Biomedical subjects

F Carnevali

Publications and source records attributed to F Carnevali.

At least 19 recordsLinked to original sources

PubMed2001

Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase.

A 1329-base pair clone isolated from a human placenta cDNA library contains a full-length 837-base pair coding region for a 31.9-kDa protein whose deduced primary structure exhibits high homology to consensus sequences found in other NMN adenylyltransferases. Northern blotting detected a major 3.1-kilobase mRNA transcript as well as a minor 4.1-kilobase transcript in all human tissues examined. In several cancer cell lines, lower levels of mRNA expression were clearly evident. The gene encoding the human enzyme was mapped to chromosome band 1p32-35. High efficiency bacterial expression yielded 1.5 mg of recombinant enzyme/liter of culture medium. The molecular and kinetic properties of recombinant human NMN adenylyltransferase provide new directions for investigating metabolic pathways involving this enzyme.

Amino Acid Sequence↗

PubMed1999

Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme.

The enzyme nicotinamide mononucleotide (NMN) adenylyltransferase (EC 2.7.7.1) catalyzes the transfer of the adenylyl moiety of ATP to NMN to form NAD. A new purification procedure for NMN adenylyltransferase from Saccharomyces cerevisiae provided sufficient amounts of enzyme for tryptic fragmentation. Through data-base search a full matching was found between the sequence of tryptic fragments and the sequence of a hypothetical protein encoded by the S. cerevisiae YLR328W open reading frame (GenBank accession number U20618). The YLR328W gene was isolated, cloned into a T7-based vector and successfully expressed in Escherichia coli BL21 cells, yielding a high level of NMN adenylyltransferase activity. The purification of recombinant protein, by a two-step chromatographic procedure, resulted in a single polypeptide of 48 kDa under SDS-PAGE, in agreement with the molecular mass of the hypothetical protein encoded by YLR328W ORF. The N-terminal sequence of the purified recombinant NMN adenylyltransferase exactly corresponds to the predicted sequence. Molecular and kinetic properties of recombinant NMN adenylyltransferase are reported and compared with those already known for the enzyme obtained from different sources.

Amino Acid Sequence↗

PubMed1999

Negative regulation of axis formation and Wnt signaling in Xenopus embryos by the F-box/WD40 protein beta TrCP.

Screening a maternal Xenopus expression library for activities that synergize with low levels of injected beta-catenin, we have isolated a clone encoding the C-terminal end of x-beta TrCP-2, a highly conserved protein belonging to the F-box/WD40 family of ubiquitin-ligase specificity factors. We show that x-beta TrCP-2 expression reduces dorsal axis formation in Xenopus embryos. A dominant negative mutant lacking the F-box triggers the opposite effect, inducing secondary axes and activating the expression of Wnt responsive genes in ectodermal explants. In light of the existence of beta TrCP transcripts associated with the vegetal cortex, we propose that beta TrCP plays a fundamental role in the establishment of the dorsal determinants during cortical rotation in Xenopus.

Animals↗

PubMed1998

FGF-mediated mesoderm induction involves the Src-family kinase Laloo.

During embryogenesis, inductive interactions underlie the development of much of the body plan. In Xenopus laevis, factors secreted from the vegetal pole induce mesoderm in the adjacent marginal zone; members of both the transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) ligand families seem to have critical roles in this process. Here we report the identification and characterization of laloo, a novel participant in the signal transduction cascade linking extracellular, mesoderm-inducing signals to the nucleus, where alteration of cell fate is driven by changes in gene expression. Overexpression of laloo, a member of the Src-related gene family, in Xenopus embryos gives rise to ectopic posterior structures that frequently contain axial tissue. Laloo induces mesoderm in Xenopus ectodermal explants; this induction is blocked by reagents that disrupt the FGF signalling pathway. Conversely, expression of a dominant-inhibitory Laloo mutant blocks mesoderm induction by FGF and causes severe posterior truncations in vivo. This work provides the first evidence that a Src-related kinase is involved in vertebrate mesoderm induction.

Amino Acid Sequence↗

PubMed1998

Electrocardiographic alterations in chronically Trypanosoma cruzi-infected persons exposed to cardiovascular factors.

