Kline, B C

Nearly 800 nucleotides from the 5' terminus of the 28S ribosomal gene of Paracoccidioides brasiliensis were sequenced, and a 14-base DNA probe specific for this species was identified. Hybridization results showed that the probe identified P. brasiliensis ribosomal DNA in a panel of ribosomal DNAs representing a total of 48 species of fungi.

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.

Plasmid F replication is controlled by a plasmid-specified Rep protein with both autorepressor and initiator functions. The mechanism by which these two functions of a Rep protein are balanced to achieve stable replication is unknown; however, we speculated in prior work that Rep protein modification could be involved. We report here that naturally proteolyzed F RepE protein has been detected and characterized. The processed molecule lost the first 17 N-terminal aminoacyl residues and initiator function but acquired increased specific DNA-binding affinity in the presence of Escherichia coli chromosomal DNA. When supplied in trans, the altered protein acts as an incompatibility substance and eliminates maintenance of F'lac. These findings indicate that protein processing has the potential to contribute to the overall control of DNA replication.

The F sex factor plasmid of Escherichia coli contains a pair of genes, ccdA and ccdB, whose protein gene products are involved in an unusual feature of plasmid maintenance. The CcdB protein is a cytotoxin that becomes activated when the F plasmid is lost, thereby killing the F- segregant cells. In F+ cells, the CcdA protein protects against the lethal effects of CcdB. In the present study we show that ccdA and ccdB expressions are negatively autoregulated at the level of transcription. Genetic studies showed that repression required at least ccdB; ccdA alone was without effect, and ccdB alone was not examined because it is lethal. Ccd-operator complexes were purified and contained a mixture of both CcdA and CcdB proteins; however, we could not conclude from our results whether CcdA was necessary for DNA binding or autorepression. By using restriction fragments of the promoter-operator region, we obtained results indicating that at least two DNA-binding sites existed for the Ccd protein(s). Subsequent footprinting of the binding sites showed protection over about a 113-base-pair region encompassing the putative promoter-operator and the beginning of the ccdA gene.