The Gene Targeting Approach of Small Fragment Homologous Replacement (SFHR) Alters the Expression Patterns of DNA Repair and Cell Cycle Control Genes

Cellular responses and molecular mechanisms activated by exogenous DNA that invades cells are only partially understood. This limits the practical use of gene targeting strategies. Small fragment homologous replacement (SFHR) uses a small exogenous wild-type DNA fragment to restore the endogenous wild-type sequence; unfortunately, this mechanism has a low frequency of correction. In this study, we used a mouse embryonic fibroblast cell line with a stably integrated mutated gene for enhanced green fluorescence protein. The restoration of a wild-type sequence can be detected by flow cytometry analysis. We quantitatively analyzed the expression of 84 DNA repair genes and 84 cell cycle control genes. Peculiar temporal gene expression patterns were observed for both pathways. Different DNA repair pathways, not only homologous recombination, as well as the three main cell cycle checkpoints appeared to mediate the cellular response. Eighteen genes were selected as highly significant target/effectors of SFHR. We identified a wide interconnection between SFHR, DNA repair, and cell cycle control. Our results increase the knowledge of the molecular mechanisms involved in cell invasion by exogenous DNA and SFHR. Specific molecular targets of both the cell cycle and DNA repair machineries were selected for manipulation to enhance the practical application of SFHR

A Genotypic-oriented View of CFTR Genetics Highlights Specific Mutational Patterns Underlying Clinical Macro-categories of Cystic Fibrosis.

Cystic Fibrosis (CF) is a monogenic disease caused by mutations of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. The genotype-phenotype relationship in this disease is still unclear, and diagnostic, prognostic and therapeutic challenges persist. We enrolled 610 patients with different forms of CF and studied them from a clinical, biochemical, microbiological and genetic point of view. Overall, 125 different mutated alleles (11 of which with novel mutations and 10 of which complex) and 225 genotypes were found. A strong correlation between mutational patterns at the genotypic level and phenotypic macro-categories emerged. This specificity appears to be largely dependent on rare and individual mutations, as well as on the varying prevalence of common alleles in different clinical macro-categories. However, 19 genotypes appeared to underlie different clinical forms of the disease. The dissection of the pathway from the CFTR mutated genotype to the clinical phenotype allowed to identify at least two components of the variability usually found in the genotype - phenotype relationship. One component seems to depend on the genetic variation of CFTR, the other component on the cumulative effect of variations in other genes and cellular pathways independent from CFTR. The experimental dissection of the overall biological CFTR pathway appears to be a powerful approach for a better comprehension of the genotype - phenotype relationship. However, a change from an allele-oriented to a genotypic-oriented view of CFTR genetics is mandatory, as well as a better assessment of sources of variability within the CFTR pathway

Intracellular immunization with cytosolic recombinant antibodies

We report the application of a strategy to inactivate cellular proteins in vertebrate cells based on the intracellular expression of immunoglobulin genes. We have selected, in this instance, the p21 protein, encoded by the ras proto-oncogene, as a target protein. The variable regions of the neutralizing anti-p21ras monoclonal antibody Y13-259 were cloned in vectors for the expression of either the whole antibody molecule or its single-chain Fv fragment (ScFv) derivative. In order to target the recombinant antibodies to the cytosol, their hydrophobic leader sequence for secretion was mutated or deleted. When these proteins are expressed in the cytosol of Xenopus laevis oocytes they colocalize with the endogenous p21ras protein in the cytoplasmic face of the oocyte plasma membrane, and they markedly inhibit the H1 kinase activity induced by insulin. Moreover, cytosolic anti-p21ras ScFv fragments block the ensuing meiotic maturation. Thus the intracellular expression of both whole antibodies and antibody domains can be used to block a biological function

A template for mutational data analysis of the CFTR gene

Background: Automated DNA sequencing produces large amounts of data that need to be analyzed by appropriate software. Personalization of software can be a difficult and time-consuming task, especially if a large number of mutations have to be analyzed. Methods: The Applied BioSystems SeqScape software, based on the KB basecaller algorithm, is a versatile tool that can be used for mutational analysis and for data quality assessment of sequences belonging to any gene of interest. Using this software we analyzed over 1400 sequences of CFTR exons and adjacent intronic zones, representing over 500,000 bases. Results: We present an up to date specific template and a linked set of instructions for automated labeling of all point mutations and polymorphisms of the CFTR gene, whose mutations cause cystic fibrosis (the most common genetic disease among Caucasian individuals). We also describe our refined software settings for mutational analysis, in order to keep to a minimum the need of manual validation. Conclusions: The use of our template greatly simplifies the mutational analysis of the CFTR gene, reducing human intervention. In our opinion, it might not only be useful to researchers that already perform CFTR mutational analysis by sequencing methods but it should also improve the approach in those laboratories that already use ABI PRISM instrumentation for a limited mutational analysis of the CFTR gene. Similar mutational templates can also be used for other disease causing genes, thus improving molecular genetics protocols

The Impact on Genetic Testing of Mutational Patterns of CFTR Gene in Different Clinical Macrocategories of Cystic Fibrosis.

More than 2000 sequence variations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are known. The marked genetic heterogeneity, poor functional characterization of the vast majority of sequence variations, and an uncertain genotype-phenotype relationship complicate the definition of mutational search strategies. We studied the effect of the marked genetic heterogeneity detected in a case series comprising 610 patients of cystic fibrosis (CF), grouped in different clinical macrocategories, on the operative characteristics of the genetic test designed to fully characterize CF patients. The detection rate in each clinical macrocategory and at each mutational step was found to be influenced by genetic heterogeneity. The definition of a single mutational panel that is suitable for all clinical macrocategories proved impossible. Only for classic CF with pancreas insufficiency did a reduced number of mutations yield a detection rate of diagnostic value. All other clinical macrocategories required an extensive genetic search. The search for specific mutational classes appears to be useful only in specific CF clinical forms. A flowchart defining a mutational search that may be adopted for different CF clinical forms, optimized in respect to those already available, is proposed. The findings also have consequences for carrier screening strategies