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G Paolella

Publications and source records attributed to G Paolella.

16 recordsLinked to original sources
PubMed1992

Nuclease resistant ribozymes with high catalytic activity.

Hammerhead ribozymes are efficient RNA enzymes characterized by a typical hammerhead secondary structure and a number of conserved bases. Little is known about the role of the ribose-phosphate backbone, although it is obviously important since a DNA molecule with the same base sequence is not a catalyst. Here we describe the synthesis of artificial ribozymes where modified (2'-O-allyl- and 2'-O-methyl-) ribonucleotides substitute for the corresponding ribonucleotides. A systematic analysis of partially substituted polymers identified a minimum set of six non-contiguous positions where insertion of modified ribonucleotides strongly affects catalytic activity. Surprisingly, ribozymes completely substituted except for these six ribonucleotides are still very active. These molecules efficiently cleave in trans target RNAs in a sequence-specific way, but, unlike RNA ribozymes, are very resistant to nuclease degradation and are very stable in serum. These properties make such synthetic polymers potentially useful for in vivo gene expression studies and therapeutic applications.

Base Sequence↗
PubMed1989

Cell type specific trans-acting factors are involved in alternative splicing of human fibronectin pre-mRNA.

ED-A and ED-B are facultative type III homologies of fibronectin, encoded by alternatively spliced exons, described in man and in rat. A hybrid alpha-globin-fibronectin minigene containing the ED-B region from the human gene has been transfected in human cell lines derived from various tissues, in order to study the processing of the generated precursor RNA in the different cell environments. In most tested lines the pre-RNA is alternatively spliced and produces two mature RNAs, with and without the ED-B exon, in different ratios that closely resemble the corresponding endogenous fibronectin RNAs. In a hepatoma cell line, Hep 3B, only one RNA is produced, in which the ED-B exon is absent; the same pattern of splicing is observed in liver. The data show that all the information required to produce accurate and regulated alternative splicing of the ED-B exon is contained in the fragment used and cell specific factors are necessary for the pre-RNA to be differentially spliced in the various cell lines. In contrast, expression in Hep 3B of a similar gene containing the ED-A area failed to reproduce the liver specific splicing pattern. Therefore regulation of ED-A processing is likely to involve different mechanisms to those responsible for control of ED-B splicing.

Base Sequence↗
PubMed1989

Assignment of human aldolase C gene to chromosome 17, region cen----q21.1.

The mapping of the gene coding for human aldolase C has been studied using a specific cDNA probe and genomic blots from a panel of human-hamster somatic cell hybrids. The results show that the aldolase C gene is on chromosome 17. In situ experiments have restricted the mapping to the region 17cen----q21.1. Using the same panel of human-hamster somatic cell hybrids, we have confirmed the localization of aldolase A and B on chromosomes 16 and 9, respectively.

Animals↗
PubMed1988

Human aldolase A gene. Structural organization and tissue-specific expression by multiple promoters and alternate mRNA processing.

The complete nucleotide sequence of the human aldolase A isoenzyme gene is reported. The cloned gene sequence, spanning 7530 bp, includes twelve exons and occurs as a single copy per haploid human genome. The structural organization of the gene is quite complex: eight exons containing the coding sequence are common to all mRNAs extracted from human and other mammalian sources; four additional exons are present in the 5' untranslated region, of these one is contained in the ubiquitous type of mRNA, the second is in the muscle-specific type of mRNA and the third and fourth are in a minor species of mRNA found in human liver tissue. Furthermore, the determined sequence includes 1000 nucleotides upstream from the first exon (exon I) in the 5' flanking region, and 400 nucleotides, which include the polyadenylation signal, downstream from the termination codon. S1-nuclease-protection analysis of the 5' end of mRNA extracted from human cultured fibroblasts, muscle and hepatoma cell lines indicates the existence of four different transcription-initiation sites. The latter are also supported by the presence of conventional sequences for eukaryotic promoters. Therefore, the four promoters on the same gene generate different tissue-specific transcripts, which share the translated sequence, but each has a unique 5' untranslated region as a result of differential mRNA processing. The nucleotide homology at the coding region and the intron-exon organization of the three human and mammalian aldolase A, B and C genes confirm that they arose from a common ancestral gene, and that aldolase B diverged first.

