Interallelic complementation at the Drosophila melanogaster gastrulation defective locus defines discrete functional domains of the protein.

The gastrulation defective (gd) locus encodes a novel serine protease that is involved in specifying the dorsal-ventral axis during embryonic development. Mutant alleles of gd have been classified into three complementation groups, two of which exhibit strong interallelic (intragenic) complementation. To understand the molecular basis of this interallelic complementation, we examined the complementation behavior of additional mutant alleles and sequenced alleles in all complementation groups. The data suggest that there are two discrete functional domains of Gd. A two-domain model of Gd suggesting that it is structurally similar to mammalian complement factors C2 and B has been previously proposed. To test this model we performed SP6 RNA microinjection to assay for activities associated with various domains of Gd. The microinjection data are consistent with the complement factor C2/B-like model. Site-directed mutagenesis suggests that Gd functions as a serine protease. An allele-specific interaction between an autoactivating form of Snake (Snk) and a gd allele altered in the protease domain suggests that Gd directly activates Snk in a protease activation cascade. We propose a model in which Gd is expressed during late oogenesis and bound within the perivitelline space but only becomes catalytically active during embryogenesis.

Gastrulation defective, a complement factor C2/B-like protease, interprets a ventral prepattern in Drosophila.

gastrulation defective (gd) encodes a serine protease required for specification of dorsal-ventral cell fates during Drosophila embryogenesis. Using RNA microinjection, I show that wild-type gd RNA can restore ventrolateral pattern elements with correct polarity with respect to egg shape in embryos lacking gd function. While low RNA concentrations restore ventrolateral pattern elements, higher concentrations ventralize the embryo. Gastrulation defective concentration has a rate-limiting effect on the domain of high Dorsal concentration but little effect upon the slope of the gradient. In embryos from pipe-null females, much higher RNA concentrations generate an ectopic axis oriented with respect to the site of injection. The data suggest that the Dorsal gradient is not directly determined by asymmetric cues in the eggshell but arises de novo within the perivitelline space as a consequence of self-regulatory properties of the protease cascade. A homology to the mammalian complement factors C2 and B is also described.

Autoproteolysis and feedback in a protease cascade directing Drosophila dorsal-ventral cell fate.

Three serine protease zymogens, Gastrulation defective (GD), Snake (Snk) and Easter (Ea), and a nerve growth factor-like growth factor ligand precursor, Spaetzle, are required for specification of dorsal- ventral cell fate during Drosophila embryogenesis. The proteases have been proposed to function in a sequential activation cascade within the extracellular compartment called the perivitelline space. We examined biochemical interactions between these four proteins using a heterologous co-expression system. The results indicate that the three proteases do function in a sequential activation cascade, that GD becomes active and initiates the cascade and that interaction between GD and Snk is sufficient for GD to cleave itself autoproteolytically. The proteolytically active form of Ea cleaves GD at a different position, revealing biochemical feedback in the pathway. Both GD and Snk bind to heparin-Sepharose, providing a link between the pipe-defined ventral prepattern and the protease cascade. Our results suggest a model of the cascade in which initiation is by relief from inhibition, and spatial regulation of activity is due to interaction with sulfated proteoglycans.

Multiple isoforms of the Drosophila Spätzle protein are encoded by alternatively spliced maternal mRNAs in the precellular blastoderm embryo.

The spätzle gene is required for proper specification of positional information along the dorsal-ventral axis of the Drosophila embryo and for induction of the innate immune response to fungal infection. It has been shown to encode a precursor of a Nerve Growth Factor-like ligand which is also a member of the cysknot protein superfamily. In dorsal-ventral patterning, the most widely accepted model of the pathway places Spätzle at the end of a ventrally restricted protease cascade that results in the proteolytic processing of the precursor form of Spätzle to an active ligand which is thought to bind to the Toll receptor. Here we show that the spätzle gene encodes at least ten different protein isoforms as a result of complex alternative splicing in precellular blastoderm embryos. Multiple transcripts are clearly present up until the time of cellularization, at which point most transcripts can no longer be detected. Nine isoforms were expressed and at least five are efficiently secreted in a heterologous protein expression system. RNA microinjection experiments demonstrate that three isoforms completely rescue embryos from spätzle null mothers, while most of the others rescue to a lesser extent. The phenotypic rescue activities of several isoforms and the relevance of these isoforms to the generation of the ventralizing signal are discussed.

Conserved Spätzle/Toll signaling in dorsoventral patterning of Xenopus embryos.

The Spätzle/Toll signaling pathway controls ventral axis formation in Drosophila by generating a gradient of nuclear Dorsal protein. Dorsal controls the downstream regulators dpp and sog, whose patterning functions are conserved between insects and vertebrates. Although there is no experimental evidence that the upstream events are conserved as well, we set out to ask if a vertebrate embryo can respond to maternal components of the fly Dorsal pathway. Here we demonstrate a dorsalizing activity for the heterologous Easter, Spätzle and Toll proteins in UV-ventralized Xenopus embryos, which is inhibited by a co-injected dominant Cactus variant. We conclude that the Dorsal signaling pathway is a component of the conserved dorsoventral (d/v) patterning system in bilateria.

