(Received for publication, December 9, 1996, and in revised form, February 11, 1997)
From the Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Canto Blanco, 28049 Madrid, Spain
Phosphorylation of the subunit of the
eukaryotic initiation factor 2 (eIF-2
) is one of the
best-characterized mechanisms for downregulating protein synthesis in
mammalian cells in response to various stress conditions. In
Drosophila, such a regulatory mechanism has not been
elucidated. We report the molecular cloning and characterization of
DGCN2, a Drosophila eIF-2
kinase related to yeast GCN2
protein kinase. DGCN2 contains all of the 12 catalytic subdomains
characteristic of eukaryotic Ser/Thr protein kinases and the conserved
sequence of eIF-2
kinases in subdomain V. A large insert of 94 amino
acids, which is characteristic of eIF-2
kinases, is also present
between subdomains IV and V. It is particularly notable that DGCN2
possesses an amino acid sequence related to class II aminoacyl-tRNA
synthetases, a unique feature of yeast GCN2 protein kinase. DGCN2
expression is developmentally regulated. During embryogenesis,
DGCN2 mRNA is dynamically expressed in several tissues.
Interestingly, at later stages this expression becomes restricted to a
few cells of the central nervous system. Affinity-purified antibodies,
raised against a synthetic peptide based on the predicted DGCN2
sequence, specifically immunoprecipitated an eIF-2
kinase activity
and recognized an ~175 kDa phosphoprotein in Western blots of
Drosophila embryo extracts.
Protein synthesis is mainly regulated at the initiation of
mRNA translation. The best-characterized mechanism of translational regulation in eukaryotes involves the phosphorylation of the
-subunit of eukaryotic initiation factor 2 (eIF-2
)1 (for review, see Refs. 1 and
2). Three distinct protein kinases regulate protein synthesis in
eukaryotic cells by phosphorylating the
subunit of eIF-2 at serine
51 (3-4). They are two mammalian eIF-2
kinases, the heme-regulated
inhibitor (HRI) and the double-stranded RNA-dependent
kinase (PKR), and the yeast GCN2 (3-4). These kinases contain twelve
conserved subdomains characteristic of all eukaryotic Ser/Thr protein
kinases (5) and reveal similarities that make them distinguishable from
other eukaryotic protein kinases. It is notable that eIF-2
kinases
possess relatively large insert sequences between catalytic subdomains
IV and V (3, 5). Even though the subdomain V is less conserved among
the eukaryotic protein kinases (5), the known eIF-2
kinases share
significant homology in this domain.
In contrast to the catalytic domains, the regulatory regions of the
eIF-2 kinases are very different (6, 7). HRI from rabbit and rat
contains two heme regulatory motifs, ACPYVM and RCPAQA, located within
the HRI kinase domain and that are not found in either PKR or GCN2 (6,
7). PKR has been cloned from human, mouse, and rat cells (7). The PKR
amino-terminal half contains three clusters of basic amino acids. The
first two contain two divergent copies of a double-stranded RNA binding motif that are required for RNA binding (3, 8). Yeast GCN2 contains a
530-amino acid sequence related to histidyl-tRNA synthetases (HisRS) in
the carboxyl-terminal region, adjacent to the kinase domain. This
domain is required for GCN2 activation in vivo (4, 9).
Phosphorylation of eIF-2 prevents initiation of protein synthesis by
sequestration of the guanine nucleotide exchange factor eIF-2B in an
inactive complex (1). The phosphorylation of eIF-2
was first
detected in rabbit reticulocyte lysates deprived of hemin. The absence
of hemin results in the activation of HRI (1, 6). PKR, constitutively
expressed in reticulocytes and inducible by interferon in other
mammalian cells, is activated by low concentrations of double-stranded
RNA in the presence of ATP (1, 8). The yeast GCN2 kinase is activated
by amino acid deprivation (9).
Until recently, phosphorylation of eIF-2 was believed to
nonspecifically down-regulate translation initiation, but recent results have suggested that it can up-regulate the synthesis of specific proteins (4, 7). In vivo and in vitro
experiments indicated that the activity of the yeast GCN2 kinase is
regulated through the interaction of the HisRS-related domain with
uncharged tRNAs that accumulate when cells are starving for amino
acids. In this case, eIF-2
phosphorylation enhances the translation of GCN4, a transcriptional activator of genes involved in
the biosynthesis of amino acids (4, 9, 10).
