Differential anaphase-I behaviour between wheat and rye univalents in triticale-wheat hybrid plants

The chromosomes of the D genome of wheat and the genome R of rye can be distinguished at meiosis by C-banding in triticale-wheat hybrid plants. All members of both genomes almost exclusively formed univalents at metaphase I. However, at anaphase I the frequencies of equationally dividing chromosomes were higher for rye than for wheat chromosomes. The differential centromere behaviour at anaphase I is ascribed to differences in the time at which wheat and rye univalents are formed during the first meiotic prophase.     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

The stimulatory effect of actinomycin D on avian reovirus replication in L cells suggests that translational competition dictates the fate of the infection.

Indirect immunostaining of avian reovirus S1133-infected L-cell monolayers showed that most of the cells can support viral replication. However, the number of cells in which the virus was actually replicating depended on the multiplicity of virus infection. The presence of actinomycin D during infection increased viral protein synthesis, viral growth, and the number of actively infected cells at late infection times. The antibiotic elicited these effects by triggering viral replication in cells that already contained unproductive cytoplasmic virus but that would not get productively infected in the absence of the drug. From these results, we propose a model for the interaction between L cells and avian reovirus S1133 in which viral versus host mRNA competition for the translational machinery determines the fate of the virus infection.     

Relationship between the levels of wheat-rye metaphase I chromosomal pairing and recombination revealed by GISH

The metaphase I and anaphase I stages of meiosis of wheat×rye hybrids carrying the ph1b mutation were analyzed by genomic in situ hybridization. This technique allows distinction between three different types of wheat-rye associations in metaphase I configurations as well as detection of wheat-rye recombinant chromosomes in anaphase I cells. The frequency of associations between wheat and rye chromosomes greatly exceeded the level of wheat-rye recombination found in the three hybrids examined. Extremely distal associations, which account for about 50% of the total wheat-rye metaphase I chromosomal pairing, can explain such a discrepancy between metaphase I and anaphase I data. It is further discussed whether these associations reflect very distally located chiasmata or nonchiasmatic pairing. The sizes of the segments exchanged in wheat-rye recombinant chromosomes provide cytological evidence that wheat-rye recombination is restricted to the distal chromosomal regions. Received: 24 August 1995; in revised form: 27 February 1996 / Accepted: 28 March 1996     

Immunocytochemical localization of DNA in synaptonemal complexes of rat and mouse spermatocytes,and of chick oocytes

The distribution of DNA in synaptonemal complexes of rat and mouse spermatocytes, and of chick oocytes was investigated by immunogold electron microscopy. Except for a few specific sites, DNA was not immunolocalized in the space between lateral elements of the complex. Some labeled fibrils connecting the lateral elements with the central element were observed associated with recombination nodules or near them. However, other labeled fibrils in the space between lateral elements did not appear to present any relationship to recombination nodules. The immunocytochemical approaches used here confirmed the presence of significant amounts of DNA in the lateral elements as previously indicated by preferential DNA staining methods. Furthermore, our findings support the view that recombination nodules are the site of chiasma formation.     

Genetic diversity of the Plasmodium falciparum GTP-cyclohydrolase 1, dihydrofolate reductase and dihydropteroate synthetase genes reveals new insights into sulfadoxine-pyrimethamine antimalarial drug resistance

Plasmodium falciparum parasites resistant to antimalarial treatments have hindered malaria disease control. Sulfadoxine-pyrimethamine (SP) was used globally as a first-line treatment for malaria after wide-spread resistance to chloroquine emerged and, although replaced by artemisinin combinations, is currently used as intermittent preventive treatment of malaria in pregnancy and in young children as part of seasonal malaria chemoprophylaxis in sub-Saharan Africa. The emergence of SP-resistant parasites has been predominantly driven by cumulative build-up of mutations in the dihydrofolate reductase (pfdhfr) and dihydropteroate synthetase (pfdhps) genes, but additional amplifications in the folate pathway rate-limiting pfgch1 gene and promoter, have recently been described. However, the genetic make-up and prevalence of those amplifications is not fully understood. We analyse the whole genome sequence data of 4,134 P. falciparum isolates across 29 malaria endemic countries, and reveal that the pfgch1 gene and promoter amplifications have at least ten different forms, occurring collectively in 23% and 34% in Southeast Asian and African isolates, respectively. Amplifications are more likely to be present in isolates with a greater accumulation of pfdhfr and pfdhps substitutions (median of 1 additional mutations; P<0.00001), and there was evidence that the frequency of pfgch1 variants may be increasing in some African populations, presumably under the pressure of SP for chemoprophylaxis and anti-folate containing antibiotics used for the treatment of bacterial infections. The selection of P. falciparum with pfgch1 amplifications may enhance the fitness of parasites with pfdhfr and pfdhps substitutions, potentially threatening the efficacy of this regimen for prevention of malaria in vulnerable groups. Our work describes new pfgch1 amplifications that can be used to inform the surveillance of SP drug resistance, its prophylactic use, and future experimental work to understand functional mechanisms.     

High‐resolution melting of the cytochrome B gene in fecal DNA: A powerful approach for fox species identification of the Lycalopex genus in Chile

Easy, economic, precise species authentication is currently necessary in many areas of research and diagnosis in molecular biology applied to conservation studies of endangered species. Here, we present a new method for the identification of three fox species of the Lycalopex genus in Chile. We developed an assay based on high‐resolution melt analysis of the mitochondrial cytochrome B gene, allowing a simple, low cost, fast, and accurate species determination. To validate the assay applicability for noninvasive samples, we collected fecal samples in the Atacama Desert, finding unexpectedly one species outside of its known distribution range. We conclude that the assay has a potential to become a valuable tool for a standardized genetic monitoring of the Lycalopex species in Chile.     

Possible involvement of the double-stranded RNA-binding core protein sigmaA in the resistance of avian reovirus to interferon

Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein sigmaA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein sigmaA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins.     

Comparative analysis of the meiotic effects of wheat ph1b and ph2b mutations in wheat×rye hybrids

 Wheat-wheat and wheat-rye homoeologous pairing at metaphase I and wheat-rye recombination at anaphase I were examined by genomic in situ hybridization (GISH) in wild-type (Ph1Ph2) and mutant ph1b and ph2b wheat×rye hybrids. The metaphase-I analysis revealed that the relative contribution of wheat-rye chromosome associations in ph2b wheat×rye was similar to that of the wild-type hybrid genotype but differed from the effect of the ph1b mutation. The greater pairing promotion effect of the ph1b mutation appears to be relatively more on distant homoeologous partner metaphase-I associations, whereas the lower promoting effect of ph2b is evenly distributed among all types of homoeologous associations. This finding reveals that distinct mechanisms are involved in the control of wheat homoeologous pairing by the two Ph genes. The frequency of wheat-rye recombination calculated from anaphase-I analysis was lower than expected from the metaphase-I data. A greater discrepancy was found in ph2b than in ph1b wheat×rye hybrids, which may suggest a more distal chiasma localization in the former hybrid genotype. Received: 20 June 1997 / Accepted: 9 December 1997