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Biomedical subjects

A Bank

Publications and source records attributed to A Bank.

At least 145 records · Page 8Linked to original sources
PubMed1978

Organization of human delta--and beta-globin genes in cellular DNA and the presence of intragenic inserts.

We have analyzed human cellular DNA for its delta--and beta-globin gene sequence content by separation of restriction enzyme fragments by agarose gel electrophoresis; transfer of the DNA fragments to nitrocellulose filters; hybridization of filters with 32P--beta-globin cDNA; and analysis by autoradiography. A short cDNA has been used to identify specifically the 3' end of the genes and to orient the fragments. A comparison of the globin gene fragments generated by normal and Lepore DNA has been used to distinguish fragments representing DNA sequences between the delta and beta genes and those containing sequences flanking either 5' to the delta gene or 3' to the beta gene. The results indicate that unique restriction fragments are presented in normal DNA and absent in Lepore DNA, and allow preliminary ordering of these fragments on a restriction enzyme map. In addition, the Lepore, delta--and beta-globin genes have been found to contain at least one inserted nucleotide sequence of about 1000 bases which is not represented in mature globin mRNA.

Cell Line↗
PubMed1978

Changes in restricted human cellular DNA fragments containing globin gene sequences in thalassemias and related disorders.

Human cellular DNA fragments from cells of normal subjects and patients with thalassemia obtained by restriction enzyme digestion were analyzed for their globin gene content. The fragments were separated on agarose gels, transferred to nitrocellulose filters, hybridized to globin [(32)P]cDNA, and radioautographed. One to ten picograms of globin gene sequences were detectable. With EcoRI digestion, eight to nine cellular DNA fragments were found to contain globin genes. Three of these contained beta-like gene sequences assayed with beta globin cDNA probe. One beta-like fragment was absent in DNA from a homozygous subject for hemoglobin Lepore. Two of the three beta gene-containing fragments present in normal DNA were absent in DNA from a patient with hereditary persistence of fetal hemoglobin. The same two fragments containing beta-like genes were absent from deltabeta thalassemic DNA and one new fragment containing beta-like genes was found. Together with results obtained by hybridization of these DNAs in solution, the data are consistent with deletion of specific restriction human DNA fragments in subjects with these disorders and a greater deletion of beta-like gene sequences in subjects with hereditary persistence of fetal hemoglobin than in those with deltabeta thalassemia.

Base Sequence↗
PubMed1978

Synthesis of globin mRNA in relation to the cell cycle during induced murine erythroleukemia differentiation.

The relationship between the synthesis of globin mRNA and the phase of cell cycle was examined in synchronized murine erythroleukemia cells. Cells were synchronized with respect to the cell division cycle either by culture with 2 mM thymidine or 2 mM thymidine followed by 0.5 mM hydroxyurea, which caused cells to accumulate in late G1 or early S (referred to as G1/S boundary). Cells were induced to erythroid differentiation by culture with 280 mM dimethyl sulfoxide or 4 mM hexamethylene bisacetamide. These inducers do not alter the progression of cells from the G1/S boundary through S, G2, and M, but do cause prolongation of the subsequent G1 phase. Accumulation of newly synthesized globin mRNA is first detected when cells are in this G1 phase.

Acetamides↗
PubMed1978

Absence of beta mRNA in beta0-thalassemia in Kurdish Jews.

We report the characterization of the amount of beta mRNA in a Kurdish Jewish population with beta0-thalassemia using the same methods employed for characterization of the Catania and Ferrara beta0 patients. We found very low amounts of beta mRNA sequences, consistent with the presence of beta0-thalassemia of the beta mRNA-negative population type. In addition, no globin gene deletion was detected that could account for the absence of beta mRNA.

Adolescent↗
PubMed1977

Preparative polyacrylamide gel electrophoretic purification of human alpha- and beta-globin messenger RNAs.

Previous studies have reported the use of globin chain-specific complementary DNAs to quantitate the amount of human globin mRNA and human globin genes in normal and abnormal cells. In order to obtain larger amounts and more purified globin mRNAs as templates for these experiments, preparative polyacrylamide gel electrophoresis in formamide has been used to separate alpha- and beta-globin mRNA from polyadenylate containing RNA of human reticulocytes. Fifty to one hundred-fifty micrograms of RNA can be applied to the preparative gel and the recovery of the globin mRNA is about 50%. The isolated alpha- and beta-globin mRNAs were assayed in a wheat germ cell-free system, and the alpha- and beta-globin synthesized quantitated by cellulose acetate electrophoresis. The purified alpha- and beta-globin mRNAs direct globin synthesis which is more than 90% either alpha- or beta-globin, respectively. The cDNAs prepared using each of the isolated mRNAs as template are also shown to be specific for alpha- or beta-mRNA sequences. The gel electrophoresis technique used permits the relatively large scale isolation of alpha- or beta-globin mRNAs from human cells.

Anemia, Hemolytic↗
PubMed1977

Accumulation of alpha- and beta-globin messenger RNAs in mouse erythroleukemia cells.

