Antenatal diagnosis of the hemoglobinopathies.
Explore the source record for details and available documents.
DNA↗
Biomedical subjects
E J Benz
Publications and source records attributed to E J Benz.
c-myc gene inactivation during induced maturation of HL-60 cells. Transcriptional repression and loss of a specific DNAse I hypersensitive site.
The c-myc proto-oncogene is amplified and expressed at high levels in HL-60 cells, a cell line derived from a patient with acute promyelocytic leukemia. Upon induction to terminal maturation, expression of c-myc is greatly reduced. We have studied the level of gene expression at which the change in c-myc expression is controlled and the changes in chromatin configuration that accompany the repression of myc expression. We report here that the repression of myc expression with induced maturation is controlled at the level of transcription, and that reduced expression is accompanied by the loss of a single DNAse I hypersensitive site 0.9 kilobase pair upstream from the gene.
Cell Differentiation↗
Expression of genes for the alpha and beta subunits of the Na,K-ATPase in normal and drug resistant cells.
The studies described in this report show that the genes for sodium, potassium-ATPase exhibit more complexity than was originally expected on the basis of studies of the Na,K-ATPase proteins or their transport activities. First, there are at least three isoforms of the alpha subunit. Second, expression of the beta subunit, although apparently always leading to the same protein product, involves complex variability in the sizes and predominance of different mRNA classes. The biologic basis for this heterogeneity of mRNA transcripts is not known. Third, ouabain resistance in at least one cell line, the human Hela C+ cell line, involves amplification of two independent genetic units, the alpha and beta subunit genes, as well as high levels of expression of the mRNAs. These results serve as strong evidence for the importance of the beta subunit, although no discrete functions have yet been assignable to this protein. It is otherwise difficult to understand why the alpha and beta subunits are both amplified in these cells, and both decline upon withdrawal of the selective agent. On the basis of evidence gathered in our laboratory and that of other groups, we conclude that the genetic basis for sodium transport in eukaryotic tissues is far more complex than previously anticipated. There appears to be a need for specialization of sodium, potassium-ATPases in different tissues and for complex generation of multiple beta-subunit mRNA species. Our molecular genetic approach has also allowed us to demonstrate that there is probably an important role played by the beta-subunit in Na,K-ATPase function.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Isolation of lactoferrin cDNA from a human myeloid library and expression of mRNA during normal and leukemic myelopoiesis.
Lactoferrin is a major constituent of polymorphonuclear leukocyte granules and is present in mature neutrophils but not in blasts or promyelocytes. We have isolated a cDNA probe for lactoferrin and used it to study the synthesis of lactoferrin mRNA by normal and leukemic granulocyte precursors. The probe pHL-41 has been subcloned in phage m13 and characterized by restriction endonuclease analysis and nucleic acid sequencing. pHL-41 contains approximately 40% of the coding sequence of the lactoferrin gene. The 3' untranslated region includes a stop codon and a possible polyadenylation signal. There is a greater than 98% agreement between the cDNA-deduced amino acid sequence and that determined by analysis of the protein. Myeloid cells from normal bone marrow and circulating leukocytes from patients with chronic granulocytic leukemia contain lactoferrin mRNA transcripts that are indistinguishable in size and relative quantity. The human promyelocytic leukemia cell line HL-60 contains no lactoferrin mRNA. Induction of monocytic or granulocytic differentiation fails to induce the synthesis of detectable lactoferrin message. Similarly, studies with the human myeloblastic leukemia cell line PLB-985 reveal the inability of these cells to produce lactoferrin mRNA even under conditions that bring about morphologically demonstrable granulocytic differentiation. These data suggest that granulocytic differentiation in the leukemic cell lines is incomplete or defective. The presence of lactoferrin may play a role in the orderly expression of the genetic program leading to the development of the normal mature granulocyte.
Base Sequence↗
Equal stabilities of normal beta globin and nontranslatable beta0 -39 thalassemic transcripts in cell-free extracts.
