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B G Forget

Publications and source records attributed to B G Forget.

At least 73 records · Page 4Linked to original sources
PubMed1991

Genomic organization of the human erythropoietin receptor gene.

Erythropoietin (EPO) mediates the growth and differentiation of erythroid progenitors through its interaction with a specific receptor. Using a partial cDNA clone for the murine erythropoietin receptor, we isolated a human genomic clone containing the erythropoietin receptor gene. The coding region of the human EPO receptor gene is contained within eight exons spanning approximately 6 kb. The human gene has a great deal of structural similarity and sequence homology with the murine gene. The murine gene also has eight exons, although the size of each intron is somewhat different. The locations at which the introns interrupt the coding sequence are conserved precisely. The genomic organization of the EPO receptor gene is also shown to be homologous to the genomic organization of the IL-2 receptor beta chain gene. The sequence of 1.1 kb of 5' flanking DNA was characterized and contains consensus sequences for both Sp1 and GATA-1 binding sites and an initiator (Inr)-like element, but lacks both a canonical TATA box and the CACCC consensus sequence found in the murine gene.

Amino Acid Sequence↗
PubMed1991

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.

Amino Acid Sequence↗
PubMed1991

Ankyrin binds to the 15th repetitive unit of erythroid and nonerythroid beta-spectrin.

Ankyrin mediates the attachment of spectrin to transmembrane integral proteins in both erythroid and nonerythroid cells by binding to the beta-subunit of spectrin. Previous studies using enzymatic digestion, 2-nitro-5-thiocyanobenzoic acid cleavage, and rotary shadowing techniques have placed the spectrin-ankyrin binding site in the COOH-terminal third of beta-spectrin, but the precise site is not known. We have used a glutathione S-transferase prokaryotic expression system to prepare recombinant erythroid and nonerythroid beta-spectrin from cDNA encoding approximately the carboxy-terminal half of these proteins. Recombinant spectrin competed on an equimolar basis with 125I-labeled native spectrin for binding to erythrocyte membrane vesicles (IOVs), and also bound ankyrin in vitro as measured by sedimentation velocity experiments. Although full length beta-spectrin could inhibit all spectrin binding to IOVs, recombinant beta-spectrin encompassing the complete ankyrin binding domain but lacking the amino-terminal half of the molecule failed to inhibit about 25% of the binding capacity of the IOVs, suggesting that the ankyrin-independent spectrin membrane binding site must lie in the amino-terminal half of beta-spectrin. A nested set of shortened recombinants was generated by nuclease digestion of beta-spectrin cDNAs from ankyrin binding constructs. These defined the ankyrin binding domain as encompassing the 15th repeat unit in both erythroid and nonerythroid beta-spectrin, amino acid residues 1,768-1,898 in erythroid beta-spectrin. The ankyrin binding repeat unit is atypical in that it lacks the conserved tryptophan at position 45 (1,811) within the repeat and contains a nonhomologous 43 residue segment in the terminal third of the repeat. It also appears that the first 30 residues of this repeat, which are highly conserved between the erythroid and nonerythroid beta-spectrins, are critical for ankyrin binding activity. We hypothesize that ankyrin binds directly to the nonhomologous segment in the 15th repeat unit of both erythroid and nonerythroid beta-spectrin, but that this sequence must be presented in the context of a properly folded spectrin "repeat unit" structure. Future studies will identify which residues within the repeat unit are essential for activity, and which residues determine the specificity of various spectrins for different forms of ankyrin.

Amino Acid Sequence↗
PubMed1991

Negative regulation of globin gene expression during megakaryocytic differentiation of a human erythroleukemic cell line.

The human erythroleukemic cell line K562 was used as a model for analysis of the mechanisms responsible for alterations in gene expression during differentiation. K562 cells normally synthesize fetal hemoglobin (gamma-globin), but treatment with tumor-promoting phorbol esters (phorbol myristate acetate and tetradecanoyl phorbol acetate) results in the loss of the erythroid phenotype of the cells and causes a shift toward a megakaryocytic phenotype. This shift involves markedly decreased production of fetal hemoglobin and de novo synthesis of a number of proteins specific for megakaryocytes. The results of this work indicate that negative regulation of fetal hemoglobin during megakaryocytic differentiation of K562 cells occurs at the level of down regulation of gamma-globin mRNA accumulation. This effect consists of at least two components: reduction in the rate of transcription of the gamma-globin gene and decrease in stability of the normally very stable gamma-globin mRNA. We have developed two assay systems that permit investigation of the transcriptional and posttranscriptional effects of phorbol myristate acetate independently from each other. These assay systems make use of a heterologous reporter gene for the transcriptional analysis and a marked gamma-globin gene for the analysis of mRNA stability. The DNA sequences located in the 3' flanking region of the A gamma-globin gene were found to be responsible for the decrease in transcription rate.

