Exploiting double exchange Diels-Alder cycloadditions for immobilization of peptide nucleic acids on gold nanoparticles

The generation of PNA-decorated gold nanoparticles (AuNPs) has revealed to be more difficult as compared to the generation of DNA-functionalized ones. The less polar nature of this artificial nucleic acid system and the associated tendency of the neutral poly-amidic backbone to aspecifically adsorb onto the gold surface rather than forming a covalent bond through gold-thiol interaction, combined with the low solubility of PNAs itself, form the main limiting factors in the functionalization of AuNP. Here, we provide a convenient methodology that allows to easily conjugate PNAs to AuNP. Positively charged PNAs containing a masked furan moiety were immobilized via a double exchange Diels-Alder cycloaddition onto masked maleimide-functionalized AuNPs in a one-pot fashion. Conjugated PNA strands retain their ability to selectively hybridize with target DNA strands. Moreover, the duplexes resulting from hybridization can be detached through a retro-Diels-Alder reaction, thus allowing straightforward catch-and-release of specific nucleic acid targets

Furan-PNA : a mildly inducible irreversible interstrand crosslinking system targeting single and double stranded DNA

We here report on the design and synthesis of tailor-made furan-modified peptide nucleic acid (PNA) probes for covalent targeting of single stranded DNA through a crosslinking strategy. After introducing furan-containing building blocks into a PNA sequence, hybridization and furan-oxidation based crosslinking to DNA is investigated. The structure of the crosslinked products is characterized and preliminary investigations concerning the application of these systems to double stranded DNA are shown

Cytogenetic analysis on the holocentric chromosomes of the cabbage aphid Brevicoryne brassicae

Chromatin organization in the holocentric chromosomes of the aphid Brevicoryne brassicae has been investigated at a cytological level after C-banding, NOR, Giemsa, DAPI and CMA(3) staining. C-banding technique showed the presence of heterochromatic bands on both telomeres of the two X chromosomes, whereas only the longest pair of autosomes show distinct intercalary C-positive bands. Moreover, silver staining and fluorescent in situ hybridization (FISH) with a 28S rDNA probe localized rDNA genes on one telomere of each X chromosome; these are the only brightly fluorescent C-positive regions revealed after CMA(3) staining, whereas all other heterochromatic bands are DAPI positive

Distribution and molecular composition of heterochromatin in the holocentric chromosomes of the aphid Rhopalosiphum padi (Hemiptera: Aphididae)

In order to study the structure of holocentric chromosomes in aphids, the localization and the composition of Rhopalosiphum padi heterochromatin and rDNA genes have been evaluated at cytogenetic and molecular level. In particular, heterochromatin resulted located on all the chromosomes both in intercalary and telomeric positions. Moreover, enzymatic digestion of R. padi genome put in evidence a DraI satellite DNA which has been isolated, cloned and sequenced. FISH experiments showed that this satellite DNA clusters in an intercalary C-positive band on the two X chromosomes

Chromosomal mapping reveals a dynamic organization of the histone genes in aphids (Hemiptera: Aphididae)

Despite their involvement in different processes, histone genes have been analysed in few insects. In order to improve the knowledge about this important gene family, genes coding for histones have been analysed in the aphid Acyrthosiphon pisum showing that at the amino acid level, aphid histones are highly conserved. In particular, data from A. pisum confirm that H1 is the most variable of the five histones, whereas histones H3 and H4 are highly conserved with the H3 almost identical from insects to vertebrates. A. pisumhistone genes are organized in a quintet with the H1 gene followed by H2A and H2B genes that are adjacent and transcribed in same directions, in the opposite strand in respect to the H1 gene. At the 3’ end of the histone cluster, genes H3 and H4 constitute an oppositely transcribed pair. The span of the aphid histone genes (more than 7 kb) is greater than the average length of the histone cluster till now reported in insects (about 5 kb). Furthermore, spacers that separate the aphid histone genes vary in length. The histone genes have been mapped in A. pisum and successively in the aphids Myzus persicae and Rhopalosiphum padi showing that they are present in a single large cluster located in an interstitial position of autosomes 1, differently from what reported in the Russian wheat aphid Diuraphis noxia,where histone genes have been localized in a telomere of the two X chromosomes suggesting a dynamic organization of this multigene family in aphids

