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J L Casey

Publications and source records attributed to J L Casey.

47 records · Page 3Linked to original sources
PubMed1989

Iron regulation of transferrin receptor mRNA levels requires iron-responsive elements and a rapid turnover determinant in the 3' untranslated region of the mRNA.

Post-transcriptional regulation of transferrin receptor mRNA levels by iron is mediated by a portion of the 3' untranslated region (UTR) of the mRNA. We have previously shown that a 678 nucleotide fragment of the 3'UTR contains the regulatory element(s). Within this region are five RNA structures which resemble the iron-responsive element (IRE) in the 5' untranslated region of the ferritin mRNA which is regulated translationally by iron. The IREs from the ferritin and transferrin receptor mRNAs compete in an in vitro assay for interaction with a cytoplasmic protein; the activity of this IRE-binding protein is dependent upon the iron status of the cells. Based on further deletion analysis reported here, the sequence required for iron regulation of the transferrin receptor have been limited to 250 nucleotides which we have produced synthetically and cloned. This sequence, which contains three IREs, is capable of producing iron-dependent regulation of transferrin receptor levels. Removal of the three IREs from the synthetic element results in loss of iron regulation. Moreover, deletion of a single cytosine residue from each of the three IREs in the synthetic regulatory element eliminates high-affinity binding to the IRE-binding protein in vitro and results in low levels of iron-independent transferrin receptor expression, consistent with production of a constitutively unstable mRNA. These data indicate that the ability of the mRNA to interact with the IRE-binding protein is required for regulation of transferrin receptor mRNA levels by iron.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

A cytosolic protein binds to structural elements within the iron regulatory region of the transferrin receptor mRNA.

The level of mRNA encoding the transferrin receptor (TfR) is regulated by iron, and this regulation is mediated by a portion of the 3' untranslated region (UTR) of the TfR transcript. This portion of 3' UTR of the human TfR mRNA contains five RNA elements that have structural similarity to the iron-responsive element (IRE) found as a single copy in the 5' UTR of the mRNA for ferritin, whose translation is regulated by iron. Moreover, five very similar elements are also contained in the 3' UTR of the chicken TfR mRNA. Cytosolic extracts of human cell lines are shown by a gel shift assay involving RNase T1 protection to contain an IRE-binding protein capable of specific interaction with the human TfR 3' UTR. When the protecting protein is removed, the protected RNA can be digested with RNase T1 to yield oligoribonucleotide fragments characteristic of two of the IREs contained in the TfR 3' UTR. As judged by cross-competition experiments, the same IRE-binding protein interacts with the ferritin IRE. The apparent affinity of RNA sequence elements for the IRE-binding protein is shown to depend upon the sequence of the RNA. A comprehensive secondary structure for the regulatory region of the TfR mRNA is proposed based on the experimentally demonstrated presence of at least two IRE-like structural elements.

Binding Sites↗
PubMed1988

A model for the structure and functions of iron-responsive elements.

Most eukaryotic cells express two proteins, whose biosynthetic rates are determined by the intracellular iron status. The genes for both these proteins, ferritin and the transferrin receptor (TfR), are regulated at the post-transcriptional level, but by entirely different mechanisms. Ferritin mRNA levels are not affected by acute changes in iron availability. Ferritin biosynthesis is regulated translationally via a defined element contained within the 5' untranslated region (UTR) of the ferritin mRNA. This element has been highly conserved during evolution and has been termed an iron-responsive element (IRE). In contrast to ferritin, the regulation of TfR biosynthesis is mirrored by equivalent changes in TfR mRNA levels. The genetic information for this regulation is mostly located in the region of the gene encoding the 3' UTR of the TfR mRNA. Five elements that closely resemble the ferritin IRE are contained within the region which is critical for TfR regulation. The IRE is suggested to function by forming a specific stem-loop structure that interacts with a transacting factor in an iron-dependent fashion. We present a model that accommodates the mediation of distinct post-transcriptional regulatory phenomena via IREs.

Animals↗
PubMed1988

Iron-responsive elements: regulatory RNA sequences that control mRNA levels and translation.

The biosynthetic rates for both the transferrin receptor (TfR) and ferritin are regulated by iron. An iron-responsive element (IRE) in the 5' untranslated portion of the ferritin messenger RNA (mRNA) mediates iron-dependent control of its translation. In this report the 3' untranslated region of the mRNA for the human TfR was shown to be necessary and sufficient for iron-dependent control of mRNA levels. Deletion studies identified a 678-nucleotide fragment of the TfR complementary DNA that is critical for this iron regulation. Five potential stem-loops that resemble the ferritin IRE are contained within the region critical for TfR regulation. Each of two of the five TfR elements was independently inserted into the 5' untranslated region of an indicator gene transcript. In this location they conferred iron regulation of translation. Thus, an mRNA element has been implicated in the mediation of distinct regulatory phenomena dependent on the context of the element within the transcript.

Animals↗
PubMed1988

Deletional analysis of the promoter region of the human transferrin receptor gene.

