Human cytomegalovirus (HCMV), a herpesvirus, is a ubiquitously distributed pathogen that causes severe disease in immunosuppressed patients and infected newborns. Efforts are underway to prepare effective subunit vaccines and therapies including antiviral antibodies. However, current vaccine efforts are hampered by the lack of information on protective immune responses against HCMV. Characterizing the B-cell response in healthy infected individuals could aid in the design of optimal vaccines and therapeutic antibodies. To address this problem, we determined, for the first time, the B-cell repertoire against glycoprotein B (gB) of HCMV in different healthy HCMV seropositive individuals in an unbiased fashion. HCMV gB represents a dominant viral antigenic determinant for induction of neutralizing antibodies during infection and is also a component in several experimental HCMV vaccines currently being tested in humans. Our findings have revealed that the vast majority (>90%) of gB-specific antibodies secreted from B-cell clones do not have virus neutralizing activity. Most neutralizing antibodies were found to bind to epitopes not located within the previously characterized antigenic domains (AD) of gB. To map the target structures of these neutralizing antibodies, we generated a 3D model of HCMV gB and used it to identify surface exposed protein domains. Two protein domains were found to be targeted by the majority of neutralizing antibodies. Domain I, located between amino acids (aa) 133–343 of gB and domain II, a discontinuous domain, built from residues 121–132 and 344–438. Analysis of a larger panel of human sera from HCMV seropositive individuals revealed positivity rates of >50% against domain I and >90% against domain II, respectively. In accordance with previous nomenclature the domains were designated AD-4 (Dom II) and AD-5 (Dom I), respectively. Collectively, these data will contribute to optimal vaccine design and development of antibodies effective in passive immunization.

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It was previously shown that a 1.5 kb fragment located in the non‐transcribed spacer (NTS) is the earliest replicating region of pea (Pisum sativum) rDNA in synchronized root cells. In the present report the structure of this region was characterized. It contains a cluster of four 11 bp near matches to the Saccharomyces cerevisiae ARS consensus sequence (ACS). These near matches are embedded in an A+T rich domain located upstream from the transcription initiation site. We identified and mapped an intrinsic DNA bending locus 5′ to the cluster of near matches. Several eukaryotic origins including the ARS from the budding yeast show very similar structural features. This observation strengthens the notion that pea rDNA replication initiates at or near this region. Replication of the entire pea rDNA repeat was analysed by two‐dimensional (2D) agarose gel electrophoresis. The results obtained indicate that only a small fraction of the potential origins is used in each replication round. Forks moving in the direction opposite to rRNA transcription are stalled at a polar replication fork barrier (RFB), which mapped near the 3′ end of the transcription unit. Consequently, most of pea rDNA appears to replicate in a unidirectional manner. These results show that the strategy used to replicate pea and yeast rRNA genes is very similar, suggesting that it has been conserved and might be common to most eukaryotes.

In mouse embryo, the early induction of the head region depends on signals from the anterior visceral endoderm (AVE) and the anterior primitive streak. Subsequently, node derivatives, including anterior definitive endoderm and axial mesendoderm, are thought to play a role in the maintenance and elaboration of anterior neural character. Foxa2 encodes a winged-helix transcription factor expressed in signaling centers required for head development, including the AVE, anterior primitive streak, anterior definitive endoderm, and axial mesendoderm. To address Foxa2 function during formation of the head, we used conditional mutants in which Foxa2 function is preserved in extraembryonic tissues during early embryonic stages and inactivated in embryonic tissues after the onset of gastrulation. In Foxa2 conditional mutants, the anterior neural plate and anterior definitive endoderm were initially specified. In contrast, the axial mesendoderm failed to differentiate. At later stages, specification of the anterior neural plate and anterior definitive endoderm was shown to be labile. As a result, head truncations were observed in Foxa2 conditional mutants. Our results therefore indicate that anterior definitive endoderm alone is not sufficient to maintain anterior head specification and that an interaction between the axial mesendoderm and the anterior definitive endoderm is required for proper specification of the endoderm. Foxa2 therefore plays an integral role in the formation of axial mesendoderm, which is required to maintain the specification of the forebrain and the anterior definitive endoderm.