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Results for “Mutagenesis, Insertional”

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At least 19 recordsLinked to original sources
PubMed2004

Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Acetyltransferases↗
PubMed1995

Virulent non-capsulate Yersinia pestis variants constructed by insertion mutagenesis.

Insertion mutagenesis with the help of the plasmid pFS23 was used to generate Yersinia pestis fra mutants. The results of pFra- strain production under non-selective conditions suggested that such Y. pestis variants may be generated in natural plague foci at high frequency and may participate in supporting the epizootic process. Present data suggest that the reduction of virulence in Fra- strains reported by the majority of investigators was connected with the use of Y. pestis variants carrying additional unidentified mutations. It was shown that the loss of the ability to produce capsular antigen (FI) alone or in combination with absence of murine toxin production did not lead to an increase in LD50 absolute values. Simultaneous loss of these two virulence determinants did not influence the duration of survival of the infected animals. However, absence of only FI antigen production in the infecting strain resulted in prolonged survival of the infected animals. Conversion of plague infection from acute to chronic form is probably dependent on the animal host species and the Y. pestis parent strain subjected to mutagenesis.

Animals↗
PubMed2004

[Development of retrovirus-mediated insertional mutagenesis in zebrafish and its application in saturation mutagenesis and gene screening].

The strategies of large-scale mutagenesis and gene screening include chemical mutagenesis, insertional mutagenesis and gene trap. Insertional mutagenesis is a method for identifying genes by using the integration of DNA as the mutagen, thereby facilitating the cloning of the mutated gene. The use of retrovirus-mediated insertional mutagenesis in zebrafish has led to the mutation and rapid identification of hundreds of genes required for embryonic development and cell growth. Gene trap elements are also used in the construction of retrovirus vectors. The whole system is likely to make zebrafish the first vertebrate to achieve saturation mutagenesis and gene screening.

Animals↗
PubMed1991

Functional mapping of anthrax toxin lethal factor by in-frame insertion mutagenesis.

Linker insertion mutagenesis was employed to create structural disruptions of the lethal factor (LF) protein of anthrax toxin to map functional domains. A dodecameric linker was inserted at 17 blunt end restriction enzyme sites throughout the gene. Paired MluI restriction sites within the linker allowed the inserts to be reduced from four to two amino acids. Shuttle vectors containing the mutated genes were transformed into the avirulent Bacillus anthracis UM23C1-1 for expression and secretion of the gene products. Mutations at five sites in the central one-third of the sequence made the protein unstable, and purified protein could not be obtained. Mutated LF proteins with insertions at the other sites were purified and assessed for toxic activity in a macrophage lysis assay and for their ability to bind to the protective antigen (PA) component of anthrax toxin, the receptor binding moiety. Most insertions located in the NH2-terminal one-third of the LF protein eliminated both toxicity and binding to PA, while all four insertions in the COOH-terminal one-third of the protein eliminated toxicity without affecting binding to PA. These data support the hypothesis that the NH2-terminal domain contains the structures required for binding to PA and the COOH-terminal domain contains the putative catalytic domain of LF.

Antigens, Bacterial↗
PubMed2003

Insertional mutagenesis in zebrafish.

Insertional mutagenesis is a method for identifying genes essential for a given biological process by using the integration of DNA as the mutagen, thereby facilitating the cloning of the mutated gene. The use of retrovirus-mediated insertional mutagenesis in zebrafish has led to the mutation and rapid identification of hundreds of genes required for embryonic development and cell viability and growth, revealing the diversity of gene products required for the development of this vertebrate. Here, I will review the methodology of this approach and the results to date, as well as other potential ways to use insertional mutagenesis for genetic screens.

Animals↗
PubMed2001

Analysis of apicoplast targeting and transit peptide processing in Toxoplasma gondii by deletional and insertional mutagenesis.

Deletion and insertion mutagenesis was used to analyze the targeting sequence of the nuclear encoded apicoplast protein, the ribosomal protein small subunit 9 of Toxoplasma gondii. Previous studies have shown that nuclear encoded apicoplast proteins possess bipartite leaders having characteristic signal sequences followed by serine/threonine rich transit sequences. Deletion analysis demonstrated that the first 55 amino acids of the rps9 leader were sufficient for apicoplast targeting. Insertional mutagenesis tagging the leader sequence with a hemagglutinin (HA) tag was used to study the events involved in the targeting pathway. Transfectants with insertions near the N-terminus of the transit displayed HA tagged precursors outside of the apicoplast, in the perinuclear region. In contrast, transfectants with the HA tag inserted near the carboxyl end of the transit-like region had apicoplast labeling. Western blot analysis of HA tagged stable isolates suggested that processing of the HA tagged leaders was a multi-step process, with processing occurring both outside of and at or within the apicoplast.

