Cloning and characterisation of the sagA gene of Aspergillus nidulans: a gene which affects sensitivity to DNA-damaging agents

Mutations within the sagA gene of Aspergillus nidulans cause sensitisation to DNA-damaging chemicals but have no effect upon spontaneous or damage-induced mutation frequency. The sagA gene was cloned on a 19-kb cosmid-derived fragment by functional complementation of a sagA1 sagC3 double mutant; subsequently, a fragment of the gene was also isolated on a 3.9-kb genomic subclone. Initial sequencing of a small section of the 19-kb fragment allowed the design of primers that were subsequently used in RTPCR experiments to show that this DNA is transcribed. A 277-bp fragment derived from the transcribed region was used to screen an A. nidulans cDNA library, resulting in the isolation of a 1.4-kb partial cDNA clone which had sequence overlap with the genomic sagA fragment. This partial cDNA was incomplete but appeared to contain the whole coding region of sagA. The sagA1 mutant was shown to possess two mutations; a G-T transversion and a+1 frameshift due to insertion of a T, causing disruption to the C-terminal region of the SagA protein. Translation of the sagA cDNA predicts a protein of 378 amino acids, which has homology to the Saccharomyces cerevisiae End3 protein and also to certain mammalian proteins capable of causing cell transformation. Received: 1 August 1998 / Accepted: 9 November 1998     

Multiple interactions between nuclear proteins of Zea mays and the promoter of the Shrunken gene

Summary Nuclear proteins were extracted from isolated nuclei of immature maize kernels. The promoter region (1.5 kb) of the Shrunken gene, which is highly transcribed in the developing endosperm of the kernel, was scanned for protein-DNA interactions. Several promoter fragments showed protein-DNA complex formation in gel retardation experiments. Two different nucleo-protein complexes (MNP1 and MNP2) have been distinguished in competition and DNase I footprinting experiments. Both nuclear DNA-binding activities are able to recognize multiple sites distributed over a 1.5 kb upstream region of the Shrunken gene. Some of the binding sites established in the in vitro reconstitution experiments are located near to DNase I hypersensitive sites found in the promoter of the Shrunken gene (Frommer and Starlinger 1988).     

Genetic analysis of a transposon carrying toluene degrading genes on a TOL plasmid pWWO

Summary Toluene degrading (xyl) genes on a Pseudomonas TOL plasmid pWWO are located within a 39-kb DNA portion. The 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pACYC184 and R388 in escherichia coli recA strains. Thus the 56- and 17-kb regions were designated Tn4651 and Tn4652, respectively. Genetic analysis of Tn4652 demonstrated that its transposition occurs by a two-step process, namely, cointegrate formation and its subsequent resolution. The presence in cis of DNA sequences of no more than 150 bp at both ends of Tn4652 was prerequisite for cointegrate formation, and this step was mediated by a trans-acting factor, transposase, which was encoded in a 3.0-kb segment at one end of the transposon. Cointegrate resolution took place site-specifically within a 200-bp fragment, which was situated 10 kb away from the transposase gene. Based on the stability of cointegrates formed by various mini-Tn4652 derivatives, it was shown that the cointergrate resolution requires two trans-acting factors encoded within 1.0- and 1.2-kb fragments that encompass the recombination site involved in the resolution.     

Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus.

We have mapped DNase I-hypersensitive sites and topoisomerase II (topo II) sites in the chicken beta-globin locus, which contains four globin genes (5'-rho-beta H-beta A-epsilon-3'). In the 65 kilobases (kb) mapped, 12 strong hypersensitive sites were found clustered within the 25-kb region from 10 kb upstream of rho to just downstream of epsilon. The strong sites were grouped into several classes based on their tissue distribution, developmental pattern, and location. (i) One site was present in all cells examined, both erythroid and nonerythroid. (ii) Three sites, located upstream of the rho-globin gene, were present at every stage of erythroid development, but were absent from nonerythroid cells. (iii) Four sites at the 5' ends of each of the four globin genes were hypersensitive only in the subset of erythroid cells that were transcribing or had recently transcribed the associated gene. (iv) Another three sites, whose pattern of hypersensitivity also correlated with expression of the associated gene, were found 3' of rho, beta H, and epsilon. (v) A site 3' of beta A and 5' of epsilon was erythroid cell specific and present at all developmental stages, presumably reflecting the activity of this enhancer throughout erythroid development. We also mapped the topo II sites in this locus, as determined by teniposide-induced DNA cleavage. All strong teniposide-induced cleavages occurred at DNase I-hypersensitive sites, while lesser amounts of cleavage were observed in transcribed regions of DNA. Most but not all of the DNase I-hypersensitive sites were topo II sites. These data are consistent with the hypothesis that, in vivo, topo II preferentially acts on nucleosome-free regions of DNA but suggest that additional topo II regulatory mechanisms must exist.     

Characterization of a Rhizobium etli chromosomal gene required for nodule development on Phaseolus vulgaris L.

A chromosomal gene, required for nodule development on Phaseolus bean, was characterized from Rhizobium etli strain TAL182. MLC640 is a Tn5 insertion mutant of TAL182 which shows decreased motility in soft TY agar and is defective in nodule development. The site of Tn5 insertion in MLC640 mapped to a 3.6-kb EcoRI chromosomal fragment. The 3.6-kb fragment was subcloned from the cosmid pUHR80 which complemented MLC640. Further subcloning and site-directed Tn5 mutagenesis localized the gene for nodule development to a 1.7-kb region within the 3.6-kb EcoRI fragment. Southern hybridization using the 3.6-kb EcoRI fragment as the probe against genomic DNA of several Rhizobium spp. indicated that this gene is conserved in different rhizobia.The authors are with the Department of Plant Molecular Physiology, University of Hawaii, 3050 Maile Way, Gimore 402, Honolulu, Hawaii 96822. USA;     

Asymmetrical progression of replication forks just after initiation on Mycoplasma capricolum chromosome revealed by two-dimensional gel electrophoresis

Previously, we mapped the replication initiation site of the Mycoplasma capricolum chromosome into a region containing the dnaA gene [M. Miyata et al., 1993a. Nucleic Acids Res. 21, 4816–4823]. In this study, various regions including this functional domain were analyzed by two complementary two-dimensional (2D) gel electrophoretic methods. Sizes of nascent strands in a 10.7-kb and a 5.6-kb region were examined by a neutral/alkaline (N/A) method. The shortest nascent strand was detected in an 875-bp region composed of the 3′ end of the dnaA gene and its downstream non-coding sequence. The shortest nascent strand detected became longer in an asymmetrical manner as position of the probe became further from the putative initiation site in both directions. The intermediate forms of eight regions restricted at different sites were examined by a neutral/neutral (N/N) method. Bubble arcs were observed in four regions including the 875-bp region. The region containing the 875-bp region at about its center showed an asymmetrical arc, although that containing the 875-bp region at its end showed a symmetrical arc. These results show that the replication forks develop in the 875-bp region and proceed bidirectionally in an asymmetrical manner around the initiation site. The results of N/A analysis of the 5.6-kb region showed a shift of intensity in the nascent strand signal, which suggests an upshift of fork progression velocity.