Genomic and cytogenetic localization of the carotenoid genes in the aphid genome

Data published in the scientific literature suggests a possible link between chromosomal rearrangements involving autosomes 1 and 3 and the presence of red morphs in the peach-potato aphid Myzus persicae (Sulzer). In order to begin a study of this relationship, we analysed the genomic and chromosomal location of genes involved in carotenoid biosynthesis in M. persicae and the pea aphid, Acyrthosiphon pisum (Harris), since carotenoids are the basis of the colour in many aphid species. Genomic analysis identified a DNA sequence containing carotenoid genes in synteny between the 2 species. According to the results obtained using in situ PCR, carotenoid genes were located in a subterminal portion of autosome 1 in both species. The same localization has also been observed in the onion aphid Neotoxoptera formosana Takahashi that, as M. persicae and A. pisum, belongs to the tribe Macrosiphini, thereby suggesting a synteny of this chromosomal region in aphids. In situ PCR experiments performed on 2 M. persicae asexual lineages bearing heterozygous translocations involving autosomes 1 and 3 revealed that carotenoid genes were located within chromosomal portions involved in recurrent rearrangements. We also verified by bioinformatics analyses the presence of fragile sites that could explain these recurrent rearrangements in M. persicae

Karyotype rearrangements and telomere analysis in Myzus persicae (Hemiptera, Aphididae) strains collected on Lavandula sp. plants

Karyotype analysis of nine strains of the peach-potato aphid Myzus persicae (Sulzer, 1776), collected on Lavandula sp. plants, evidenced showed that five of them had a standard 2n = 12 karyotype, one possessed a fragmentation of the X chromosome occurring at the telomere opposite to the NOR-bearing one and three strains had a chromosome number 2n = 11 due to a non-reciprocal translocation of an autosome A3 onto an A1 chromosome. Interestingly, the terminal portion of the autosome A1 involved in the translocation was the same in all the three strains, as evidenced by FISH with the histone cluster as a probe. The study of telomeres in the M. persicae strain with the X fission evidenced that telomerase synthesised de novo telomeres at the breakpoints resulting in the stabilization of the chromosomal fragments. Lastly, despite the presence of a conserved telomerase, aphid genome is devoid of genes coding for shelterin, a complex of proteins involved in telomere functioning frequently reported as conserved in eukaryotes. The absence of this complex, also confirmed in the genome of other arthropods, suggests that the shift in the sequence of the telomeric repeats has been accompanied by other changes in the telomere components in arthropods in respect to other metazoans

Cytogenetic and molecular characterization of the MBSAT1 satellite DNA in holokinetic chromosomes of the cabbage moth, Mamestra brassicae (Lepidoptera)

Digestion of Mamestra brassicae DNA with DraI produced a prominent fragment of approximately 200 by and a ladder of electrophoretic bands with molecular weights which are a multiple of 200 bp. Southern blotting revealed that this ladder is composed of DNA fragments that are multimers of the 200-by DraI band suggesting that DraI isolated a satellite that has been called Mamestra brassicae satellite DNA 1 (MBSAT1). MBSAT1 is the first satellite DNA isolated in Lepidoptera. In-situ DraI digestion of chromosome spreads, together with fluorescent in-situ hybridization, showed that MBSAT1 sequences are clustered in heterochromatin of the sex chromosomes, Z and W. MBSAT1 was 234 bp long with an AT content of 60.7%. The curvature-propensity plot suggested a curvature in the MBSAT1 structure

Starting at the end: telomeres and telomerase in arthropods

Telomere composition and structure have been studied in several arthropods allowing us to better understand the evolution of such an important portion of the eukaryotic chromosomes. Genes coding for telomerase reverse transcriptase (TERT) have been sequenced and studied in few arthropod species only,where they resulted highly transcribed also in somatic tissues suggesting a different TERT regulation in respect to vertebrates. Contrary to the strict conservation of telomeres,subtelomeric regions were more polymorphic and heterogeneous in composition and frequently contained retrotransposable elements that strongly influenced subtelomere evolution