Previous studies have indicated that in vitro
phosphorylation of Drosophila eIF-2 by reticulocyte HRI
inhibits the exchange reaction catalyzed by mammalian eIF-2B (11). More
recent results have provided evidence that translational regulation by
phosphorylation of eIF-2
and sequestration of eIF-2B can operate in
insect cells (12), suggesting that an endogenous eIF-2
kinase may be
present in such cells. However, to date, no eIF-2
kinase activity
has been detected in Drosophila. Here we report the cloning
of a Drosophila melanogaster eIF-2
kinase (DGCN2). Amino
acid sequence analysis indicates that DGCN2 is closely related to yeast
GCN2 kinase.
Genomic DNA was
prepared from adult flies as described (13). Sense
(5-CTBYWYATYCARATG-3
) and antisense (5
-CCAAARTCWCCDATYTT-3
) degenerated oligonucleotide primers were synthesized based on regions
of homology between all known eIF-2
kinases. Polymerase chain
reaction was performed using each primer at 5 µM and 500 ng of genomic DNA. A "hot start" PCR was performed by adding the DNA polymerase after an initial denaturation step of 5 min at 95 °C
and then 35 cycles consisting of 95 °C for 1 min, 37 °C for 2 min, and 72 °C for 2 min, followed by a final step of 72 °C for
10 min. The amplification was carried out in an Ericomp TwinBlock System (Ericomp, San Diego, CA). The reaction products were gel purified and subcloned into a pCRII vector using a TA cloning kit
(Invitrogen).
A cDNA library in Uni-Zap
XR, made from 2- to 14-h-old embryos (Stratagene), was screened
according to standard procedures (14). 1 × 106
plaque-forming units were transferred to Hybond-C nitrocellulose membranes (Amersham Corp.). The 278-bp DGCN2 genomic DNA
fragment was radiolabeled with [-32P]dCTP using a
random primed DNA labeling kit (Boehringer Mannheim). The membranes
were prehybridized 2 h at 42 °C in a solution containing 5 × saline/sodium/phosphate/EDTA, 50% formamide, 0.1% SDS, 5 × Denhardt's solution, and 100 µg/ml denaturated salmon sperm DNA. Hybridization was carried out overnight at 42 °C in this solution containing a 32P-labeled DGCN2 probe (1 × 107 cpm/ml). The membranes were subjected to high
stringency washings and processed for autoradiography. Positive phages
were purified by successive rounds of screening. Insert cDNAs in
positive phages were excised in vivo by plasmid rescue of a
pBlueScript SK
phagemid, after superinfection with a
helper phage, as recommended by the manufacturer (Stratagene). A second
cDNA library in a
ZAPII vector, made from 0- to 4-h-old embryos
(Stratagene), was screened, as described above, using the 1131-bp
DGCN2 cDNA isolated from the first library.
The cDNA inserts were sequenced across both strands by the dideoxy chain termination method (15) using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase FS and the Automatic Sequencer System 373A (Applied Biosystem). All primers for sequencing were from Isogen Bioscience (Amsterdam). Sequence homology searches were performed using BLAST (16) and FASTA (17) programs. Similarity and identity values were obtained by using the BESTFIT and GAP programs (Wisconsin Package, Genetics Computer Group, University of Wisconsin, Madison).
Rapid Amplification of cDNA 51 µg of poly(A)+ RNA of 0-12 h
Drosophila Oregon R embryos, obtained according to Campuzano
et al. (18), was reverse-transcribed using the Marathon
cDNA amplification kit (CLONTECH) according to
the instructions of the manufacturer. To avoid amplification of
nonspecific products, "hot start" PCR was performed by adding TaqStart antibody (CLONTECH) to the DNA polymerase
(Expand long template PCR system, Boehringer Mannheim). Amplifications
were performed in a GeneAmp 2400 thermal cycler (Perkin-Elmer) as
recommended by the Marathon kit protocol. The antisense primer
(5-ACATTCTCATGATTCAGCCGCGAAAGAAGC-3
), complementary to nucleotides
2033-2062 of DGCN2 cDNA was used to amplify the 5
-end,
in combination with the adaptor primer AP1 supplied with the Marathon
kit. The sense primer (5
-ACTCACCGCTCTTATCCCCACTTTCCGCAA-3
) complementary to nucleotides 3289-3318 of DGCN2 cDNA
was used to amplify the 3
-end in combination with the adaptor primer
AP1. The 5
and 3
RACE products were subcloned into the pCRII vector and sequenced across both strands. The sequence of three independent cDNA clones of each product were compared to detect errors that could have occurred during the reverse-transcription and the PCR amplification.
