Repairing the sickle cell mutation. I. Specific covalent binding of a photoreactive third strand to the mutated base pair.

A DNA third strand with a 3'-psoralen substituent was designed to form a triplex with the sequence downstream of the T.A mutant base pair of the human sickle cell beta-globin gene. Triplex-mediated psoralen modification of the mutant T residue was sought as an approach to gene repair. The 24-nucleotide purine-rich target sequence switches from one strand to the other and has four pyrimidine interruptions. Therefore, a third strand sequence favorable to two triplex motifs was used, one parallel and the other antiparallel to it. To cope with the pyrimidine interruptions, which weaken third strand binding, 5-methylcytosine and 5-propynyluracil were used in the third strand. Further, a six residue "hook" complementary to an overhang of a linear duplex target was added to the 5'-end of the third strand via a T(4) linker. In binding to the overhang by Watson-Crick pairing, the hook facilitates triplex formation. This third strand also binds specifically to the target within a supercoiled plasmid. The psoralen moiety at the 3'-end of the third strand forms photoadducts to the targeted T with high efficiency. Such monoadducts are known to preferentially trigger reversion of the mutation by DNA repair enzymes.     

Structure of subepidermal tissue of gamasid mites (electron microscopy data)

Fine structure of the subepidermal tissue was investigated in the adult predatory mite Hypoaspis miles (Gamasina: Laelapidae) and bloodsucking mite Dermanyssus gallinae (Gamasinae: Dermanyssidae) of both sexes. This tissue is analogous to the fat body of other arthropodes. The subepidermal tissue of females consists of two types of cells: cells containing a synthetic apparatus and secretory granules; cells containing storage substances. In the subepidermal tissue of H. miles, cells being similar to hemocytes were also observed. As the males of both species have only one cell type in this tissue--the storage one and the females have the secretory cells, it is suggested that the subepidermal tissue of females could probably take part in the vitellogenesis.     

The consensus sequence for self-catalyzed site-specific G residue depurination in DNA

The sequence variation tolerated within the stem-loop-forming genomic consensus sequence for self-catalyzed site-specific depurination of G residues is explored. The variation in self-depurination kinetics with sequence changes in the loop residues and stem base pairs, as well as with pH, provides insights into the self-catalytic mechanism. The observations suggest that self-catalyzed depurination of the 5' G residue of the loop consensus sequence 5'-G(T/A)GG-3' probably involves formation of some intraloop hydrogen-bonded base pair with the 3'-terminal G residue; although the electronic structure of both these G residues is retained, their 2-amino substituents are not critical for that interaction. The strong dependence of the self-depurination kinetics on stem stability suggests that the lifetime of some strained form of the loop is controlled by the integrity of the stem. In addition to the effects of length and base pair sequence on stem stability, there is a base pair requirement at the base of the loop: self-depurination is suppressed by 5'-C·G-3', 5'-A·T-3', or a mismatch but is most favored by 5'T·A3' and less so by 5'-G·C-3'. The occurrence in T and G of a similarly located carbonyl capable of hydrogen-bonding to the water molecule required for glycosyl bond hydrolysis may explain this sequence requirement. In toto, the more complete definition of the consensus sequence provided by this investigation enables a more accurate estimation of their number in the human genome and their distribution among different genes.     

Genomic Markers Associated with Cold-Hardiness in Brassica rapa L.

Molecular Biology - Brassica rapa L. is a valuable and widespread species, but its cultivation in risk farming areas requires high-quality cold-hardy varieties to be developed. Mechanisms of the...     

Phylogenetic reconstruction of Aegilops section Sitopsis and the evolution of tandem repeats in the diploids and derived wheat polyploids.

The evolution of 2 tandemly repeated sequences Spelt1 and Spelt52 was studied in Triticum species representing 2 evolutionary lineages of wheat and in Aegilops sect. Sitopsis, putative donors of their B/G genomes. Using fluorescence in situ hybridization we observed considerable polymorphisms in the hybridization patterns of Spelt1 and Spelt52 repeats between and within Triticum and Aegilops species. Between 2 and 28 subtelomeric sites of Spelt1 probe were detected in Ae. speltoidies, depending on accession. From 8 to 12 Spelt1 subtelomeric sites were observed in species of Timopheevi group (GAt genome), whereas the number of signals in emmer/aestivum accessions was significantly less (from 0 to 6). Hybridization patterns of Spelt52 in Ae. speltoides, Ae. longissima, and Ae. sharonensis were species specific. Subtelomeric sites of Spelt52 repeat were detected only in T. araraticum (T. timopheevii), and their number and chromosomal location varied between accessions. Superimposing copy number data onto our phylogenetic scheme constructed from RAPD data suggests 2 major independent amplifications of Spelt52 and 1 of Spelt1 repeats in Aegilops divergence. It is likely that the Spelt1 amplification took place in the ancient Ae. speltoides before the divergence of polyploid wheats. The Spelt52 repeat was probably amplified in the lineage of Ae. speltoides prior to divergence of the allopolyploid T. timopheevii but after the divergence of T. durum. In a separate amplification event, Spelt52 copy number expanded in the common ancestor of Ae. longissima and Ae. sharonensis.     

