Recombinant modified vaccinia virus Ankara expressing a soluble form of glycoprotein B causes durable immunity and neutralizing antibodies against multiple strains of human cytomegalovirus

Human cytomegalovirus (CMV) is a viral pathogen that infects both genders, who remain asymptomatic unless they receive immunosuppressive drugs or acquire infections that cause reactivation of latent virus. CMV infection also causes serious birth defects following primary maternal infection during gestation. A safe and effective vaccine to limit disease in this population continues to be elusive. A well-studied antigen is glycoprotein B (gB), which is the principal target of neutralizing antibodies (NAb) towards CMV in humans and has been implicated as the viral partner in the receptor-mediated infection by CMV in a variety of cell types. Antibody-mediated virus neutralization has been proposed as a mechanism by which host immunity could modify primary infection. Towards this goal, an attenuated poxvirus, modified vaccinia virus Ankara (MVA), has been constructed to express soluble CMV gB (gB680-MVA) to induce CMV NAb. Very high levels of gB-specific CMV NAb were produced after two doses of the viral vaccine. NAb were durable within a twofold range for up to 6 months. Neutralization titers developed in immunized mice are equivalent to titers found clinically after natural infection. This viral vaccine, expressing gB derived from CMV strain AD169, induced antibodies that neutralized CMV strains of three different genotypes. Remarkably, preexisting MVA and vaccinia virus (poxvirus) immunity did not interfere with subsequent immunizations of gB680-MVA. The safety characteristics of MVA, combined with the robust immune response to CMV gB, suggest that this approach could be rapidly translated into the clinic.     

Comparison of FDR- and SDR-derived tetraploid progeny from 2x×4x crosses using haploids of Solanum tuberosum L. that produce mixed modes of 2n eggs

 The relationship between heterozygosity and heterosis in tetraploid potato (Solanum tuberosum subsp. tuberosum L., 2n=4x=48) was examined in a series of first-division restitution (FDR)- and second-division restitution (SDR)-derived tetraploid subpopulations. The subpopulations were constructed using two 2n egg-producing, mixed-mode haploids (2n=2x=24) crossed to three tetraploid (2x= 4x=48) potato clones. Half-tetrad analysis using a co-dominant electrophoretic marker (Pgm-2), which is closely linked to the centromere, discriminated between FDR- and SDR-derived 4x progeny. The FDR:SDR ratio of the 4x progeny observed was dependent upon the haploid parent used in the 2x×4x cross. Field studies were conducted between 1992 and 1996 to compare the yield and specific gravity of the two subpopulations and their parents from three crosses. There was no difference in the total tuber yield or specific gravity between the FDR- and SDR-subpopulations based upon family means, despite the expectation that FDR-derived progeny would transmit a greater portion of the genome’s heterozygosity intact than SDR-derived progeny. The 4x parent in each family had a higher yield than either 4x progeny subpopulation. Inbreeding, as a consequence of the haploidization process and a lack of genetic diversity, may have negated any advantage of the FDR-derived progenies over the SDR-derived progenies. Received: 14 April 1998 / Accepted: 12 May 1998     

Stable Ribonucleic Acid Synthesis in Stringent (rel+) and Relaxed (rel−) Polyamine Auxotrophs of Escherichia coli K-12

The relationship of polyamines to stable ribonucleic acid (RNA) synthesis under conditions of amino acid withdrawal or chloramphenicol treatment was examined with the use of a closely related rel(+), rel(-) pair conditionally incapable of synthesizing putrescine. Under conditions of polyamine starvation, the cellular sperimidine level fell to one-third to one-half of the value observed in putrescine-supplemented cultures and putrescine became undetectable; cadaverine was synthesized by both strains, but the relaxed strain, MA 252, accumulated less cadaverine per cell than its stringent twin, MA 254. Upon amino acid withdrawal, the stringent strain remained stringent whether starved of or supplemented with polyamines. Similarly, the relaxed strain was capable of making RNA either with or without polyamine starvation. On the addition of chloramphenicol or upon amino acid withdrawal in the relaxed strain, supplementation with spermidine had no effect on the initial rate of RNA synthesis, although RNA accumulation was greater in the presence of added spermidine. Spermidine added at the conclusion of RNA synthesis prompted additional synthesis, although preincubation with spermidine again had no effect on the initial rate. All forms of stable RNA species were made with polyamine supplementation. The present data appear to rule out the possibility that polyamines are primary causative agents in stimulating RNA synthesis, but rather suggest an indirect or secondary role for spermidine in which the polyamines "stimulate" stable RNA synthesis probably by relieving RNA product inhibition of RNA synthesis.     

