Murine Gammaherpesvirus 68 LANA Acts on Terminal Repeat DNA To Mediate Episome Persistence

Murine gammaherpesvirus 68 (MHV68) ORF73 (mLANA) has sequence homology to Kaposi''s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA). LANA acts on the KSHV terminal repeat (TR) elements to mediate KSHV episome maintenance. Disruption of mLANA expression severely reduces the ability of MHV68 to establish latent infection in mice, consistent with the possibility that mLANA mediates episome persistence. Here we assess the roles of mLANA and MHV68 TR (mTR) elements in episome persistence. mTR-associated DNA persisted as an episome in latently MHV68-infected tumor cells, demonstrating that the mTR elements can serve as a cis-acting element for MHV68 episome maintenance. In some cases, both control vector and mTR-associated DNAs integrated into MHV68 episomal genomes. Therefore, we also assessed the roles of mTRs as well as mLANA in the absence of infection. DNA containing both mLANA and mTRs in cis persisted as an episome in murine A20 or MEF cells. In contrast, mTR DNA never persisted as an episome in the absence of mLANA. mLANA levels were increased when mLANA was expressed from its native promoters, and episome maintenance was more efficient with higher mLANA levels. Increased numbers of mTRs conferred more efficient episome maintenance, since DNA containing mLANA and eight mTR elements persisted more efficiently in A20 cells than did DNA with mLANA and two or four mTRs. Similar to KSHV LANA, mLANA broadly associated with mitotic chromosomes but relocalized to concentrated dots in the presence of episomes. Therefore, mLANA acts on mTR elements to mediate MHV68 episome persistence.     

Genetic analysis of transpositions in the lac region of Escherichia coli

The lac region of Escherichia coli, carried on an F′ lacproB episome, was used as a target for the transposition of several transposable elements. Tn9 shows a preferential integration (by a factor of 50) into a region extending from the end of the Z gene through the Y gene. Throughout the remainder of the lacI, Z and Y genes one other short region, located in the middle of the I gene, is favored for integration. Within these favored regions many different integration points are evident. Inspection of the DNA sequence for the I and Y genes, and parts of the Z gene, shows a strong correlation between A + T richness and regions of preferential integration. Tn5 insertions follow a similar pattern, although with less preference; whereas Tn10 insertions (provided by T. J. Foster), also favor the Y gene and the end of Z, but are distributed among fewer integration points. Most of the Tn3 insertions into the episome are accompanied by a nearby or adjacent deletion.     

Isolation of P22 Specialized Transducing Phage following F''-Episome Fusion

A P22 specialized transducing phage has been constructed which carries the structural gene for aspartate transcarbamylase (ATCase). This gene (pyrB) was first brought close to the P22 attachment site by fusing an F' pyrB⁺ episome to an F' prolac episome which carries a P22 prophage attachment site. A prophage was added to these fused F' episomes and the lysogen was UV-induced. The specialized transducing phage was isolated from the resulting lysate. The phage also carries argI, the structure gene for ornithine transcarbamylase.     

Differential use of multiple replication origins in the ribosomal DNA episome of the protozoan parasite Entamoeba histolytica

The factors that control the initiation of eukaryotic DNA replication from defined origins (oris) on the chromosome remain incompletely resolved. Here we show that the circular rDNA episome of the human pathogen Entamoeba histolytica contains multiple potential oris, which are utilized in a differential manner. The primary ori in exponentially growing cells was mapped close to the promoter of rRNA genes in the upstream intergenic spacer (IGS) by two-dimensional gel electrophoresis. Replication initiated predominantly from the upstream IGS and terminated in the downstream IGS. However, when serum-starved cells were allowed to resume growth, the early oris which became activated were located in other parts of the molecule. Later the ori in the upstream IGS became activated, with concomitant silencing of the early oris. When the upstream IGS was located ectopically in an artificial plasmid, it again lost ori activity, while other parts of the rDNA episome could function as oris in this system. Therefore, the activation or silencing of the ori in this episome is context dependent, as is also the case with many eukaryotic replicons. This is the first replication origin to be mapped in this primitive protozoan and will provide an opportunity to define the factors involved in differential ori activity, and their comparison with metazoans.     

Characterization of V-Prime Factors in Escherichia coli K-12

An episome derived from an Hfr_v (Hfr isolated from a V colicinogenic parent) strain of Escherichia coli K-12 was isolated and characterized. The direction of gene transfer was inverted from that in the original parental strain.     

