Beng Guat Ooi

Baculovirus vectors for multiple gene expression and for occluded virus production

The nature and properties of a variety of plasmids are described that facilitate the construction of baculovirus vectors for expression of one or more heterologous genes. The plasmids are designed for use with Autographa californica nuclear polyhedrosis virus, AcMNPV, as a vector for protein production in insect cells and/or insect larvae. Several plasmids described here facilitate the simultaneous insertion and expression of two different genes. Some vector systems allow high and equal levels of transcription of both genes while others employ two different promoters for differential transcription. Four of the plasmids described here are designed for expression of both the viral polyhedrin-encoding gene and a heterologous gene. Such recombinants form polyhedral occlusion bodies which serve as visible markers of recombination and facilitate oral infection of insect larvae for mass-scale protein production. A synthetic promoter with a unique sequence can be used at a variety of sites in the viral genome and avoids sequence duplication. A series of plasmids are also described that supply an N terminus with an efficient translational initiation signal and convenient multiple cloning sites in the three different translational reading frames. The modular nature of all the constructs allows the use of other promoters with different temporal regulation to be utilized in the construction of additional plasmids for customized expression work.

Eight base pairs encompassing the transcriptional start point are the major determinant for baculovirus polyhedrin gene expression

The effects of mutations within the 92-bp region immediately upstream from the translational initiation ATG of the polyhedrin gene of the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV) were determined by plasmid transient expression assays in the presence of wild-type (wt) AcMNPV DNA. Clustered point mutations were generated by substitution of 10-bp stretches of the polyhedrin promoter/leader region with a 10-bp HindIII linker. Three of these linker scan (LS) mutations in the region from nucleotides (nt) -62 to -84 (relative to the original polyhedrin ATG at +1, +2, +3) had no effect or a mild stimulatory effect on reporter gene expression. One mutation immediately upstream (nt -52 to -60) from the transcription start point (at nt -50) reduced expression four-fold. Three overlapping mutations affecting 8 bp from nt -44 to -51 (encompassing the transcriptional start point) reduced expression by 1000-fold. A 1000-fold reduction was also observed for a total deletion of nt -1 and -92. Five LS mutations between nt -1 and -43 each reduced expression two- to five-fold, whereas combining an LS mutation and a 9-bp deletion mutation in the leader reduced expression approx. nine-fold. Reversing the orientation of the reporter gene along with the wt 92-bp upstream polyhedrin promoter/leader sequences resulted in slightly higher expression levels than those observed for the normal orientation indicating that all the essential cis-acting promoter elements, with the possible exception of long-range enhancer sequences, are located downstream from nt -92. Sequences of the AcMNPVhr5 enhancer (homologous region No. 5 of AcMNPV) had only a minor effect on expression from the polyhedrin promoter in transient assays. The results show that 8 bp encompassing the transcriptional start point, a sequence which is conserved in all late AcMNPV promoters, is essential for polyhedrin gene expression. Additional nucleotides within the leader region are necessary for optimal expression.

Downstream sequences augment transcription from the essential initiation site of a baculovirus polyhedrin gene

A series of recombinant baculoviruses containing linker-substituted polyhedrin promoters attached to a reporter gene encoding chloramphenicol acetyl transferase (CAT) were constructed and tested for expression of the gene. The major determinant for promoter activity was narrowed to within eight nucleotides, TAAGTATT, at the start point of polyhedrin mRNA transcription. Mutations within TAAGTATT blocked initiation of transcription from this site and resulted in a 2000-fold decrease in CAT activity. Linker mutations from 12 to 22 bases upstream from the TAAGTATT sequence increased the steady-state levels of RNAs initiated within TAAGTATT and increased CAT expression by up to 50%. Mutations downstream from TAAGTATT and within the region specifying the untranslated RNA leader diminished transcriptional initiation at TAAGTATT and decreased CAT activity two- to 20-fold. The half-lives of CAT RNAs were not noticeably affected by mutations in the untranslated RNA leader region and thus RNA turn-over was not responsible for the reduced levels of these CAT RNAs. Nuclear run-on analysis of two mutant viruses showed that these mutations decrease the rate of transcriptional initiation. Transcriptional initiation thus appears to be the major means of polyhedrin gene regulation. The data define promoter-related roles for TAAGTATT and the sequences specifying the untranslated mRNA leader in transcriptional initiation. A model by which the viral-induced RNA polymerase distinguishes late and very late initiation sites is proposed.

