We constructed a chimeric plasmid carrying a complete copy of the trifunctional trpC gene from the Ascomycete fungus Aspergillus nidulans. This plasmid, designated pHY201, replicates in Escherichia coli, where it confers resistance to ampicillin and chloramphenicol and complements thpC mutants lacking phosphoribosylanthranilate isomerase activity. We used pHY201 to transform an A. nidulans trpCstrain to trpC' at frequencies of >20 stable transformants per ,ug of DNA. Southern blot analysis of DNA from transformants showed that pHY201 DNA had integrated into the A. nidulans chromosomes in a majority of cases. Most of the integration events appeared to occur at the site of the tipC-allele of the recipient strain. In several instances, we succeeded in recovering pHY201, or derivatives thereof, from A. nidulans transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of E. coli.The Ascomycete fungus Aspergillus nidulans has been used extensively for the study of eukaryotic gene structure, organization, and regulation (1-4) because features of its life cycle make it particularly amenable to biochemical and genetic analysis. A. nidulans has been especially valuable for investigating the genetic and molecular processes controlling fungal cell differentiation (5-9). In contrast to the situation with its close relatives Saccharomyces cerevisiae (yeast) and Neurospora crassa, however, investigations of A. nidulans have not been facilitated by the availability of a tractable DNA-mediated transformation system.We recently isolated the trifunctional trpC gene from A. nidulans (10) for use as a selective marker in transformation experiments with this organism. Here we report construction of a chimeric plasmid, designated pHY201, consisting of a complete wild-type copy of the Aspergillus trpC gene inserted into the unique Sal I site of pBR329 (11). We find that pHY201 DNA, either in circular or linear form, transforms a trpC-A. nidulans strain to trpC+ at frequencies of >20 stable transformants per pg of DNA. The transforming DNA becomes integrated into the host genome, frequently at the site of the resident gene. In several instances, we were able to recover pHY201 in native or altered form from Aspergillus transformants by restriction endonuclease digestion of chromosomal DNA, ligation, and transformation of Escherichia coli to ampicillin resistance. Thus, pHY201 has properties that suggest that it may serve as a valuable prototype for the development of more sophisticated Aspergillus cloning vectors. (15) of E. coli MC1066 by using a recombinant phage library formed between A. nidulans nuclear DNA and X Charon 4 (9, 10). We determined that a 4.1-kilobase Xho I restriction fragment present in a phage designated XAn trpC12 contained the entire trpC gene plus about 0.4 kilobase of 3' and 5' flanking sequences (unpublished results). This fragment was recloned into the unique Sal I site of pBR329 (11) to form pHY201 (Fig. 1). MATERIALS AND METHODS MaterialsIsolation and Labeli...

We have determined the structure and complete nucleotide sequence of the trifunctional trpC gene from the Ascomycetous fungus Aspergillus nidulans. Results from RNA gel blot analyses showed that this gene encodes two size classes of polyribosomal, poly (A)+RNAs with approximate lengths of 2,400 and 2,600 nucleotides. S1 nuclease protection studies demonstrated that the distribution into the two size classes is due to selection of alternative sites for polyadenylation. The transcription units contain a single open translation reading frame of 2,304 nucleotides. The sequence of this reading frame is approximately 40% divergent from the sequence of the functionally analogous trp-1 gene from Neurospora crassa (Schechtman, M.G. and Yanofsky, C., J. Mol. Appl. Gen. 2:83-99). The predicted amino acid sequence of the A. nidulans trpC polypeptide is also 40% divergent from the predicted amino acid sequence of the N. crassa trp-1 polypeptide. The A. nidulans gene has considerably less bias in codon selection than observed for the N. crassa gene. Discrete regions of DNA homology were also found in similar positions in the 5' and 3' flanking sequences of the Aspergillus and Neurospora genes. Similar regions of homology were not observed in other Aspergillus or Neurospora genes that have been sequenced. Thus, if these evolutionarily conserved sequences act as signals for transcription initiation or polyadenylation, or are involved in gene regulation, their functions are restricted to a subset of protein coding genes in these two closely related fungi.

