Isolation and Properties of rex? Mutants of Bacteriophage Lambda

Twenty-five rex(-) mutants of phage lambda have been isolated. Three of the mutants, including one amber mutant, map at three distinct sites within the rex region of the lambda genetic map. The existence of the amber mutant provides further evidence that rex and cI are distinct genes, since it seems to be identical to wild-type lambda in its ability to establish or maintain lysogeny.     

Protein Synthesis Directed by an RNA? Temperature-Sensitive Mutant of Sindbis Virus

The structural proteins of wild-type Sindbis virus were shown to arise by posttranslational cleavage of larger precursors. The proteins synthesized in wildtype infection were compared with those specified by ts-11, a temperature-sensitive mutant unable to synthesize viral RNA at the restrictive temperature. Abnormally large, virus-specific proteins were found in the mutant-infected cells after the shift from 28 C to 41.5 C. These large polypeptides were presumably precursors which were cleaved too rapidly to be detected in the wild-type infection. The largest had a molecular weight of 133,000 and was the same size as the apparent precursor detected during infection with a group of Sindbis mutants which could not form nucleocapsids at the nonpermissive temperature. The stability of ts-11-specific RNA synthesis, after shift from permissive to restrictive conditions, differed from that in cells infected by wild-type virus, indicating that the virus had a genetic lesion in an enzyme involved in RNA synthesis. This mutation might have caused the precursor to fold incorrectly so that it could not be cleaved. The possibility cannot be excluded, however, that a second lesion in an uncharacterized viral function, such as a protease, was the cause of the accumulation of the precursors.     

λ-Carrageenan P32 Is a Potent Inhibitor of Rabies Virus Infection

Rabies, caused by rabies virus (RABV), is an acute, fatal encephalitic disease that affects many warm-blooded mammals. Currently, post-exposure prophylaxis regimens are effective for most rabies cases, but once the clinical signs of the disease appear, current treatment options become ineffective. Carrageenan has been reported as a potent inhibitor of many viruses. In this study, the λ-carrageenan (λ-CG) P32 was investigated for its potential role in inhibiting RABV infection. Our results show that P32 specifically inhibits the replication of several RABV strains but not vesicular stomatitis virus in multiple cell lines and shows low cytotoxicity. P32 mainly abrogated viral replication during the early stage of the post-adsorption period. Further studies demonstrated that P32 could affect not only viral internalization but also viral uncoating by blocking cell fusion mediated by RABV glycoprotein. Moreover, P32 can fully inhibit RABV infection in vitro during the post-adsorption period, whereas heparin and heparan sulfate, which possess similar structures to P32, showed significant but not complete inhibition of RABV infectivity. Collectively, our results indicate that λ-CG P32 is a promising agent that can inhibit RABV infection mainly by inhibiting viral internalization and glycoprotein-mediated cell fusion and can be used for the development of novel anti-RABV drugs.     

Temperature-Sensitive DNA Synthesis Mutants of BACILLUS SUBTILIS—APPENDIX: Theory of Density Transfer for Symmetric Chromosome Replication

A simple method for characterizing temperature-sensitive DNA synthesis mutants is described. The method uses density transfer and transformation techniques and is based on expected theoretical behavior of the chromosome population. A direct proof of inhibition of initiation of DNA replication is provided. The mutant dna-1, showing quick inhibition of initiation, is further characterized and mapped. An independent method for mapping genetic markers close to the origin, based on their transfer behavior after inhibition of initiation, is presented.     

Immunogenicity of Rabies Virus Inactivated by β-Propiolactone, Acetylethyleneimine, and Ionizing Irradiation

Ionizing radiation, beta-propiolactone, and acetylethyleneimine were compared for their ability as virus-inactivating agents for the preparation of rabies vaccine. Each agent reduced viral infectivity exponentially; ionizing radiation also destroyed viral hemagglutinin. The vaccine prepared by ionizing radiation was equal or superior to that prepared by beta-propiolactone in its ability to protect mice from rabies infection. The acetylethyleneimine-treated vaccine was a less potent immunogen.     

