Transgenic animal mutagenesis assays using lambda shuttle vectors have recently been described for isolation and characterization of spontaneous and chemical induced DNA mutations. Extensive information on lambda and E. coli genetics provides a wealth of techniques to allow selection of mutant target genes. Here we describe the modification of an E. coli host which permits two methods for the direct selection of mutant genes. These methods reduce the number of plates needed to be screened for a comparable amount of frequency data by 20-100-fold and thus provide a significant savings of the materials and time required for the screening of mutations. In addition, mutants selected by these approaches described here may alter or broaden the spectrum of mutations that are recoverable. Ultimately, a combination of selective and nonselective techniques may prove valuable for the analysis of mutations produced in vivo in transgenic animals.
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http://dx.doi.org/10.1016/0165-7992(93)90031-p | DOI Listing |
Appl Microbiol Biotechnol
January 2025
Chair of Microbiology, Technical University of Munich, TUM School of Life Science, Emil-Ramann-Str. 4, 85354, Freising, Germany.
The anaerobic bacterium Clostridium cellulovorans is a promising candidate for the sustainable production of biofuels and platform chemicals due to its cellulolytic properties. However, the genomic engineering of the species is hampered because of its poor genetic accessibility and the lack of genetic tools. To overcome this limitation, a protocol for triparental conjugation was established that enables the reliable transfer of vectors for markerless chromosomal modification into C.
View Article and Find Full Text PDFCell Commun Signal
January 2025
Department of Biosciences and Medical Biology, Paris-Lodron University Salzburg, Hellbrunner Strasse 34, Salzburg, 5020, Austria.
FLT3 mutations occur in approximately 25% of all acute myeloid leukemia (AML) patients. While several FLT3 inhibitors have received FDA approval, their use is currently limited to combination therapies with chemotherapy, as resistance occurs, and efficacy decreases when the inhibitors are used alone. Given the highly heterogeneous nature of AML, there is an urgent need for novel targeted therapies that address the disease from multiple angles.
View Article and Find Full Text PDFSci Rep
January 2025
Biological Engineering Program, Faculty of Engineering, King Mongkut's University of Technology Thonburi, Bangkok, 10140, Thailand.
Nanobodies (Nbs) hold great potential to replace conventional antibodies in various biomedical applications. However, conventional methods for their discovery can be time-consuming and expensive. We have developed a reliable protein selection strategy that combines magnetic activated cell sorting (MACS)-based screening of yeast surface display (YSD) libraries and functional ligand-binding identification by Tat-based recognition of associating proteins (FLI-TRAP) to isolate antigen-specific Nbs from synthetic libraries.
View Article and Find Full Text PDFProtein Sci
February 2025
Yusuf Hamied Department of Chemistry, University of Cambridge, Cambridge, UK.
We have recently demonstrated a novel anaerobic NADH-dependent haem breakdown reaction, which is carried out by a range of haemoproteins. The Yersinia enterocolitica protein, HemS, is the focus of further research presented in the current paper. Using conventional experimental methods, bioinformatics, and energy landscape theory (ELT), we provide new insight into the mechanism of the novel breakdown process.
View Article and Find Full Text PDFBreed Sci
September 2024
Guizhou Institute of Upland Food Crops, Guizhou Academy of Agricultural Sciences, Guiyang 550006, Guizhou Province, China.
To explore the molecular mechanism behind maize grain quality and use of different gene stacking to improve the nutritional quality of grain, marker-assisted selection (MAS) was used to select three recessive mutant lines containing , along with the double-recessive mutant lines containing , , and . The resulting seeds were taken for transcriptome sequencing analysis 18 days after pollination (DAP). Results: Compared with the recurrent parent genes, in the lysine synthesis pathway, the gene pyramiding lines (, , and ) revealed that the gene encoding aspartate kinase (AK) was up-regulated and promoted lysine synthesis.
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