Robert F. Weaver

Identifying the RNA polymerases that synthesize specific transcripts of the Autographa californica nuclear polyhedrosis virus

Nuclear run-on assays carried out in the presence and absence of the RNA polymerase II inhibitor, alpha-amanitin, were used to determine the exact timing of the switch from inhibitor-sensitive transcription catalysed by host RNA polymerase II, to inhibitor-resistant transcription catalysed by the baculovirus-induced RNA polymerase. These studies revealed that the onset of alpha-amanitin-resistant transcription is just after 6 h post-infection, simultaneous with the beginning of the late phase of infection. They also showed that transcripts from the p26 gene in the HindIII Q/P region and the p35 gene in the HindIII K/Q region of the viral genome are synthesized by the host RNA polymerase II both early and late in infection. On the other hand, transcripts of the p10 gene in the HindIII Q/P region and the gamma transcripts in the HindIII K region are synthesized by the alpha-amanitin-resistant, virus-induced RNA polymerase late in infection.

Structural comparison of the Autographa californica nuclear polyhedrosis virus-induced RNA polymerase and the three nuclear RNA polymerases from the host, Spodoptera frugiperda

A partial subunit structure has been determined for the novel RNA polymerase that is induced in fall armyworm (Spodoptera frugiperda) cells upon infection with the Autographa californica nuclear polyhedrosis virus (AcNPV). The putative structure includes nine polypeptides; the complexity of this structure is in accord with the high sedimentation coefficient (15S) estimated for this enzyme. A comparison of the putative structure of the virus-induced polymerase with those of the three host nuclear RNA polymerases shows that the structure of the viral polymerase is apparently unlike any of the host nuclear polymerases. This conclusion is reinforced by immunoblot experiments that show no cross-reactivity between the virus-induced polymerase and an antiserum directed against Drosophila RNA polymerase II. The virus-induced RNA polymerase appears at the onset of the late phase of infection and still appears when viral DNA synthesis is blocked by aphidicolin. Thus, the virus-induced polymerase seems to be composed of early viral products.

Categorizing some early and late transcripts directed by the Autographa californica nuclear polyhedrosis virus.

Using an S1 mapping assay on RNA from Spodoptera frugiperda cells infected by the Autographa californica nuclear polyhedrosis virus in the presence and absence of cycloheximide and aphidicolin, we can distinguish three classes of transcripts. First, there are those whose synthesis is blocked by the DNA synthesis inhibitor aphidicolin and which are therefore late transcripts. These include the late transcript of the 39K gene and a late leftward transcript across the XhoI site in the HindIII-F region. Second, there are those whose synthesis is not blocked by aphidicolin, but whose accumulation is inhibited by the protein synthesis inhibitor cycloheximide and which are therefore presumably delayed early genes. These include the p26 transcript(s), the early 39K transcript and the alpha 2 transcript in the HindIII-K/Q region. Third, there are those whose accumulation is not affected or is enhanced by cycloheximide. These are not necessarily immediate early transcripts, but their response to cycloheximide is clearly different from that of those in the second class. They include the alpha 1 and alpha 3 transcripts in the HindIII-K/Q region and the early leftward transcript across the XhoI site in the HindIII-F region.

Construction of a recombinant expression plasmid encoding a staphylococcal protein A-ricin A fusion protein

A recombinant plasmid has been constructed containing the coding regions for a functional fragment of staphylococcal protein A and the entire ricin A chain in tandem in the same reading frame. The recombinant gene has been expressed in Escherichia coli cells to produce a protein with both staphylococcal protein A activity and ricin A chain activity. Such a fusion protein could be used for producing immunotoxins.A recombinant plasmid has been constructed containing the coding regions for a functional fragment of staphylococcal protein A and the entire ricin A chain in tandem in the same reading frame. The recombinant gene has been expressed in Escherichia coli cells to produce a protein with both staphylococcal protein A activity and ricin A chain activity. Such a fusion protein could be used for producing immunotoxins.

