[Molecular biomarkers in workers and population exposed to inorganic arsenic: preliminary study in vitro]. / Biomarcatori molecolari in lavoratori professionalmente esposti ad arsenico inorganico e nella popolazione generale: studio preliminare in vitro.

The inorganic Arsenic (iAs) is a metalloid widely diffuse in all environmental matrices. The iAs has been classified as a Group 1 carcinogen by the International Agency for Research on Cancer. The microRNAs (miRNAs) are small non coding RNAs that negatively regulate the expression of hundreds of target genes in many key physiological and pathological cell processes, including stress response, differentiation, proliferation, apoptosis and cancer, miRNA expression profiles appear altered in most human cancers and it has been highlighted the potential of miRNA profiling in cancer diagnosis and prognosis. The present study evaluates the effect of iAs exposure on global miRNA expression in Jurkat cells. Treated cells show a reproducible increase in the expression levels of three miRNAs: miR-663, miR-222 and miR-638. This study supports the importance to proceeding in the investigation aimed to the possible application of some miRNAs as biomarkers in the environmental and occupational exposure to iAs.

Characterization of HuH6, Hep3B, HepG2 and HLE liver cancer cell lines by WNT/ß - catenin pathway, microRNA expression and protein expression profile.

Somatic mutations in the genes members of WNT/ß-catenin pathway, especially in CTNNB1 codifying for ß-catenin, have been found to play an important role in hepatocarcinogenesis. The purpose of this work is to characterize alterations of the WNT/ß-catenin signalling pathway, and to study the expression pattern of a panel of microRNAs and proteins potentially involved in the pathogenesis of liver cancer. In this respect, the molecular characterization of the most used liver cancer cell lines HuH6, Hep3B, HepG2, and HLE, could represent a useful tool to identify novel molecular markers for hepatic tumour. A significant modulation of FZD7, NLK, RHOU, SOX17, TCF7L2, TLE1, SLC9A3R1 and WNT10A transcripts was observed in all the four liver cancer cell lines. The analysis of selected microRNAs showed that miR-122a, miR-125a and miR-150 could be suitable candidates to discriminate tumoural versus normal human primary hepatocytes. Finally, Grb-2 protein expression resulted to be increased more than two-fold in liver cancer cell lines in comparison to normal human primary hepatocytes. These advances in the knowledge of molecular mechanisms involved in the pathogenesis of liver cancer may provide new potential biomarkers and molecular targets for the diagnosis and therapy.

Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase.

The phenomenon of posttranscriptional gene silencing (PTGS), which occurs when a transgene is introduced into a cell, is poorly understood. Here, the qde-3 gene, which is required for the activation and maintenance of gene silencing in the fungus Neurospora crassa, was isolated. Sequence analysis revealed that the qde-3 gene belongs to the RecQ DNA helicase family. The QDE3 protein may function in the DNA-DNA interaction between introduced transgenes or with an endogenous gene required for gene-silencing activation. In animals, genes that are homologous to RecQ protein, such as the human genes for Bloom's syndrome and Werner's syndrome, may also function in PTGS.

Post-transcriptional gene silencing across kingdoms.

Post-transcriptional gene silencing (PTGS) as a consequence of the introduction of either transgenes or double-stranded RNA molecules has been found to occur in a number of species. In the past year, studies in different systems have greatly enhanced our understanding of the molecular mechanisms of these phenomena. The ubiquitous presence of PTGS in both the plant and animal kingdoms and the finding of common genetic mechanisms suggest that PTGS is a universal gene-regulation system fundamental in biological processes such as protection against viruses and transposons.

Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.

Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme.

Homology-dependent gene silencing is a phenomenon that occurs in a broad range of organisms and has implications for both basic and applied science. Gene silencing is a mechanism that controls invading transposons and provides protection against virus infections. It also has evolutionary implications in genome maintenance. Recent studies have begun to unravel the molecular mechanisms of this puzzling phenomenon.

White collar proteins: PASsing the light signal in Neurospora crassa.

