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The term "5´ end mRNA artifact" refers to the incorrect assignment of the first AUG codon in an mRNA, due to the incomplete determination of its 5´ end sequence (Casadei et al., 2003). Since the '70s, the amino acid sequence of gene products has been routinely deduced from the nucleotide sequence of the relative cloned cDNA (DNA complementary to mRNA), according to rules for recognition of the start codon (first-AUG rule, optimal sequence context) and the genetic code (Kozak, 2002). All standard methods for the cloning of cDNA are affected by a potential inability to effectively clone the 5´ region of mRNA. This is due to the reverse transcriptase failure to extend first-strand cDNA along the full length of the mRNA template toward its 5´ end (Sambrook, 2001). The identification of a more complete mRNA 5´ end could reveal an additional upstream AUG – in-frame with the previously determined one – thus extending the predicted amino terminus sequence of the product and avoiding subsequent relevant errors in the experimental study of the relative cDNA (Casadei et al., 2003). The continuous incorporation of information derived from individual and large-scale cDNA sequencing projects, including those specifically designed to characterize mRNA 5´ end (Carninci et al., 1996; Suzuki et al., 2000; Porcel et al., 2004), in the last few years led to continuous improvement of completeness of mRNA reference sequences (e.g., RefSeq), and also to the corresponding protein coding sequences. However, genome browsers do not appear to systematically extract useful information from the ever-increasing vast quantity of EST (expressed sequence tag) data. To date, EST data remain invaluable due to significantly longer continuous RNA sequences they may provide in comparison with the very short fragments typically deposited in current high-throughput nucleotide sequencing databases. We previously used individual EST-based gene model refinement by classic in silico sequence analysis to revise the mRNA sequence of 109 human chromosome 21 protein-coding genes (Casadei et al., 2003). The success of this approach encouraged us to develop a piece of software ("5'_ORF_Extender" software) in order to automate the steps that were previously performed manually, applying it to the Danio rerio (zebrafish) genome (Frabetti et al., 2007). In the present work, we present a modified strategy able to analyze the much more numerous human sequences. Firstly, we fully revised the software algorithm by using pre-computed coordinates of the UCSC-downloaded RefSeqs and ESTs genome alignment data and specific UCSC-downloaded EST sequence entries. Furthermore, we adopted an original quality filter which was able to test if each single EST candidate with sequence information of possible use for extending a known mRNA, was attributed to the same locus of that mRNA by an updated, complete and embedded version of UniGene. Lastly, we automated data summarization for an analyzed genome. Following these improvements, parsing more than 7 million BLAT alignment, 5'_ORF_Extender 2.0 recognized a total of 477 loci, out of the 18,665 human loci represented in the mRNA reference set, as bona fide candidates for extension. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1 (guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1), QARS (glutaminyl-tRNA synthetase) and TDP2 (tyrosyl-DNA phosphodiesterase 2) cDNAs, and the consequences for the functional studies of these loci are discussed. In addition, we generated a list of 20,775 human mRNAs in which the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5´ in the current form. Bibliografia: R. Casadei et al., mRNA 5’ region sequence incompleteness: a potential source of systematic errors in translation initiation codon assignment in human mRNAs, Gene 321 (2003) 185–193. M. Kozak, Pushing the limits of the scanning mechanism for initiation of translation, Gene 99 (2002) 1–34. P. Carninci et al., High-efficiency fulllength cDNA cloning by biotinylated CAP trapper, Genomics 37 (1996) 327–336. F. Frabetti et al., Systematic analysis of mRNA 5’ coding sequence incompleteness in Danio rerio: an automated EST-based approach, Biol. Direct 2 (2007) 34.

Among perinatal stem cells of the umbilical cord, human Wharton’s jelly mesenchymal stem cells (hWJ-MSCs) are of great interest for cell-based therapy approaches in regenerative medicine, showing some advantages over other MSCs. In fact, hWJ-MSCs, placed between embryonic and adult MSCs, are not tumorigenic and are harvested with few ethical concerns. Furthermore, these cells can be easily cultured in vitro, maintaining both stem properties and a high proliferative rate for several passages, as well as trilineage capacity of differentiation. Recently, it has been demonstrated that cytoskeletal organization influences stem cell biology. Among molecules able to modulate its dynamics, Cytochalasin B (CB), a cyto-permeable mycotoxin, influences actin microfilament polymerization, thus affecting several cell properties, such as the ability of MSCs to differentiate towards a specific commitment. Here, we investigated for the first time the effects of a 24 h-treatment with CB at different concentrations (0.1–3 μM) on hWJ-MSCs. CB influenced the cytoskeletal organization in a dose-dependent manner, inducing changes in cell number, proliferation, shape, and nanomechanical properties, thus promoting the osteogenic commitment of hWJ-MSCs, as confirmed by the expression analysis of osteogenic/autophagy markers.

Opioids are considered the oldest drugs known by humans and have been used for sedation and pain relief for several centuries. Nowadays, endogenous opioid peptides are divided into four families: enkephalins, dynorphins, endorphins, and nociceptin/orphanin FQ. They exert their action through the opioid receptors (ORs), transmembrane proteins belonging to the super-family of G-protein-coupled receptors, and are expressed throughout the body; the receptors are the δ opioid receptor (DOR), μ opioid receptor (MOR), κ opioid receptor (KOR), and nociceptin/orphanin FQ receptor (NOP). Endogenous opioids are mainly studied in the central nervous system (CNS), but their role has been investigated in other organs, both in physiological and in pathological conditions. Here, we revise their role in stem cell (SC) biology, since these cells are a subject of great scientific interest due to their peculiar features and their involvement in cell-based therapies in regenerative medicine. In particular, we focus on endogenous opioids’ ability to modulate SC proliferation, stress response (to oxidative stress, starvation, or damage following ischemia–reperfusion), and differentiation towards different lineages, such as neurogenesis, vasculogenesis, and cardiogenesis.

