Globin Sequences Research Articles

Assignment of fluorine nuclear magnetic resonance signals from rabbit cyanomethemoglobin.

A fluorine NMR study of cyanomethemoglobin prepared from hemoglobin isolated from rabbits maintained on a diet containing DL-p-fluorophenylalanine is described. The results indicate that substitution of fluorophenylalanine occurs essentially randomly at all phenylalanine positions of the alpha- and beta-globin chains; a set of hybrid hemoglobins in which only the alpha- or only the beta-chains contain the fluorinated amino acid was prepared and used to ascertain the fluorine NMR signals arising from each chain. The temperature and pH dependences of chemical shifts, spin-lattice relaxation times, 19F(1H) nuclear Overhauser effects, and the effect of chemical modification of the beta-93 sulfhydryl groups were examined. When considered in light of presently available X-ray structures of human and horse hemoglobins, the available data permit a tentative assignment of most signals to particular fluorophenylalanine/phenylalanine positions in the globin sequences.

Preferential transcription and nuclear transport of globin gene sequences, as control steps leading to final differentiation of murine erythroleukemic cells.

Murine erythroleukemic (MEL) cells undergo a specific program of differentation in vitro, which is mainly characterized by accumulation of globin mRNA. These cells serve as a model system to study in detail the expression of a specific gene product at the transcriptional and post-transcriptional level. In this report we describe experiments in which the transcription rate of globin and non-globin genes, as well as their cytoplasmic appearance, was measured during the differentiation process. Two independent steps for regulating the abundance of globin mRNA were observed. On the transcriptional level we have observed that, in contrast to the transcription of globin genes, the transcription rate of non-globin genes is dramatically reduced throughout the period of induction. When the rate of cytoplasmic appearance was measured newly synthesized globin RNA molecules were found to be preferentially transported into the cytoplasm. It was shown that the reduction in cytoplasmic appearance of nonglobin genes is not a result of a shut-off in their transcriptional activity. In cells treated with 12-O-tetradecanoylphorbol 13-acetate the transcription rate remains constant while a continuous reduction in the cytoplasmic appearance is observed. These two independent phenomena which affect the non-globin genes, i.e. the suppression of their transcription and reduced cytoplasmic appearance, lead to the reduction in the relative amounts of the stable poly(A)-rich mRNA population and to the accumulation of globin sequences in the cytoplasm of the differentiated erythroid cells. These observations are in agreement with our previous model, which claimed that disappearance of the stable poly(A)-rich mRNA population is an obligatory process leading to the final differentiation of MEL cells.

Amino acid sequence of the smallest polypeptide chain containing heme of extracellular hemoglobin from the polychaete tylorrhynchus heterochaetus

The complete amino acid sequence of 139 residues of the smallest polypeptide chain of extracellular hemoglobin from the polychaete Tylorrhynchus heterochaetus was determined. Mainly owing to the absence of seven residues in the G-helical region, this polypeptide chain is the second shortest, after Chironomus III hemoglobin (136 residues), of all vertebrate and invertebrate globin sequences so far reported. The residues at 42 sites (30%) of the Tylorrhynchus chain are identical with those of Lumbricus extracellular hemoglobin (AIII), which was sequenced very recently (Garlick, R.L. and Riggs, A.F. (1982) J. Biol. Chem. 257, 9005–9015). The C-terminal 20 residues of the Tylorrhynchus chain also showed 35% homology with those of Glycera intracellular hemoglobin, whereas the C-terminal sequence of Tylorrhynchus chain showed little correspondence to that of Lumbricus chain. The molecular mass of the polypeptide chain was determined to be 16327 including the heme group.

Minor early embryonic chick hemoglobin M. Amino acid sequences of the epsilon and alpha D chains.

