As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we Consequently, we set out to examine additional genes that might be involved. The identification of pcnB, the structural gene for poly(A) polymerase I (PAP I) (5), has allowed us to determine whether polyadenylylation affects mRNA decay in E. coli the same way it does in eukaryotes (6). For many eukaryotic mRNAs, the degradation of poly(A) tails initiates mRNA decay (7-9). If it does so in E. coli, then, presumably, mRNA stability would increase in the absence of PAP I.To test whether the degradation of poly(A) tails initiates mRNA decay in E. coli, a series of isogenic strains containing mutations in PAP I (10), RNase E(mne) (1), PNPase (pnp) (1), and RNase II (mb) (1) were constructed. Northern analysis of the trxA, ompA, and lpp mRNAs showed that when polyadenylylation was almost completely eliminated, their half-lives always increased significantly and their decay patterns changed. We also determined the number and size of poly(A) tails in the total bacterial RNA population. In wild-type E. coli, poly(A) tails ranged from 10 to >50 nt. Where there was no PNPase and there was reduced RNase II activity, both the number and the size of the tails increased significantly. More than 90% of the poly(A) tails were absent in ApcnB mutants. We discuss the implications of these results below.MATERIALS AND METHODS Bacterial Strains. All strains were derived from E. coli MG1693 (thyA715), provided by B. Bachmann (E. coli Genetic Stock Center, Yale University). SK5704 (pnp-7 mb-500 me-i thyA 715) (1) was constructed by phage P1-mediated transduction (11). SK7988 (ApcnB thyA715) and SK8901 (lApcnBpnp-7 mb-500 me-I thyA 715) were derived from MG1693 and SK5704, respectively. A kanamycin-resistance (KmR) determinant in the ApcnB gene (10) was used as a selectable transduction marker. SK7988 and SK8901 each grew more slowly than their pcnB+ parents. The absence of poly(A) polymerase in SK8901 did not affect the conditional lethality associated with inactivating RNase II, RNase E, and PNPase.RNA Isolation. Seven-milliliter samples were mixed with an equal volume of crushed frozen TM buffer (10 mM Tris, pH 7.2/5 mM MgCl2) containing 20 mM NaN3 and chloramphenicol at 0.4 mg/ml. The cells were then centrifuged and the pellet was suspended in 0.34 ml of TM buffer with lysozyme at 0.3 mg/ml and DNase I at 32 units/ml. After three freezethaw cycles, one-sixth volume of 20 mM acetic acid was added, followed by an equal volume of Catrimox-14 (Iowa Biotechnology, Oakdale, IA).The resulting precipitate of protein, DNA, and RNA was spun in a microcentrifuge at medium speed to form a soft pellet. That pellet was completely suspended in 1 ml of 2 M LiCl in 35% ethanol. In this solution protein and DNA dissolve but RNA does not. The resulting RNA precipitate was centrifuged at high speed for 5 min to form a pellet. It was then suspended in 2 M LiCl in distilled water and centrifuged again. The pellet was washed with 70% ethanol and resuspended in distilled w...

Mutational activation of cellular proto-oncogenes is an important event in the pathogenesis of chemically induced tumors. We have used the ori P-tk shuttle vector, pHET, to analyze the types of DNA sequence changes induced after treating mammalian cells with the carcinogen N-ethyl-N-nitrosourea (ENU). This shuttle vector contains the putative replication origin of the Epstein-Barr virus (EBV) and is stably maintained as a plasmid in EBV-transformed human lymphoblastoid cells. Populations of plasmid-bearing cells were treated with ENU, and plasmid DNA was isolated approximately 7-8 population doublings after treatment for analysis of mutations induced at the herpes simplex virus type 1 thymidine kinase (HSV-tk) target gene. After ENU treatment, frequencies of four of the six possible base substitution mutations significantly increased. Transition mutations were the most common sequence change: 48% of the 46 mutants sequenced were GC----AT transitions and 17% were AT----GC transitions. In addition, the number of AT----TA (20%) and AT----CG (9%) transversion mutations significantly increased after ENU treatment. Based on the comparison of mutations induced by ENU in human cells with the types of base pair changes previously reported for other alkylating agents, we propose that the O2-ethylthymine adduct may be a significant premutagenic lesion in mammalian cells, capable of resulting in AT base pair transversion mutations. Studies from other laboratories have demonstrated the importance of AT----TA transversion mutations in the activation of cellular proto-oncogenes by ENU.

