Analysis of sequences involved in IE2 transactivation of a baculovirus immediate-early gene promoter and identification of a new regulatory motif.

Opep-2 is a unique baculovirus early gene that has only been identified in the Orgyia pseudotsugata multiple capsid nucleopolyhedrovirus (OpMNPV). Previous analyses have shown this gene is expressed at very early times post-infection (p.i.) but is shut down by 36-48 h p.i. The promoter of opep-2 therefore, represents a class of early genes that is temporally regulated. In this study, a detailed analysis of the opep-2 promoter is performed to analyze the role individual motifs play in early gene expression. A new 13 base pair regulatory element was identified and shown to be essential in controlling high-level expression of this gene. In addition, mutational analysis revealed that GATA and CACGTG motifs, which have been shown to bind cellular factors in Sf9 and Ld652Y cells, played minor roles in influencing opep-2 expression in the absence of other viral factors. The OpMNPV transactivator IE2 causes a significant activation of the opep-2 promoter. Cotransfection of an extensive number of promoter deletions and mutations did not show any sequence specificity for IE2 transactivation. This is the first detailed analysis of the sequence requirements for IE2 transactivation, and these results suggest that IE2 does not bind directly to specific elements in the opep-2 promoter.

The baculovirus transcriptional transactivator ie0 produces multiple products by internal initiation of translation.

Ie0 is the only gene of the baculovirus Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) that is known to be spliced. In this study, cDNAs of ie0 were isolated, cloned, and sequenced. It was observed that IE0 contains 35 amino acids (aa) added to the N-terminus of IE1. In addition, it was found that the leader sequence of ie0 contains a 4-aa minicistron. To functionally characterize IE0, ie0 cDNAs were expressed under control of either the ie1 or the ie0 promoter. Unexpectedly, examination of ie0 translation products revealed that the predominant product from ie0 mRNAs was not IE0, but IE1. Mutation analysis showed that IE1 translation was preferentially initiated from either of two AUGs found in the first 15 nucleotides (nt) of the ie1 ORF that are internal to the ie0 ORF. It is unknown whether the internal translation initiation occurs via a leaky scanning mechanism or by an internal ribosomal entry site. Transactivation analysis with constructs that had point mutations in the ie1 AUGs and were translated only as IE0 revealed that OpMNPV IE0 is a 14- to 15-fold stronger transactivator than IE1. IE0 was also shown to be autoregulatory and to transactivate early genes in an enhancer-independent or -dependent manner. These results suggest that differential expression of baculovirus early genes can be obtained by coexpression of IE0 and IE1 in infected cells, which may permit subtle regulation of specific sets of viral genes.

Characterization of the acidic domain of the IE1 regulatory protein from Orgyia pseudotsugata multicapsid nucleopolyhedrovirus.

This study presents a detailed analysis of the acidic N-terminal region of the Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) transactivator IE1. The N-terminal region of IE1 is rich in acidic amino acids and has been hypothesized to be an acidic activation domain. Removal of the N-terminal 126 amino acids containing the acidic domain of IE1 resulted in complete loss of transactivation activity, indicating that this region is essential for transactivation. The OpMNPV acidic domain was replaced with the archetype acidic activation domain from VP16 and the acid-rich region of Autographa californica multicapsid NPV (AcMNPV) IE1. These chimeric constructs were fully capable of transactivation in transient assays. The chimeric OpMNPV IE1s containing the herpes simplex virus VP16 and AcMNPV IE1 acidic activation domains consistently transactivated a reporter gene to higher levels than the OpMNPV IE1 acidic activation domain. Transactivation by the chimeric constructs is enhanced synergistically when cotransfected with IE2 into Lymantria dispar and Spodoptera frugiperda cells. Both N- to C-terminal and C- to N-terminal deletions of the OpMNPV acidic activation domain were constructed to define functional domains within the OpMNPV IE1 acidic activation domain. At least two potential activation domains were identified. Within each of these domains, two core regions at amino acids 28-43 and amino acids 113-124 were identified that were similar to core regions of VP16 and GAL4, which contain predominately acidic and bulky hydrophobic amino acids.

Protease evolution in Streptomyces griseus. Discovery of a novel dimeric enzymes.

