Biomedical subjects

Chiara Conti

Publications and source records attributed to Chiara Conti.

6 recordsLinked to original sources

PubMed2006

Methods to study replication fork collapse in budding yeast.

Replication of the eukaryotic genome is a difficult task, as cells must coordinate chromosome replication with chromatin remodeling, DNA recombination, DNA repair, transcription, cell cycle progression, and sister chromatid cohesion. Yet, DNA replication is a potentially genotoxic process, particularly when replication forks encounter a bulge in the template: forks under these conditions may stall and restart or even break down leading to fork collapse. It is now clear that fork collapse stimulates chromosomal rearrangements and therefore represents a potential source of DNA damage. Hence, the comprehension of the mechanisms that preserve replication fork integrity or that promote fork collapse are extremely relevant for the understanding of the cellular processes controlling genome stability. Here we describe some experimental approaches that can be used to physically visualize the quality of replication forks in the yeast S. cerevisiae and to distinguish between stalled and collapsed forks.

Blotting, Southern↗

PubMed2005

Human ribosomal RNA gene arrays display a broad range of palindromic structures.

The standard model of eukaryotic ribosomal RNA (rRNA) genes involves tandem arrays with hundreds of units in clusters, the nucleolus organizer regions (NORs). A first genomic overview for human cells is reported here for these regions, which have never been sequenced in their totality, by using molecular combing. The rRNA-coding regions are examined by fluorescence on single molecules of DNA with two specific probes that cover their entire length. The standard organization assumed for rDNA units is a transcribed region followed by a nontranscribed spacer. While we confirmed this arrangement in many cases, unorthodox patterns were also observed in normal individuals, with one-third of the rDNA units rearranged to form apparently palindromic structures (noncanonical units) independent of the age of the donors. In cells from individuals with a deficiency in the WRN RecQ helicase (Werner syndrome), the proportion of palindromes increased to one-half. These findings, supported by Southern blot analyses, show that rRNA genes are a mosaic of canonical and (presumably nonfunctional) palindromic units that may be altered by factors associated with genomic instability and pathology.

Blotting, Southern↗

PubMed2005

Genomic organization of amplified MYC genes suggests distinct mechanisms of amplification in tumorigenesis.

Integration of the human papillomavirus (HPV) genome into the host genome is associated with the disruption of the HPV E2 gene and with amplification and rearrangement of the viral and flanking cellular sequences. Molecular characterization of the genomic structures of coamplified HPV sequences and oncogenes provides essential information concerning the mechanisms of amplification and their roles in carcinogenesis. Using fluorescent hybridization on stretched DNA molecules in two cervical cancer-derived cell lines, we have elucidated the genomic structures of amplified regions containing HPV/myc genes over several hundreds of kilobases. Direct visualization of hybridization signals on individual DNA molecules suggests that overreplication and breakage-fusion-bridge-type mechanisms are involved in the genomic instability associated with HPV cervical cancers. Further analysis from two other genital cancer-derived cell lines reveals a recurrent motif of amplification, probably generated by a common mechanism involving overreplication upon viral integration. Interestingly, different amplification patterns seem to be correlated with the disease outcome, thus providing new insights into HPV-related cancer development and tumor progression.

Female↗

PubMed2003

Paroxysmal dyskinesias in childhood.

We report on clinical features of a large series of patients with paroxysmal dyskinesias. Fourteen patients had paroxysmal kinesigenic dyskinesia, with a mean age at onset of 7.1 years. In thirteen children the condition was idiopathic and nine of them had a positive family history; the remaining one had a Chiari malformation. Response to antiepileptic drugs was good in 60% of the treated patients. Six children had paroxysmal non-kinesigenic dyskinesia, with a mean age at onset of 8.1 years. Five children were symptomatic because of cerebral palsy (two patients), basal ganglia stroke (one patient), and acute inflammatory encephalopathy (one patient); the remaining patient's condition was familial with autosomal-dominant mode of inheritance. Response to medical treatment was unsuccessful contrasting with paroxysmal kinesigenic dyskinesia. Six children had paroxysmal exercise-induced dyskinesia, with a mean age at onset of 5 years. Their condition was idiopathic, with a positive family history in four. Two of these patients had also rolandic epilepsy and writer's cramp, and the syndrome had been previously mapped to chromosome 16. Although there have been great advances in the genetics of paroxysmal dyskinesias in which an ion channel dysfunction has been hypothesized, the diagnosis is still based on clinical grounds. The precise classification of the patients with paroxysmal dyskinesias is important for therapeutic decisions.

Adolescent↗

PubMed2002

A combinatorial approach for fast, high-resolution mapping.

High-resolution physical maps can be used as a scaffold for several subsequent studies, such as sequencing projects and positional cloning of disease genes and genetic elements that regulate gene expression. Here we describe a method for fast, high-resolution physical mapping on stretched DNA molecules, based on a combinatorial multi-FISH approach. Fluorescent labels are assigned to a binary code and probes are identified by a binary tag according to their labeling. To validate the approach, we have mapped eight probes covering a region of about 300 kb on human chromosome 11 with three hybridization assays. This approach enables one to determine the structural organization of a large region by means of the order of its clones, without ambiguities. The structure established in a control cell constitutes a reference for further studies, to detect rearrangements displayed by disease cells and to find differences shown by different cell types and organisms.

Humans↗

PubMed2002

Combing the genome for genomic instability.

Genomic instability is one of the major features of cancer cells. The clinical phenotypes associated with several human diseases have been linked to recurrent DNA rearrangements and dysfunction of DNA replication processes that involve unstable genomic regions. Analysis of these rearrangements, which are frequently submicroscopic and can lead to loss or gain of dosage-sensitive genes or gene disruption, requires the development of sensitive, high-resolution techniques. This will lead to a better understanding of the mechanisms underlying genome instability and a greater awareness of the role of chromosomal rearrangements in disease. A new technology that involves molecular combing, a method that permits straightening and aligning molecules of genomic DNA, should make possible a detailed analysis of genomic events at the level of single DNA molecules. Such a single molecule approach could help to elucidate important properties that are masked in bulk studies.

Cell Transformation, Neoplastic↗