METHODS: We analyzed the potential influence that associated risk factors (ARF), such as smoking, alcoholism, overweight, and hypertension, could have on the establishment of chronic chagasic cardiomyopathy (CC). The sample was comprised of 124 individuals, 69 males and 55 females (mean age +/- SD, 41 +/- 9.5 years), who were born in en demic areas of Northern Argentina and migrated further to Rosario City, an area where autochthonous cases of Chagas' disease have never been registered. Assessments included the following: clinical examination to discard previous cardiomyopathies; search for the presence of ARF according to standard criteria; specific serology; frontal chest X-ray, and 12-lead resting electrocardiogram (ECG). Subjects were classified on the basis of their serological status and presence of ARF into four groups: Tc+ARF+ T. cruzi-infected persons with ARF (n = 41); Tc-ARF+ seronegativity in presence of ARF (n = 27); Tc+ARF- individuals showing positive serology that lacked ARF (n = 27), and Tc-ARF- seronegative individuals having no ARF (n = 29). RESULTS: Except for a higher female/male ratio in groups presenting no ARF (p < 0.02), no statistical differences as to age, length of residence in endemicity areas (LR), and ARF distribution were recorded among groups. Forty-one persons presented abnormal ECG tracings, distributed thus: Tc+ARF+, 18/41; Tc-ARF+, 14/27, Tc+ARF-, 14/27, and Tc-ARF, 4/29 (p < 0.01, in relation to the latter group). Subjects from the Tc+ARF+, Tc-ARF+, and Tc+ARF- groups had 4.89-, 6.7-, and 6.7-fold increases, respectively, if having an abnormal ECG when compared with Tc-ARF- individuals. Comparisons on the frequency of abnormal ECG between seropositives carrying ARF or not yielded a non-significant odds ratio, be it estimated as crude, or after adjusting for sex, age, and LR in multivariate analysis. CONCLUSIONS: Presence of ARF was not associated with an increasing risk of cardiac affectation in chronically T. cruzi-infected persons, but resulted in chagasic-compatible ECG abnormalities in those seronegative individuals.

Adult↗

PubMed1995

Cloning, expression and functional role of XrpFI alpha and beta subunits in Xenopus laevis oocyte.

The cDNAs coding for the alpha and beta components of XrpFI, an activator of ribosomal protein genes transcription, were isolated. RNA analysis showed that transcripts of the beta genes are present at constant level throughout Xenopus development unlike the beta proteins which are undetectable in the early stages of embryogenesis when transcription is silent. On the contrary, alpha transcripts and proteins were found in all the embryo stages examined. The role of the XrpFI alpha and beta subunits in rp-gene transcription has been studied by mRNA microinjection into X.laevis oocytes. The presence of the beta subunit appears to be critical for the proper rp-gene promoter function.

Animals↗

PubMed1994

Characterization of FIII/YY1, a Xenopus laevis conserved zinc-finger protein binding to the first exon of L1 and L14 ribosomal protein genes.

The cDNA coding for the Xenopus laevis homolog of the transcriptional activator/repressor protein delta/YY1 was isolated from a lambda gt11 oocyte cDNA library. The deduced aminoacid sequence shows that the four zinc fingers of the DNA binding domain are 99% conserved when compared to the mouse (delta) and 95% to the human (YY1) proteins, while differences are found in the N-terminal region. In particular, the long run of consecutive glycines and histidines of delta and YY1 is missing. The protein, named FIII/YY1, was overexpressed into Xenopus oocytes from the cDNA under direction of the L14 rp-promoter and found to share antigenic and DNA-binding properties with the oocyte endogenous protein binding to the first exon of the X.laevis ribosomal protein genes (rp-genes) L1 and L14.

Amino Acid Sequence↗

PubMed1993

XrpFI, an amphibian transcription factor composed of multiple polypeptides immunologically related to the GA-binding protein alpha and beta subunits, is differentially expressed during Xenopus laevis development.

XrpFI, first identified in the extract of Xenopus laevis oocyte nuclei, binds to a proximal sequence of the L14 ribosomal protein gene promoter. Its target sequence, 5'-TAACCGGAAGTTTGT-3', is required to fully activate the promoter, and the two G's of the central motif are essential for factor binding and transcriptional activation; our data also suggest that XrpFI may play a role in cap site positioning. The binding site of XrpFI is homologous to the sequence recognized by the family of ets genes. Antibodies specific for Ets-1 and Ets-2 proteins did not react with XrpFI, but those raised against the rat alpha and beta GA-binding proteins both supershifted the retarded bands formed by XrpFI. The Xenopus polypeptides related to GA-binding protein alpha interact with DNA both as monomers and as heterodimers associated with beta-related proteins. Oocyte nuclei contain multiple forms of alpha- and beta-related proteins: the alpha-like proteins remain throughout development, while the pattern of the beta species changes in the embryonic stages examined. beta-like proteins are undetectable in the cleavage period up to the neurula stage, but at later stages, when ribosomal protein genes are actively transcribed, two beta-related polypeptides reappear.