Fragments of human genomic DNA corresponding to the promoter region of the gene for the transferrin receptor have been cloned upstream of the bacterial gene for chloramphenicol acetyltransferase and these constructs used to assess promoter activity following transfection into a human rhabdomyosarcoma cell line. Progressive 5' deletions as well as internal linker-substitution constructs support a critical role in gene expression of a sequence element approximately 70 bp upstream of the mRNA start site. In this region, the receptor gene was found to contain 11bp that are identical to a segment of the enhancers of polyoma virus and adenovirus. A fragment encompassing this element was shown to increase gene expression when the fragment was placed in either orientation upstream of the remainder of the transferrin receptor promoter but the same fragment did not activate an enhancer-less SV40 promoter. Removal from within the receptor promoter of three potential binding sites for the transcription factor Sp1 did not decrease the promoter's activity.

Acetyltransferases↗
PubMed1988

Two genetic loci participate in the regulation by iron of the gene for the human transferrin receptor.

Iron regulation of the human transferrin receptor gene was examined in murine cells transformed with chimeric constructs containing the human transferrin receptor gene's promoter and either the structural gene for bacterial chloramphenicol acetyltransferase or the human transferrin receptor cDNA. The activity of the transferrin receptor gene's promoter with the heterologous indicator gene was found to be approximately equal to 3-fold higher in cells treated with the iron chelator desferrioxamine than in cells treated with the iron source, hemin. A higher degree of iron regulation was seen in the expression of the human transferrin receptor cDNA driven by its own promoter. The receptor cDNA under the control of the simian virus 40 early promoter was also iron-regulated. Several human transferrin receptor transcripts differing in their 3' end were produced in the murine cells regardless of the promoter used, with the shorter transcripts being relatively unregulated by iron. Deletion of cDNA corresponding to most of the 3' untranslated portion of the mRNA for the receptor ablated the iron regulation. We conclude that at least two genetic elements exist for the regulation of the transferrin receptor gene by iron. One has its locus in the DNA upstream of the transferrin receptor gene's transcription start site, and the other is dependent upon the integrity of the sequences in the 3' end of the gene.

Acetyltransferases↗
PubMed1988

The promoter region of the human transferrin receptor gene.

Genomic DNA fragments corresponding to the promoter region of the human transferrin receptor were linked to either the full-length receptor cDNA or to the bacterial enzyme chloramphenicol acetyltransferase. These constructs were transfected into mouse and human cells, respectively. Gene expression was monitored 40-48 hours after transfection. Bal31 exonuclease was employed to produce 5' to 3' deletions of the promoter region. Deletion of DNA between -86 and -70 upstream of the receptor's mRNA start site resulted in a greater than 80% reduction in apparent promoter activity. DNA sequencing of the 150 bp upstream of the start site revealed that the promoter region contained several sequence elements more than 90% homologous to the consensus sequence for binding of the transcription factor Sp1. In addition, an 11 bp sequence identical to a segment of the enhancers of polyoma virus and adenovirus was located between -80 and -70. Internal deletions confirmed that this enhancer homologue was critical for full promoter activity. A 66 bp fragment encompassing the -80/-70 element augmented gene expression when the fragment was placed in either orientation upstream of the remainder of the transferrin receptor promoter.

Animals↗
PubMed1981

Thermodynamics of the binding of D-glucose to yeast hexokinase.

The binding of D-glucose to baker's yeast hexokinase (EC 2.7.1.1, ATP:D-hexose 6-phosphotransferase) was studied by isothermal and differential scanning calorimetry (DSC) and by fluorometric titration. The enthalpy and heat capacity changes associated with the binding of glucose were found to be nearly zero at both low and high ionic strengths over the temperature range from 7 to 29 degrees C. Thus, the free-energy change, amounting to -5.1 kcal mol(-1) at 25 degrees C and high ionic strength, is nearly independent of the temperature and is primarily of entropic origin. DSC study of the thermal unfolding of the free enzyme at low ionic strength gave an excess heat capacity curve with two maxima. This result appears to reflect a difference in thermal stability of the two domains in the hexokinase molecule which are indicated by X-ray crystallography [Bennett, W.S., & Steitz, T. A. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4848-4852]. In contrast, the unfolding of free enzyme at high ionic strength was fully cooperative. The excess heat capacity curve for the unfolding of the glucose-bound enzyme had only one peak at both low and high ionic strengths. This is consistent with the X-ray result that the binding of glucose induces a conformational change in the enzyme which brings the two lobes into close proximity. It is interesting that such a significant, molecule-wide conformational change is accompanied by only very small net changes in enthalpy and heat capacity.

Binding Sites↗
PubMedDate not supplied

An outbreak of fulminant hepatitis delta in the Waorani, an indigenous people of the Amazon basin of Ecuador.

An outbreak of delta hepatitis occurred during 1998 among the Waorani of the Amazon basin of Ecuador. Among 58 people identified with jaundice, 79% lived in four of 22 Waorani communities. Serum hepatitis B surface antigen (HBsAg) was found in the sera of 54% of the jaundiced persons, and 14% of asymptomatic persons. Ninety-five percent of 105 asymptomatic Waorani had hepatitis B core (HBc) IgG antibody, versus 98% of 51 with jaundice. These data confirm that hepatitis B virus (HBV) infection is highly endemic among the Waorani. Sixteen of 23 (70%) HBsAg carriers identified at the onset of the epidemic had serologic markers for hepatitis D virus (HDV) infection. All 16 were jaundiced, where as only two of seven (29%) with negative HDV serology were jaundiced (P = .0006). The delta cases clustered in families, 69% were children and most involved superinfection of people chronically infected with HBV. The data suggest that HDV spread rapidly by a horizontal mode of transmission other than by the sexual route.

Adolescent↗