Rhizobium japonicum USDA191 is a member of a new group of Rhizobium japonicum strains found in China. This strain is one of several strains shown to be salt-tolerant and fast-growing; it is unique in being the only strain of this group that effectively nodulates American soybean cultivars. For these reasons strain USDA 191 was chosen for further study and comparison to the common American Rhizobium japonicum isolate USDA 1 10. Strain USDA 19 1 has a doubling time of 3.2 h in complex medium and grows in concentrations of up to 0.4 M-NaCl, while strain USDAl 10, which has a doubling time of 12 h, is severely inhibited in media containing 0.1 MNaCl. Under salt stress conditions, intracellular levels of K+ and glutamate were shown to increase. A comparison based on carbohydrate metabolism, DNA homology and protein patterns on polyacrylamide gels reveals that strain USDA191 is more closely related to the fastgrowing rhizobia than to Rhizobium japonicum. However, the strain retains capacity to nodulate American soybean and cowpea cultivars effectively.

We constructed a 9.9-kilobase cloning vector, designated pKBY2, for isolating genes by complementation of mutations in Aspergillus nidulans. pKBY2 contains the bacteriophage X cos site, to permit in vitro assembly of phage particles; a bacterial origin of replication and genes for resistance to ampicillin and chloramphenicol, to permit propagation in Escherichia coli; the A. nidulans trpC' gene, to permit selection in Aspergillus; and a unique BamHI restriction site, to permit insertion of DNA fragments produced by digestion with restriction endonucleases BamHI, Bgl II, Mbo I, or Sau3A. We used this cosmid to form a quasirandom recombinant DNA library containing 35-to 40-kilobase DNA fragments from a wild-type strain of A. nidulans. DNA from this library transformed a yellow-spored (yA ) pabaA-tpC-Aspergillus strain (FGSC237) to tipC' at frequencies of approximately 10 transformants per pg of DNA. Three of approximately 1000 tipC' pabaA-colonies obtained were putative yA+ transformants, because they produced wild-type (green) spores. DNA from each of the green-spored transformants contained pKBY2 sequences and DNA from two transformants transduced E. coli to ampicillin resistance following treatment in vitro with a X packaging extract. The cosmids recovered in E. coli had similar restriction patterns and both yielded tipC' transformants of A. nidulans FGSC237, 85% of which produced green spores. Several lines of evidence indicate that the recovered cosmids contain a wild-type copy of the yA gene.Complementation of mutations in Saccharomyces cerevisiae (yeast) and in animal cells by transformation with recombinant DNA libraries provides a convenient and valuable method for the isolation of specific eukaryotic genes (1-4). Plasmid "shuttle" vectors for this purpose typically include genes for selection in both Escherichia coli and the eukaryotic host and one or more unique restriction sites for insertion of foreign DNA fragments. In addition, these vectors must transform the eukaryotic host at a frequency sufficient to allow screening of entire genomes and should provide a simple way to recover the complementing DNA in the prokaryotic host. Shuttle vectors for S. cerevisiae frequently contain sequences mediating autonomous replication in yeast to enhance the frequency of transformation and to allow recovery of plasmids by direct transformation of E. coli with total DNA isolated from yeast cells (5-9). Generally, analogous vectors have been constructed for use with cultured mammalian cells (10, 11). However, because of restrictions in the size of DNA fragments that can be introduced into these plasmids, it is difficult to screen entire animal genomes. Some shuttle vectors for mammalian cells have been constructed that contain one or two bacteriophage X cos sites to allow in vitro assembly of phage particles containing large (30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) kilobase) cloned inserts, in order to reduce this problem (3, 4).Aspergillus nidulans is an Ascomycetous fungus related to S. cerevisiae and,...