Biomedical subjects

J Pelletier

Publications and source records attributed to J Pelletier.

At least 163 records · Page 9Linked to original sources

PubMed1991

Expression of the Wilms' tumor gene WT1 in the murine urogenital system.

The Wilms' tumor gene WT1 is a recessive oncogene that encodes a putative transcription factor implicated in nephrogenesis during kidney development. In this report we analyze expression of WT1 in the murine urogenital system. WT1 is expressed in non-germ-cell components of the testis and ovaries in both young and adult mice. In situ mRNA hybridization studies demonstrate that WT1 is expressed in the granulosa and epithelial cells of ovaries, the Sertoli cells of the testis, and in the uterine wall. In addition to the 3.1-kb WT1 transcript detected by Northern blotting of RNA from kidney, uterus, and gonads, there is an approximately 2.5-kb WT1-related mRNA species in testis. The levels of WT1 mRNA in the gonads are among the highest observed, surpassing amounts detected in the embryonic kidney. During development, these levels are differentially regulated, depending on the sexual differentiation of the gonad. Expression of WT1 mRNA in the female reproductive system does not fluctuate significantly from days 4 to 40 postpartum. In contrast, WT1 mRNA levels in the tesis increase steadily after birth, reaching their highest expression levels at day 8 postpartum and decreasing slightly as the animal matures. Expression of WT1 in the gonads is detectable as early as 12.5 days postcoitum (p.c.). As an initial step toward exploring the tissue-specific expression of WT1, DNA elements upstream of WT1 were cloned and sequenced. Three putative transcription initiation sites, utilized in testis, ovaries, and uterus, were mapped by S1 nuclease protection assays. The sequences surrounding these sites have a high G + C content, and typical upstream CCAAT and TATAA boxes are not present. These studies allowed us to identify the translation initiation site for WT1 protein synthesis. We have also used an epitope-tagging protocol to demonstrate that WT1 is a nuclear protein, consistent with its role as a transcription factor. Our results demonstrate regulation of WT1 expression during development of the gonads, implicate WT1 in genitourinary development, and provide a molecular framework toward understanding genitourinary defects observed among hereditary cases of Wilms' tumor.

Amino Acid Sequence↗

PubMed1991

Structural and functional analysis of the ribosome landing pad of poliovirus type 2: in vivo translation studies.

The naturally uncapped genomic and mRNAs of poliovirus initiate translation by an internal ribosome-binding mechanism. The mRNA 5' untranslated region (UTR) of poliovirus is approximately 750 nucleotides in length and has seven to eight (depending on the serotype) AUG codons upstream of the initiator AUG. The sequence required for internal ribosome binding has been termed the ribosome landing pad (RLP). To better understand the mechanisms of internal initiation, we have determined the boundaries and critical elements of the RLP of poliovirus type 2 (Lansing strain) in vivo. By using deletion analysis, we demonstrate the existence of a core RLP in the poliovirus mRNA 5' UTR whose boundaries are between nucleotides 134 and 155 at the 5' end and nucleotides 556 and 585 at the 3' end. Sequences flanking the core RLP affect translational activity. The importance of several stem-loop structures in the RLP for internal initiation has been determined. Mutation of the phylogenetically conserved loop sequences in the proximal stem-loop structure of the RLP (stem-loop structure III; nucleotides 127 to 165) abolished internal translation. However, deletion of the second stem-loop in the RLP (stem-loop structure IV; nucleotides 189 to 223) reduced internal translation by only 50%. Internal deletions encompassing nucleotides 240 to 300, 350 to 380, or 450 to 480, predicted to disrupt stem-loop structure V and possibly VI, also abrogated internal initiation. Small point mutations within a short polypyrimidine sequence, highly conserved among all picornaviruses, abolished translation. A conservation of distance between the conserved polypyrimidine tract and a downstream AUG could play an important role in the mechanism of internal initiation.

Animals↗

PubMed1991

Isolation, characterization, and expression of the murine Wilms' tumor gene (WT1) during kidney development.

The human Wilms' tumor predisposition gene, WT1, is a Cys-His zinc finger polypeptide which appears to be a transcription factor controlling gene expression during embryonic kidney development. In order to analyze the role of the WT1 gene in nephroblast differentiation, we have isolated the murine homolog of human WT1. An extremely high level of amino acid sequence conservation (greater than 95%) extends throughout all regions of the predicted mouse and human WT1 polypeptides. Two alternative splices within the WT1 transcript have been conserved between mice and humans, suggesting that these have functional significance. Expression of the mouse WT1 mRNA in fetal kidney increases during late gestation, peaks just prior to or shortly after birth, and declines dramatically by 15 days postpartum. Developmental regulation of WT1 expression appears to be selective for the kidney. The restriction of WT1 expression to a limited number of tissues is in contrast to previously described tumor suppressor genes. In addition, the narrow window of time during which WT1 is expressed at high levels in the kidney is consistent with the origin of Wilms' tumor from primitive nephroblasts and the postulated role of this gene as a negative regulator of growth.