Animals↗
PubMed2006

Detecting statistically significant common insertion sites in retroviral insertional mutagenesis screens.

Retroviral insertional mutagenesis screens, which identify genes involved in tumor development in mice, have yielded a substantial number of retroviral integration sites, and this number is expected to grow substantially due to the introduction of high-throughput screening techniques. The data of various retroviral insertional mutagenesis screens are compiled in the publicly available Retroviral Tagged Cancer Gene Database (RTCGD). Integrally analyzing these screens for the presence of common insertion sites (CISs, i.e., regions in the genome that have been hit by viral insertions in multiple independent tumors significantly more than expected by chance) requires an approach that corrects for the increased probability of finding false CISs as the amount of available data increases. Moreover, significance estimates of CISs should be established taking into account both the noise, arising from the random nature of the insertion process, as well as the bias, stemming from preferential insertion sites present in the genome and the data retrieval methodology. We introduce a framework, the kernel convolution (KC) framework, to find CISs in a noisy and biased environment using a predefined significance level while controlling the family-wise error (FWE) (the probability of detecting false CISs). Where previous methods use one, two, or three predetermined fixed scales, our method is capable of operating at any biologically relevant scale. This creates the possibility to analyze the CISs in a scale space by varying the width of the CISs, providing new insights in the behavior of CISs across multiple scales. Our method also features the possibility of including models for background bias. Using simulated data, we evaluate the KC framework using three kernel functions, the Gaussian, triangular, and rectangular kernel function. We applied the Gaussian KC to the data from the combined set of screens in the RTCGD and found that 53% of the CISs do not reach the significance threshold in this combined setting. Still, with the FWE under control, application of our method resulted in the discovery of eight novel CISs, which each have a probability less than 5% of being false detections.

Chromosome Mapping↗
PubMed2004

Genetic aspects of targeted insertion mutagenesis in yeasts.

Targeted insertion mutagenesis is a main molecular tool of yeast science initially applied in Saccharomyces cerevisiae. The method was extended to fission yeast Schizosaccharomyces pombe and to "non-conventional" yeast species, which show specific properties of special interest to both basic and applied research. Consequently, the behaviour of such non-Saccharomyces yeasts is reviewed against the background of the knowledge of targeted insertion mutagenesis in S. cerevisiae. Data of homologous integration efficiencies obtained with circular, ends-in or ends-out vectors in several yeasts are compared. We follow details of targeted insertion mutagenesis in order to recognize possible rate-limiting steps. The route of the vector to the target and possible mechanisms of its integration into chromosomal genes are considered. Specific features of some yeast species are discussed. In addition, similar approaches based on homologous recombination that have been established for the mitochondrial genome of S. cerevisiae are described.

Gene Expression Regulation, Fungal↗
PubMed1991

A novel approach to insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of the Haemophilus influenzae chromosome was accomplished by a novel method employing a 2.2-kbp element, TSTE. This element, consisting of the neo gene of Tn5 flanked by Haemophilus-specific uptake sequences, was ligated to circularized chromosomal fragments before transformation into the homologous strain. Eight mutants defective in the production of haemocin were detected. This strategy provides an efficient mechanism for the insertional mutagenesis of H. influenzae.

DNA Transposable Elements↗
PubMed1998

Insertional mutagenesis of pathogenic fungi.

Screening insertional mutants for loss of virulence is an effective method for investigating the molecular genetic basis of bacterial pathogenesis, but has only recently been applied to fungal pathogens. For many pathogenic fungi transformation with heterologous plasmid DNA results in complex integration events. This problem can now be circumvented for some species using restriction enzyme mediated integration. Insertional mutagenesis of Fusarium oxysporum using the naturally occurring fungal transposon impala has been described, but transposon tagging for other fungi has yet to be developed. Although insertional mutagenesis has recently identified important virulence determinants of fungal phytopathogens, the lack of suitable screening strategies has so far limited its applicability for fungal pathogens of humans.

Fungi↗
PubMed2005

Retroviral insertional mutagenesis identifies oncogene cooperation.

Retroviral insertional mutagenesis has been applied to identify oncogenes that are important for both human and rodent carcinogenesis. The method reveals not only primary oncogenes but also cooperative genes that might be affected as second or third hits in multistep carcinogenesis. The use of genetically engineered mice such as NUP98-HOXA9 transgenic mice enabled efficient identification of cooperative genes, which provides important information for the molecular pathway in carcinogenesis/leukemogenesis. With use of the retrovirus mediated gene transfer system, retroviral insertional mutagenesis will provide invaluable information to understand genetic interaction in complex mechanisms of carcinogenesis.

