scholarly journals Optimization of a Lambda-RED Recombination Method for Rapid Gene Deletion in Human Cytomegalovirus

2021 ◽  
Vol 22 (19) ◽  
pp. 10558
Author(s):  
Estéfani García-Ríos ◽  
Julia Gata-de-Benito ◽  
Mireia López-Siles ◽  
Michael J. McConnell ◽  
Pilar Pérez-Romero
Keyword(s):  
Human Cytomegalovirus ◽  
Gene Function ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Artificial Chromosome ◽  
Deletion Mutants ◽  
Transplant Patients ◽  
Recombination System ◽  
Rapid Generation ◽  
Red Recombination

Human cytomegalovirus (HCMV) continues to be a major cause of morbidity in transplant patients and newborns. However, the functions of many of the more than 282 genes encoded in the HCMV genome remain unknown. The development of bacterial artificial chromosome (BAC) technology contributes to the genetic manipulation of several organisms including HCMV. The maintenance of the HCMV BAC in E. coli cells permits the rapid generation of recombinant viral genomes that can be used to produce viral progeny in cell cultures for the study of gene function. We optimized the Lambda-Red Recombination system to construct HCMV gene deletion mutants rapidly in the complete set of tested genes. This method constitutes a useful tool that allows for the quick generation of a high number of gene deletion mutants, allowing for the analysis of the whole genome to improve our understanding of HCMV gene function. This may also facilitate the development of novel vaccines and therapeutics.

scholarly journals Use of Recombination-Mediated Genetic Engineering for Construction of Rescue Human Cytomegalovirus Bacterial Artificial Chromosome Clones

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Kalpana Dulal ◽  
Benjamin Silver ◽  
Hua Zhu
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Homologous Recombination ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Bac Clone ◽  
Dna Viruses ◽  
E Coli ◽  
Recombination System ◽  
Capture Method

Bacterial artificial chromosome (BAC) technology has contributed immensely to manipulation of larger genomes in many organisms including large DNA viruses like human cytomegalovirus (HCMV). The HCMV BAC clone propagated and maintained insideE. coliallows for accurate recombinant virus generation. Using this system, we have generated a panel of HCMV deletion mutants and their rescue clones. In this paper, we describe the construction of HCMV BAC mutants using a homologous recombination system. A gene capture method, or gap repair cloning, to seize large fragments of DNA from the virus BAC in order to generate rescue viruses, is described in detail. Construction of rescue clones using gap repair cloning is highly efficient and provides a novel use of the homologous recombination-based method inE. colifor molecular cloning, known colloquially as recombineering, when rescuing large BAC deletions. This method of excising large fragments of DNA provides important prospects forin vitrohomologous recombination for genetic cloning.

scholarly journals A Gateway platform for functional genomics inHaloferax volcanii: deletion of three tRNA modification genes

Archaea ◽  
2009 ◽  
Vol 2 (4) ◽  
pp. 211-219 ◽  
Author(s):  
Basma El Yacoubi ◽  
Gabriela Phillips ◽  
Ian K. Blaby ◽  
Crysten E. Haas ◽  
Yulien Cruz ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Genetic Manipulation ◽  
Model Organism ◽  
Rna Modification ◽  
Deletion Mutants ◽  
Trna Modification ◽  
Gtp Cyclohydrolase I ◽  
Phenotypic Analysis ◽  
Targeted Gene Deletion ◽  
Folate Biosynthesis

In part due to the existence of simple methods for its cultivation and genetic manipulation,Haloferax volcaniiis a major archaeal model organism. It is the only archaeon for which the whole set of post-transcriptionally modified tRNAs has been sequenced, allowing for anin silicoprediction of all RNA modification genes present in the organism. One approach to check these predictions experimentally is via the construction of targeted gene deletion mutants. Toward this goal, an integrative “Gateway vector” that allows gene deletion inH. volcaniiuracil auxotrophs was constructed. The vector was used to delete three predicted tRNA modification genes: HVO_2001 (encoding an archaeal transglycosyl tranferase or arcTGT), which is involved in archeosine biosynthesis; HVO_2348 (encoding a newly discovered GTP cyclohydrolase I), which catalyzes the first step common to archaeosine and folate biosynthesis; and HVO_2736 (encoding a member of the COG1444 family), which is involved inN4-acetylcytidine (ac4C) formation. Preliminary phenotypic analysis of the deletion mutants was conducted, and confirmed all three predictions.

scholarly journals Rapid Genetic Engineering of Human Cytomegalovirus by Using a Lambda Phage Linear Recombination System: Demonstration that pp28 (UL99) Is Essential for Production of Infectious Virus

2004 ◽  
Vol 78 (1) ◽  
pp. 539-543 ◽  
Author(s):  
William J. Britt ◽  
Michael Jarvis ◽  
Jun-Young Seo ◽  
Derek Drummond ◽  
Jay Nelson
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Intracellular Localization ◽  
Point Mutations ◽  
Artificial Chromosome ◽  
Base Change ◽  
Chromosomal Dna ◽  
Lambda Phage ◽  
Recombination System

ABSTRACT A highly efficient lambda phage recombination system previously utilized for studies of bacterial artificial chromosome (BAC)-maintained mouse chromosomal DNA was adapted for the study of the role of human cytomegalovirus (HCMV)-encoded pp28 (UL99) in virus replication. Incorporating a two-step mutagenesis strategy with blue/white selection in Escherichia coli containing a HCMV AD169 BAC, we have shown that we can rapidly introduce point mutations into the HCMV BAC using linear PCR fragments. All manipulations were carried out in bacteria, which greatly accelerated the introduction and analysis of mutations in the viral genome. Our results indicated that HCMV pp28 was essential for the production of infectious virus and that introduction of a single base change that resulted in loss of the myristylation site on pp28 was also associated with the lack of production of infectious virus. Although the block in the viral morphogenesis cannot be determined from these studies, the latter finding suggested that authentic intracellular localization of pp28, not only the expression of the protein, is required for virus assembly.

