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Biomedical subjects

A Viotti

Publications and source records attributed to A Viotti.

At least 19 recordsLinked to original sources
PubMed2001

Methylation of the Opaque2 box in zein genes is parent-dependent and affects O2 DNA binding activity in vitro.

In the maize endosperm, the expression of the subfamily 4 (SF4) of the zein genes is under the transcriptional control of the Opaque2 (O2) basic leucine zipper transcriptional activator, which binds to the O2-box 5'-TCCACGTAGA-3'. Southern experiments showed that the O2-box core sequence ACGT is heavily methylated in sporophytic tissues but becomes hypomethylated in the endosperm. DNA analyses of two inbred lines and their reciprocal crosses indicate that the hypomethylation state is established on the maternal complements of the endosperm. Electrophoretic mobility shift assay (EMSA) and southwestern experiments with endosperm protein extracts and recombinant O2 using oligonucleotides with methylated and unmethylated cytosines in the O2-box indicate an inhibitory effect of modified sequences on O2-binding activity. These results suggest that DNA methylation modulates O2 activity in vivo and shed light on molecular mechanisms involved in the parent-dependent zein gene expression in maize endosperm.

Base Sequence↗
PubMed2000

Differential expression of chitin synthase III and IV mRNAs in ascomata of Tuber borchii Vittad.

A full-length genomic clone encoding a class III chitin synthase (CHS) and one DNA fragment corresponding to a class IV CHS were isolated from the mycorrhizal fungus Tuber borchii and used for an extensive expression analysis, together with a previously identified DNA fragment corresponding to a class II CHS. All three Chs mRNAs are constitutively expressed in vegetative mycelia, regardless of the age, mode of growth, and proliferation capacity of the hyphae. A strikingly different situation was observed in ascomata, where class III and IV, but not class II, mRNAs are differentially expressed in a maturation stage-dependent manner and accumulate, respectively, in sporogenic and vegetative hyphae. These data, the first on the expression of distinct Chs mRNAs during fruitbody development, point to the different cellular roles that can be played by distinct chitin synthases in the differentiation of spores of sexual origin (CHS III) or in ascoma enlargement promoted by the growth of vegetative hyphae (CHS IV).

Amino Acid Sequence↗
PubMed2000

Specific combinations of zein genes and genetic backgrounds influence the transcription of the heavy-chain zein genes in maize opaque-2 endosperms.

The transcript levels of heavy-chain zein genes (zH1 and zH2) and the occurrence of the zH polypeptides in different opaque-2 (o2) lines were investigated by RNA-blot analyses and by sodium dodecylsulfate-polyacrylamide gel electrophoresis or two-dimensional gel electrophoresis protein fractionations. Four mutant alleles o2R, o2T, o2It, and o2-676 introgressed into different genetic backgrounds (GBs) were considered. The mono-dimensional gel electrophoresis zein pattern can be either conserved or different among the various GBs carrying the same o2 allele. Likewise, in the identical GB carrying different o2 alleles, the zein pattern can be either conserved or differentially affected by the different mutant allele. Zein protein analysis of reciprocal crosses between lines with different o2 alleles or the same o2 showed in some case a more than additive zH pattern in respect to the o2 parent lines. Electrophoretic mobility shift assay approaches, with O2-binding oligonucleotide and endosperm extracts from the above o2 lines, failed to reveal o2-specific retarded band in any of the o2 extracts. The results suggest that the promoter of some zH1 and zH2 contains motif(s) that can respond to factors other than O2.

Alleles↗
PubMed1999

Variety identification in maize lines via capillary electrophoresis of zeins in isoelectric acidic buffers.

Zeins (the prolamins or seed storage proteins in maize) have been used to characterize and identify different genotypes. Zeins were fractionated by capillary zone electrophoresis in acidic, amphoteric buffers, which represent a medium of moderate conductivity and are thus compatible with higher voltage gradients. The running buffer consisted of 40 mM isoelectric aspartic acid, in presence of 6 M urea and 0.5% hydroxyethyl cellulose (apparent pH: 3.8; pI in the absence of urea: 2.77). Thirty-one different zein peaks were mapped out of a total of 21 different maize genotypes. Each of them typically exhibited seven to twelve peaks, with some genotypes showing up to 20 zein bands. Due to slightly changing elution times, caused by a lack of reproducibility of the electroendoosmotic flow in uncoated silica surfaces, correct peak assignment and alignment among different runs was obtained by multivariate statistical analysis. The present method compares well, both in resolution and total number of peaks, with current protocols adopted for screening of maize inbreds, which consist of isoelectric focusing in agarose gels.

