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Biomedical subjects

E J Benz

Publications and source records attributed to E J Benz.

At least 109 records · Page 6Linked to original sources
PubMed1978

Hemoglobin switching in sheep: only the gamma gene is in the active conformation in fetal liver but all the beta and gamma genes are in the active conformation in bone marrow.

Differential expression of the closely linked gamma, beta(A) (or beta(B)), and beta(C) globin genes in sheep results in the production of fetal hemoglobin (Hb F, alpha(2)gamma(2)) during gestation and the adult hemoglobins (Hb A, alpha(2)beta(2) (A), and Hb B, alpha(2)beta(2) (B)) after birth. Erythropoietic stress in certain animals leads to production of Hb C (alpha(2)beta(2) (C)). The molecular mechanism of differential expression of these genes in nuclei of fetal and adult erythroid cells has been investigated by analysis of their susceptibility to digestion by DNase I (genes that are in the conformation associated with active transcription are sensitive to this nuclease). The concentration of globin gene sequences in DNA from control and DNase I-digested nuclei was determined by annealing to synthetic DNAs and analogous cDNA probes derived from recombinant plasmids containing one of the sheep globin genes. In nuclei from sheep fetal liver erythroid cells, the gamma genes but not the beta genes were digested by DNase I; the gamma locus was open but the beta(A) or beta(C) loci was closed, consistent with synthesis of only Hb F by these cells. DNase I digestion of nuclei from bone marrow of anemic sheep making only Hb C or Hb B resulted in equivalent digestion of the beta and gamma gene sequences, although gamma mRNA was not detected in these cells. Digestion by DNase I did not decrease the globin gene sequence concentration in residual DNA of spleen nuclei. As a further control, DNA from digested bone marrow and spleen nuclei were shown to anneal equally well to a cDNA prepared from liver polysomal mRNA. Differential expression of the gamma and beta globin genes in sheep fetal erythroid cell appears to be based on differences in chromatin structure. The gamma globin gene remains in the active conformation in adult erythroid cells; failure of gamma mRNA to accumulate in these cells probably reflects transcriptional or post-transcriptional regulation.

Animals↗
PubMed1978

Studies of globin chain synthesis and globin mRNA content in a patient homozygous for hemoglobin Lepore.

Globin chain synthesis and globin mRNA content were studied in blood cells of a patient homozygous for Hb Lepore. Peripheral blood cells incubated with tritiated leucine synthesized approximately 1.5 to 3% as many Lepore globin chains as alpha chains. Globin mRNA in peripheral blood cell RNA was assayed by molecular hybridization assays using human alpha and beta cDNA, and the results indicated the presence of approximately 1% to 2% as much beta-like mRNA (presumably deltabeta Lepore mRNA) as alpha mRNA. The amount of Lepore deltabeta chain mRNA in peripheral blood cells is therefore proportional to the amount of Lepore globin chain synthesis in the same cells. An incidental observation was the finding that peripheral blood cell RNA of this patient, at a time when she was being heavily transfused, contained substantially higher levels of beta-like mRNA (relative to alpha mRNA) than in subsequent studies. Cell-free translation of this mRNA however revealed that it contained authentic beta chain mRNA which must have been derived in some way from the transfused blood cells.

Child↗
PubMed1977

Regulation of hemoglobin synthesis during the development of the red cell (third of three parts).

In this article we have surveyed the current state of knowledge regarding the accumulation of globin mRNA and hemoglobin in red cells. We have attempted to examine the interplay of numerous processes that seem to be necessary to achieve this highly differentiated state. Finally, we have made an effort to formulate some of the mechanisms whereby individual red cells may come to contain varying proportions of specific hemoglobins. The past several years have been characterized by a veritable explosion of knowledge concerning the globin structure genes, and the structure, transcription, processing and function of globin mRNA in erythroid cells. It now seems possible to analyze the earlier stages of erythropoiesis by cultivation and examination of erythroid colonies in vitro. The primary differentiation events leading to the production of specific globins, especially for hemoglobin F production in man, are now experimentally accessible. There is good reason to hope that these advances will soon permit achievement of the long desired therapeutic goal of enhancing hemoglobin F synthesis in patients with severe beta-chain hemoglobinopathies. Our aim has been to review the scientific information that might provide the rationable for amelioration of the clinical phenotypes in patients inheriting abnormal globin genes.

Adult↗
PubMed1977

Hemoglobin switching in sheep and goats. Preparation and characterization of complementary DNAs specific for the alpha-, beta-, and gamma-globin messenger RNAs of sheep.

Specific complementary DNAs (cDNAs) for the messenger RNAs coding for sheep alpha-, betaA-, betaB-, betaC-, and gamma-globins were prepared by thermal denaturation of heterologous hybrids (e.g. alphabetaB-cDNA-alphagamma-mRNA) followed by hydroxylapatite chromatography. Each cDNA represented a nearly full-length copy of its globin mRNA complement as determined by electrophoretic analysis in polyacrylamide gels containing 98% formamide. The purity of each cDNA fraction was estimated by hybridization analysis and thermal denaturation. The beta- and gamma-cDNAs contained 5 to 20% contaminating alpha-cDNA while the alpha-cDNA was 25 to 30% contaminated with non-alpha-cDNA. The melting temperatures (Tm) of homologous duplexes between each non-alpha chain cDNA and its mRNA complement ranged from 69.5-71.5 degrees in 50% formamide while alpha-alpha duplexes melted with a Tm of 75-76 degrees. The Tm values of heterologous duplexes formed between each non-alpha-cDNA and the various globin mRNAs (e.g. betaB-cDNA-Hb C mRNA) ranged between 64.5 degrees and 68 degrees and thus were only 1.5-5.0degrees below that of homologous duplexes. These results suggest that the nucleotide sequence divergence among the various non-alpha-mRNAs (or cDNAs) is not greatly different from the minimum predicted from the amino acid sequence differences of the corresponding globins. When annealing reactions were performed above the Tm of the heterologous hybrids (68 degrees), each non-alpha-cDNA hybridized only to its own complementary mRNA. Thus the purified cDNAs provide molecular probes for the quantitation of alpha-, beta-, and gamma-globin-specific nucleotide sequences.

