Arginine 30 and asparagine 74 have functional roles in the glutamine dependent activities of Escherichia coli asparagine synthetase B.

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

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Anti-Bacterial Effect of CpG-DNA Involves Enhancement of the Complement Systems

International Journal of Molecular Sciences ◽

10.3390/ijms20143397 ◽

2019 ◽

Vol 20 (14) ◽

pp. 3397 ◽

Cited By ~ 1

Author(s):

Kim ◽

Park ◽

Kim ◽

Gautam ◽

Akauliya ◽

...

Keyword(s):

Escherichia Coli ◽

Immune System ◽

Bacterial Infections ◽

Bacterial Activity ◽

Complement C3 ◽

Host Immune System ◽

Cpg Dna ◽

Functional Roles ◽

E Coli ◽

Bacterial Effect

CpG-DNA activates the host immune system to resist bacterial infections. In this study, we examined the protective effect of CpG-DNA in mice against Escherichia coli (E. coli) K1 infection. Administration of CpG-DNA increased the survival of mice after E. coli K1 infection, which reduces the numbers of bacteria in the organs. Pre-injection of mice with CpG-DNA before E. coli K1 infection increased the levels of the complement C3 but not C3a and C3b. The survival of the mice after E. coli K1 infection was significantly decreased when the mice were pre-injected with the cobra venom factor (CVF) removing the complement compared to the non-CVF-treated mice group. It suggests that the complement has protective roles against E. coli K1 infection. In addition, the survival of complement-depleted mice was increased by CpG-DNA pre-administration before E. coli K1 infection. Therefore, we suggest that CpG-DNA enhances the anti-bacterial activity of the immune system by augmenting the levels of complement systems after E. coli K1 infection and triggering other factors as well. Further studies are required to investigate the functional roles of the CpG-DNA-induced complement regulation and other factors against urgent bacterial infection.

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Recovery of active beta-lactamases from Proteus vulgaris and RTEM-1 hybrid by random mutagenesis by using a dnaQ strain of Escherichia coli.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.9.2152 ◽

1996 ◽

Vol 40 (9) ◽

pp. 2152-2159 ◽

Cited By ~ 6

Author(s):

S M Hosseini-Mazinani ◽

E Nakajima ◽

Y Ihara ◽

K Z Kameyama ◽

K Sugimoto

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Random Mutagenesis ◽

Amino Acid Residues ◽

Proteus Vulgaris ◽

Coding Region ◽

Beta Lactamases ◽

Functional Roles ◽

Hybrid Genes ◽

Lactamase Gene

Proteus vulgaris and RTEM-1 beta-lactamases that belong to molecular class A with 37% amino acid similarity were examined to find the relationship between amino acid residues and activity of enzymes. MICs of ampicillin were > 2,000 micrograms/ml for Escherichia coli cells producing these enzymes. We have made 18 hybrid genes by substituting the coding region of the P. vulgaris beta-lactamase gene with the equivalent portions from the RTEM-1 gene. Most of these hybrids produced inactive proteins, but a few hybrid enzymes had partial or trace activity. From one of the hybrid genes (MIC of ampicillin, 100 micrograms/ml), we recovered three kinds of active mutants which provided ampicillin MICs of 1,000 micrograms/ml by the selection of spontaneous mutations in a dnaQ strain of E. coli. In these mutants, Leu-148, Met-182, and Tyr-274 were replaced with Val, Thr, and His, respectively. These amino acids have not been identified as residues with functional roles in substrate hydrolysis. Furthermore, from these hybrid mutants, we obtained a second set of mutants which conferred ampicillin MICs of 1,500 micrograms/ml. Interestingly, the second mutations were limited to these three amino acid substitutions. These amino acid residues which do not directly interact with substrates have an effect on enzyme activity. These mutant enzymes exhibited lower K(m) values for cephaloridine than both parental enzymes.

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Transformation of Lotus corniculatus Plants with Escherichia coli Asparagine Synthetase A: Effect on Nitrogen Assimilation and Plant Development

Plant Cell Tissue and Organ Culture (PCTOC) ◽

10.1023/b:ticu.0000022554.27490.3d ◽

2004 ◽

Vol 78 (2) ◽

pp. 139-150 ◽

Cited By ~ 13

Author(s):

Michele Bellucci ◽

Luisa Ederli ◽

Francesca De Marchis ◽

Stefania Pasqualini ◽

Sergio Arcioni

Keyword(s):

Escherichia Coli ◽

Plant Development ◽

Nitrogen Assimilation ◽

Lotus Corniculatus ◽

Asparagine Synthetase

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Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells.

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1623 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1623-1628 ◽

Cited By ~ 18

Author(s):

M Cartier ◽

M W Chang ◽

C P Stanners

Keyword(s):

Escherichia Coli ◽

Cell Lines ◽

Animal Cell ◽

Chinese Hamster ◽

Simian Virus 40 ◽

Simian Virus ◽

Asparagine Synthetase ◽

Translational Initiation ◽

Shuttle Vectors ◽

Advantages And Disadvantages

A new dominant amplifiable selective system for use in bacterium-animal cell shuttle vectors was developed by the insertion of a 2-kilobase genomic fragment containing the cloned Escherichia coli gene for asparagine synthetase (AS) into the pBR322-simian virus 40 recombinant vector pSV2 so as to place the translational initiator codon for the bacterial AS about 1,000 base pairs downstream from the simian virus 40 early promoter. This new construct, pSV2-AS, retains bacterial sequences for transcriptional and translational initiation and so can express AS in bacteria. The construct can also complement AS- mutants of mammalian cells, giving AS+ transfectants capable of growth in medium lacking asparagine, with relatively high efficiency (about 300 colonies per microgram of DNA per 10(6) cells exposed). The vector can be amplified up to 100-fold in such AS+ transfectants by selection in asparagine-free medium containing increasing concentrations of the AS inhibitor beta-aspartyl hydroxamate. AS+ transfectants were found to be much more resistant to a second AS inhibitor, Albizziin, than were normal AS+ animal cell lines. This difference, which may indicate a strong resistance of the bacterial AS enzyme to Albizziin, was exploited to develop an effective selection for bacterial AS transfectants of a number of wild-type AS+ cell lines of rat, Chinese hamster, mouse, and human origin. LR-73 cells, a Chinese hamster AS+ cell line, were transfected with pSV2-AS with an efficiency of about 1,000 colonies per 0.5 microgram of DNA per 10(6) cells. The integrated construct in these cells was amplified by incubation of the transfectants in increasing concentrations of beta-aspartyl hydroxamate. Advantages and disadvantages of this new dominant, selectable, and amplifiable marker over markers commonly used in shuttle vectors are discussed.

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