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N Marmiroli

Publications and source records attributed to N Marmiroli.

At least 19 recordsLinked to original sources
PubMed2006

A branched-chain amino acid aminotransferase gene isolated from Hordeum vulgare is differentially regulated by drought stress.

Differential display was used to isolate cDNA clones showing differential expression in response to ABA, drought and cold in barley seedling shoots. One drought-regulated cDNA clone (DD12) was further analyzed and found to encode a branched-chain amino acid aminotransferase (HvBCAT-1). A genomic clone was isolated by probing the Morex BAC library with the cDNA clone DD12 and the structure of Hvbcat-1 was elucidated. The coding region is interrupted by six introns and contains a predicted mitochondrial transit peptide. Hvbcat1 was mapped to chromosome 4H. A comparison was made to rice and Arabidopsis genes to identify conserved structural patterns. Complementation of a yeast (Saccharomyces cerevisiae) double knockout strain revealed that HvBCAT-1 can function as the mitochondrial (catabolic) BCATs in vivo. Transcript levels of Hvbcat-1, increased in response to drought stress. As the first enzyme in the branched-chain amino acid (BCAA) catabolic pathway, HvBCAT-1 might have a role in the degradation of BCAA. Degradation of BCAA could serve as a detoxification mechanism that maintains the pool of free branched-chain amino acids at low and non toxic levels, under drought stress conditions.

Abscisic Acid↗
PubMed2005

Development of a peptide nucleic acid polymerase chain reaction clamping assay for semiquantitative evaluation of genetically modified organism content in food.

In the present study a peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method was developed and applied to the detection of genetically modified organisms (GMO), to test PCR products for band identity and to obtain a semiquantitative evaluation of GMO content. The minimal concentration of PNA necessary to block the PCR was determined by comparing PCRs containing a constant amount of DNA in the presence of increasing concentration of target-specific PNA. The lowest PNA concentration at which specific inhibition took place, by the inhibition of primer extension and/or steric hindrance, was the most efficient condition. Optimization of PCR clamping by PNA was observed by testing five different PNAs with a minimum of 13 bp to a maximum of 15 bp, designed on the target sequence of Roundup Ready soybean. The results obtained on the DNA extracted from Roundup Ready soybean standard flour were verified also on DNA extracted from standard flours of maize GA21, Bt176, Bt11, and MON810. A correlation between the PNA concentration necessary for inducing PCR clamping and the percentage of the GMO target sequence in the sample was found.

DNA Primers↗
PubMed2005

Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods.

A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in genetically modified soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the efficiency of all reactions. This multiplex PCR has constituted the basis for an efficient platform for genetically modified organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High specificity and sensitivity were obtained.

DNA, Plant↗
PubMed2003

Analysis of protein profiles of genetically modified potato tubers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Traceability of genetically modified (GM) foods demands the development of appropriate reliable techniques in order to identify and quantify peptide or nucleic acid residues in GM plants and food products through the food chain. In this study the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was demonstrated for the characterization of proteins of transformed and untransformed potato (Solanum Tuberosum L.) tubers. In GM tubers the expression level of the G1-1 gene, which regulates transition from dormancy to sprouting tubers, was inhibited by antisense technology. The analysis of antisense transformed lines showed that several of them exhibited a significant delay in sprouting relative to the control lines, in accordance with a decrease in the transcript level. Preliminary attempts to compare the protein patterns obtained from transformed and control lines using traditional electrophoresis were not able to reveal differences in the low-kDa range. Instead, MALDI-TOFMS applied to total peptide extract without any purification was able to distinguish spectral patterns of transformed and untransformed lines. In particular, several characteristic peaks from m/z 4373 to 4932 were detected only in the mass spectra of GM tuber samples.

Food, Genetically Modified↗
PubMed2002

Genomic analysis of cultivated barley (Hordeum vulgare) using sequence-tagged molecular markers. Estimates of divergence based on RFLP and PCR markers derived from stress-responsive genes, and simple-sequence repeats (SSRs).

Three types of molecular markers have been compared for their utility in evaluating genetic diversity among cultivars of Hordeum vulgare. Restriction fragment length polymorphisms at 71 sites were scored with the aid of probes corresponding to stress-responsive genes from barley and wheat, coding for a low-molecular-weight heat shock protein, a dehydrin, an aldose reductase homolog, and a 18.9-kDa drought-induced protein of unknown function. Indexes of genetic diversity computed in the total sample and within groups of cultivars (two-rowed and six-rowed, winter and spring varieties) indicated high values of genetic differentiation ( F (ST) >15%). A second assessment of genetic diversity was performed by PCR amplification of genomic DNA using as primers 13 arbitrary oligonucleotides derived from sequences of the same stress-responsive genes. A high degree of polymorphism was uncovered using these markers also, but they yielded low values for F (ST) (<7%) among groups of cultivars. Finally, 15 different simple-sequence repeats (AC or AG) were amplified with primers based on unique flanking sequences. Levels of polymorphism and differentiation between groups of cultivars revealed by these markers were quite high. Ordination techniques applied to measures of genetic distance among cultivars demonstrated a remarkable ability of the RFLPs associated with stress-responsive genes to discriminate on the basis of growth habit. The correlation with production data for the cultivars in different environments was also significant. This "functional genomics" strategy was therefore as informative as the "structural genomics" (SSR-based) approach, but requires the analysis of fewer probes.