The Genetics of Dopa Decarboxylase in DROSOPHILA MELANOGASTER . II. Isolation and Characterization of Dopa-Decarboxylase-Deficient Mutants and Their Relationship to the α-Methyl-Dopa-Hypersensitive Mutants

Of 84 lethals isolated over the dopa decarboxylase (DDC) deficiency Df(2L)50, 8 have been identified as DDC-deficient alleles on the basis of their effect on DDC activity when heterozygous over the CyO balancer chromosome with activities ranging from 28% to 53% of controls. Some of the Ddc-deficient alleles exhibit intracistronic complementation. Most of the complementing pairs of alleles are much reduced in viability, e.g. < 5% of expected, and express a common syndrome of mutant phenes which can reasonably be inferred to derive from inadequately sclerotinized cuticle. Individuals heterozygous for the noncomplementing allele, Ddcⁿ⁷, over the 12-band DDC deficiency, Df(2L)130, die at the end of embryogenesis as unhatched larvae with unpigmented mouth parts.

The Ddc alleles and the l(2)amd α-methyl dopa (αMD) hypersensitive alleles are both located within the 11 band region 37B10-C7. The l(2)amd locus is immediately to the right of hk(2–53.9).Ddc has been mapped within 0.004 Map Units to the right of l(2)amd with a maximum estimated recombination frequency of 0.01%. None of the Ddc/CyO strains are sensitive to the dietary administration of α-methyl dopa (αMD), and complementation occurs between the Ddc deficient alleles and the l(2)amd alleles both on the basis of viability and DDC activity. No effect on DDC by the amd alleles has been found to date. Even in the complementing heterozygote, amd^H1/amd ^H89, the level of activity, thermostability, and in vitro αMD inhibition of DDC remains unaffected. Although no biochemical phene has yet been established for the αMD hypersensitive amd alleles, it seems likely that the two groups of mutants are functionally related.

     

Temperature-Sensitive Simian Virus 40-Transformed Cells: Phenomena Accompanying Transition from the Transformed to the “Normal” State

Temperature-sensitive simian virus (SV 40)-transformed 3T3 cells (tsSV3T3), which express the transformed phenotype when growing at 32 C but not at 39 C, were used to study changes in growth behavior during shift-up or shift-down experiments. In cultures of tsSV3T3 cells which had reached or were beyond monolayer density at 32 C, DNA synthesis reached very low levels within 24 to 48 h after shift-up. When cells which had been allowed to grow to high densities at 32 C were shifted to 39 C, not only cell growth stopped, but within two to three days the cultures shed a large number of cells into the medium. These cells were nonviable, and shedding stopped only when the number of cells attached had been reduced to that characteristic of the saturation density at 39 C. The remaining attached cells were viable and after the shift to 32 C were again able to grow from the monolayer to high cell densities. This behavior has been compared with that of normal 3T3 and wild-type SV3T3 cells under different conditions. We have also isolated new tsSV3T3 lines, using cells which had been infected with non-mutagenized wild-type SV40. This further demonstrates that the temperature sensitivity of these lines is due to a cellular rather than a viral mutation.     

Isolation of protoplasts by means of a “species-specific” autolysine inChlamydomonas

Summary Prior to fusion, gametes ofChlamydomonas reinhardii discard their cell walls. This naturally occuring phenomenon has provided the basis for a method of protoplast isolation from both gametes and vegetative cells within the genusChlamydomonas. When synchronized cultures of compatibleChlamydomonas gametes are mixed it is possible, after removal of the cells, to obtain a solution having a high cell wall lytic activity. That vegetative and gamete cells after treatment with this gamete-autolysine are indeed protoplasts has been proved by various light and electron microscopical methods.The species specifity of this autolysine, its difference to the previously described sporangialautolysine (Schlösser 1966) and furthermore its use in the large scale production of protoplasts is also described. Since this wall autolysine is a factor produced by the cells themselves at a particular stage in their life cycle it represents a non-foreign agent in contrast to all other enzymic methods previously employed for protoplast isolation.     

