Multifunctional magnetic nanoparticles for enhanced intracellular drug transport.

In this paper we report the synthesis and characterization of biocompatible multi-functional magnetic nanoparticles (MNPs) able to enhance the intracellular transport of N-methylated drugs. The Fe3O4 magnetic core was first functionalized with a mixed monolayer consisting of two different phosphonic acids having terminal acetylenic and amino groups, which provide an active platform for further functionalization with organic molecules. Then, a tetraphosphonate cavitand receptor (Tiiii) bearing an azide moiety and the N-hydroxysuccinimide (NHS) activated forms of poly(ethylene glycol) (PEG), folic acid (FA) and carboxy-X-rhodamine (Rhod) were covalently anchored on alkyne and amine moieties respectively, through 1,3-dipolar cycloaddition and EDC/NHS coupling reactions. The obtained MNPs are biocompatible and possess magnetic, luminescence and recognition properties which make them suitable for multimodal theranostic applications. In particular, combined confocal microscopy and cytotoxicity experiments showed that these multi-functional MNPs are able to recognize a specific drug "in situ" and promote its cellular internalization, thus enhancing its efficiency.

Genetic variation in the CHRNA5 gene affects mRNA levels and is associated with risk for alcohol dependence.

Alcohol dependence frequently co-occurs with cigarette smoking, another common addictive behavior. Evidence from genetic studies demonstrates that alcohol dependence and smoking cluster in families and have shared genetic vulnerability. Recently a candidate gene study in nicotine dependent cases and nondependent smoking controls reported strong associations between a missense mutation (rs16969968) in exon 5 of the CHRNA5 gene and a variant in the 3'-UTR of the CHRNA3 gene and nicotine dependence. In this study we performed a comprehensive association analysis of the CHRNA5-CHRNA3-CHRNB4 gene cluster in the Collaborative Study on the Genetics of Alcoholism (COGA) families to investigate the role of genetic variants in risk for alcohol dependence. Using the family-based association test, we observed that a different group of polymorphisms, spanning CHRNA5-CHRNA3, demonstrate association with alcohol dependence defined by Diagnostic and Statistical Manual of Mental Disorders, 4th edn (DSM-IV) criteria. Using logistic regression we replicated this finding in an independent case-control series from the family study of cocaine dependence. These variants show low linkage disequilibrium with the SNPs previously reported to be associated with nicotine dependence and therefore represent an independent observation. Functional studies in human brain reveal that the variants associated with alcohol dependence are also associated with altered steady-state levels of CHRNA5 mRNA.

Linkage disequilibrium maps constructed with common SNPs are useful for first-pass disease association screens.

To develop an efficient strategy for mapping genetic factors associated with common diseases, we constructed linkage disequilibrium (LD) maps of human chromosomes 5, 7, 17, and X. These maps consist of common single nucleotide polymorphisms at an average intermarker distance of 100 kb. The genotype data from these markers in a panel of American samples of European descent were analyzed to produce blocks of markers in strong pair-wise LD. Power calculations were used to guide block definitions and predicted that high-level LD maps would be useful in initial genome scans for susceptibility alleles in case-control association studies of complex diseases. As anticipated, LD blocks on the X chromosome were larger and covered more of the chromosome than those found on the autosomes.

[Localization of DNA sequences tightly associated with synaptonemal complex in compositional fractions of golden hamster]. / Lokalizatsiia posledovatel'nostei DNK, prochno assotsirovannykh s sinaptonemym kompleksom, v kompozitsionnykh fraktsiiakh zolotistogo khomiachka.

The synaptonemal complex isolated from the spermatocyte nuclei by exhaustive hydrolysis of the latter by DNase II contains tightly associated DNA sequences (SCAR DNA). Here we studied the compositional properties of a cloned family of SCAR DNA of golden hamster, namely we performed the localization of 27 SCAR DNA clones on compositionally fractionated genomic DNA from golden hamster. We observed that sequences of the SCAR DNA family are mainly localized in the GC-poor isochore families L1 and L2, that showed 63% hybridization signals. This means that 37% of signals is referred to the GC-rich isochores, indicating the presence of SCAR DNA overall the genome, even if each isochore family presents differences in density and sequence type. Moreover, the SCAR DNA sequences containing regions of homology with LINE/SINE repeats were observed in all the isochore families. The compositional localization of SCAR DNA is in agreement with the hypothesis that SC and SCAR DNA participate in the chromatin organization during the meiosis prophase I, which should result in the attachment of chromatin loops to lateral elements of SC along the whole length of the latter.

[Comparative compositional mapping of chicken and quail chromosomes]. / Sravnitel'noe kompozitsionnoe kartirovanie khromosom kuritsy i perepela.

