Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Genetic tools”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources
PubMed2001

An animal model for the molecular genetics of CADASIL. (Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy).

BACKGROUND: CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is an inherited condition that causes repeated small-scale strokes in adults. CADASIL is caused only by mutations in the human NOTCH3 gene that increase or decrease the number of cysteines within the epidermal growth factor (EGF) repeats of the NOTCH3 protein. Drosophila: lethal-Abruptex is a similar condition because it is also caused only by mutations that increase or decrease the number of cysteines within the EGF repeat portion of the Notch protein. SUMMARY OF COMMENT: Drosophila: lethal-Abruptex and human CADASIL are precisely analogous at the molecular level, and both are genetically dominant. These precise similarities, together with the fact that the structure and function of Notch has been highly conserved throughout the animal kingdom, provide an animal model for the molecular and genetic aspects of human CADASIL. It also provides support for Spinner's proposal that CADASIL results from dominant inhibition of the Notch pathway. CONCLUSIONS: Because the phenotypes of Notch mutations are cell-autonomous, the symptoms of CADASIL indicate that adult vascular smooth muscle cells require the continuing function of the NOTCH3 pathway in the adult. For this reason, further analysis of the NOTCH3 pathway may provide more general insights into the biology of vascular smooth muscle cells. In the case of CADASIL, the powerful genetic tools available in Drosophila: should help to facilitate future research.

Animals↗
PubMed2006

Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway.

BACKGROUND: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. RESULTS: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. CONCLUSION: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.

Actinobacteria↗
PubMed2001

Isolation and characterization of a temperature-sensitive generalized transducing bacteriophage for Vibrio cholerae.

CP-T1 is the only described generalized transducing bacteriophage for the intestinal pathogen Vibrio cholerae, yet many of its basic biological parameters remain unknown. Due to low frequencies of transduction and pseudolysogen formation, CP-T1 has not been widely used as a genetic tool. To overcome these limitations, we have isolated a conditional mutant of CP-T1 that exhibits temperature-sensitive plaque formation. Several biological properties of CP-T1ts were determined, including its restrictive temperature, adsorbance profile to host cells, burst time, and burst size. Based on these properties, an optimized transduction protocol was designed which resulted in several fold higher transduction frequencies for a variety of genetic markers from a number of chromosomal loci. Generalized transduction was also demonstrated between classical and E1 Tor biotype strains of V. cholerae.

Bacteriophages↗
PubMed2001

Ischemic injury and faulty gene transcripts in the brain.

The brain has the highest metabolic rate of all organs and depends predominantly on oxidative metabolism as a source of energy. Oxidative metabolism generates reactive oxygen species, which can damage all cellular components, including protein, lipids and nucleic acids. The processes of DNA repair normally remove spontaneous gene damage with few errors. However, cerebral ischemia followed by reperfusion leads to elevated oxidative stress and damage to genes in brain tissue despite a functional mechanism of DNA repair. These critical events occur at the same time as the expression of immediate early genes, the products of which trans-activate late effector genes that are important for sustaining neuronal viability. These findings open the possibility of applying genetic tools to identify molecular mechanisms of gene repair and to derive new therapies for stroke and brain injury.

Animals↗
PubMed2001

Hermes-mediated germ-line transformation of the Mediterranean fruit fly Ceratitis capitata.

We report the use of the Hermes transposable element for germ-line transformation of the Mediterranean fruit fly, Ceratitis capitata. Hermes was able to genetically transform this insect at an estimated frequency between 0.6 and 1.1%, which is comparable to the transformation frequencies obtained for this species when using other transposable elements. Hermes integrates into the medfly genome by a cut-and-paste mechanism and the sequences integrated into the genome are delimited by the terminal nucleotides of the Hermes inverted terminal repeats. Integration resulted in the generation of 8 bp target site duplications, the sequences of which conformed to the target site duplications generated by hAT element transposition in insects. The Hermes element is one additional genetic tool that can be deployed in manipulating and characterizing the medfly genome.

Animals↗
PubMed2000

The Drosophila genome sequence: implications for biology and medicine.

The 120-megabase euchromatic portion of the Drosophila melanogaster genome has been sequenced. Because the genome is compact and many genetic tools are available, and because fly cell biology and development have much in common with mammals, this sequence may be the Rosetta stone for deciphering the human genome.

Animals↗
PubMed2000

Genetic variation of susceptibility to Mycobacterium avium subsp. paratuberculosis infection in dairy cattle.