Rhythmic oscillatory patterns sustain cellular dynamics, driving the concerted action of regulatory molecules, microtubules, and molecular motors. We describe cellular microtubules as oscillators capable of synchronization and swarming, generating mechanical and electric patterns that impact biomolecular recognition. We consider the biological relevance of seeing the inside of cells populated by a network of molecules that behave as bioelectronic circuits and chromophores. We discuss the novel perspectives disclosed by mechanobiology, bioelectromagnetism, and photobiomodulation, both in term of fundamental basic science and in light of the biomedical implication of using physical energies to govern (stem) cell fate. We focus on the feasibility of exploiting atomic force microscopy and hyperspectral imaging to detect signatures of nanomotions and electromagnetic radiation (light), respectively, generated by the stem cells across the specification of their multilineage repertoire. The chance is reported of using these signatures and the diffusive features of physical waves to direct specifically the differentiation program of stem cells in situ, where they already are resident in all the tissues of the human body. We discuss how this strategy may pave the way to a regenerative and precision medicine without the needs for (stem) cell or tissue transplantation. We describe a novel paradigm based upon boosting our inherent ability for self-healing.

A wide variety of peptides not only interact with the cell surface, but govern complex signaling from inside the cell. This has been referred to as an “intracrine” action, and the orchestrating molecules as “intracrines”. Here, we review the intracrine action of dynorphin B, a bioactive end-product of the prodynorphin gene, on nuclear opioid receptors and nuclear protein kinase C signaling to stimulate the transcription of a gene program of cardiogenesis. The ability of intracrine dynorphin B to prime the transcription of its own coding gene in isolated nuclei is discussed as a feed-forward loop of gene expression amplification and synchronization. We describe the role of hyaluronan mixed esters of butyric and retinoic acids as synthetic intracrines, controlling prodynorphin gene expression, cardiogenesis, and cardiac repair. We also discuss the increase in prodynorphin gene transcription and intracellular dynorphin B afforded by electromagnetic fields in stem cells, as a mechanism of cardiogenic signaling and enhancement in the yield of stem cell-derived cardiomyocytes. We underline the possibility of using the diffusive features of physical energies to modulate intracrinergic systems without the needs of viral vector-mediated gene transfer technologies, and prompt the exploration of this hypothesis in the near future.

Metal-on-metal (MoM) hip prostheses are known to release chromium and cobalt (Co), which negatively affect the health status, leading to prosthesis explant. Albumin (ALB) is the main serum protein-binding divalent transition metals. Its binding capacity can be affected by gene mutations or modification of the protein N-terminal region, giving the ischaemia-modified albumin (IMA). This study evaluated ALB, at gene and protein level, as marker of individual susceptibility to Co in MoM patients, to understand whether it could be responsible for the different management of this ion. Co was measured in whole blood, serum and urine of 40 MoM patients. A mutational screening of ALB was performed to detect links between mutations and metal binding. Finally, serum concentration of total ALB and IMA were measured. Serum total ALB concentration was in the normal range for all patients. None of the subjects presented mutations in the investigated gene. Whole blood, serum and urine Co did not correlate with serum total ALB or IMA, although IMA was above the normal limit in most subjects. The individual susceptibility is very important for patients’ health status. Despite the limited results of this study, we provide indications on possible future investigations on the toxicological response to Co.

Metal ion release and accumulation is considered to be a factor responsible for the high failure rates of metal-on-metal (MoM) hip implants. Numerous studies have associated the presence of these ions, besides other factors, including a hypoxia‑like response and changes in pH due to metal corrosion leading to the induction of the oxidative stress response. The aim of the present study was to verify whether, in patients with a MoM hip prosthesis, mRNA and protein expression of HMOX‑1 was modulated by the presence of metal ions and whether patients without prostheses exhibit a different expression pattern of this enzyme. The study was conducted on 22 matched pairs of patients with and without prostheses, for a total of 44 samples. Ion dosage was determined using inductively coupled plasma mass spectrometry equipped with dynamic cell reaction. HMOX‑1 gene expression was quantified by reverse transcription-quantitative polymerase chain reaction and HMOX‑1 protein expression was analyzed using an enzyme-linked immunosorbent assay. The results demonstrated that although there were significant differences in the metallic ion concentrations amongst the two groups of patients, there was no correlation between circulating levels of cobalt (Co) and chromium (Cr), and HMOX‑1 gene and protein expression. Additionally, there was no significant difference in the protein expression levels of HMOX‑1 between the two groups. In conclusion, it was demonstrated that circulating Co and Cr ions released by articular prosthetics do not induce an increase in HMOX‑1 mRNA and protein expression at least 3.5 years after the implant insertion. The present study suggests that involvement of HMOX‑1 may be excluded from future studies and suggests that other antioxidant enzymes, including superoxide dismutase, glutathione peroxidase and reductase should be investigated.

Down syndrome critical region gene 1-like 2 (DSCR1L2) belongs to the human DSCR1-like gene family, which also includes DSCR1 and DSCR1L1. Both DSCR1 and DSCR1L1 proteins interact with calcineurin, a calcium/calmodulin-dependent phosphatase. To date, no interactor has been described for DSCR1L2. The aim of this work was to perform a first functional study of DSCR1L2 using yeast two-hybrid analysis conducted on a human heart cDNA library. Here, we report the interaction between DSCR1L2 and the human cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus. This interaction was confirmed by both yeast cotransformation and GST (glutathione-sepharose transferase) fusion protein assay. Moreover, a new DSCR1L2 mRNA isoform, generated by alternative splicing, was identified and cloned in different tissues: it lacks two central exons, encoding the most conserved domains among the DSCR1-like protein family. A quantitative relative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that in heart tissue the normalized expression level ratio for DSCR1L2 and DSCR1L2-E2E5 mRNA isoforms is 3.5 : 1, respectively. The yeast cotransformation and GST fusion protein assay demonstrated the interaction between this new DSCR1L2 variant and the human cardiac troponin I and the prominent role of DSCR1L2 exon 2 in determining binding between both DSCR1L2 isoforms and TNNI3. These data indicate an entirely new role for a DSCR1-like family gene, suggesting a possible involvement of DSCR1L2 in cardiac contraction.

