Atomic Force Microscope nanolithography on chromosomes to generate single-cell genetic probes.

BACKGROUND: Chromosomal dissection provides a direct advance for isolating DNA from cytogenetically recognizable region to generate genetic probes for fluorescence in situ hybridization, a technique that became very common in cyto and molecular genetics research and diagnostics. Several reports describing microdissection methods (glass needle or a laser beam) to obtain specific probes from metaphase chromosomes are available. Several limitations are imposed by the traditional methods of dissection as the need for a large number of chromosomes for the production of a probe. In addition, the conventional methods are not suitable for single chromosome analysis, because of the relatively big size of the microneedles. Consequently new dissection techniques are essential for advanced research on chromosomes at the nanoscale level. RESULTS: We report the use of Atomic Force Microscope (AFM) as a tool for nanomanipulation of single chromosomes to generate individual cell specific genetic probes. Besides new methods towards a better nanodissection, this work is focused on the combination of molecular and nanomanipulation techniques which enable both nanodissection and amplification of chromosomal and chromatidic DNA. Cross-sectional analysis of the dissected chromosomes reveals 20 nm and 40 nm deep cuts. Isolated single chromosomal regions can be directly amplified and labeled by the Degenerate Oligonucleotide-Primed Polymerase Chain Reaction (DOP-PCR) and subsequently hybridized to chromosomes and interphasic nuclei. CONCLUSIONS: Atomic force microscope can be easily used to visualize and to manipulate biological material with high resolution and accuracy. The fluorescence in situ hybridization (FISH) performed with the DOP-PCR products as test probes has been tested succesfully in avian microchromosomes and interphasic nuclei. Chromosome nanolithography, with a resolution beyond the resolution limit of light microscopy, could be useful to the construction of chromosome band libraries and to the molecular cytogenetic mapping related to the investigation of genetic diseases.

Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation.

The Marek's disease virus (MDV) integration may induce a novel organization of chromatin architecture with a modified genetic expression. In our opinion it is worthwhile trying to relate cytogenetic stability to functional modifications. Recently, atomic force microscopy technique was applied to study the structure of chromosomes at a nanoscale level. This high resolution allows to investigate the different structure of chromatin in order to study cytogenetic stability and chromosome aberrations due to MDV insertion. In this paper data are presented indicating a duplication [78,WZ,dup(1p)(p22-p23)] and a deletion [78,WZ cht del(3)(q2.10)] of Chromosomes 1 and 3 relatively. Relationships between GTG (G-bands by Trypsin using Giemsa) bands and the topography of chromosomes are also discussed, naming them Topographic Banding. The architecture of chromosomes observed by AFM can be related to the data obtained with classic banding techniques thus overcoming the optical resolution limits. The presence of chromatin bridges between sister chromatids at most of the heterochromatic regions is also evidenced. Besides, we present different studies of the longitudinal and transversal symmetry of the hetero and euchromatic regions to clearly demonstrate a different underlying architecture of these regions. It is indeed evident that the heterochromatic bands are more symmetrical than euchromatic bands.

Effects of transferrins and cytokines on nitric oxide production by an avian lymphoblastoid cell line infected with Marek's disease virus.

Marek's disease virus (MDV), the causative agent of Marek's disease (MD), is a herpesvirus that infects poultry causing T lymphomas. Although vaccination may prevent lymphomas formation, it is not known whether it controls viral replication and spreading in the environment. Ovotransferrin (Otrf), a member of the transferrin family, is known to exert in vitro antiviral activity in primary cultures of chicken embryo fibroblasts (CEF). In addition Otrf is produced by CEF and by an avian lymphoblastoid cell line (MDCC-MSB1) following infection/reinfection with MDV. The present work was designed to investigate the effects of reinfection and of Otrf and lactoferrin (Lf) on the production by MDCC-MSB1 of nitric oxide (NO), a molecule naturally exerting an antiviral activity. These effects were also tested with two cytokines (IL-8 and IFN-gamma), alone and in association with transferrins. Synergy was found between Otrf and IFN-gamma, thus suggesting a possible role in a complementary or alternative strategy against MDV spreading.

Ovotransferrin expression and release by chicken cell lines infected with Marek's disease virus.

