carried out
using three sets of oligonucleotide primers made to overlapping
segments of the norpA cDNA (nucleotides 594-1956,
1796-2551, 2417-4051; numbering according to Bloomquist et al. (1988)). During synthesis of the PCR primers,
restriction endonuclease sites were added to the 5`-ends to facilitate
cloning into plasmids (Kaufman and Evans, 1990). Standard methods
(Sambrook et al., 1989) were used for cloning the amplified norpA cDNA fragments into plasmid vectors, transforming the
recombinant plasmids into Escherichia coli, and determining
the nucleotide sequence of the cloned inserts. A set of 34
oligonucleotide sequencing primers made against various regions along
the norpA cDNA were used to facilitate sequencing of the
cloned DNAs.
Northern Blots
Northern blots were
carried out as described by Zhu et al.(1993) except that DNA
probes were used. Probes were constructed by amplifying an 80-bp region
of norpA cDNA corresponding to either exon 4 or exon 4A by the
polymerase chain reaction (Innis et al., 1990). The amplified
DNA fragments were purified by fractionation on a 2% agarose gel and
extraction of the DNA from an excised plug of agarose (Sambrook et
al., 1989). Radiolabeling of the DNA probes were carried out by
primer extension reactions using Klenow polymerase in the presence of
[P]dCTP (Sambrook et al., 1989).
RT-PCR Analyses
RNA used as template for
RT-PCR was prepared by grinding approximately 100 µg of tissue in 1
volume of homogenizing buffer (0.15 M sodium acetate, 5 mM EDTA, 1% SDS, 50 mM Tris-Cl, pH 9.0), volume of phenol,
and volume of a mixture of chloroform/isoamyl alcohol (100:1) (Montell et al., 1985) using Teflon pestles in 1.5-ml microcentrifuge
tubes (Kontes). The homogenates were extracted once with
chloroform/isoamyl alcohol, four times with phenol/chloroform (1:1),
and once with chloroform/isoamyl alcohol, followed by precipitation of
the RNA in ethanol (Sambrook et al., 1989). RNA was
resuspended in water, and its concentration was estimated from the
absorbance at 260 nm (Sambrook et al., 1989). Approximately 5
µg of RNA was used as template for reverse transcription. Next, a
1,363-bp norpA cDNA fragment (nucleotides 594-1956) that
encompasses the site of alternative splicing was amplified by PCR, and
the amplified products were analyzed by Southern blotting after
fractionating on 1% agarose gels (Sambrook et al., 1989). The
alternatively spliced norpA products were detected by
hybridizing to digoxygenin-labeled 80-bp probes that comprise either norpA exon 4 or exon 4A. Digoxygenin labeling of probes,
hybridization reactions, and visualization in nitro blue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl phosphate was carried out using the
Genius system (Boehringer Mannheim) according to the
manufacturer's instructions.
In Situ Hybridization
10-micron-thick
frozen sections of adult head (0-4 days after eclosion) were
prepared on glass slides as described by Zhu et al.(1993). In situ hybridization of norpA exon 4 or exon 4A
probes to the tissue sections were carried out using modifications of
the procedure of Tautz and Pfeifle(1989). Tissue sections were air
dried for approximately 4 h after cutting followed by fixation for 20
min in a freshly made solution of 4% paraformaldehyde in 1 PBS
(150 mM NaCl, 10 mM sodium phosphate, pH 7.22). After
fixing, tissues were rinsed in 3
PBS and then 1
PBS for
5 min each followed by dehydration in a graded (30-100%) ethanol
series (Hafen et al., 1983) and air drying for 2 h. The dried
tissue sections were treated by rehydrating for 5 min in 1
PBS
and then immediately incubating in 0.2 N HCl for 20 min. The
treated sections were then incubated in 2
SSC (1
SSC is
0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) for 30
min at 65 °C followed by digesting for 3 min in 1
PBS
containing 50 µg/ml proteinase K. Following proteinase K digestion,
sections were incubated for 2 min in 1
PBS containing 2 mg/ml
glycine and then fixed as before in a solution of 4% paraformaldehyde
in PBS. After rinsing in PBS, tissues were acetylated by incubation in
500 ml of 0.1 M triethanolamine-Cl, pH 8.0, to which 0.625 ml
of acetic anhydride had freshly been added. Sections were then rinsed
twice in PBS for 5 min each, 2
SSC for 5 min, and then
dehydrated in a graded ethanol series as before.