Summary: Extracting the desired data from a database entry for later analysis is a constant need in the biological sequence analysis community; GeneRecords 1.0 is a solution for GenBank biological flat file parsing, as it implements a structured representation of each feature and feature qualifier in GenBank following import in a common database managing system usable in a personal computer (Macintosh and Windows environments). This collection of related databases enables the local management of GenBank records, allowing indexing, retrieval and analysis of both information and sequences on a personal computer. Availability: the current release, including the FileMaker Pro runtime application (built for Windows and Macintosh environments), is freely available at http://apollo11.isto.unibo.it/software/

Regulator of calcineurin 3 (RCAN3) belongs to the human RCAN gene family, which also includes RCAN1 and RCAN2. All three members interact with and inhibit calci- neurin. Based on this effect, several studies have demonstrated a role for RCAN1 and RCAN2 on inflammation, using human umbilical vein endothelial cells (HUVECs) as a model. RCAN1 and 2 are strongly induced by vascular endothelial growth factor (VEGF), inhibit cell proliferation and down- regulate many pro-inflammatory and pro-angiogenic genes. The present work is the first study to investigate the role of RCAN3 on inflammation in HUVECs. RCAN3 isoforms have been characterized and quantified in HUVECs; only those with the same frame are expressed and show a peculiar expression pattern. RCAN3 inhibits HUVEC proliferation both basally and under VEGF or phorbol 12-myristate 13- acetate-stimulated conditions, however it does not modulate gene expression of the chosen inflammatory genes. Results indicate an interesting role for RCAN3 in modulating HUVEC proliferation, independently from the inflammatory and angiogenic processes.

Various tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as an input. TRAM (Transcriptome Mapper) is a new general software tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources, as well as to perform intra-sample and inter-sample data normalization methods, including an original variant of quantile normalization (scaled quantile) useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if chromosomal segments of defined length are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches the genome for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a test biological model, based on a meta-analysis comparison between a human CD34+ hematopoietic progenitor cells sample pool and a megakaryocytic cells sample pool, identifying biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte. The large agreement with classical biological knowledge about megakaryocytopoiesis of TRAM results, obtained without any a priori specific assumption, shows that TRAM can perform integrated analysis of expression data from multiple platforms producing high confidence lists of over/under-expressed chromosomal segments and clustered genes. TRAM is the first complete software usable in a personal computer (Macintosh and Windows environments) designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. In conjunction with our previous implementation of a GenBank [1, 2] and UniGene [3] formats full parsing systems, TRAM may also contribute to the building of a novel, relational, multi-purpose, user-friendly and modular platform for the large-scale integrated analysis of genomic and post-genomic data.

Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, "reference" isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3.

Background CYYR1 is a recently identified gene located on human chromosome 21 whose product has no similarity to any known protein and is of unknown function. Analysis of expressed sequence tags (ESTs) have revealed high human CYYR1 expression in cells belonging to the diffuse neuroendocrine system (DNES). These cells may be the origin of neuroendocrine (NE) tumors. The aim of this study was to conduct an initial analysis of sequence, splicing and expression of the CYYR1 mRNA in human NE tumors. Methods The CYYR1 mRNA coding sequence (CDS) was studied in 32 NE tumors by RT-PCR and sequence analysis. A subtle alternative splicing was identified generating two isoforms of CYYR1 mRNA differing in terms of the absence (CAG- isoform, the first described mRNA for CYYR1 locus) or the presence (CAG+ isoform) of a CAG codon. When present, this specific codon determines the presence of an alanine residue, at the exon 3/exon 4 junction of the CYYR1 mRNA. The two mRNA isoform amounts were determined by quantitative relative RT-PCR in 29 NE tumors, 2 non-neuroendocrine tumors and 10 normal tissues. A bioinformatic analysis was performed to search for the existence of the two CYYR1 isoforms in other species. Results The CYYR1 CDS did not show differences compared to the reference sequence in any of the samples, with the exception of an NE tumor arising in the neck region. Sequence analysis of this tumor identified a change in the CDS 333 position (T instead of C), leading to the amino acid mutation P111S. NE tumor samples showed no significant difference in either CYYR1 CAG- or CAG+ isoform expression compared to control tissues. CYYR1 CAG- isoform was significantly more expressed than CAG+ isoform in NE tumors as well as in control samples investigated. Bioinformatic analysis revealed that only the genomic sequence of Pan troglodytes CYYR1 is consistent with the possible existence of the two described mRNA isoforms. Conclusion A new "subtle" splicing isoform (CAG+) of CYYR1 mRNA, the sequence and the expression of this gene were defined in a large series of NE tumors.

Background All standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5' region of mRNA. The aim of this work was to estimate mRNA open reading frame (ORF) 5' region sequence completeness in the model organism Danio rerio (zebrafish). Results We implemented a novel automated approach (5'_ORF_Extender) that systematically compares available expressed sequence tags (ESTs) with all the zebrafish experimentally determined mRNA sequences, identifies additional sequence stretches at 5' region and scans for the presence of all conditions needed to define a new, extended putative ORF. Our software was able to identify 285 (3.3%) mRNAs with putatively incomplete ORFs at 5' region and, in three example cases selected (selt1a, unc119.2, nppa), the extended coding region at 5' end was cloned by reverse transcription-polymerase chain reaction (RT-PCR). Conclusion The implemented method, which could also be useful for the analysis of other genomes, allowed us to describe the relevance of the "5' end mRNA artifact" problem for genomic annotation and functional genomic experiment design in zebrafish. Open peer review This article was reviewed by Alexey V. Kochetov (nominated by Mikhail Gelfand), Shamil Sunyaev, and Gáspár Jékely. For the full reviews, please go to the Reviewers' Comments section.