Patients with beta zero thalassemia arising from premature terminator codon mutations in the gene for beta globin do not produce beta globin protein; these individuals also exhibit a decreased amount of beta globin mRNA in their erythroid cells. The absence of beta globin protein is readily explained by the inability of the beta zero-39 mRNA to be translated. The decrease in beta globin mRNA has been attributed to either decreased cytoplasmic stability of the nontranslatable decreased cytoplasmic stability of the nontranslatable mRNA or to an undefined nuclear lesion. To compare directly the relative stabilities of normal and beta zero-39 thalassemic globin transcripts, we prepared normal and thalassemic beta globin pre-mRNAs and mRNAs using cloned DNA templates and the SP6 promoter-polymerase system. The stability of the transcripts was assessed by incubation in various cell-free extracts. Our results indicate that although the stabilities of the beta globin transcripts varied considerably from one extract to another the stabilities of the beta zero-39 thalassemic pre-mRNAs and mRNAs were equal to those of normal beta globin mRNAs in every extract tested.
Animals↗
Amino acid sequence of the plasma membrane ATPase of Neurospora crassa: deduction from genomic and cDNA sequences.
The plasma membrane of Neurospora crassa contains an electrogenic H+-ATPase (EC 3.6.1.35), for which we have isolated and sequenced both genomic and cDNA clones. The ATPase gene is interrupted by four small introns (58-124 base pairs). It encodes a protein of 920 amino acids (Mr, 99,886) possessing as many as eight transmembrane segments. The Neurospora ATPase shows significant amino acid sequence homology with the Na+,K+- and Ca2+-transporting ATPases of animal cells, particularly in regions that appear to be involved in ATP binding and hydrolysis.
Amino Acid Sequence↗
Rat-brain Na,K-ATPase beta-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells.
We deduced the complete amino acid sequence of the rat brain Na,K-ATPase beta-subunit from cDNA. The rat brain beta-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo beta-subunit polypeptides. Analysis of rat tissue RNA reveals that the beta-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, beta-subunit DNA sequences are amplified (approximately 20-fold) and beta-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the beta-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.
Amino Acid Sequence↗
Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.
We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.
Base Sequence↗
Serous retinal detachments in thrombotic thrombocytopenic purpura.
Serous retinal detachments are a rare ocular complication of thrombotic thrombocytopenic purpura (TTP), with only six previous case reports in the literature. We have recently observed two patients with relapsing TTP who developed bilateral serous retinal detachments during acute exacerbations of their disease. In contrast to all but one of the previously described patients, both of our patients survived the episode during which retinal detachments occurred. Hypertension appears to contribute to the development of retinal detachments in TTP, and vigorous efforts at blood pressure control are indicated. Serous retinal detachments may be a more frequent component of chronic relapsing TTP than has been suspected.
Adult↗
Polyadenylation of a human mitochondrial ribosomal RNA transcript detected by molecular cloning.
We have identified by molecular cloning a polyadenylated RNA transcript in the human leukemia cell line, K562, which is complementary to a portion of the gene-encoding mitochondrial 16S ribosomal RNA (mt 16S rRNA). The cloned portion of the transcript corresponds to positions 2191-2395 of the human mt genome. The clone represents a cDNA copy of an RNA transcript from the H strand and carries an additional poly(A) tail 21 residues long at its 3'-end. Our data provide direct evidence for polyadenylation of some mt 16S rRNA transcripts.
Base Sequence↗
Analysis of gene expression during hematopoiesis: present and future applications.
Recombinant DNA technology now provides the strategies required to identify genes whose expression controls the development of normal and pathologic blood cells. Characterization of the gene families responsible for synthesis of hemoglobins, immunoglobulins, histocompatibility antigens, and cellular enzymes have already, or are about to, provide major insights into the mechanisms producing normal erythroid cells, immunocytes, and immune surface features. Hemoglobinopathies, leukemias, and autoimmune diseases of the bone marrow can now be examined to a degree of detail previously inaccessible to investigators. Oncogene translocation analysis is shedding new light on the pathogenesis of leukemias and lymphomas. Recent basic advances now permit direct cloning and identification of genes in host organisms which express their protein products, thus allowing isolation of genes coding for the hematopoietic surface markers and growth factors which characterize and regulate blood cell progenitors. This review summarizes the molecular genetic approach to analysis of normal and pathologic hematopoiesis, surveys major findings which have resulted, and examines the potential use of refined gene cloning strategies for improved understanding of blood cell development.
Antibody Diversity↗
Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA.
