Preliminary experiments established that a 0.5-ml inoculum that is introduced directly into the stomach of mice was cleared rapidly into the small intestine. Bicarbonate buffer, but not skim milk, protected such an inoculum from stomach acid until at least 90% of it had entered the small intestine. Passage and survival of various Escherichia coli strains through the mouse gut were tested by introducing a buffered bacterial inoculum directly into the stomach, together with the following two intestinal tracers: Cr 51 Cl 3 and spores of a thermophilic Bacillus sp. Quantitative recovery of excreted bacteria was accomplished by collecting the feces overnight in a refrigerated cage pan. The data show that wild-type E. coli strains and E. coli K-12 are excreted rapidly (98 to 100% within 18 h) in the feces without overall multiplication or death. E. coli ϰ1776 and DP50supF, i.e., strains certified for recombinant DNA experiments underwent rapid death in vivo, such that their excretion in the feces was reduced to approximately 1.1 and 4.7% of the inoculum, respectively. The acidity of the stomach had little bactericidal effect on the E. coli K-12 strain tested, but significantly reduced the survival of more acidsensitive bacteria ( Vibrio cholerae ) under these conditions. Long-term implantation of E. coli strains into continuous-flow cultures of mouse cecal flora or into conventional mice was difficult to accomplish. In contrast, when the E. coli strain was first inoculated into sterile continuous-flow cultures or into germfree mice, which were subsequently associated with conventional mouse cecal flora, the E. coli strains persisted in a large proportion of the animals at levels resembling E. coli populations in conventional mice. Metabolic adaptation contributed only partially to the success of an E. coli inoculum that was introduced first. A mathematical model is described which explains this phenomenon on the basis of competition for adhesion sites in which an advantage accrues to the bacterium which occupies those sites first. The mathematical model predicts that two or more bacterial strains that compete in the gut for the same limiting nutrient can coexist, if the metabolically less efficient strains have specific adhesion sites available. The specific rate constant of E. coli growth in monoassociated gnotobiotic mice was 2.0 h −1 , whereas the excretion rate in conventional animals was −0.23 h −1 . Consequently, limitation of growth must be regarded as the primary mechanism controlling bacterial populations in the large intestine. The beginnings of a general hypothesis of the ecology of the large intestine are proposed, in which the effects of the competitive metabolic interactions described earlier are modified by the effects of bacterial association with the intestinal wall.

A previous study had established that anaerobic continuous-flow (CF) cultures of conventional mouse cecal flora were able to maintain the in vivo ecological balance among the indigenous bacterial species tested. This paper describes experiments designed to determine the mechanisms which control the population sizes of these species in such CF cultures. One strain each of Escherichia coli, Fusobacterium sp., and Eubacterium sp. were studied. Growth of these strains in filtrates of CF cultures was considerably more rapid than in the CF cultures themselves, indicating that the inhibitory activity had been lost in the process of filtration. Growth rates to match those in CF cultures could be obtained, however, by restoring the original levels of H2S in the culture filtrates. The inhibitory effect of H2S in filtrates and in dialysates of CF cultures could be abolished by adding glucose or pyruvate, but not formate or lactate. The fatty acids present in CF cultures matched those in the cecum of conventional mice in both quality and concentration. These acids could not account for the slow rates of growth of the tested strains in CF cultures, but they did cause a marked increase in the initial lag phase of E. coli growth. The results obtained are compatible with the hypothesis that the populations of most indigenous intestinal bacteria are controlled by one or a few nutritional substrates which a given strain can utilize most efficiently in the presence of H2S and at the prevailing conditions of pH and anaerobiosis. This hypothesis consequently implies that the populations of enterobacteria, such as the E. coli strain tested, and those of the predominant anaerobes are controlled by analogous mechanisms.

p53-mediated increase in cyclin-dependent kinase inhibitor p21(WAF1) protein is thought to be the major mediator of cell cycle arrest after DNA damage. Previously p21 protein levels have been reported to increase or to decrease after UV irradiation. We show that p21 protein is degraded after irradiation of a variety of cell types with low but not high doses of UV. Cell cycle arrest occurs despite p21 degradation via Tyr(15) inhibitory phosphorylation of cdk2 and differs from the classical p21-dependent checkpoint elicited by ionizing radiation. In contrast to the basal turnover of p21, degradation of p21 switches to ubiquitin/Skp2-dependent proteasome pathway following UV irradiation. ATR activation after UV irradiation is essential for signaling p21 degradation. Finally, UV-induced p21 degradation is essential for optimal DNA repair. These results provide novel insight into regulation of p21 protein and its role in the cellular response to DNA damage.

p21(WAF1/CIP1), a cyclin-dependent kinase inhibitor and a critical regulator of cell cycle, is controlled transcriptionally by p53-dependent and -independent mechanisms and posttranslationally by the proteasome. We have identified WISp39, a tetratricopeptide repeat (TPR) protein that binds p21. WISp39 stabilizes newly synthesized p21 protein by preventing its proteasomal degradation. WISp39, p21, and hsp90 form a trimeric complex in vivo. The interaction of WISp39 with Hsp90 is abolished by point mutations within the C-terminal TPR domain of WISp39. Although this WISp39 TPR mutant binds p21 in vivo, it fails to stabilize p21. Our results suggest that WISp39 recruits Hsp90 to regulate p21 protein stability. WISp39 downregulation by siRNA prevents the accumulation of p21 and cell cycle arrest after ionizing radiation. The results demonstrate the importance of posttranslational stabilization of p21 protein by WISp39 in regulating cellular p21 activity.

Replication factor C (RF‐C), a complex of five polypeptides, is essential for cell‐free SV40 origin‐dependent DNA replication and viability in yeast. The cDNA encoding the large subunit of human RF‐C (RF‐Cp145) was cloned in a Southwestern screen. Using deletion mutants of RF‐Cp145 we have mapped the DNA binding domain of RF‐Cp145 to amino acid residues 369–480. This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP‐ribose) polymerases and is absent in other subunits of RF‐C. The PCNA binding domain maps to amino acid residues 481–728 and is conserved in all five subunits of RF‐C. The PCNA binding domain of RF‐Cp145 inhibits several functions of RF‐C, such as: (i) in vitro DNA replication of SV40 origin‐containing DNA; (ii) RF‐C‐dependent loading of PCNA onto DNA; and (iii) RF‐C‐dependent DNA elongation. The PCNA binding domain of RF‐Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells. In contrast, the DNA binding domain of RF‐Cp145 does not inhibit DNA synthesis in vitro or in vivo. We therefore conclude that amino acid residues 481–728 of human RF‐Cp145 are critical and act as a dominant negative mutant of RF‐C function in DNA replication in vivo.