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R Dell'Arciprete

Publications and source records attributed to R Dell'Arciprete.

5 recordsLinked to original sources
PubMed2001

Cyclin D1 gene contains a cryptic promoter that is functional in human cancer cells.

A novel cyclin D1 (CCND1)-TROP2 fusion oncogene has been isolated from human cancer cells. Unexpectedly, the chimeric cDNA was found to express TROP2 in the absence of exogenous promoters. Mutagenesis of the TROP2 and CCND1 sequences and in vitro transcription/translation show that a cryptic promoter is present in the 3' coding region of CCND1. The CCND1 cryptic promoter is functional in luciferase assays, where it augments the basal expression levels by eightfold and efficiently cooperates with an SV-40 enhancer. The transcription start sites of the cryptic promoter map at bases 797 and 935 of CCND1, as determined by RNase protection assays. The cryptic promoter possesses canonical binding sites for ubiquitous transcription factors and W/S, X1, and CAAT/Y boxes that are characteristic of major histocompatibility complex class II gene promoters. Remarkably, the cryptic CCND1 promoter is active in human cancer cells and generates a truncated transcript that contains CCND1 instability sequences. Thus, this novel CCND1 transcription unit may play a role in the regulation of the expression of cyclin D1 and in tumor cell growth.

3T3 Cells↗
PubMed2001

Green fluorescent protein variants fold differentially in prokaryotic and eukaryotic cells.

Better-folding Green Fluorescent Protein (GFP) mutants selected from bacterial screenings are commonly used in widely different cellular environments. However, it is unclear if the folding efficiency of GFPs is invariant in different cell types. In this work, we have analysed the folding properties of GFP variants in bacteria versus mammalian cells. Remarkably, S65T was found to fold at comparable levels with the wild type GFP in bacteria, but at 10-fold lower levels in mammalian cells. On the other hand, Bex1 folded 3-4 times better than the wtGFP or S65T in E. coli, and 10-20-fold or more than 95-fold better, respectively, in mammalian cells. The Vex1 mutant demonstrated similar properties to Bex1. No evidence of differential GFP unfolding in vivo or of preferential degradation of unfolded GFP molecules was found. Moreover, no relationship between GFP folding efficiency and expression levels, or protein stability was detected. Trivial Aconfounding factors, like GFP unfolding caused by different pH or fluorescence quenching due to molecular crowding, were also excluded. In summary, our results demonstrate that specific GFP variants follow different folding trajectories in mammalian versus bacterial cells. The specificity of this differential folding supports a role of chaperones in guiding the folding of GFP in vivo. J. Cell. Biochem. Suppl. 36: 117-128, 2001.

Animals↗
PubMed1996

High-efficiency expression gene cloning by flow cytometry.

Our goal was to develop a convenient and widely applicable procedure for gene cloning based on flow cytometry. To this purpose, we have developed an efficient protocol for DNA transfection and selection of rare transfectants. Transfection by calcium phosphate co-precipitation was extensively investigated. The use of specific batches of calcium chloride, of carrier DNA purified in guanidinium thiocyanate, and of plasmid DNA banded in cesium chloride proved crucial for high efficiency of transfection. Several tissue culture parameters were also found critical. With the optimized procedure we can transfect almost 100% of the COS-7 cells with cDNA encoding cell surface antigens or green fluorescent protein. Moreover, we routinely obtain high average levels of expression. Efficient cell sorting in flow cytometry was achieved by subtracting the cell autofluorescence background, by displacing stained cells in the red dimension, and by combining fluorescein-conjugated primary and secondary antibodies. Efficient recovery of the transfected DNA constructs was obtained from 2500-3000 cells directly sorted in Hirt lysis buffer. Using the above protocol we have cloned by expression the gene encoding Trop-2, a cell surface glycoprotein expressed by human carcinomas.

Animals↗
PubMed1995

Cloning of the gene encoding Trop-2, a cell-surface glycoprotein expressed by human carcinomas.

We have cloned by expression the cDNA encoding Trop-2, a cell-surface glycoprotein expressed by most human carcinomas. Formal proof of the identity of the clone is the hybridization to DNA and RNA from genomic TROP2 transfectants. TROP2 is a single-copy gene in human cells, hybridizes to a single 1.8-kb mRNA from expressing sources and encodes a 35,709 Da type-1 transmembrane protein with a single transmembrane domain. TROP2 is essentially identical to GA733-1. Thus, we have proven that GA733-1, for which a protein product had not been identified, is a functional gene. TROP2 is also homologous to TROP1/KSA/GA733-2, confirming the serological similarities between the 2 molecules. The homology between the Trop-1 and Trop-2 peptides is clustered in 2 extracytoplasmic domains and in the transmembrane/cytoplasmic region. Twelve cysteines and a potential cytoplasmic tyrosine phosphorylation site are also conserved. Trop-1 and Trop-2 are homologous to serum IGF-II-binding proteins and appear as signal transducers. Thus, they likely represent novel cell-surface receptors and may play a role in regulating the growth of carcinoma cells. On the other hand, we have found no evidence for a role of Trop-2 and Trop-1 as homophilic adhesion molecules.

Amino Acid Sequence↗