Construction of an IS-Free Corynebacterium glutamicum ATCC 13 032 Chassis Strain and Random Mutagenesis Using the Endogenous ISCg1 Transposase

Mobile genetic elements (MGEs) contribute to instability of the host genome and plasmids. Previously, removal of the prophages in the industrial amino acid producer Corynebacterium glutamicum ATCC 13 032 resulted in strain MB001 which showed better survival under stress conditions and increased transformability. Still, eight families of Insertion Sequence (IS) elements with 27 potentially active members remain in MB001, two of which were demonstrated to be detrimental in biotechnological processes. In this study, systematical deletion of all complete IS elements in MB001 resulted in the MGE-free strain CR101. CR101 shows growth characteristics identical to the wildtype and the increased transformability of MB001. Due to its improved genome stability, we consider this strain to be an optimal host for basic research and biotechnology. As a “zero-background” host, it is also an ideal basis to study C. glutamicum IS elements. Re-sequencing of CR101 revealed that only five spontaneous point mutations had occurred during the construction process, highlighting the low mutation rate of C. glutamicum on the nucleotide level. In a second step, we developed an easily applicable ISCg1-based transposon mutagenesis system to randomly transpose a selectable marker. For optimal plasmid stability during cloning in Escherichia coli, the system utilizes a genetic switch based on the phage integrase Bxb1. Use of this integrase revealed the presence of a functional attB site in the C. glutamicum genome. To avoid cross-talk with our system and increase ease-of-use, we removed the attB site and also inserted the Bxb1 encoding gene into the chromosome of CR101. Successful insertion of single markers was verified by sequencing randomly selected mutants. Sequencing pooled mutant libraries revealed only a weak target site specificity, seemingly random distribution of insertion sites and no general strand bias. The resulting strain, ML103, together with plasmid pML10 provides a easily customizable system for random mutagenesis in an otherwise genomically stable C. glutamicum. Taken together, the MGE-free C. glutamicum strain CR101, the derivative ML103, and the plasmid pML10 provide a useful set of tools to study C. glutamicum in the future.

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Constitutive Expression of AID Leads to Tumorigenesis

Journal of Experimental Medicine ◽

10.1084/jem.20030275 ◽

2003 ◽

Vol 197 (9) ◽

pp. 1173-1181 ◽

Cited By ~ 323

Author(s):

Il-mi Okazaki ◽

Hiroshi Hiai ◽

Naoki Kakazu ◽

Shuichi Yamada ◽

Masamichi Muramatsu ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

Genome Stability ◽

Somatic Hypermutation ◽

Cytidine Deaminase ◽

Point Mutations ◽

Constitutive Expression ◽

Cell Receptor ◽

Genetic Alterations ◽

Artificial Substrates

Genome stability is regulated by the balance between efficiencies of the repair machinery and genetic alterations such as mutations and chromosomal rearrangements. It has been postulated that deregulation of class switch recombination (CSR) and somatic hypermutation (SHM), which modify the immunoglobulin (Ig) genes in activated B cells, may be responsible for aberrant chromosomal translocations and mutations of non-Ig genes that lead to lymphocyte malignancy. However, the molecular basis for these genetic instabilities is not clearly understood. Activation-induced cytidine deaminase (AID) is shown to be essential and sufficient to induce both CSR and SHM in artificial substrates in fibroblasts as well as B cells. Here we show that constitutive and ubiquitous expression of AID in transgenic mice caused both T cell lymphomas and dysgenetic lesions of epithelium of respiratory bronchioles (micro-adenomas) in all individual mice. Point mutations, but not translocations, were massively introduced in expressed T cell receptor (TCR) and c-myc genes in T lymphoma cells. The results indicate that AID can mutate non-Ig genes including oncogenes, implying that aberrant AID expression could be a cause of human malignancy.