scholarly journals Mutability of demographic noise in microbial range expansions

2021 ◽  
Author(s):  
QinQin Yu ◽  
Matti Gralka ◽  
Marie-Cécilia Duvernoy ◽  
Megan Sousa ◽  
Arbel Harpak ◽  
...  
Keyword(s):  
Gene Deletion ◽  
Genetic Manipulation ◽  
Single Gene ◽  
Population Level ◽  
Growth Conditions ◽  
Establishment Probability ◽  
In The Wild ◽  
Order Of Magnitude ◽  
Demographic Noise ◽  
Single Gene Deletion

AbstractDemographic noise, the change in the composition of a population due to random birth and death events, is an important driving force in evolution because it reduces the efficacy of natural selection. Demographic noise is typically thought to be set by the population size and the environment, but recent experiments with microbial range expansions have revealed substantial strain-level differences in demographic noise under the same growth conditions. Many genetic and phenotypic differences exist between strains; to what extent do single mutations change the strength of demographic noise? To investigate this question, we developed a high-throughput method for measuring demographic noise in colonies without the need for genetic manipulation. By applying this method to 191 randomly-selected single gene deletion strains from the E. coli Keio collection, we find that a typical single gene deletion mutation decreases demographic noise by 8% (maximal decrease: 81%). We find that the strength of demographic noise is an emergent trait at the population level that can be predicted by colony-level traits but not cell-level traits. The observed differences in demographic noise from single gene deletions can increase the establishment probability of beneficial mutations by almost an order of magnitude (compared to in the wild type). Our results show that single mutations can substantially alter adaptation through their effects on demographic noise and suggest that demographic noise can be an evolvable trait of a population.

scholarly journals Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Timo W. M. De Groof ◽  
Elizabeth G. Elder ◽  
Eleanor Y. Lim ◽  
Raimond Heukers ◽  
Nick D. Bergkamp ◽  
...  
Keyword(s):  
Gene Expression ◽  
Human Cytomegalovirus ◽  
Early Gene ◽  
Latent Reservoir ◽  
Solid Organ ◽  
Cell Transplant ◽  
Viral Receptor ◽  
Transplant Patients ◽  
Latently Infected ◽  
Infected Cells

AbstractLatent human cytomegalovirus (HCMV) infection is characterized by limited gene expression, making latent HCMV infections refractory to current treatments targeting viral replication. However, reactivation of latent HCMV in immunosuppressed solid organ and stem cell transplant patients often results in morbidity. Here, we report the killing of latently infected cells via a virus-specific nanobody (VUN100bv) that partially inhibits signaling of the viral receptor US28. VUN100bv reactivates immediate early gene expression in latently infected cells without inducing virus production. This allows recognition and killing of latently infected monocytes by autologous cytotoxic T lymphocytes from HCMV-seropositive individuals, which could serve as a therapy to reduce the HCMV latent reservoir of transplant patients.

scholarly journals Mutational Mapping of UL130 of Human Cytomegalovirus Defines Peptide Motifs within the C-Terminal Third as Essential for Endothelial Cell Infection

2010 ◽  
Vol 84 (18) ◽  
pp. 9019-9026 ◽  
Author(s):  
Andrea Schuessler ◽  
Kerstin Laib Sampaio ◽  
Laura Scrivano ◽  
Christian Sinzger
Keyword(s):  
Endothelial Cell ◽  
Human Cytomegalovirus ◽  
Artificial Chromosome ◽  
Protein Domain ◽  
Cell Infection ◽  
Peptide Motifs ◽  
Charged Amino Acids ◽  
C Terminus ◽  
Charge Cluster ◽  
A Charge

ABSTRACT The UL130 gene is one of the major determinants of endothelial cell (EC) tropism of human cytomegalovirus (HCMV). In order to define functionally important peptides within this protein, we have performed a charge-cluster-to-alanine (CCTA) mutational scanning of UL130 in the genetic background of a bacterial artificial chromosome-cloned endotheliotropic HCMV strain. A total of 10 charge clusters were defined, and in each of them two or three charged amino acids were replaced with alanines. While the six N-terminal clusters were phenotypically irrelevant, mutation of the four C-terminal clusters each caused a reduction of EC tropism. The importance of this protein domain was further emphasized by the fact that the C-terminal pentapeptide PNLIV was essential for infection of ECs, and the cell tropism could not be rescued by a scrambled version of this sequence. We conclude that the C terminus of the UL130 protein serves an important function for infection of ECs by HCMV. This makes UL130 a promising molecular target for antiviral strategies, e.g., the development of antiviral peptides.

Pleiotropic drug-resistance attenuated genomic library improves elucidation of drug mechanisms

2015 ◽  
Vol 11 (11) ◽  
pp. 3129-3136 ◽  
Author(s):  
Namal V. C. Coorey ◽  
James H. Matthews ◽  
David S. Bellows ◽  
Paul H. Atkinson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Drug Resistance ◽  
Mechanism Of Action ◽  
Gene Deletion ◽  
Genomic Library ◽  
Deletion Mutants ◽  
Pleiotropic Drug Resistance ◽  
Genome Wide

Identifying Saccharomyces cerevisiae genome-wide gene deletion mutants that confer hypersensitivity to a xenobiotic aids the elucidation of its mechanism of action (MoA).

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