Paratuberculosis is an infectious disease that is not easily amenable to classical control methods such as treatment and vaccination. Experimental animal models suggest that there could be genetic factors responsible for susceptibility or resistance to infection with the causative agent, Mycobacterium avium subsp. paratuberculosis. The aim of this study was to estimate genetic variation in susceptibility to paratuberculosis in Dutch dairy cattle. Data collected during a vaccination trial, conducted from 1984 to 1994, was used. A total of 3020 cows, with complete pedigree records and infection status at slaughter, were available for analysis. A standard polygenic statistical probit model was used to estimate heritabilities. The estimated heritability of susceptibility to M. avium. subsp. paratuberculosis infection was 0.06 for the overall population. In the subpopulation of vaccinated animals the estimated heritability was 0.09. Other calculations based on the model used in this study argue against a prominent role for vertical transmission. Because the establishment of genetic variation is one of the first steps towards the exploration of the possible use of selection for genetic improvement, the present study provides evidence for the presence of genetic variation in the susceptibility of cattle to paratuberculosis. Because the economic impact of the disease is substantial, the development and application of genetic tools, along with other control methods, could be instrumental in the eradication of paratuberculosis.

Animals↗
PubMedDate not supplied

The c-Harvey-ras-1 oncogene in chromosome mediated gene transfer.

Transfection of DNA derived from a variety of tumours can induce morphological transformation of certain immortalised but normally contact-inhibited cell lines. This important technique has been instrumental in the identification and subsequent molecular cloning of a number of oncogenes, including Harvey-ras. We can extend this approach for studying neoplastic potential by performing chromosome-mediated, as distinct from DNA-mediated, gene transfer. This modification offers two potentially important advantages, both of which stem from the fact that sub-chromosomal lengths of DNA are transferred. Firstly, the expression of the oncogene can be studied in its normal chromosomal milieu; potential modifying effects of linked and unlinked sequences can be evaluated. Secondly, new chromatin segments with transforming potential but too large to be transferred as naked DNA may be revealed. Our experiments illustrate some of the new insights into the molecular basis of neoplastic change which can be gained by this technique. They also demonstrate the power of the technique as a genetic tool for the isolation and detailed molecular analysis of oncogene-associated, sub-chromosomal regions of the human genome.

Animals↗
PubMed2000

Applicability of yeast genetics to neurologic disease.

As advances in gene mapping technology reveal genes associated with neurologic diseases, the need to identify a gene's normal function arises often. Experimental genetics is very useful in identifying a gene's function. It relies on model organisms both because it is not appropriate in humans, and because many processes are remarkably similar among eukaryotes. Many cellular processes have evolved once, and species differences are variations on a theme. Molecular genetic tools available in the yeast Saccharomyces cerevisiae provide a means to more rapidly reach an understanding of gene function, yielding substantial insight into the same process in humans. Yeast will never complain of headache or "spells," but do have expansions of trinucleotide repeats, prions, and other processes very much analogous to those underlying many neurologic diseases. In spite of the absence of a nervous system in yeast, yeast genetics has contributed substantial insight into neurologic diseases mechanisms. The real strength of yeast in studying human disease is in genetic analysis of gene function and in providing genetically powerful functional assays. Arch Neurol. 2000;57:1129-1134

Humans↗
PubMed2001

The role of chemotherapy in early non-small-cell lung cancer management.

Great advances have been made in chemotherapy in advanced and metastatic non-small-cell lung cancer (NSCLC), and a major milestone was reached with the administration of neoadjuvant chemotherapy in stage IIIA N2 disease. The systemic nature of lung cancer has been confirmed by many genetic analyses documenting micrometastases in negative lymph nodes and bone marrow, and mRNA gene overexpression as a surrogate of cancer cells has been identified in peripheral blood. Furthermore, serum or plasma cell-free tumor DNA has been observed even in tumors with a diameter of less than 2 cm. Pharmacogenetic screening can lead to tailored chemotherapy even in patients with early disease through the use of a genetic tool kit that will allow us to optimize the use of chemotherapy by using serial measurements of serum DNA that can help to detect residual disease and re-assess the chemosensitivity of sub-clinical micrometastatic disease. The ongoing (neo)adjuvant taxol/carboplatin hope (NATCH) trial is testing the value of three cycles of chemotherapy given pre- or post-operatively compared with surgery alone and will analyze genetic abnormalities in serum DNA at three different points during patient follow-up. Our major concern in this review is to analyze the pros and cons of chemotherapy in NSCLC. Although this review is not a formal meta-analysis, we have discussed the most relevant published studies in this field. We conclude that not only is there no evidence of detrimental effects of chemotherapy, in fact, there are many indications that chemotherapy induces response in up to 80% of patients and downgrades N2 disease in up to 50% of patients. This translates into significantly better survival when accompanied by complete resection. Since at least 50% of patients with stage IB disease develop distant metastases, it seems logical to explore the role of chemotherapy in early disease.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1999

The year of the worm.