Poly(A)+ RNA was prepared as described (18). Five µg of poly(A)+ RNA from each developmental stage were separated on a formaldehyde/agarose gel, transferred to nylon membrane, hybridized, and exposed as described (14).
Whole-mount Embryo RNA in Situ HybridizationLocalization of RNA in whole-mount embryos with antisense digoxigenin-labeled probes was performed essentially as described (19).
Antibodies Against a Synthetic DGCN2 PeptideBased on the
DGCN2 cDNA coding sequence, a synthetic peptide
(CG-SQSQQDLSVKPAK) was made, corresponding to amino acids 610-622 (see
Fig. 1) with two additional residues (CG) in its
NH2-terminal end. The peptide was synthesized by Fmoc
(N-(9-fluorenyl)methoxycarbonyl) chemistry using an Applied
Biosystems 431A automated solid-phase synthesizer in the Protein
Chemistry Facility of the Centro de Biología Molecular
"Severo Ochoa." The purity of the peptide, monitored by reverse
phase high performance liquid chromatography, was 95%, and peptide
identity was confirmed by mass spectrometry. This peptide was coupled
by the terminal cysteine residue to keyhole limpet hemocyanin
(Calbiochem), and rabbits were immunized as described (20). Serum was
purified by affinity chromatography by coupling synthetic peptide
through the cysteine terminal residue to Sulfolink beads, according to
the instructions of the manufacturer (Pierce). The specific IgGs were
further purified by affinity chromatography on a protein A-Sepharose
column as described (21). For simplicity, these affinity purified
anti-DGCN2-peptide antibodies will be named in the future as anti-DGCN2
antibodies.
Immunoprecipitation and Drosophila eIF-2
One g of dechorionized 0- to 12-h-old
Drosophila Oregon R embryos was suspended in 5 ml of lysis
buffer containing 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 2 mM
EDTA, 20 µg/ml DNAase, and 20 µg/ml RNAase. The embryos were
homogenized with 20 strokes in a Dounce homogenizer (Type B pestle) and
incubated 30 min on ice, and then the homogenate was centrifuged for 10 min at 4 °C, at 14,000 × g. The supernatant was
precleared with 30 µl of protein A-Sepharose (Sigma) that was
equilibrated in TBS buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl) containing 1% Nonidet P-40. After incubation for
1 h at 4 °C with continuous mixing, the supernatant was
recovered and incubated with anti-DGCN2 antibodies (10 µg of IgG) for
1 h at 4 °C. In control experiments, the peptide immunogen (90 µg/ml) was also included in this incubation just before the antibody
was added. Protein A-Sepharose (40 µl), equilibrated in TBS
containing 2% bovine serum albumin and 1% Nonidet P-40, was then
added, and the reaction mixture was incubated for 1 h at 4 °C
with continuous mixing. The immunoprecipitates were washed three times
with lysis buffer and three more times with 20 mM Tris-HCl,
pH 7.5. The immunoprecipitates were then assayed for their ability to
phosphorylate eIF-2. Assay mixtures (30 µl) containing 10 µl of
pellet suspension were preincubated for 10 min at 30 °C in the
presence of buffer containing 20 mM Tris-HCl, pH 7.5, 0.6 mg/ml bovine serum albumin, 50 µM ATP, and 5 mM Mg(OAc)2. All samples were
subsequently incubated for 20 min at 30 °C in the presence of
purified rabbit reticulocyte eIF-2 (0.5 µg) as substrate and 5 µCi
of [-32P]ATP (3000 Ci/mmol). Incubations were
terminated by the addition of SDS sample buffer, and samples were
analyzed by electrophoresis on 7.5% SDS-PAGE as described (22). After
the electrophoresis, the proteins were transferred to an Immobilon-P
membrane (Millipore). The membrane was exposed to x-ray film for
visualizing phosphorylated proteins and subsequent immunodetection with
anti-DGCN2 antibodies.
