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Biomedical subjects

M G Farace

Publications and source records attributed to M G Farace.

36 records · Page 2Linked to original sources
PubMed1983

Desferrioxamine inhibits induced erythroid differentiation of human leukemic K-562 cells.

The iron chelator desferrioxamine reversibly inhibits heme accumulation and globin synthesis in human leukemic K-562 cells induced to express erythroid genes by butyric acid. These results suggest that iron metabolism can modulate globin gene expression. In addition we describe experimental conditions (6.25 micrograms/ml desferrioxamine) which do not suppress transcription of globin genes and translation of globin mRNA but prevent heme synthesis. Therefore expression of globin genes in butyric acid induced K-562 cells does not require accumulation of heme molecules. Human leukemic K-562 cells cultured with different inducers and treated with desferrioxamine should be used as a useful model system to further analyse the relationship between expression of erythroid genes, iron metabolism and heme accumulation.

Butyrates↗
PubMed1981

The nucleotide sequence of the mouse embryonic beta-like y-globin messenger RNA as determined from cloned cDNA.

We have determined the nucleotide sequence of two cloned cDNAs corresponding to the mRNA of mouse embryonic y2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acido residue 21 and extending to the AATAAA sequence in the 3' untranslated region. Therefore, when the amino acid sequence encoded by the cDNA is combined with the available amino acid sequence, a complete y2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known beta- and beta-like globin sequences and the y2 sequence show that the embryonic, fetal-adult duplication occurred approx. 160 million years (MY) ago and that the embryonic-fetal duplication occurred approx. 100 MY ago.

Amino Acid Sequence↗
PubMed1981

Molecular cloning of DNA sequences coding for mouse embryonic globins.

Yolk sac derived erythroid cells in mouse embryos synthesize four embryonic globins of which two are alpha-like and two are beta-like. Pure globin messenger RNAs from these cells were used as templates for two successive polymerizing reactions and a mixture of double stranded cDNAs coding for the four globins was obtained. These molecules were blunt-end ligated to an ECoR1 digested pBR322 plasmid and the recombinant plasmids were used to transform E. coli Hb101. Bacterial clones which proved positive upon hybridization with 32P-labelled embryonic globin cDNA were amplified and their plasmid DNA was isolated. Three different plasmids were studied, namely no. 2, 16 and 54. The restriction map of these plasmids showed that: 1) plasmid no. 2 and 54 had lost extensive DNA sequences comprising the genes responsible for tetracycline resistance; 2) the size of inserted sequences ranges from 427 base pairs of plasmid no. 16 to about 280 base pairs of plasmid no. 54; 3) plasmid no. 2 does not share any of the studied restriction sites with the other plasmids, while no. 2 and 54 have at least one site in common. The coding properties of inserted DNA were determined by positive hybrid translation showing that no. 2 codes for the alpha-like embryonic chain x, while no. 16 and 54 code for a beta-like embryonic chain, either y or z.

Animals↗
PubMed1981

Embryonic hemoglobins in man and other mammals.

This report attempts to review the present state of research on embryonic hemoglobins in humans and other mammals from a cytologic, molecular, and clinical point of view. In all mammals, the yolk sac is the site responsible for the "primitive" erythropoiesis, which produces an erythroid population characterized by peculiar cytologic features and globin gene expression. Morphological and molecular events that underlie prenatal erythropoiesis are described herein giving rise to questions regarding biology at large (i.e., the differential activity of genes capable of similar functions); molecular biology of eukaryotic genes (i.e., globin gene organization and structural subtleties); and clinical hematology (i.e., syndromes associated with the appearance of embryonic hemoglobins.

Aneuploidy↗
PubMed1981

Control of globin gene expression in mouse yolk sac erythroid cells.

In the present paper are reported results from our laboratory and data of the literature on the structure and the expression of embryonic globin genes in mouse fetuses. The following aspects are presented: 1. Erythropoiesis and synthesis of globin chains in fetal mice. 2. The transcriptional and post-transcriptional control of embryonic globin gene expression. 3. The chromosomal location and organization of mouse embryonic globin genes.

Animals↗
PubMed1981

Translational discrimination between embryonic and adult mouse globin mRNAs in the rabbit reticulocyte lysate system.

