Margarida O. Krause

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

SummaryThe 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 ofSpodoptera 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.

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.

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. J. Cell. Biochem. 64:313–327.

Histones from exponential and stationary L-cells: Evidence for metabolic heterogeneity of histone fractions retained after isolation of nuclei

Abstract Histones of exponential and G 1 -arrested mouse L-cells were double-labeled with either [ 3 H]lysine and [ 32 P]phosphate or with [ 14 C]arginine and [ 3 H]acetate in order to investigate cell cycle-dependent changes in rates of synthesis and metabolic modifications as well as the effect of the method of nuclear isolation on retention of the labeled fractions. Results indicate that newly synthesized lysine-rich histones incorporate 32 P at the highest rate. However, phosphorylation can also be detected in G 1 -arrested cells and in the case of F 2b , F 1 0 , and F 1 1 , with no detectable new synthesis. On the other hand, acetylation proceeded at similar rates in both exponential and stationary cells with the exception of F 2a 2 and F 3 which showed higher acetylation levels in the latter. In relation to the method used for nuclear isolation, we observed that, even in cases where no difference in the relative amount of a given histone could be detected, the species recovered were not identical. We conclude that none of the methods commonly employed retains all of the histones. In general, the acetylated species appear to be best preserved at a higher divalent cation concentration while the newly synthesized ones are better conserved at lower ionic strengths.

Low molecular weight nuclear RNA from SV40-transformed WI38 cells; effect on transcription of WI38 chromatin in vitro

Abstract Nuclei isolated from normal and SV40-transformed WI38 cells were used as templates for RNA synthesis in vitro . Comparison of template properties showed marked differences between the two chromatins with both homologous and heterologous enzymes. Addition of the 0.35 M NaCl extract from the chromatin of the SV40-transformed cells, revealed two separate activities: one stimulating the template activity of normal WI38 chromatin and one affecting the stability of the homologous RNA polymerase. Separation of the protein and SnRNA fractions in the extract demonstrated that the stabilization effect is due to chromosomal protein(s). However, SnRNA alone was found to be responsible for the stimulation of the transcriptional activity of normal cells to a level undistinguishable from that of the transformed cells.

Histones from exponential and stationary L-cells: Evidence for differential binding of lysine-rich and arginine-rich fractions in chromatin☆☆☆

Abstract Histones from exponential and stationary-phase mouse L-cells were quantitated after acrylamide gel electrophoresis in order to investigate cell cycle-dependent changes in the mode of binding of the various fractions in chromatin. By introducing various concentrations of citrate and divalent cations in the medium used for cell lysis and isolation of nuclei prior to histone extraction it was possible to demonstrate that certain histone fractions are preferentially retained in either exponential or stationary-phase nuclei. Differential retention of lysine-rich F 1 was most evident when the lysing medium contained 1 m m Mg 2+ and Ca 2+ and 5 m m citrate (pH 2.75). In these conditions twice as much F 1 is retained in stationary as in exponential nuclei. Differential retention of arginine-rich histones was most evident when the lysing medium contained 10 m m Mg 2+ and Ca 2+ and no citrate. In these conditions more F 2a 1 is retained in exponential than in stationary nuclei while the opposite is true for F 3 . However, the total amount of arginine-rich fractions (F 2a 1 + F 3 ) retained was found to be the same in both cell phases. The results are discussed in relation to known structural features of the histones.

RNA synthesis and chromatin structure in mammalian cells: In situ detection of template changes in living and formalin fixed L cell nuclei

Abstract An autoradiographic study was carried out to compare the mean rates of RNA synthesis in exponential and stationary L cell monolayer cultures, with the ability of the cell nuclei to rapidly bind 3H-actinomycin D (AMD) in vivo and to serve as templates for polyadenylic acid, poly(A), synthesis in vitro. A correlation was found between AMD binding and 3H-uridine incorporation for both cell phases under study, indicating that the increase in RNA synthetic activity from stationary to exponentially growing L cells occurs as a result of structural modifications in the chromatin which make new sites on the DNA available for AMD binding. Poly(A) synthesis by bacterial RNA polymerase, with intact formalin-fixed nuclei as templates, demonstrated a similar increase in the number of template sites from stationary to exponential phase nuclei. Treatment of the fixed nuclei with trypsin or low concentrations of HCl increased the template activity of the nuclei but eliminated the differences between the two growth phases. The similarity of the results obtained by means of two fundamentally distinct approaches reinforces the hypothesis that the control of RNA synthesis in L cells largely resides in the structural conformation of the chromatin.

Arginine-rich histone synthesis and acetylation in WI 38 cells stimulated to proliferate

Abstract Confluent WI 38 human diploid fibroblasts were stimulated to proliferate by a change of medium containing 10% fresh serum. Synthesis and acetylation of arginine-rich histones in contactinhibited cells (G 0 stage) and in cells 60 min after serum stimulation (G 1 stage) as well as in exponentially growing cells were investigated using a method for isolation of nuclei which has been demonstrated to result in optimal recovery of arginine-rich fractions. Pulse-labelling with [3H]arginine-[3H]lysine or [3H]acetate was combined with treatment with either actinomycin D or cycloheximide in order to investigate whether histone turnover and acetylation following serum stimulation are controlled at the transcriptional and/or at the translational level. Results revealed that the rate of 3H-labeled amino acid incorporation into arginine-rich histone fractions is higher in G 0 than in G 1 cells and is sensitive to both actinomycin D and cycloheximide. On the other hand histone acetylation proceeds at similar rates in both G 0 and G 1 cells und is not affected by either inhibitor. Differences in the extractability of arginine-rich histones with 0.25 M HCl are also reported; H 4 was found to be more resistant to HCl extraction than any other fraction in G 0, G 1 or exponential populations. However fraction H 3 is extracted more rapidly from G 0 cells. These differences are presumed to reflect cell-cycle dependent modifications in the binding of histone fractions in chromatin.

Distinct nuclear 7S RNAs hybridize to regulatory regions of two oncogenes.

The role of 7SK nuclear RNA remains obscure despite indications that it might be involved in the control of gene expression and associated with transformation. Here we identify distinct 7S nuclear transcripts by hybridization with DNA and RNA probes derived from regulatory regions of SV40 and c-myc genes. 7SK RNA hybridized to both early and late strands within the 21bp repeat region of SV4C and only to the sense strand within the first c-myc intron. Another nuclear 7S RNA, distinct from cytoplasmic 7SL, hybridized selectively to the SV40 late strand within the 72bp region. This RNA, designated 7SN, also hybridized to the c-myc first intron but with opposite polarity to 7SK. The identification of a novel 7S transcript and the observed selective hybridization of nuclear 7S RNAs with oncogene promoters raises interesting possibilities about their function.

Small nuclear RNAs from Drosophila KC-H cells; characterization and comparison with mammalian RNAs

Small molecular weight nuclear RNAs were extracted from cultured Drosophila KC-H cells and characterized by their electrophoretic mobilities in 5–15% gradient acrylamide gels or in 10% acrylamide-7 M urea gels. Comparison between the electrophoretic profiles of these SnRNAs with those from human and mouse cells revealed striking similarities and allowed for assignation of band nomenclatures as established for mammalian cells. Comparison of mobilities in the two gel systems also permitted correspondence between the different nomenclatures established by various groups for this class of RNAs, as well as an approximate estimate of their molecular sizes.