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This study focuses on the molecular mechanisms of a noncoding RNA-mediated chromosome organization in Escherichia coli. We show for the first time that noncoding RNA, naRNA4, a critical element at least in part of chromosome structural organization (DNA condensation), may form DNA–RNA complexes in an uncommon way. Surprisingly, we found that HU protein catalyzes the DNA–RNA interactions without being part of the “final” complexes. There are several important examples of noncoding RNA playing critical roles in eukaryotic chromatin functioning, e.g., heterochromatin assembly, gene silencing, and X-inactivation, although nothing is known about the nature of DNA–RNA interaction(s) in these processes. Our findings will be of great interest in the chromatin field, providing mechanistic insights into the large-scale organization of DNA.

We determined the crystal structure of the Escherichia coli nucleoid-associated HUαβ protein by x-ray diffraction and observed that the heterodimers form multimers with octameric units in three potential arrangements, which may serve specialized roles in different DNA transaction reactions. It is of special importance that one of the structures forms spiral filaments with left-handed rotations. A negatively superhelical DNA can be modeled to wrap around this left-handed HUαβ multimer. Whereas the wild-type HU generated negative DNA supercoiling in vitro , an engineered heterodimer with an altered amino acid residue critical for the formation of the left-handed spiral protein in the crystal was defective in the process, thus providing the structural explanation for the classical property of HU to restrain negative supercoils in DNA.

To determine whether the spacer region between the -35 and -10 elements plays any sequence-specific role, we randomized the GC-rich sequence ( -20 CCGGCTCG -13 ) within the spacer region of the cAMP-dependent lac promoter and selected an activator-independent mutant, which showed extraordinarily high intrinsic activity. The hyperactive promoter is obtained by incorporation of a specific 10-bp-long AT-rich DNA sequence within the spacer, referred to as the -15 sequence, which must be juxtaposed to the upstream end of the -10 sequence for the hyperactivity. The transcription enhancement functions only in the presence of a -35 element. The spacer sequence enhanced both RNA polymerase binding and open complex formation. Isolated in the lac promoter, it also enhanced transcription when placed at two other unrelated promoters. Sequence analysis shows a low GC content and an abundance of stereochemically flexible TG:CA and TA:TA dimeric steps in the -18/-9 region and a strong correlation between the presence of flexible dimeric steps in this region and the intrinsic strength of the promoter.

Genetic instability is thought to be responsible for the numerous genotypic changes that occur during neoplastic transformation and metastatic progression. To explore the role of genetic instability at the level of point mutations during mammary tumor development and malignant progression, we combined transgenic mouse models of mutagenesis detection and oncogenesis. Bitransgenic mice were generated that carried both a bacteriophage lambda transgene to assay mutagenesis and a polyomavirus middle T oncogene, mammary gland-targeted expression of which led to metastatic mammary adenocarcinomas. We developed a novel assay for the detection of mutations in the lambda transgene that selects for phage containing forward mutations only in the lambda cII gene, using an hfl- bacterial host. In addition to the relative ease of direct selection, the sensitivity of this assay for both spontaneous and chemically induced mutations was comparable to the widely used mutational target gene, lambda lacI, making the cII assay an attractive alternative for mutant phage recovery for any lambda-based mouse mutagenesis assay system. The frequencies of lambda cII- mutants were not significantly different in normal mammary epithelium, primary mammary adenocarcinomas, and pulmonary metastases. The cII mutational spectra in these tissues consisted mostly of G/C-->A/T transitions, a large fraction of which occurred at CpG dinucleotides. These data suggest that, in this middle T oncogene model of mammary tumor progression, a significant increase in mutagenesis is not required for tumor development or for metastatic progression.

The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.

We have previously described temperature sensitive rho mutants of Escherichia coli (e.g., rho15) that are defective in transcription termination at various signals, including an IS2 DNA insertion in the gal operon [Das, A., Court, D. & Adhya, S. (1976) Proc. Natl. Acad. Sci. USA, 73, 1959-1963]. In this paper, we report the isolation of mutants altered in the beta subunit of RNA polymerase (a class of Rifampicin-resistant mutants), which restore gal IS2 polarity in the rho 15 strain. It has been shown that one of these suppressor RNA polymerases (rpoB101) requires rho to terminate transcription of phage lambda mRNA. In contrast to the wild type RNA polymerase, the suppressor RNA polymerase also terminates lambda mRNA transcription in the presence of rho15 protein. We have isolated new rho mutants (e.g., rho112) that are defective in transcription termination in the rpoB101 strain. These results strongly support the notion that rho and RNA polymerase interact functionally during transcription termination. We have shown that rho15 catalyzes ATP hydrolysis during transcription with rpoB101 RNA polymerase, but not with wild-type RNA polymerase. Because rho 15 protein hydrolyzes ATP in the presence of free RNA, we suggest that rho may recognize the 3'-OH end of RNA. During transcription, this recognition involves an interaction with RNA polymerase, resulting in the displacement of the polymerase and the release of the nascent mRNA.

A cDNA clone for human interleukin 2 (IL-2) has been fused to the 5' end of a modified Pseudomonas exotoxin (PE) gene that lacks the sequences encoding the cell recognition domain. The chimeric protein IL-2-PE40 was produced in Escherichia coli. It was extremely toxic to IL-2 receptor-positive cells but had no measurable effect on cells lacking the IL-2 receptor. IL-2-PE40 might be a useful cytotoxic agent in the treatment of diseases involving IL-2 receptor-positive cells and in the treatment of allograft rejection.