Animals↗

PubMed1990

HrpF, a human sequence-specific DNA-binding protein homologous to XrpFI, a Xenopus laevis oocyte transcription factor.

The identification in HeLa nuclei of a novel DNA-binding protein, designated HrpF, is presented. This factor recognizes and binds a sequence of the Xenopus laevis L14 ribosomal protein (r-p) gene promoter bound by the Xenopus r-p transcription factor I (XrpFI). We show here that XrpFI and HrpF share a conserved DNA-binding domain. We also present evidences suggesting that the two factors perform similar functions in the cell. We discuss the hypothesis that closely related factors might be involved in the control of rp-gene transcription in vertebrates.

Animals↗

PubMed1990

Evidence for a direct negative effect of estradiol at the level of the pituitary gland in sheep.

The purpose of this experiment was to determine if pituitary stores of LH could be replenished by administration of GnRH when circulating concentrations of both progesterone and estradiol-17 beta (estradiol) were present at levels observed during late gestation. Ten ovariectomized (OVX) ewes were administered estradiol and progesterone via Silastic implants for 69 days. One group of 5 steroid-treated OVX ewes was given GnRH for an additional 42 days (250 ng once every 4 h). Steroid treatment alone reduced (p less than 0.01) the amount of LH in the anterior pituitary gland by 77%. Pulsatile administration of GnRH to steroid-treated ewes resulted in a further decrease (p less than 0.01) in pituitary content of LH. Compared to the OVX ewes, concentrations of mRNAs for alpha- and LH beta-subunits were depressed (p less than 0.01) in all steroid-treated ewes, whether or not they received GnRH. The ability of the dosage of GnRH used to induce release of LH was examined by collecting blood samples for analysis of LH at 15 days and 42 days after GnRH treatment was initiated. Two of 5 and 3 of 5 steroid-treated ewes that received pulses of GnRH responded with increased serum concentrations of LH after GnRH administration during the first and second bleedings, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Nuclear factors specifically bind to upstream sequences of a Xenopus laevis ribosomal protein gene promoter.

The upstream region of the Xenopus laevis L14 ribosomal protein gene was deleted starting from the 5' extremity in order to define the promoter length necessary to express a linked reporter CAT gene. The functional analysis indicated that a sequence located between -63 and -49 from the capsite is important for an efficient promoter activity. Band shift and ExoIII protection assays evidenced the binding to this region of a factor, called XrpFI, present in the crude nuclear extract from X.laevis oocytes. Methylation interference analysis localized the contacts in the G residues belonging to a short box, 5' CTTCC 3', positioned between -53 and -49 from the capsite. An additional factor, XrpFII, makes contacts with the sequence 5'GCCTGTTCGCC 3' located between -27 and -17 from the capsite. The deletion mutant still containing this sequence is poorly transcribed, but resumes activity when a short fragment containing the binding site for factor XrpFI is cloned in an upstream position.

Animals↗

PubMed1989

Large scale isolation of nuclei from oocytes of Xenopus laevis.

We describe an improvement on the procedure of Scalenghe et al. (F. Scalenghe, M. Buscaglia, C. Steinheil, and M. Crippa (1978) Chromosoma 66, 299-308) for the large scale isolation of nuclei from Xenopus laevis oocytes. The nuclear extract obtained was tested for its ability to transcribe a cloned Xenopus 5 S RNA gene and for the presence of nuclear factors interacting with a X. laevis ribosomal protein gene promoter. Efficiency of accurate transcription and of factor binding is comparable with that of an extract prepared from manually isolated nuclei.

Animals↗

PubMed1985

Activation of in vitro transcription and topology of closed DNA domains.

Activation of in vitro transcription of otherwise inert DNA sequences by purified yeast RNA polymerase II has been observed following the introduction in closed DNA domains of fragments of various origin. This enhancer-like effect on the in vitro transcriptional capacity is only detected in negatively supercoiled DNA domains and is characterized for each chimaeric plasmid by the superhelical density (- sigma) at which a sharp transition toward activation takes place. We have analyzed the topological state (as defined by localization and evaluation of the relative occurrence of secondary structures sensitive to S1 endonuclease) of the activated closed domains as a function of the conditions that determine the transcriptional enhancer effect, i.e. superhelical density, size, and nature of the components of the domains. We observe that variations in transcriptional capacity coincide with a defined pattern of secondary structures. These observations support a cause-effect relation between topology and regulation of transcription.

