Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

Godiska, Ronald; Mead, David A.; Dhodda, Vinay K.; Wu, Chun-Ta; Hochstein, Rebecca; Karsi, Attila; Usdin, Karen; Entezam, Ali; Ravin, Nikolai V.

doi:10.1093/nar/gkp1181

Cited by 92 publications

(106 citation statements)

References 40 publications

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“…As a result, we have encountered instances where, due to the foreign gene products produced, particular constructs were unstable in E. coli (unpublished results). Likewise, cloned heterologous DNA from extremely AT-rich organisms is also known to be exceptionally unstable in E. coli, due to frequent deletions and rearrangements (11). Thus, a cloning-independent methodology should be particularly useful for constructing markerless mutations in these species.…”

Section: Discussionmentioning

confidence: 99%

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

Xie

Okinaga

et al. 2011

Appl Environ Microbiol

119

156

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Insertion duplication mutagenesis and allelic replacement mutagenesis are among the most commonly utilized approaches for targeted mutagenesis in bacteria. However, both techniques are limited by a variety of factors that can complicate mutant phenotypic studies. To circumvent these limitations, multiple markerless mutagenesis techniques have been developed that utilize either temperature-sensitive plasmids or counterselectable suicide vectors containing both positive-and negative-selection markers. For many species, these techniques are not especially useful due to difficulties of cloning with Escherichia coli and/or a lack of functional negative-selection markers. In this study, we describe the development of a novel approach for the creation of markerless mutations. This system employs a cloning-independent methodology and should be easily adaptable to a wide array of Gram-positive and Gram-negative bacterial species. The entire process of creating both the counterselection cassette and mutation constructs can be completed using overlapping PCR protocols, which allows extremely quick assembly and eliminates the requirement for either temperature-sensitive replicons or suicide vectors. As a proof of principle, we used Streptococcus mutans reference strain UA159 to create markerless in-frame deletions of 3 separate bacteriocin genes as well as triple mutants containing all 3 deletions. Using a panel of 5 separate wild-type S. mutans strains, we further demonstrated that the procedure is nearly 100% efficient at generating clones with the desired markerless mutation, which is a considerable improvement in yield compared to existing approaches.Streptococcus mutans is a Gram-positive bacterial species that resides within multispecies oral biofilms formed on human tooth surfaces. It is also considered to be one of the principal species associated with dental caries initiation (6,7,34,35,44, 47,52). S. mutans genetic research has benefited tremendously from the many genetic tools that have been adapted for use in studies of the organism (4,5,13,15,22,25,26,29,33,45,51). For genetic studies of S. mutans, defined mutations are usually engineered in either of two ways: insertion duplication mutagenesis via single-crossover homologous recombination or marked allelic replacement mutagenesis using double-crossover homologous recombination (25,27,41). Both approaches are highly reliable strategies for mutagenesis and are simple to engineer, but they also have the potential to create unwanted artifacts that could influence the outcome of a genetic study. For example, insertion duplication mutations often result in the production of truncated proteins. Rarely is it known with certainty whether the protein fragments actually influence mutant phenotypes. Furthermore, due to significant polar effects downstream of the mutation site, both insertion duplication mutagenesis and allelic replacement mutagenesis are of limited utility within operons. In some cases, these issues have been addressed by creating allelic replacement mut...

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Section: Discussionmentioning

confidence: 99%

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

Xie

Okinaga

et al. 2011

Appl Environ Microbiol

119

156

View full text Add to dashboard Cite

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“…Strategies such as insertion of introns into the viral genome (25,26) or maintaining the genome in multiple small fragments (27-31) have been used to overcome the inherent instability of the ZIKV and other flavivirus genomes. Here, using a novel linear vector (32) (Fig. 1A) with bacterial transcription terminators flanking the cloning site, we generated stable full-length cDNA clones of the ZIKV genome.…”