Animals↗
PubMed1977

Relative stability of alpha- and beta-globin messenger RNAs in homozygous beta+ thalassemia.

The relative concentrations of alpha-, beta-, and gamma-globin mRNA sequences were measured in bone marrow nuclear and cytoplasmic RNA and in RNA from peripheral blood reticulocytes of three patients with homozygous beta+ thalassemia. Our results suggest that the quantitative deficiency in beta-globin mRNA may arise because of abnormal metabolism of molecules containing beta mRNA sequences. Complementary DNAs specific for each of the globins were synthesized. Variable quantities of RNA were incubated to equilibrium with 3H-labeled alpha- and 32P-labeled beta- or gamma-enriched cDNA. We found for each of the patients that the alpha/beta mRNA sequence ratio was more nearly normal in the nuclear RNA than in either cytoplasmic or reticulocyte RNA. Conversely, gamma mRNA sequences were very low in the nucleus with an increase in the relative concentration in both cytoplasm and reticulocyte RNA. The thermal stability of nucleic acid duplexes formed between beta cDNA and nuclear RNA from one patient with beta+ thalassemia was equivalent to that of duplexes formed with normal nuclear RNA. Approximately equal amounts of thalassemic alpha and beta mRNA were retained by oligo(dT)-cellulose, indicating that the 3' poly(A) segment was present on both. Our results indicate that beta-globin mRNA, although grossly normal in structure, fails to accumulate in beta+ thalassemic erythroid cells in amounts equivalent to the mRNA for alpha-globin.

Bone Marrow↗
PubMed1975

Synthesis of DNA complementary to separated human alpha and beta globin messenger RNAs.

Human globulin messenger RNA, purified by oligo(dT)-cellulose column chromatography, is reproducibly separated into two bands by polyacrylamide gel electrophoresis in the presence of 99% formamide. The more rapidly migrating (fast) band is somewhat more abundant than the slow band in normal (nonthalassemic) total reticulocyte globin messenger RNA. In alpha-thalassemic (Hb H disease) messenger RNA, the slow band is 6.5 times more abundant than the fast band, whereas in beta-thalassemic messenger RNA the fast band is three times more abundant than a second band, which has a slightly greater mobility than the slow band of normal and alpha-thalassemic RNA. The RNA bands of nonthalassemic globin messenger RNA were eluted from the gel and efficiently transcribed into DNA copies by use of the RNA-dependent DAN polymerase of avian myeloblastosis virus. Hybridization of these copy DNAs to fast and slow band RANs and to nonfractionated normal, alpha-thalassemic, and geta-thalassemic messenger RNAs revealed that the eluted fast band RNA contains predominantly alpha-chain specific sequences, whereas the eluted slow band RNA contains predominantly beta-chain specific sequences. Nucleotide sequence analysis of 32-P-labeled RNA transcribed from the slow band copy DNA also indicated that the slow band RNA is beta messenger RNA.

Avian Myeloblastosis Virus↗
PubMed1975

Absence of functional messenger RNA activity for beta globin chain synthesis in beta 0-thalassemia.

Functional human globin messenger RNA was isolated from reticulocytes of two patients with homozygous beta 0-thalassemia, three patients with sickle cell beta 0-thalassemia, and one patient doubly heterozygous for beta 0-thalassemia and hemoglobin Lepore. When incubated in the Krebs type II mouse ascites tumor-cell-free system, messenger RNA from these patients actively directed the synthesis of human beta s and/or alpha- and gamma-globin chains but failed to stimulate the synthesis of any beta A-chains, even though nonthalassemic human globin mRNA preparations consistently stimulated two to four times as much beta A- or beta S-globin chain synthesis as alpha-chain synthesis when incubated in the same system under the same conditions. These results strongly suggest that functional beta A-chain-specific globin mRNA is absent in beta 0-thalassemia.

Anemia↗
PubMed1973

Quantitative deficiency of chain-specific globin messenger ribonucleic acids in the thalassemia syndromes.

A hybridization assay procedure was devised that makes possible quantitation of the ratio of mRNA of alpha to mRNA of beta globin chains in an RNA sample. The assay uses the radioactive synthetic DNA copies obtained by incubation of RNA-dependent DNA polymerase of avian myeloblastosis virus with rabbit globin mRNA that is 80-90% enriched in mRNA specific for synthesis of alpha or beta globin chains. The rabbit alpha-chain mRNA is obtained from the postribosomal supernatant of rabbit reticulocyte lysates; the rabbit beta-chain mRNA is obtained from the largest polysomes of rabbit reticulocytes treated with L-O-methylthreonine. Sufficient homology exists between rabbit and human globin chains and globin mRNAs that the synthetic DNA copies of chain-specific rabbit globin mRNA hybridize with human globin mRNA. Applied to the study of globin mRNA isolated from reticulocytes of humans with alpha and beta thalassemia, the technique revealed marked quantitative deficiency of alpha-chain mRNA relative to beta-chain mRNA in alpha thalassemia and similar deficiency of beta-chain mRNA relative to alpha-chain mRNA in beta thalassemia. The thalassemia syndromes are therefore characterized by true quantitative deficiency of the mRNA specific for the affected globin chain.

Animals↗