Base Sequence↗
PubMed2001

Tuberization in potato involves a switch from apoplastic to symplastic phloem unloading.

Phloem unloading was studied in potato plants in real time during the early stages of tuberization using carboxyfluorescein (CF) as a phloem-mobile tracer, and the unloading pattern was compared with autoradiography of tubers that had transported (14)C assimilates. In stolons undergoing extension growth, apoplastic phloem unloading predominated. However, during the first visible signs of tuberization, a transition occurred from apoplastic to symplastic transport, and both CF and (14)C assimilates subsequently followed identical patterns of phloem unloading. It is suggested that the switch to symplastic sucrose unloading may be responsible for the upregulation of several genes involved in sucrose metabolism. A detailed analysis of sugar levels and (14)C sugar partitioning in tuberizing stolons revealed a distinct difference between the apical region of the tuber and the subapical region. Analysis of invertase activity in nontuberizing and tuberizing stolons revealed a marked decline in soluble invertase in the subapical region of swelling stolons, consistent with the switch from apoplastic to symplastic unloading. However, cell wall-bound invertase activity remained high in the apical 1 to 2 mm of tuberizing stolons. Histochemical analysis of potato lines transformed with the promoter of an apoplastic invertase gene (invGE) linked to a reporter gene also revealed discrete gene expression in the apical bud region. Evidence is presented that the apical and lateral tuber buds function as isolated domains with respect to sucrose unloading and metabolism.

Carbohydrate Metabolism↗
PubMed2000

A simple sequence repeat-based linkage map of barley.

A total of 568 new simple sequence repeat (SSR)-based markers for barley have been developed from a combination of database sequences and small insert genomic libraries enriched for a range of short simple sequence repeats. Analysis of the SSRs on 16 barley cultivars revealed variable levels of informativeness but no obvious correlation was found with SSR repeat length, motif type, or map position. Of the 568 SSRs developed, 242 were genetically mapped, 216 with 37 previously published SSRs in a single doubled-haploid population derived from the F(1) of an interspecific cross between the cultivar Lina and Hordeum spontaneum Canada Park and 26 SSRs in two other mapping populations. A total of 27 SSRs amplified multiple loci. Centromeric clustering of markers was observed in the main mapping population; however, the clustering severity was reduced in intraspecific crosses, supporting the notion that the observed marker distribution was largely a genetical effect. The mapped SSRs provide a framework for rapidly assigning chromosomal designations and polarity in future mapping programs in barley and a convenient alternative to RFLP for aligning information derived from different populations. A list of the 242 primer pairs that amplify mapped SSRs from total barley genomic DNA is presented.

Chromosome Mapping↗
PubMed1998

DNA fingerprinting analysis by a PCR based method for monitoring the genotoxic effects of heavy metals pollution.

Environmental pollutants can have deleterious effects on living organisms. At high concentrations, or at high activities, they can cause acute toxicity damaging cells, tissues and organs. Chronic toxification, on the other hand, can also cause serious damage from bio-accumulation. Plants, as biological indicators, can measure both the actual and the potential effects of pollutants, when they are used to measure effects of heavy metals. We have applied a system of "molecular fingerprinting" based on PCR (RAPD: Random Amplified Polymorphic DNA) to the evaluation of the genotoxic effects of heavy metals in order to estimate the environmental risk connected with their potential mutagenic effects in the model plant Arabidopsis thaliana, ecotype Columbia. Genomic DNA was utilised for RAPD analysis using random primers (10-mers). DNA from plants exposed to heavy metals solution displayed polymorphic bands which were not detectable in DNA of unexposed plants. The enhanced formation of RAPD polymorphisms was also observed in DNA of plant exposed in situ to an industrial pollution source. The comparison between "unexposed" and "exposed" genomes show that RAPD analysis can be used to evaluate how the environmental pollutants modify the structure of DNA in living organisms.

Biosensing Techniques↗
PubMed1997

Differential display-mediated isolation of a genomic sequence for a putative mitochondrial LMW HSP specifically expressed in condition of induced thermotolerance in Arabidopsis thaliana (L.) heynh.