The distribution of various isochore families on mitotic chromosomes of domestic chicken and Japanese quail was studied by the method of fluorescence in situ DNA--DNA hybridization (FISH). DNA of various isochore families was shown to be distributed irregularly and similarly on chromosomes of domestic chicken and Japanese quail. The GC-rich isochore families (H2, H3, and H4) hybridized mainly to microchromosomes and a majority of macrochromosome telomeric regions. In chicken, an intense fluorescence was also in a structural heterochromatin region of the Z chromosome long arm. In some regions of the quail macrochromosome arms, hybridization was also with isochore families H3 and H4. On macrochromosomes of both species, the pattern of hybridization with isochores of the H2 and H3 families resembled R-banding. The light isochores (L1 and L2 families) are mostly detected within macrochromosome internal regions corresponding to G bands, whereas microchromosomes lack light isochores. Although mammalian and avian karyotypes differ significantly in organization, the isochore distribution in genomes of these two lineages of the warm-blooded animals is similar in principle. On macrochromosomes of the two avian species studied, a pattern of isochore distribution resembled that of mammalian chromosomes. The main specific feature of the avian genome, a great number of microchromosomes (about 30% of the genome), determines a compositional specialization of the latter. This suggests the existence of not only structural but also functional compartmentalization of the avian genome.

Crossing over in chicken oogenesis: cytological and chiasma-based genetic maps of the chicken lampbrush chromosome 1.

Chiasmata in diplotene bivalents are located at the points of physical exchange (crossing-over) between homologous chromosomes. We have studied chiasma distribution within chicken lampbrush chromosome 1 to estimate the crossing-over frequency between chromosome landmarks. The position of the centromere and chromosome region 1q3.3-1q3.6 on lampbrush chromosome 1 were determined by comparative physical mapping of the TTAGGG repeats in the chicken mitotic and lampbrush chromosomes. The comparison of the chiasma (=crossing over)-based genetic distances on chicken chromosome 1 with the genetic linkage map obtained in genetic experiments showed that current genetic distances estimated by the high-resolution genetic mapping of the East Lansing, Compton, and Wageningen chicken reference populations are 1.2-1.9 times longer than those based on chiasma counts. Conceivable reasons for this discrepancy are discussed.

Human chromosomal banding by in situ hybridization of isochores.

Further to the classical methods that involve different chromosome treatments followed by staining, in situ hybridization of isochores represents a novel approach to chromosomal banding. Isochores are long compositionally homogeneous DNA segments that, in the human genome, belong to five families, two GC-poor families (L1 and L2) representing 30% and 33% of the genome, respectively, and three GC-rich families (H1, H2 and H3) representing 24%, 7.5% and 4-5- of the genome, respectively. Gene concentration increases with increasing GC levels, reaching an up to 20-fold higher level in H3 compared to L1 isochores. In situ hybridization of DNA from different isochore families on metaphase chromosomes allow to distinguish different sets of Giemsa and Reverse bands. In addition, it also provides information on the chromosomal distribution of genes.

Compositional mapping of chicken chromosomes and identification of the gene-richest regions.

'Compositional chromosomal mapping', namely the assessment of the GC level of chromosomal bands, led to the identification, in the human chromosomes, of the GC-richest H3+ bands and of the GC-poorest L1+ bands, which were so called on the basis of the isochore family predominantly present in the bands. The isochore organization of the avian genome is very similar to those of most mammals, the only difference being the presence of an additional, GC-richest, H4 isochore family. In contrast, the avian karyotypes are very different from those of mammals, being characterized, in most species, by few macrochromosomes and by a large number of microchromosomes. The 'compositional mapping' of chicken mitotic and meiotic chromosomes by in-situ hybridization of isochore families showed that the chicken GC-richest isochores are localized not only on a large number of microchromosomes but also on almost all telomeric bands of macrochromosomes. On the other hand, the GC-poorest isochores are generally localized on the internal regions of macrochromosomes and are almost absent in microchromosomes. Thus, the distinct localization of the GC-richest and the GC-poorest bands observed on human chromosomes appears to be a general feature of chromosomes from warm-blooded vertebrates.

Genes, isochores and bands in human chromosomes 21 and 22.

The recently available DNA sequences from chromosomes 21 and 22 enabled us to define the relationships of different band types with isochores and with gene concentration and to compare these relationships with previous results. We showed that chromosomal bands appear as Giemsa or Reverse bands depending not on their absolute GC level, but on the composition GC level relative to those of adjacent contiguous bands. We also demonstrated that the GC-richest, and gene-richest H3+ bands are characterized by a lower DNA compaction compared with the GC-poorest, gene-poorest L1+ bands. Moreover, our results indicate that the human genome contains about 30,000 genes.

Purification of the aldehyde oxidase homolog 1 (AOH1) protein and cloning of the AOH1 and aldehyde oxidase homolog 2 (AOH2) genes. Identification of a novel molybdo-flavoprotein gene cluster on mouse chromosome 1.