Denhardt's
solution (0.2 mg/ml Ficoll, 0.2 mg/ml bovine serum albumin, 0.2 mg/ml
polyvinylpyrrolidone), 5
SSC, 100 µg/ml salmon testes DNA,
and 25 µg/ml E. coli tRNA), to which the
digoxygenin-labeled DNA probe was added. Following hybridization, the
slides were washed by incubating at 37 °C in fresh hybridization
solution for 20 min followed by incubations in 50% hybridization
solution in 1
PBS and then 1
PBS for 20 min each.
Visualization of the digoxygenin-labeled probe was carried out
according to instructions in the Genius Kit (Boehringer Mannheim) using
nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate as
a chromogen.
Cloning and Sequence Analysis of Splice-variant
Subtypes of norpA
Zhu et al.(1993) proposed that
the norpA-encoded PLC is not exclusively utilized in
phototransduction because norpA expression is not solely found
in retina but is also present in tissues not known to contain visual
system machinery. Moreover, Zhu et al.(1993) identified at
least three major sizes of norpA transcripts (one in adult eye
and two in adult body), suggesting that norpA RNA is
alternatively spliced. To ascertain whether norpA RNA encodes
other subtypes of norpA protein, we utilized RT-PCR (Fuqua et
al., 1990) to obtain norpA cDNA from body (thorax and
abdomen) and leg and then compared the nucleotide sequences of the
amplified products to the head-specific cDNA sequence described by
Bloomquist et al. (1988).
Figure 1:
Nucleotide sequences of alternatively
spliced regions of the norpA gene. The nucleotide sequences of
exon 4 and exon 4A are shown in capitalletters with
intervening sequences at the junction sites shown in lowercaseletters. Numbersbelow the sequences correspond to the location of these exons in the norpA cDNA reported by Bloomquist et al. (1988).
Consensus sequences at intron/exon junctions in Drosophila (Mount et al., 1992) are shown above the
proposed intron/exon boundary regions. M indicates A or C, R indicates A or G, Y indicates C or T, W indicates A or T, and N is any nucleotide. Invariant
positions within the splicing consensus are overlined, and the angled line marks the splice site. Notice that the nucleotides
at the 5`- and 3`-boundaries of exon 4A match splicing consensus
sequences exactly, strongly supporting the proposal that exon 4A is an
alternatively spliced exon.
To determine whether
the putative exon 4A occurs within the norpA gene, we
performed DNA sequence analysis on the norpA genomic clone,
COSM1 (Bloomquist et al., 1988). Indeed, there is a region of
COSM1 DNA that matches exon 4A exactly (Fig. 1). Moreover, the
nucleotide sequences surrounding exon 4A match consensus sequences
found at intron/exon boundaries in other Drosophila genes (Fig. 1) (Mount et al., 1992). Furthermore, exon 4A
localizes approximately 300 bp upstream from exon 4 in the genomic DNA (Fig. 2, panelA). Since exon 4A is present
within norpA genomic DNA as well as norpA cDNAs from
body and leg, and the nucleotides surrounding exon 4A match those at
splice sites in other Drosophila genes, we conclude that exon
4A is an alternatively spliced exon that is retained in some norpA transcripts when exon 4 is excluded.