Rabies and Canine Distemper Virus Epidemics in the Red Fox Population of Northern Italy (2006–2010)

Since 2006 the red fox (Vulpes vulpes) population in north-eastern Italy has experienced an epidemic of canine distemper virus (CDV). Additionally, in 2008, after a thirteen-year absence from Italy, fox rabies was re-introduced in the Udine province at the national border with Slovenia. Disease intervention strategies are being developed and implemented to control rabies in this area and minimise risk to human health. Here we present empirical data and the epidemiological picture relating to these epidemics in the period 2006–2010. Of important significance for epidemiological studies of wild animals, basic mathematical models are developed to exploit information collected from the surveillance program on dead and/or living animals in order to assess the incidence of infection. These models are also used to estimate the rate of transmission of both diseases and the rate of vaccination, while correcting for a bias in early collection of CDV samples. We found that the rate of rabies transmission was roughly twice that of CDV, with an estimated effective contact between infected and susceptible fox leading to a new infection occurring once every 3 days for rabies, and once a week for CDV. We also inferred that during the early stage of the CDV epidemic, a bias in the monitoring protocol resulted in a positive sample being almost 10 times more likely to be collected than a negative sample. We estimated the rate of intake of oral vaccine at 0.006 per day, allowing us to estimate that roughly 68% of the foxes would be immunised. This was confirmed by field observations. Finally we discuss the implications for the eco-epidemiological dynamics of both epidemics in relation to control measures.     

Isolation of large amounts of highly pure mitochondria for “omics” studies

Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.     

Spontaneous and Engineered Deletions in the 3′ Noncoding Region of Tick-Borne Encephalitis Virus: Construction of Highly Attenuated Mutants of a Flavivirus