We report the cloning of the AOH1 and AOH2 genes, which encode two novel mammalian molybdo-flavoproteins. We have purified the AOH1 protein to homogeneity in its catalytically active form from mouse liver. Twenty tryptic peptides, identified or directly sequenced by mass spectrometry, confirm the primary structure of the polypeptide deduced from the AOH1 gene. The enzyme contains one molecule of FAD, one atom of molybdenum, and four atoms of iron per subunit and shows spectroscopic features similar to those of the prototypic molybdo-flavoprotein xanthine oxidoreductase. The AOH1 and AOH2 genes are 98 and 60 kilobases long, respectively, and consist of 35 coding exons. The AOH1 gene has the potential to transcribe an extra leader non-coding exon, which is located downstream of exon 26, and is transcribed in the opposite orientation relative to all the other exons. AOH1 and AOH2 map to chromosome 1 in close proximity to each other and to the aldehyde oxidase gene, forming a molybdo-flavoenzyme gene cluster. Conservation in the position of exon/intron junctions among the mouse AOH1, AOH2, aldehyde oxidase, and xanthine oxidoreductase loci indicates that these genes are derived from the duplication of an ancestral precursor.

Telomeres in warm-blooded vertebrates are composed of GC-rich isochores.

We have hybridized the vertebrate telomeric sequence (TTAGGG)n on DNA compositional fractions from 13 mammalian species and 3 avian species, representing 9 and 3 orders, respectively. Our results indicate that the 50- to 100-kb fragments derived from telomeric regions are composed of GC-rich and GC-richest isochores. Previous works from our laboratory demonstrated that single-copy sequences from the human H3 isochore family (the GC-richest and gene-richest isochore in the human genome) share homology with compositionally correlated compartments of warm-blooded vertebrates. This correlation suggested that the GC-richest isochores are, as in the human genome, the gene-richest regions of warm-blooded vertebrates' genome. Moreover, this evidence suggests that telomeric regions are the most gene-dense region of all warm-blooded vertebrates. The implications of these findings are discussed.

Molecular structure and evolution of DNA sequences located at the alpha satellite boundary of chromosome 20.

We have isolated and characterised one PAC clone (dJ233C1) containing a linkage between alphoid and non-alphoid DNA. The non-alphoid DNA was found to map at the pericentromeric region of chromosome 20, both on p and q sides, and to contain homologies with one contig (ctg176, Sanger Centre), also located in the same chromosome region. At variance with the chromosome specificity shown by the majority of non-alphoid DNA, a subset of alphoid repeats derived from the PAC yielded FISH hybridisation signals located at the centromeric region of several human chromosomes, belonging to three different suprachromosomal families. The evolutionary conservation of this boundary region was investigated by comparative FISH experiments on chromosomes from great apes. The non-alphoid DNA was found to have undergone events of expansion and transposition to different pericentromeric regions of great apes chromosomes. Alphoid sequences revealed a very wide distribution of FISH signals in the great apes. The pattern was substantially discordant with the data available in the literature, which is essentially derived from the central alphoid subset. These results add further support to the emerging opinion that the pericentromeric regions are high plastics, and that the alpha satellite junctions do not share the evolutionary history with the main subsets.

Gene density in the Giemsa bands of human chromosomes.

The human genome is formed by isochores belonging to five families, L1, L2, H1, H2 and H3, that are characterized by increasing GC levels and gene concentrations. In-situ hybridization of DNA from different isochore families provides, therefore, information not only on the correlation between isochores and chromosomal bands, but also on the distribution of genes in chromosomes. Three subsets of R(everse) bands were identified: H3+, H3* and H3-, that contain large, moderate, and no detectable amounts, respectively, of the gene-richest H2 and H3 isochores, and replicate very early and early, respectively, in S phase of the cell cycle. Here, we investigated the GC levels, replication timings and DNA compaction of G(iemsa) bands. We showed that G bands comprise two different subsets of bands, one of which is predominantly composed of L1 isochores, replicates at the end of the S phase, has a higher DNA compaction relative to H3+ bands and corresponds to the darkest G bands of Francke (1994). In contrast, the other subset is composed of L2 and H1 isochores, has less-extreme properties in replication and composition and corresponds to the less-dark G bands of Francke.

Identification of the gene-richest bands in human prometaphase chromosomes.