Il gene umano RCAN3 (Regulator of Calcineurin 3, anche conosciuto come DSCR1L2), localizzato sul cromosoma 1 (1p36.11), appartiene alla famiglia genica RCAN, di cui gli altri membri sono i geni RCAN1 ed RCAN2. Recentemente il Comitato internazionale HUGO ha proposto per i geni e le proteine di questa famiglia la nomenclatura presentata per la prima volta in questo lavoro; nomenclatura che è stata ampiamente discussa anche nell’ambito di un “Forum article” in via di pubblicazione. Il gene RCAN3 è costituito da 5 esoni e ad oggi sono state identificate due isoforme alternative: RCAN3-2,3,4b,5, che manca di 30 nucleotidi all’inizio dell’esone 4 e codifica per una proteina priva di 10 amminoacidi nella sua porzione centrale (RCAN3-2,3,4b,5) ed RCAN3-2,5 che manca degli esoni 3-4 e codifica per una proteina di 115 amminoacidi, con una porzione proteica C-terminale diversa da RCAN3, a causa di uno scivolamento del modulo di lettura a partire dall’esone 5 (RCAN3-2,5). Ad oggi più di un centinaio di lavori sono stati pubblicati sulla famiglia RCAN e nella maggior parte sono presentati studi sulle funzioni di RCAN1 ed RCAN2. Solo due lavori si sono occupati dei possibili ruoli di RCAN3. Recentemente il nostro gruppo di ricerca, mediante un saggio dei due ibridi condotto in una genoteca di cDNA di cuore umano, ha dimostrato che RCAN3 ed RCAN3-2,5 sono in grado di interagire con la troponina inibitoria cardiaca (TNNI3), una delle principali componenti dell’apparato contrattile muscolare; inoltre Mulero e colleghi hanno identificato nella calcineurina, una fosfatasi calcio-calmodulina dipendente, un altro possibile interattore di RCAN3. Lo scopo del presente lavoro è stato condurre una analisi delle diverse isoforme di RCAN3 e delle rispettive proteine da un punto di vista molecolare, bioinformatico, di espressione genica e funzionale. In primo luogo sono state identificate grazie ad un clonaggio mediante RT-PCR due nuove isoforme di RCAN3, in accordo con l’elevato numero di splicing dimostrato nei geni umani: RCAN3-2,4,5 che manca dell’esone 3 ed RCAN3-2,3,5, che manca dell’esone 4. Sequenze di cDNA per le isoforme alternative di RCAN3 sono state identificate solo in Homo sapiens, utilizzando come strumenti per tale ricerca il programma BLASTN, ECgene Browser e Genome Browser; solo per l’isoforma RCAN3-2,4,5 sono state ritrovate 2 EST. In seguito è stata condotta una RT-PCR quantitativa relativa per analizzare il pattern di espressione delle 5 isoforme note in 7 tessuti umani normali (cuore, cervello, polmone, prostata, testicolo, leucociti, intestino tenue), basandosi su dati precedentemente raccolti che dimostravano come il gene RCAN3 fosse espresso in diversi tessuti.Tutte le isoforme sono risultate espresse nei singoli tessuti e in tutti i tessuti si è dimostrato che l’isoforma più espressa è RCAN3. Inoltre il livello di espressione dell’intero locus RCAN3 è risultato confrontabile in tutti i tessuti, dimostrandone l’ubiquitarietà. Infine, grazie ad un saggio di cotrasformazione in lievito e ad un saggio in vitro di legame alla GST, è stata dimostrata l’interazione delle due nuove isoforme identificate RCAN3-2,4,5 e RCAN3-2,3,5 con la TNNI3, così come si poteva ipotizzare grazie alla presenza dell’esone 2, il cui prodotto è ritenuto essere sufficiente per il legame a tale proteina cardiaca.

L’analisi del trascrittoma (l’insieme degli RNA trascritti) di un tessuto, o di un campione cellulare, è uno degli strumenti fondamentali per comprendere le proprietà biologiche globali del tessuto in esame (System Biology). Attualmente, la metodica più utilizzata per tale analisi è lo studio dei profili di trascrizione mediante microarray, che si basa sulla creazione di sonde specifiche per RNA noti da utilizzare per valutare la quantità degli RNA stessi presenti nel tessuto in esame. Le sonde possono essere costituite da cDNA oppure da oligonucleotidi creati tramite la tecnologia brevettata dalla ditta Affymetrix, che ne fornisce anche la piattaforma d’analisi. Uno degli scopi prioritari degli studi attuali dei profili di trascrizione è determinare quali regioni cromosomiche sono trascritte (mappe di trascrizione) al fine di comprendere i meccanismi di regolazione genica. Abbiamo sviluppato il software “TRAnscript Map Analizer” (TRAMA) per generare mappe di trascrizione genica umana da dati d’espressione, ottenuti da esperimenti sia mediante la piattaforma Affymetrix sia mediante array a cDNA. Il software richiede per funzionare la previa importazione delle informazioni riguardanti i cDNA umani noti, e se necessario quelle relative alle sonde Affymetrix del set umano U133, tutte prelevabili dal sito web “Genome Browser”. Inoltre, occorrono i dati aggiornati della banca dati UniGene, ottenibili anche utilizzando il software “UniGene Tabulator”. In seguito alla importazione dei dati di espressione da analizzare, il software genera una tabella per ogni tipo di cromosoma umano (per un totale di 24 tabelle) in cui ogni riga (record) contiene i dati di un segmento cromosomico di lunghezza fissata dall’utente, in bp, a cui sono associati i dati dell’espressione di quella regione e le informazioni sui geni in essa contenuti (indicati mediante il loro simbolo genico, o il relativo cluster di UniGene se il simbolo non è disponibile). L’espressione di ciascun segmento è visualizzabile come barra colorata dal verde al rosso proporzionalmente all’espressione media del cromosoma, rispettivamente più alta o più bassa. Successivamente, il software genera una seconda tabella per ogni cromosoma, contenente i segmenti con un’espressione pari ad almeno la media dell’espressione genica per quel cromosoma nell’esperimento analizzato. Inoltre il software genera, in una tabella separata, una singola mappa genomica virtuale che dispone in maniera contigua tutti i cromosomi umani, per analizzare l’espressione complessiva del genoma in un singolo esperimento. Il software può elaborare due diversi profili di espressione, generando in questo caso anche una mappa di trascrizione basata sul rapporto di espressione tra i due profili. In questo modo è possibile applicare il programma allo studio dei dati di espressione del genoma umano per la identificazione di "cluster" di geni coespressi (moduli funzionali) localmente definiti sulla mappa. Il software TRAMA è basato su FileMaker Pro 8.5, un gestore di basi di dati (databases) di semplice utilizzo, ed è distribuito come applicazione stand-alone per le piattaforme Macintosh e Windows corredato di una guida descrittiva per l’uso.