Cell Differentiation↗
PubMed1991

Spectrin Rouen (beta 220-218), a novel shortened beta-chain variant in a kindred with hereditary elliptocytosis. Characterization of the molecular defect as exon skipping due to a splice site mutation.

The molecular defect responsible for the shortened beta-spectrin chain variant, spectrin Rouen, was identified by analysis of cDNA and genomic DNA of affected individuals after amplification by the polymerase chain reaction. Peripheral blood reticulocyte RNA was transcribed into cDNA and amplified using primers corresponding to the 3' end of beta-spectrin cDNA. Agarose gel electrophoresis of cDNA amplification products from affected individuals revealed the expected band of 391 bp as well as a shortened band of 341 bp. Nucleotide sequencing of the shortened cDNA amplification product revealed that the sequences corresponding to the penultimate exon of the beta-spectrin gene (exon Y) were absent. This result was confirmed by hybridization of a Southern blot of amplification products with a labeled probe specific for exon Y. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the beta-spectrin gene revealed a mutation in the 5' donor consensus splice site of the intron downstream of the Y exon, TGG/GTGAGT to TGG/GTTAGT, in one allele. We postulate that this mutation leads to the splicing out or skipping of exon Y, thus producing a shortened beta-spectrin chain. To our knowledge, this is the first documented example of exon skipping as the cause of a shortened beta-spectrin chain in a case of hereditary elliptocytosis. The exon skip results in the loss of the 17 amino acids of exon Y and creates a frameshift with the synthesis of 33 novel amino acids prior to premature chain termination 14 residues upstream of the normal carboxy terminus of the beta-spectrin chain, giving a mutant beta-spectrin chain that is 31 amino acids shorter than the normal chain.

Amino Acid Sequence↗
PubMed1991

Delta beta thalassemia and hereditary persistence of fetal hemoglobin.

Delta beta Thalassemia and hereditary persistence of fetal hemoglobin (HPFH) constitute a heterogeneous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Coinheritance of these disorders with other beta chain hemoglobinopathies, such as beta thalassemia and the sickle cell (beta s) gene, can result in attenuation of the clinical severity of these hemoglobinopathies owing to the increased Hb F levels. The molecular basis of these disorders is quite heterogeneous and consists of both deletion and nondeletion types of mutations. The characterization of these molecular defects has provided new insights on the structure and function of important regulatory elements that are involved in the normal control of expression of the beta- and gamma-globin genes and in hemoglobin switching.

Animals↗
PubMed1990

Beta spectrin in human skeletal muscle. Tissue-specific differential processing of 3' beta spectrin pre-mRNA generates a beta spectrin isoform with a unique carboxyl terminus.

Spectrin, an important component of the mammalian erythrocyte membrane skeleton, is a heterodimeric protein with alpha and beta subunits of 280 and 246 kDa, respectively. Spectrin-like proteins have also been demonstrated in a wide variety of nonerythroid cells. To examine the hypothesis that nonerythroid beta spectrins may be encoded by the "erythroid" beta spectrin gene, we have isolated cDNA clones from a human fetal skeletal muscle library by hybridization to a previously described red cell beta spectrin cDNA. Detailed comparison of muscle and erythroid beta spectrin cDNAs has revealed sequence identity over the majority of their lengths, confirming that they are the product of the same gene. However, there is a sharp divergence in sequence at their 3' ends. A consequence of this divergence is the replacement of the carboxyl terminus of erythroid beta spectrin with a different, longer carboxyl-terminal domain in skeletal muscle. We hypothesize that tissue-specific differential polyadenylation leads to the selective activation of a donor splice site within the beta spectrin coding sequence, splicing downstream nonerythroid exons into the mature muscle beta spectrin mRNA. We predict that replacement, in nonerythroid cells, of the beta spectrin carboxyl terminus, known to participate in spectrin self-association and phosphorylation, has significant functional consequences. These data may explain previously reported nonerythroid beta spectrin isoforms that resemble red cell beta spectrin by immunochemical analysis.

Amino Acid Sequence↗
PubMed1990

Full-length sequence of the cDNA for human erythroid beta-spectrin.