Degenerate oligonucleotide
primers were synthesized based on regions of homology between all
previously known eIF-2 kinases. These primers correspond to
conserved sequences L(F/H)IQM (kinase subdomain V) and KIGDFG (kinase
subdomain VII). Despite the fact that subdomain VII is well conserved
in all protein kinases, any one of the amplified products had to belong
to an eIF-2
kinase gene because the subdomain V is specific to
eIF-2
kinases (23). Following PCR with the indicated primers on
genomic DNA, a product of 278 bp was subcloned and sequenced. The
nucleotide and the amino acid analyses of this product revealed a gene
fragment that showed an ORF (nucleotides 2397-2608) displaying 73.3%
similarity to yeast GCN2. This amplified gene fragment contained an
intron of 64 nucleotides interrupting the ORF. The intron boundaries presented the donor/acceptor splice signals (GT/AG) and were inserted after the position 2437 of the DGCN2 cDNA (data not
shown). Because of its potential authenticity, this gene fragment was
used to screen a cDNA library from Drosophila embryos.
From 1 × 106 phages, one positive plaque (clone N1)
was purified, excised into pBlueScript SK
and sequenced.
It contained an ORF of 1131 bp, that was 56% similar to yeast GCN2.
The N1 cDNA was used to screen a second embryo cDNA library,
leading to the isolation of three independent positives. One of the
clones (S24), that overlapped 534 nucleotides with the 3
-N1 clone,
contained an ORF of 1060 bp. This ORF shared homology with eIF-2
kinase catalytic domains and with motifs m1 and m2 of HisRS.
Amino acid sequence analyses of N1 and S24 cDNA clones revealed
that they lacked the full coding sequence. To get the missing 5- and
3
-ends, additional sequences were obtained by 5
and 3
RACE
experiments producing two additional cDNA clones, AR5 and DR3, of
2062 and 2433 bp, respectively. Taken together, all the overlapping
DGCN2 cDNA clones, (AR5, N1, S24, DR3) span a total of
5721 nucleotides (GenBankTM accession number U80223[GenBank]). The
resulting full-length cDNA displays a large ORF of 4767 bp
(nucleotides 318-5084) flanked by 317 bp of a 5
non-coding sequence
and 637 bp of a 3
non-coding sequence, which contains a putative
polyadenylation signal (AATAAA, nucleotides 5679-5684). The first
methionine codon of the ORF (ATG, nucleotides 318-320) is likely to
represent the translational start since it matches the
Drosophila consensus for translational initiators at
positions -2, -1, +4, and +5 (24). Moreover, in-frame termination
codons are present upstream from the initiation codon, suggesting that
this is indeed the authentic start site. The full-length DGCN2 cDNA encodes for a protein of 1589 amino acids
(Fig. 1), with a predicted molecular mass of 178.7 kDa.
Note that yeast GCN2 is a 1590-amino acid protein with a molecular mass
of about 180 kDa (9). Southern blot analysis of Drosophila
genomic DNA revealed that DGCN2 is present as a single copy
gene (data not shown).
The eIF-2 kinase domain of DGCN2 (residues 523-898)
contains the 12 conserved catalytic subdomains of Ser/Thr protein
kinases (Fig. 1). This domain, as in HRI and yeast GCN2, possesses a
large (94 residues) insert between subdomains IV and V (Fig.
2). DGCN2 kinase domain is more closely related to the
rat PKR (there are 40.2% identities and 61.3% similarities) and the
yeast GCN2 (39.4% identities and 60.3% similarities) although it
shares high homology with all of the known eIF-2
kinases (37 to 40%
identities and 56 to 61% similarities).
Downstream from the eIF-2 kinase domain, DGCN2 contains a 519 amino
acid sequence (residues 919-1437) related to histidyl-tRNA synthetases
(HisRS) (Fig. 2). HisRS enzymes share three relatively short sequence
motifs (m1, m2, and m3) (Fig. 1; Ref. 25). Like yeast GCN2 (26), the
highest degree of similarity between DGCN2 and HisRS occurs in the
roughly 360 amino acids stretching from motif 1 to motif 3 (47 to 50%
similarities and 22 to 24% identities).
