PubMed Health⌕ Search

Biomedical subjects

T J Ley

Publications and source records attributed to T J Ley.

At least 91 records · Page 5Linked to original sources
PubMed1989

Tissue-specific expression and developmental regulation of the human fgr proto-oncogene.

In this study, we show that c-fgr proto-oncogene expression is limited to normal peripheral blood granulocytes, monocytes, and alveolar macrophages, all of which contain 50 to 100 copies of c-fgr mRNA per cell. The c-fgr RNA molecules in these cells consisted of partially spliced transcripts containing intron 7 and completely spliced molecules capable of encoding the predicted p55 c-fgr protein. The splicing of intron 7 appeared to occur after the splicing of most of the other introns; partially spliced molecules containing intron 7 did not appear to be transported into the cytoplasm. Very low levels of fgr transcripts were also present in U937 promonocytic cells and increased in abundance with 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced differentiation. The level of fgr transcripts began to increase 2 to 4 h after TPA addition, peaked at 8 h, and subsequently declined. Since we found that the half-life of fgr mRNA was longer than 8 h, these changes are best explained by transient transcriptional activation of fgr during TPA-induced differentiation, although nuclear runoff experiments were not sensitive enough to detect this event. Cycloheximide also caused accumulation of c-fgr transcripts in U937 cells; no superinduction was observed when TPA and cycloheximide were added at the same time. Induction by either agent was blocked with actinomycin D. These results demonstrate that the c-fgr gene is expressed in a tissue- and development-specific fashion and suggest that constitutive expression of c-fgr in U937 cells is regulated by a labile transcriptional repressor.

Endonucleases↗
PubMed1989

Globin gene expression in erythroid human fetal liver cells.

We measured steady-state levels of the human globin mRNAs in liver samples from several mid-gestational fetuses. RNA from the epsilon, gamma, beta, zeta, theta, and alpha globin genes were present in fetal liver samples isolated from 10-25-wk embryos. The abundance of all human globin mRNAs declined in older fetuses, presumably because of a gradual reduction in the proportion of erythroid precursors in the liver as development proceeds. The gamma:beta globin mRNA ratio in 10-18-wk fetal erythroblasts was 6-7:1, and in adult erythroid bone marrow the ratio was 0.02:1. In fetal liver samples, the relative abundance of epsilon transcripts was less than 1% that of gamma, and zeta transcripts less than 5% that of alpha. Embryonic transcripts declined in abundance during late fetal development and were not detected in newborn liver or adult erythroid bone marrow. theta globin mRNA also represented a minor species (less than 1% that of alpha) in fetal liver samples, but in contrast to the embryonic mRNAs, was most abundant in adult marrow samples obtained from patients with erythroid hyperplasia. These results support the hypothesis that globin protein levels are regulated by the relative amounts of each globin mRNA at various stages of erythropoietic development.

DNA Probes↗
PubMed1988

High-resolution analysis of the human gamma-globin gene promoter in K562 erythroleukemia cell chromatin.

We performed high-resolution mapping studies of the DNAse I-hypersensitive sites located just 5' to the human G gamma- and A gamma-globin genes of K562 erythroleukemia cells, in which these genes are constitutively expressed at low levels. This analysis revealed that the hypersensitive site extends from approximately -210 +/- 5 to -25 +/- 5 base pairs (bp) upstream from the transcription initiation site. Within this region, a GC-rich region located between the proximal CCAAT box and the TATA box is particularly accessible to nuclease digestion; however, the 5' end of the hypersensitive site is less accessible to nucleases. The pattern of DNAse I cleavage does not change on either strand with hemin induction of K562 cells, which increases the rate of gamma-globin gene transcription about threefold. The region within the hypersensitive site includes all the consensus promoter elements of the gamma-globin genes as well as an octamer sequence located between -182 and -175, and a region associated with a variety of mutations that may cause hereditary persistence of fetal hemoglobin (HPFH).

Chromatin↗
PubMed1987

An enhancer element lies 3' to the human A gamma globin gene.