Figure 2:
Splice-variant subtypes of phospholipase C
encoded by the norpA gene. PanelA schematically illustrates the proposed mutually exclusive splicing
of norpA RNA and relative location of the exons within genomic
DNA. Exon 4A is located in the genomic DNA approximately 300
nucleotides upstream of exon 4. PanelB schematically
illustrates the deduced subtypes of norpA protein and the location of
amino acid differences. The highly conserved box X and box Y regions
(Rhee and Choi, 1992a, 1992b) that are present in major types of PLC
are represented by cross-hatched rectangles. Numbersabove the schematic of subtype I protein refer to
locations of significant regions within norpA protein (Bloomquist et al., 1988). PanelC shows an alignment of
amino acids within the alternatively spliced region of norpA protein
with corresponding regions in other PLC- isozymes (Dros plc21 (Shortridge et al., 1991), bovine PLC-
1 (Suh et
al., 1988), human PLC-
2 (Park et al., 1992), rat
PLC-
3 (Jhon et al., 1993), and bovine PLC-
4
(Ferreira et al., 1993, Lee et al., 1993a)). There
are 12 amino acids in this region that are identical in norpA subtype I
and subtype II proteins, and 4 of these are invariantly conserved among
the PLC-
enzymes (indicated by asterisksabove the alignment). Numbers refer to the location of the
amino acids within each PLC-
enzyme, and those in parentheses refer to tentative numbering assigned to rat PLC-
3 because
the N terminus of the mature protein is not yet identified (Jhon et
al., 1993).
We have designated the
putative norpA protein encoded by an exon 4-containing transcript as
subtype I protein and one that is encoded by an exon 4A-containing
transcript as subtype II. The differences between deduced norpA subtype
I and subtype II proteins occur within a small domain of 26 amino
acids(130-155), which localizes outside of the highly conserved
box X and box Y domains (Rhee et al., 1989) shared among major
types of PLC (Fig. 2, panelB). Within the
domain of 26 amino acids, 12 are identical in both subtypes of norpA
protein. Moreover, alignment of this region with similar domains in
other PLC- enzymes reveals that four of the conserved residues
occur at invariant positions (Fig. 2, panelC).
Northern Analysis of Adult RNA
When
radiolabeled norpA cDNA probes are hybridized to blots of
poly(A) RNA, three major transcripts can be
identified. As shown in Fig. 3(panelA), a
major norpA transcript that is 7.5 kb in length is easily
detected in wild-type head but is absent from head of eya mutant. The absence of the 7.5-kb transcript from eya head suggests that it is expressed in the compound eye. Two other
transcripts, one that is 5.5 kb and one that is 5.0 kb in length, are
visible in body. None of these transcripts are detectable in head or
body of norpA
mutant (Zhu et
al., 1993), suggesting that they are encoded by the norpA gene.
Figure 3:
Northern blot analysis of norpA transcripts in adult Drosophila tissues. Approximately 5
µg of poly(A) RNA was loaded in each lane and
probed with a 3.4-kb norpA cDNA fragment (nucleotides
1-3453) (A), an 80-bp exon 4 cDNA fragment (B),
or an 80-bp exon 4A cDNA fragment (C). Lane designations indicate RNA isolated from adult head or body (thorax
and abdomen) of wild-type (WT) Drosophila, eyes
absent (eya) mutant, or norpA mutant.
Mobility of RNA size standards (in kilobases) are indicated on the right. PanelsD-F show the result of
reprobing the blots with Drosophila RP49 cDNA (O'Connell
and Rosbash, 1984) as a control to test for RNA loading. The 3.4-kb norpA cDNA probe detects three major transcripts in adults (arrows in panelA). The largest is
approximately 7.5-kb in length and is visible in the wild-type head RNA lane but not in head of eya mutant (panelA). Two other transcripts of 5.5 and 5.0 kb (arrows) appear in wild-type body as well as body of eya mutant. None of these bands are visible in RNA obtained from head
or body of norpA mutant (data not shown). Notice that norpA subtype I (exon 4) probe detects the 7.5-kb transcript
in head but not the 5.5- or 5.0-kb transcripts in body (panelB). Conversely, the subtype II (exon 4A) probe detects
the 5.5- and 5.0-kb transcripts in body but not the 7.5-kb transcript
in head (panelC). None of these transcripts are
detectable in norpA mutant tissues. The significance of two
major bands (5.0 and 4.4 kb) in wild-type head (panelA) or smearing in the head RNA lanes is unknown
(Zhu et al., 1993). However, these other bands and smearing
are not visible in head RNA prepared from Canton-S strain (data not
shown). Differences in signal intensity between body lanes (panelsA and C) should not be taken to
reflect relative amounts of RNA but is likely due to experimental
fluctuations augmented by weak signals and extensive autoradiographic
exposure times.