Proteolytic processing of the Drosophila Spätzle protein by easter generates a dimeric NGF-like molecule with ventralising activity.

Biochemical interactions underlying the generation of the ventralising signal during Drosophila embryogenesis were investigated by the expression of recombinant Easter and Spätzle proteins. An active form of Easter protease cleaves the Spätzle protein, generating a carboxyterminal polypeptide fragment which, when microinjected into the perivitelline space of a spätzle deficient embryo, directs production of ventrolateral pattern elements. This Spätzle carboxyterminal fragment is a disulfide-linked dimer and modelling suggests that the core disulfide bonds and dimer arrangement of this fragment are highly similar to vertebrate nerve growth factor. Thus Spätzle is a member of a new family of neurotrophin-like signalling molecules in invertebrate development.

Horseshoe crab coagulogen is an invertebrate protein with a nerve growth factor-like domain.

The rapid clotting of the horseshoe crab hemolymph is essential for both its host defense and hemostasis. It is mediated by the clotting cascade system which consists of four serine proteinase zymogens and the clottable protein coagulogen. Coagulogen, the target protein of the cascade, is converted to an insoluble gel upon activation of the cascade, giving rise to clot formation. Thus this cascade is reminiscent of the mammalian blood coagulation leading to fibrin clot. The structural analysis of coagulogen revealed a polypeptide fold and disulfide bridge pattern in the C-terminal half of the molecule very similar to nerve growth factor (NGF). This finding assigns coagulogen as the first structurally characterized invertebrate protein which belongs to the cystine knot superfamily. The putative structural similarity of coagulogen and the Drosophila morphogen Spaetzle as well as the homology of its processing proteinases suggests a common origin of the two functionally different cascades. This would exemplify a divergent evolution of two proteinase cascades having totally different functions from common ancestors in a long history of evolution.

Spatial regulation of Drosophila snake protease activity in the generation of dorsal-ventral polarity.

Positional information along the dorsal-ventral axis of the Drosophila embryo is acquired through a signal transduction pathway which employs a extracellular protease cascade. The sequential activation of serine protease zymogens results in the ventrally localized production of a ligand in the perivitelline space of the embryo. Snake is one of several serine proteases which function in generating the ventralizing signal. Here, we investigate the biochemical properties of Snake in vivo and in vitro using recombinant forms of the protease. Wild-type Snake zymogen completely rescues embryos from snake null females when microinjected into the perivitelline space. Biochemical evidence for a covalently associated two-chain form of the activated protease is presented. The contribution of the activation peptide region to zymogen activation was addressed using site-directed mutagenesis. The phenotypic rescue properties of an autoactivated form of Snake reveal that the covalently associated proenzyme polypeptide chain suppresses a dominant effect associated with the activated catalytic chain alone. Recombinant active catalytic chain was produced and found to be short lived as a recombinant protein. These results suggest a model in which the proenzyme polypeptide both stabilizes and targets the Snake catalytic chain to a ventrally localized activation complex within the perivitelline space.

Mutational analysis of the Drosophila snake protease: an essential role for domains within the proenzyme polypeptide chain.

Two genes involved in the generation of dorsoventral asymmetry in the developing Drosophila melanogaster embryo, snake and easter, encode the zymogen form of serine proteases. Mutant alleles of snake were cloned and sequenced revealing two types of lesions: point mutations which alter the amino acid sequence (snk073 and snkrm4) and point mutations which alter the splicing (snk229 or snk233) of intron 1 of the mRNA from the normal 3' end of the intron to a cryptic site. snake mutant embryos derived from homozygous mothers can be fully rescued by injection of RNA transcripts of the wild-type snake cDNA. RNA phenotypic rescue and site-directed mutagenesis experiments indicate that snake requires the serine, histidine and aspartic acid of the catalytic triad for normal activity. Deletion experiments show that an acidic proenzyme domain is required for snake rescue activity to be uniformly distributed throughout the embryo. A second proenzyme domain, called the disulfide knot, appears to be essential for normal regulation of activity of the snake catalytic chain. Transcripts encoding only the proenzyme polypeptides of either snake or easter can dorsalize wild type embryos. We propose a model in which the proenzyme determinants of both the snake and easter enzymes mediate interaction between the serine proteases and other components of the dorsal-ventral patterning system.

Ventralizing signal determined by protease activation in Drosophila embryogenesis.