The activation of the insulin-like growth factor 1/IGF1 receptor system (IGF1/IGF1R) is a critical event in the transformation and tumorigenicity processes in a wide variety of human tumors. The IGF1/IGF1R system has been recently studied in carcinoid tumors that often arise in the gastrointestinal tract; these tumors are characterized by hypersecretion of bioamines and neuropeptides, leading to functional tumor disease. Two alternatively spliced IGF1R mRNA transcripts have been described to differ by only three nucleotides (CAG) in the coding sequence, resulting in an amino-acid change from the originally described Thr-Gly to an Arg in the extracellular portion of the receptor beta subunit. In transfected Chinese hamster ovary cells, the form without CAG (CAG-) exhibited an approximate 2-fold increase in IGF1 stimulation of activities required for its mitogenic properties. In this study, we examine the relative expression of the two IGF1R mRNA isoforms by a semiquantitative RT-PCR approach using highly standardized conditions, beta-2 microglobulin (B2M) as a reference gene and gel imaging analysis. We analyzed a large series of human neuroendocrine tumors (32 samples) and 9 normal tissues. A significant higher expression of both isoforms in the tumor samples (approximately 2-fold increase) was found, while a constant CAG+/CAG- IGF1R mRNA isoforms of an approximate 3:1 ratio was observed in all tumoral and normal cell types studied. The phylogenetic study of the IGF1R locus in several species suggests that human IGF1R CAG- mRNA isoform is evolutionarily more recent compared to the IGF1R CAG+ mRNA isoform and it could be used by the splicing apparatus at this intron/exon junction with a lower efficiency. This study highlights the relevance of IGF1R mRNA expression in neuroendocrine tumor cells, and the constant presence of 'subtle' alternative splicing for the IGF1R locus.

UniGene è un sistema sperimentale per ripartire in maniera non ambigua le sequenze nucleotidiche contenute in GenBank ed attribuirle a gruppi di sequenze (cluster) rappresentativi di geni trascritti. Nella costruzione di UniGene, ad ogni locus genico è assegnato un gruppo di sequenze di RNA che possono essere determinate sperimentalmente in modo accurato “finished”, etichette di sequenze espresse (EST) o sequenze predette. Come conseguenza, UniGene risulta fondamentale in tutte le analisi bioinformatiche che coinvolgono dati a partire da sequenze nucleotidiche, come l’attribuzione di sonde nucleotidiche utilizzate nell’analisi del profilo d’espressione genica, l’integrazione dei dati tra banche dati differenti, l’identificazione di nuovi geni e la predizione della funzione o, infine, l’analisi di profili d’espressione per specifici tessuti o per posizione di mappa genica. Esistono due modalità per l’accesso ai dati UniGene: la ricerca di specifiche schede attraverso il portale web “Entrez” selezionando UniGene tra la lista di banche dati, o il prelievo dell’intera banca dati dal sito “ftp” abilitato. In entrambi i casi, il risultato è l’ottenimento di un file di testo semplice (flat file) da cui le informazioni necessarie devono essere estratte appositamente per essere utilizzate. Il processo di estrazione dati da un file di testo (parsing) richiede l’applicazione di conoscenze informatiche complesse, come i linguaggi BioPerl, JAVA o C++ utilizzati per analizzare le banche dati MedLine (Oliver et al. 2004) ed Entrez Gene (Liu and Grigoriev, 2005), o l’utilizzo di pacchetti software mirati, ad esempio GeneRecords (D’Addabbo et al. 2004) per analizzare il formato GenBank. Tuttavia, ad oggi, non esistono pubblicazioni riguardanti algoritmi o applicazioni dedicati al parsing del formato UniGene. UniGene Tabulator è stato da noi sviluppato per gestire i dati contenuti nel formato UniGene: il file di testo semplice viene importato direttamente in una banca dati apposita, al cui interno avviene il parsing delle informazioni. La banca dati è costituita da sei tabelle, una per ogni tipo di informazione presente nella scheda UniGene (informazioni generali, somiglianze con altre proteine note, sequenze STS note, dati di relativi alla mappa di trascrizione ed informazioni sulle genoteche utilizzate per la creazione delle EST, associate nel cluster UniGene) relazionate tra loro mediante l’identificativo del cluster UniGene. Ogni “record” di una tabella contiene i dati di una singola sequenza, o di un cluster solo per le informazioni generali, ed è suddiviso in campi che contengono i valori estratti. Ogni campo è indicizzato per eseguire rapidamente ricerche specifiche ed il risultato può essere esportato in un file di testo per essere utilizzato per ulteriori analisi o altre ricerche. UniGene tabulator è basato su FileMaker Pro 8, un gestore di basi di dati di semplice utilizzo, ed è distribuito come applicazione stand-alone per piattaforme Macintosh e Windows corredato di una guida descrittiva delle operazioni utili ad importare il file UniGene con i dati per la specie animale in studio.

