The Biochemistry, Molecular Biology, and Genetic Manipulation of Primary Ammonia Assimilation

2002 ◽  
pp. 71-92 ◽  
Author(s):  
Bertrand Hirel ◽  
Peter J. Lea
Keyword(s):  
Molecular Biology ◽  
Genetic Manipulation ◽  
Ammonia Assimilation

scholarly journals Selectable Markers for Use in Genetic Manipulation of Extensively Drug-Resistant (XDR) Acinetobacter baumannii HUMC1

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Brian M. Luna ◽  
Amber Ulhaq ◽  
Jun Yan ◽  
Paul Pantapalangkoor ◽  
Travis B. Nielsen ◽  
...  
Keyword(s):  
Molecular Biology ◽  
Acinetobacter Baumannii ◽  
Genetic Manipulation ◽  
Multidrug Resistant ◽  
Molecular Techniques ◽  
Drug Resistant ◽  
Selectable Markers ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Selection Of

ABSTRACT Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics. Acinetobacter baumannii is one of the most antibiotic-resistant pathogens in clinical medicine, and extensively drug-resistant (XDR) strains are commonly isolated from infected patients. Such XDR strains are already resistant to traditional selectable genetic markers, limiting the ability to conduct pathogenesis research by genetic disruption. Optimization of selectable markers is therefore critical for the advancement of fundamental molecular biology techniques to use in these strains. We screened 23 drugs that constitute a broad array of antibiotics spanning multiple drug classes against HUMC1, a highly virulent and XDR A. baumannii clinical blood and lung isolate. HUMC1 is resistant to all clinically useful antibiotics that are reported by the clinical microbiology laboratory, except for colistin. Ethical concerns about intentionally establishing pan-resistance, including to the last-line agent, colistin, in a clinical isolate made identification of other markers desirable. We screened additional antibiotics that are in clinical use and those that are useful only in a lab setting to identify selectable markers that were effective at selecting for transformants in vitro. We show that supraphysiological levels of tetracycline can overcome innate drug resistance displayed by this XDR strain. Last, we demonstrate that transformation of the tetA (tetracycline resistance) and Sh ble (zeocin resistance), but not pac (puromycin resistance), resistance cassettes allow for selection of drug-resistant transformants. These results make the genetic manipulation of XDR A. baumannii strains easily achieved. IMPORTANCE Multidrug-resistant (MDR), extensively drug-resistant (XDR), and pan-drug-resistant (PDR) strains of Acinetobacter baumannii have frequently been characterized. The ability of A. baumannii to develop resistance to antibiotics is a key reason this organism has been difficult to study using genetic and molecular biology approaches. Here we report selectable markers that are not only useful but necessary for the selection of drug-resistant transformants in the setting of drug-resistant backgrounds. Use of these selectable markers can be applied to a variety of genetic and molecular techniques such as mutagenesis and transformation. These selectable markers will help promote genetic and molecular biology studies of otherwise onerous drug-resistant strains, while avoiding the generation of pathogenic organisms that are resistant to clinically relevant antibiotics.

Genome-scale genetic manipulation methods for exploring bacterial molecular biology

2012 ◽  
Vol 8 (6) ◽  
pp. 1626 ◽  
Author(s):  
Alla Gagarinova ◽  
Andrew Emili
Keyword(s):  
Molecular Biology ◽  
Genetic Manipulation ◽  
Genome Scale

scholarly journals Molecular Biology of Chili Pepper Anthocyanin Biosynthesis

2017 ◽  
Vol 56 (1) ◽  
Author(s):  
César Aza-González ◽  
Héctor Gordon Núñez-Palenius ◽  
Neftalí Ochoa-Alejo
Keyword(s):  
Molecular Biology ◽  
Biosynthetic Pathway ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Anthocyanin Biosynthesis ◽  
Chili Pepper ◽  
Horticultural Crop ◽  
Research Areas ◽  
Capsicum Spp ◽  
Chili Peppers

Chili pepper (<em>Capsicum spp.</em>) is an important horticultural crop worldwide. Chili pepper fruits from different <em>Capsicum </em>species have been highly consumed in Mexico since pre-Columbian times. Some <em>Capsicum</em> species synthesize and accumulate anthocyanins in different tissues and organs. Although the anthocyanin biosynthetic pathway has been established for different plant species, very few studies on anthocyanin chemistry, biochemistry and molecular biology of these pigments produced by chili peppers have been reported. In this review we describe the information on the type of anthocyanins synthesized and accumulated in chili pepper, and also on the molecular biology of the biosynthetic pathway. Additionally, we discuss the applications of current knowledge for the genetic manipulation, through genetic engineering, of this trait, and also the future anthocyanin-related research areas in <em>Capsicum.</em>

scholarly journals Development of a System for Genetic Manipulation ofBartonella bacilliformis

1999 ◽  
Vol 65 (8) ◽  
pp. 3441-3448 ◽  
Author(s):  
James M. Battisti ◽  
Michael F. Minnick
Keyword(s):  
Molecular Biology ◽  
Genetic Manipulation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Restriction Enzymes ◽  
Kanamycin Resistance ◽  
Broad Host Range ◽  
Wild Type ◽  
Site Specific ◽  
Suicide Vector

