A model system for increased meiotic nondisjunction in older oocytes

For at least 5% of all clinically recognized human pregnancies, meiotic segregation errors give rise to zygotes with the wrong number of chromosomes. Although most aneuploid fetuses perish in utero, trisomy in liveborns is the leading cause of mental retardation. A large percentage of human trisomies originate from segregation errors during female meiosis I; such errors increase in frequency with maternal age. Despite the clinical importance of age-dependent nondisjunction in humans, the underlying mechanisms remain largely unexplained. Efforts to recapitulate age-dependent nondisjunction in a mammalian experimental system have so far been unsuccessful. Here we provide evidence that Drosophila is an excellent model organism for investigating how oocyte aging contributes to meiotic nondisjunction. As in human oocytes, nonexchange homologs and bivalents with a single distal crossover in Drosophila oocytes are most susceptible to spontaneous nondisjunction during meiosis I. We show that in a sensitized genetic background in which sister chromatid cohesion is compromised, nonrecombinant X chromosomes become vulnerable to meiotic nondisjunction as Drosophila oocytes age. Our data indicate that the backup pathway that normally ensures proper segregation of achiasmate chromosomes deteriorates as Drosophila oocytes age and provide an intriguing paradigm for certain classes of age-dependent meiotic nondisjunction in humans.     

Interaction and assembly of murine pre-replicative complex proteins in yeast and mouse cells

Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time.     

Tumor vaccination after allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned tumor-bearing murine host

Allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned host is supposed to provide an optimized platform for tumor vaccination. We recently showed that an allogeneic T cell-depleted graft was well accepted if the tumor-bearing host was NK depleted. Based on this finding, a vaccination protocol in tumor-bearing, nonmyeloablatively conditioned, allogeneically reconstituted mice was elaborated. Allogeneically reconstituted mice, bearing a renal cell carcinoma, received tumor-primed donor lymph node cells (LNC), which had or had not matured in the allogeneic host. Primed LNC were supported by tumor lysate-pulsed dendritic cells, which were donor or host derived. Optimal responses against the tumor were observed with host-tolerant, tumor-primed LNC in combination with host-derived dendritic cells. High frequencies of tumor-specific proliferating and CTLs were recorded; the survival time of tumor-bearing mice was significantly prolonged, and in >50% of mice the tumor was completely rejected. Notably, severe graft-vs-host disease was observed in reconstituted mice that received tumor-primed LNC, which had not matured in the allogeneic host. However, graft-vs-host was not aggravated after vaccination with tumor-primed, host-tolerant LNC. Thus, the LNC were tolerant toward the host, but not toward the tumor. The finding convincingly demonstrates the feasibility and efficacy of tumor vaccination after allogeneic reconstitution of the nonmyeloablatively conditioned host.     

Support for association of schizophrenia with genetic variation in the 6p22.3 gene,dysbindin, in sib-pair families with linkage and in an additional sample of triad families

Genetic variants in a gene on 6p22.3, dysbindin, have been shown recently to be associated with schizophrenia (Straub et al. 2002a). There is no doubt that replication in other independent samples would enhance the significance of this finding considerably. Since the gene is located in the center of the linkage peak on chromosome 6p that we reported earlier, we decided to test six of the most positive DNA polymorphisms in a sib-pair sample and in an independently ascertained sample of triads comprising 203 families, including the families for which we detected linkage on chromosome 6p. Evidence for association was observed in the two samples separately as well as in the combined sample (P=.00068 for SNP rs760761). Multilocus haplotype analysis increased the significance further to .00002 for a two-locus haplotype and to .00001 for a three-locus haplotype. Estimation of frequencies for six-locus haplotypes revealed one common haplotype with a frequency of 73.4% in transmitted, and only 57.6% in nontransmitted, parental haplotypes. All other six-locus haplotypes occurring at a frequency of >1% were less often transmitted than nontransmitted. Our results represent a first successful replication of linkage disequilibrium in psychiatric genetics detected in a region with previous evidence of linkage and will encourage the search for causes of schizophrenia by the genetic approach.     

A 65-kilobase pathogenicity island is unique to Philadelphia-1 strains of Legionella pneumophila

Nucleotide sequence analysis of an approximately 80-kb genomic region revealed an approximately 65-kb locus that bears hallmarks of a pathogenicity island. This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors. Comparative studies with other Legionella pneumophila strains and serogroups indicated that this approximately 65-kb locus is unique to L. pneumophila serogroup 1 Philadelphia-1 strains.     

Antiviral activity of tenofovir (PMPA) against nucleoside-resistant clinical HIV samples

The presence of the lamivudine-associated M184V RT mutation increases tenofovir susceptibility in multiple HIV genotypes. Tenofovir is uniquely active against multinucleoside-resistant HIV expressing the Q151M mutation, but shows reduced susceptibility to the T69S insertion mutations. HIV with common forms of zidovudine and lamivudine resistance are susceptible to tenofovir, corroborating phase II clinical results demonstrating the activity of tenofovir DF in treatment-experienced patients.     

Comprehensive detection of genomic duplications and deletions in the DMD gene,by use of multiplex amplifiable probe hybridization

Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening.     

Reversible and irreversible adhesion of motile Escherichia coli cells analyzed by total internal reflection aqueous fluorescence microscopy

The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively.     

Evolutionary fate of an unstable human minisatellite deduced from sperm-mutation spectra of individual alleles

Although mutation processes at some human minisatellites have been extensively characterized, the evolutionary fate of these unstable loci is unknown. Minisatellite instability is largely germline specific, with mutation rates up to several percent and with expansion events predominating over contractions. Using allele-specific small-pool polymerase chain reaction, we have determined sperm-mutation spectra of individual alleles of the highly unstable human minisatellite CEB1 (i.e., D2S90). We show that, as allele size increases, the proportion of contractions rises from <5% to 50%, with the average size of deletion increasing and eventually exceeding the average size of expansion. The expected net effect of these trends after many generations is an equilibrium distribution of allele sizes, and allele-frequency data suggest that this equilibrium state has been reached in some contemporary human populations.