In addition, the amino-terminal portion of DGCN2 contains a sequence (residues 304-518) related to subdomains III to XI of eukaryotic protein kinases (5). The partial kinase segment (Fig. 2) is comparable with that observed in yeast GCN2 kinase, although in the case of yeast, the sequences in the amino-terminal segment are only related to subdomains VIb to XI (10). Taken together, these data indicate that DGCN2 is very closely related to the yeast GCN2 kinase.
Developmental Expression of DGCN2 TranscriptsNorthern blot
analysis of poly(A)+ RNA isolated from different
developmental stages revealed a unique transcript of approximately 6.5 kb. The transcript is expressed throughout development and with two
major peaks of accumulation, one during early embryogenesis and the
second in the adult stage (Fig. 3).
To determine the embryonic transcriptional regulation of the
DGCN2 gene, we performed in situ hybridization on
embryos with antisense digoxigenin-labeled probes. Fig.
4 depicts the spatio-temporal pattern of
DGCN2 expression in embryonic stages classified according to
Campos-Ortega and Hartenstein (27). DGCN2 mRNA can be
first detected at high levels during stages 2 and 3 prior to
cellularization of blastoderm nuclei (Fig. 4A). Later, at
the stage of syncytial blastoderm (late 4), the mRNA concentrates
peripherally in the so-called cortex (Fig. 4B). By stage 5, when the cellularization is complete, the concentration of the mRNA
in the cortex is more evident (Fig. 4, C and D).
By the onset of gastrulation, DGCN2 expression concentrates
in several areas of cell movement, including all the furrows and the
proctodeal primordium (Fig. 4, E and F). During
the stages of germ band elongation, the mRNA accumulates in the
primordia of the gut (anterior and posterior) and in the mesoderm (Fig.
4, G and H). This mesodermal staining diffuses during stages 11 and 12, corresponding to the reorganization of this
mesodermal germ layer. By the end of the stage 12, the mRNA accumulates in the anterior and posterior midgut as well as in areas of
the primitive brain (supraesophageal ganglion) and in the central
nervous system (CNS) (Fig. 4I). During stage 13 and extending into
later stages, accumulation of DGCN2 transcripts are seen
throughout the CNS including the supraesophageal ganglia and the
ventral cord. Although by stage 14 where many cells of the ventral cord
express DGCN2 transcripts (Fig. 4J), in later stages, this
expression becomes restricted to a few cells (four per neuromer) that
probably correspond to neurons (Fig. 4, K and L).
Characterization of an eIF-2
To determine that DGCN2 is an eIF-2 kinase, we
immunoprecipitated DGCN2 from 0- to 12-h-old Drosophila
embryo extracts by using the anti-DGCN2 antibodies. The isolated immune
complexes were incubated with [
-32P]ATP in the
presence or absence of highly purified rabbit reticulocyte eIF-2 as a
substrate, and the radiolabeled products were analyzed by SDS-PAGE and
Western blot as described under "Experimental Procedures." In
control experiments, the reticulocyte heme-reversible HRI (20) was
incubated in the presence (Fig. 5, A and
B, lane 1) or absence (lane 2) of
eIF-2. In addition to the eIF-2
phosphorylation, the phosphorylated
HRI was observed. Note that the eIF-2 preparation is free of endogenous
protein kinases (Fig. 5A, lane 3). As expected, the anti-DGCN2 antibodies did not recognize either HRI (Fig.
5B, lanes 1 and 2) or eIF-2
(lane 3).
A single labeled polypeptide with a molecular mass of about 175 kDa was
produced by DGCN2 immune complexes (Fig. 5A, lane 5). This phosphopolypeptide was recognized by the anti-DGCN2
antibodies in Western assays (Fig. 5B, lanes 4 and 5). More importantly, DGCN2 immune complexes
phosphorylated the subunit of the eIF-2 (Fig. 5A,
lane 4). The immunoprecipitation was specific because it was
prevented by addition of the peptide immunogen in the
immunoprecipitation assay (Fig. 5, A and B,
lanes 6 and 7). All together, our results indicate that DGCN2 is, indeed, an eIF-2
kinase of Drosophila melanogaster and is phosphorylated in the immune complexes. This reaction may occur by autophosphorylation as in yeast GCN2 (28).
