Insertion mutagenesis of the lac repressor and its implications for structure-function analysis

Nelson, B.; Manoil, Colin; Traxler, Beth

doi:10.1128/jb.179.11.3721-3728.1997

Cited by 26 publications

(25 citation statements)

References 50 publications

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“…The most likely explanation for such discrepancies is the fact that the Mu transposition system used does not substitute or delete any amino acid residues, and the original amino acids were retained in the mutated virus. Several insertional mutagenesis studies indicate that insertions do not always perturb protein function (Manoil and Bailey 1997;Nelson et al 1997;Neuveglise et al 1998;Laurent et al 2000). Another explanation may be that in trans complementation (e.g., Li and Carrington 1995) between two viral genomes carrying insertions at different positions and coinfecting protoplasts might mask mutant phenotypes and interfere with detection of certain essential sites.…”

Section: Discussionmentioning

confidence: 99%

Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Kekarainen¹,

Savilahti²,

Valkonen³

2002

Genome Res.

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Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient in vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate in tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles

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Section: Discussionmentioning

confidence: 99%

Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Kekarainen¹,

Savilahti²,

Valkonen³

2002

Genome Res.

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“…Previous work with the well-defined LacI repressor protein using these transposons identified the important functional motifs, validating their use in structure-function analysis of less-well-defined proteins (22). Briefly, prgB, carried on a shuttle plasmid, was targeted by TnlacZ/in or TnphoA/in in E. coli strain CC160.…”

Section: Construction Of Prgb Insertion Mutantsmentioning

confidence: 99%

“…Digestion of the insertion with BamHI removes most of the transposon but leaves an in-frame 31-amino-acid insertion. These transposons have been successfully used to analyze the structure-function relationship of a number of membrane and cytosolic proteins (14,15,19,22,23), but this is the first attempt to use them in the analysis of a gram-positive surface protein.…”

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confidence: 99%

Analysis of Functional Domains of the Enterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

Waters

Dunny

2001

J Bacteriol

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Pheromone-inducible aggregation substance (AS) proteins of Enterococcus faecalis are essential for highefficiency conjugation of the sex pheromone plasmids and also serve as virulence factors during host infection. A number of different functions have been attributed to AS in addition to bacterial cell aggregation, including adhesion to host cells, adhesion to fibrin, increased cell surface hydrophobicity, resistance to killing by polymorphonuclear leukocytes and macrophages, and increased vegetation size in an experimental endocarditis model. Relatively little information is available regarding the structure-activity relationship of AS. To identify functional domains, a library of 23 nonpolar 31-amino-acid insertions was constructed in Asc10, the AS encoded by the plasmid pCF10, using the transposons TnlacZ/in and TnphoA/in. Analysis of these insertions revealed a domain necessary for donor-recipient aggregation that extends further into the amino terminus of the protein than previously reported. In addition, insertions in the C terminus of the protein also reduced aggregation. As expected, the ability to aggregate correlates with efficient plasmid transfer. The results also indicated that an increase in cell surface hydrophobicity resulting from AS expression is not sufficient to mediate bacterial aggregation.Enterococcus faecalis has become a growing health concern as a mediator of the spread of antibiotic resistance and a leading agent of nosocomial infections (for review, see reference 10). The surface protein aggregation substance (AS) appears to play a role in both antibiotic resistance spread and in the pathogenesis of enterococcal infections. Expression of AS, which is encoded on the sex pheromone plasmids of E. faecalis, is induced by small 7-to 8-amino-acid peptide pheromones (26). AS on the surface of the donor cell then binds its receptor, enterococcal binding substance, on the recipient cell, mediating close cell contact that leads to conjugative transfer of the plasmid. It is thought that AS has no role in forming the DNA channel machinery, as efficient conjugation can occur if AS is expressed on either the donor or recipient cells (26).Over 20 different pheromone plasmids have been identified. Often, these pheromone plasmids express antibiotic resistance genes and other virulence factors, and many clinical isolates have multiple pheromone plasmids (36). The AS genes from the three most-studied plasmids, Asa1 from pAD1, Asp1 from pPD1, and Asc10 from pCF10 (encoded by the prgB gene), have been sequenced and show high identity (see below). The gene encoding Asa373, the AS protein of the pheromone plasmid pAM373, has also been sequenced but shows little homology with the other known AS proteins and appears to aggregate through a different mechanism (21). Expression of AS, which is normally tightly controlled in laboratory cultures, is induced in serum (13).A number of functions of AS that may contribute to virulence have been identified. A major function of AS is host cell adhesion. Kreft et al. fou...

