PubMed HealthSearch

PubMed · 8224901

A novel phage lambda replacement Cre-lox vector that has automatic subcloning capabilities.

Abstract

We have developed a novel phage lambda replacement cloning vector, lambda pAn. lambda pAn allows one to automatically subclone the insert as a plasmid using the Cre-loxP site-specific recombination system. This eliminates the need to subclone insert fragments and permits the rapid structural analysis of insert DNA. lambda pAn is similar to other phage lambda replacement vectors taking inserts ranging in size from 5 to 19 kb. We have placed the pyrG gene of Aspergillus nidulans on the vector as a nutritional selective marker for transformation. We have developed this vector as part of an overall plan to facilitate the cloning of dominant extragenic suppressor mutations from A. nidulans, but also know that it is a generally useful vector for the purposes of isolating genomic clones without the need to subclone from the phage lambda vector.

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

C L Holt, G S May. 1993-10-29. A novel phage lambda replacement Cre-lox vector that has automatic subcloning capabilities.. https://doi.org/10.1016/0378-1119(93)90230-z

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

Structural Characterization and Engineering of a GH134 β-Mannanase from Aspergillus nidulans for Enhancement of Activity and Stability.

Mannans are abundant plant hemicelluloses, and endo-β-mannanases are important biocatalysts for their conversion into functional manno-oligosaccharides. Here, we report the structural and functional characterization of a glycoside hydrolase family 134 β-mannanase from Aspergillus nidulans (AnGH134) and a structure-guided engineering strategy to improve its performance on locust bean gum. The 1.75 Å crystal structure reveals the conserved lysozyme-like fold of GH134 enzymes and supports an inverting catalytic mechanism with Glu43 and Asp55 as the putative catalytic residues. Docking, mutational, and molecular dynamics analyses indicate that AnGH134 uses an extended substrate-binding groove and that groove-exit residues and the C-terminal region contribute to productive catalysis. Guided by these findings, N-terminal fusion of CBM10 enhanced catalytic efficiency and thermal stability, whereas C-terminal fusion was detrimental. These results provide a framework for engineering GH134 mannanases.

Aspergillus nidulans

In-Silico and Functional Characterization of EcdLp, an ABC Transporter of Aspergillus nidulans NRRL11440.

Echinocandin B (ECB) biosynthesis in Aspergillus nidulans is primarily governed by multiple genes located within the biosynthetic echinocandin (ecd) gene cluster. The contributory functions of many genes, including transcription factors and tailoring enzymes of the ecd gene cluster, have been previously studied. The present study focused on determining the role of transporter proteins, EcdLp, EcdCp, and EcdDp, in ECB efflux using in silico and biochemical approaches. The molecular docking analysis revealed that ECB relatively showed higher binding affinity for EcdLp than the other co-clustered MFS transporters EcdCp and EcdDp, suggesting a preferred substrate of EcdLp. These results were further confirmed by heterologous integration of the ecdL gene in the ABC transporters-deficient Saccharomyces cerevisiae AD1-8u⁻, confirming active efflux. However, the binding of ECB in EcdLp is distinct from the R6G binding, overlapping the promiscuous site of farnesol, resulting in inhibition of R6G efflux in a dose-dependent manner. In conclusion, these results decipher the ECB binding and efflux mechanism and unveil the evolutionarily specialized architecture of EcdLp that permits targeted metabolite export in addition to environmental responsiveness, and lay the groundwork for optimizing ECB production via transporter engineering.

Aspergillus nidulans