PubMed Health⌕ Search

Biomedical subjects

E Sbisà

Publications and source records attributed to E Sbisà.

At least 19 recordsLinked to original sources
PubMed1999

Guinea pig p53 mRNA: identification of new elements in coding and untranslated regions and their functional and evolutionary implications.

We report the sequence of the guinea pig p53 cDNA. The comparative analysis of the coding and noncoding regions of p53 cDNAs of all available complete vertebrate sequences has allowed us to single out new conserved signals possibly involved in p53 functional activity. We have focused our attention on the most variable region of the protein, the proline (P)-rich domain, suggested to play a fundamental role in antiproliferative pathways. In this domain we have identified the PXXXXP repeated motif and singled out a common consensus sequence that can be considered a signature for mammalian p53: PXXXXPX{0,4}PX{0,9}PA(T,P,I,)(S,P)WPL. We have demonstrated the significance of the PXXXXP motif in SH3-binding protein and suggested its structure to be a loop. Also, the 5' and 3' untranslated regions (UTRs) of the guinea pig were sequenced, and this study represents the first detailed structural analysis of the UTRs of the p53 mRNAs available in literature. The 5' UTR of guinea pig (233 nt) can be folded into a stable secondary structure resembling that predicted in mouse. The 3' UTR of guinea pig is 771 nt long and shows higher similarity with human than with rodent sequences, having a region of about 350 nt that is deleted in rat and mouse. In the 3' UTR we have identified the presence of a mammalian-wide interspersed repeat sequence and of a cytoplasmic polyadenylation element, which could be involved in translational activation by promoting polyadenylation of mRNA, providing information about a possible mechanism of regulation of p53 expression mediated by the 3' UTR of the mRNA. The observations presented here could open new avenues to targeted mutations and experimental approaches useful in investigating new regulation mechanisms of p53 translation, activity, and stability.

3' Untranslated Regions↗
PubMed1998

Folding and binding activity of the 3'UTRs of Paracentrotus lividus bep messengers.

Bep mRNAs are localized at the animal pole of P. lividus eggs. In the present communication the secondary structures of the 3'UTRs of the bep1, bep3 and bep4 mRNAs are reported. The minimal lengths of these regions required to bind the 54-kDa protein, previously shown to be involved in localization and anchoring of these RNAs, is estimated. Microinjection of the bep3 3'UTR into egg shows that this RNA fragment is also able to become localized to one of the egg poles, as happens for the entire bep3 RNA.

Amino Acid Sequence↗
PubMed1998

p53 mutation is a poor prognostic indicator for survival in patients with hepatocellular carcinoma undergoing surgical tumour ablation.

Forty-two patients with hepatocellular carcinoma (HCC) were resected and their tumours were analysed for p53 mutations by GC-clamped denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) and gene sequencing. All the exons have been analysed in this study. Eight of 12 HCCs with cirrhosis due to viral hepatitis and the two patients with sarcomatoid changes displayed p53 mutations. In contrast, no mutation was observed in the fibrolamellar variant (n = 9), non-cirrhotics (n = 13) and alcoholic cirrhosis (n = 6). The mutations observed were in exons 5-8. Two mutations were observed in codons 136 and 213 as well as a T insertion between residues 156 and 157 (exon 5) and these are reported for the first time in HCC. Likewise, the silent mutation polymorphism in codon 213 was noticed in 3 of the 42 patients. Survival analysis of these patients after surgery showed the mean and median survival in patients with wild-type p53 to be 60 and 43 months respectively. In the group with p53 mutations, the mean and median survival was 15 and 12 months. The difference was statistically significant (P= 0.003).

Adolescent↗
PubMed1997

Mammalian mitochondrial D-loop region structural analysis: identification of new conserved sequences and their functional and evolutionary implications.