ABSTRACT Lack of a system for site-specific genetic manipulation has severely hindered studies on the molecular biology of allBartonella species. We report the first site-specific mutagenesis and complementation for a Bartonella species. A highly transformable strain of B. bacilliformis, termed JB584, was isolated and found to exhibit a significant increase in transformation efficiency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type strains. Restriction analyses of genomic preparations with the methylation-sensitive restriction enzymesClaI and StuI suggest that strain JB584 possesses a dcm methylase mutation that contributes to its enhanced transformability. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, was constructed. An internal 508-bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into pUB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB508 into JB584 by electroporation generated eight Kanr clones of B. bacilliformis. Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 andfla had occurred. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded byfla and secretion/assembly of flagella were abolished. Complementation of fla in trans was accomplished with a pBBR1MCS recombinant containing the entire wild-type fla gene (pBBRFLAG). These data conclusively show that inactivation of fla results in a bald, nonmotile phenotype and that pMB1 and REP replicons make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.

Electron Microscopy in Molecular Biology

1967 ◽  
Vol 25 ◽  
pp. 2-3
Author(s):  
Cecil E. Hall
Keyword(s):  
Electron Microscopy ◽  
Molecular Biology ◽  
Noise Level ◽  
Molecular Structures ◽  
Material Structure ◽  
The Arts ◽  
Shadow Casting ◽  
Electron Image ◽  
High Degree ◽  
Very High

The visualization of organic macromolecules such as proteins, nucleic acids, viruses and virus components has reached its high degree of effectiveness owing to refinements and reliability of instruments and to the invention of methods for enhancing the structure of these materials within the electron image. The latter techniques have been most important because what can be seen depends upon the molecular and atomic character of the object as modified which is rarely evident in the pristine material. Structure may thus be displayed by the arts of positive and negative staining, shadow casting, replication and other techniques. Enhancement of contrast, which delineates bounds of isolated macromolecules has been effected progressively over the years as illustrated in Figs. 1, 2, 3 and 4 by these methods. We now look to the future wondering what other visions are waiting to be seen. The instrument designers will need to exact from the arts of fabrication the performance that theory has prescribed as well as methods for phase and interference contrast with explorations of the potentialities of very high and very low voltages. Chemistry must play an increasingly important part in future progress by providing specific stain molecules of high visibility, substrates of vanishing “noise” level and means for preservation of molecular structures that usually exist in a solvated condition.

An introduction to the themed issue on RNA biology in China

2020 ◽  
Vol 64 (6) ◽  
pp. 863-866
Author(s):  
Zhe Wu
Keyword(s):  
Molecular Biology ◽  
Chinese Society ◽  
Fortieth Anniversary ◽  
Rna Biology ◽  
Research And Education ◽  
Education In China

Abstract The year 2019 marked the fortieth anniversary of the Chinese Society of Biochemistry and Molecular Biology (CSBMB), whose mission is to promote biomolecular research and education in China. The last 40 years have witnessed tremendous growth and achievements in biomolecular research by Chinese scientists and Essays in Biochemistry is delighted to publish this themed issue that focuses on exciting areas within RNA biology, with each review contributed by key experts from China.

Metabolic engineering of CHO cells to prepare glycoproteins

2018 ◽  
Vol 2 (3) ◽  
pp. 433-442 ◽  
Author(s):  
Qiong Wang ◽  
Michael J. Betenbaugh
Keyword(s):  
Metabolic Engineering ◽  
Cho Cells ◽  
Genetic Manipulation ◽  
Chinese Hamster ◽  
Post Translational Modification ◽  
Effector Functions ◽  
Recombinant Glycoproteins ◽  
Production Platform ◽  
Chemical Inhibitors ◽  
Amino Acid Mutations

As a complex and common post-translational modification, N-linked glycosylation affects a recombinant glycoprotein's biological activity and efficacy. For example, the α1,6-fucosylation significantly affects antibody-dependent cellular cytotoxicity and α2,6-sialylation is critical for antibody anti-inflammatory activity. Terminal sialylation is important for a glycoprotein's circulatory half-life. Chinese hamster ovary (CHO) cells are currently the predominant recombinant protein production platform, and, in this review, the characteristics of CHO glycosylation are summarized. Moreover, recent and current metabolic engineering strategies for tailoring glycoprotein fucosylation and sialylation in CHO cells, intensely investigated in the past decades, are described. One approach for reducing α1,6-fucosylation is through inhibiting fucosyltransferase (FUT8) expression by knockdown and knockout methods. Another approach to modulate fucosylation is through inhibition of multiple genes in the fucosylation biosynthesis pathway or through chemical inhibitors. To modulate antibody sialylation of the fragment crystallizable region, expressions of sialyltransferase and galactotransferase individually or together with amino acid mutations can affect antibody glycoforms and further influence antibody effector functions. The inhibition of sialidase expression and chemical supplementations are also effective and complementary approaches to improve the sialylation levels on recombinant glycoproteins. The engineering of CHO cells or protein sequence to control glycoforms to produce more homogenous glycans is an emerging topic. For modulating the glycosylation metabolic pathways, the interplay of multiple glyco-gene knockouts and knockins and the combination of multiple approaches, including genetic manipulation, protein engineering and chemical supplementation, are detailed in order to achieve specific glycan profiles on recombinant glycoproteins for superior biological function and effectiveness.

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