Erythrocytes of the early chick embryo contain four hemoglobins, two major and two minor. In this paper, we present amino acid sequences for the beta-like and alpha-like chains of HbM, the least abundant of the four early chick hemoglobins. The complete amino acid sequence of the beta-like chain of HbM is identical with that of the epsilon chain of HbE, the other minor early embryonic hemoglobin in the domestic chicken. Analysis of the alpha-like chain of HbM (92 of 141 residues) reveals a globin sequence closely related to the minor adult alpha D chain. Comparison of our sequence data with the nucleotide sequence of the alpha D globin gene suggests that a single gene encodes both the embryonic and adult alpha D globin polypeptides. We discuss the structure, possible function, and evolution of the HbM globin chains.

The nucleotide sequence of the mouse embryonic β-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 y 2 globin. The combined overlapping sequences span a total of 480 bp, beginning at the codon corresponding to amino acid 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 y 2 protein sequence can be obtained. Comparisons, at the nucleotide level, between the known β- and β-like globin sequences and the y 2 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.

Adult chicken alpha-globin genes alpha A and alpha D: no anemic shock alpha-globin exists in domestic chickens.

Three alpha-type globin genes have been identified in the alpha-globin linkage group of chickens. No other alpha-type genes hae been directly shown to be within 10 kilobase pairs of any of these three closely linked genes. These three genes have been conclusively identified by DNA sequence analysis. The gene at the 5' end of the linkage group is an embryonic alpha-type globin gene, pi or pi', and the central gene corresponds to the minor adult alpha- globin, alpha D. The 3'-terminal gene sequence corresponds to the sequence of cDNA clones previously described as "alpha S", presumed anemic shock-induced alpha-globin gene [Salser, W. A., Cummings, I., Liu, A., Strommer, J., Padayatty, J. & Clarke, P. (1979) in Cellular and Molecular Regulation of Hemoglobin Switching, eds. Stamatoyannopoulos, G. & Nienheis, A. (Grune and Stratton, New York), pp. 621-643; Richards, R. I. & Wells, J. R. E. (1980) J. Biol. Chem. 255, 9306-9311]. Several groups of workers have isolated alpha S-type cDNA clones but no one has identified a cDNA clone corresponding to the published amino acid sequence of the major chicken alpha-globin, alpha A. We have identified the alpha S-type sequence as the only abundant alpha-like globin sequence in cDNA clones made from reticulocyte mRNA isolated from nonanemic chickens. Therefore, we suggest that the alpha S-type sequence corresponds to the true alpha A-globin species.

The asynchrony of γ- and β-chain synthesis during human erythroid cell maturation: III. γ- and β-mRNA in immature and mature erythroid clones

Globin chain synthesis and the accumulation of globin mRNAs were examined in immature and mature erythroid clones produced by circulating erythroid progenitors (burst forming units) of the neonate. Immature clones were composed of early erythroblasts containing little hemoglobin while the mature clones appeared at later days in culture and were composed of well hemoglobinized erythroblasts. There was a strikingly higher γ γ + β chain synthetic ratio in the nonhemoglobinized clones compared to their fully hemoglobinized counterparts. RNA was prepared from immature and from mature colonies and α-, β-, and γ-mRNA content was measured by solution hybridization to α-, β-, and γ-[ 32P]DNA probes. The probes were sense strand coding globin sequences prepared from chimeric plasmids by isolation of nick-translated [ 32P]DNA which annealed to cord blood or adult reticulocyte globin mRNA. In three experiments, the γ γ + β mRNA ratios in immature bursts were 0.58, 0.55, and 0.70 while ratios in the mature counterparts were 0.21, 0.38, and 0.45. There was excellent agreement between globin chain synthetic ratios and globin mRNA ratios suggesting that the globin mRNA species present in immature and mature erythroblasts are translated with similar efficiencies. These data suggest that the maturational modulation of non-α-globin chain synthesis is not under translational control. The absolute mRNA values (molecules per cell) consistently showed similar amounts of γ-mRNA per cell in both the immature and the mature cell populations. The most striking effect of maturation was the increase in the amount of β-mRNA per cell (and the concomitant increase in α-mRNA). These data, together with previously published results on globin chain biosynthesis suggest that the maturation related decline in γ/γ + β chain ratios is due to asynchrony in γ- and β-mRNA accumulation. Apparently γ-mRNA accumulates early (at or near the onset of globin synthesis) and the accumulation of β-mRNA follows. This asynchrony in mRNA accumulation results in a decline in the relative synthesis of fetal hemoglobin during maturation.