mentioning

confidence: 99%

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Annamalai

Pattnaik

Sahoo

et al. 2017

J Virol

111

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Zika virus (ZIKV), a mosquito-transmitted flavivirus responsible for sporadic outbreaks of mild and febrile illness in Africa and Asia, reemerged in the last decade causing serious human diseases, including microcephaly, congenital malformations, and Guillain-Barré syndrome. Although genomic and phylogenetic analyses suggest that genetic evolution may have led to the enhanced virulence of ZIKV, experimental evidence supporting the role of specific genetic changes in virulence is currently lacking. One sequence motif, VNDT, containing an N-linked glycosylation site in the envelope (E) protein, is polymorphic; it is absent in many of the African isolates but present in all isolates from the recent outbreaks. In the present study, we investigated the roles of this sequence motif and glycosylation of the E protein in the pathogenicity of ZIKV. We first constructed a stable full-length cDNA clone of ZIKV in a novel linear vector from which infectious virus was recovered. The recombinant ZIKV generated from the infectious clone, which contains the VNDT motif, is highly pathogenic and causes lethality in a mouse model. In contrast, recombinant viruses from which the VNDT motif is deleted or in which the N-linked glycosylation site is mutated by single-amino-acid substitution are highly attenuated and nonlethal. The mutant viruses replicate poorly in the brains of infected mice when inoculated subcutaneously but replicate well following intracranial inoculation. Our findings provide the first evidence that N-linked glycosylation of the E protein is an important determinant of ZIKV virulence and neuroinvasion.IMPORTANCE The recent emergence of Zika virus (ZIKV) in the Americas has caused major worldwide public health concern. The virus appears to have gained significant pathogenicity, causing serious human diseases, including microcephaly and Guillain-Barré syndrome. The factors responsible for the emergence of pathogenic ZIKV are not understood at this time, although genetic changes have been shown to facilitate virus transmission. All isolates from the recent outbreaks contain an N-linked glycosylation site within the viral envelope (E) protein, whereas many isolates of the African lineage virus lack this site. To elucidate the functional significance of glycosylation in ZIKV pathogenicity, recombinant ZIKVs from infectious clones with or without the glycan on the E protein were generated. ZIKVs lacking the glycan were highly attenuated for the ability to cause mortality in a mouse model and were severely compromised for neuroinvasion. Our studies suggest glycosylation of the E protein is an important factor contributing to ZIKV pathogenicity.

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“…Their high GC content causes amplification failure. A high concentration of housekeeping genes impedes the survival of such DNAs in BACs because of the transcription of the cloned DNA and selection against open reading frames [Godiska et al, 2010]. That is probably why chicken chromosome-specific BAC markers for the smallest MIs are missing in the available chicken genomic BAC-clone collections (CHORI-261 [https://bacpacresources.org], WAG [http://www.us.lifesciences.sourcebioscience.com]).…”

Section: Discussionmentioning

confidence: 99%

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description

Galkina¹,

Fillon²,

Saifitdinova³

et al. 2017

Cytogenet Genome Res

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Lampbrush chromosomes are giant, transcriptionally active, meiotic chromosomes found in oocytes of all vertebrates with the exception of mammals. Lampbrush chromosomes offer a convenient tool for cytogenetic mapping and, in particular, have been instrumental in mapping genes and linkage groups on chicken (GGA) chromosomes. Whereas cytogenetic maps of macrochromosome GGA1-10 and microchromosome GGA11-16 lampbrush bivalents have been established, identification and description of smaller microchromosome bivalents are still missing. In this work, we used specific FISH probes for the identification of 12 chicken lampbrush chromosomes formed by GGA17-28. Our observations on chromomere and lateral loop arrangement and chiasma position allowed us to construct the respective cytogenetic maps for these microchromosomes. For the 10 smallest chicken microchromosomes, GGA29-38, no individual molecular tags are available, yet they can be collectively marked using the PO41 repeat. The reported results contribute to building of working cytogenetic maps of the chicken karyotype.

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Linear plasmid vector for cloning of repetitive or unstable sequences in Escherichia coli

Cited by 92 publications

References 40 publications

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

Cloning-Independent and Counterselectable Markerless Mutagenesis System in Streptococcus mutans

Zika Virus Encoding Nonglycosylated Envelope Protein Is Attenuated and Defective in Neuroinvasion

Chicken Microchromosomes in the Lampbrush Phase: A Cytogenetic Description

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