Base Sequence↗
PubMed1988

Sequence analysis and in vivo expression show that alternative splicing of ED-B and ED-A regions of the human fibronectin gene are independent events.

The structure of two alternatively spliced regions. ED-A and ED-B, of human fibronectin gene, was determined, in order to show whether any similarity was present between the two. Although some interesting features are present in each, no obvious common structure or sequence homology was found. Functional analysis of the alternative splicing events was carried out by transient expression in Hela cells. A hybrid gene was constructed by inserting the ED-B region into the third exon of the human alpha 1-globin gene. The transfected hybrid gene is expressed and produces, in Hela cells, two alternatively spliced RNAs, showing a pattern very similar to that observed for the endogenous fibronectin gene in fibroblasts. Cotransfection of this gene with a similar gene containing the ED-A region, shows that no interference is present between the two alternative splicing processes.

Amino Acid Sequence↗
PubMed1987

A new human species of aldolase A mRNA from fibroblasts.

A full-length cDNA aldolase A clone was isolated from a human fibroblast cDNA library and completely sequenced. Excluding the poly(A) tail, the clone covers 1095 base pairs (bp) of the coding region, plus 199 bp downstream for the termination codon and 146 bp upstream for the initiation codon, within a total of 1440 bp. Primer extension experiments performed with human cultured fibroblast mRNA indicate an elongated product of a further 40 bp. These results evaluated together with those obtained in a concurrent study concerning aldolase A mRNA isolated from human liver are direct evidence of aldolase A mRNA multiplicity in man. The data also suggest the existence in mammals of three different classes of aldolase A mRNA, which would account for tissue specificity and resurgence of foetal expression in tumors.

Amino Acid Sequence↗
PubMed1987

Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon.

Purification and amino acid sequence analysis of a proteolytic fragment of fibronectin (FN) from transformed human cells demonstrated that a high percentage of these FN molecules contains an extra amino acid sequence which is present only in a very low percentage of FN molecules from normal fibroblasts and is undetectable in plasma FN. This new amino acid sequence introduces into the FN molecule a site very sensitive to a number of proteolytic enzymes. By analyzing the cellular mRNA and genomic clones, we have demonstrated that this sequence derives from a differential splicing pattern of the FN mRNA precursors, which leads in transformed cells to a high-level expression of an extra type III homology repeat (ED-B) coded for by a previously unobserved exon. Here we also report the complete sequence of this new exon. These results demonstrate that in malignant cells the mechanisms regulating the splicing of FN mRNA precursors are altered.

Amino Acid Sequence↗
PubMed1987

Mapping of a restriction fragment length polymorphism within the human aldolase B gene.

Peripheral blood DNA was hybridized to the full-length cDNA and the cloned structural gene of human aldolase B. With PvuII endonuclease a restriction fragment length polymorphism was detected that was present in the heterozygous state in about 21% of the individuals tested. A map of the human aldolase gene was constructed for the two groups of individuals found to produce different fragments after PvuII digestion. This allowed the localization of the polymorphic site within the gene, which was found to be due to the loss of a PvuII site in the last intron upstream from the 3' end. This polymorphism may be used as a genetic marker to study individuals affected by hereditary fructose intolerance.

Chromosome Mapping↗
PubMed1986

Structure and expression of mouse aldolase genes. Brain-specific aldolase C amino acid sequence is closely related to aldolase A.