Amino Acid Sequence↗

PubMed1990

Translation initiation factor-dependent extracts from Saccharomyces cerevisiae.

Translation initiation factor 4A- and 4E-dependent extracts were developed from Saccharomyces cerevisiae and used to study factor requirements for translation of individual mRNAs in vitro. Whereas all mRNAs tested required eIF-4A, mRNAs devoid of secondary structure in their 5' untranslated region did not require exogenous eIF-4E for translation. The latter included alfalfa mosaic virus RNA4, mRNA containing the untranslated region of tobacco mosaic virus RNA and mRNA containing part of the untranslated region of poliovirus RNA. Furthermore, initiation of translation on mRNAs containing part of the untranslated region of poliovirus RNA is most likely internal.

Animals↗

PubMed1990

The candidate Wilms' tumour gene is involved in genitourinary development.

Wilms' tumour is an embryonic kidney tumour thought to arise through aberrant mesenchymal stem cell differentiation and to result from loss of function of a 'tumour suppressor' gene(s). Both sporadic and syndrome-associated Wilms' tumours are accompanied by an increased frequency of abnormalities of the urinary tract and genitalia. Deletional analysis of individuals with the WAGR syndrome (for, Wilms' tumour, aniridia, genitourinary abnormalities and mental retardation) showed that a Wilms' tumour gene lies at chromosomal position 11p13. This led to the isolation of a candidate Wilms' tumour gene, encoding a zinc-finger protein which is likely to be a transcription factor. To gain insight into the role of this candidate gene in normal development and tumorigenesis, we have now performed in situ messenger RNA hybridization on sections of human embryos and Wilms' tumours. The candidate Wilms' tumour gene is expressed specifically in the condensed mesenchyme, renal vesicle and glomerular epithelium of the developing kidney, in the related mesonephric glomeruli and in cells approximating these structures in tumours. The other main sites of expression are the genital ridge, fetal gonad and mesothelium. These data suggest that (1) this candidate is indeed a Wilms' tumour gene, (2) the associated genital abnormalities are pleiotropic effects of mutation in the Wilms' tumour gene itself, in support of recent genetic analysis, and (3) this gene has a specific role in kidney development and a wider role in mesenchymal-epithelial transitions.

Blotting, Northern↗

PubMed1990

An internal deletion within an 11p13 zinc finger gene contributes to the development of Wilms' tumor.

We have recently described the isolation of a candidate for the Wilms' tumor susceptibility gene mapping to band p13 of human chromosome 11. This gene, primarily expressed in fetal kidney, appears to encode a DNA binding protein. We now describe a sporadic, unilateral Wilms' tumor in which one allele of this gene contains a 25 bp deletion spanning an exon-intron junction and leading to aberrant mRNA splicing and loss of one of the four zinc finger consensus domains in the protein. The mutation is absent in the affected individual's germline, consistent with the somatic inactivation of a tumor suppressor gene. In addition to this intragenic deletion affecting one allele, loss of heterozygosity at loci along the entire chromosome 11 points to an earlier chromosomal nondisjunction and reduplication. We conclude that inactivation of this gene, which we call WT1, is part of a series of events leading to the development of Wilms' tumor.

Alleles↗

PubMed1990

Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus.

We have isolated a series of genomic and cDNA clones mapping within the boundaries of constitutional and tumor deletions that define the Wilms' tumor locus on human chromosome 11 (band p13). The transcription unit corresponding to these clones spans approximately 50 kb and encodes an mRNA approximately 3 kb long. This mRNA is expressed in a limited range of cell types, predominantly in the kidney and a subset of hematopoietic cells. The polypeptide encoded by this locus has a number of features suggesting a potential role in transcriptional regulation. These include the presence of four zinc finger domains and a region rich in proline and glutamine. The amino acid sequence of the predicted polypeptide shows significant homology to two growth regulated mammalian polypeptides, EGR1 and EGR2. The genetic localization of this gene, its tissue-specific expression, and the function predicted from its sequence lead us to suggest that it represents the 11p13 Wilms' tumor gene.

Amino Acid Sequence↗

PubMed1990

The sheep pars tuberalis: an immunohistochemical study. Demonstration of the presence of glycoprotein and lipotropin hormones.

Using indirect immunofluorescence with fourteen different antisera raised against pituitary hormones and peptides, we characterized immunochemically the cells of the sheep pars tuberalis. The presence of LH- and FSH-containing cells, shown in previous studies, was also observed in the present investigation. In addition, we found TSH-containing cells, never observed in sheep, and beta LPH-containing cells. The latter hormone has never been found in any studied species. It appeared that a small amount of perikarya (less than 20%) were immunolabelled and, that the sheep pars tuberalis contained a majority of immunonegative cells as in the guinea-pig rabbit and rhesus monkey. This study may contribute to a better knowledge of the function of the sheep pars tuberalis.