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S3440P Substitution in C-Terminal Region of Human Reelin Dramatically Impairs Secretion of Reelin from HEK 293T Cells

10.20944/preprints201907.0025.v1 ◽

2019 ◽

Author(s):

Elika Esmaeilzadeh-Gharehdaghi ◽

Ehsan Razmara ◽

Amirreza Bitaraf ◽

Mahdi Mahmoudi ◽

Masoud Garshasbi

Keyword(s):

Tandem Repeats ◽

Point Mutations ◽

Random Mutagenesis ◽

In Vitro Study ◽

Terminal Region ◽

Main Role ◽

Mutant Type ◽

Secretion Efficiency ◽

Retzius Cells

Background: Reelin is a large extracellular glycoprotein secreted by Cajal–Retzius cells and has a main role during brain development, especially in neuronal migration. Reelin is comprised of N-terminal F-spondin like domain, eight tandem repeats, and a highly conserved basic C-terminal region (CTR). The CTR main role in the secretion of Reelin has been investigated by advertently inducing deletion in whole or a part of this region; however, the role of CTR point mutations on the secretion of Reelin is shrouded in mystery. Materials and Methods: In this study, we performed experimental analyses on a subregion of Human Reelin containing 5th and 6th repeats (R5-R6), a part of 8 th repeat and the CTR which were amplified from cDNA of K562 and HEPG2 cells and cloned into a mammalian expressional plasmid (pVP22/myc-His). Bioinformatics investigation was performed on the CTR at both level of nucleotide and amino acid as well as mutant type. Random mutagenesis by error-prone PCR method was utilized to induce mutation in the CTR. The secretion efficiency of recombinant wild-type and mutant Reelin constructs compared in cell lysate and supernatant isolated from the transiently transfected HEK 293T cells using 6XHistag ELISA method. Results: In-vitro study demonstrated that the CTR alteration(S3440P) leads to impairment of Reelin secretion even after overexpression. Conclusions: Our results indicate that S3440P substitution in highly conserved structure of the CTR has an important effect on Reelin secretion.

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Transcriptional Mutagenesis Prevents Ribosomal DNA Deterioration: The Role of Duplications and Deletions

Genome Biology and Evolution ◽

10.1093/gbe/evz235 ◽

2019 ◽

Vol 11 (11) ◽

pp. 3207-3217

Author(s):

Enrico Sandro Colizzi ◽

Paulien Hogeweg

Keyword(s):

Genome Stability ◽

Evolutionary Dynamics ◽

Point Mutations ◽

Gene Duplications ◽

Genetic Operators ◽

Theory Of Evolution ◽

Long Term Stability ◽

Number Variation

Abstract Clashes between transcription and replication complexes can cause point mutations and chromosome rearrangements on heavily transcribed genes. In eukaryotic ribosomal RNA genes, the system that prevents transcription–replication conflicts also causes frequent copy number variation. Such fast mutational dynamics do not alter growth rates in yeast and are thus selectively near neutral. It was recently found that yeast regulates these mutations by means of a signaling cascade that depends on the availability of nutrients. Here, we investigate the long-term evolutionary effect of the mutational dynamics observed in yeast. We developed an in silico model of single-cell organisms whose genomes mutate more frequently when transcriptional load is larger. We show that mutations induced by high transcriptional load are beneficial when biased toward gene duplications and deletions: they decrease mutational load even though they increase the overall mutation rates. In contrast, genome stability is compromised when mutations are not biased toward gene duplications and deletions, even when mutations occur much less frequently. Taken together, our results show that the mutational dynamics observed in yeast are beneficial for the long-term stability of the genome and pave the way for a theory of evolution where genetic operators are themselves cause and outcome of the evolutionary dynamics.

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Mutational analysis of the consensus sequence of a replication origin from yeast chromosome III.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3917 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3917-3925 ◽

Cited By ~ 106

Author(s):

J V Van Houten ◽

C S Newlon

Keyword(s):

