A solution hybridization assay for ribosomal RNA from bacteria using biotinylated DNA probes and enzyme-labeled antibody to DNA:RNA.

Rapid, convenient and non-isotopic nucleic-acid hybridization methods are needed for this technology to have practical use in clinical diagnostic tests. A method for hybridization of RNA with a DNA probe in solution followed by capture and measurement of the hybrid is described. DNA probes complementary to 23S rRNAs from Escherichia coli and Bacillus subtilis were labeled with a photoactivable biotin reagent. Hybridization of the biotinylated probes with rRNA was complete in less than 5 min. The resultant hybrids were allowed to bind simultaneously to succinylated avidin immobilized on latex and to beta-galactosidase-labeled Fab' fragments of a monoclonal antibody-specific for DNA:RNA. Finally, beta-galactosidase associated with the captured hybrids was measured colorimetrically. The hybridization method can detect less than 1000 bacteria per assay and has broad specificity to permit detection of the various genera of bacteria that infect the urinary tract.

Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids.

Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity was selected. This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.[3H]RNA. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody. The observed association constant for the antibody and DNA.[3H]RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites. The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA. The colorimetric response of this assay increased linearly with the amount of hybrid formed.

Purification and characterization of an extracellular protease from Flavobacterium arborescens.

An extracellular protease from Flavobacterium arborescens has been purified to an apparent homogeneity and characterized. The enzyme is most active at pH 8-10.5, requires no metal cofactor, and is inhibited by diisopropyl fluorophosphate. The protease is nonspecific, is active at temperatures up to 60 degrees C, and is completely free of nucleases. The ease of purification and freedom from nucleolytic contaminants make the protease a useful deproteinizing agent in DNA and RNA manipulations.

Specific RNA-cleaving activities from HeLa cells.

Subcellular fractionation of HeLa cells was carried out under gentle conditions to isolate enzymes that cleave RNA precursors in a specific manner. Four separate activities--cleavage of HeLa cell heterogeneous nuclear RNA, the HeLa cell 45S rRNA precursor, RNA . DNA hybrids (RNase H), and the Escherichia coli tRNATyr precursor (RNase P)--were revealed by these studies. The specificity and limited nature of these cleavages suggest that they are due to eukaryotic RNA-processing enzymes. The virtual absence of random nucleases from these enzymes was demonstrated by their inability to cleave the 8000-base early mRNA precursor of bacteriophage T7, E. coli 30S rRNA precursor, or HeLa cytoplasmic poly(A)-containing RNA.

Genome-linked protein associated with the 5' termini of bacteriophage phi29 DNA.

A DNA-protein complex was isolated from Bacillus subtilis bacteriophage phi29 by sucrose gradient sedimentation or gel filtration in the presence of agents known to break noncovalent bonds. A 28,000-dalton protein was released from this complex by subsequent hydrolysis of the DNA. The DNA-protein complex was examined for its susceptibility to enzymes which act upon the 5' and 3' termini of DNA molecules. It was susceptible to exonucleolytic degradation from the 3' termini by exonuclease III but not from the 5' termini by lambda exonuclease. Attempts to label radioactively the 5' termini by phosphorylation with T4 polynucleotide kinase were unsuccessful despite prior treatment with alkaline phosphatase or phosphatase treatment of denatured DNA. Removal of the majority of the bound protein by proteolytic digestion did not increase susceptibility. These results suggest that the linked protein is covalently attached to the 5' termini of phi29 DNA.

Deoxyribonucleic acid synthesis in bacteriophage SPO1-infected Bacillus subtilis. I. Bacteriophage deoxyribonucleic acid synthesis and fate of host deoxyribonucleic acid in normal and polymerase-deficient strains.

The effect of bacteriophage SPO1 infection of Bacillus subtilis and a deoxyribonucleic acid (DNA) polymerase-deficient (pol(-)) mutant of this microorganism on the synthesis of DNA has been examined. Soon after infection, the incorporation of deoxyribonucleoside triphosphates into acid-insoluble material by cell lysates was greatly reduced. This inhibition of host DNA synthesis was not a result of host chromosome degradation nor did it appear to be due to the induction of thymidine triphosphate nucleotidohydrolase. Examination of the host chromosome for genetic linkage throughout the lytic cycle indicated that no extensive degradation occurred. After the inhibition of host DNA synthesis, a new polymerase activity arose which directed the synthesis of phage DNA. This new activity required deoxyribonucleoside triphosphates as substrates, Mg(2+) ions, and a sulfhydryl reducing agent, and it was stimulated in the presence of adenosine triphosphate. The phage DNA polymerase, like that of its host, was associated with a fast-sedimenting cell membrane complex. The pol(-) mutation had no effect on the synthesis of phage DNA or production of mature phage particles.

Lack of nucleotide sequence relationship among the temperate bacteriophage SPO2, Bacillus subtilis, and virulent bacteriophages beta 3 and beta 22.

Radiolabeled phage SPO2 fragments were tested for the abiity to form interspecies deoxyribonucleic acid (DNA) duplexes with DNA from Bacillus subtilis and from phages beta3 and beta22. No reassociation, above control values, occurred between the DNA of phage SPO2 and that of its host or either of the virulent B. subtilis phages. It appears that extensive nucleotide sequence similarities between portions of host and phage DNA species are not necessary for the formation of the lysogenic state. The thermal elution profile of reassociated SPO2 DNA exhibited a normal distribution, indicating no heterogeneity in base composition. This differs from temperate enterophages whose DNA molecules contain regions of differing base composition.

Differential expression of bacteriophage genomes in vegetative and sporulating cells of Bacillus subtilis.

Two antigenically distinct bacteriophages, beta3 and beta22, have been isolated and characterized with Bacillus subtilis strain W23 as a host. They differ in plaque morphology, single-step growth characteristics, host range, and thermal stability. The deoxyribonucleic acids isolated from beta3 and beta22 differ in base composition, density in CsCl and Cs(2)SO(4), sedimentation coefficient, molecular weight, and thermal denaturation temperature. These phages have been used to analyze the ability of B. subtilis to sporulate despite infection by virulent phages. When development of phages beta3 and beta22 in sporulating cultures was compared with that in log cultures, an increase in the latent periods of infection and a decrease in the burst sizes for the two phages were observed. Sporulating cultures infected with beta3 yielded the usual percentage (85%) of mature spores; 80% of these contained phage determinants and 20% were uninfected. However, cultures infected with beta22 lysed. Of the small fraction (0.01%) which sporulated, 83% were uninfected and 17% were infected. Phage beta3-infected and uninfected spores were examined to distinguish any chemical or physical differences. Preparations of both types of spore contained 81.4 mug of dipicolinic acid per mg (dry weight), and examination by phase-contrast microscopy gave no evidence of any difference in outward appearance. A 20% decrease in infected spore count was observed upon heating at 80 C for 10 min. Differences in the infection processes of the two phages prompted an analysis of the transcription process after infection. Deoxyribonucleic acid-ribonucleic acid hybrid analysis of relative amounts of phage-specific and host-specific messenger ribonucleic acid (mRNA) present in infected cells suggested that beta3 was unable to repress the synthesis of host mRNA and that beta3-specific mRNA synthesis was repressed in sporulation-phase cultures. Phage beta22, in contrast, was able to repress host-specific mRNA synthesis in both log-infected and sporulation-infected cells. The results suggest that the differential expression of the phage genomes is due to the relative ability of the phages to repress the host genome.