This report describes the cloning and sequencing of a novel protease gene derived from Streptomyces griseus. Also described is the heterologous expression of the gene in Bacillus subtilis and characterization of the gene product. The sprD gene encodes a prepro mature protease of 392 amino acids tentatively named S. griseus protease D (SGPD). A significant component of the enzyme preregion was found to be homologous with the mitochondrial import signal of hsp60. The sprD gene was subcloned into an Escherichia coli/B. subtilis shuttle vector system such that the pro mature portion of SGPD was fused in frame with the promoter, ribosome binding site, and signal sequences of subtilisin. The gene fusion was subsequently expressed in B. subtilis DB104, and active protease was purified. SGPD has a high degree of sequence homology to previously described S. griseus proteases A, B, C, and E and the alpha-lytic protease of Lysobacter enzymogenes, but unlike all previously characterized members of the chymotrypsin superfamily, the recombinant SGPD forms a stable alpha 2 dimer. The amino acid sequence of the protein in the region of the specificity pocket is similar to that of S. griseus proteases A, B, and C. The purified enzyme was found to have a primary specificity for large aliphatic or aromatic amino acids. Nucleotide sequence data were used to construct a phylogenetic tree using a method of maximum parsimony which reflects the relationships and potentially the lineage of the chymotrypsin-like proteases of S. griseus.

Streptomyces griseus protease C. A novel enzyme of the chymotrypsin superfamily.

In this report we describe a novel chymotrypsin-like serine protease produced by Streptomyces griseus. The enzyme has been tentatively named S. griseus protease C (SGPC). The gene encoding the enzyme (sprC) was identified and isolated on the basis of its homology to the previously characterized S. griseus protease B (SGPB). The sprC gene encodes a 457-amino acid prepro-mature protein of which only the 255 carboxyl-terminal amino acids are present in the mature enzyme. Mature SGPC contains two distinct domains connected by a 19-amino acid linker region rich in threonines and prolines. While the amino-terminal domain is homologous to S. griseus proteases A, B, and E and the alpha-lytic protease of Lysobacter enzymogenes, the carboxyl-terminal domain is not homologous with any known protease. However, the carboxyl-terminal domain shares extensive homology with chitin-binding domains of Bacillus circulans chitinases A1 and D, suggesting that the enzyme is specialized for the degradation of chitin-linked proteins. Recombinant expression and preliminary characterization of the catalytic properties of the enzyme are also reported. The primary specificity of SGPC is similar to that of SGPB; both enzymes preferentially cleave peptide bonds following large hydrophobic side chains.

Phylogenetic relationships in the honeybee (genus Apis) as determined by the sequence of the cytochrome oxidase II region of mitochondrial DNA.

The complete nucleotide sequence of the mitochondrial cytochrome oxidase II (COII) gene was determined for five species of the honeybee (Genus: Apis): A. andreniformis, A. cerana, A. dorsata, A. florea, and A. koschevnikovi; these were then compared to the known sequence of the A. millifera gene from Crozier et al. (1989, Mol. Biol. Evol., 6: 399-411) and the wasp Excristes roborator (Liu and Beckenbach, 1992, Mol. Phylogenet. Evol., 1:41-52). Phylogenetic relationships were derived using the parasimony methods DNAPARS and PROTPARS of Felsenstein ("PHYLIP Manual Version 3.4, "University Herbarium, Univ. of California, Berkeley). The results suggest that A. dorsata is the most ancestral species, followed by the branching of A. florea/A. andreniformis and A. koschevnikovi, and then A. mellifera and A. cerana. This inference differs from the currently accepted view that considers the A. florea/A. andreniformis line to be the most ancestral.

A model for disease heterogeneity in systemic lupus erythematosus. Relationships between histocompatibility antigens, autoantibodies, and lymphopenia or renal disease.

We composed a model from autoimmune serologic findings, HLA antigens, and clinical findings that explains, at least partially, the clinical heterogeneity of 40 patients with systemic lupus erythematosus (SLE). In these patients, anti-RO (SS-A) was related to the HLA-DQ1/DQ2 heterozygotes, anti-La (SS-B) was related to HLA-B8 and HLA-DR3, and anti-nuclear RNP (Sm) was related to HLA-DR4. Lymphopenia was associated with anti-Ro (SS-A) and, secondarily, with anti-single-stranded DNA. Renal disease in these SLE patients was inversely associated with anti-La (SS-B) and was positively associated with anti-double-stranded DNA. There were no associations between the HLA antigens and these clinical manifestations. The results support a model of disease expression in which individuals are nonspecifically potentiated for SLE. Their HLA antigen composition influences the production of particular autoantibodies that are related in complex ways to the different particular clinical findings of SLE manifested in individual patients.

Adult-onset cyclic bicytopenia: a case report and review of treatment of cyclic hematopoiesis.

A unique case of adult-onset synchronous cyclic neutropenia and thrombocytopenia occurring at six-week intervals is presented. Periods of cytopenia were associated with fever, myalgias, gastrointestinal symptoms, and mild mucocutaneous bleeding. Alternate-day steroid treatment failed to correct the periodic fluctuations in peripheral blood counts but ameliorated symptoms during cytopenia. The treatment of cyclic hematopoiesis is reviewed.