Comparative study of genomes of two species of flax by C-banding of chromosomes

C-banding patterns of the karyotypes of two closely related wild flax species, Linum austriacum L. (2n = 18) and Linum grandiflorum Desf. (2n = 16), were studied. The karyotypes of both species were similar in the chromosome morphology and size. In each species, metacentric and acrocentric chromosomes (1.7-4.3 microns) and one satellite chromosome were observed. In the karyotypes of the species studied, all homologous chromosome pairs were identified, and quantitative ideograms were constructed. Eight chromosome pairs in the two species had similar C-banding patterns. A low level of intraspecific polymorphism in the intercalary and telomeric C-bands was shown in both species. The results indicate that the genomes of two flax species originated from one ancestral genome with the main chromosome number of 8 or 9. Apparently, the doubling of chromosome number or loss of one chromosome with subsequent redistribution of the chromosome material in the ancestral form resulted in the divergence into two species, L. austriacum L. and L. grandiflorum Desf. A considerable similarity of chromosomes in these species provides evidence for their close phylogenetic relatedness, which makes it possible to place them in one section within the Linum genus.     

Peritrophic matrix in the midgut of tick females of the genus Ixodes (Acari: Ixodidae)

The formation of the peritrophic matrix in the midgut of females of 5 ixodid tick species (Ixodes pacificus, I. pavlovskyi, I. persulcatus, I. ricinus and I. scapularis) was studied by means of light and electron microscopy in different periods of the feeding and after detachment. The formation of the peritrophic matrix started when the first food portions came into the gut lumen, 9-12 hours after the attachment. Renovation of the peritrophic matrix took place during the whole feeding period; every new generation of midgut cells synthesized their own matrix. It was deposited on the apical surface of every midgut cell in the beginning of differentiation, and was functioning during the life of the cell. The peritrophic matrix separates spaces of the cavitary and cytozoic digestions.     

Transovarial and transphasic transmissions of Borrelia by the taiga tick Ixodes persulcatus (Ixodidae)

Three generations of the taiga tick Ixodes persulcatus, the descendants from naturally infected females, have been examined by means of dark field and phase contrast microscopies and indirect immunofluorescent reactions with monoclonal antibodies. Location of borreliae in oocytes was examined by means of electron microscopy. The examined ticks derived from 9 females collected in the Novgorod Province, from 6 females of 1st laboratory generation and 5 females of the 2nd generation. In total, 250 larvae, 178 nymphs, 59 females and 70 males of three consequent generation have been examined. Almost 100% of descendants of naturally infected females were infected with Borrelia burgdorferi s. l. and similar infection rate was observed in unfed tick larvae collected in field conditions. The borreliae received transovarially to larvae of the 1st generation then were transmitted to 100% nymphs and imago of this generation and two next generations.     

Comparative cytogenetic study of the forms of Macleaya cordata (Willd.) R. Br. from different localities

A comparative cytogenetic study of two introduced forms of Makleaya cordata (Willd.) R. Br. = syn. Bocconia cordata Willd. grown in different ecological and geographical regions (Moscow and Donetsk areas) was carried out. In the study, a complex of methods utilizing various chromosomal markers, i.e., C- and DAPI-banding technique, fluorescence in situ hybridization (FISH) with probes of26S and 5S rDNA, as well as estimation of the total area of C-positive regions (C-HCH) in prophase nucleoli and meiosis analysis, was used. In the karyotypes (2n = 20), each chromosome was identified on the basis of C-banding and FISH patterns and the chromosome ideograms were built. Pericentrometric and telomeric C-positive bands in chromosomes of the Moscow form karyotype were found to be smaller and intercalary bands, larger than the corresponding bands in the M. cordata form grown in Donetsk. It was found that the content of C-HCH in prophase nucleoli in the form of M. cordata grown in Donetsk was higher than in the form grown in Moscow. In both forms sites of 26S rDNA and 5s rDNA were localized on satellite chromosome 1 and on chromosome 4 respectively but the signals were more intensive in the plant form grown in Donetsk. The results of this study enable selecting M. cordata forms for use in pharmacology and recommending them for cultivation in various ecological and geographical regions.