Relationships of the Col plasmids E2, E3, E4, E5, E6, and E7: Restriction mapping and colicin gene fusions

Thirteen ColE plasmids representing the E2-E7 types have been compared by restriction mapping. Over 80% of their restriction sites were found to be similarly positioned, indicating that these plasmids share a common structure. Three variants are ColE2-CA42 and ColE7-K317, both of which contain 1.8-kb DNA segments in place of a 2.5-kb segment common to the other plasmids, and ColE6-CT14, which has an additional 5.0-kb DNA segment compared to the other plasmids. The colicin (col), immunity (imm), and colicin release (hic) genes of these plasmids have been localized to regions corresponding to those known for ColE3-CA38 and ColE2-P9, with the imm and hic genes adjacent to the 3' end of the col gene. Active colicin is produced from hybrid col genes containing 5' and 3' ends from different E-type plasmids. The 3'-termini of the fused col genes specify the colicin type.     

Mode of Action of Colicins of Types E1, E2, E3, and K

The effect of colicins on deoxyribonucleic acid and protein synthesis, and also their effect on the ability of T4 phage to replicate in Escherichia coli K-12, were studied. Colicins of type K inhibited deoxyribonucleic acid synthesis, protein synthesis, and phage growth. Among colicins of type E, there was an absolute correlation between mode of action and subdivision into types E(1), E(2), and E(3).     

Negative regulation of the bovine papillomavirus E5, E6, and E7 oncogenes by the viral E1 and E2 genes.

Papillomaviruses induce benign squamous epithelial lesions that infrequently are associated with uncontrolled growth or malignant conversion. The virus-encoded oncogenes are clearly under negative regulation since papillomaviruses can latently infect cells and since different levels of viral oncogene expression are seen within the layers of differentiating infected epitheliomas. We used bovine papillomavirus type 1 (BPV-1) to investigate the mechanisms involved in the negative regulation of transformation. We found that the following two distinct and interacting mechanisms negatively regulate BPV-1 transformation effected by virally encoded trans-acting factors: (i) E2 repressors suppress transformation by the E6 and E7 oncogenes, and (ii) E1 and the E2 transactivator suppress transformation by the E6, E7, and E5 oncogenes. These systems interact in that the E2 repressors function to relieve the transformation suppression effected by the E1 and E2 transactivator genes. A BPV-1 mutant that lacked E2 repressors and E1 had greatly augmented transformation capacity. Analysis of this mutant revealed that the enhanced transformation was due to expression of the E6 and E7 genes in the absence of E5, revealing a previously unappreciated potency and synergy for the BPV-1 E6 and E7 oncogenes.     

Incompatibility Exhibited by Colicin Plasmids E1, E2, and E3 in Escherichia coli

Escherichia coli strains were made multiply colicinogenic for the colicin plasmids E1, E2, or E3 (Col E1, Col E2, or Col E3, respectively) by both a deoxyribonucleic acid transformation system and bacterial conjugation. The multiply colicinogenic bacteria constructed exhibited an immunity to the colicins produced by all the plasmids they carried and also produced colicins corresponding to all the plasmids they carried. An incompatibility was observed among the plasmids. In doubly colicinogenic cells where the presence of two plasmids was established, Col E2 was lost more frequently than Col E3. In triply colicinogenic cells, Col E1, Col E2, and Col E3 were lost, with Col E3 being lost least frequently. A significant reduction in the acquisition of a conjugationally transferred Col E1 plasmid by cells colicinogenic for Col E1 was demonstrated.     

Synthesis of (S)-13-Hydroxy (2E,4E,8E)- and (2E,4E,8Z)-Tetradecatrienoic Acids

The syntheses of (S)-13-hydroxy-(2E,4E,8E)-tetradecatrienoic acid (1) and (2E,4E,8Z)-tetradecatrienoic acid (2) were carried out by using the Wittig reaction as the key step. The asymmetric center at C-13 and the double bond between C-8 and C-9 for natural compound 1 were reconfirmed as being of (S) configuration and E, respectively.

The relationship between the structure of the unsaturated hydroxy fatty acids and their inhibitory effect on the growth of lettuce was investigated.     

Vesicular stomatitis virus-based therapeutic vaccination targeted to the E1, E2, E6, and E7 proteins of cottontail rabbit papillomavirus

Persistent human papillomavirus (HPV)-associated benign and malignant lesions are a major cause of morbidity and mortality worldwide. Vaccination against HPV early proteins could provide an effective means of treating individuals with established infections. Recombinant vesicular stomatitis virus (VSV) vectors have been used previously to elicit strong humoral and cellular immune responses and develop prophylactic vaccines. We have shown that VSV vectors also can be used to elicit therapeutic immunity in the cottontail rabbit papillomavirus (CRPV)-rabbit model of high-risk HPV infection. In the present study, three new VSV vectors expressing the CRPV E1, E2, or E7 protein were produced and compared to the previously generated VSV-E6 vector for therapeutic efficacy. To determine whether vaccine efficacy could be augmented by simultaneous vaccination against two CRPV proteins, the four vaccines were delivered individually and in all possible pairings to rabbits 1 week after CRPV infection. Control rabbits received the recombinant wild-type VSV vector or medium only. Cumulative papilloma volumes were computed for analysis of the data. The analyses showed that VSV-based vaccination against the E1, E2, E6, or E7 protein significantly reduced papilloma volumes relative to those of the controls. Furthermore, VSV-based CRPV vaccination cured all of the papillomas in 5 of 30 rabbits. Of the individual vaccines, VSV-E7 was the most effective. The VSV-E7 vaccine alone was the most effective, as it reduced cumulative papilloma volumes by 96.9% overall, relative to those of the controls, and ultimately eliminated all of the disease in all of the vaccinees. Vaccine pairing was not, however, found to be beneficial, suggesting antigenic competition between the coexpressed CRPV proteins. These preclinical results, obtained in a physiologically relevant animal model of HPV infection, demonstrate that VSV vectors deserve serious consideration for further development as therapeutic antitumor vaccines.     

Two new E colicins, E8 and E9, produced by a strain of Escherichia coli

We have isolated a strain of Escherichia coli from chicken caeca which produces two E colicins and colicin M. This strain has seven plasmids, five of which have been transferred to E. coli K12. Two E. coli K12 derivatives which produce the two E colicins separately have been tested against seven standard E colicin producing strains which define seven different immunity groups. Our results indicate that these new E colicins define two further immunity groups, E8 and E9.     

Natural variation in the genes responsible for maturity loci E1, E2, E3 and E4 in soybean

Background and Aims

The timing of flowering has a direct impact on successful seed production in plants. Flowering of soybean (Glycine max) is controlled by several E loci, and previous studies identified the genes responsible for the flowering loci E1, E2, E3 and E4. However, natural variation in these genes has not been fully elucidated. The aims of this study were the identification of new alleles, establishment of allele diagnoses, examination of allelic combinations for adaptability, and analysis of the integrated effect of these loci on flowering.

Methods

The sequences of these genes and their flanking regions were determined for 39 accessions by primer walking. Systematic discrimination among alleles was performed using DNA markers. Genotypes at the E1–E4 loci were determined for 63 accessions covering several ecological types using DNA markers and sequencing, and flowering times of these accessions at three sowing times were recorded.

Key Results

A new allele with an insertion of a long interspersed nuclear element (LINE) at the promoter of the E1 locus (e1-re) was identified. Insertion and deletion of 36 bases in the eighth intron (E2-in and E2-dl) were observed at the E2 locus. Systematic discrimination among the alleles at the E1–E3 loci was achieved using PCR-based markers. Allelic combinations at the E1–E4 loci were found to be associated with ecological types, and about 62–66 % of variation of flowering time could be attributed to these loci.

Conclusions

The study advances understanding of the combined roles of the E1–E4 loci in flowering and geographic adaptation, and suggests the existence of unidentified genes for flowering in soybean,     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

Rubella virus contains one capsid protein and three envelope glycoproteins, E1, E2a, and E2b.

We have analyzed the structure of rubella virus proteins labeled metabolically with [35S]methionine, [3H]mannose, and [3H]glucosamine or externally with [3H]borohydride after galactose oxidase treatment. Four structural proteins, with MrS of about 58,000 (E1), 47,000 (E2a), 42,000 (E2b), and 33,000 (C), were resolved on sodium dodecyl sulfate-polyacrylamide gels. Tryptic peptide maps obtained from [35S]methionine-labeled proteins indicated that E1 and C were unrelated to each other and to E2a and E2b, whereas the latter two gave similar, if not identical, maps. E1, E2a, and E2b were associated with the envelope and were located externally on the virus particle, whereas the C protein was associated with the RNA in the nucleocapsid. Solubilization of the virus with Triton X-100, followed by removal of the nucleocapsid and the detergent, resulted in the formation of soluble envelope protein complexes (rosettes) containing E1, E2a, and E2b. Although external labeling with [3H]borohydride and metabolic labeling with [3H]glucosamine suggested that all three proteins were glycosylated, only E1 and E2b were efficiently labeled with [3H]mannose. It is thus possible that the difference in migration between E2a and E2b is due to differences in glycosylation. Analysis by immunoprecipitation and sodium dodecyl sulfate-gel electrophoresis of intracellular [35S]methionine-labeled structural proteins synthesized in the presence and absence of tunicamycin supported the conclusion that E1 and E2 are glycoproteins. Unglycosylated E1 and E2 had an Mr of about 53,000 and 30,000, respectively.     

Lyconadins D and E, and complanadine E, new Lycopodium alkaloids from Lycopodium complanatum

Three new Lycopodium alkaloids, lyconadins D (1) and E (2), and complanadine E (3), were isolated from the club moss Lycopodium complanatum. Lyconadin D (1) was the first example of fastigiatine-type alkaloid isolated from Lycopodium complanatum. The structures and relative stereochemistry of 1-3 were elucidated on the basis of spectroscopic data. Complanadine E (3) enhanced mRNA expression for NGF.     

Simple sequence repeat (SSR) markers linked to E1, E3, E4, and E7 maturity genes in soybean.

Soybean near isogenic lines (NILs), contrasting for maturity and photoperiod sensitivity loci, were genotyped with approximately 430 mapped simple sequence repeats (SSRs), also known as microsatellite markers. By analysis of allele distributions across the NILs, it was possible to confirm the map location of the Dt1 indeterminate growth locus, to refine the SSR mapping of the T tawny pubescence locus, to map E1 and E3 maturity loci with molecular markers, and to map the E4 and E7 maturity loci for the first time. Molecular markers flanking these loci are now available for marker-assisted breeding for these traits. Analysis of map locations identified a putative homologous relationship among four chromosomal regions; one in the middle of linkage group (LG) C2 carrying E1 and E7, one on LG I carrying E4, one at the top of LG C2, at which there is a reproductive period quantitative trait locus (QTL), and the fourth on LG B1. Other evidence suggests that homology also exists between the E1 + E7 region on LG C2 and a region on LG L linked to a pod maturity QTL. Homology relationships predict possible locations in the soybean genome of additional maturity loci, as well as which maturity loci may share a common evolutionary origin and similar mechanism(s) of action.     

Production and Characterization of Improved Adenovirus Vectors with the E1, E2b, and E3 Genes Deleted

Adenovirus (Ad)-based vectors have great potential for use in the gene therapy of multiple diseases, both genetic and nongenetic. While capable of transducing both dividing and quiescent cells efficiently, Ad vectors have been limited by a number of problems. Most Ad vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication-defective vector can be propagated only in human 293 cells that supply the deleted E1 gene functions in trans. Unfortunately, the use of high titers of E1-deleted vectors has been repeatedly demonstrated to result in low-level expression of viral genes still resident in the vector. In addition, the generation of replication-competent Ad (RCA) by recombination events with the E1 sequences residing in 293 cells further limits the usefulness of E1-deleted Ad vectors. We addressed these problems by isolating new Ad vectors deleted for the E1, E3, and the E2b gene functions. The new vectors can be readily grown to high titers and have several improvements, including an increased carrying capacity and a theoretically decreased risk for generating RCA. We have also demonstrated that the further block to Ad vector replication afforded by the deletion of both the E1 and E2b genes significantly diminished Ad late gene expression in comparison to a conventional E1-deleted vector, without destabilization of the modified vector genome. The results suggested that these modified vectors may be very useful both for in vitro and in vivo gene therapy applications.     