È noto che i metodi classici di clonaggio del cDNA sono influenzati da una potenziale incapacità di amplificare efficacemente la regione 5´ dell’mRNA. Dato che la sequenza amminoacidica dei prodotti genici è di norma dedotta dalla sequenza dei nucleotidi del relativo cDNA clonato, è importante verificare che una sequenza codificante identificata (o open reading frame, ORF) sia quanto più possibile completa al relativo 5´. In studi precedenti abbiamo dimostrato, mediante analisi bioinformatica accurata e successivo clonaggio del cDNA (reazione a catena della polimerasi dopo retrotrascrizione, RT-PCR), che in 4 su 109 geni noti localizzati sul cromosoma 21 umano, le ORF annotate erano incomplete all’estremità 5´. Scopo del presente lavoro è stato valutare la completezza nella regione 5´ delle sequenze ORF di mRNA di Danio rerio (zebrafish), organismo modello sempre più utilizzato in studi di genomica. Abbiamo sviluppato un innovativo metodo automatizzato che consente di: 1) confrontare in modo sistematico le EST (expressed sequence tags, etichette di sequenze espresse) disponibili con tutte le sequenze di mRNA conosciute di Danio rerio; 2) identificare eventuali tratti di sequenza da aggiungere nella regione 5´; 3) verificare la presenza di tutte le condizioni necessarie a dimostrare l’esistenza di una nuova ORF estesa. Queste condizioni sono, in breve, la presenza di un nuovo “primo codone AUG” in fase con quello precedentemente descritto e la mancanza di uno qualsiasi dei tre codoni di stop in fase tra il codone di recente identificazione e quello inizialmente annotato. Il nostro programma ha analizzato gli 8.528 mRNAs di Danio rerio estratti dal database RefSeq (disponibili al 31/12/2005) e ha permesso di identificare 265 (3,1%) mRNA con ORF potenzialmente incomplete nella regione 5´. Attraverso RT-PCR e sequenziamento automatico abbiamo dimostrato per tre geni (selt1a, unc119, nppa), scelti tra quelli candidati, che la regione codificante all'estremità 5' era stata caratterizzata in modo incompleto nelle descrizioni originali. La conoscenza delle ORF complete dell’mRNA assume una particolare valenza in zebrafish per gli studi di localizzazione del prodotto genico, di sovraespressione genica nonché per la realizzazione di organismi knockdown mediante oligonucleotidi morfolinici. Il metodo sviluppato, la cui utilizzazione è applicabile anche all’analisi di altri genomi, ha consentito di descrivere in questo lavoro la rilevanza del problema dell’incompleta caratterizzazione delle estremità 5´ degli mRNA e di sottolinearne le possibili conseguenze nell’ambito dell'annotazione genomica e degli esperimenti funzionali eventualmente progettati in zebrafish.

Summary: UniGene Tabulator 1.0 provides a solution for full parsing of UniGene flat file format; it implements a structured graphical representation of each data field present in UniGene following import into a common database managing system usable in a personal computer. This database includes related tables for sequence, protein similarity, sequence-tagged site (STS) and transcript map interval (TXMAP) data, plus a summary table where each record represents a UniGene cluster. UniGene Tabulator enables full local management of UniGene data, allowing parsing, querying, indexing, retrieving, exporting and analysis of UniGene data in a relational database form, usable on Macintosh (OS X 10.3.9 or later) and Windows (2000, with service pack 4, XP, with service pack 2 or later) operating systems-based computers. Availability: The current release, including both the FileMaker runtime applications, is freely available at Contact: pierluigi.strippoli@unibo.it Supplementary information: We also distribute a precalculated implementation for current Homo sapiens (build #190, March 2006) and Danio rerio (zebrafish, build #90, March 2006) UniGene data.

Tramite analisi di sequenze EST (Expressed Sequence Tags) è stato osservato che il gene umano CYYR1 (cysteine/tyrosine-rich 1) mostra un’alta espressione in cellule appartenenti al sistema APUD (Amine Precursor Uptake and Decarboxylation). E’ noto che anomalie del differenziamento cellulare in cellule APUD sono spesso alla base di alcuni tipi di tumore di origine neuroendocrina (APUDomi), come le neoplasie endocrine multiple, i tumori carcinoidi, il feocromocitoma e il melanoma. E’ stata analizzata, mediante RT-PCR specifica e successivo sequenziamento, l’intera sequenza codificante (CDS) dell’mRNA di CYYR1 umano in 32 tumori neuroendocrini (NE). In tutti i campioni analizzati la CDS di CYYR1 è risultata sovrapponibile a quella di riferimento con eccezione della sequenza ottenuta dall’unico tumore localizzato nel collo, un carcinoma scarsamente differenziato con aspetti neuroendocrini. In quest’ultimo caso, l’analisi ha evidenziato la presenza di una mutazione, in eterozigosi, in posizione 333 della CDS (T al posto di C) che porta alla sostituzione amminoacidica P111S. L’analisi di sequenza, inoltre, ha mostrato chiaramente l’esistenza di due isoforme di mRNA dovuta alla presenza di due possibili siti accettori di splicing all’estremo 3¢ dell’introne 3 che termina con la sequenza CAGCAG (splicing alternativo “sottile”). Tale splicing alternativo è presente anche nei tessuti normali. Le due isoforme di mRNA prodotte differiscono per una tripletta CAG (CAG-: assenza della tripletta, CAG+: presenza della tripletta); ne derivano due prodotti proteici putativi diversi per assenza o presenza di un residuo di alanina nel punto di giunzione fra esone 3 ed esone 4. E’ stata successivamente quantificata l’espressione relativa delle due isoforme messaggeriali (CAG- e CAG+) in 29 tumori NE e in 8 tessuti normali tramite un metodo che si basa su una RT-PCR condotta in condizioni altamente standardizzate e stringenti. Il metodo impiega la beta-2 microglobulina (B2M) come gene di riferimento e i risultati della elettroforesi dei prodotti di PCR sono elaborati con un analizzatore di immagini (Gel Doc 2000). Nei campioni tumorali è stata ritrovata una espressione significativamente più bassa della isoforma CAG- rispetto ai controlli normali in un rapporto di 2:3 (p=0,022). Infine abbiamo condotto un’analisi bioinformatica per verificare o meno la presenza delle due isoforme degli ortologhi di CYYR1 in altre specie: solo in Pan troglodites si può ipotizzare l’esistenza delle due isoforme per la presenza, nella sequenza genomica, di un confine introne-esone analogo a quello umano.

