A new method of in situ hybridization

A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry.     

Isolation and properties of a ferredoxin from Anabaena Flos-Aquae

Ferredoxin was isolated from the blue-green alga Anabaena flos-aquae. Its homogeneity was shown by conventional and SDS-polyacrylamide gel electrophoresis, and isoelectric focusing on polyacrylamide gel columns, the latter indicating a pI at ca pH 3·7. The absorption spectrum had, in the oxidized state, maxima at 462, 421, 327 and 276 nm, with a shoulder at 284 nm, a spectrum characteristic of plant-type ferredoxins. The 421 : 276 nm absorbance ratio was typically 0.49. The ferredoxin effectively mediated the photoreduction of NADP⁺ by barley chloroplasts depleted of native ferredoxin. The MW obtained by sedimentation-equilibrium and sedimentation velocity-diffusion coefficient studies was ca 12 000 daltons, a value somewhat higher than suggested by amino acid composition data. The ferredoxin contained 2Fe and 2S per molecule.     

Tissue antigens from the third instar larva of Drosophila melanogaster

Antibodies were prepared against the soluble proteins from six tissues of Drosophila larvae. These were used to analyse the antigens in different tissues and at different developmental stages. The results suggest (1) the pattern of antigens determines the characteristics of a tissue, (2) salivary gland antigens are sequestered by the imaginal disks, (3) not all pupal glue antigens are synthesized in the salivary glands, and (4) most larval serum antigens are synthesized by the fat body.     

Differential fertility depending on the age of puberty

229 women, aged 41 to 60 years, belonging to five villages from Maramures, were divided into three groups, according to their type of puberty (early, median, delayed). We found a higher degree of fertility in the women of early puberty than in those of delayed puberty. The slight age difference in marriage recorded only in the group of delayed puberty cannot account for the big difference found in the three groups of women in respect to the number of children. In the past, a positive correlation between fertility and mortality limited the descendants born from women of early puberty (i.e. the selective value). At present, in the absence of differential mortality the differential fertility related to the type of puberty offers new means for explaining some aspects of the acceleration of the age of puberty.     

Next-Generation Sequencing of Connective Tissue Genes in Patients with Classical Ehlers-Danlos Syndrome

Background: Ehlers-Danlos syndrome (EDS) is a common non-inflammatory, congenital connective tissue disorder. Classical type (cEDS) EDS is one of the more common forms, typically caused by mutations in the COL5A1 and COL5A2 genes, though causative mutations in the COL1A1 gene have also been described. Material and methods: The study group included 59 patients of Polish origin, diagnosed with cEDS. The analysis was performed on genomic DNA (gDNA) with NGS technology, using an Illumina sequencer. Thirty-five genes related to connective tissue were investigated. The pathogenicity of the detected variants was assessed by VarSome. Results: The NGS of 35 genes revealed variants within the COL5A1, COL5A2, COL1A1, and COL1A2 genes for 30 of the 59 patients investigated. Our panel detected no sequence variations for the remaining 29 patients. Discussion: Next-generation sequencing, with an appropriate multigene panel, showed great potential to assist in the diagnosis of EDS and other connective tissue disorders. Our data also show that not all causative genes giving rise to cEDS have been elucidated yet.