To differentiate between which of the bands appearing on
the Northern analysis are norpA subtype I or subtype II
transcripts, we radiolabeled 80-mer DNA probes that correspond to norpA exon 4 or exon 4A and used them individually as probe in
the hybridization reactions. As shown in Fig. 3(panelB), exon 4 hybridizes to a 7.5-kb transcript that is
present in wild-type head but is absent from body as well as eya mutant head and norpA mutant head. This 7.5-kb transcript
appears to be the same 7.5-kb transcript identified by hybridization to
the much longer norpA DNA probes (Fig. 3, panelA). Conversely, exon 4A probe specifically hybridizes to
5.5- and 5.0-kb transcripts in wild-type body and eya mutant
body but does not detect transcripts in wild-type head or tissues of norpA mutant (Fig. 3, panelC). Again,
these appear to be the same transcripts detected by the longer norpA DNA probes. These data suggest that the distributions of
the two subtypes of norpA RNA are mutually exclusive and that norpA subtype I (exon 4) transcripts are expressed in eye and
that subtype II (exon 4A) transcripts are expressed in body. In similar
Northern analyses, norpA transcripts could not be detected at
earlier times of development when the 80-mer exon-specific DNA probes
were used (data not shown).
RT-PCR Analysis of Tissue and Developmental
Distribution of norpA RNA
To corroborate the results from
the Northern analyses and to identify tissues expressing norpA transcripts in too low abundance to detect on Northern blots, we
utilized RT-PCR (Fuqua et al., 1990) to amplify the norpA products from tissue- and developmentally specific RNA pools. RNA
used in the RT-PCR analyses was prepared from 0-12-h-old embryo,
12-24-h embryo, 3-, 5-, 7-, and 9-day-old developing organisms,
and head, leg, thorax, and abdomen of adult. The 3-, 5-, 7-, and
9-day-old Drosophila correspond approximately to first and
second instar larvae, third instar larvae, early pupae, and late pupae,
respectively (Zhu et al., 1993). A single primer set was used
in the PCR amplification reactions so that both splice-variant products
would be amplified as 1,363-bp DNA fragments (see ``Materials and
Methods''). Differentiation between subtype I and subtype II
amplification products was carried out by hybridization to individual
80-bp exon 4 or exon 4A probes.
Figure 4:
RT-PCR analyses showing tissue and
developmental distribution of norpA subtype I and II RNA.
Total RNA was extracted from tissues, reverse transcribed into cDNA,
and used as template for PCR to generate 1,363-bp norpA cDNA
products. The amplified DNAs were separated on 1% agarose gels, blotted
to nylon filters, and probed with a digoxygenin-labeled norpA exon 4 or exon 4A probe (see ``Materials and Methods''). Lanes are norpAHead, norpA, mutant
head; eyaHead, eyes absent mutant head; WT
Head, wild-type head; Thorax, wild-type thorax; Abdomen, wild-type abdomen; Leg, wild-type legs; 9Day, 7Day, 5Day, and 3Day, developing Drosophila (wild type) collected on the days indicated after
egg laying; 12-24 Hour, 12-24-h-old (wild-type)
embryos; 0-12 Hour, 0-12-h-old (wild-type)
embryos. Notice that the norpA subtype I (exon 4) probe easily
detects amplification products (arrow) from wild-type head and
to a lesser extent from head of norpA mutant and eya mutant (upperpanel). Conversely, subtype II
(exon 4A) probe detects amplification products from all of the tissues
tested, but signals are most intense in thorax and abdomen of adults (arrow, lowerpanel).