Base Sequence↗

PubMed1984

Transitions in topological organization of supercoiled DNA domains as a potential regulatory mechanism.

We present an analysis of the influence of DNA superhelicity on the topology of chimeric plasmids. A correlation is found between topological variations and in vivo/in vitro functions. Data refer to the topological transitions observed in function of variations of the superhelical density in the three closed DNA domains that represent the three extreme examples, from the point of view of the topological organization, among many chimeric systems analyzed. The plasmids studied are ADR2-BS-pBR322 (the vector pBR322 + the yeast gene ADR2), ADR3-5c-yRp7 (containing yeast TRP1, a constitutive mutant of yeast ADR2, a fragment of 5 kilobases of unanalyzed chromosomal DNA and the vector pBR322), and p31 (pBR322 + a yeast DNA fragment encompassing the right moiety of the Ty1 element and its in vivo promoter). In the model systems analyzed, the topological transitions observed in the eukaryotic sequences (relevant in one case for activation of selective in vitro transcription (Ty), in another (ADR2) for the in vivo expression) take place in the range of superhelical densities predicted on theoretical ground (Vologodskii, A. V., Lukashin, A. V. Anshelevich, V. V. & Frank-Kamenetskii, M. D. (1979) Nucleic Acids Res. 6, 967-982).

Alcohol Dehydrogenase↗

PubMed1983

Topological modifications and template activation are induced in chimaeric plasmids by inserted sequences.

The effect of the insertion of foreign genes or gene systems in closed DNA domains has been investigated in vitro in purified systems. We observe that in chimaeric plasmids two apparently independent classes of modifications, (1) functional and (2) topological, do take place in defined instances. (1) Among the screened yeast gene systems, examples have been found of DNA sequences that upon insertion cause activation of in vitro transcription of distant genes. (2) Foreign DNA sequences may lead to new topological features of the harbouring plasmids; it is shown that more than one S1-sensitive secondary structure may be contemporaneously present on the same chimaeric plasmid. DNA superhelicity is a prerequisite of these modifications. The two classes of effects (1) functional and (2) topological are not a priori directly related one to the other but appear to be two independent consequences of the same cause: the insertion of foreign DNA sequences into closed DNA domains. These observations suggest a regulatory model of gene expression based on alternative topologies of closed DNA domains.

Chimera↗

PubMed1982

In vitro transcription by purified yeast RNA polymerase II. Coarse promoter mapping on homologous cloned genes.

Clones of the yeast Tyl element and 2 microns plasmid have been selectively transcribed in vitro by partially or completely purified yeast RNA polymerase II. Electrophoretic analysis of whole and restricted ternary transcription complexes allows the localization of the in vitro actively transcribed regions of the analyzed genes. The DNA regions that actively promote in vitro transcription correspond to the nucleotide sequence that in the Tyl element encompasses the in vivo transcription initiation sites and that in the 2 micrometer plasmid encompasses the starting codons of two oppositely oriented potential protein coding frames. The transcription assay that we describe herein may be applied to analyze rapidly the in vitro transcription of clones genes, to localize transcription initiation sites on supercoiled templates and to evaluate the differential in vitro promoter strength in RNA polymerase II served genes. Data obtained with RNA polymerase II at two different stages of purification are presented in parallel. Studies with a completely purified enzyme should certainly be preferred although the use of a partially purified RNA polymerase II may be convenient and may reveal factors which affect specificity.

Base Sequence↗

PubMed1981

Selective in vitro transcription by purified yeast RNA polymerase II on cloned 2 micron DNA.

The in vitro transcription properties of purified yeast RNA polymerase II have been analyzed on prokaryotic plasmids (pBR322 and pBR313) and chimaeric plasmids bearing yeast 2 micron sequences (BTYP 1, BTYH 2 and BTYH 3). Conditions for selective transcription of the 2 micron DNA sequences in chimaeric plasmids have been determined. pBR322 and pBR313 are not transcribed by the purified RNA polymerase II when not bearing eukaryotic inserts. We show that the agarose gel electrophoretic analysis of ternary transcription complexes allows the localization of nascent RNA chains. The RNA produced has been visualized by electron microscopy (nascent RNA hybridization loops) and by gel electrophoretic analysis. All the observed properties are shared by RNA polymerase II purified by a conventional method (1) and by a rapid alternative procedure described herein. The peculiar properties of a partially purified form of RNA polymerase II are reported.

DNA, Fungal↗

PubMed1981

Single-stranded molecules in DNA preparations from cultured mammalian cells at different moments of cell cycle.

Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.

Animals↗