Inactivation of the eIF-2 function by phosphorylation of its subunit is the best-characterized mechanism for regulating total
protein synthesis in mammalian cells (1, 2). The regulation of
GCN4 expression in yeast represents gene-specific
translational control by phosphorylation of eIF-2
. This unique
response depends on four short upstream ORFs in the leader sequence of
GCN4 mRNA (9). As mentioned above, previous studies
provided evidence that protein synthesis in insect cells can be
regulated by phosphorylation of eIF-2
and inhibition of eIF-2B
activity (11, 12).
We have cloned a Drosophila eIF-2 kinase through a
PCR-based strategy. The data reported here strongly suggest that we
have succeeded in characterizing the first homologue of yeast GCN2 from
Drosophila melanogaster.
The amino-terminal portion of yeast GCN2 contains a sequence related to subdomains VIb to XI of eukaryotic protein kinases. Similarly, DGCN2 contains a sequence, in the amino-terminal region, related to subdomains III to XI of eukaryotic protein kinases (5). It is especially noteworthy that this partial kinase domain of DGCN2 (Fig. 2) displays a significant homology to mammalian Raf proto-oncogene and to yeast BCK1, which are involved in the mitogen-activated protein kinase signaling cascades in vertebrates and Saccharomyces cerevisiae, respectively (29). Our finding raises the possibility that DGCN2 may also play additional roles in the signal transduction pathways. In this respect, it has been reported that interleukin 3 stimulates protein synthesis by regulating PKR (30) and that PKR is autophosphorylated in vivo in response to platelet-derived growth factor (31).
DGCN2 mRNA is dynamically expressed during embryogenesis. During gastrulation, high levels are detected in the furrows, active areas of cell rearrangements during which cells change their neighbors and environments, allowing interactions and inductive processes between various regions of the developing body (32). Since these morphogenetic movements represent the first morphological manifestation of cell fate and differentiation programs, the localized expression of DGCN2 in these areas may indicate a putative role in some of these processes, for example, helping to differentiate the three different germ layers of mesoderm, endoderm, and ectoderm. In fact, the early expression in the mesoderm suggests that DGCN2 could be involved in determining mesoderm germ layer identity during early stages of development.
At late stages of embryogenesis, during the formation of the axon scaffold, DGCN2 expression concentrates in the nervous system. Interestingly, when the commissures are almost completely formed, this expression is selectively restricted to a few cells of the ventral cord (two pairs of cells per neuromer) that probably correspond to neurons. This surprising selectivity is also consistent with the idea that DGCN2 might be involved in determining neural cell identity for these cells.
Anti-DGCN2 antibodies specifically immunoprecipitated a polypeptide of
about 175 kDa from Drosophila embryo extracts that specifically phosphorylated the subunit of eIF-2 (Fig.
5A). This polypeptide is a phosphoprotein and is identified
by Western blotting with the antibodies (Fig. 5B). The same results
were obtained previously when yeast GCN2 immune complexes were used (28, 33).
Collectively, our results indicate that DGCN2 is a
Drosophila eIF-2 kinase homologue of the yeast GCN2
kinase. In the future, it will be important to know whether uncharged
tRNA or other activators act as a signal to regulate DGCN2. The fact
that HRI, as well as PKR, functionally substituted for GCN2 in the
GCN4 translational control of yeast (34) may suggest that
DGCN2 will play a similar role. It is noteworthy that amino acid
starvation or a defective aminoacyl-tRNA synthetase leads to increased
levels of eIF-2
phosphorylation in mammalian cells (35) although the
kinase responsible for that has yet to be identified. It will also be interesting to see whether translation of any Drosophila
embryonic gene is modulated by a system of short upstream ORFs, like
that present at GCN4, in response to modest changes in the
level of eIF-2
phosphorylation.
Several recent studies suggest a role for eIF-2 phosphorylation in
the control of cell growth and differentiation (7). Although they
mostly concern PKR, it will be of great interest to determine whether
DGCN2 is involved in the translational control of mRNAs that encode
key growth regulating proteins and to determine whether eIF-2
phosphorylation has any role in cell-cycle control and cellular
differentiation in Drosophila.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U80223[GenBank].
We are grateful to S. Campuzano for poly(A)+ RNA fractions and to E. Martínez-Salas and J.M. Sierra for critical reading of the manuscript. We also thank J. Vázquez and E. Madueño for valuable technical advice.