Buffers↗
PubMed1999

The activity of the maize Opaque2 transcriptional activator is regulated diurnally.

The maize (Zea mays L.) Opaque2 (O2) protein is an endosperm-specific transcriptional activator whose DNA-binding activity is regulated diurnally by a phosphorylation/dephosphorylation mechanism. We show that the O2 transcript undergoes pronounced oscillations during the day-night cycle. The highest level of the O2 message is present at midday and the lowest level at midnight. The level of O2 transcript follows a diurnal rhythm that appears controlled by the circadian clock. Two different endosperm-expressed DNA-binding proteins, PBF (prolamin box-binding factor) and OHP1 (O2-heterodimerizing protein 1), were also analyzed. While the PBF message levels oscillate diurnally, the steady-state levels of OHP1 transcript were constant through the day and night. We present data showing that the seed is not directly involved in the perception of the light signal, but presumably responds to diurnal fluxes of nutrients into the endosperm. Moreover, we show that the O2 protein is not involved in the regulation of its own transcript levels. These data indicate that O2 activity is down-regulated at night by both a reduction in O2 transcript and by hyperphosphorylation of residual O2 protein, and suggest that regulatory gene activity during endosperm development may be acutely sensitive to a diurnal signal(s) emanating from the plant and passing into the developing seeds.

Circadian Rhythm↗
PubMed1998

Identification of maize lines via capillary electrophoresis of zeins in isoelectric, acidic buffers.

Modified capillary zone electrophoresis of zeins in strongly acidic, isoelectric buffers is reported for screening of maize (Zea mays L.) lines. The optimized background electrolyte contained 40 mM aspartic acid (pH = pI = 2.77), 8 M urea and 0.5% short-chain hydroxyethylcellulose (Mn 27000 Da) (apparent pH in 8 M urea: 3.9). Due to the low conductivity of such a buffer (0.7 mmhos), separations can be carried out at 800 V/cm, even in relatively large bore capillaries (50 microm ID), ensuring good sensitivity. The zein patterns thus obtained are species-specific and allow easy identification of all maize lines tested. No adsorption of proteins to the silica wall is observed and high reproducibility in peak areas and transit times is obtained. The relatively slow migration time (ca. 30 min), even under such a high voltage gradient, is attributed to the paucity of basic amino acid residues typical of zeins, leading to an unfavorable charge/ mass ratio due to the modest net positive charge at the operative pH.

Acids↗
PubMed1997

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns to a single band corresponding to the nonphosphorylated component. In vivo and in vitro labeling confirmed that O2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated O2 polypeptides were able to bind an oligonucleotide containing the O2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime to nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that O2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that O2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears to be influenced by environmental conditions.

Circadian Rhythm↗
PubMed1997

Experimental use of a pedicle gallbladder graft for the repair of large duodenal defects.

The use of a gallbladder patch to cover duodenal wall defects was attempted on 36 dogs. This defect was surgically induced on the second part of the duodenum. All the dogs survived and had an uneventful postoperative course. The healing of the defect was satisfactory and the structure and thickness of the graft remained unchanged This method could be a suitable alternative in the treatment of large duodenal defects, especially in the presence of other intra-abdominal injuries, and when the use of other graft material is not feasible.

Animals↗
PubMed1995

Endosperm-specific demethylation and activation of specific alleles of alpha-tubulin genes of Zea mays L.

We have investigated the methylation status of the alpha-tubulin genes, and the degree of accumulation of their mRNAs in endosperm, embryo and seedling tissues of Zea mays L. We have found that many of the alpha-tubulin genes are differentially demethylated in the endosperm relative to the embryo and seedling. However, only for tub alpha 2 and tub alpha 4 could a correlation between DNA demethylation and increased RNA accumulation be detected. By analyzing the inbred lines W64A and A69Y and their reciprocal crosses, we have also identified in the endosperm two alpha-tubulin genes, tub alpha 3 and tub alpha 4, that are differentially demethylated if transmitted by the maternal germline, but that remain hypermethylated when transmitted by the paternal germline.

Alleles↗
PubMed1995

Maternal-specific demethylation and expression of specific alleles of zein genes in the endosperm of Zea mays L.

