DQ α and β RFLP reveals the composition of the DQ molecule recognized by T-cell clones

Pst I RFLP, revealed with DQ and DQ probes, was compared with Taq I RFLP using a panel of DR-homozygous cell lines and HLA-typed family members. Taq I patterns, characteristic for each DR-associated DQ and allelic forms, were recognized in the homozygous state and then proven to segregate in the heterozygous members of informative families. The presence of both specific and chains was found to be necessary to form the type of DQ molecule specifically recognized by two alloreactive T-cell clones. Particular and associations also seem to be responsible for some Dw splits of the DRw6-positive cells. Taq I RFLP analysis may be more complex than the Pst I analysis, but is certainly more informative and complete, considering the type of information we were seeking by performing these types of experiments.Abbreviations used in this paper BSA bovine serum albumin - GLO glyoxalase - kb kilobase(s) - LCL lymphoblastoid cell line - MHC major histocompatibility complex - PBL peripheral blood lymphocyte - PLT primed lymphocyte test - RFLP restriction fragment length polymorphism - SDS sodium dodecyl sulfate - SSC standard sodium citrate - SSCP sodium, sodium citrate, sodium phosphate - TBE Tris-borate, boric acid, ethylenediaminetetraacetate (EDTA) - TCGF T-cell growth factor     

High-frequency conversion to a "fluffy" developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene.

Transient exposure of mycelia from Aspergillus niger and Aspergillus nidulans to the cytidine analog 5-azacytidine, leading to no more than 0.3 to 0.5% substitution for cytosine by 5-azacytosine in A. nidulans DNA, resulted in the conversion of a high fraction of the cell population (more than 20%) to a mitotically and meiotically stable "fluffy" developmental phenotype. The phenotypic variants are characterized by the developmentally timed production of a profuse fluffy network of undifferentiated aerial hyphae that seem to escape signals governing vegetative growth. Genetic analysis with six different fluffy clones reveals that this trait is not cytoplasmically coded, is recessive in heterozygous diploids but codominant in heterokaryons, and exhibits a 1:1 Mendelian segregation pattern upon sexual sporulation of heterozygous diploids. Complementation and mitotic haploidization studies indicated that all variants are affected in the same gene, which can be tentatively located on chromosome VIII of A. nidulans. Molecular analysis to search for modified bases showed that DNA methylation is negligible in in both A. niger and A. nidulans and that no differences could be detected among DNAs from wild-type cells, fluffy clones, or mycelia exposed to 5-azacytidine. It thus appears that high-frequency conversion of fungal mycelia to a stable, variant developmental phenotype by 5-azacytidine is the result of some kind of target action on a single nuclear gene and that this conversion can occur in organisms virtually devoid of DNA methylation.     

Endogenous polyamine content during in vivo maturation and in vitro culture of maize pollen

Changes in polyamine content during in vivo maturation and in vitro culture of maize (Zea mays L.) pollen were studied. The endogenous content of free, conjugated and bound polyamines was analyzed during 30 days of pollen evolution, in both developmental pathways (microsporogenesis and androgenesis). The induction of androgenesis from cold-pretreated uninucleate pollen results, in most of cases, in a lower total polyamine content than that of the in vivo uninucleate pollen. These differences indicate that polyamine metabolism is altered during the induction of androgenesis, and this could be a consequence of increased polyamine assimilation. In general, pollen stages that involve cell division (tetrades, pre-anthesis pollen and four-day cultured pollen) are characterized by a predominance of free Spd. The increase of Spd and Spm in 15-day cultured pollen, when the first embryoids are formed, outline the possible implication of these polyamines in embryogenetic processes. Furthermore, these findings may contribute to the improvement of maize androgenesis yield, especially in recalcitrant genotypes, by the exogenous application of polyamines or polyamine-inhibitors to the culture medium.Abbreviations PAs polyamines - Put putrescine - Spd spermidine - Spm spermine - S free polyamine fraction - SH conjugated polyamine fraction - PH bound polyamine fraction