The filamentous fungus Neurospora crassa is an excellent paradigm for the study of blue light signal transduction. The isolation and characterization of the genes for two central regulators of the blue light response, white collar-1 and white collar-2, have begun to shed light on the mechanism of blue light signal transduction in fungi. These proteins are not only proposed to encode blue-light-activated transcription factors but also to be elements of the blue light signal transduction pathway.

Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs.

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.

Two intervening sequences in the ATPase subunit 6 gene of Neurospora crassa. A short intron (93 base-pairs) and a long intron that is stable after excision.

A 3590 base-pair region of the mitochondrial genome of Neurospora crassa, including the gene for ATPase subunit 6 (oli2), has been sequenced. The oli2 gene is interrupted by two intervening sequences. The first intron, situated after the third codon of the gene, is 93 base-pairs long; two-thirds of this intron consist of a palindromic sequence. The second intron is 1370 base-pairs long and contains an extended open reading frame that is continuous and in frame with the upstream exon sequence. This intron has structural homology with most other fungal mitochondrial introns. Transcript analysis has yielded a complex pattern of RNA species and demonstrated that the second intron is quite stable after excision. An unknown reading frame (homologous to reading frames of other mitochondrial genomes) is located 1000 base-pairs upstream from the oli2 coding sequence.

Cloning and characterization of PKC-homologous genes in the truffle species Tuber borchii and Tuber magnatum.

The protein kinases C (PKCs) define a growing family of ubiquitous signal transducting serine/threonine kinases that control ion conductance channels, release of hormones and cell growth and proliferation. Degenerated oligonucleotides were used as primers for polymerase chain reactions to amplify PKC-related sequences from the white truffle species Tuber magnatum and Tuber borchii. The deduced amino acid sequences of cloned sequences reveal domains homologous to the regulatory and kinase domains of PKC-related proteins, but lack typical Ca(2+)-binding domain and therefore should be classified as nPKCs. Both contain a large extended N-terminus which is found exclusively in fungi PKCs. Phylogenetic analysis of the kinase domain demonstrates high homology with known filamentous fungi isoenzymes.

Circadian clocks: what makes them tick?

In the not too distant past, it was common belief that rhythms in the physical environment were the driving force, to which organisms responded passively, for the observed daily rhythms in measurable physiological and behavioral variables. The demonstration that this was not the case, but that both plants and animals possess accurate endogenous time-measuring machinery (i.e., circadian clocks) contributed to heightening interest in the study of circadian biological rhythms. In the last few decades, flourishing studies have demonstrated that most organisms have at least one internal circadian timekeeping device that oscillates with a period close to that of the astronomical day (i.e., 24h). To date, many of the physiological mechanisms underlying the control of circadian rhythmicity have been described, while the improvement of molecular biology techniques has permitted extraordinary advancements in our knowledge of the molecular components involved in the machinery underlying the functioning of circadian clocks in many different organisms, man included. In this review, we attempt to summarize our current understanding of the genetic and molecular biology of circadian clocks in cyanobacteria, fungi, insects, and mammals.

Photoregulation of the albino-3 gene in Neurospora crassa.

In this paper we describe the light-regulated expression of albino-3 (al-3), a carotenoid biosynthetic gene of Neurospora crassa, in the wild-type strain. Our results suggest that the al-3 gene expression is regulated by the transcriptional activation of the gene and the low stability of its mRNA. The activation of the al-3 gene does not require protein synthesis to occur. The kinetic analysis of the al-3 mRNA reveals that the gene is transiently expressed even in continuous light, suggesting the presence of an adaptative process.

Photoinduction of albino-3 gene expression in Neurospora crassa conidia.

The synthesis of carotenoids is induced by blue light in Neurospora crassa mycelia, while in conidia (the vegetative spores) the accumulation of carotenoids also occurs in the dark. The expression of the albino-3 (al-3) gene (coding for the carotenogenic enzyme geranyl-geranyl pyrophosphate synthetase) in isolated conidia was analysed. The level of al-3 mRNA was shown to be increased in light-induced wild type (wt) conidia. This light response was elicited by blue light and was under the control of the white collar-1 (wc-1) and white collar-2 (wc-2) gene products. This indicates that the blue-light photoreceptor and the light transduction pathway which activate al-3 gene expression in mycelia are probably the same as in conidia.