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“…The location of the 15-bp insertion was identified by a genetic footprinting analysis. Random linker-insertion mutagenesis has been employed in structure-function studies of proteins, and the results indicate a minimal effect on protein conformation (18,21,24,30). Therefore, to perform genome-scale functional profiling analysis of a herpesvirus, we have developed a high-throughput mutational analysis platform by combining a Mu transposonmediated 15-bp random insertion mutagenesis system and a quantitative, genetic profiling method with capillary electrophoresis.…”

Section: Human Gammaherpesviruses Epstein-barr Virus (Ebv) Andmentioning

confidence: 99%

High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome

Arumugaswami

Sitapara

Hwang

et al. 2009

J Virol

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Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a model to develop the functional profiling approach. The mutant murine gammaherpesvirus 68 viral library, containing 15-bp random insertions in the RTA locus, was passaged in murine fibroblast cells for multiple rounds of selection. The effect of each 15-bp insertion was characterized using fluorescent-PCR profiling. We identified 1,229 insertions in the 3,845-bp RTA locus, of which 393, 282, and 554 were critically impaired, attenuated, and tolerated, respectively, for viral growth. The functional profiling phenotypes were verified by examining several individual RTA mutant clones for transactivating function of the RTA promoter and transcomplementing function of the RTA-null virus. Thus, the profiling approach enabled us to identify several novel functional domains in the RTA locus in the context of the herpesvirus genome. Importantly, our study has demonstrated a novel system to conduct high-density functional genetic mapping. The genome-scale expansion of the genetic profiling approach will expedite the functional genomics research on herpesvirus. Human gammaherpesviruses Epstein-Barr virus (EBV) andKaposi's sarcoma-associated herpesvirus (KSHV) are involved in the development of epithelial, mucosal, hematopoietic, and endothelial cell cancers (7, 28). Murine gammaherpesvirus 68 (MHV-68), a pathogen affecting wild rodents, has been used as an animal model to study the fundamental principles underlying replication of gammaherpesviruses (32,35,37). For gamma-2 herpesviruses, including KSHV and MHV-68, the balance between latent and lytic phases of the viral life cycle is controlled by the immediate-early viral protein, RTA (replication and transcription activator) (11,25,26,34,41); hence, RTA plays a pivotal role in the viral life cycle. RTA is a b-zipper protein consisting of N-terminal DNA binding (DBD) and dimerization domains and a C-terminal transactivation (TA) domain (9,10,13,15,36). Understanding both the regulation of RTA transcription and the mechanism of RTA function is important. The promoter of RTA contains many regulatory cis-elements that are bound by viral and cellular factors, resulting in transcriptional activation or repression of RTA. Protein kinase C, cyclic AMP/protein kinase A, AP-1, Ras/Raf/ MEK/ERK/Ets-1, and Notch signaling pathways have been demonstrated to induce KSHV reactivation through direct or indirect activation of RTA expression (8,23,38,43,45). NF-B and LANA negatively regulate the RTA expression (4,19,20).The promoter elements of RTA have been studied extensively using transient transfe...

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Insertion mutagenesis of the lac repressor and its implications for structure-function analysis

Cited by 26 publications

References 50 publications

Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Analysis of Functional Domains of the Enterococcus faecalis Pheromone-Induced Surface Protein Aggregation Substance

High-Resolution Functional Profiling of a Gammaherpesvirus RTA Locus in the Context of the Viral Genome

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