We have surveyed 22 kb of DNA from the region surrounding the human fetal (gamma) globin genes and have identified one fragment that meets all of the criteria for a non-tissue specific enhancer element. The enhancer-containing fragment starts approximately 400 bp 3' to the polyadenylation signal of the A gamma gene and is less than 750 bp in length. Addition of this fragment to plasmids containing a 'gamma-CAT' hybrid gene [consisting of the gamma globin gene promoter fused to the chloramphenicol acetyl transferase (CAT) gene] increases CAT expression 6-23-fold in K562 erythroleukemia cells, depending upon the method of transfection. The increase in expression is essentially independent of the orientation or position of the fragment with respect to the gamma-CAT hybrid gene. The 3' gamma enhancer activates heterologous promoters in erythroleukemia cells, and is also active in non-erythroid cell lines. The enhancer acts by increasing the number of transcripts initiated from the normal gamma globin gene transcription initiation site. The enhancer region contains two DNase I hypersensitive sites in erythroleukemia cells but none in nonerythroid human leukemia cell lines. The 3' gamma globin gene enhancer contains a unique element that is similar to sequences found in an enhancer 3' to the chicken beta globin gene, suggesting that this conserved element may have a role in enhancer function.

Base Sequence↗
PubMed1986

Hydroxyurea induction of hemoglobin F production in sickle cell disease: relationship between cytotoxicity and F cell production.

Initial alterations in fetal hemoglobin (HbF) production among eight sickle cell anemia subjects treated with hydroxyurea (Hu) are summarized. Four of these subjects had been previously treated with 5-azacytidine (5-aza). All subjects treated with Hu (50 mg/kg/d for three to five days) had suppression of their total reticulocyte counts by seven days, whereas the four subjects previously treated with 5-aza (2 mg/kg/d for three to five days) had increased reticulocyte counts at day 7. The effect of Hu on increasing the number of HbF-containing reticulocytes (F reticulocytes) is extremely variable, ranging from ten-to less than onefold differences in maximal posttherapy v pretherapy levels. Recovery from marrow suppression did not result in greater than twofold increases in F reticulocyte counts. Mean day 7 F reticulocyte levels in the four subjects treated with both Hu and 5-aza were 4.1 X 10/microL and 15.4 X 10(4)/microL, respectively. Among Hu-treated subjects, increased F reticulocyte production was correlated with low serum creatinine levels and rapid removal of Hu from the plasma. Furthermore, suppression of CFU-E colony formation on day 2 of therapy with Hu was inversely correlated with maximal F reticulocyte response. We conclude that where Hu treatment results in marrow toxicity (decreased reticulocyte counts, decreased CFU-E colony formation) HbF production is less likely to increase. Those sickle cell anemia subjects with minimal renal dysfunction (serum creatinine level, greater than 1.0 mg/dL) exhibit the most cytotoxicity and least F reticulocyte response to Hu.

Anemia, Sickle Cell↗
PubMed1985

Induction of hemoglobin F synthesis in patients with beta thalassemia.

5-Azacytidine transiently augments fetal hemoglobin production in patients with beta-thalassemia or sickle cell anemia. This change would probably be beneficial to such patients (e.g. a normal fetal gene product is substituted for a deficient or defective adult gene product) if HbF production could be sustained at high levels for prolonged periods. Even though the clinical use of 5-azacytidine is limited because of its presumed potential to cause cancer, studies with this drug have provided new insights into globin gene regulation and have stimulated the development of other strategies to increase HbF synthesis.

Animals↗
PubMed1985

Disseminated infection caused by Cunninghamella bertholletiae in a patient with beta-thalassemia. Case report and review of the literature.

Rare cases of acute mucormycosis in immunocompromised persons have been attributed to Cunninghamella bertholletiae. We report the first known case of this organism causing a chronic, indolent, systemic disease clinically similar to entomophthoramycosis. Cunninghamella bertholletiae is an opportunistic pathogen whose morbidity may be underappreciated because it is usually considered to be a laboratory contaminant.

Adult↗
PubMed1984

Human globin gene expression in hybrid 2S MEL X human fibroblast cells.