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB ؉ ). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3 end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I ؊ polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.In Escherichia coli, mRNA decay has been studied extensively in vivo with a variety of approaches. The identification of exo-and endoribonucleases and the use of mutant forms of these enzymes (1-5) has resulted in the recognition of decay intermediates in vivo (6-9) and hypotheses to explain the temporal process of decay for several messages (9-11). In addition, careful analysis of some messages has demonstrated physical characteristics of both the 5Ј-and 3Ј-untranslated regions that profoundly affect the accessibility of these mRNAs to ribonucleolytic attack (12-15). More recent evidence has indicated that factors other than ribonucleases and RNA structure may modulate message decay, particularly the role of translating ribosomes (16) and the activity of poly(A) polymerase (PAP) I (17, 18).Although our understanding of mRNA decay has progressed considerably, many questions remain regarding the overall biochemical mechanism. In other complex biological pathways, such as DNA replication and protein synthesis, in vitro studies have provided invaluable assistance for isolating and characterizing important system components. In the case of mRNA decay, however, no useful in vitro system has been described. To be of value, an in vitro reaction should accurately reflect the relative decay rates of short-and long-lived messages (19) and generate the same breakdown products observed in vivo. The use of labeled run-off mRNA transcripts in the presence of E. coli extracts has not met these criteria (unpublished results).As a first step in developing a reliable in vitro system, we present studies using mRNAs associated with polysomes as substrates for decay. This approach was adopted because in vivo decay has been shown to be affected by translation rates (16,20). In addition, we hypothesized that many of the factors necessary for mRNA decay might be associated with polyribosomes. Because of the complexity of such a system, we chose to focus initially on whether polyadenylylation affected mRNA decay rates in vitro.PAP activity was firs...

The fixation of DNA lesions induced in Escherichia coli by N-ethyl-N-nitrosourea (ENU) occurs by both SOS-dependent and SOS-independent pathways. To determine whether these pathways result in differential processing of ENU-induced lesions, we have analyzed the DNA sequence changes of mutations induced at a plasmid-encoded herpes simplex virus type 1 thymidine kinase gene by ENU treatment of plasmid-bearing RecA- and RecA+ bacteria, and by transformation of RecA-, RecA+ and SOS-induced RecA+ bacteria with ENU-modified plasmid DNA. Transition mutations were the predominant types of base substitution mutations observed for wild-type and RecA- E. coli, consistent with the SOS-independent mispairing of O6-ethylguanine and O4-ethylthymine adducts during DNA replication. Under conditions of SOS processing of ENU lesions, however, we observed the frequent induction of A:T----C:G transversion mutations. The proportion of A:T----C:G transversion mutations (42%) observed after transformation of SOS-induced bacteria with ENU modified DNA was approximately equal to that of the G:C----A:T transitions (46%). The frequencies of these mutations were increased 20- and 5-fold respectively over that observed for non-induced RecA+ cells. We suggest that ethylated DNA lesions which normally block DNA replication can be processed to yield A:T----C:G transversion mutations in SOS-induced E. coli.

We have identified a common restriction fragment length polymorphism of the alpha fibrinogen gene with the enzyme TaqI. This polymorphism is probably due to a single base change that creates or destroys a TaqI recognition site about 1000 basepairs from the 3' end of the alpha fibrinogen géne. The frequency of the rare allele in 83 unrelated healthy individuals is 0.33. We have used in situ hybridisation of the alpha fibrinogen cDNA to localise the gene on chromosome 4q29-31. We have confirmed this regional localisation by restriction fragment detection in a human X Chinese hamster somatic cell hybrid which contains a translocated human chromosome 4 with a breakpoint at 4q26. The alpha, beta, and gamma fibrinogen genes are all present on human chromosome 4q26-qter.