We have isolated a cDNA clone for the rat brain Na,K-ATPase alpha subunit. A lambda gt11 cDNA expression library constructed from mRNA of 1- and 2-week-old rat brains was screened with an antibody reactive with rat brain Na,K-ATPase. A positive phage clone, lambda rb5, containing a 1200-base-pair cDNA insert expressed a beta-galactosidase-cDNA fusion protein that was reactive by immunoblotting with the Na,K-ATPase antibody. This fusion protein was also reactive in ELISA with a monoclonal antibody directed against the alpha subunit of the Na,K-ATPase. A 27S mRNA species exhibiting sequence hybridization to the cDNA insert of lambda rb5 was identified in rat brain, kidney, and liver, as well as in dog kidney. This 27S mRNA exhibited a tissue-specific pattern of abundance consistent with the relative abundance of Na,K-ATPase polypeptides in vivo: kidney greater than brain greater than liver. In a ouabain-resistant HeLa cell line, C+, which contains minute chromosomes and at least a 10-fold greater number of sodium pumps than parental HeLa cells, DNA sequences complementary to lambda rb5 cDNA were amplified approximately 40-fold. Analysis of the lambda rb5 cDNA sequence demonstrated a perfect nucleotide sequence match between a portion of the cDNA and the amino acid sequence of the Na,K-ATPase alpha-subunit fluorescein isothiocyanate binding site. Taken together, the data presented here demonstrate that the lambda rb5 cDNA clone is a portion of the gene coding for the rat brain Na,K-ATPase alpha subunit. The ATPase gene appears to be present in one or very few copies in the rat and human genomes and to be transcriptionally regulated in different rat tissues. In a ouabain-resistant human cell line, on the other hand, ouabain resistance appears to involve an increase in the number of gene copies coding for the Na,K-ATPase.
Animals↗
Metabolism of structurally abnormal mRNAs resulting from beta-thalassemia mutations.
Explore the source record for details and available documents.
Base Sequence↗
Beta globin messenger RNA content of bone marrow erythroblasts in heterozygous beta-thalassemia.
RNA from bone marrow erythroblasts and peripheral blood reticulocytes of patients with heterozygous beta-thalassemia was analyzed for relative content of alpha and beta globin messenger RNA by molecular hybrization. Erythroblasts from nonthalassemic patients exhibited approximately the same alpha and beta globin mRNA content (beta/alpha mRNA ratio = 0.8-1.0) as circulating reticulocytes (beta/alpha mRNA ratio = 0.74-1.2). The mRNA ratios corresponded well to levels of globin synthesis observed in bone marrow and peripheral blood. Erythroblasts from four patients with heterozygous beta-thalassemia also exhibited approximately the same beta/alpha mRNA ratios in bone marrow erythroblasts (0.34-0.59) as in reticulocytes (0.34-0.4): beta globin mRNA was clearly deficient in bone marrow erythroblasts. Globin biosynthesis by erythroblasts of beta-thalassemia heterozygotes was balanced despite the mRNA deficiency (beta/alpha = 0.9-1.0), suggesting that post-translational phenoma (eg, proteolysis of free globin chains), rather than instability of beta mRNA, accounts for the balanced globin chain synthesis frequently observed in bone marrow erythroblasts of patients with beta-thalassemia trait.
Adult↗
An immediate hemolytic transfusion reaction due to anti-C and a delayed hemolytic transfusion reaction due to anti-Ce+e: hemoglobinemia, hemoglobinuria and transient impaired renal function.
A patient with phenotype R2r and anti-C has a hemolytic transfusion reaction (HTR) with hemoglobinemia and hemoglobinuria which occurred within 2 h of receiving an R1r transfusion. Transient impaired renal function ensued. A patient with phenotype R2R2 and anti-Ce+e had the same experience on day 4 after receiving three R1r and one rr units. 2 other patients, 1 R2r with anti-C who received one R1r unit and the other R2R2 with anti-Ce+e who received two R1r units, showed no clinical evidence of HTR. Both anti-C antibodies were entirely IgG while both anti-Ce+e antibodies initially were predominantly IgM. IgG subclassing was unsuccessful and red blood cell-mononuclear phagocyte assays were normal. These cases occurred from 1979 to 1981.
Aged↗
Molecular genetics of the sickling syndromes: evolution of new strategies for improved diagnosis.