Developmental biology has almost come full circle. Initially aimed at description at the organismal level, in the last 25 years it has zoomed in on individual genes that are involved in specific steps in development. Now, complete genome sequences are becoming available--and to gain a full understanding of the relevance of the complete genome, experimental developmental biology will hold centre stage again, but now armed with large genome databases, and with a new set of refined genetic tools. The first multicellular organism to be sequenced is the nematode C. elegans. This review aims to recognise some new avenues in C. elegans experimental biology that are opened by the genome sequence.

Amino Acid Sequence↗
PubMed1990

Infectious measles virus from cloned cDNA.

The study of measles virus (MV) and of negative strand RNA viruses in general has been hampered by the lack of an experimental system for genetic manipulation. Here we describe a procedure for generating infectious MV from cloned MV cDNA. First we assembled a genetically marked DNA copy of the MV genome in plasmids, under the control of phage T3 or T7 promoters, allowing production of transcripts almost identical to the MV genome or antigenome. Incubation of these linearized plasmid DNAs with the appropriate phage polymerase and only two ribonucleoside triphosphates yielded committed transcription complexes. Microinjection of these complexes into the cytoplasm of helper cells which provide the proteins necessary for MV genome encapsidation and transcription/replication, reproducibly give rise to lytic MVs. The transcripts of one of these viruses were analysed by sequencing after reverse transcription followed by DNA amplification, and found to contain the genetic tags. The described procedure permits the analysis of a negative strand RNA virus with the same genetic tools previously applicable only to positive strand RNA viruses and retroviruses.

Base Sequence↗
PubMed2006

Germ-line transformation and RNAi of the ladybird beetle, Harmonia axyridis.

To elucidate the molecular mechanisms underlying the tremendous diversity of insect wing colour patterns, it is imperative to identify and functionally characterize the genes involved in this developmental process. Here we report the first successful germ-line transformation using the transposable element vector piggyBac in the ladybird beetle Harmonia axyridis, which demonstrates typical genetic polymorphism in its wing colour patterns. The transformation efficiency by piggyBac was 3.7% per fertile G(0). We investigated the effectiveness of RNAi in Harmonia by injecting EGFP (enhanced green fluorescent protein) dsRNA into early transgenic EGFP-expressing embryos and observed substantial reduction of EGFP fluorescence in 87.2% of hatched larvae. Application of these new genetic tools to non-model insects such as Harmonia will facilitate the broad understanding of developmental mechanisms and evolutionary processes that are inaccessible using established model systems.

Animals↗
PubMed2000

Development of shuttle vectors for spirochetes.

Constructions of Escherichia coli-spirochete shuttle vectors are based on naturally occurring plasmids, broad host range plasmids or bacteriophages. This review primarily focuses on genetic tools for Treponema denticola which is associated with periodontal diseases. The T. pallidum FlaA protein, E. coli beta-galactosidase, and the green fluorescent protein were successfully expressed in T. denticola from a shuttle vector system.

Flagellin↗
PubMed2026

Single-organ proteomics in Drosophila melanogaster larva.

The combination of genetic accessibility, organ complexity, evolutionary conservation, and cost-efficiency makes Drosophila melanogaster (Dm) a well-known model system for biomedical and fundamental biological research. Proteomic analysis of single organs enables the identification and quantification of proteins expressed in specific organs. This will help to uncover specific biological functions and unique protein profiles that are not detectable in whole-organism analyses. In this study we have isolated single organs form Dm larvae, and we have performed a deep proteomics mapping by following a minimal manipulation preparation procedure. The combined dataset across all organs comprised 9132 identified proteins. As anticipated, principal component analysis (PCA) revealed clear separation between the proteomes of most organs, confirming distinct protein profiles. These findings demonstrate the applicability of the sample preparation strategy for high-resolution proteomic characterization of individual organs in Drosophila. Given the extensive genetic tools available for this model organism, our approach has the potential to open new avenues for proteomic studies in Drosophila melanogaster and any other biological systems where the sample amount is limiting. SIGNIFICANCE STATEMENT: Drosophila melanogaster is a well-known model system for biomedical and fundamental biological research that serves as a valuable in vivo model organism due to its high degree of evolutionary conservation with higher vertebrates, tractable genetics, and logistical efficiency. However, the proteome of Drosophila at single organ level has been elusive to date, due to several factors like low sensitivity of previous generation mass spectrometers and sample preparation procedures, difficult isolation of some organs. In this study we have applied a compilation of advanced methods including minimal sample manipulation together with simple, straightforward and efficient protein extraction and digestion methods. Obtained peptides were minimally handled to be analyzed by applying specific and sensitive nLC methods coupled on-line to state-of-the-art MS/MS system. Altogether, the applied strategy allowed us to get the first single organ study to date for this animal. These datasets represent a significative resource for future genomic, transcriptomic and proteomic studies in Drosophila, as multi-omic integration requires deep proteomics to translate data into functional biochemistry, and serves as a critical bridge and an indispensable standalone resource across the genomic, transcriptomic, and proteomic landscapes.