Characterization of a cDNA encoding ricin E, a hybrid ricin-Ricinus communis agglutinin gene from the castor plant Ricinus communis

Two classes of ricin cDNA clones have been identified and sequenced. The cDNA clone pBL-1 closely matches in nucleotide sequence the ricin genomic clone pAKG previously described by Halling et al., 1985 (Nucl. Acids Res. 13:8019). A second group of cDNA clones, represented by pBL-3, encode a hybrid protein (ricin E), having a ricin-like A chain and N-terminal half of the B chain and an RCA (Ricinus communis agglutinin)-like C-terminal half of the B chain.

Vitellogenin: its structure, synthesis and processing in the cockroach Periplaneta americana.

The vitellogenin in the oocyte of the American cockroach contains three different polypeptides with approximate molecular weights of 123 000, 118 000 and 57 000. These are present in a molar ratio of 2 : 2 : 2; all three contain carbohydrate. The same subunit structure is observed in vitellogenin isolated from hemolymph and in that secreted by fat body in organ culture. The only protein found within fat body by precipitation with female-specific antiserum is a large polypeptide, with a molecular weight of approx. 250 000. No mature vitellogenin subunits are found in fat body, even after pulsing with labeled amino acids and chasing with unlabeled substrates for up to 4 h. These data suggest that vitellogenin is synthesized in the fat body as a large precursor, which is processed to smaller polypeptides upon secretion into the hemolymph.

Structural comparison of deoxyribonucleic acid-dependent ribonucleic acid polymerases I and II from the slime mold Physarum polycephalum

Abstract RNA Polymerases I and II have been purified to homogeneity from the slime mold Physarum polycephalum . When subjected to ultracentrifugation in separate sucrose gradients, or together, the two enzymes migrate with S values estimated at 14.5 S. Polyacrylamide gel electrophoresis under denaturing conditions reveals that the two enzymes have distinct but similar subunit structures. The probable subunit structure of polymerase II is as follows: 175,000 1 ; 140,000 1 ; 24,000 2 ; 17,000 1 .

Structural analysis of the matrix protein from the nuclear polyhedrosis virus of heliothis zea

Polyhedrin from the nuclear polyhedrosis virus of Heliothis zea was analyzed. Alkali-solubilized polyhedrin consists of a 12 S aggregate of 27,000 MW subunits. Results from chemical crosslinking experiments suggest that 12 subunits are present in the 12 S aggregate. In isoelectric focusing gels, the aggregate migrates as a single entity with an isoelectric point of 5.9. Under denaturing conditions, four charge isomers of the subunits are revealed. The presence of alkaline protease activity in the Heliothis virus is confirmed.

Isolation of three forms of DNA-dependent RNA polymerase from Heliothis zea fat body tissue.

Abstract Three different forms of DNA-dependent RNA polymerase activity have been isolated from Heliothis zea fat body tissue. The enzyme activities have been partially characterized and identified as the cognate enzymes to forms I, II, and (probably) III found in other eukaryotic sources. The catalytic activities and chromatographic behaviour of the H. zea RNA polymerases I and II are compared to those of RNA polymerases isolated from other insects.

An improved method for isolation of Heliothis zea DNA-dependent RNA polymerase: Separation and characterization of a form III RNA polymerase activity

Abstract An improved method for the isolation of DNA-dependent RNA polymerase enzyme activities from fat body tissue of Heliothis zea is described. This method involves polyethyleneimine precipitation of a crude tissue homogenate followed by ammonium sulphate elution of the RNA polymerase activity from the precipitate. After chromatography of the salt eluate on DEAE-Sephadex, three major and two minor peaks of the H. zea RNA polymerase activity are resolved. On the basis of their chromatographic and catalytic behaviour and α-amanitin sensitivities, the three major peaks have been identified as RNA polymerase forms I, II and III. Characteristics of the possible form III enzyme activity are reported.