Plants of Arabidopsis thaliana pre-treated at 37 degrees C for 2 h can survive an otherwise lethal heat shock at 45 degrees C. Differential display reverse transcriptase-PCR (DDRT-PCR) was utilized to clone DNA fragments corresponding to mRNAs specifically expressed in conditions of induced thermotolerance or of expression of thermotolerance. One of these DDRT-PCR fragments enabled the isolation of a genomic clone pAt1.3EX, containing the sequence Athsp23.5, the gene for a low-molecular-weight (LMW) heat shock protein (HSP), AtHSP23.5. Athsp23.5 is low- or single-copy in the Arabidopsis genome and its open reading frame is interrupted by a 137 bp intron. Analysis of the sequence suggests AtHSP23.5 is targeted to the mitochondrion. The steady-state level of the AtHSP23.5 mRNA varied significantly according to the heat treatment, increasing on heat shock (transfer from 22 degrees C to 37 degrees C), with a further increase during expression of thermotolerance (transfer from 22 degrees C to 37 degrees C and then to 45 degrees C). Expression was low after an abrupt stress (from 22 degrees C to 45 degrees C). This behaviour was different from that observed for other LMW HSP mRNAs that were present at high level at 37 degrees C, but did not increase significantly in condition of expression of thermotolerance, and reached a considerable steady-state level also during the abrupt stress at 45 degrees C. The retrotranscription of AtHSP23.5 mRNA followed by amplification with two primers encompassing the intron allowed for the isolation of an almost full-length cDNA sequence. The sequence analysis of the two cDNAs obtained from condition 22 degrees C-->37 degrees C and condition 22 degrees C-->45 degrees C suggested that in both cases the intron had been correctly spliced. The importance of correct intron splicing in survival at high temperatures and the role of mitochondrial HSP in induction and expression of thermotolerance are discussed.

Amino Acid Sequence↗
PubMed1993

Identification, characterization, and analysis of cDNA and genomic sequences encoding two different small heat shock proteins in Hordeum vulgare.

In vitro translation of mRNAs prepared from barley (Hordeum vulgare) seedlings (cv. Onice) exposed at 40 degrees C directed the synthesis of major heat shock proteins (HSPs) with molecular masses of 80-90, 70, 42 and 16-22 kDa. A cDNA library prepared from the 40 degrees C mRNAs and screened by differential hybridization led to the isolation of heat shock specific sequences. One of these (Hv hsp18) was confirmed by hybrid-arrested and hybrid-released translation as encoding for an 18-kDa HSP. The barley hsp18 sequence has an open reading frame encoding a 160 amino acid residue 18-kDa protein that is 63% identical to wheat 16.9-kDa HSP (clone C5-8), 54% identical to soybean (Glycine max) 17.5-kDa HSP, and 49% identical to Arabidopsis thaliana 17.6-kDa HSP. Lower similarities were found with class II plant small HSPs such as soybean 17.9-kDa HSP (27%), Pisum sativum 17.7-kDa HSP (30%), wheat (Triticum aestivum) 17.3-kDa HSP (clone Ta hsp 17.3) (30%), and with animal small HSPs and alpha-crystallins. The Hv hsp18 sequence was used to pick up Hv hsp17 genomic sequence encoding for another class I 17-kDa HSP. By computer analysis of the nucleotide sequence the TATA box, two heat shock promoter elements, a metal-ion response element, and the polyadenylation signals were identified. Barley HSP18 has an additional cysteine-rich region when compared with HSP17 mapping at the carboxy terminal end.

Amino Acid Sequence↗
PubMed1988

Germination of Saccharomyces cerevisiae ascospores without trehalose mobilization as revealed by in vivo 13C nuclear magnetic resonance spectroscopy.

Saccharomyces cerevisiae ascospores germinate in the presence of acetate without any detectable trehalose degradation, as revealed by high-resolution nuclear magnetic resonance spectroscopy and by a standard colorimetric assay. The results presented here substantiate the hypothesis that in S. cerevisiae trehalose supplies energy during dormancy of the spores and not during the germination process.

Acetates↗
PubMed1987

Cell division age dependency of meiosis in an apomictic variant of Saccharomyces cerevisiae.

The cell division age dependency of sporulation was investigated in a diploid strain of Saccharomyces cerevisiae (19el) which undergoes a single equational nuclear division during sporulation with consequent formation of asci containing two uninucleate diploid spores (apomictic dyads). Under modified nutritional conditions which partially restore meiosis and hence normal tetrad formation, newly formed (age 0) daughter cells were observed to be capable of formation of apomictic dyads but not of meiotic tetrads. Even under conditions in which only apomictic dyads developed, approximately 20% of the asci resulted from differentiation of newborn 'inexperienced' cells. Thus, the data indicated production of at least one bud to be a prerequisite for meiosis but not for apomixis; however, occurrence of at least one complete mitotic cell division cycle was evidently insufficient for the morphogenetic switch from diploid to haploid spore formation, since older cells bearing several bud scars often underwent apomictic dyad development, and some produced no spores.