Base Pair ◽

High Frequency ◽

Mutational Analysis ◽

Plasmid Stability ◽

Consensus Sequence ◽

Point Mutations ◽

Autonomously Replicating Sequence ◽

Frequency Transformation ◽

The Core ◽

High Frequency Transformation

Yeast autonomously replicating sequence (ARS) elements contain an 11-base-pair core consensus sequence (5'-[A/T]TTTAT[A/G]TTT[A/T]-3') that is required for function. The contribution of each position within this sequence to ARS activity was tested by creating all possible single-base mutations within the core consensus sequence of ARS307 (formerly called the C2G1 ARS) and testing their effects on high-frequency transformation and on plasmid stability. Of the 33 mutations, 22 abolished ARS function as measured by high-frequency transformation, 7 caused more than twofold reductions in plasmid stability, and 4 had no effect on plasmid stability. Mutations that reduced or abolished ARS activity occurred at each position in the consensus sequence, demonstrating that each position of this sequence contributes to ARS function. Of the four mutations that had no effect on ARS activity, three created alternative perfect matches to the core consensus sequence, demonstrating that the alternate bases allowed by the consensus sequence are, indeed, interchangeable. In addition, a change from T to C at position 6 did not perturb wild-type efficiency. To test whether the essential region extends beyond the 11-base-pair consensus sequence, the effects on plasmid stability of point mutations one base 3' to the T-rich strand of the core consensus sequence (position 12) and deletion mutations that altered bases 5' to the T-rich strand of the core consensus sequence were examined. An A at position 12 or the removal of three T residues 5' to the core consensus sequence severely diminished ARS efficiency, showing that the region required for full ARS efficiency extends beyond the core consensus sequence in both directions.

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Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study

Blood ◽

10.1182/blood-2006-01-015347 ◽

2007 ◽

Vol 109 (11) ◽

pp. 5011-5015 ◽

Cited By ~ 93

Author(s):

Arghya Ray ◽

Sandra W. Cowan-Jacob ◽

Paul W. Manley ◽

Jürgen Mestan ◽

James D. Griffin

Keyword(s):

Imatinib Mesylate ◽

Kinase Inhibitor ◽

Single Point ◽

Point Mutations ◽

Random Mutagenesis ◽

Kinase Domain ◽

Site Directed Mutagenesis ◽

Multiple Point ◽

Resistance Mutations ◽

Mutagenesis Study

Abstract Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 μM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

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Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft.

Molecular Biology of the Cell ◽

10.1091/mbc.7.7.1123 ◽

1996 ◽

Vol 7 (7) ◽

pp. 1123-1136 ◽

Cited By ~ 58

Author(s):

K M Ruppel ◽

J A Spudich

Keyword(s):

Active Site ◽

Three Dimensional ◽

Point Mutations ◽

Random Mutagenesis ◽

Motor Domain ◽

Actin Binding ◽

Dimensional Structure ◽

Null Cell ◽

Myosin Motor Domain ◽

Cell Phenotypes

We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement.

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A single V317A or V317M substitution in Enzyme II of a newly identified β-glucoside phosphotransferase and utilization system of Corynebacterium glutamicum R extends its specificity towards cellobiose

Microbiology ◽

10.1099/mic.0.26053-0 ◽

2003 ◽

Vol 149 (6) ◽

pp. 1569-1580 ◽

Cited By ~ 57

Author(s):

Pavel Kotrba ◽

Masayuki Inui ◽

Hideaki Yukawa

Keyword(s):

Corynebacterium Glutamicum ◽

Carbon Catabolite Repression ◽

Point Mutations ◽

Spontaneous Mutant ◽

Specific Enzyme ◽

Phosphotransferase System ◽

Transcription Antitermination ◽

Transcription Regulators ◽

Membrane Spanning ◽

Component Gene

A catabolic system involved in the utilization of β-glucosides in Corynebacterium glutamicum R and its spontaneous mutant variants allowing uptake of cellobiose were investigated. The system comprises a β-glucoside-specific Enzyme IIBCA component (gene bglF) of the phosphotransferase system (PTS), a phospho-β-glucosidase (bglA) and an antiterminator protein (bglG) from the BglG/SacY family of transcription regulators. The results suggest that transcription antitermination is involved in control of induction and carbon catabolite repression of bgl genes, which presumably form an operon. Functional analysis of the bglF and bglA products revealed that they are simultaneously required for uptake, phosphorylation and breakdown of methyl β-glucoside, salicin and arbutin. Although cellobiose is not normally a substrate for BglF permease and is not utilized by C. glutamicum R, cellobiose-utilizing mutants can be obtained. The mutation responsible was mapped to the bgl locus and sequenced, and point mutations were found in codon 317 of bglF. These led to substitutions V317A and/or V317M near the putative PTS active-site H313 in the membrane-spanning IIC domain of BglF and allowed BglF to act on cellobiose. Such results strengthen the evidence that the IIC domains can be regarded as selectivity filters of the PTS.