Animals↗

PubMed1990

Intraventricular choroid plexus "arachnoid" cyst. MRI findings.

A young women presented with chronic headaches associated with a cyst of the right lateral ventricle. The diagnosis of intraventricular so-called "arachnoid" cyst was supported by CT scan, MRI and stereotactic puncture. MRI was of great value for demonstrating that the cyst was located within the lateral ventricule, that it was delineated by a thin wall adherent to the choroid plexus and that the cyst content was CSF-like.

Adult↗

PubMed1990

Characterization of melatonin receptors in the ram pars tuberalis: influence of light.

The present study was conducted to assess the binding of [125I]melatonin to frozen unfixed sections of pars tuberalis/median eminence tissue from Ile-de-France rams exposed or not exposed to light before slaughter. The specificity of [125I]melatonin binding to the pars tuberalis tissue was revealed by autoradiography and the magnitude of binding as related to the pars tuberalis area was determined after incubation and counting of pars tuberalis/median eminence sections. Subsequent studies with sections incubated with [125I]melatonin indicated that 1. the binding sites were saturable; 2. binding was stable for 24 h at 20 degrees C, but unstable at 28 or 37 degrees C; 3. melatonin and [127I]melatonin had a similar potency to compete with [125I]melatonin for binding sites, whereas other ligands such as serotonin or N-acetylserotonin were devoid of activity, and 4. by Scatchard analysis, the constant affinity Ka was found to be high in the 10(10) l/mol range. Rams exposed to light throughout the night prior to slaughter presented a significant increase in the apparent number of [125I]melatonin binding sites in comparison to animals maintained under darkness (2.25 +/- 0.30 vs 1.01 +/- 0.17 fmol/mm2 pars tuberalis, p less than 0.01), whereas Ka values were similar in both groups. These results indicate the presence of true melatonin receptors in the pars tuberalis of the ram. Furthermore, they suggest that their apparent number is light-dependent.

Animals↗

PubMed1989

A mammalian translation initiation factor can substitute for its yeast homologue in vivo.

The translation initiation factor 4E (eIF-4E) is involved in the recognition of the cap structure at the 5' end of eukaryotic mRNA and facilitates ribosome binding. Subsequently, additional initiation factors mediate ribosomal scanning of mRNA and initiator AUG recognition (Shatkin, A. J. (1985) Cell 40, 223-224; Rhoads, R. E. (1988) Trends Biochem. Sci. 13, 52-56; Edery, I., Pelletier, J., and Sonenberg, N. (1987) in Translational Regulation of Gene Expression (Ilan, J., ed) pp. 335-366, Plenum Publishing Corp., New York). We show here that initiation factor 4E is functionally conserved between the unicellular eukaryote Saccharomyces cerevisiae and mammals. Although the amino acid identity of the factors from both species is limited to only 33%, mouse eIF-4E can substitute for yeast eIF-4E in vivo without major effects on cell viability, growth, and mating. This finding provides a starting point for new experimental strategies to investigate the structure-function relationship of eukaryotic translation initiation factor eIF-4E.

Amino Acid Sequence↗

PubMed1989

Melatonin binding sites in the sheep pars tuberalis.

The localization of [125I]melatonin binding sites has been studied by autoradiography on frozen unfixed sections of the pituitary stalk, the suprachiasmatic area, the pineal and the pituitary glands in sheep. Dense specific labelling has been found exclusively in the pars tuberalis of the pituitary stalk but not in the part of the median eminence surrounded by the pars tuberalis. The labelling was completely excluded by a 200-fold excess of cold melatonin. No comparable labelling was found in the suprachiasmatic nucleus, the pineal and the pituitary glands. These results constitute the first report of melatonin-specific labelling in the ovine species.

Animals↗

PubMed1989

Fused bacterial luciferase subunits catalyze light emission in eukaryotes and prokaryotes.

A monocistronic luxAB gene containing the luxA and luxB genes coding for bacterial luciferase has been generated by site-specific mutagenesis so that it is now possible to express luciferase under control of a single promoter in eukaryotes as well as in prokaryotes. The fused luciferase subunits (alpha and beta), linked by a decapeptide, were synthesized in yeast under the Gal-4 promoter, in Escherichia coli under the T7-phage promoter, and in vitro in a rabbit reticulocyte lysate after transcription and capping of the mRNA under the SP6 phage promoter. Replacement of the ATG codon initiating the luxB sequence in the fused luxAB gene with CAG prevented internal initiation and allowed purification of a highly active fused luciferase in the absence of the beta-luciferase subunit. Consequently luciferase activity can be directly attributed to the fused luciferase alone and does not require complementation with free beta subunit of luciferase. Light emission could be measured in yeast and bacterial cells without the need for cell lysis providing the basis for measuring gene expression directly in vivo. These results demonstrate the potential applicability of the fused bacterial luciferase genes as a reporter of gene expression both in prokaryotic and eukaryotic systems.

