Duplication of insertion element IS50 associated with Tn5 transposition in Azospirillum brasilense

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

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Molecular cloning and characterization of mutant and wild-type human beta-actin genes.

Molecular and Cellular Biology ◽

10.1128/mcb.4.10.1961 ◽

1984 ◽

Vol 4 (10) ◽

pp. 1961-1969 ◽

Cited By ~ 45

Author(s):

J Leavitt ◽

P Gunning ◽

P Porreca ◽

S Y Ng ◽

C S Lin ◽

...

Keyword(s):

Homologous Recombination ◽

Human Fibroblasts ◽

Actin Gene ◽

Actin Genes ◽

Beta Actin ◽

Gene Libraries ◽

Actin Mrna ◽

Dna Fragment ◽

Specific Sequences

There are more than 20 beta-actin-specific sequences in the human genome, many of which are pseudogenes. To facilitate the isolation of potentially functional beta-actin genes, we used the new method of B. Seed (Nucleic Acids Res. 11:2427-2446, 1983) for selecting genomic clones by homologous recombination. A derivative of the pi VX miniplasmid, pi AN7 beta 1, was constructed by insertion of the 600-base-pair 3' untranslated region of the beta-actin mRNA expressed in human fibroblasts. Five clones containing beta-actin sequences were selected from an amplified human fetal gene library by homologous recombination between library phage and the miniplasmid. One of these clones contained a complete beta-actin gene with a coding sequence identical to that determined for the mRNA of human fibroblasts. A DNA fragment consisting of mostly intervening sequences from this gene was then used to identify 13 independent recombinant copies of the analogous gene from two specially constructed gene libraries, each containing one of the two types of mutant beta-actin genes found in a line of neoplastic human fibroblasts. The amino acid and nucleotide sequences encoded by the unmutated gene predict that a guanine-to-adenine transition is responsible for the glycine-to-aspartic acid mutation at codon 244 and would also result in the loss of a HaeIII site. Detection of this HaeIII polymorphism among the fibroblast-derived clones verified the identity of the beta-actin gene expressed in human fibroblasts.

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Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping

Plasmid ◽

10.1016/0147-619x(79)90028-3 ◽

1979 ◽

Vol 2 (3) ◽

pp. 446-453

Author(s):

Lesley W. Coggins ◽

Eva McCluskey

Keyword(s):

Electron Microscope ◽

Restriction Endonuclease ◽

Restriction Endonuclease Mapping ◽

Endonuclease Mapping

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Construction and Characterization of an Acapsular Mutant of Mannheimia haemolytica A1

Infection and Immunity ◽

10.1128/iai.70.5.2622-2629.2002 ◽

2002 ◽

Vol 70 (5) ◽

pp. 2622-2629 ◽

Cited By ~ 14

Author(s):

Linda J. McKerral ◽

Reggie Y. C. Lo

Keyword(s):

Southern Hybridization ◽

Capsular Polysaccharide ◽

Calf Serum ◽

Mannheimia Haemolytica ◽

Knockout Mutant ◽

Chloramphenicol Resistance ◽

Polysaccharide Biosynthesis ◽

Dna Fragment ◽

Microscopy Examination

ABSTRACT The nmaA and nmaB genes, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in Mannheimia haemolytica A1. A chloramphenicol resistance (Cmr) cassette cloned behind an M. haemolytica A1 promoter, plpcat, was created and used to interrupt nmaA and nmaB. A 1.3-kbp DNA fragment that encompasses part of nmaA and nmaB was replaced by the 1.0-kbp plpcat, resulting in a knockout mutant which is Cmr and unable to synthesize N-acetylmannosamine (ManNAc) and N-acetylmannosaminuronic acid (ManNAcA). The DNA replacement was confirmed by Southern hybridization and PCR analyses of the nmaA and nmaB loci. Electron microscopy examination of the mutant showed the absence of capsular materials compared to the parent strain. The loss of NmaA and NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis. Serum sensitivity assays indicated that the acapsular mutant is as resistant as the encapsulated parent to complement-mediated killing by colostrum-deprived calf serum but is more sensitive to killing by immune bovine serum. Analysis of lipopolysaccharide prepared from the acapsular mutant and encapsulated parent confirmed that these strains have long O-polysaccharide chains, possibly conferring resistance to serum-mediated killing.

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Establishment and characterization of hamster cell lines transformed by restriction endonuclease fragments of adenovirus 5.

Journal of Virology ◽

10.1128/jvi.49.1.162-170.1984 ◽

1984 ◽

Vol 49 (1) ◽

pp. 162-170 ◽

Cited By ~ 11

Author(s):

D T Rowe ◽

P E Branton ◽

S P Yee ◽

S Bacchetti ◽

F L Graham

Keyword(s):

Restriction Endonuclease ◽

Cell Lines ◽

Adenovirus 5

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Cloning and Characterization of a Chromosomal Class C β-Lactamase and Its Regulatory Gene in Laribacter hongkongensis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.5.1957-1964.2005 ◽

2005 ◽

Vol 49 (5) ◽

pp. 1957-1964 ◽

Cited By ~ 23

Author(s):

Susanna K. P. Lau ◽

Pak-leung Ho ◽

Maria W. S. Li ◽

Hoi-wah Tsoi ◽

Raymond W. H. Yung ◽

...

Keyword(s):

Genomic Dna ◽

Southern Hybridization ◽

Regulatory Gene ◽

Kinetic Properties ◽

Restriction Fragments ◽

Regulatory Systems ◽

Laribacter Hongkongensis ◽

Consensus Motifs ◽

Class C

ABSTRACT Laribacter hongkongensis, a newly discovered bacterium recently shown to be associated with community-acquired gastroenteritis, is generally resistant to most β-lactams except the carbapenems. We describe the cloning and characterization of a novel chromosomal class C β-lactamase and its regulatory gene in L. hongkongensis. Two genes, ampC and ampR, were cloned by inserting restriction fragments of genomic DNA from L. hongkongensis strain HLHK5 into pBK-CMV to give the recombinant plasmid pBK-LHK-5. The ampR and ampC genes and their promoters were divergently oriented, with the ampR gene immediately upstream of the ampC gene and an intercistronic Lys-R motif, typical of inducible ampC-ampR regulatory systems. The deduced amino acid sequence of the cloned AmpC β-lactamase (pI 8.1) contained consensus motifs characteristic of class C β-lactamases but had identities no greater than 46% to known class C β-lactamases. The kinetic properties of this AmpC were also compatible with those of a class C β-lactamase. PCR of 20 clinical isolates of L. hongkongensis, including HLHK5, showed the presence of both ampC and ampR genes in all isolates. Southern hybridization suggested that the ampC gene of HLHK5 was chromosomally encoded. Subcloning experiments showed that the expression of the ampC gene of HLHK5 was regulated by its ampR gene, which acts as a repressor. The β-lactamase characterized from strain HLHK5 was named LHK-5 (gene, bla LHK-5) and represents the first example of AmpC β-lactamase in the β subdivision of proteobacteria.

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Engineering nucleosomes for generating diverse chromatin assemblies

Nucleic Acids Research ◽

10.1093/nar/gkab070 ◽

2021 ◽

Author(s):

Zenita Adhireksan ◽

Deepti Sharma ◽

Phoi Leng Lee ◽

Qiuye Bao ◽

Sivaraman Padavattan ◽

...

Keyword(s):

Dynamic Properties ◽

Dna Nanostructures ◽

Macromolecular Assemblies ◽

Maximum Resolution ◽

Different Types ◽

Chromatin Structural ◽

Dna Fragment

Abstract Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.

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