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The ARS consensus sequence is required for chromosomal origin function in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.12.10.4305 ◽

1992 ◽

Vol 12 (10) ◽

pp. 4305-4313 ◽

Cited By ~ 49

Author(s):

A M Deshpande ◽

C S Newlon

Keyword(s):

Dna Sequences ◽

Plasmid Stability ◽

Single Point ◽

Consensus Sequence ◽

Point Mutations ◽

Base Pairs ◽

Chromosomal Origin ◽

Chromosomal Copy ◽

Origin Function ◽

Derivatives Of

Replication origins have been mapped to positions that coincide, within experimental error (several hundred base pairs), with ARS elements. To determine whether the DNA sequences required for ARS function on plasmids are required for chromosomal origin function, the chromosomal copy of ARS306 was deleted and the chromosomal copy of ARS307 was replaced with mutant derivatives of ARS307 containing single point mutations in domain A within the ARS core consensus sequence. The chromosomal origin function of these derivatives was assayed by two-dimensional agarose gel electrophoresis. Deletion of ARS306 deleted the associated replication origin. The effects on chromosomal origin function of mutations in domain A paralleled their effects on ARS function, as measured by plasmid stability. These results demonstrate that chromosomal origin function is a property of the ARS element itself.

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σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

10.1101/505099 ◽

2018 ◽

Cited By ~ 1

Author(s):

Rémi Hocq ◽

Maxime Bouilloux-Lafont ◽

Nicolas Lopes Ferreira ◽

François Wasels

Keyword(s):

Point Mutations ◽

Random Mutagenesis ◽

Value Added ◽

Clostridium Beijerinckii ◽

Phenotypic Traits ◽

Wild Type ◽

Genomic Comparison ◽

Alcohol Synthesis

Microbial production of butanol and isopropanol, two high value-added chemicals, is naturally occurring in the solventogenic Clostridium beijerinckii DSM 6423. Despite its ancient discovery, the precise mechanisms controlling alcohol synthesis in this microorganism are poorly understood. In this work, an allyl alcohol tolerant strain obtained by random mutagenesis was characterized. This strain, designated as the AA mutant, shows a dominant production of acids, a severely diminished butanol synthesis capacity, and produces acetone instead of isopropanol. Interestingly, this solvent-deficient strain was also found to have a limited consumption of two carbohydrates and to be still able to form spores, highlighting its particular phenotype. Sequencing of the AA mutant revealed point mutations in several genes including CIBE_0767 (sigL), which encodes the σ54 sigma factor. Complementation with the wild-type sigL gene fully restored solvent production and sugar assimilation, demonstrating that σ54 plays a central role in regulating these pathways in C. beijerinckii DSM 6423. Genomic comparison with other strains further revealed that these functions are probably conserved among the C. beijerinckii strains. The importance of σ54 in C. beijerinckii was further assessed by the characterization of a sigL deletion mutant of the model strain NCIMB 8052 obtained with a CRISPR/Cas9 tool. The resulting mutant exhibited phenotypic traits similar to the AA strain, and was subsequently complemented with the sigL gene from either the wild type or the AA strains. The results of this experiment confirmed the crucial role of σ54 in the regulation of both solventogenesis and sugar consumption pathways in C. beijerinckii.

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Cyclin B1 scaffolds MAD1 at the corona to activate the spindle assembly checkpoint

10.1101/726224 ◽

2019 ◽

Cited By ~ 2

Author(s):

Lindsey A Allan ◽

Magda Reis ◽

Yahui Liu ◽

Pim Huis in ’t Veld ◽

Geert JPL Kops ◽

...

Keyword(s):

Genome Stability ◽

Spindle Assembly Checkpoint ◽

Point Mutations ◽

Spindle Assembly ◽

Cyclin B1 ◽

Cyclin B ◽

Mitotic Progression ◽

Mitotic Kinase ◽

Binding Interface

ABSTRACTThe Cyclin B:CDK1 kinase complex is the master regulator of mitosis that phosphorylates hundreds of proteins to coordinate mitotic progression. We show here that, in addition to these kinase functions, Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminus of MAD1, and point mutations in this region remove corona MAD1 and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness, in this context, arises because Cyclin B1-MAD1 localisation becomes MPS1-independent after the corona has been established. We demonstrate that this allows corona-MAD1 to persist at kinetochores when MPS1 activity falls, ensuring that it can still be phosphorylated on a key C-terminal catalytic site by MPS1. Therefore, this study explains how corona MAD1 generates a robust SAC signal and why stripping of this pool by dynein is essential for SAC silencing. It also reveals that the key mitotic kinase, Cyclin B1-Cdk1, scaffolds the pathway that inhibits its own degradation.

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