The flavivirus genome is a positive-strand RNA molecule containing a single long open reading frame flanked by noncoding regions (NCR) that mediate crucial processes of the viral life cycle. The 3′ NCR of tick-borne encephalitis (TBE) virus can be divided into a variable region that is highly heterogeneous in length among strains of TBE virus and in certain cases includes an internal poly(A) tract and a 3′-terminal conserved core element that is believed to fold as a whole into a well-defined secondary structure. We have now investigated the genetic stability of the TBE virus 3′ NCR and its influence on viral growth properties and virulence. We observed spontaneous deletions in the variable region during growth of TBE virus in cell culture and in mice. These deletions varied in size and location but always included the internal poly(A) element of the TBE virus 3′ NCR and never extended into the conserved 3′-terminal core element. Subsequently, we constructed specific deletion mutants by using infectious cDNA clones with the entire variable region and increasing segments of the core element removed. A virus mutant lacking the entire variable region was indistinguishable from wild-type virus with respect to cell culture growth properties and virulence in the mouse model. In contrast, even small extensions of the deletion into the core element led to significant biological effects. Deletions extending to nucleotides 10826, 10847, and 10870 caused distinct attenuation in mice without measurable reduction of cell culture growth properties, which, however, were significantly restricted when the deletion was extended to nucleotide 10919. An even larger deletion (to nucleotide 10994) abolished viral viability. In spite of their high degree of attenuation, these mutants efficiently induced protective immune responses even at low inoculation doses. Thus, 3′-NCR deletions represent a useful technique for achieving stable attenuation of flaviviruses that can be included in the rational design of novel flavivirus live vaccines.The genus Flavivirus (family Flaviviridae) consists of small, enveloped, mainly mosquito- or tick-transmitted viruses with an unsegmented positive-stranded RNA genome (34). Some of these viruses are human pathogens of global medical importance, most notably yellow fever virus, the dengue (DEN) viruses, Japanese encephalitis virus, and tick-borne encephalitis (TBE) virus (22). In spite of the availability of vaccines against several of these viruses, flavivirus infections continue to be a major health problem in many countries of the world. Elucidation of the molecular basis of the pathogenicity of these viruses and identification of specific determinants of virulence are therefore a major focus of flavivirus research.The approximately 11-kb flavivirus genome (for a review, see reference 3) encodes three structural proteins (the capsid protein C, the small membrane protein M, which is formed by proteolytic cleavage from its precursor protein prM, and the large envelope protein E) and seven nonstructural proteins (the glycoprotein NS1, the protease component NS2A, NS2B, the protease/helicase NS3, NS4A, NS4B, and the RNA polymerase NS5). All of the viral proteins are encoded within a single long open reading frame which is flanked by noncoding regions (NCR) believed to carry regulatory elements involved in replication, translation, and packaging of the genome. Molecular analyses of natural low-virulence strains and strains attenuated in vitro by passaging procedures or, more recently, by specific mutagenesis techniques, have shown that genetic determinants that govern the virulence of flaviviruses are located within the coding regions of both structural and nonstructural proteins as well as within the flanking NCRs (2, 21, 26; for reviews, see references 20 and 22). In this study, we focus on the effects of deletions in the 3′ noncoding region (3′ NCR) of TBE virus.TBE virus causes widespread human disease in many European and Asian countries, and its molecular biology has been studied in some detail (29; for a review, see reference 9). The length of the 3′ NCR of TBE virus was previously found to be remarkably heterogeneous even among closely related strains, ranging from approximately 450 to almost 800 nucleotides (31). A more detailed analysis indicated that the 3′ NCR can be divided into a 3′-terminal core element of approximately 340 nucleotides in length and a variable region located between the core element and the open reading frame. The core element is present in all strains investigated so far, and its nucleotide sequence is highly conserved among strains. The entire core element is predicted to fold into a well-defined secondary structure independent of the sequence of the adjacent variable genomic element (27). The variable region is characterized by low sequence conservation, extensive size variability between strains, repetitive sequence elements, and an internal poly(A) tract in certain TBE virus strains (15, 31). Evidence for 3′-NCR size heterogeneity and specific RNA-folding patterns for the 3′-terminal approximately 400 nucleotides have also been observed with several other flaviviruses (5, 24, 25, 33). A similar organization of the 3′ NCRs also appears to be shared by members of the other two genera of the family Flaviviridae, pestiviruses and hepaciviruses (13, 23, 30, 35).Although the functional importance of the flavivirus 3′ NCR is generally acknowledged, the assumed involvement of particular sequence elements in replication, modulation of translation, or packaging is largely hypothetical. Evidence for functionality is so far based mostly on the identification of highly conserved RNA sequence elements or folding patterns by computer techniques. A few studies have provided direct evidence for the binding of protein factors to the stem-loop structure closest to the 3′ terminus (1, 4). Moreover, Men et al. (21) demonstrated that certain deletions introduced into the 3′ NCR of DEN-4 virus resulted in viable mutants with significantly restricted growth properties. By this approach, these researchers were able to identify particular sequences that are required for viability and others that can be deleted without apparent impact on the biology of DEN-4 virus. Studying replicons of Kunjin virus, Khromykh and Westaway (14) found that parts of the 3′ NCR could be deleted or even replaced by a foreign protein expression cassette without loss of replication competence. The 3′-NCR sequences of these flaviviruses, however, exhibit very little homology to the sequences of the tick-borne flaviviruses, which even lack the sequence boxes CS1 and CS2 that are conserved among all mosquito-borne flaviviruses (7, 16).The establishment of an efficient and stable infectious cDNA clone system for TBE virus European subtype prototypic strain Neudoerfl (17) has enabled us to test the functional importance of 3′-NCR sequence elements of this virus by a directed mutagenesis approach. As reported in this communication, spontaneous deletions in the variable region of strain Neudoerfl occur frequently during viral growth in cell culture or in infected animals. This prompted us to construct 3′-NCR deletion mutants of variable lengths to study the influence of these deletions on the biological properties of TBE virus. Our results demonstrate a correlation between the presence of certain RNA sequences or secondary structures and growth properties, viability, and attenuation of the resulting virus mutants. We present several 3′-NCR deletion mutants that are 4 orders of magnitude less virulent than wild-type TBE virus.With regard to vaccine development, the most desirable mutations are ones that are genetically stable and cause significant attenuation but maintain adequate replication properties in cell culture and strong immunogenicity in animals even at low inoculation doses. The evaluation of the TBE virus mutants presented in this article indicates that certain deletions in the 3′ NCR can indeed meet these criteria.     