Animals↗
PubMed2003

Hypomutable regions of yeast TFIIB in a unigenic evolution test represent structural domains.

As genome sequences of many organisms - including humans - are being decoded, there is a great need for genetic tools to analyze newly discovered genes/proteins. A 'unigenic evolution' approach has been previously proposed for dissecting protein domains, which is based on the assumption that functionally important regions of a protein may tolerate missense mutations less well than other regions. We describe a unigenic evolution analysis of general transcription factor IIB (TFIIB) - a protein that is well characterized both structurally and functionally - to better understand the molecular basis of this genetic approach. The overall distribution profile of hypomutable regions within yeast TFIIB correlates extremely well with the known compact structural domains, suggesting that the unigenic evolution approach can help reveal structural properties of a protein. We further show that a small region located immediately carboxyl-terminal to the zinc ribbon motif is functionally important despite its strong hypermutability. Our study further demonstrates the usefulness of the unigenic evolution approach in dissecting protein domains, but suggests that the mutability of different regions of a protein in such a test is determined primarily by their structural properties.

Amino Acid Sequence↗
PubMed2024

Development of SacB-based counterselection for efficient allelic exchange in Fusobacterium nucleatum.

Fusobacterium nucleatum, prevalent in the oral cavity, is significantly linked to overall human health. Our molecular comprehension of its role in oral biofilm formation and its interactions with the host under various pathological circumstances has seen considerable advancements in recent years, primarily due to the development of various genetic tools for DNA manipulation in this bacterium. Of these, counterselection-based unmarked in-frame mutation methods have proved notably effective. Under suitable growth conditions, cells carrying a counterselectable gene die, enabling efficient selection of rare, defined allelic exchange mutants. The sacB gene from Bacillus subtilis, encoding levansucrase, is a widely used counterselective marker partly due to the easy availability of sucrose. Yet, its potential application in F. nucleatum genetic study remains untested. We demonstrated that F. nucleatum cells expressing sacB in either a shuttle or suicide plasmid exhibit a lethal sensitivity to supplemental sucrose. Utilizing sucrose counterselection, we created an in-frame deletion of the F. nucleatum tonB gene, a critical gene for energy-dependent transport processes in Gram-negative bacteria, and a precise knock-in of the luciferase gene immediately following the stop codon of the hslO gene, the last gene of a five-gene operon possibly related to the natural competence of F. nucleatum. Post-counterselection with 5% sucrose, chromosomal plasmid loss occurred in all colonies, leading to gene alternations in half of the screened isolates. This sacB-based counterselection technique provides a reliable method for isolating unmarked gene mutations in wild-type F. nucleatum, enriching the toolkit for fusobacterial research.IMPORTANCEInvestigations into Fusobacterium nucleatum's role in related diseases significantly benefit from the strategies of creating unmarked gene mutations, which hinge on using a counterselective marker. Previously, the galk-based allelic exchange method, although effective, faced an inherent limitation-the need for a modified host. This study aims to surmount this limitation by substituting galK with sacB for gene modification in F. nucleatum. Our application of the sacB-based methodology successfully yielded a tonB in-frame deletion mutant and a luciferase gene knock-in at the precise chromosomal location in the wild-type background. The new method augments the existing toolkit for F. nucleatum research and has far-reaching implications due to the easy accessibility to the counterselection compound sucrose. We anticipate its broader adoption in further exploration, thereby reinforcing its critical role in propelling our understanding of F. nucleatum.

Fusobacterium nucleatum↗
PubMed2001

Strategies using the immune system for therapy of brain tumors.

Phase II immunotherapy and gene therapy studies should be pursued because of encouraging results in many phase I studies. Future testing in this field may consider modifications of some of the above-mentioned combined strategies. For instance, in the immunization and adoptive transfer studies performed by Holladay et al and by Plautz et al, the systemic adoptive transfer could be altered to intratumoral placements of effector cells. This permutation may be more efficacious because local adoptive immunotherapy approaches involve placement of effector cells where they are needed. Additionally, new avenues of gene therapy are being explored that may offer added beneficial effects for immunization, local or systemic adoptive immunotherapy, or combined chemotherapy and adoptive immunotherapy of tumors. With new genetic tools, such as microarray analyses, SEREX, and creation of cDNA libraries from tumor cells, significant progress in the treatment of neoplasms in the immunologically privileged brain should be forthcoming.

Animals↗