Functional identification of al-3 from Neurospora crassa as the gene for geranylgeranyl pyrophosphate synthase by complementation with crt genes, in vitro characterization of the gene product and mutant analysis.

In this work the Neurospora crassa al-3 gene function was determined. Geranylgeranyl pyrophosphate (GGPP) synthase activity was measured in al-2 FGSC 313 and al-3 RP100 FGSC 2082 mutant strains by in vitro synthesis methods. This experiment showed that al-3 RP100 mutant expresses a reduced GGPP synthase activity. The mutated al-3 gene was cloned and sequenced; a single missense mutation was found changing serine into asparagine. Genetic complementation was performed by Escherichia coli transformation, with clusters of crt genes from Erwinia uredovora. Carotenoid accumulation was observed in E. coli transformants when the N. crassa al-3 gene substitutes the GGPP synthase gene (crtE) in the carotenogenic crt cluster. Cell-free studies with E. coli transformants gave direct evidence of the function of the al-3 protein as GGPP synthase and indicated that a short-chain prenylpyrophosphate, such as dimethylallyl pyrophosphate, is the genuine substrate.

Mapping of mitochondrial structural genes in Neurospora crassa.

A hybridization method has been employed to study the organization of the mitochondrial genome of Neurospora crassa. The method involves the use of 5' end-labeled single-stranded restriction fragments obtained from cytoplasmic "petite" strains of Saccharomyces cerevisiae known to contain single mitochondrial genes. The presence and localization of genes homologous to Subunits 1, 2, and 3 of cytochrome oxidase, cytochrome b and Subunit 6 of the ATPase is thus established for the mitochondrial genome of N. crassa.

Characteristics of post-transcriptional gene silencing.

A number of gene silencing phenomena that inactivate genes at the post-transcriptional level have been identified. Due to its potential for studying gene function, post-transcriptional gene silencing (PTGS) has become an intense area of research. In this review we describe the different means of inducing PTGS and discuss the possible biological roles of these artificially induced phenomena. We also discuss other features of PTGS such as the mechanism of mRNA degradation, the nature of the silencing signal and the mechanism of PTGS inhibition by viral proteins.

Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences.

Up to 36% of Neurospora crassa transformants showing an albino phenotype were recovered by transforming a wild-type strain with different portions of the carotenogenic albino-3 (al-3) and albino-1 (al-1) genes. The presence of the exogenous sequences (which were randomly integrated in ectopic locations) provoked a severe impairment in the expression of the endogenous al-1 or al-3 genes. This phenomenon, which we have termed 'quelling', was found to be spontaneously and progressively reversible, leading to wild-type or intermediate phenotypes. The phenotypic reversion is characterized by a progressive release of the transcriptional inhibition and seems to correlate with a reduction of the number of the ectopic integrated sequences. Moreover, quelling appears to be monodirectional, as, once relieved, it cannot take place again, despite the continuing presence of some of the ectopic sequences in the genome.

Assembly of the mitochondrial membrane system: partial sequence of a mitochondrial ATPase gene in Saccharomyces cerevisiae.

The nucleotide sequence of mitochondrial DNA of a cytoplasmic "petite" mutant of Saccharomyces cerevisiae is reported. The DNA has a repeat length of 1060 base pairs and contains a genetic marker (oli-1) for the ATPase proteolipid. The nucleotide sequence reveals the presence of part of the structural gene of the subunit-9 proteolipid of the ATPase complex and an extended A+T-rich region adjacent to the carboxyl-terminal end of the gene. The structural gene sequence agrees with the primary structure of the protein. These studies point out the feasibility of using the DNA of appropriately marked "petite" mutants to obtain the sequence of mitochondrial genes.