Regulation of host RNA levels during baculovirus infection

During infection of the permissive host insect cell line Spodoptera frugiperda IPLB-SF-21 by the baculovirus Autographa californica nuclear polyhedrosis virus (AcMNPV), the levels of host actin, histone, and heat shock 70 (hsp70) RNAs are reduced substantially. Reduction of the host RNA levels occurs primarily during a narrow window of the replication process, from approximately 12 to 18 hr postinfection (p.i.), corresponding to the phase in which the extracellular form of the virus buds into the media. A late viral protein appears to be required for this reduction since cycloheximide, an inhibitor of cytosolic protein synthesis, and aphidicolin, an inhibitor of host and viral DNA polymerases, inhibit the reduction of actin and histone RNA levels. A cDNA corresponding to the carboxyl half of the S. frugiperda mitochondrial cytochrome c oxidase subunit III (COIII) gene was isolated, sequenced, and characterized. Two differentially regulated mitochondrial transcripts of this gene are observed. The level of the larger of these transcripts, which is dependent on active cytosolic protein synthesis, is reduced during virus infection in a fashion similar to that of the nuclear host genes. The smaller COIII transcript is stable until at least 24 hr p.i. but the level of this RNA eventually declines by 48 hr p.i.

Amino acid substitutions in mitochondrial ATPase subunit 9 of Saccharomyces cerevisiae leading to oligomycin or venturicidin resistance.

A series of isonuclear oligomycin‐resistant mutants of Saccharomyces cerevisiae carrying mutations in the mitochondrial olil gene has been studied. DNA sequence analysis of this gene has been used to define the amino acid substitutions in subunit 9 of the mitochondrial ATPase complex. A domain of amino acids involved in oligomycin resistance can be recognized which encompasses residues in each of the two hydrophobic portions of the subunit 9 polypeptide that are thought to span the inner mitochondrial membrane. Certain amino acid substitutions also confer cross‐resistance to venturicidin: these residues define an inner domain for venturicidin resistance. The expression of venturicidin resistance resulting from one particular substitution is modulated by nuclear genetic factors.

The oli1 gene and flanking sequences in mitochondrial DNA of Saccharomyces cerevisiae: the complete nucleotide sequence of a 1.35 kilobase petite mitochondrial DNA genome covering the oli1 gene.

SummaryAs part of our genetic and molecular analysis of mutants of Saccharomyces cerevisiae affected in the oli1 gene (coding for mitochondrial ATPase subunit 9) we have determined the complete nucleotide sequence of the mtDNA genome of a petite (23-3) carrying this gene. Petite 23-3 (1,355 base pairs) retains a continuous segment of the relevant wild-type (J69-1B) mtDNA genome extending 983 nucleotides upstream, and 126 nucleotides downstream, of the 231 nucleotide oli1 coding region. There is a 15-nucleotide excision sequence in petite 23-3 mtDNA which occurs as a direct repeat in the wild-type mtDNA sequence flanking the unique petite mtDNA segment (interestingly, this excision sequence in petite 23-3 carries a single base substitution relative to the parental wild-type sequence). The putative replication origin of petite 23-3 is considered to lie in its single G,C rich cluster, which differs in just one nucleotide from the standard oris sequence. The DNA sequences in the intergenic regions flanking the oli1 gene of strain J69-1B (and its derivatives) have been systematically compared to those of the corresponding regions of mtDNA in strains derived from the D273-10B parent (sequences from the laboratory of A. Tzagoloff). The nature and distribution of the sequence divergencies (base substitutions, base deletions or insertions, and more extensive rearrangements) are considered in the context of functions associated with mitochondrial gene expression which are ascribed to specialized sequences in the intergenic regions of the yeast mitochondrial genome.