In the present study mouse adult (alpha, beta) and embryonic globin (x, y, z) mRNas are compared for their capacity of being translated in a micrococcus nuclease treated rabbit reticulocyte lysate. The results of these experiments indicate that: 1) alpha and beta adult RNAs are translated with a higher efficiency as compared to the alpha-like and beta-like embryonic mRNAs; 2) adult beta and embryonic beta-like messengers are translated more efficiently than their corresponding adult alpha and embryonic alpha-like messengers; 3) the alpha/beta synthetic ratio, both for adult and embryonic globins is highly dependent upon the concentration of globin mRNAs. For embryonic globins the ratio decreases from 0.21 at low mRNA concentration to 0.11 at high mRNA concentration. For adult globin mRNAs the alpha/beta chain synthetic ratio decreases from 1.4 to 0.9 within the same range of concentration.

Animals↗
PubMed1979

Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

Animals↗
PubMed1979

Nuclear RNA sequences coding for alpha and beta globins in erythroid cells: evidence for multiple intermediate molecules.

The poly (A)-containing nuclear RNA from dimethylsulfoxide-induced Friend leukemia cells was fractionated by acrylamide gel electrophoresis in denaturing conditions and analyzed for alpha and beta globin RNA sequences. The results indicate that nuclear RNA contains one species of large-size RNA (0.6 X 10(6) daltons), which is the putative precursor for beta globin mRNA only. In addition, it was shown by electrophoretic analysis that the complex of RNA molecules not resolved by sucrose gradient centrifugation (11S) comprises sequences of decreasing size (0.34, 0.28, and 0.26 X 10(6) daltons), which might be the precursors of alpha and beta globin mRNA.

Erythrocytes↗
PubMed1978

Isolation and characterization of the messenger RNAs for mouse embryonic globin chains.

Messenger RNAs for mouse embryonic globins were purified from yolk sac erythroid cells by oligodeoxythymidilate-cellulose chromatography and sucrose density centrifugation. Full-sized complementary DNA copies of embryonic globin mRNAs were synthesized. Acrylamide gel electrophoresis of these RNAs indicate an average molecular weight of 220 000, including a polyadenylated sequence of about 35 residues, as determined by hybridization to [3H]polyuridylate. The wheat-germ translation products of mRNAs have the size and the ionic characteristics of the four embryonic globin chains alpha, x, y and z. Hybridization kinetics in vast RNA excess were performed and compared to standard r0t curves of adult globin messengers, demonstrating a total base sequence complexity of about 880 000 daltons, that is four different RNA sequences of 220 000 molecular weight. The titration of embryonic globin cDNAs with increasing amounts of their complementary RNA templates indicates that the embryonic globin messengers were isolated at a high degree of purity.

Animals↗
PubMed1978

Terminal stages of erythroid differentiation: persistent production of globin messenger RNA is necessary to sustain globin synthesis.

The kinetic relationship between the globin mRNA accumulation and the rate of synthesis of globin chains was studied during the terminal stages of differentiation in erythroid cells derived from the yolk sac of mouse fetuses. RNA derived from the whole cells and from different cell compartments were hybridized to DNA complementary to embryonic globin mRNA. The relative proportion of embryonic globin RNA molecules and their absolute number per cell were estimated on the 11th, 12th, and 13th days of mouse fetal development. During erythroid terminal differentiation globin mRNA became progressively predominant on polyribosomes along with the progressive specialization of cell functions. The number of embryonic globin RNA molecules per cell remained constant while yolk sac erythroid cells underwent two rounds of cell division. These data indicate that the transcription of globin genes is operative throughout the last stages of terminal differentiation and that there is no detectable storage of globin RNA sequences in these cells. The rates of accumulation of mRNA molecules and of globin synthesis both seem correlated to the length of the cell cycle.

Animals↗
PubMed1976

No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed.

Alleles↗
PubMedDate not supplied

Mouse foetal development: translational inactivation of globin genes during the terminal differentiation of yolk sac erythroid cells.

The molecular mechanism of globin genes inactivation has been explored at the translational level in yolk sac erythroid cells from mouse foetuses. Ageing of the messenger molecules is paralleled by a progressive decrease of the poly(A) tail. Poly(A) less messengers are less active when tested in a wheat germ cell free system and become uncapable to terminate globin chain elongation.

Age Factors↗