Specification of dorsal-ventral cell fate during Drosophila embryogenesis is mediated by a signal transduction pathway. Asymmetry of cell fates arises through the spatially restricted production of a ligand in an extracellular compartment called the perivitelline space. The snake and easter genes are required for the production of the ligand and they encode the proenzyme form of secreted extracellular serine proteases. We have examined the effect of producing a preactivated form of the snake protease on the generation of dorsal-ventral polarity. SP6 RNA microinjection experiments reveal that different cell fates acquired at cellular blastoderm can be specified by the amount and spatial distribution of activated snake protein. Our results support a protease cascade model in which localized activation of uniformly distributed protease proenzymes leads to the spatially restricted production of ligand in the perivitelline space on the ventral side of the embryo.

A gene required for the specification of dorsal-ventral pattern in Drosophila appears to encode a serine protease.

The maternal effect gene snake is required for the establishment of the dorsal-ventral axis during the embryonic development of Drosophila. The molecular cloning of the gene and analysis of a complementary DNA sequence suggest that the gene encodes a serine protease which is structurally similar to proteases involved in blood clotting, peptide processing, and complement fixation pathways.

A Drosophila melanogaster transfer RNA gene cluster at the cytogenetic locus 90BC.

We report the isolation and characterization of a 31 X 10(3) base-pair DNA segment containing a cluster of Drosophila melanogaster transfer RNA genes from the cytogenic locus 90BC. Seven distinct coding regions have been identified in a 15 X 10(3) base-pair DNA segment. These coding regions contain at least ten tRNA structural genes and include sequences encoding the following tRNAs: tRNAval, tRNAPro, tRNAAla and tRNAThr. We have determined the nucleotide sequence of six of these structural genes and their flanking regions. These genes do not contain intervening sequences nor do they encode the terminal CCA. The tRNA genes from the locus also appear to be functional when assayed in a Xenopus germinal vesicle in vitro transcriptional system.

Internal promoter elements of transfer RNA genes are preferentially exposed in chromatin.

We have examined the chromatin organization of a cluster of transfer RNA genes at the cytogenic locus 90BC on the right arm of the third Drosophila melanogaster chromosome. We find that the internal promoter sequences are preferentially exposed to micrococcal nuclease and DNase I in chromatin. Moreover, these exposed sequences have an unusual single strand nuclease-sensitive conformation.

Intimate association of 5S RNA and tRNA genes in Drosophila melanogaster.

In this communication, we report the isolation of seven new recombinants derived from the 5S gene locus of Drosophila melanogaster. These recombinants can be divided into three different classes. There are four clones derived from within the 5S gene cluster, two which contain non-5S sequences representing the 5'flanking segment of the gene array and have a structure similar to 12D8 described by Artavanis-Tsakonas et al. (1977), and finally one (22A8) which is shown to contain a DNA segment that is located adjacent to the 3' end of the 5S gene cluster. Analysis of these recombinants supports the model in which all 5S genes of our D. melanogaster Oregon-R wild type strain are arranged in one uninterrupted cluster and are transcribed in the same direction. Interestingly, the 2.5 kb of non-5S RNA coding sequences on the recombinant derived from the 3' edge of the cluster contains at least four genes coding for tRNAs and one of these is located less than 300 bp downstream from the last 5S transcription unit. These tRNA genes are shown to be functional on the basis of the ability of 22A8 DNA to direct the synthesis of tRNA in an in vitro transcription system.

Changes in leucine aminotransferase isozymes by viral transformation and its correlation with the isozyme changes occurring during differentiation.

The isozyme pattern of leucine aminotransferase (EC 2.6.1.6) in various cell lines and their viral-transformed derivatives were examined. The Wistar 3C rat liver cell line was found to contain only isozyme I, while its simian virus 40-transformed counterpart had isozyme III in addition to isozyme I. A spontaneously transformed late passage clone of these liver cells was also found to have acquired isozyme III. Polyoma virus-transformed baby hamster kidney cells were also found to contain a greater predominance of isozyme III than their normal untransformed counterpart. Examination of the isozymes in a cloned normal rat kidney cell line transformed by a mutant of Rous sarcoma virus which is temeprature-sensitive for transformation indicated that, in fact, such an isozyme change does correlate with transformation. When grown at 36 degrees these cells contained predominantly isozyme III; however, upon reacquiring normal morphology and lowered glucose transport activity when grown at 40 degrees, their isozyme pattern was now found to be changed and consisted predominantly of isozyme I, as is found in normal adult rat kidney tissue. Isozyme III was found to be present in neonatal kidney tissue of the rat and hamster, and its predominance in the virus-transformed normal rat and baby hamster kidney cells was interpreted as indicative of the dedifferentiation of these cells upon viral transformation. A similar change of the isozyme pattern of leucine aminotransferase in chicken embryos during their development was observed, such that in 5-day-old embryos Form III was predominant, while in the more mature differentiated chicken embryo of Day 17, Form I was predominant.