This paper reports the first comprehensive analysis of Displacement loop (D-loop) region sequences from ten different mammalian orders. It represents a systematic evolutionary study at the molecular level on regulatory homologous regions in organisms belonging to a well defined class, mammalia, which radiated about 150 million years ago (Mya). We have aligned and analyzed 26 complete D-loop region sequences available in the literature and the fat dormouse sequence, recently determined in our laboratory. The novelty of our alignment consists of the extensive manual revision of the preliminary output obtained by computer program to optimize sequence similarity, particularly for the two peripheral domains displaying heterogeneity in length and the presence of repeated sequences. The multialignment is available at the WWW site: http://www.ba.cnr.it/dloop.html. Our comparative study has allowed us to identify new conserved sequence blocks present in all the species under consideration and events of insertion/deletion which have important implications in both functional and evolutionary aspects. In particular we have detected two blocks, about 60 bp long, extended termination associated sequences (ETAS1 and ETAS2) conserved in all the organisms considered. Evaluation against experimental work suggests a possible functional role of ETAS1 and ETAS2 in the regulation of replication and transcription and targeted experimental approaches. The analyses on conserved sequence blocks (CSBs) clearly indicate that CSB1 is the only very essential element, common to all mammalian mt genomes, while CSB2 and CSB3 could be involved in different though related functions, probably species specific, and thus more linked to nuclear mitochondrial coevolutionary processes. Our hypothesis on the different functional implications of the conserved elements, CSBs and TASs, reported so far as main regulatory signals, would explain the different conservation of these elements in evolution. Moreover the intra-order comparison of the D-loop regions highlights peculiar features useful to define the evolutionary dynamics of this region in closely related species.

Animals↗
PubMed1997

A 54-kDa protein specifically associates the 3' untranslated region of three maternal mRNAs with the cytoskeleton of the animal part of the Paracertrotus lividus egg.

Bep mRNAs, i.e., maternal messengers coding for cell surface proteins, are localized in the animal part of Paracertrotus lividus egg and embryos. Here we have examined the involvement of the cytoskeleton in asymmetric distribution of bep3 mRNA. Moreover, in order to understand whether and how cis- and trans-acting factors are necessary for bep3 mRNA localization, we have looked for in vitro-specific interactions between egg proteins and bep3 mRNA. By northwestern assay we have identified a 54-kDa protein that binds to the 3'UTR of bep3 mRNA. This 54-kDa protein also permits association of 3'UTR of bep3 with cytoskeleton elements, indicating its involvement in the localization process. Binding of 54-kDa protein to 3'UTR of bep1 and bep4 has also been demonstrated, suggesting that a binding motif is shared with these other two mRNAs of the same gene family. Northwestern analyses carried out utilizing proteins extracted from different developmental stages indicate that the 54-kDa protein is the only protein able to bind to the 3'UTR of bep3.

Animals↗
PubMed1996

The evolution of the RNase P- and RNase MRP-associated RNAs: phylogenetic analysis and nucleotide substitution rate.

We report a detailed evolutionary study of the RNase P- and RNase MRP- associated RNAs. The analyses were performed on all the available complete sequences of RNase MRP (vertebrates, yeast, plant), nuclear RNase P (vertebrates, yeast), and mitochondrial RNase P (yeast) RNAs. For the first time the phylogenetic distance between these sequences and the nucleotide substitution rates have been quantitatively measured.The analyses were performed by considering the optimal multiple alignments obtained mostly by maximizing similarity between primary sequences. RNase P RNA and MRP RNA display evolutionary dynamics following the molecular clock. Both have similar rates and evolve about one order of magnitude faster than the corresponding small rRNA sequences which have been, so far, the most common gene markers used for phylogeny. However, small rRNAs evolve too slowly to solve close phylogenetic relationships such as those between mammals. The quicker rate of RNase P and MRP RNA allowed us to assess phylogenetic relationships between mammals and other vertebrate species and yeast strains. The phylogenetic data obtained with yeasts perfectly agree with those obtained by functional assays, thus demonstrating the potential offered by this approach for laboratory experiments.

Animals↗
PubMed1995

Human mitochondrial tRNA processing.

tRNA processing is a central event in mammalian mitochondrial gene expression. We have identified key enzymatic activities (ribonuclease P, precursor tRNA 3'-endonuclease, and ATP(CTP)-tRNA-specific nucleotidyltransferase) that are involved in HeLa cell mitochondrial tRNA maturation. Different mitochondrial tRNA precursors are cleaved precisely at the tRNA 5'- and 3'-ends in a homologous mitochondrial in vitro processing system. The cleavage at the 5'-end precedes that at the 3'-end, and the tRNAs are substrates for the specific CCA addition in the same in vitro system. Using a comparative enzymatic approach as well as biochemical and immunological techniques, we furthermore demonstrate that human cells contain two distinct enzymes that remove 5'-extensions from tRNA precursors, the previously characterized nuclear and the newly identified mitochondrial ribonuclease P. These two cellular isoenzymes have different substrate specificities that seem to be well adapted to their structurally disparate mitochondrial and nuclear tRNA substrates. This kind of approach may also help to understand the structural diversities and commonalities of tRNAs.