Spectrin is the major molecular consituent of the red cell membrane skeleton. We have isolated overlapping human erythroid beta-spectrin cDNA clones and determined 6773 base pairs of contiguous nucleotide sequence. This includes the entire coding sequence of beta-spectrin. The sequence translates into a 2137 amino acid, 246-kDa peptide. beta-Spectrin is found to consist of three distinct domains. Domain I, at the N terminus, is a 272-amino acid region lacking resemblance to the spectrin repetitive motif. Sequences in this region exhibit striking sequence homology, at both nucleotide and amino acid levels, to the N-terminal "actin-binding" domains of alpha-actinin and dystrophin. Between residues 51 and 270 there is 55% amino acid identity to human dystrophin, with only four single amino acid gaps in alignment. Domain II consists of 17 spectrin repeats. Several sequence variations are observed in typical repeat structure. Homology to alpha-actinin extends beyond domain I into the N-terminal portion of domain II. Domain III, 52 amino acid residues at the C terminus, does not adhere to the spectrin repeat motif. Combining knowledge of spectrin primary structure with previously reported functional studies, it is possible to make several inferences regarding structure/function relationships within the beta-spectrin molecule.

Amino Acid Sequence↗
PubMed1990

The gene for the human erythropoietin receptor: analysis of the coding sequence and assignment to chromosome 19p.

The full-length coding sequence of the human erythropoietin receptor has been assembled from cDNA and genomic DNA. The derived 508 amino acid sequence is 82% identical to the murine erythropoietin (Epo) receptor with one single residue gap in alignment. There is no major structural difference between the human and murine receptor molecules. Nucleotide sequence homology is, as expected, very high within the coding domain. Unexpectedly, there are two distinct, short stretches of 3' untranslated sequence homology between human and murine cDNAs. The functional significance of this sequence conservation is unknown. The human Epo receptor gene is localized to human chromosome 19p by in situ hybridization. This chromosome assignment is confirmed by hybridization to a panel of sorted human chromosomes.

Amino Acid Sequence↗
PubMed1990

Reverse transcription and direct amplification of cellular RNA transcripts by Taq polymerase.

We report the ability of Taq polymerase to directly transcribe RNA templates in vitro. We have made use of this finding to develop a single-step protocol for amplification of RNA transcripts. The method was shown to require only subnanogram amounts of total cellular RNA as starting material. A microassay was developed in which RNA can be extracted from one drop of blood or 1000 cultured cells, and analyzed for the expression of a specific gene.

Base Sequence↗
PubMed1990

Sequence analysis of the gamma-globin gene locus from a patient with the deletion form of hereditary persistence of fetal hemoglobin.

The gamma-globin genes from a patient homozygous for a deletion form of hereditary persistence of fetal hemoglobin (HPFH-1) have been cloned and sequenced. The DNA sequence of the patient's gamma-globin genes corresponds to a previously identified sequence framework (chromosome A) with the exception of 10 base changes. Seven of these base changes can be attributed to normal allelic variation generated by small gene conversion events. The remaining three base changes are present in a 0.76 kb HindIII fragment containing a putative enhancer located 3' to the A gamma-globin gene. The same three base changes have also been described in the Seattle variant of nondeletion HPFH. We have analyzed 16 alleles from non-HPFH individuals and five alleles from individuals with nondeletion or deletion HPFH for the presence of these base changes by polymerase chain reaction amplification of cloned or chromosomal DNA and hybridization to allele-specific oligonucleotide probes. Although these base changes were found in an individual with HPFH-2, they were not found in the DNA from two patients with nondeletion HPFH. More importantly, all three base changes were detected in DNA from five non-HPFH individuals and appear to be common in blacks. We conclude that these base changes do not correlate with an HPFH phenotype and that the significant mutation in HPFH-1 is the deletion of over 100 kb of genomic DNA.

Base Sequence↗
PubMed1990

Evidence that expression of Sp alpha I/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous alpha-spectrin allele.

Many cases of hereditary elliptocytosis (HE) result from mutated spectrin alpha-chains. It has repeatedly been observed that the amount of a mutant alpha-chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the alpha-spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp alpha I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp alpha I/65 variant, suggesting the existence, in trans, of a low percentage alpha-allele. The alpha-spectrin gene haplotype associated with the latter was constantly - + -, based on the XbaI, PvuII, and MspI polymorphic sites. In contrast, a basal level of expression of the Sp alpha I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage alpha-allele. The haplotype corresponding to this other alpha-allele was + - +. Study of another generation of the family showed, however, that the - + - haplotype could also be linked to a normal percentage alpha-allele. These results are consistent with the view that the expression level of alpha I/65 spectrin (and of other types of alpha-variants) is compounded by a genetic factor that is linked to the normal alpha-allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.

Elliptocytosis, Hereditary↗
PubMed1990

Expression of human globin genes in transgenic mice carrying the beta-globin gene cluster with a mutation causing G gamma beta + hereditary persistence of fetal hemoglobin.