The accumulation of alpha- and beta-globin mRNA sequences in murine erythroleukemia cells (MELC) treated with various inducers has been studied using specific alpha- and beta-globin complementary DNAs (cDNAs). In cells cultured with dimethylsulfoxide (Me2SO), hexamethylene bisacetamide (HMBA) or butyric acid, accumulation of alpha-globin mRNA is detectable after 16, 12 and 8 hr of culture, respectively. An increase in beta-globin mRNA sequences is not detected until 20-24 hr after culture. In cells exposed to hemin, both alpha- and beta-globin mRNAs are detectable by 6 hr of culture, and a constant ratio of alpha/beta-mRNA is maintained during induction. In maximally induced cells, the alpha/beta-globin mRNA ratios are approximately 1 in cells induced by Me2SO and HMBA, and 0.66 and 0.3-0.50 in cells induced by butyric acid and hemin, respectively. Thus different inducers of erythroid differentiation in MELC lead to different times of onset of the expression of alpha- and beta-like genes. In addition, the relative accumulation of alpha- and beta-globulin mRNAs in induced cells differs with various types of inducers.

Acetamides↗
PubMed1977

Effect of endotoxin-induced shock on the reticuloendothelial system. Phagocytic activity and DNA-synthesis of reticuloendothelial cells following endotoxin treatment.

There was a marked decrease in the RES phagocytic activity during the first 12 hours after injection of high concentrations of endotoxin in rats. Phagocytic activity then increased considerably, reaching maximum values on days 3 to 5 and it was still higher than in control animals 20 days later. Parallel studies on 3H thymidine incorporation showed a significant increase in the rate of DNA-synthesis of reticulum cells of the liver during the 5 days period following endotoxin injection. Peak values were obtained on day 2 when the number of labelled cell were 50 times higher than in the controls. A likely reason for the increased DNA-synthesis is a repair of RES following endotoxin induced damage but it may also represent an endotoxin induced proliferation of reticulum cells that may at least partly account for the enhanced phagocytic activity.

Animals↗
PubMed1977

Differential effects of chemical inducers on expression of beta globin genes in murine erythroleukemia cells.

Murine erythroleukemia cells are induced to erythrodifferentiate by polar compounds such as dimethyl sulfoxide and hexamethylene bisacetamide as well as by fatty acids such as butyric acid and propionic acid. The effect of these inducers on the expression of two beta globin genes, betamaj and betamin, during the course of differentiation of the cells has been examined. After 4 days of culture with hexamethylene bisacetamide or dimethyl sulfoxide, the betamaj-containing hemoglobin (Hbmaj) predominates. By contrast, in the presence of butyric acid or propionic acid, after 4 days of culture, relatively equal amounts of Hbmaj and Hbmin are found. When cultured with dimethyl sulfoxide or hexamethylene bisacetamide, murine erythroleukemia cells synthesize more betamaj than betamin, while about equal amounts of the two globins are synthesized in the presence of butyric acid. When poly(A)-containing RNA from the cells exposed to different inducers is translated in a wheat germ cell-free system, the ratio of betamaj to betamin synthesized reflects that in whole cells. In a strain of murine erythroleukemia cells resistant to dimethyl sulfoxide (DR-10), the preferential stimulation of betamaj synthesis by hexamethylene bisacetamide of the betamin synthesis by butyric acid is more pronounced than with the dimethyl sulfoxide-sensitive cells (DS-19). These data suggest that polar compounds and fatty acids cause different expression of the betamaj and betamin genes in murine erythroleukemia cells.

Acetamides↗
PubMed1977

Sickle cell anemia in the newborn.

The clinical presentation of homozygous sickle cell disease is unusual in the neonatal period. Recently, we have encountered a newborn infant whose disease was apparent at birth and who died at 5 days of age. The findings at autopsy suggested a sickle cell crisis with multisystem involvement that was present prior to birth. Laboratory findings confirmed homozygous sickle cell disease without the presence of elevated levels of hemoglobin S. The cause of the unusually severe clinical course of the disease in our patient is the object of the discussion.

Adult↗
PubMed1976

Erythroid cell differentiation: murine erythroleukemia cell variant with unique pattern of induction by polar compounds.

The murine-virus-infected erythroleukemia cell system provides an opportunity to examine regulatory mechanisms controlling cytodifferentiation. A cloned cell line (DR10c3) resistant to the erythropoiesis-inducing effect of dimethylsulfoxide (Me2SO) was isolated from the Me2SO-sensitive line DS19. DR10c3 is characterized as follows: (1) the uptake of [3H]Me2SO is similar to that in DS19; (2) cell growth with and without Me2SO is similar to that of DS19; (3) resistance is relatively stable; (4) the karyotype of DR10c3 reveals an average loss of five chromosomes per cell, but is otherwise similar to that of DS19; (5) total protein and globin synthesis by cells cultured 4 days with or without Me2SO is similar to these syntheses in DS19 cultured without Me2SO; (6) virtually no globin mRNA is detectable after 3 days in Me2SO, as assayed both by RNA-complementary DNA hybridization and by the heterologous cell-free protein-synthesizing system; (7) other polar compounds, N-methylpyrrolidinone, 1-methyl-2-piperidone, N, N-dimethylacetamide, and N-methylacetamide, induce erythroid differentiation in DR10c3, and the accumulation of alpha- and beta-globin chains is indistinguishable from that in DS19; and (8) the concentration optima for induction of differentiation by all these compounds are identical for DR10c3 and DS19.

