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The vaccinia virus/bacteriophage T7 hybrid transient expression system employs a recombinant vaccinia virus that encodes the T7 RNA polymerase gene, a plasmid vector with a gene of interest regulated by a T7 promoter, and any cell line suitable for infection and transfection. Although high expression in a majority of cells is achieved. the severe cytopathic effects of vaccinia virus and the safety precautions required for use of infectious agents are undesirable features of the system. Here, we report the construction of a highly attenuated and avian host-restricted vaccinia virus recombinant that encodes the T7 RNA polymerase gene (MVA/T7 pol) and demonstrate the use of the virus for transient expression in mammalian cells. MVA/T7 pol has reduced cytopathic effects compared to the previously used replication-competent vaccinia virus, while providing a high level of gene expression in multiple mammalian cell lines.

The coding sequence deduced from two overlapping cDNA inserts obtained from a pathogenic strain of Entamoeba histolytica revealed a striking homology (greater than 85%) with elongation factor EF-1 alpha from Saccharomyces cerevisiae and Artemia salina. The deduced amino acid sequence predicted a size of 49 kDa, and antibodies raised against the S. cerevisiae EF-1 alpha cross-reacted with an amoebic protein of similar size (45-47 kDa). Sequence analysis of the cDNA revealed that the 5' untranslated region contained a stretch of 190 nucleotides which was perfectly complementary to a segment of the 3' terminal coding region situated 1015 bases downstream of the methionine initiation codon. Electron microscopy of self-renatured cDNA confirmed the potential of such molecules to form a stem-loop secondary structure. The presence of the complementary sequences was confirmed at the genomic level by sequence analysis of polymerase chain reaction-amplified segments which span both the 3' and 5' terminal complementary regions. Comparison of the deduced amino acid sequence of E. histolytica EF-1 alpha with Ef-Tu from Escherichia coli and EF-1 alpha from different sources, suggested that the major functional domains of the protein are located within the loop structure.

Subacute sclerosing panencephalitis (SSPE) is a fatal disease affecting the human central nervous system several years after acute measles infection. Measles virus (MV) genomes replicating in SSPE brains do not give rise to budding particles and present various defects in gene expression, mostly concerning the matrix (M) protein. For one SSPE case (K), shown previously to be devoid of M protein expression, we examined here in detail the features involved in this defect. In the brain of patient K the normal, monocistronic MV M mRNA was completely substituted by a bicistronic RNA containing the coding sequence of the preceding phosphoprotein (P) gene in addition to the M coding sequence. Analysis of the P-M intercistronic region by direct cDNA sequencing showed that the consensus sequence at this RNA processing site was unaltered but revealed several distant point mutations. cDNA cloning and sequencing of the entire M coding region established that one of the point mutations leads to a stop codon at triplet 12 of the M reading frame. It is unknown whether this defect, explaining by itself the lack of M protein, is related also to the block of M mRNA formation. In addition we note that as much as 1% of the nucleotides different between two overlapping clones from the same brain. This high sequence variability could possibly account for the diversity of defects observed in MV gene expression in SSPE brains and may be a general phenomenon associated with RNA virus persistence.

A cDNA clone (subclone B) previously isolated from the human parasite Entamoeba histolytica was characterized. DNA sequence analysis of subclone B identified the DNA as that encoding apoferredoxin. E. histolytica ferredoxin cDNA contains unusually short 5′ and 3′ noncoding regions of 9 and 25 nucleotides, respectively. A genomic ferredoxin clone was isolated from E. histolytica DNA, and comparison of genomic and cDNA sequences revealed that the ferrodoxin gene is unspliced. The deduced amino acid sequence of E. histolytica ferredoxin resembles clostridial type of ferredoxins, and shows an arrangement of cysteines characteristic for the coordination of 2[4Fe-4S] centres. Of interest is the absence of an aromatic amino acid in the N-terminal region of the protein, a feature which is conserved in clostridial ferredoxins. Southern blot analysis of three different E. histolytica strains (200:NIH, Rahman and HM-1:IMSS) demonstrated the presence of a family of at least two ferredoxin genes. One of these genes is marked by restriction length polymorphisms in different strains of E. histolytica.