Phage-associated mutator phenotype in group A streptococcus

Defects in DNA mismatch repair (MMR) occur frequently in natural populations of pathogenic and commensal bacteria, resulting in a mutator phenotype. We identified a unique genetic element in Streptococcus pyogenes strain SF370 that controls MMR via a dynamic process of prophage excision and reintegration in response to growth. In S. pyogenes, mutS and mutL are organized on a polycistronic mRNA under control of a common promoter. Prophage SF370.4 is integrated between the two genes, blocking expression of the downstream gene (mutL) and resulting in a mutator phenotype. However, in rapidly growing cells the prophage excises and replicates as an episome, allowing mutL to be expressed. Excision of prophage SF370.4 and expression of MutL mRNA occur simultaneously during early logarithmic growth when cell densities are low; this brief window of MutL gene expression ends as the cell density increases. However, detectable amounts of MutL protein remain in the cell until the onset of stationary phase. Thus, MMR in S. pyogenes SF370 is functional in exponentially growing cells but defective when resources are limiting. The presence of a prophage integrated into the 5′ end of mutL correlates with a mutator phenotype (10⁻⁷ to 10⁻⁸ mutation/generation, an approximately a 100-fold increase in the rate of spontaneous mutation compared with prophage-free strains [10⁻⁹ to 10⁻¹⁰ mutation/generation]). Such genetic elements may be common in S. pyogenes since 6 of 13 completed genomes have related prophages, and a survey of 100 strains found that about 20% of them are positive for phages occupying the SF370.4 attP site. The dynamic control of a major DNA repair system by a bacteriophage is a novel method for achieving the mutator phenotype and may allow the organism to respond rapidly to a changing environment while minimizing the risks associated with long-term hypermutability.     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

The thermophilic protease aqualysin I (AQI) gene (aqul), derived from Thermus aquaticus YT-I, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid. The plasmid was introduced into two strains of E. coli JMI09 (DE3), one carrying and one lacking an F’ episome, which carries the lacI_q gene. Upon cultivation the strain carrying an F’ episome produced AQI as an insoluble fusion protein (74 kDa) with the T7 gene 10 protein. This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity. On the other hand, when the strain lacking an F’ episome was used as a host cell for aqul expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form. This soluble protein could be processed into active AQI by heat treatment. Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction.     

Structural Genes for Ornithine Transcarbamylase in Salmonella typhimurium and Escherichia coli K-12

Mutations of Salmonella typhimurium affecting the structural gene for ornithine transcarbamylase (argl) have been isolated and mapped. The two ornithine transcarbamylase loci in Escherichia coli K-12 have been demonstrated by F′ episome transfer.     

Robust reporter system based on chalcone synthase rppA gene from Saccharopolyspora erythraea

Industrial overproducing strains present unique hosts for expression of heterologous gene clusters encoding secondary metabolite biosynthesis. For this purpose, efficient gene expression tools and methods are needed. A robust and versatile reporter system based on the rppA gene from Saccharopolyspora erythraea is presented as the method of choice when studying gene expression in actinomycete hosts. The method is easily scalable to accommodate high-throughput procedure, and collected samples can be easily stored and re-tested when needed. The product of RppA is an inert 1,3,6,8-tetrahydroxynaphthalene which spontaneously oxidises to a dark-red quinone flaviolin providing a qualitative visual assessment of gene expression on an agar plate as well as a quantitative spectrophotometric measurement in liquid broth without the need for invasive procedures or external substrate addition. The applicability of the reporter system has been demonstrated by expressing the rppA gene under the control of the heterologous promoters actII-ORF4/P_actI, ermE and its upregulated variant ermE*. The model streptomycete Streptomyces coelicolor, and three industrially important species, Streptomyces tsukubaensis (FK506), Streptomyces cinnamonensis (monensin) and Streptomyces rimosus (oxytetracycline) were used as hosts. The reporter system has shown its utility independently of cultivation conditions or composition of growth medium, from simple laboratory to complex industrial media. The simplicity and robustness of the system, demonstrated even in industrial settings, shows great potential for wider use in different microbial hosts and applications, and may thus represent a new generic and versatile tool useful to a wider scientific community.     

Penicillin-Binding Protein 1 of Staphylococcus aureus Is Essential for Growth

pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in an Escherichia coli MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.     

Entamoeba histolytica: gene linkage groups and relevant features of its karyotype

We identified some gene linkage groups in Entamoeba histolytica using a 4-M urea improved transversal alternating field electrophoresis (TAFE) method. Complex rosette-structured DNA molecules were found trapped along the gel lanes, explaining the fuzziness of the patterns. Using several episomal probes, including 16 S, 5.8 S, and 25 S ribosomal (r)Dna genes, an autonomous replication sequence (ARS), and EhVR1, we identified a complete ribosomal episome linkage group (CELG) at the 1.2-Mb position. Three other incomplete groups were found: IELG-1, formed by EhVR1,16 S, 5.8 S, and 25 S genes; IELG-2 formed by EhVR1, 16 S and 25 S; and IELG-3 formed only by 5.8 S. Ehadh3, Ehpfo, and Ehredox genes migrated at the 1.8-Mb position, forming the non-ribosomal linkage group, NRLG-1.8, while the Ehenl-1 gene migrated at 1.6 Mb forming the NRLG-1.6 group. Ehhk was located at 1.2, 0.8, and 0.17 Mb in three different groups: NRLG-1.2, IELG-3-0.8, and NRLG-0.17. Putative lineal chromosomes were also identified using an heterologous telomeric probe. By in situ hybridization experiments, the rDNA and Ehhk genes were located in both nucleus and cytoplasm, while the Ehpfo and Ehredox genes were found mainly in the nucleus. We propose a model hypothezising that the 16 S and 25?S genes are in a linear molecule, duplicated in two inverted repeats, which may be looped out of the linear DNA to form an episome probably lacking or not the 5.8 S sequence, which could be added later by recombination.     

High efficiency, site-specific excision of a marker gene by the phage P1 cre–loxP system in the yellow fever mosquito, Aedes aegypti

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre–loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre–loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.