Acetamides↗
PubMed1976

Translation and stability of human globin mRNA in Xenopus oocytes.

Human globin messenger RNA (mRNA) prepared from erythroid cells of patients with sickle cell anemia has been translated in Xenopus laevis oocytes. Addition of hemin to the injected mRNA causes total globin synthesis to increase and the ratio of alpha- to betas-globin synthesis (alpha/betas ratio) to approach unity. To determine the effect of the length of the poly-(A) segment on human globin mRNA stability, 10 S globin mRNA was fractionated into poly-(A)-poor fractions by oligo (dT)-cellulose column chromatography. When oocytes are injected with each of these fractions, translation of the poly-(A)-rich globin mRNA is sustained for a longer period than that of the poly-(A)-poor mRNA. Regardless of the mRNA fraction injected, the alpha/betas ratio of the synthesized globin decreases as the injected oocytes are incubated for longer periods. The results indicate that in frog oocytes poly-(A)-rich mRNA has greater translational stability than poly-(A)-poor mRNA, AND beta-mRNA has greater stability than alpha-mRNA with comparable poly-(A) content.

Animals↗
PubMed1976

Quantitation of human gamma globin genes and gamma globin mRNA with purified gamma globin complementary DNA.

Complementary DNA (cDNA) specific for gamma-globin nucleotide sequences has been prepared by hybridizing total cDNA made from cord blood messenger RNA (mRNA) as template to an excess of normal adult human globin mRNA and recovering the single-stranded cDNA from hydroxylapatite. The specificity of the gamma cDNA for gamma mRNA sequences is strongly supported by the hybridization of this cDNA at low Cot values (Co, concentration of RNA and t, time in seconds) to RNA samples containing large amounts of functional gamma globin mRNA and the lack of hybridization to RNA samples containing little, if any, gamma-globin mRNA. The absence of cross-hybridization of gamma cDNA with alpha, beta, and delta mRNAs is demonstrated by the complete hybridization of the gamma cDNA to mRNA samples completely lacking either alpha or beta and delta mRNA. An estimate of the number of gamma-globin genes in human cellular DNA was obtained by hybridization of purified gamma cDNA to DNA from spleen and white blood cells of normal and beta-thalassemia subjects and measurement of the percent of gamma cDNA hybridized at saturation. The results indicate that there are between one and two gamma-globin genes per total haploid gene DNA equivalent obtained from both normal and beta-thalassemia subjects. These values are consistent with genetic evidence for the presence of multiple gamma gene loci in human cells. The finding that the number of gamma-globin genes in beta-thalassemia DNA is similar to that in nonthalassemia DNA indicates that a deletion of gamma-globin genes cannot account for either the inadequate gamma-globin synthesis or indirectly for the decreased or absent beta-globin synthesis in beta-thalassemia cells.

Amino Acid Sequence↗
PubMed1976

Isolation and in vitro differentiation of human erythroid precursor cells.

There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia.

Anemia, Sickle Cell↗
PubMed1976

Decreased alpha globine mRNA in nucleated red cell precursors in alpha thalassemia.

The alpha thalassemias are associated with a decrease in alpha chain synthesis. Hemoglobin H (HbH) disease is a moderately severe form of alpha thalassemia characterized by the production of 5%--20% of HbH, while alpha thalassemia trait is a milder form of alpha thalassemia. In two patients with HbH disease, the ratio of alpha chain synthesis to beta chain synthesis (alpha/beta ratio) was decreased in both bone marrow cells and reticulocytes. When isolated mRNA from bone marrow cells and reticulocytes was translated in a heterologous cell-free system, the alpha/beta ratios were lower than the intact cell ratios. These findings were confirmed by hybridization of the mRNA of both marrow cells and reticulocytes using purified alpha and beta cDNA probes. In the intact cells of two patients with alpha thalassemia trait, the alpha/beta ratios were also decreased and were similar in marrow cells and reticulocytes. Cell-free studies of translatable mRNA also demonstrated decreased alpha/beta ratios, but, unlike the HbH studies, the cell-free alpha/beta ratios were similar to the intact cell ratios. One hybridization study utilizing peripheral blood mRNA had an alpha/beta ratio consistent with the cell-free ratios. These results indicated that, in both HbH disease and alpha thalassemia trait, there was decreased alpha globin mRNA present in both nucleated red cell precursors and reticulocytes. In addition, the data suggested that there may be translational mechanisms that operate in intact HbH cells which attempt to balance globin chain production. In alpha thalassemia trait cells, no such controls appeared to be active and globin chain synthesis was directly proportional to the amount of alpha and beta globin mRNA in the cells.

Adult↗