Alpha-Globulin sequences are located in a region of early-replicating DNA in murine erythroleukemia cells.

The time of replication in the S phase of regions of the mouse genome including the alpha-globin genes was determined in the murine erythroleukemia cell line transformed by Friend virus. Cells grown for short times in the presence of BrdUrd were fractionated into synchronous populations by centrifugal elutriation. The DNA was cleaved by restriction endonucleases, and fragments containing bromouracil (BrU-DNA) were isolated in density gradients of Cs2SO4. BrU-DNA fractions replicated during selected S-phase intervals were subjected to electrophoresis in agarose gels, transferred to diazobenzyloxymethyl-paper, and hybridized to an alpha-globin probe. Reconstruction experiments using a cloned mouse EcoRI fragment including one alpha-globin gene demonstrated that the extent of hybridization provides an accurate measurement that the extent of hybridization provides an accurate measurement of the concentrations of specific fragments in a DNA sample. The alpha-globin fragments were detected primarily in the BrU-DNA replicated during early S phase (approximately the first quarter of S). This result was confirmed in other synchrony experiments and by Cot analysis. (Cot is the initial concentration of DNA in mol of nucleotide per liter multiplied by the time in sec.) The temporal replication of mouse satellite sequences, already known from previous studies, was used as an internal control for cell synchrony. To show that the globin sequences were not lost from the cells in late S phase during isolation of the DNA, we quantitated th alpha-globin fragments in BrU-DNA prepared from a mixture of cells in early and late S phase. The results demonstrate that the alpha-globin gene regions in these cells are replicated during early S phase.

The high mobility group proteins and transcribed nucleosomes

Monomer nucleosomes released from nuclei during brief micrococcal nuclease digestions are enriched in transcribed sequences ( Bloom and Anderson, 1978). These nucleosomes are depleted in H1 and enriched in three high mobility group proteins HMG14, HMG17 and another HMG-like protein. Analysis of such nucleosomes by polyacrylamide gel electrophoresis reveal that they are heterogenous. Similarly, monomer nucleosomes soluble in 0.1 M NaCl separate on polyacrylamide gels into mainly two types of particle, one of which has HMG14 and HMG17 bound. However, the DNA of the HMG-nucleosomes from chick erythrocytes is not enriched in globin sequences, suggesting that protein rearrangement may have occurred.

Modification of the methylation pattern in the vicinity of the chicken globin genes in avian erythroblastosis virus transformed cells.

Having previously found a reduced transcription of globin genes and an abortive processing of the already transcribed globin pre-mRNA in Avian Erythroblastosis Virus (AEV) transformed cells (1), we compared the genomic DNA of these cells with that of normal chicken erythroblasts, using 32-P-labelled cDNA probes specific for the beta, alpha A and alpha D globin sequences. Restriction endonuclease digestion, electrophoresis of digests in agarose gels, Southern blotting and hybridization were carried out. Our results show that the overall genome organization is not disturbed in the immediate neighbourhood of the adult globin genes; the observed restriction fragments are identical for both DNAs after EcoRI, HindIII, BamHI and XbaI digestion, using the beta, alpha A and alpha D globin cDNA probes. However, we observe specific modifications at some methylation sites in the beta, beta-like and alpha D regions: after HpaII or MspI digestion in the alpha D region and after HhaI digestion in the beta and beta-like region, heavier bands appear in the transformed cell DNA in addition to the ones observed in normal DNA. This implies that, at some specific sites, the transformed cell DNA is more methylated than the normal erythroblast DNA. The possible significance of this observation is discussed.

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library.

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.