Background Formal description of a cell's genetic information should provide the number of DNA molecules in that cell and their complete nucleotide sequences. We pose the formal problem: can the genome sequence forming the genotype of a given living cell be known with absolute certainty so that the cell's behaviour (phenotype) can be correlated to that genetic information? To answer this question, we propose a series of thought experiments. Results We show that the genome sequence of any actual living cell cannot physically be known with absolute certainty, independently of the method used. There is an associated uncertainty, in terms of base pairs, equal to or greater than μs (where μ is the mutation rate of the cell type and s is the cell's genome size). Conclusion This finding establishes an "uncertainty principle" in genetics for the first time, and its analogy with the Heisenberg uncertainty principle in physics is discussed. The genetic information that makes living cells work is thus better represented by a probabilistic model rather than as a completely defined object.

‘Successful aging’, i.e. the ability to attain old age in relatively good health, is believed to be related to the capability to cope with different environmental stresses. Independently of their specific differentiation, all body cells respond to hyperthermia and other stresses with the production of Heat Shock Proteins (HSPs) that play an important role in cell survival. We investigated the heat shock response in B-lymphoid cell lines from 44 centenarians and 23 younger subjects, by studying both HSP70 synthesis and cell survival after hyperthermic treatment. Interestingly, no significant difference could be found between the two age groups as far as HSP70 synthesis was concerned; moreover, cell lines from centenarians appeared to be less prone to heat-induced apoptosis than lines from younger controls. These results, which are in contrast with previous findings showing an age-related decrease of the HSP70 synthesis and of hyperthermic response, corroborate the above mentioned hypothesis that the biological success of centenarians is due to the preservation of the capability to cope with stresses. An A/C polymorphism identified in the promoter region of HSP70-1 gene had been previously shown to affect the probability to attain longevity in females. To investigate if this effect was related to any influence of this polymorphism on HSP70 protein synthesis the correlation between A/C polymorphism and protein synthesis was investigated. We found that cells from AA centenarian females displayed a lower synthesis of HSP70.

Nel corso dello studio del gene umano CYYR1 (Vitale et al., 2002), localizzato sul cromosoma 21, abbiamo isolato e clonato in vettore un cDNA corrispondente a una isoforma di splicing che differisce di 3 basi (CAG) dalla forma precedentemente da noi identificata e descritta. La sequenza del cDNA della isoforma permette di prevedere la codifica di un prodotto polipeptidico con un amminoacido aggiuntivo (alanina) inserito tra P111 e G112. Lo studio mediante RT-PCR (reazione a catena della polimerasi dopo retrotrascrizione) semiquantitativa ha permesso di dimostrare che le due isoforme sono espresse differenzialmente in diversi tessuti normali studiati. L’analisi genomica ha rivelato che l’origine dello splicing alternativo risiede nella sequenza terminale dell’introne 3 del gene CYYR1 (CAGCAG), che presenta due siti accettori di splicing canonici “AG” distanti tre basi tra loro ed entrambi utilizzabili dalla cellula. E’ stata quindi eseguita una revisione sistematica, basata su analisi bioinformatica e in alcuni casi anche sul clonaggio mediante RT-PCR, degli mRNA umani che si presentano in due possibili isoforme differenti per la presenza o l’assenza di una tripletta CAG. In particolare, abbiamo analizzato con uno specifico programma da noi sviluppato le sequenze di circa 30.000 introni umani (banca dati SRS), dimostrando che in 39 casi (0,13 %) un introne umano termina con la sequenza CAGCAG. In 10 casi, l’analisi della banca dati EST (expressed sequence tags, etichette di sequenze espresse) mostrava l’effettiva esistenza di mRNA differenti per la tripletta CAG. In alcuni casi, la tripletta aggiuntiva si trova nella regione codificante, permettendo la previsione di una sequenza amminoacidica del prodotto variante più lunga di 1 amminoacido. Abbiamo dimostrato la presenza di splicing alternativo, mediante RT-PCR, per i geni: SGNE1 (secretory granule, neuroendocrine protein 1), G6PD (glucose-6-phosphate dehydrogenase), PKD1 (polycystic kidney disease 1 gene). Nel caso di IGF1R (codificante il recettore per l’insulin-like growth factor 1), dati di letteratura precedenti hanno mostrato che i prodotti polipeptidici tradotti a partire dalle due differenti isoforme di mRNA e differenti per un singolo amminoacido possono svolgere una diversa funzione biologica (in termini di oncogenicità in saggi di trasfezione cellulare). La identificazione sistematica di isoforme di splicing minimamente differenti con produzione di catene polipeptidiche varianti, oltre ad aver permesso l’identificazione rapida di nuove isoforme di splicing di geni umani noti da tempo e svolgenti azioni biologiche fondamentali, amplia la nozione di variabilità dei prodotti di trascrizione genica che possono essere originati da un singolo locus e prelude alla ricerca di meccanismi simili operanti a livello dei siti donatori di splicing o di altre analoghe sequenze a livello dei siti accettori.