A somatic cell hybrid line, called M11-X, was developed in order to study the expression and regulation of the human beta-like globin genes in a mouse erythroid environment. M11-X cells were obtained by fusing the human fibroblast cell line GM3552 (which contains the translocation chromosome t(11;X) that carries the human beta-like globin genes) with hypoxanthine phosphoribosyltransferase (HPRT) -negative tetraploid (2S) mouse erythroleukemia (MEL) cells. After induction with 5 mM hexamethylene bisacetamide (HMBA), these cells contain approximately 300-600 copies per cell of correctly initiated, processed, and terminated human beta-globin mRNA; however, neither human epsilon- nor gamma-globin mRNAs were detected. Carboxymethylcellulose chromatography followed by SDS-polyacrylamide gel electrophoresis and Western blotting revealed that normal human beta-globin protein was also present. These results suggest that the human beta-globin gene, when present in mouse erythroid cells, can be transcribed and its mRNA translated into normal products, but at a much lower level than the mouse beta-globin genes. Analysis of the frequency of cytosine methylation near the human gamma-globin genes indicated that these genes are heavily methylated in M11-X cells. The inability to express the human gamma-globin genes of these cells might be accounted for, at least in part, by DNA methylation.

Animals↗
PubMed1984

DNA methylation and regulation of the human beta-globin-like genes in mouse erythroleukemia cells containing human chromosome 11.

The human beta-globin gene is expressed--but the human fetal (gamma) and embryonic (epsilon) globin genes are not--in an induced mouse erythroleukemia cell line (M11-X) that contains most of human chromosome 11. A 24-hr exposure of M11-X cells to 5-azacytidine before induction causes "global" DNA hypomethylation but selective activation of the human gamma-globin genes. Genomic DNA is remethylated 2-3 days after exposure to 5-azacytidine, but sequences near the human and mouse globin genes remain hypomethylated, suggesting that the remethylation process is inhibited in these regions.

Acetamides↗
PubMed1984

Beta O-39 thalassemia gene: a premature termination codon causes beta-mRNA deficiency without affecting cytoplasmic beta-mRNA stability.

The function of a beta O-thalassemia globin gene with a premature termination codon at position 39 was studied in tissue culture cells using plasmid expression vectors. The thalassemic globin gene was isolated by molecular cloning from the bone marrow DNA of an Italian patient with severe thalassemia. Sequences upstream of the normal beta-globin gene and the beta O-39 globin gene were removed to 127 nucleotides (nt) 5' to the start site of transcription, thereby eliminating uncharacterized DNA sequences but preserving promoter function. To create a hybrid gene differing from the normal by only the single nt change in codon 39, the truncated 5' end of the beta O-39 gene was recombined with the 3' end of the normal gene. The beta O-39 substitution resulted in a 6-14-fold reduction in cytoplasmic RNA accumulation in transfected monkey kidney or HeLa cells. This decrease in mRNA did not appear to be due to instability, as the beta-mRNA present 24-36 hours after transfection was stable for up to 24 hours in the presence of actinomycin D. In the presence of actinomycin D, the globin mRNA precursor disappeared, suggesting that globin gene transcription had been effectively blocked. We also examined a second globin mRNA with a premature termination codon; this RNA that arises from incorrect splicing of the transcript of a beta + -24 thalassemia gene was as stable as the correctly spliced species. Thus, the presence of a premature termination codon does not necessarily alter cytoplasmic mRNA stability, nor does cytoplasmic instability account for the quantitative deficiency of beta-globin mRNA. Our observations suggest that a premature termination codon alters intranuclear stability and/or nuclear to cytoplasmic transport of the beta-globin mRNA.

Codon↗
PubMed1983

A weak upstream promoter gives rise to long human beta-globin RNA molecules.

The 5' ends of most normal human beta globin mRNA molecules correspond to a single transcription initiation site, often referred to as the "CAP" site (1-4). Using S1 nuclease mapping and primer extension techniques, we have determined that a minority of beta globin gene transcripts are longer at the 5' ends. These longer molecules comprise about 10% of total beta globin RNA molecules in normal human bone marrow cells and in peripheral blood reticulocytes. The long molecules are transcribed only from the sense strand of DNA and are probably spliced correctly. A DNA segment that includes imperfect "CCAAT" and "TATA" promoter-like sequences begins approximately 150 base pairs (bp) upstream from the normal beta globin gene promoter; this "pseudo-promoter" may function in the initiation of the long globin RNA molecules.

Bone Marrow↗