Cell Division↗
PubMed1986

Germination conditions that require mitochondrial function in Saccharomyces cerevisiae: utilization of acetate and galactose.

Ascospores of Saccharomyces cerevisiae inherited at least one functioning mitochondrion as shown by their ability to germinate on nonfermentable carbon sources. After transfer to germination medium, the optical density of the culture at 600 nm decreased (phase-dark), reaching a minimum within 60 min in the presence of glucose and within 180 min after transfer to acetate medium; thereafter, the optical density increased. Budding cells first appeared 90 min after transfer to glucose and 150 min after transfer to acetate. Augmentation of respiratory components, respiratory activity, and macromolecular synthesis (except for DNA synthesis) started at about the same time on glucose and on acetate, although the highest values for all these processes were reached in the presence of glucose. Mitochondrial inhibitors which affected germination on acetate did not arrest germination on glucose. However, mitochondrial activity was required for germination on galactose in a strain carrying the mutated allele imp1 of the nucleomitochondrion-connecting gene IMP1.

Acetates↗
PubMed1985

Partial restoration of meiosis in an apomictic strain of Saccharomyces cerevisiae: a model system for investigation of nucleomitochondrial interactions during sporulation.

In an apomictic strain of Saccharomyces cerevisiae (ATCC 4117-H2) which undergoes a single nuclear division during sporulation and consequently forms asci containing two uninucleate diploid spores, a study was undertaken to investigate the effects of cultivation in three presporulation media (YPA; YNB; SMM) on nuclear division and ascosporogenesis in sporulation medium. Comparison of effects of presporulation culture in these media on the number of spores formed per ascus showed that a marked induction (30 +/- 4.3 per cent) of three- and four-spored asci could occur in sporulation medium following cultivation in a defined YNB medium supplemented with a 1 per cent solution of vitamins and containing decreased ammonium sulphate and increased glucose levels. Experiments in which the concentrations of glucose and of ammonium sulphate were varied simultaneously indicated that the initial presporulation carbon to nitrogen source ratio is an important factor in determining tetrad formation in sporulation medium. Nuclear staining demonstrated two classes of asci: binucleate (one- and two-spored) and tetranucleate (three- and four-spored). Genetic evidence and data concerning effects of inclusion in sporulation medium of a meiotic inhibitor (glucose) indicated spores in tetrads were haploid rather than diploid. This ability to condition a significant number of cells for meiotic rather than apomictic differentiation made possible investigation of effects of mitochondrial inhibitors on both developmental processes simultaneously. It was found possible to selectively inhibit meiotic development by inclusion in sporulation medium of appropriate concentrations of specific inhibitors. Moreover, the data suggest meiotic sporulation is more strictly dependent than apomictic sporulation on mitochondrial function.

Acetates↗
PubMed1985

Relationship between growth inhibition and mitochondrial function in petite-negative yeasts. I. Effects of antibiotics and dyes upon pathogenic and non-pathogenic Candida species.

Antibiotics and dyes which preclude growth of Saccharomyces cerevisiae in media containing oxidizable carbon sources arrested the growth of Candida albicans, Candida tropicalis and Candida utilis even in glucose medium. The growth in the presence of sub-inhibitory concentrations of the various antibiotics and dyes determined a reduction in the cell survival but with no accumulation of respiratory deficient mutants. Under these culture conditions, the total respiration declined leaving a residual antimycin A-resistant--hydroxamate-sensitive O2 uptake, and the amount of the respiratory cytochromes aa3 and b synthesized was reduced. SDS gel electrophoresis of soluble proteins prepared from the antibiotic-treated cells showed some bands in the MW range 92-100 K, which became faint after the cells were grown in the presence of some mitochondrial inhibitors. The ultrastructural analysis of these cells evidenced disappearance of the mitochondrial cristae and their replacement by unfolded membranes. The data obtained suggest that the petite negative trait of Candida could depend on the non-viability or on the very low viability of those cells which have lost their mitochondrial function.

Anti-Bacterial Agents↗
PubMed1985

Relationship between growth inhibition and mitochondrial function in petite-negative yeasts. II. Effects of central nervous system drugs upon pathogenic and non-pathogenic Candida species.

Six nervous system drugs which inhibited vegetative reproduction of S. cerevisiae arrested also mitotic division of C. utilis, C. albicans and C. tropicalis. Chlorpromazine and chlorpheniramine which proved to be the most effective, affected respiration and cytochrome biosynthesis. Electrophoretic bands with MW congruent to 100 K were faint in silver-stained electrophoregrams of proteins from cells grown in the presence of a sub-inhibitory concentration of chlorpromazine.

Amitriptyline↗