Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A comparison was undertaken of poxvirus promoters in vaccinia and fowlpox virus (FPV) recombinants using the level of beta-galactosidase expressed from the LacZ gene as a measure of promoter function. In this study a comparison was made of the vaccinia virus promoters, P 7.5 and P L11, the major late promoter of cowpox virus, P CPX (expressing the abundant inclusion body protein), and the FPV promoters, P E/L and P L. In vaccinia virus recombinants the FPV P E/L promoter expressed one-third to one-half the level of beta-galactosidase expressed by the P L11 promoter. In comparison with the P 7.5 promoter, the FPV P E/L promoter expressed four to five times the level of beta-galactosidase. In FPV recombinants beta-galactosidase activity expressed was equal for the P E/L and P CPX promoters. Levels expressed by P L11 and P L were one-half and one-fifth that level, respectively. The temporal regulation of the promoters was maintained in both vaccinia virus and FPV recombinants. The P E/L promoter of FPV has the TAAATG sequence characteristic of late poxvirus promoters at the transcription initiation site. In an attempt to enhance the utility of this promoter for the expression of foreign genes in FPV and vaccinia virus recombinants, the effect upon promoter function of changing the G of the ATG to A, T, or C was determined using transient expression assays with vaccinia virus. Substitution of A, T, or C for the G abolished promoter function. Because of its early/late function, the level of expression and the presence of the oppositely oriented late P L promoter, the FPV P E/L promoter will be valuable for the expression of foreign genes in poxvirus recombinants.
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PMID:Quantitative assessment of poxvirus promoters in fowlpox and vaccinia virus recombinants. 132 41

Vaccina virus (VV) and cowpox virus (CPV) differ in their abilities to replicate in Chinese hamster ovary (CHO) cells because VV has a disrupted host range (hr) gene. To facilitate an examination of the molecular events associated with abortive infection of CHO cells with VV, we constructed two sets of recombinant viruses that contain a viral early promoter regulating the cat gene encoding chloramphenicol acetyltransferase and viral intermediate or late promoters regulating the lacZ gene encoding beta-galactosidase. The first set has the disrupted hr gene and the second set has the intact CPV homolog, allowing replication in CHO cells. Reporter chloramphenicol acetyltransferase and beta-galactosidase assays demonstrated that early gene expression was unperturbed, whereas intermediate and late gene expression were severely inhibited under abortive conditions. Metabolic labeling studies confirmed the absence of viral late protein synthesis. The accumulation of viral DNA under abortive conditions was consistent with the synthesis of viral early proteins and established that inhibition of late protein synthesis was not primarily due to a replicative block. Analysis of steady state levels of viral mRNAs revealed substantial quantities of early and intermediate species but only very small amounts of late mRNAs under nonpermissive conditions. Despite the presence of viral intermediate mRNAs, the corresponding intermediate proteins, which function as late transcription factors, were not detected by immunoprecipitation of lysates from metabolically labeled infected CHO cells. Furthermore, when expression of lacZ was regulated by an intermediate promoter, no beta-galactosidase was detected even though lacZ transcripts were present. Thus, the abortive phenotype in CHO cells can be explained by a block to translation of intermediate mRNAs which prevents the synthesis of late transcription factors.
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PMID:Restriction of vaccinia virus replication in CHO cells occurs at the stage of viral intermediate protein synthesis. 785 9

Genes encoding virus-specific proteins with molecular masses of 36 kDa and 12 kDa were mapped in HindIII-P and HindIII-U DNA fragments of vaccinia strain LIVP and ectromelia strain K-1 viruses, respectively, by hybrid selection of RNA to cloned DNA fragments followed by in vitro translation. The 36K translation initiation codon was detected in the HindIII-J fragment. The nucleotide sequences of corresponding genes from vaccinia, ectromelia, cowpox and variola virus genomes were determined. The 12K protein has similarity to mammalian glutaredoxins. The derived amino acid sequence of the 36K polypeptide was compared with the protein bank PIR. No homology was found between the 36K protein and known structures of proteins. The 36K protein genes of vaccinia and ectromelia viruses were cloned in pUR290, which led to the production of E. coli chimeric proteins, consisting of the sequence of beta-galactosidase and the viral protein on their C-ends. The chimeric proteins were shown to possess viral antigenic specificity. To identify the protein product of the 36K gene monospecific antisera to chimeric proteins were obtained. The late 36K protein is associated with virosomes but is not incorporated into the virions of orthopoxviruses.
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PMID:Comparative analysis of the conserved region of the orthopoxvirus genome encoding the 36K and 12K proteins. 823 12

The group B entomopoxvirus (EPV) from Amsacta moorei (AmEPV) productively infects only insect cells. A series of AmEPV-lacZ recombinants was constructed in which the lacZ gene was regulated by either late (the AmEPV spheroidin or the cowpox virus A-type inclusion [ATI]) or early (the AmEPV esp [early strong promoter; derived from a 42-kDa AmEPV protein] or the Melolontha melolontha EPV fusolin, fus) virus promoters. When the AmEPV recombinants were used to infect vertebrate cells, beta-galactosidase expression occurred (in >30% of the cells) when lacZ was regulated by either the fus or esp early promoters but not when lacZ was regulated by the late promoters (spheroidin or ATI). Therefore, AmEPV enters vertebrate cells and undergoes at least a partial uncoating and early, but not late, viral genes are expressed. Neither viral DNA synthesis nor cytopathic effects were observed under any infection conditions. When an AmEPV recombinant virus containing the Aequorea victoria green fluorescent protein gene (gfp) under the control of the esp promoter was used to infect vertebrate cells at a low multiplicity of infection, single fluorescent cells resulted, which continued to divide over a period of several days, ultimately forming fluorescent cell clusters, suggesting that vertebrate cells survive the infection and continue to grow. Therefore, AmEPV may prove to be a highly efficient, nontoxic method of gene delivery into vertebrate cells for transient gene expression.
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PMID:Transient, nonlethal expression of genes in vertebrate cells by recombinant entomopoxviruses. 937 19

Spheroidin (SPH) is the most highly expressed gene of the entomopoxvirus isolated from Amsacta moorei (AmEPV). The level of expression of poxvirus genes is believed to be governed in large part by the promoter. Poxvirus promoters generally consist of approximately 40 bp which frequently terminate at the 3' end with a translation initiating TAAATG sequence. We have examined the requirements for high levels of SPH gene expression by constructing AmEPV recombinants containing either the SPH promoter or the late vertebrate poxvirus promoter derived from the cowpox virus A-type inclusion (ATI) gene. In addition, we have examined SPH promoter derivatives which extend beyond the 3' TAAATG to include 2 or 20 bp of the 5' coding sequence of the SPH gene. Examination of insect cells infected with these AmEPV ATI-lacZ or SPH-lacZ recombinants suggests that ATI-lacZ expression begins 12 h before and is essentially complete prior to any SPH-lacZ expression, allowing functional distinction between the ATI and SPH promoters and implying that different factors regulate the two promoters within the insect environment. SPH promoter-regulated expression is significantly enhanced within infected insect cells by including the additional 20 bp of the N-terminal SPH coding sequences as part of the promoter. However, when any of the SPH promoter constructs, including those containing the downstream sequences, were inserted into vaccinia virus, only very low levels of beta-galactosidase expression were observed. These results imply that downstream coding sequences within the SPH gene enhance SPH gene expression only within the insect environment.
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PMID:High-level expression of Amsacta moorei entomopoxvirus spheroidin depends on sequences within the gene. 951 42