Production of Pectolytic Enzymes by Rhizopus stolonifer Sporangiospores After “Lethal” Gamma Irradiation

Irradiated sporangiospores of Rhizopus stolonifer excreted pectolytic enzymes, which hydrolyzed pectin and macerated potato tuber discs, into the suspending medium. Pectin glycosidase, but not pectin methylesterase, activity developed regardless of the amount of radiation the spores had received, unless the dose exceeded about 1 megarad. The ability to produce pectolytic enzymes was found to be more radiation-resistant than the potential for colony formation or the ability to germinate. Spores made incapable, through irradiation, of forming colonies continued to produce pectolytic enzymes after a 6-day period following irradiation treatment.     

Transformation of Murine Cells by Two “Slow Viruses,” Visna Virus and Progressive Pneumonia Virus

Visna and progressive pneumonia virus (PPV), two antigenically related, non-oncogenic "slow viruses" which have ribonucleic acid (RNA)-dependent deoxyribonucleic acid (DNA) polymerase activity, were examined for their ability to transform cells. Murine cells which had been exposed to either visna or PPV developed foci of altered, spindle-shaped cells 3 to 4 weeks after infection. Visna and PPV transformed lines were established from these cultures. There was no evidence that other oncogenic DNA or RNA viruses were involved in the observed transformation. Visna or PPV could be "rescued" from all transformed lines by co-cultivation with normal sheep testis cells. "Rescued" virus was identified as visna or PPV, and they retained the capacity to transform mouse cells. These experiments may have important implications in the understanding of both viral carcinogenesis and "slow" viral infections.     

Analysis of Minimal Functions of Simian Virus 40 III. Evidence for “Host Cell Repair” of Oncogenicity and Infectivity of UV-Irradiated Simian Virus 40

The in vitro transforming capacity of simian virus 40 (SV40) for Syrian hamster cells is highly resistant to inactivation by UV light in comparison to infectivity. In the same cell system, we demonstrated a "host cell repair mechanism" sensitive to caffeine which is, to a large extent, responsible for the high resistance to UV inactivation of the transforming capacity of SV40. The survival of infectivity of UV-irradiated SV40 in CV-1 cells was also sensitive to caffeine, again indicating host cell repair. On the other hand, depression of normal cell DNA synthesis by hydroxyurea during the first 24 h postinfection only modestly reduced, and to a similar extent, the transforming capacity of UV-irradiated and nonirradiated SV40.     

Immunization of Knock-Out α/β Interferon Receptor Mice against High Lethal Dose of Crimean-Congo Hemorrhagic Fever Virus with a Cell Culture Based Vaccine

Crimean-Congo hemorrhagic fever (CCHF) is an acute tick-borne zoonotic disease. The disease has been reported in many countries of Africa, Asia, the Middle East, and in Eurasia. During the past decade, new foci of CCHF have emerged in the Balkan Peninsula, southwest Russia, the Middle East, western China, India, Africa, and Turkey. CCHF virus produces severe hemorrhagic manifestations in humans with fatality rates up to 30%. Vaccine development efforts have been significantly hampered by a lack of animal models and therefore, no protective vaccine has been achieved. Lately, IFN α/β receptor deficient (IFNAR^−/−) mice have been established as a novel small animal model of CCHF virus infection. In the present study, we found that IFNAR^−/− mice highly susceptible to CCHF virus Turkey-Kelkit06 strain. Immunization with the cell culture based vaccine elicited a significant level of protection against high dose challenge (1,000 PPFU) with a homologous CCHF virus in IFNAR^−/− mice.     