Brain-specific aldolase C amino acid sequence (greater than 75% of the coding region) was determined for the first time. Two cDNA clones, pAM1 and pAM2, were identified, from a mouse brain library, by using human aldolase B cDNA as a probe. The larger one, pAM2, identified as a cDNA for aldolase C, has been completely sequenced and covers the 5'-untranslated region of the mRNA and the codons for amino acids 1-227 of the protein. The sequence indicates that aldolase C is more akin to aldolase A than to aldolase B. A cDNA library from mouse muscle was also screened, allowing the identification of clones pAM3 and pAM4, which contain cDNAs for aldolase A. The sequence obtained from pAM3 covers 70% of the coding sequence (amino acids 99-355) from the -COOH part of the protein. The cDNAs for the three aldolases, A, B and C, have been hybridized to RNA from various rat tissues. The results confirm the tissue specificity of the expression of the mRNA for the different isoenzymes and support the hypothesis that aldolase C expression, as aldolase A and B, is regulated at the transcriptional level or, in any case, via mRNA concentration.

Amino Acid Sequence↗
PubMed1986

Primary and secondary structural homologies between the HIS4 gene product of Saccharomyces cerevisiae and the hisIE and hisD gene products of Escherichia coli and Salmonella typhimurium.

A detailed comparative analysis of the Escherichia coli and Salmonella typhimurium hisIE and hisD gene products and the functionally equivalent, single, HIS4 gene product of Saccharomyces cerevisiae permitted several insights concerning the relationship between these genes. Our analysis supports the idea that HIS4 results from the fusion of his IE and hisD. The comparison permitted a more precise definition of the functional domains of hisI/HIS4A and hisE/HIS4B as well as the two functional domains of hisD/HIS4C. The homologies between the bacterial and yeast sequences suggest a region of the hisD/HIS4C protein that may constitute one of the active centres. A large fragment at the amino terminal region of the yeast protein is missing from the bacterial hisIE gene product and is probably not needed for catalytic activity. Another region of non-homology in the yeast protein is probably a peptide bridge connecting the HIS4AB domain to HIS4C. Although the overall homology at the level of amino acid sequence is modest (about 38%) there is a striking similarity when the hydropathic patterns and predicted secondary structural configurations of these proteins are compared.

Amino Acid Sequence↗
PubMed1985

A fast DNA sequence handling program for Apple II computer in BASIC and 6502 assembler.

A fast general purpose DNA handling program has been developed in BASIC and machine language. The program runs on the Apple II plus or on the Apple IIe microcomputer, without additional hardware except for disk drives and printer. The program allows file insertion and editing, translation into protein sequence, reverse translation, search for small strings and restriction enzyme sites. The homology may be shown either as a comparison of two sequences or through a matrix on screen. Two additional features are: (i) drawing restriction site maps on the printer; and (ii) simulating a gel electrophoresis of restriction fragments both on screen and on paper. All the operations are very fast. The more common tasks are carried out almost instantly; only more complex routines, like finding homology between large sequences or searching and sorting all the restriction sites in a long sequence require longer, but still quite acceptable, times (generally under 30 s).

Base Sequence↗
PubMed1985

A microcomputer program for the identification of tRNA genes.

A microcomputer program which locates tRNA genes within long DNA sequences is described. The search is performed either by identifying tRNA-like secondary structures or by locating eukaryotic RNA polymerase III promoter consensus sequences. The program is also useful in finding inverted repeats allowing the formation of stem-loop secondary structures in tRNA. The program has been developed in BASIC and 6502 Assembler and runs on the Apple II plus and IIe microcomputers. The execution is quite fast; all the operations are carried out in 1-90 s, depending on the required task and on the sequence length.

Base Sequence↗
PubMedDate not supplied

Human aldolase A gene: structural organization and expression in tumor cell lines.

The structural intron/exon organization of the cloned human aldolase A gene is reported. The gene is composed of twelve exons, including the coding and the 5' and 3' non-coding regions. Four mRNAs, different at the 5' non-coding region, are transcribed from this gene in a tissue-specific manner. The study of the expression of aldolase A gene in normal liver and neoplastic cell lines demonstrated the resurgence of foetal pattern of expression in all tumor liver cell lines and HeLa cells, thus supporting the foetalism theory for this gene system.

Carcinoma, Hepatocellular↗