Zeins constitute 60-70% of maize endosperm protein. Zein genes are specifically transcribed in the endosperm, and a correlation has been established between tissue-specific expression and demethylation. Three inbred lines and their reciprocal crosses were analysed to assess for allele-specific differences in methylation, transcription and translation. DNAs from endosperm, embryo and seedling tissues analysed by cleavage with methylation-sensitive restriction enzymes and Southern blot hybridization with zein cDNA and genomic sequences show that specific demethylation of zein sequences occurs only in endosperm and is restricted to the maternal complements. Steady-state transcript accumulation of zein mRNA assessed by RNase protection assay reveals qualitative and quantitative differences among endosperm RNAs of the inbreds and of their reciprocal hybrids. Moreover, two-dimensional gel electrophoresis of zein proteins identified polypeptides that are maternally imprinted in reciprocal crosses. These results indicate that endosperm-specific expression of specific zein alleles may occur via parental imprinting and disclose a possible role of methylation in regulating the expression of genes differently contributed in the endosperm by the maternal and paternal genomes.

Alleles↗
PubMed1994

Molecular analysis of wild-type and mutant alleles at the Opaque-2 regulatory locus of maize reveals different mutations and types of O2 products.

The expression of the various members of the zein multigene family in maize endosperm is controlled by different regulatory loci. One of these loci, Opaque-2, coding for a bZIP transcriptional factor, controls the expression of a subset of zein genes. Analysis of genomic DNA from plants carrying wild-type (O2) or mutant o2 alleles shows specific DNA restriction patterns that correlate with transcript types and their various gene products. Northern and western analyses show the presence in different wild types of a 1.7 kb transcript coding for different sizes of normal O2 proteins that migrate as doublets in the 68-72 kDa range. Among the various o2 mutants analysed we showed the occurrence of various null-transcript alleles, the presence of alleles with a normal size transcript which, however, produce a different-sized o2 protein, and a mutant producing both a normal size transcript and a longer transcript, but generating only a single o2 product migrating around 40 kDa. Analysis of other mutations (o7, fl2) known to affect zein polypeptide synthesis shows no interference of these mutations in the expression of the O2 gene products. The overall results indicate the occurrence of micro heterogeneity in the O2 wild-type genes and a broad spectrum of o2 mutations, both producing different sizes of O2 or o2 proteins. A nomenclature of the O2 and o2 genes based on the RFLP, transcripts and products of the various alleles is presented.

Alleles↗
PubMed1994

[Zinc deficiency syndrome during TPN].

The zinc deficiency syndrome, also called enteropathic acrodermatitis, has been mostly observed in those pathologies of the gastroenteric system characterized by grave food shortages during long term TPN in association with inflammatory intestinal pathologies. The authors believe this to be caused of such syndrome, that in the case in question is demonstrated before the normal period described in the literature to be caused by both increased request related to TPN and a greater loss or less absorption due to intestinal phlogosis. The case described has been noted in a general surgery division in a young patient suffering from Crohn's disease for many years in treatment with medical therapy and now complicated by perianal abscess following burrowing on the outside subjected and therefore in treatment with artificial parenteral nutrition pre and post operative. Such pathology to be due when to begin a symptomatology characterized by consciousness alteration, diarrhoea, vesicular squamous cutaneous lesions around orifices, often infected by bacteria and mycosis in patients in treatment nutritional artificial continued for digestive apparatus diseases. The knowledge of this syndrome and its diagnosis lead, through integrative therapy, to its resolution in a short time. The authors describe the course of a clinical case occurred to then and ended with the patient's recovery. They underline the risks of ignoring this pathology.

Acrodermatitis↗
PubMed1992

Functional analysis of the regulatory region of a zein gene in transiently transformed protoplasts.

The transcription of zein genes in maize is tissue-specific and developmentally regulated. The 5' regulatory region of many zein genes contains two promoters, P1 and P2, lying approximately 1000 bases apart. The promoter/enhancer activity of various fragments of the two promoter regions of the zein gene E19 have been analysed by means of transient expression experiments. The results indicate that the various regions differentially affect the expression of the GUS reporter gene activity in protoplasts from tobacco leaves, maize immature endosperms and in vitro endosperm cell cultures. In tobacco protoplasts only the proximal promoter region, P2, activates GUS expression, while in endosperm culture cells only the distant promoter, P1, gives significant activity. The P1 region, both in direct and opposite orientation, stimulates a low level of GUS expression in protoplasts from immature endosperms.

