Abstract: In human genomics, the high throughput analysis of general databases promises to provide relevant information and to produce novel biolgical knowledge. This strategy might be applied as well to the reconstruction of transcriptional profiles of different tissues or of the same tissue in different developmental stages (Bortoluzzi and Danieli, 1998). We developed a novel software tool, in order to mine the UniGene database, to retrieve extended datasets and to merge them. By applying this tool, information on 4,080 UniGene clusters were retrieved, belonging to three adult human skeletal muscle cDNA libraries, selected for being unnormalised or unsubtracted. The software processed the resulting records, which were sorted out according to specific criteria. In particular, for the present work, the field 'ESTs SEQUENCES' was considered. For each entry, the number of ESTs obtained from skeletal muscle cDNA libraries was annotated. If additional ESTs, obtained from different tissues, were reported in the record, their presence was also automatically annotated by the program, to produce information on the expression of the corresponding gene in different human tissues. The level of expression of each gene was estimated as a percentage of the total transcriptional activity, by computing the number of the skeletal muscle ESTs corresponding to a given entry, over the total number of skeletal muscle ESTs reported for all the entries included in the catalogue. The basic assumption is that the number of detected ESTs per gene is a function of the transcript frequency in the population of mRNAs. The expressed genes were classified according to their different levels of expression. Only 10% of genes expressed in muscle resulted transcribed at high level. They contribute to more than 50% of the total transcriptional activity of the tissue. The large majority of genes expressed in the adult skeletal muscle appear to be active also in several different tissues. Most skeletal muscle genes were found in at least one additional tissue and about 89% of them in more than 4 additional tissues. Forty-seven entries (1.2% of the total) were found only in cDNA libraries obtained from human skeletal muscle. The validation of this "in silico" approach was attempted by a comparison with SAGE data on genes expressed in skeletal muscle, reported in the Rochester SAGE catalogue (Welle et al, 1999), release July 1999. We considered the expression data concerning the 295 tags corresponding to fully annotated genes highly expressed in skeletal muscle. The reciprocal correspondence between 120 genes belonging to both catalogues was established, by using the GenBank ID and the levels of expression of each gene were compared. The results obtained by the two methods showed a statistically significant concordance (Bortoluzzi et al., 2000). Transcriptional profiles of the adult human skeletal muscle and of the adult human retina, produced in our laboratory, are published at the dedicated web site GETProfiles (http://telethon.bio.unipd.it/GETProfiles/index.html).

Abstract: Uno dei problemi fondamentali della ricerca biologica e' la comprensione dei meccanismi fondamentali che permettono alle cellule differenziate, sebbene totipotenti, di poter selettivamente utilizzare solo una piccola e peculiare porzione del loro enorme potenziale genetico. Infatti, ognuna di esse e' caratterizzata da una particolare configurazione di geni attivi ed inattivi, che subiscono una stretta regolazione della loro espressione sia spaziale che temporale. Per es., le cellule della mano sono tali in quanto esprimono un 'set' di geni in larga misura differente da quello espresso da altri tipi cellulari, come, ad es., le cellule del cervello. Analogamente, le cellule neoplastiche presentano un caratteristico pannello d'espressione genica, che e' alterato rispetto alle cellule sane. Questo controllo dell'attivita' trascrizionale e' in larga misura affidato a delle proteine, i fattori trascrizionali (FT), che controllano, spesso in combinazione con altri FT, l'attivita' di svariate centinaia di geni. I FT esercitano la loro azione legandosi specificamente a piccole sequenze di DNA (i cosiddetti siti di legame), localizzate nelle sequenze regolatrici dei loro geni bersaglio, la cui attivita' viene, di consequenza, diminuita o intensificata. Un metodo per poter analizzare il meccanismo di azione di un FT consiste quindi nella identificazione dei suoi geni-bersaglio. Purtroppo, questa procedura si rileva spesso complessa e costosa da un punto di vista sperimentale e non e' sempre coronata da successo. Un attraente approccio alternativo al problema consiste nel cercare i potenziali siti di legame dei FT direttamente negli enormi databases di sequenze di DNA accumulatisi nel corso degli anni. La natura elusiva e degenerata dei siti di legame rende comunque quest'approccio poco attuabile. Una ricerca nei databases per queste piccole sequenze porta, infatti, a dei risultati in cui la predominanza del 'rumore di fondo' (cioe' l'elevato numero di siti non biologicamente rilevanti) renderebbe virtualmente impossibile la formulazione di ipotesi testabili poi sperimentalmente. Abbiamo quindi sviluppato un software, TargetFinder, che fosse in grado di diminuire notevolmente questo rumore di fondo, preservando allo stesso tempo i reali geni-bersaglio individuati nella ricerca in banca dati. L'euristica utilizzata dal programma si basa sull'incorporazione nella ricerca di semplice considerazioni che tengono conto del contesto biologico ove la ricerca stessa viene effettuata. Sara' illustrato come TargetFinder e' stato utilizzato dal nostro laboratorio per la ricerca di potenziali geni bersaglio del fattore trascrizionale Otx2 murino.