Animals↗
PubMed2005

Insertional mutagenesis in mice: new perspectives and tools.

Insertional mutagenesis has been at the core of functional genomics in many species. In the mouse, improved vectors and methodologies allow easier genome-wide and phenotype-driven insertional mutagenesis screens. The ability to generate homozygous diploid mutations in mouse embryonic stem cells allows prescreening for specific null phenotypes prior to in vivo analysis. In addition, the discovery of active transposable elements in vertebrates, and their development as genetic tools, has led to in vivo forward insertional mutagenesis screens in the mouse. These new technologies will greatly contribute to the speed and ease with which we achieve complete functional annotation of the mouse genome.

Animals↗
PubMed1998

Construction of antibiotic resistance cassettes with multiple paired restriction sites for insertional mutagenesis of Haemophilus influenzae.

Insertional mutagenesis of cloned genes coupled with site specific recombination into the genome of the parent organism is an ideal method for characterizing gene function. In this paper we describe the production and utility of two antibiotic resistance cassettes for use in Haemophilus influenzae. The mutagenic elements encode resistance to chloramphenicol or spectinomycin. Multiple paired restriction enzyme sites bound both cassettes. Use of these constructs to create mutants in H. influenzae demonstrated that the cassettes are readily incorporated into the genome in single copy and allow easy detection of mutant constructs. The insertions are stable following repeated in vitro passage. In addition, the elements are compatible with each other and allow the construction of multiple mutations within a single strain.

Chloramphenicol O-Acetyltransferase↗
PubMed2006

Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice.

Retroviral insertional mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by insertional mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by insertional mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.

Adenosine Triphosphatases↗
PubMed2004

Identification of apoptosis regulatory genes using insertional mutagenesis.

This chapter describes a retroviral insertion mutagenesis approach using replication-deficient myeloproliferative sarcoma virus retroviral vectors to identify apoptosis regulatory genes in the interleukin-3-dependent Baf-3 cell line. We describe the retroviral insertion mutagenesis protocol and the selection steps to obtain apoptosis resistant mutants. We also present several methods to isolate the cellular DNA sequences flanking the provirus to identify the gene responsible for the apoptosis-resistant phenotype.

Animals↗
PubMed1984

Construction of mutants of Moloney murine leukemia virus by suppressor-linker insertional mutagenesis: positions of viable insertion mutations.

A highly efficient method for the generation of insertion mutations is described. The procedure involves the use of a 220-base-pair (bp) EcoRI fragment bearing the SuIII+ suppressor tRNA gene as an insertional mutagen. The plasmid DNA to be mutagenized is linearized by a variety of means, and the suppressor fragment is ligated into the site of cleavage. Successful insertion mutants can be readily detected in Escherichia coli carrying lac- amber mutations on MacConkey lactose plates; virtually 100% of the red colonies contain insertions of the fragment. Subsequent removal of the SuIII+ gene and recyclization leaves a 12-bp insertion if the original cleavage was blunt-ended and a 9-bp insertion if the original cleavage generated 3-bp cohesive termini. This technique, as well as conventional linker mutagenesis with decamer and dodecamer linkers, was used to generate a large library of insertion mutations in cloned DNA copies of the genome of Moloney murine leukemia virus. A number of viable mutants were isolated bearing 9-, 10-, and 12-bp insertions in various domains of the genome. The map positions of the viable mutations suggest that the viral long terminal repeats and portions of the gag and env genes are quite insensitive to alteration. Although most of the mutations were stable for many passages, some of the mutants lost the inserted DNA; we presume that the insertion was somewhat deleterious in these mutants and that continued passage of the virus selected for overgrowth by a revertant.

Animals↗
PubMed2005

Retroviral insertional mutagenesis: past, present and future.

Retroviral insertion mutagenesis screens in mice are powerful tools for efficient identification of oncogenic mutations in an in vivo setting. Many oncogenes identified in these screens have also been shown to play a causal role in the development of human cancers. Sequencing and annotation of the mouse genome, along with recent improvements in insertion site cloning has greatly facilitated identification of oncogenic events in retrovirus-induced tumours. In this review, we discuss the features of retroviral insertion mutagenesis screens, covering the mechanisms by which retroviral insertions mutate cellular genes, the practical aspects of insertion site cloning, the identification and analysis of common insertion sites, and finally we address the potential for use of somatic insertional mutagens in the study of nonhaematopoietic and nonmammary tumour types.

Cloning, Molecular↗