Roles of ExoI and SbcCD Nucleases in “Reckless” DNA Degradation in recA Mutants of Escherichia coli

Exponentially growing recA mutant cells of Escherichia coli display pronounced DNA degradation that starts at the sites of DNA damage and depends on RecBCD nuclease (ExoV) activity. As a consequence of this “reckless” DNA degradation, populations of recA mutants contain a large proportion of anucleate cells. We have found that both DNA degradation and anucleate-cell production are efficiently suppressed by mutations in the xonA (sbcB) and sbcD genes. The suppressive effects of these mutations were observed in normally grown, as well as in UV-irradiated, recA cells. The products of the xonA and sbcD genes are known to code for the ExoI and SbcCD nucleases, respectively. Since both xonA and sbcD mutations are required for strong suppression of DNA degradation while individual mutations have only a weak suppressive effect, we infer that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells. We suggest that their roles might be in processing (blunting) DNA ends, thereby producing suitable substrates for RecBCD binding.The RecA protein plays a central role in homologous recombination and recombinational DNA repair in Escherichia coli, as well as in other bacterial species. It catalyzes the key stages of the recombination process—homologous pairing and DNA strand exchange. Cells carrying null mutations in the recA gene are completely deficient for homologous recombination and are extremely sensitive to DNA-damaging agents (for a review, see references 21, 24, and 25). Populations of recA null mutants contain a large proportion (50 to 60%) of nonviable cells, reflecting the inability of these mutants to repair spontaneously occurring DNA damage (31). Also, exponentially growing recA cells display pronounced spontaneous DNA degradation that presumably starts at the sites of DNA damage and that depends on RecBCD nuclease (ExoV) activity (5, 48). This phenotype of recA cells is aggravated after DNA-damaging treatment, such as UV irradiation (48).According to the present data, the majority of RecA-catalyzed DNA transactions in E. coli start with binding of the RecA protein onto single-stranded DNA (ssDNA) substrates. This binding is mediated by the RecBCD and/or RecFOR protein, which helps RecA to overcome hindrance imposed by the SSB protein during competition for the DNA substrate. The RecBCD and RecFOR proteins begin RecA polymerization on ssDNA, giving rise to a nucleoprotein filament that is indispensable for further recombination reactions (3, 33; reviewed in reference 44).The RecBCD enzyme is crucial for initiation of recombinational processes at double-stranded DNA (dsDNA) ends (or breaks [DSBs]) in wild-type E. coli (a set of reactions known as the RecBCD pathway) (9, 43, 44). Upon recognizing a blunt or nearly blunt dsDNA end and binding to it, RecBCD acts as a combination of powerful helicase and nuclease, thus unwinding and simultaneously degrading both strands of the DNA duplex. After encountering a specific octanucleotide sequence designated Chi, the strong 3′-5′ nuclease activity of the enzyme is attenuated and a weaker 5′-3′ nuclease activity is upregulated (1). This Chi-dependent modification allows RecBCD to create a long 3′ ssDNA tail and to direct the loading of RecA protein onto it (2, 3). In vivo data suggest that this transition of RecBCD from a nuclease to a recombinase mode of action requires the presence of the RecA protein, suggesting that the two proteins might interact (27).In wild-type E. coli cells, the RecFOR protein complex works predominantly on DNA gaps, which may arise in chromosomes due to replication forks passing over the noncoding lesions (e.g., UV-induced pyrimidine dimers) or may be present in replication forks stalled at different obstacles in DNA (44). On the other hand, the RecFOR complex has an important role in recBC sbcBC(D) mutant