Mammals posses both serum transferrin and lactoferrin, whose functions are taken over in birds by ovotransferrin, displaying both iron transport and antibacterial activities. Ovotransferrin also exerts antiviral activity towards Marek's disease virus, an avian member of the herpes family of viruses. This virus infects lymphoid organs and induces the transcription of ovotransferrin in infected chicken embryo fibroblasts. However, it has not yet been established whether ovotransferrin gene transcription is linked to the release of the protein outside the cells or whether ovotransferrin expression and release also occurs in chicken lymphoblastoid cells in which the Marek's disease viral genome is integrated. Our results indicate that both serum and egg-white isoforms of ovotransferrin are expressed and released in the supernatants of chicken embryo fibroblast and lymphoblastoid cells in the absence of infection. Viral infection of chicken embryo fibroblasts caused a slight increase of ovotransferrin release, whereas viral reinfection of lymphoblastoid cells caused a remarkable ovotransferrin release in a virus concentration-dependent manner. These findings suggest that ovotransferrin release in vivo may play a crucial role in protecting the whole organism from viral infection spreading, and support the hypothesis that the antiviral activity of ovotransferrin is an important part of the innate immune response in birds, resembling the antiviral activity of lactoferrin in mammals.

Antiviral activity of ovotransferrin derived peptides.

Ovotransferrin and lactoferrin are iron-binding proteins with antiviral and antibacterial activities related to natural immunity, showing marked sequence and structural homologies. The antiviral activity of two hen ovotransferrin fragments DQKDEYELL (hOtrf(219-227)) and KDLLFK (hOtrf(269-301) and hOtrf(633-638)) towards Marek's disease virus infection of chicken embryo fibroblasts is reported here. These fragments have sequence homology with two bovine lactoferrin fragments with antiviral activity towards herpes simplex virus, suggesting that these fragments could have a role for the exploitation of the antiviral activity of the intact proteins towards herpes viruses. NMR analysis showed that these peptides, chemically synthetized, did not possess any favourite conformation in solution, indicating that both the aminoacid sequence and the conformation they display in the intact protein are essential for the antiviral activity.

Proteolytic activity of bovine lactoferrin.

Bovine lactoferrin catalyzes the hydrolysis of synthetic substrates (i.e., Z-aminoacyl-7-amido-4-methylcoumarin). Values of Km and kcat for the bovine lactoferrin catalyzed hydrolysis of Z-Phe-Arg-7-amido-4-methylcoumarin are 50 microM and 0.03 s(-1), respectively, the optimum pH value is 7.5 at 25 degrees C. The bovine lactoferrin substrate specificity is similar to that of trypsin, while the hydrolysis rate is several orders of magnitude lower than that of trypsin. The bovine lactoferrin catalytic activity is irreversibly inhibited by the serine-protease inhibitors PMSF and Pefabloc. Moreover, both iron-saturation of the protein and LPS addition strongly inhibit the bovine lactoferrin activity. Interestingly, bovine lactoferrin undergoes partial auto-proteolytic cleavage at positions Arg415-Lys416 and Lys440-Lys441. pKa shift calculations indicate that several Ser residues of bovine lactoferrin display the high nucleophilicity required to potentially catalyze substrate cleavage. However, a definitive identification of the active site awaits further studies.

Antiviral activity of ovotransferrin discloses an evolutionary strategy for the defensive activities of lactoferrin.

Ovotransferrin (formerly conalbumin) is an iron-binding protein present in birds. It belongs to the transferrin family and shows about 50% sequence homology with mammalian serum transferrin and lactoferrin. This protein has been demonstrated to be capable of delivering iron to cells and of inhibiting bacterial multiplication. However, no antiviral activity has been reported for ovotransferrin, although the antiviral activity of human and bovine lactoferrins against several viruses, including human herpes simplex viruses, has been well established. In this report, the antiviral activity of ovotransferrin towards chicken embryo fibroblast infection by Marek's disease virus (MDV), an avian herpesvirus, was clearly demonstrated. Ovotransferrin was more effective than human and bovine lactoferrins in inhibiting MDV infection and no correlation between antiviral efficacy and iron saturation was found. The observations reported here are of interest from an evolutionary point of view since it is likely that the defensive properties of transferrins appeared early in evolution. In birds, the defensive properties of ovotransferrin remained joined to iron transport functions; in mammals, iron transport functions became peculiar to serum transferrin, and the defensive properties towards infections were optimised in lactoferrin.