Spatial Localization of norpA Transcripts in
Head
To determine the spatial localization of norpA transcripts in adult head, the 80-bp exon-specific DNAs were used
as probe in tissue in situ hybridization reactions. As shown
in Fig. 5, norpA subtype I transcripts (exon 4) appear
to localize specifically in retina. norpA subtype II
transcripts (exon 4A) are not detectable in retina, but weak signals
are visible over the cortex of brain (Fig. 5). This localization
of norpA expression in brain and retina agrees with findings
of Zhu et al.(1993) that norpA protein is localized in brain
as well as retina. Moreover, these results agree with those from the
Northern analyses that show subtype I transcripts are localized in the
compound eye.
Figure 5:
Spatial localization of subtypes of norpA RNA in adult head. Digoxygenin-labeled norpA DNA probes were used for in situ hybridization analysis
on 10-µm tissue sections cut horizontally through head. Signals
were developed with an anti-digoxygenin antibody conjugated to alkaline
phosphatase using 4-nitro blue tetrazolium chloride and
5-bromo-4-chloro-3-indolyl phosphate as chromogen. Anterior is up in
both panels. The 80-bp norpA exon 4 probe (specific
for subtype I RNA) hybridizes to transcripts in retina (r),
but no signals are seen in brain (b) (panelA). Conversely, exon 4A probe (norpA subtype II)
does not detect transcripts in retina, but weak signals do appear over
the cortex of brain (arrows, panelB).
, PLC-
, PLC-
, and PLC-
) that can
be further divided into subtype groupings (Rhee and Choi, 1992a,
1992b). Recent findings indicate that subtype groupings may be further
divided into splice-variant products that are encoded by single genes.
Including the present work, four different PLC-
encoding genes
that produce splice-variant subtypes have been identified (Shortridge et al., 1991; Ferreira et al., 1993; Bahk et
al., 1994). Comparison of the splicing differences in these PLCs
reveals that all occur outside of the box X and box Y conserved regions
shared among the major PLC types (Fig. 6). A growing body of
evidence suggests that the regions outside of the box X and box Y
conserved domains comprise regulatory sites responsible for interacting
with signaling components such as G-proteins (Lee et al.,
1993b; Wu et al., 1993a, 1993b). It is thus likely that the
differences among subtypes caused by alternative splicing are a
reflection of the need for each subtype to interact with individual
signaling components of different signal-generating pathways. This idea
is supported by the results of the present work showing that the two
subtypes of norpA likely operate in completely different types
of signaling pathways, one being phototransduction and the others,
although yet unidentified, are not phototransduction pathways.
Figure 6:
Differences in splice-variant subtypes of
PLC-. Schematic representations of four different PLC-
enzymes are shown: norpA, Drosophila norpA-encoded
PLC (Bloomquist et al., 1988); plc21, Drosophila
plc21-encoded PLC (Shortridge et al., 1991); PLC-
1,
rat PLC-
1 (Suh et al., 1988; Bahk et al., 1994);
and PLC-
4, bovine PLC-
4 (Ferreira et al., 1993; Lee et al., 1993a). The cross-hatched rectangles represent the box X and box Y regions that are highly conserved
among major types of PLC (Rhee and Choi, 1992a, 1992b). Splice-variant
differences between individual subtypes are illustrated by stippledboxesabove or below each schematic. Numbersabove each schematic represent amino acid
positions at boundaries of the splice-variant domains, and those in parenthesesbelow the names indicate total length (in
amino acids) or the range of length of the PLC subtypes. Numbering of PLC-
4 is according to Lee et al. (1993a). Note
that all of the splicing differences occur outside of the box X and box
Y conserved domains.
Transformation of a norpA subtype I mini-gene into the germ
line of norpA mutant has recently been shown to rescue the
phototransduction defect concomitant with rescuing PLC activity in
retina (McKay et al., 1995). A future experiment may include
the expression of norpA subtype II protein in mutant retina to see if
it can substitute for subtype I and rescue the visual defect. If it
turns out that norpA subtype II protein is able to provide PLC activity
in retina without rescuing the light-evoked responses of
photoreceptors, it would demonstrate that the 14 amino acids found in
subtype I protein but absent from subtype II are critical for
interaction with components of the phototransduction machinery.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.