Cell Compartmentation↗
PubMed1995

RNase mitochondrial RNA processing cleaves RNA from the rat mitochondrial displacement loop at the origin of heavy-strand DNA replication.

Ribonuclease mitochondrial RNA processing cleaves RNAs from the mammalian mitochondrial main non-coding regulatory region, called the displacement loop. Our data demonstrate that rat cells contain a site-specific ribonuclease mitochondrial RNA processing activity. We found that this enzyme processes the rat mitochondrial displacement-loop RNA substrate at the level of the conserved sequence block 1, a result which is different from that for mouse. This finding correlates with the in-vivo transcriptional analysis of the rat displacement-loop region. Processing by homologous and heterologous ribonuclease mitochondrial RNA enzymes occurs in the same manner, suggesting a conserved mode of substrate recognition.

Animals↗
PubMed1994

Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature.

Amino Acid Sequence↗
PubMed1994

Detection of novel transcripts in the human mitochondrial DNA region coding for ATPase8-ATPase6 subunits.

We have analyzed the tRNA(Lys), ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNA(Lys), ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNA(Lys) plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.

Adenosine Triphosphatases↗
PubMed1992

Transcription mapping of the Ori L region reveals novel precursors of mature RNA species and antisense RNAs in rat mitochondrial genome.

We have identified new transcripts in the region surrounding the L-strand replication origin (Ori L) of rat liver mitochondrial DNA. In particular, we have detected previously unidentified intermediates of RNA processing on both the heavy and the light strands, such as precursors of the ND2 mRNA plus the Trp-tRNA and precursors of the tRNAs clustered in the Ori L region. This indicates that the mechanism of RNA processing in mitochondria proceeds step-wise producing a variety of precursors of the mature forms. The other striking finding is the detection of antisense RNA species in the region of L-strand replication. Since a variety of antisense transcripts were also found in the D-loop region of rat mitochondrial DNA, we suggest that they might play a regulatory role in the replication and expression of the mitochondrial genome.

Animals↗
PubMed1990

The complete and symmetric transcription of the main non coding region of rat mitochondrial genome: in vivo mapping of heavy and light transcripts.

The experiments here reported demonstrate that the main non-coding region of rat mitochondrial DNA is symmetrically transcribed. We have identified stable heavy and light transcripts, whose pattern is rather complex, in the D-loop region of rat mitochondrial DNA. Their relative concentrations have been determined. We detected heavy transcripts which encompass the whole D-loop and more abundant heavy RNA species which we interpreted as transcripts terminating downstream of the 3' end of the last coded gene (Thr-tRNA). The processed heavy RNA species contain polyA, suggesting a strict association between cleavage and polyadenylation. The pattern of light transcripts shows a long RNA, which, starting from the light strand promoter, covers the whole segment, and shorter RNA species which seems to be actively processed at the level of the conserved sequence boxes, probably acting as primers. The symmetric transcription of the D-loop containing region of rat mitochondrial DNA, and in particular the presence of stable transcripts complementary to the putative RNA primers, suggest that mechanisms mediated by interaction between complementary transcripts (antisense RNAs) might play a role in the regulation of mitochondrial DNA replication and expression.

Amino Acid Sequence↗
PubMed1989

The complete nucleotide sequence of the Rattus norvegicus mitochondrial genome: cryptic signals revealed by comparative analysis between vertebrates.