Il gene DSCR1L2 (1p35.3-p33), identificato e clonato nel nostro laboratorio, appartiene alla famiglia genica umana DSCR1L (Down Sindrome critical region 1-like) di cui fanno parte anche i geni DSCR1 (21q22.12) e DSCR1L1 (6p12.3). Le sequenze del cDNA di questi geni non presentano alcuna somiglianza con quelle di membri di famiglie geniche note. L’analisi genomica mostra che, mentre gli invertebrati contengono un solo gene di tipo DSCR1-like, nei vertebrati sono presenti tre membri della famiglia. Lo studio dei loci DSCR1, DSCR1L1 e DSCR1L2 mostra l’esistenza nel genoma umano di una nuova regione di paralogia segmentale di circa 500 kb che comprende l’1,4% del cromosoma 21, nella quale si trovano nello stesso ordine tre membri delle tre famiglie geniche DSCR1L, Runt (AML, acute myeloid leukemia) e CLIC (chloride intracellular channel). L’analisi filogenetica mostra una divergenza tardiva di DSCR1L1 e DSCR1L2 dal gene DSCR1 più ancestrale, e la conservazione della paralogia segmentale fin dai vertebrati inferiori. Lo studio dell’mRNA di DSCR1L2 mostra una espressione ubiquitaria del gene, con un livello molto alto di espressione nel tessuto muscolare, in particolare in quello cardiaco. L’analisi bioinformatica e mediante reazione a catena della polimerasi dopo retrotrascrizione (RT-PCR) ha permesso di identificare tre isoforme di mRNA generate da splicing alternativo. Una delle isoforme si ottiene per perdita degli esoni 2 e 3 del gene, con conseguente perdita della regione codificante per il dominio ricco in prolina caratteristico della famiglia, una seconda forma mostra la perdita dell’esone 2, mentre la terza si caratterizza per la mancanza di 30 basi codificanti per 10 amminoacidi nella regione centrale della proteina. E’ stato recentemente dimostrato da altri Autori che le proteine DSCR1 e DSCR1L1 si legano alla Calcineurina (CnA), una fosfatasi calcio-calmodulina dipendente che modula l’espressione genica nel muscolo scheletrico e cardiaco durante lo sviluppo e in condizioni di ipertrofia provocata da stress ambientali o da patologie. Da parte di più Autori è stato supposto il legame anche di DSCR1L2 con la CnA stessa nonostante l’assenza di prove sperimentali, solo sulla base dell’omologia di sequenza. Il test dei due ibridi in lievito da noi eseguito per individuare l’interazione proteina-proteina tra DSCR1L2 e una o più proteine tradotte a partire dai cDNA presenti in una genoteca di cuore umano non ha potuto tuttavia rilevare nessuna interazione con la CnA. E’ stata invece dimostrata l’interazione tra DSCR1L2 e la troponina I (TNNI3) cardiaca umana, la subunità inibitoria del complesso delle troponine coinvolte nella contrazione muscolare. L’interazione con TNNI3 della proteina DSCR1L2 mostra un caso interessante di divergenza funzionale nell’ambito della famiglia genica DSCR1L, e contribuisce alla chiarificazione dei complessi proteici implicati nella contrazione muscolare. Inoltre, abbiamo identificato un secondo interattore proteico di DSCR1L2, corrispondente a un cDNA tradotto secondo un modulo di lettura non fisiologico; questo polipeptide non naturale di 48 amminoacidi potrebbe risultare di interesse farmacologico per il suo eventuale ruolo nella modulazione della funzione di DSCR1L2. L’interazione tra DSCR1L2 e TNNI3 è stata confermata mediante cotrasformazione in lievito dei due plasmidi contenenti DSCR1L2 e TNNI3. Una ulteriore conferma di tale interazione viene da esperimenti di coimmunoprecipitazione in vitro. La zona di interazione è stata infine mappata mediante cotrasformazione del cDNA corrispondente alla isoforma di splicing di DSCR1L2 mancante degli esoni 2 e 3 e del cDNA di TNNI3. I risultati mostrano l’assenza di interazione, individuando la zona di interazione nei domini codificati dagli esoni 2 e 3.

The amino acid sequence of gene products is routinely deduced from the nucleotide sequence of the relative cloned cDNA, according to the rules for recognition of start codon (first-AUG rule, optimal sequence context) and the genetic code. From this prediction stem most subsequent types of product analysis, although all standard methods for cDNA cloning are affected by a potential inability to effectively clone the 5′ region of mRNA. Revision by bioinformatics and cloning methods of 109 known genes located on human chromosome 21 (HC 21) shows that 60 mRNAs lack any in-frame stop upstream of the first-AUG, and that in five cases (DSCR1, KIAA0184, KIAA0539, SON, and TFF3) the coding region at the 5′ end was incompletely characterized in the original descriptions. We describe the respective consequences for genomic annotation, domain and ortholog identification, and functional experiments design. We have also analyzed the sequences of 13,124 human mRNAs (RefSeq databank), discovering that in 6448 cases (49%), an in-frame stop codon is present upstream of the initiation codon, while in the other 6676 mRNAs (51%), identification of additional bases at the mRNA 5′ region could well reveal some new upstream in-frame AUG codons in the optimal context. Proportionally to the HC 21 data, about 550 known human genes might thus be affected by this 5′ end mRNA artifact. © 2003 Elsevier B.V. All rights reserved.

Background : The ADARB1 gene is located in 21q22.3 region, previously linked to familial bipolar disorder, and its product has a documented action in the editing of the pre-mRNA of glutamate receptor B subunit. Dysfunction of glutamatergic neurotransmission could play an important role in the patophysiology of bipolar disorder (BD). Glutamate excitatory neurotransmission regulation is a possible mechanism of the initial effect of anticonvulsants in regulating mood. Methods : To investigate the hypothesis of an involvement of ADARB1 gene in the BD, the ADARB1 cDNA has been cloned and sequenced in seven selected bipolar I disorder patients with evidence of familiarity of mood disorders. A detailed investigation of the gene nucleotide sequence in the open reading frame has been performed. Results : No alteration in the sequence of the ADARB1 gene cDNA was found in any patient, except a common neutral polymorphism in three out of seven patients. Conclusions : Mutations in ADARB1 gene are not commonly associated with bipolar I disorder, therefore other genes in the 21q22 region could be associated with bipolar illness in some families, likely in the context of a multifactorial transmission model.

Background Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input. Results TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified. Conclusions TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at http://apollo11.isto.unibo.it/software/, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes.