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  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: RNA secondary structure is important in a wide variety of biological processes, but relatively little is known about the pathways and kinetics of RNA folding. When the IS 10 transposase (tnp) gene is transcribed from a promoter outside the element, little increase in tnp expression is observed. This protection from outside transcription (pot) occurs at the translational level, presumably resulting from mRNA secondary structure proposed to sequester the tnp ribosomebinding site. Here, we confirm the pot RNA structure and show that it blocks 30S ribosomal subunit binding in vitro. Point mutations that abolish protection in vivo map to the pot structure. Surprisingly, these pot mutations do not severely alter the pot secondary structure or increase 30S subunit binding in vitro, except in one case. Using an oligonucleotide hybridization assay, we show that most of the pot mutations slow the kinetics of pot structure formation, with little or no effect on the inhibitory function of the final structure. Moreover, a suppressor mutation reverses this effect. We propose a pathway for pot mRNA folding that is consistent with the mutations and implicates the formation of important kinetic intermediates. The signficance of these observations for the RNA folding problem in general is discussed.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1572-994X
    Keywords: HSV-1 ; SV40 ; promoters ; recombinant viruses
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract To determine the role of the HSV-1 genome structure and environment on the regulation of gene expression, we constructed recombinant viruses containing a heterologous gene inserted into either the immediate early ICP0 or late glycoprotein C (gC) genes of HSV-1. The heterologous gene consisted of the SV40 early promoter (without enhancer sequences) linked to the coding sequences for the bacterial chloramphenicol acetyl transferase (CAT). The expression of CAT was examined in Vero cells infected with either virus (named ICP0-CAT and Sph 6). For both recombinants, expression of CAT was not dependent upon prior viral protein synthesis. The kinetics of expression of CAT-specific mRNA resembled that of the HSV-1 genes into which CAT was inserted. Primer extension analysis revealed that the SV40 promoter is recognized and used when placed in cis in two different HSV-1 genome locations, and Northern hybridization experiments confirmed that the heterologous gene was expressed in the absence of prior viral protein synthesis. Therefore, this gene was not regulated as strictly as an HSV-1 gene, but was influenced by the environment into which it was placed, presumably by factors that are present when the normal viral gene is on.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1573-0603
    Keywords: Capsule synthesis ; Mutagenesis ; Recombination ; Streptococci ; Suicide vector
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We describe the use of suicide vectors to generate site-specific mutations in group B streptococcus. This is accomplished by cloning gene-specific sequences into a temperature sensitive plasmid and selecting for clones which have undergone homologous recombination. The recombinan clones can be easily isolated by selecting for clones which have retained the antibiotic resistance markers that are present on the vector or cloned into the gene-specific sequences. To confirm the fidelity of the recombination events, PCR analysis is performed on chromosomal DNA isolated from the recombinant clones. Using this strategy, we have generated site- specific insertions in several capsule genes and have found that these insertions occurred as expected and that the mutations result in loss of capsule expression. In this report, we specifically detail the construction of cpsB mutants by single and double cross-over recombination and demonstrate that the resulting strains are acapsular.
    Type of Medium: Electronic Resource
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