cells, replacing the RecA-loading activity of RecBCD during recombination reactions starting from dsDNA ends. Recombination reactions mediated by RecFOR proteins are termed the RecF (or RecFOR) pathway (44).Cells mutated in the recB and/or recC gene exhibit strong deficiency in conjugational and transductional recombination, as well as in the repair of DSBs (8, 21). These defects can be rectified by extragenic sbcB and sbcC(D) suppressor mutations that inactivate two nucleases, thus enabling full efficiency of the RecF pathway on dsDNA ends (21, 44). The sbcB gene (also designated xonA) encodes exonuclease I (ExoI), the enzyme that digests ssDNA in the 3′-5′ direction (23). The sbcC and sbcD genes encode subunits of the SbcCD nuclease, which acts both as an endonuclease that cleaves hairpin structures and as an exonuclease that degrades linear dsDNA molecules (10, 11). Inactivation of either of the two subunits leads to the loss of SbcCD enzyme activity (18).The exact mechanism of activation of the RecF pathway by sbc mutations is not completely understood. A plausible explanation is that inactivation of ExoI and SbcCD nucleases is necessary to prevent the degradation of recombinogenic 3′ DNA ends created in a RecBCD-independent manner (8, 23, 38, 45, 46). It was recently shown that the sbcB15 mutant allele (encoding a protein without nucleolytic activity) (37) is a better suppressor of the RecBCD⁻ phenotype than an sbcB deletion (50), suggesting that some nonnucleolytic activity of ExoI may also contribute to the efficiency of the RecF pathway (46, 50).ExoI and SbcCD are usually viewed as enzymes with inhibitory roles in recombination due to their deleterious actions on the RecF pathway. However, some results suggest that these enzymes could also have stimulatory roles in recombination reactions proceeding on the RecBCD pathway. Genetic experiments with UV-irradiated E. coli cells indicated that ExoI and SbcCD might be involved in blunting radiation-induced DNA ends prior to RecBC(D) action (38, 45, 46). Such a role of ExoI and SbcCD seems to be particularly critical in recD recF mutants, in which the majority of DSB repair depends on the RecBC enzyme (38). It was also suggested that the blunting roles of the two nucleases may be required during conjugational recombination (16, 46).In this work, we studied the effects of sbcB (xonA) and sbcD mutations on DNA degradation occurring spontaneously in exponentially growing recA mutant cells, as well as on DNA degradation induced in recA mutants by UV irradiation. We have demonstrated that in both cases DNA degradation is strongly reduced in recA mutants that carry in addition a combination of xonA and sbcD null mutations. The results described in this paper suggest that ExoI and SbcCD play partially redundant roles in regulating DNA degradation in recA cells.     

Simian Virus 40 Mutants with Deletions at the 3′ End of the Early Region Are Defective in Adenovirus Helper Function

Coinfection of monkey cells with simian virus 40 (SV40) and adenovirus type 2 (Ad2) increased the Ad2 yield 1,000-fold over that obtained by Ad2 infection alone of monkey cells (A. S. Rabson, G. T. O'Conor, I. K. Berezesky, and F. J. Paul, Proc. Soc. Exp. Biol. Med. 116:187-190, 1964). The ability of viable mutants of SV40 that contain deletions at various sites in the viral DNA to enhance Ad2 growth in monkey cells was examined. Only those mutants with deletions near the 3' end of the early region were deficient in providing this helper function. Mutants dl1265, lacking 39 base pairs at map position 0.18, and dl1263, lacking 33 base pairs at map position 0.20 (H. van Heuverswyn, C. Cole, P. Berg, and W. Fiers, J. Virol. 30:936-941, 1979), were approximately 4 and 30% as effective as wild-type SV40, respectively. The extent of enhancement of Ad2 yield depended on the multiplicity of infection by SV40, but not by Ad2 (at a multiplicity of infection of 相似文献