Mass spectrometry-based protein footprinting characterizes the structures of oligomeric apolipoprotein E2, E3, and E4

The three common isoforms of apolipoprotein E (ApoE) differ at two sites in their 299 amino acid sequence; these differences modulate the structure of ApoE to affect profoundly the isoform associations with disease. The ε4 allele in particular is strongly associated with Alzheimer's disease. The study of the structural effects of these mutation sites in aqueous media is hampered by the aggregation proclivity of each ApoE isoform. Hence, understanding the differences between isoforms has thus far relied on lower resolution biophysical measurements, mutagenesis, homology studies, and the use of truncated ApoE variants. In this study, we report two comparative studies of the ApoE family by using the mass spectrometry-based protein footprinting methods of FPOP and glycine ethyl ester (GEE) labeling. The first experiment examines the three full-length WT isoforms in their tetrameric state and finds that the overall structures are similar, with the exception of M108 in ApoE4 which is more solvent-accessible in this isoform than in ApoE2 and ApoE3. The second experiment provides clear evidence, from a comparison of the footprinting results of the wild-type proteins and a monomeric mutant, that several residues in regions 183-205 and 232-251 are involved in self-association.     

Genomic constitutions of four Chinese endemic Elymus species: E. brevipes, E. yangii, E. anthosachnoides, and E. altissimus (Triticeae, Poaceae).

The objectives of this study were to determine the genomic constitution and to explore the genomic variation within four Chinese endemic Elymus species, i.e., E. brevipes (Keng) L?ve (2n = 4x = 28) and E. yangii B.R. Lu (2n = 4x = 28), E. anthosachnoides (Keng) L?ve (2n = 4x = 28), and E. altissimus (Keng) L?ve (2n = 4x = 28). Intraspecific crosses between different populations of the four Elymus species, as well as interspecific hybridizations among the four target species, and with six analyzer species containing well-known genomes, i.e., E. caninus (L.) L. (2n = 4x = 28, SH), E. sibiricus L. (2n = 4x = 28, SH), E. semicostatus (Lees ex Steud.) Melderis (2n = 4x = 28, SY), E. parviglumis (Keng) L?ve (2n = 4x = 28, SY), E. tsukushiensis Honda (2n = 6x = 42, SHY), and E. himalayanus (Nevski) Tzvelev (2n = 6x = 42, SHY), were achieved through the aid of embryo rescue. Chromosome pairing behaviors were studied in the parental species and their hybrids. Numerical analysis on chromosome pairing was made on the interspecific hybrids. With one exception, each meiotic configuration at metaphase I in the hybrids involving the target taxa and the analyzer species containing the "SH" genomes fit a 2:1:1 model with x-values ranging between 0.91 and 1.00; chromosome pairing in the hybrids involving analyzer parents with the "SY" genomes match a 2:2 model, with x-values between 0.97 and 0.99. All pentaploid hybrids with a genomic formula "SSYYH," except for two crosses having unexpected low c-values, had pairing patterns fitting the 2:2:1 model with x-values varying between 0.96 and 1.00. It is concluded based on hybridization, fertility, and chromosome pairing data that (i) the four target Elymus species are strictly allotetraploid taxa, (ii) they are closely related species, all comprised of the "SY" genomes, (iii) minor genomic structural rearrangements have occurred within the four Elymus species, and (iv) meiotic pairing regulator(s) exists in some of the Elymus taxa studied.     