This paper reports the nucleotide sequence of rat mitochondrial DNA, only the fourth mammalian mitochondrial genome to be completely sequenced. Extensive comparative studies performed with similar genomes from other organisms revealed a number of interesting features. 1) Messenger RNA genes: the codon strategy is mainly dictated by the base compositional constraints of the corresponding codogenic DNA strand. The usage of the initiation and termination codons follows well-established rules. In general the canonical initiator, ATG, and terminators, TAA and TAG (in rat, only TAA), are always present when there is gene overlapping or when the mRNAs possess untranslated nucleotides at the 5' or 3' ends. 2) Transfer RNA genes: a number of features suggest the peculiar evolutionary behavior of this class of genes and confirm their role in the duplication and rearrangement processes that took place in the evolution of the animal mitochondrial genome. 3) Ribosomal RNA genes: accurate sequence analysis revealed a number of significant examples of complementarity between ribosomal and messenger RNAs. This suggests that they might play an important role in the regulation of mitochondrial translation and transcription mechanisms. The properties revealed by our work shed new light on the organization and evolution of the vertebrate mitochondrial genome and more importantly open up the way to clearly aimed experimental studies of the regulatory mechanisms in mitochondria.

Amino Acid Sequence↗
PubMed1987

Structural elements highly preserved during the evolution of the D-loop-containing region in vertebrate mitochondrial DNA.

A detailed comparative study of the regions surrounding the origin of replication in vertebrate mitochondrial DNA (mtDNA) has revealed a number of interesting properties. This region, called the D-loop-containing region, can be divided into three domains. The left (L) and right (R) domains, which have a low G content and contain the 5' and the 3' D-loop ends, respectively, are highly variable for both base sequence and length. They, however, contain thermodynamically stable secondary structures which include the conserved sequence blocks called CSB-1 and TAS which are associated with the start and stop sites, respectively, for D-loop strand synthesis. We have found that a "mirror symmetry" exists between the CSB-1 and TAS elements, which suggests that they can act as specific recognition sites for regulatory, probably dimeric, proteins. Long, statistically significant repeats are found in the L and R domains. Between the L and R domains we observed in all mtDNA sequences a region with a higher G content which was apparently free of complex secondary structure. This central domain, well preserved in mammals, contains an open reading frame of variable length in the organisms considered. The identification of common features well preserved in evolution despite the high primary structural divergence of the D-loop-containing region of vertebrate mtDNA suggests that these properties are of prime importance for the mitochondrial processes that occur in this region and may be useful for singling out the sites on which one should operate experimentally in order to discover functionally important elements.

Amino Acid Sequence↗
PubMed1986

Structural conservation and variation in the D-loop-containing region of vertebrate mitochondrial DNA.

The nucleotide sequences of the D-loop-containing regions of three rat mitochondrial DNAs (mtDNAs), two from the species Rattus norvegicus and one from R. rattus, were determined. Comparisons made among these sequences and with the mouse sequence showed that, on the basis of both base composition and frequency of nucleotide alterations, three domains could be defined within the D-loop-containing region: a central conserved segment, poor in L-strand adenine, flanked by two divergent, adenine-rich regions. Deletions and insertions were found to occur at an unexpectedly high frequency in these sequences and the conserved sequence block called CSB-1 was found not to be intact in the R. rattus sequence. Although in comparisons of more distantly related mtDNAs the D-loop region is the most divergent on the molecule, it does not diverge more than typical protein genes between R. norvegicus and R. rattus, and its central conserved domain appears to be one of the molecule's most conserved regions. The most variable domain borders the tRNAPhe gene and contains the L and H-strand promoters and the 5' terminus for H-strand DNA synthesis. Within this region we have found sequences in all the mtDNAs we have examined, including those of human, two artiodactyls and Xenopus, that are capable of folding into cloverleaf structures. In the other divergent domain of the same mtDNAs, we find sequences capable of assuming similar secondary structural configurations at or near the sites for the termination of D-loop DNA synthesis. The evolutionary preservation of the potential to form such structures despite the high primary-structural divergence of the regions they occur in, suggests the structures are of principal importance for some processes occurring in the D-loop-containing region.

Animals↗
PubMed1983

Non-random patterns of nucleotide substitutions and codon strategy in the mammalian mitochondrial genes coding for identified and unidentified reading frames.

The base sequence of large part of the mitochondrial DNA of Wistar rats is presented. The sequence is compared with those of other mammalian mitochondrial DNAs. The nucleotide and amino acid homologies, codon strategy, nature and patterns of substitutions are reported. It results a very high amount of silent substitutions and, in short divergence time, a predominance of transitions on transversions. In both types of substitutions a strong bias in avoiding the use of the G in the third codon position is observed.

ATP Synthetase Complexes↗