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1.
Development of a high-frequency transforming vector for Aspergillus nidulans   总被引:18,自引:0,他引:18  
D J Ballance  G Turner 《Gene》1985,36(3):321-331
The pyr4 gene of Neurospora crassa, which codes for orotidine-5'-phosphate decarboxylase, is capable of transforming an Aspergillus nidulans pyrG mutant by chromosomal integration, despite low homology between the transforming DNA and the recipient genome. Integration of pFB6, a plasmid carrying pyr4 and capable of replication in Escherichia coli, was not observed at the pyrG locus. The efficiency of transformation was considerably enhanced (50-100 fold) by inclusion in the transforming vector of a 3.5-kb A.nidulans chromosomal sequence, ans1. Although this sequence was isolated on the basis of replicating activity in Saccharomyces cerevisiae, there was no evidence for such activity in A.nidulans. Part of the ans1 fragment appears to be reiterated in the A.nidulans genome, though it is not yet clear whether this is directly responsible for the high transformation frequency. The efficiency of transformation of A.nidulans by plasmids bearing ans1, using an improved protocol, was approx. 5 X 10(3) stable transformants per microgram of plasmid DNA.  相似文献   
2.
P Gottlieb  G LaFauci  R Rudner 《Gene》1985,33(3):259-268
Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.  相似文献   
3.
W D Davies  J Pittard  B E Davidson 《Gene》1985,33(3):323-331
Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.  相似文献   
4.
Four peptides possessing both natriuretic activity and smooth muscle relaxant activity were isolated from rat atrium and their amino acid sequences determined. The four peptides designated ANF-I, ANF-II, ANF-III and ANF-IV containing 35, 31, 30 and 25 amino acid residues, respectively, were obtained in a molar ratio of 4:60:20:16. The predominant species ANF-II, which may represent the native form of ANF, has the following sequence: (H2N)-G-P-R-S-L-R-R-S-S-C-F-G-G-R-I-D-R-I-G-A-Q-S-G-L-G-C-N-S-F-R-Y-(COOH) in which Cys-10 and Cys-26 are disulfide linked. Cleavage of the aspartyl linkage at position 16 by staphylococcal protease caused complete inactivation, indicating that the ring conformation is essential. The dose-response relationships determined for the four pepdides in relaxing norepinephrine-induced contraction of rabbit thoracic aorta showed half-maximum relaxation at concentrations ranging from 1.5 × 10?9 to 2.5 × 10?9 M. Comparable dose-response relationships were observed in relaxation of carbacol-induced contraction of chick rectum strips as tested with ANF-II and ANF-IV.  相似文献   
5.
The synthesis of 1-deoxy-D-xylulose 5-phosphate (DXP), catalyzed by the enzyme DXP synthase (DXS), represents a key regulatory step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis. In plants DXS is encoded by small multigene families that can be classified into, at least, three specialized subfamilies. Arabidopsis thaliana contains three genes encoding proteins with similarity to DXS, including the well-known DXS1/CLA1 gene, which clusters within subfamily I. The remaining proteins, initially named DXS2 and DXS3, have not yet been characterized. Here we report the expression and functional analysis of A. thaliana DXS2. Unexpectedly, the expression of DXS2 failed to rescue Escherichia coli and A. thaliana mutants defective in DXS activity. Coherently, we found that DXS activity was negligible in vitro, being renamed as DXL1 following recent nomenclature recommendation. DXL1 is targeted to plastids as DXS1, but shows a distinct expression pattern. The phenotypic analysis of a DXL1 defective mutant revealed that the function of the encoded protein is not essential for growth and development. Evolutionary analyses indicated that DXL1 emerged from DXS1 through a recent duplication apparently specific of the Brassicaceae lineage. Divergent selective constraints would have affected a significant fraction of sites after diversification of the paralogues. Furthermore, amino acids subjected to divergent selection and likely critical for functional divergence through the acquisition of a novel, although not yet known, biochemical function, were identified. Our results provide with the first evidences of functional specialization at both the regulatory and biochemical level within the plant DXS family.  相似文献   
6.
7.
Contemporary enzymes are highly efficient and selective catalysts. However, due to the intrinsically very reactive nature of active sites, gratuitous secondary reactions are practically unavoidable. Consequently, even the smallest cell, with its limited enzymatic repertoire, has the potential to carry out numerous additional, very likely inefficient, secondary reactions. If selectively advantageous, secondary reactions could be the basis for the evolution of new fully functional enzymes. Here, we investigated if Escherichia coli has cryptic enzymatic activities related to thiamin biosynthesis. We selected this pathway because this vitamin is essential, but the cell's requirements are very small. Therefore, enzymes with very low activity could complement the auxotrophy of strains deleted of some bona fide thiamin biosynthetic genes. By overexpressing the E. coli's protein repertoire, we selected yjbQ, a gene that complemented a strain deleted of the thiamin phosphate synthase (TPS)-coding gene thiE. In vitro studies confirmed TPS activity, and by directed evolution experiments, this activity was enhanced. Structurally oriented mutagenesis allowed us to identify the putative active site. Remote orthologs of YjbQ from Thermotoga, Sulfolobus, and Pyrococcus were cloned and also showed thiamin auxotrophy complementation, indicating that the cryptic TPS activity is a property of this protein family. Interestingly, the thiE- and yjbQ-coded TPSs are analog enzymes with no structural similarity, reflecting distinct evolutionary origin. These results support the hypothesis that the enzymatic repertoire of a cell such as E. coli has the potential to perform vast amounts of alternative reactions, which could be exploited to evolve novel or more efficient catalysts.  相似文献   
8.
9.
M Chen  Y Li  D Birch  RD Willows 《FEBS letters》2012,586(19):3249-3254
A Chl f-containing filamentous cyanobacterium was purified from stromatolites and named as Halomicronema hongdechloris gen., sp. nov. after its phylogenetic classification and the morphological characteristics. Hongdechloris contains four main carotenoids and two chlorophylls, a and f. The ratio of Chl f to Chl a is reversibly changed from 1:8 under red light to an undetectable level of Chl f under white-light culture conditions. Phycobiliproteins were induced under white light growth conditions. A fluorescence emission peak of 748 nm was identified as due to Chl f. The results suggest that Chl f is a red-light inducible chlorophyll.  相似文献   
10.
In eubacteria, ribosome recycling factor (RRF) and elongation factor G (EFG) function together to dissociate posttermination ribosomal complexes. Earlier studies, using heterologous factors from Mycobacterium tuberculosis in Escherichia coli revealed that specific interactions between RRF and EFG are crucial for their function in ribosome recycling. Here, we used translation factors from E. coli, Mycobacterium smegmatis and M. tuberculosis, and polysomes from E. coli and M. smegmatis, and employed in vivo and in vitro experiments to further understand the role of EFG in ribosome recycling. We show that E. coli EFG (EcoEFG) recycles E. coli ribosomes with E. coli RRF (EcoRRF), but not with mycobacterial RRFs. Also, EcoEFG fails to recycle M. smegmatis ribosomes with either EcoRRF or mycobacterial RRFs. On the other hand, mycobacterial EFGs recycle both E. coli and M. smegmatis ribosomes with either of the RRFs. These observations suggest that EFG establishes distinct interactions with RRF and the ribosome to carry out ribosome recycling. Furthermore, the EFG chimeras generated by swapping domains between mycobacterial EFGs and EcoEFG suggest that while the residues needed to specify the EFG interaction with RRF are located in domains IV and V, those required to specify its interaction with the ribosome are located throughout the molecule.  相似文献   
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