Base Sequence↗
PubMed1991

Genetic and molecular analysis of Sn, a light-inducible, tissue specific regulatory gene in maize.

The Sn locus of maize is functionally similar to the R and B loci, in that Sn differentially controls the tissue-specific deposition of anthocyanin pigments in certain seedling and plant cells. We show that Sn shows molecular similarity to the R gene and have used R DNA probes to characterize several Sn alleles. Northern analysis demonstrates that all Sn alleles encode a 2.5 kb transcript, which is expressed in a tissue-specific fashion consistent with the distribution of anthocyanins. Expression of the Sn gene is light-regulated. However, the Sn: bol3 allele allows Sn mRNA transcription to occur in the dark, leading to pigmentation in dark-grown seedlings and cob integuments. We report the isolation of genomic and cDNA clones of the light-independent Sn: bol3 allele. Using Sn cDNA as a probe, the spatial and temporal expression of Sn has been examined. The cell-specific localization of Sn mRNA has been confirmed by in situ hybridization using labelled antisense RNA probes. According to its proposed regulatory role, expression of Sn precedes and, in turn, causes a coordinate and tissue-specific accumulation of mRNA of structural genes for pigment synthesis and deposition, such as A1 and C2. The functional and structural relationship between R, B, Lc and Sn is discussed in terms of an evolutionary derivation from a single ancestral gene which gave rise this diverse gene family by successive duplication events.

Acyltransferases↗
PubMed1990

The maize zein gene zE19 contains two distinct promoters which are independently activated in endosperm and anthers of transgenic Petunia plants.

The activity, tissue specificity and temporal expression of the tandem promoter region preceding a maize zein gene (zE19, encoding a 19 kDa zein protein) were tested in transgenic Petunia plants. To simplify the analysis, the tandem promoter as well as each of the two separate promoter regions were fused to the beta-glucuronidase (GUS) reporter gene. All of the three constructs directed the synthesis of GUS in the endosperm of transformed seeds indicating that both separate promoters are independently activated and show the same tissue and cell type specificity observed for zein genes in maize. The kinetics of accumulation and the localization of GUS activity are not coordinated with those of Petunia endogenous seed storage proteins during the development of transformed seeds. Unexpectedly, we detected high levels of GUS activity in anthers of transformed Petunia plants for all three constructs. This appears to reflect the expression pattern of zein genes in maize, since we detect zein transcripts in anthers. Finally, we discuss the possible origin and function of the tandem promoter arrangement on the basis of these data.

Base Sequence↗
PubMed1987

Translational regulation of the expression of zein cloned in yeast under an inducible GAL promoter.

Yeast cells transformed by a plasmid containing a zein sequence fused to an hybrid yeast promoter GAL1-10/CYC1 accumulate, during a batch growth in galactose minimal medium, large amounts of zein only during a growth-limited phase that precede the entering into the stationary phase. We found that zein is fairly stable in yeast cells and the increased accumulation of zein polypeptide depends mainly upon a marked increase of its rate of synthesis. The increase of the rate of heterologous protein synthesis is not dependent on variation in the plasmid copy number and it is not related to the relative level of zein mRNA, indicating the existence of a postranscriptional regulation that modulates the translatability of this messenger RNA in function of the growth conditions. A possible explanation of this modulation is discussed in terms of a codon bias effect that slow-down the translation of heterologous mRNAs during the exponential phase of growth.

Autoradiography↗
PubMed1986

Transcription and expression of zein sequences in yeast under natural plant or yeast promoters.

Maize genomic fragments containing the regulatory and coding regions of a zein gene for a low size class 23-kd protein have been inserted in an interspecific Escherichia coli-Saccharomyces cerevisiae expression vector in different constructions. The presence of the inducible GAL1-10 upstream activation site (UAS) allows us to regulate differentially by carbon sources the transcription of the zein gene both under the plant promoter and under the yeast CYC-1 promoter. We found that the zein promoter region is properly recognized at the correct transcription start, while different termination points occur during transcription. The yeast UAS was also shown to function as a typical eukaryotic enhancer regardless of its distance or orientation with respect to the plant promoter. Yeast cells transformed by a plasmid containing a zein sequence fused to a short piece of the CYC-1 gene produced a fused polypeptide, of expected mol. wt, in variable amount from 0.2 to 5% depending on the growth phase conditions.

Journal Article↗