Bacterial Proteins↗

PubMed1989

Poliovirus translation: a paradigm for a novel initiation mechanism.

All eukaryotic cellular mRNAs, and most viral mRNAs, are blocked at their 5' ends with a cap structure (m7GpppX, where X is any nucleotide). Poliovirus, along with a small number of other animal and plant viral mRNAs, does not contain a 5' cap structure. Since the cap structure functions to facilitate ribosome binding to mRNA, translation of polio-virus must proceed by a cap-independent mechanism. Consistent with this, recent studies have shown that ribosomes can bind to an internal region within the long 5' noncoding sequence of poliovirus RNA. Possible mechanisms for cap-independent translation are discussed. Cap-independent translation of poliovirus RNA is of major importance to the mechanism of shut-off of host protein synthesis after infection. Moreover, it is likely to play a role in determining poliovirus neurovirulence and attenuation.

Humans↗

PubMed1989

Construction of a fused LuxAB gene by site-directed mutagenesis.

Bacterial luciferases are heteropolymetric enzymes consisting of two non-identical subunits (alpha and beta). The two polypeptides are produced by transcription in the same direction of two genes, luxA and luxB, located immediately adjacent to each other and separated by only 29 base pairs in the Vibrio harveyi genome. Using site-directed mutagenesis, stop codons after luxA were eliminated and the luxB gene was placed in-frame with luxA, resulting in a fused luxAB gene. Transcription of two luxAB mutant genes from the bacteriophage T7 promoter and translation in Escherichia coli resulted in the synthesis of fused polypeptides containing the alpha and the beta subunits of luciferase linked by either a single amino acid residue or a decapeptide. E. coli synthesizing the latter fusion protein with the decapeptide linker expressed a level of luminescence comparable to E. coli containing the wild type genes while E. coli synthesizing the polypeptide with a single amino acid as a linker expressed about 2000-fold lower light. These results provide the basis for generating a bacterial luciferase system that can be expressed under the control of a single promoter in both eukaryotic and prokaryotic systems.

Base Sequence↗

PubMed1989

A cellular protein that binds to the 5'-noncoding region of poliovirus RNA: implications for internal translation initiation.

Initiation of translation on poliovirus mRNA occurs by internal binding of ribosomes to a region within the 5'-noncoding portion of the mRNA. The mechanistic details and trans-acting factors involved in this event are not understood fully. We used a mobility-shift electrophoresis assay to identify a specific RNA-protein complex, which can form between an RNA fragment that contains nucleotides 559-624 of the poliovirus 5' UTR (untranslated region) and a component or components of a HeLa cell extract. Complex formation was reduced greatly in a reticulocyte lysate or a wheat-germ extract. A 52-kD polypeptide (p52) has been identified as part of the protein-RNA complex by use of an UV cross-linking assay. This polypeptide apparently is not a known translation initiation or elongation factor. The possible involvement of p52 in translation initiation of poliovirus protein synthesis is discussed.

Base Sequence↗

PubMed1989

Internal binding of eucaryotic ribosomes on poliovirus RNA: translation in HeLa cell extracts.

Translation initiation on poliovirus mRNA in poliovirus-infected cells has been shown to occur by internal binding of ribosomes to the 5' noncoding region (J. Pelletier and N. Sonenberg, Nature [London] 334:320-325, 1988). Here we show that internal ribosome binding can occur in HeLa cell extracts in vitro. Internal binding to the 5' noncoding region of poliovirus mRNA in a bicistronic context was independent of the upstream open reading frame and did not require poliovirus proteins.

HeLa Cells↗

PubMed1989

A lysine substitution in the ATP-binding site of eucaryotic initiation factor 4A abrogates nucleotide-binding activity.

Eucaryotic initiation factor 4A (eIF-4A) is a member of a family of proteins believed to be involved in the ATP-dependent melting of RNA secondary structure. These proteins contain a derivative of the consensus ATP-binding site AXXGXGKT. To assess the importance of the consensus amino acid sequence in eIF-4A for ATP binding, we mutated the consensus amino-proximal glycine and lysine to isoleucine and asparagine, respectively. The effect of the mutations was examined by UV-induced cross-linking of [alpha-32P]dATP to eIF-4A. Mutation of the lysine residue (but not of the glycine residue) resulted in the loss of [alpha-32P]dATP cross-linking to eIF-4A, suggesting that the lysine is an important determinant in ATP binding to eIF-4A.

Adenosine Triphosphate↗