The zab domain of the human RNA editing enzyme ADAR1 recognizes Z-DNA when surrounded by B-DNA

The Zab domain of the editing enzyme ADAR1 binds tightly and specifically to Z-DNA stabilized by bromination or supercoiling. A stoichiometric amount of protein has been shown to convert a substrate of suitable sequence to the Z form, as demonstrated by a characteristic change in the CD spectrum of the DNA. Now we show that Zab can bind not only to isolated Z-forming d(CG)(n) sequences but also to d(CG)(n) embedded in B-DNA. The binding of Zab to such sequences results in a complex including Z-DNA, B-DNA, and two B-Z junctions. In this complex, the d(CG)(n) sequence, but not the flanking region, is in the Z conformation. The presence of Z-DNA was detected by cleavage with a Z-DNA specific nuclease, by undermethylation using Z-DNA sensitive SssI methylase, and by circular dichroism. It is possible that Zab binds to B-DNA with low affinity and flips any favorable sequence into Z-DNA, resulting in a high affinity complex. Alternatively, Zab may capture Z-DNA that exists transiently in solution. The binding of Zab to potential as well as established Z-DNA segments suggests that the range of biological substrates might be wider than previously thought.     

High incidence of transposon Tn3 insertions into a replication control gene of the chimeric R/Ent plasmid pCG86 of Escherichia coli.

Insertion of transposon Tn3 into the chimeric R/Ent plasmid pCG86 occurred preferentially into a replication control gene, generating mutants with increased plasmid copy number. In two mutants, the nucleotide sequence of the region of the gene containing the Tn3 insertion was determined.     

Salinity effects on CO2 assimilation and diffusive conductance of cowpea leaves

The effect of NaCl salinity at concentrations of 43–173 mM in nutrient solution on net gas exchange of attached cowpea [Vigna unguiculata (L.) Walp cv. California Black-eye No. 5 (CB5)] leaves was investigated under both greenhouse and growth chamber conditions.
There was a marked decrease in leaf conductance to water vapor after exposure to low salinity levels and a slighter decrease when salinity levels were higher. The decrease in net assimilation was much more gradual throughout the entire salinity range. The altered responses of net assimilation and leaf conductance to salinity were more evident at a high light intensity. A decrease in intercellular partial CO₂ pressure [p(CO₂)] was found at the low and intermediate salinity levels but not at the high level. These findings suggest that CO, assimilation was mainly controlled by stomatal conductance and the fixation of CO, might have been increased due to stimulated biochemical activity or to higher chlorophyll concentration per unit leaf area. A decrease in assimilation was already found one day after salinization and pro-ceeded up to 4 days when it was inhibited by 50% at 43 mM NaCl and up to 85% at 173 mM. The decrease in transpiration was larger than the decrease in net assimila-tion, and both were attributed to osmotic stress. Partial recovery was found thereaf-ter and new steady-state rates, in the range of 55 to 100% of the control, were then obtained for salinity levels between 43 and 130 mM. Inhibition of net CO, assimila-tion at this stage was attributed partly to a specific sodium effect and partly to plant water status. A linear relationship between leaf sodium content and net photosynthe-sis was also evident at this stage. Net CO, assimilation recovered more completely than transpiration when salt stress was removed, but at 173 mM NaCl recovery was neglible.     

Influence of management on the seed production and seed bank of calcareous fen species

Abstract. The dynamics of the seed production and seed bank of two dominant perennial species of calcareous fens were investigated in response to different mowing frequency, fallow or pasture. The production of fertile shoots responded strongly to management practices. On sites where mowing alternated with fallow, autumn mowing led to an increase of fertile shoot density in Schoenus ferrugineus and a decrease in Molinia caerulea in the subsequent season. Repeated annual mowing after long-term fallow raised the density in Schoenus and inhibited initiation of flowering shoots in Molinia. Seed set and viability were influenced mainly by climatic or other factors. Variation of seed production between sites and in consecutive years at the same site could thus be large. The relationship between total seed production and seed bank was weak, but it improved when germination rates were included in the calculation. As the seed bank was generally smaller than the viable seed production, low survival and a high turnover of seeds in the upper soil layers is assumed for both species. Implications for recruitment strategies are discussed.     

Characterization of the basic replicons of the chimeric R/Ent plasmid pCG86 and the related Ent plasmid P307

Restriction-enzyme fragments that can replicate autonomously after circularization were isolated from the chimeric R/Ent plasmid pCG86 and the Ent plasmid P307. Two such regions containing a basic replicon were located in each plasmid. One of the basic replicons of P307, RepFIB, is almost identical with one of the basic replicons of pCG86. The other basic replicon in P307, RepFIC, is partly homologous with the second basic replicon in pCG86, RepFIIA/RepFIC. The latter is a hybrid basic replicon and is in addition partly homologous with RepFIIA, a basic replicon present in IncFII R plasmids. By restriction-enzyme mapping and nucleotide-sequence analysis we have determined a site in the hybrid replicon where it ceases to be homologous with the RepFIIA basic replicon contained in the IncFII miniplasmid pSM1. The 2410-bp region of homology with pSM1 corresponds with a segment containing the origin of replication and all the genes responsible for replication control of pSM1.