The human genome is a mosaic of long, compositionally homogeneous DNA segments, the isochores, that can be partitioned into five families, two GC-poor families (L1 and L2), representing 63% of the genome, and three GC-rich families (H1, H2 and H3), representing 24%, 7.5% and 4-5% of the genome, respectively. Gene concentration increases with increasing GC levels, reaching a level 20-fold higher in H3 compared with L isochores. In-situ hybridization of DNA from different isochore families provides, therefore, information on the chromosomal distribution of genes. Using this approach, three subsets of reverse or Giemsa-negative bands, H3+, H3* and H3-, containing large, moderate, and no detectable amounts, respectively, of the gene-richest H3 isochores were identified at a resolution of 400 bands. H3+ bands largely coincide with the most heat-denaturation-resistant bands, the chromomycin-A3-positive, DAPI-negative bands, the bands with the highest CpG island concentrations, and the earliest replicating bands. Here, we have defined the H3+ bands at a 850-band resolution, and have thus identified the human genome regions, having an average size of 4 Mb, that are endowed with the highest gene density.

The human serum deprivation response gene (SDPR) maps to 2q32-q33 and codes for a phosphatidylserine-binding protein.

The serum deprivation response gene (SDPR, alias sdr) has been previously isolated for its high mRNA expression in serum-starved cells compared to contact-inhibited NIH3T3 cells; such regulation is not observed in single-oncogene transformed NIH3T3 cells after serum starvation. More recently Sdpr has been identified as a substrate of protein kinase C (PKC): this interaction determines the compartimentalization of PKC to caveolae, a plasma membrane invagination of which Sdpr is a major component. Lack of Sdpr-PKC interaction in transformed cells has been proposed to be involved in the alteration of PKC subcellular localization and substrate specificity. Here we report the cloning of the human SDPR homologue (HGMW-approved symbol SDPR) and its mapping to 2q32-q33 in the human genome. In analogy with the murine system, SDPR mRNA expression is increased when human fibroblasts are serum starved, it becomes down-regulated during synchronous cell-cycle reentry, but it is not induced in cells arrested by contact inhibition. Analysis of SDPR expression in human tissues reveals a near ubiquitous expression, with highest levels found in heart and lung. We show that human SDPR encodes PS-p68, a previously characterized phosphatidylserine-binding protein purified from human platelets. Accordingly, recombinant Sdpr is able to specifically bind phosphatidylserine in the absence of Ca2+. SDPR is homologous to two genes in the databank, one of which, srbc, is similarly regulated during growth arrest and encodes a phosphatidylserine-binding protein that is a substrate of PKC.

The mouse aldehyde oxidase gene: molecular cloning, chromosomal mapping and functional characterization of the 5'-flanking region.

In this article, we report on the chromosome mapping and molecular cloning of the genetic locus encoding the mouse molybdo-iron/sulfur-flavoprotein aldehyde oxidase. The aldehyde oxidase locus maps to mouse chromosome 1 band C1-C2, as determined by fluorescence in situ hybridization experiments conducted on metaphase chromosomes. The gene is approximately 83 kb long and consists of 35 exons. The exon/intron boundaries are perfectly conserved relative to the corresponding human homolog and almost completely conserved relative to the mouse xanthine oxidoreductase gene. This further supports the concept that the aldehyde oxidase and xanthine oxidoreductase loci evolved from the same ancestral precursor by a gene duplication event. The position of a major transcription start site was defined by primer extension and RNase mapping analysis. The 5'-flanking region of the mouse aldehyde oxidase gene contains a functional and orientation-dependent promoter as well as several putative binding sites for known cell-specific and general transcription factors. Deletion analysis of the 5'-flanking region defines an approximately 470 bp DNA stretch which is necessary and sufficient for the transcription of the mouse aldehyde oxidase gene.

The gene-richest bands of human chromosomes replicate at the onset of the S-phase.

Previous investigations on the correlations between isochore organization and human chromosomal bands have identified three sets of R(everse) bands: H3+, H3* and H3-, endowed with large, moderate, and no detectable amounts of the gene-richest H3 isochores, respectively. In the present work we compared the replication timing of these three sets of bands and showed that the chromosomal bands containing H3 isochores replicate almost entirely (in the case of H3+ bands) or largely (in the case of H3* bands) at the onset of S phase, whereas chromosomal bands not containing H3 isochores (H3- bands) replicate later. The existence, at a resolution of 400 bands per haploid genome, of at least three distinct subsets of R bands is, therefore, not only supported by their GC and gene concentration but also by their replication times.

cDNA characterization and chromosome mapping of the human GAS2 gene.

Murine Gas2 is a microfilament-associated protein whose expression is increased at growth arrest in mammalian cells. During apoptosis, Gas2 is specifically cleaved at its C-terminus by a still unknown ICE-like protease, and the processed protein induces dramatic rearrangements in the cytoskeleton when overexpressed in several cell types. Here we report the characterization of a cDNA encoding the human homologue of Gas2, showing high conservation with the murine counterpart at the protein level. Fluorescence in situ hybridization analysis and radiation hybrid mapping localized the GAS2 gene on human chromosome 11p14.3-p15.2, in a region homologous to the gas2 region on mouse chromosome 7.