Abstract: Prediction of protein-coding genes in newly sequenced DNA becomes very important in large genome sequencing projects. These problems are complicated due to exon-intron of the eukaryotic genes. Currently existing collections of expressed sequence tags (ESTs) are very large and thus very useful for gene mapping. Gene identification in the newly-discovered DNA sequences is an important problem in current molecular biology studies. A number of programs have been developed for predicting the protein coding genes. The most common approach is based on the combination of the potential functional signals with global statistical properties of protein coding regions. Another approach for gene structure prediction is based on the homology detection throughout the databases of nucleotide or amino acid sequences. By using the information available on homologous protein sequences, it is possible to significantly improve the accuracy of gene structure prediction. Currently existing collections of expressed sequence tags (ESTs) are very large and can be very useful for gene mapping. Homology searches against the EST Division of GenBank (dbEST) and Unigene database can be used for this purpose. ESTs (Expressed Sequence Tags) offer a rapid route to gene identification (Adams, et al, 1991, Adams , et al, 1992), analysis of expression and regulation data, and can highlight multigene family diversity and gene alternative splicing). EST matches may identify more than half of the known human genes (Hillier et al, 1996). The price of the high-volume and high-throughput nature of the data, however, is that ESTs contain high error rates (Aaronson, et al 1996), do not have a defined protein product, are not well annotated and present only a raw substrate for sequence matching. The ESTMAP system involves the following procedures: 1) Repeat masking. The repeated elements (for example, the human Alu elements) can be automatically masked in a query sequence before the homology search. Homology searches against the collection of repeated element (Jurka et al., 1992) are used for repeats detection. We implemented a program REPEAT for that purpose. A censored sequence (with 'N's instead of repeated elements) is automatically produced by REPEAT. 2) Homology searches. BLASTN (Altschul et al. 1990) is used for homology searches of the censored query sequence against the EST Division of GenBank (dbEST) and the Unigene database of sequences (www. ncbi.nlm.nih.gov) This step is most time-consuming since these EST datsets are very large. 3) EST mapping. The BLASTN output is used as input information by a EST_GENE program. Information about an EST sequence is used only when the similarity between the EST sequence and the query sequence is greater then 95%. The module EST_GENE is also able to predict the introns in DNA comparing ESTs and a query sequence based on the alignment method suggested by Huang (1994) (a linear-space divide-and-conquer strategy). The GT/AG splicing sites rule is used by EST_GENE, however non-canonical splicing signals (Milanesi and Rogozin, 1998) can also be predicted in cases of unambiguous alignment. 4) Output of results. The graphical visualization of the results is particularly important for the analysis of alternative splicing in a query sequence. By using a Java based graphical interface the user can visualize the EST maps and the sequence pattern of predicted features. Homology searches are very important for functional mapping, homology with a known functional region can suggest the function of a query sequence. In particular, when the homologous protein sequence is already known and EST matches are detected, then the gene structure can be reconstructed with high accuracy. Information about EST matches is automatically used by the GeneBuilder system (Milanesi et al., 1999). Acknowledgment This work was supported by Italian CNR Genetic Engineering Project

Abstract: Le proteine di P. falciparum mostrano una caratteristica decisamente peculiare: quando vengono confrontate con le proteine omologhe di altri organismi quasi sempre presentano lunghe porzioni di sequenza che separano domini ben conservati. E' stata effettuata una prima analisi sulla gamma-glutamil-cisteina sintetasi di cui sono note le sequenze in due specie differenti di Plasmodio (P. falciparum e P. berghei) (ref.1). I risultati hanno permesso di stabilire che pur mantenendo un carattere essenzialmente idrofilico, le porzioni centrali di queste inserzioni sono caratterizzate da un uso ripetuto di alcuni amminoacidi (zone semplici) e tendono a mutare piu' rapidamente dei loro "bordi". Allo scopo di effettuare una caratterizzazione di tali sequenze sono state prese in considerazione ed analizzate tutte le proteine presenti nel cromosoma 2. Circa l'88% delle proteine esaminate presenta domini a sequenza semplice che vengono comunemente considerati come domini non globulari estrusi dal "core" della struttura proteica senza alcuna funzione nota per la proteina. Abbiamo effettuato una prima analisi statistica su tutte queste regioni considerando la loro distribuzione in lunghezza, la loro distribuzione lungo la sequenza proteica, il numero di inserzioni per proteina, la composizone amminoacidica, la presenza di "tandem repeats" e il carattere prevalentemente idrofilico o idrofobico.