The purine biosynthesis enzyme PRAT detected in proenzyme and mature forms during development of Drosophila melanogaster.

Glutamine phosphoribosylpyrophosphate amidotransferase (PRAT; EC 2.4. 2.14) is the first and rate-limiting enzyme of de novo purine biosynthesis. PRAT expression in Drosophila development was examined to determine if it is correlated with cell proliferation and/or nutritional isolation. An antiserum, raised against the 16 carboxyl-terminal amino acids of PRAT, detects two proteins corresponding to a 60 kDa proenzyme and 55 kDa mature enzyme, consistent with a 53 amino acid propeptide predicted from the gene sequence. Mature enzyme is maternally expressed, and proenzyme appears in embryos at 2-8 h, corresponding to the interval during which zygotic transcription is initiated. Upon hatching of first instar larvae, proenzyme levels are reduced and remain low relative to mature enzyme. Adult females have higher levels of both proteins relative to males, consistent with maternal expression. Maternal expression reflects a requirement for the enzyme during embryogenesis, while reduction in expression following hatching reflects a switch to an exogenous source of purines. Prat mRNA levels follow a similar overall pattern in the same developmental stages examined for the protein. Discovery of a second gene encoding PRAT with 78% amino acid identity leads to the possibility that the antiserum raised against the carboxyl-terminus detects two enzymes.

C-myc deregulation during transformation induction: involvement of 7SK RNA.

The process of oncogenic transformation has been widely studied but is still poorly understood. We have focused on the mechanism of deregulation of the c-myc gene during transformation of a temperature-sensitive SV40-transformed mouse cell line. Run-on transcription assays showed that the two c-myc minor promoters, P1 and P3, are transiently activated following induction of transformation and that peak activation of both promoters is preceded by a large increase in transcription of a small RNA (7SK). To test the possibility that this RNA might participate in promoter activation, we transfected cells with sense and antisense oligodeoxynucleotides corresponding to different regions of the 7SK RNA predicted to be accessible within the RNP particle. Out of 14 oligos tested, inhibition of activation of P1 and/or P3 was observed with four antisense oligonucleotides corresponding to looped regions in the putative 7SK secondary structure. To identify c-myc promoter sequences which might serve as targets for 7SK activity, we carried out mobility-shift assays with either whole or 7SK-depleted cell extracts. The CT element of the c-myc promoter formed a 7SK-dependent complex which could be competed only with the same antisense 7SK oligo that suppressed P1 and P3 activation in vivo. Taken together these results suggest that 7SK RNP participates in transformation-dependent c-myc deregulation.

Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.

Attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. However, it is often difficult to ensure correct processing or secretion of the encoded peptides. Here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. We selected the c-myc gene for two main reasons: (i) its protein is known to be well conserved in evolution and to have multiple essential functions during development; and (ii) c-myc family genes have yet to be characterized in insects, thus blockage of essential genes by anti-sense transcripts from a strong virus promoter could provide a sensitive test for the existence of myc-like gene products. An appropriate fragment of the human c-myc gene was inserted downstream from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus and tested in bioassays on Spodoptera frugiperda larvae. Western blot analysis with a human c-myc antibody revealed an endogenous protein band which bound specifically to these antibodies. This band disappeared more rapidly from cells infected with the antisense c-myc recombinant virus than from those infected with c-myc-negative virus. Results of bioassays showed that the antisense construct stopped feeding as soon as the polyhedrin promoter-driven transcripts accumulated, followed shortly by death of the larvae. These results suggest that c-myc-like protein(s) exist in insects and that the antisense strategy is an effective approach to virus insecticide productions.

A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.

The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.