In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A,or E1/E4 Deleted

Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.Replication-deficient human adenoviruses (Ad) have been widely investigated as ex vivo and in vivo gene delivery systems for human gene therapy. The ability of these vectors to mediate the efficient expression of candidate therapeutic or vaccine genes in a variety of cell types, including postmitotic cells, is considered an advantage over other gene transfer vectors (3, 28, 49). However, the successful application of currently available E1-defective Ad vectors in human gene therapy has been hampered by the fact that transgene expression is only transient in vivo (2, 15, 16, 33, 36, 46). This short-lived in vivo expression of the transgene has been explained, at least in part, by the induction in vivo of cytotoxic immune responses to cells infected with the Ad vector. Studies with rodent systems have suggested that cytotoxic T lymphocytes (CTLs) directed against virus antigens synthesized de novo in the transduced tissues play a major role in eliminating cells containing the E1-deleted viral genome (56–58, 61). Consistent with the concept of cellular antiviral immunity, expression of transgenes is significantly extended in experimental rodent systems that are deficient in various components of the cellular immune system or that have been rendered immunocompromised by administration of pharmacological agents (2, 33, 37, 48, 60, 64).Based on the assumption that further reduction of viral antigen expression may lower the immune response and thus extend persistence of transgene expression, previous studies have investigated the consequences of deleting both E1 and an additional viral regulatory region, such as E2A or E4. The E2A region encodes a DNA binding protein (DBP) with specific affinity for single-stranded Ad DNA. The DNA binding function is essential for the initiation and elongation of viral DNA synthesis during the early phase of Ad infection. During the late phase of infection, DBP plays a central role in the activation of the major late promoter (MLP) (for a recent review, see reference 44). The E4 region, located at the right end of the viral genome, encodes several regulatory proteins with pleiotropic functions which are involved in the accumulation, splicing, and transport of early and late viral mRNAs, in DNA replication, and in virus particle assembly (reviewed in reference 44). The simultaneous deletion of E1 and E2A or of E1 and E4 should therefore further reduce the replication of the virus genome and the expression of early and late viral genes. Such multidefective vectors have been generated and tested in vitro and in vivo (9, 12, 17, 19–21, 23, 24, 26, 34, 40, 52, 53, 59, 62, 63). Recombinant vectors with E1 deleted and carrying an E2A temperature-sensitive mutation (E2Ats) have been shown in vitro to express much smaller amounts of virus proteins, leading to extended transgene expression in cotton rats and mice (19, 20, 24, 59). To eliminate the risks of reversion of the E2Ats point mutation to a wild-type phenotype, improved vectors with both E1 and E2A deleted were subsequently generated in complementation cell lines coexpressing E1 and E2A genes (26, 40, 63). In vitro analysis of human cells infected by these viruses demonstrated that the double deletion completely abolished viral DNA replication and late protein synthesis (26). Similarly, E1/E4-deleted vectors have been generated in various in vitro complementation systems and tested in vitro and in vivo (9, 17, 23, 45, 52, 53, 62). These studies showed that deletion of both E1 and E4 did indeed reduce significantly the expression of early and late virus proteins (17, 23), leading to a decreased anti-Ad host immune response (23), reduced hepatotoxicity (17, 23, 52), and improved in vivo persistence of the transduced liver cells (17, 23, 52).Interpretation of these results is difficult, however, since all tested E1- and E1/E4-deleted vectors encoded the bacterial β-galactosidase (βgal) marker, whose strong immunogenicity is known to influence the in vivo persistence of Ad-transduced cells (32, 37). Moreover, the results described above are not consistent with the conclusions from other studies showing, in various immunocompetent mouse models, that cellular immunity to Ad antigens has no detectable impact on the persistence of the transduced cells (37, 40, 50, 51). Furthermore, in contrast to results of earlier studies (19, 20, 59), Fang et al. (21) demonstrated that injection of E1-deleted/E2Ats vectors into immunocompetent mice and hemophilia B dogs did not lead to an improvement of the persistence of transgene expression compared to that with isogenic E1-deleted vectors. Similarly, Morral et al. (40) did not observe any difference in persistence of transgene expression in mice injected with either vectors deleted in E1 only or vectors deleted in both E1 and E2A. Finally, the demonstration that some E4-encoded products can modulate transgene expression (1, 17, 36a) makes the evaluation of E1- and E1/E4-deleted vectors even more complex when persistence of transgene expression is used for direct comparison of the in vivo persistence of cells transduced by the two types of vectors.The precise influence of the host immune response to viral antigens on the in vivo persistence of the transduced cells, and hence the impact of further deletions in the virus genome, therefore still remains unclear. To investigate these questions, we generated a set of isogenic vectors with single deletions (AdE1°) and double deletions (AdE1°E2A° and AdE1°E4°) and their corresponding complementation cell lines and compared the biologies and immunogenicities of these vectors in vitro and in vivo. To eliminate any possible influence of transgene-encoded products on the interpretation of the in vivo results, we used E1-, E1/E2A-, and E1/E4-deleted vectors with no transgenes.     

Electrophoretic and immunologic characterization of proteins of merozoites of Eimeria acervulina, E. maxima, E. necatrix, and E. tenella.

Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.     

Apolipoprotein E, amyloid, and Alzheimer disease

Despite important inroads into the molecular pathology of Alzheimer disease, effective long-term treatment for the condition remains elusive. Among the many gene products that are recognized as factors in the disease is apolipoprotein ( (apoE). The risk that specific isoforms of apoE pose with regard to Alzheimer Disease clearly varies, and so the roles that apoE plays in the brain will be crucial to a full understanding of the disease and to efforts to develop effective therapies.