We have introduced into the mouse germ line the 40-kilobase (kb) Kpn I fragment containing the beta-globin gene cluster from an individual with a non-deletion form of hereditary persistence of fetal hemoglobin (HPFH) believed to be due to a point mutation at position -202 of the G gamma-globin gene. The G gamma-globin gene, as well as the beta-globin gene, was expressed in adult erythroid tissues of the resulting transgenic mice. The level of expression of the G gamma-globin gene was about 20% of that of the beta-globin gene. Others have previously shown that cloned individual normal human beta- and gamma-globin genes containing a limited amount of 5'- and 3'-flanking DNA are expressed in a manner similar to that of their corresponding murine homologs during development in transgenic mice. In contrast, we have observed that the pattern of expression of the normal (non-mutated) A gamma- and beta-globin genes in the 40-kb insert was different from that of their corresponding murine homologs. The beta-globin gene remained inactive at the fetal stage, whereas the normal A gamma-globin gene was expressed beyond the embryonic (yolk sac) stage into the fetal stage of development and then became inactive in adult erythroid cells. The pattern of expression of the human globin transgenes during mouse development resembles that observed during human development. These results suggest that the gross organization of the human beta-like globin gene cluster is important for stage-specific expression of each human globin gene during development.

Animals↗
PubMed1990

Assignment of the gene for beta-spectrin (SPTB) to chromosome 14q23----q24.2 by in situ hybridization.

Type I hereditary spherocytosis results from a molecular defect in the beta-polypeptide of the erythrocyte cytoskeletal protein spectrin. Using a cDNA probe, we had previously assigned the gene for human erythrocyte beta-spectrin (SPTB) to chromosome 14 based upon analysis of its segregation in panels of human x rodent somatic cell hybrids (Winkelmann et al., 1988). Here we report the regional localization of this gene by in situ hybridization to 14q23----q24.2.

Chromosome Mapping↗
PubMed1990

Two elliptocytogenic alpha I/74 variants of the spectrin alpha I domain. Spectrin Culoz (GGT----GTT; alpha I 40 Gly----Val) and spectrin Lyon (CTT----TTT; alpha I 43 Leu---Phe).

Spectrin alpha I/74 elliptocytosis results from abnormalities involving the "head" region of spectrin dimer. Increased susceptibility to trypsin enhances cleavage of the alpha spectrin chain, yielding an increased amount of the alpha I 74-kD fragment at the expense of the alpha I 80-kD parent fragment. Recently we showed that the mutations causing the Sp alpha I/74 abnormality may lie in the alpha- or the beta-chain, and that spectrin Culoz and spectrin Lyon were two (alpha I/74) alpha-variants, respectively. We now show that the spectrin Culoz alpha I domain undergoes prominent tryptic cleavage after Lys 42, whereas cleavage prevails after Arg 39 in spectrin Lyon. Applying the polymerase chain reaction (PCR) technique to exon 2 of the spectrin alpha I domain, we have established that the mutation responsible for spectrin Culoz is alpha I 40 Gly----Val; GGT----GTT. Applying the PCR technique to the cDNA derived from reticulocyte mRNA, we have shown that the mutation responsible for spectrin Lyon is alpha I 43 Leu----Phe; CTT----TTT. Studies of normal controls and of family members using dot blot hybridization with allele-specific oligonucleotide probes confirmed these results. Variants such as spectrin Culoz and spectrin Lyon should provide insight into a region that participates in spectrin dimer self-association and whose susceptibility to proteolysis must reflect subtle conformational changes.

Amino Acid Sequence↗
PubMed1990

Point mutation in the beta-spectrin gene associated with alpha I/74 hereditary elliptocytosis. Implications for the mechanism of spectrin dimer self-association.

alpha I/74 hereditary elliptocytosis (HE) is a subgroup of HE in which patients exhibit an impaired self-association of spectrin dimers and an abnormal proteolytic cleavage of the alpha I domain of spectrin. We studied a family in which the proband presented with a severe neonatal hemolytic anemia with poikilocytosis. Biochemical analysis of erythrocytes from the proband and his family members allowed us to ascertain a diagnosis of homozygosity for alpha I/74 HE in the proband and heterozygosity in his parents and several of their offspring. Results of polymorphism linkage analysis suggested that the defect in this family was located in beta rather than alpha spectrin. We analyzed the 3' end of the beta-spectrin gene of the proband and detected a mutation that changes a codon for alanine to one for proline. Allele-specific oligomer hybridization on slot blots of DNA from other family members confirmed the presence of the mutation only in members heterozygous for the disorder. This is the first example of a point mutation in the beta-spectrin chain that is associated with defective spectrin dimer self-association and an abnormal proteolytic cleavage of the alpha chain. Based on this finding, we propose a model for the mechanism of interaction between the alpha- and beta-spectrin chains.

Amino Acid Sequence↗