This survey is intended to illustrate some major areas relevant to the diagnosis and treatment of sickle cell syndromes that have benefited by the input of molecular genetic approaches. The development of gene mapping techniques has permitted the direct examination of the effect of co-inheritance of alpha-thalassemia and sickle cell anemia on clinical severity, providing, for the first time, a direct strategy for investigation of the clinical heterogeneity of these syndromes. In addition, antenatal diagnosis of these disorders is now best done by direct gene mapping whenever appropriate facilities are available. Treatment by manipulation of gamma-globin gene expression has been shown to be an effective means of achieving at least partial reversal of the hemoglobin F to hemoglobin A switch. Whether the magnitude of this reversal is sufficient to interfere with the clinical phenotype of sickle cell disease remains to be determined. Moreover, the agent currently available to accomplish this goal, 5-azacytidine, remains unsuitable for wide-spread clinical application for a variety of reasons. Nonetheless, the molecular geneticist has already demonstrated that desirable effects can be achieved by building on the knowledge of globin gene physiology. This knowledge is best acquired by application of the concepts and methodologies of molecular genetics.
Anemia, Sickle Cell↗
Lactoferrin biosynthesis during granulocytopoiesis.
We examined the synthesis of lactoferrin, an iron binding protein that, among hematopoietic cells, is restricted to secondary granules of polymorphonuclear leukocytes. Lactoferrin biosynthesis was absent from leukemic myeloblasts and promyelocytes but abundant in normal bone marrow and both the bone marrow and peripheral blood of patients with chronic myelogenous leukemia (CGL) if the samples contained substantial numbers of myelocytes and metamyelocytes. Lactoferrin was present in the steady state in normal or CGL bands and polymorphonuclear leukocytes, but no lactoferrin biosynthesis was detectable in these samples. Taken together, these results suggest that lactoferrin accumulation begins with the onset of biosynthesis at the myelocyte stage and is largely complete by the beginning of the band stage of maturation. HL-60 cells, a permanent promyelocytic leukemia cell line, synthesized no lactoferrin. Translation of messenger RNA in Xenopus laevis oocytes revealed that mRNA from patients with chronic myelogenous leukemia and abundant myelocytes and metamyelocytes directed the synthesis of readily detectable amounts of lactoferrin, whereas HL-60 cells contained no translatable lactoferrin mRNA. We thus hypothesize that lactoferrin is a useful marker of gene expression restricted to the terminal stages of granulocyte maturation. Biosynthesis of this protein appears to be mediated by appearance of translatable mRNA at the myelocyte stage, coincident with development of secondary granules. Absence of lactoferrin production by HL-60 cells is due to absence of translatable lactoferrin mRNA, either because of lineage infidelity of these transformed cells or because of arrest before the developmental stage at which secondary granules appear.
Animals↗
Intranuclear defect in beta-globin mRNA accumulation due to a premature translation termination codon.
We have analyzed a cloned beta O-thalassemia (beta O-thal) gene from a patient doubly heterozygous for hemoglobin Lepore and beta O-thalassemia. Studies of 3H-uridine incorporation into beta-globin mRNA in this patient's erythroblasts suggested an intranuclear defect in both beta and Lepore (delta beta) mRNA synthesis, as did S1 nuclease analysis of nuclear RNA. However, the nucleotide sequence of the beta O-thal gene revealed only a single base change in codon 39 (CAG----UAG), which created a premature translation termination codon. The 5' flanking sequence, including transcription promotor boxes and the mRNA initiation (CAP) site, were normal. The unexpected effect of this mutation on intranuclear beta-mRNA synthesis in vivo was studied by insertion of the cloned gene into a plasmid expression vector and transfection into tissue culture (COS-1) cells. beta-Globin mRNA produced by the transfected cells was assessed by S1 nuclease analysis. The beta O-39 thalassemia gene generated five- to tenfold less beta-mRNA than a normal beta-gene in both nuclear and cytoplasmic RNA, simulating the results observed in vivo. Moreover, the small amount of beta O-39 mRNA produced was as stable as normal beta-mRNA during an actinomycin D chase, ruling out rapid cytoplasmic turnover as a cause of the reduced accumulation. Cotransfection of the beta O-39 thalassemia gene with a mutant tyrosine suppressor tRNA gene resulted in restoration of the beta O-39 mRNA accumulation to near-normal levels. On the basis of these results, we suggest that the low levels of beta-mRNA known to exist in the common form of beta O-thalassemia, beta O-39 thalassemia, result from a lesion in transcription, or early posttranscriptional processes; the defect appears to be corrected by restoration of proper translational potential to the mutant mRNA, at least in a gene transfer-expression system in tissue-culture cells.
Base Sequence↗