Molecular cloning and characterization of the human β-like globin gene cluster

The genes encoding human embryonic (ϵ), fetal ( Gγ, Aγ) and adult (δ, β) β-like globin polypeptides were isolated as a set of overlapping cloned DNA fragments from bacteriophage λ libraries of high molecular weight (15–20 kb) chromosomal DNA. The 65 kb of DNA represented in these overlapping clones contains the genes for all five β-like polypeptides, including the embryonic ϵ-globin gene, for which the chromosomal location was previously unknown. All five genes are transcribed from the same DNA strand and are arranged in the order 5′-ϵ-(13.3 kb)- Gγ-(3.5 kb)- Aγ-(13.9 kb)-δ-(5.4 kb)-β-3′. Thus the genes are positioned on the chromosome in the order of their expression during development. In addition to the five known β-like globin genes, we have detected two other β-like globin sequences which do not correspond to known polypeptides. One of these sequences has been mapped to the Aγ-δ intergenic region while the other is located 6–9 kb 5′ to the ϵ gene. Cross hybridization experiments between the intergenic sequences of the gene cluster have revealed a nonglobin repeat sequence ( ∗) which is interspersed with the globin genes in the following manner: 5′- ∗∗ϵ- ∗Gγ- Aγ ∗- ∗∗δ-β ∗-3′. Fine structure mapping of the region located 5′ to the δ-globin gene revealed two repeats with a maximum size of 400 bp, which are separated by approximately 700 bp of DNA not repeated within the cluster. Preliminary experiments indicate that this repeat family is also repeated many times in the human genome.

Enrichment of estradiol-receptor complexes in a transcriptionally active fraction of chromatin from MCF-7 cells.

We have examined the interaction of the estradiol receptor molecule with chromatin in MCF-7 cells, a human breast tumor cell line responsive to estradiol. Receptor was found associated with the various nucleosomal products produced by digestion with micrococcal nuclease. In order to determine whether these receptor binding sites were distributed in a random or nonrandom manner within the chromatin, we have fractionated MCF-7 cell chromatin into transcriptionally active and inactive fractions by limited micrococcal nuclease digestion followed by Mg(2+) precipitation. A comparison of the Mg(2+)-soluble and insoluble chromatin fractions showed that the Mg(2+)-soluble fraction: (i) was composed predominantly of mononucleosomes; (ii) was enriched in nonhistone proteins; (iii) apparently lacked histone H1; (iv) was enriched approximately 5-fold in transcribed sequences as measured by a cDNA probe to cytoplasmic poly(A)-RNA sequences; and (v) was depleted at least 5-fold of globin sequences, which is presumably a nontranscribed gene in these cells. When these cells were stimulated with beta-[(3)H]estradiol, the Mg(2+)-soluble fraction showed a significant enrichment in chromatin-bound estradiol receptor: the Mg(2+)-soluble mononucleosomes showed a 3- to 4-fold enrichment and the di- and trinucleosomes, a 7- to 19-fold enrichment, when compared to the corresponding subunits in the Mg(2+)-insoluble chromatin fraction. This cofractionation of chromatin enriched in transcribed sequences and bound estradiol receptor indicated that receptor binding to MCF-7 cell chromatin was not random but, rather, occurred preferentially in specific regions of the chromatin.

In situ hybridization to lampbrush chromosomes: a potential source of error exposed.

Denatured 3H-labelled DNAs containing Xenopus or human globin sequences hybridize to RNA transcripts on a single pair of lateral loops on lampbrush chromosome IX of Triturus cristatus carnifex, and to no other loops on this chromosome or the rest of the complement. However they do so, not because of the globin sequences in the probes, but rather because the plasmids from which the probes were prepared were constructed with G.C homopolymer tails. Simple sequence poly d(C/G)n probes also hybridize with RNA transcripts on this same pair of loops, and with no others.

Electrophoretic separation of a class of nucleosomes enriched in HMG 14 and 17 and actively transcribed globin genes

Monomer nucleosomes from chick erythrocytes can be fractionated according to their electrophoretic mobility in (comparatively) high salt acrylamide gels. We show that the fractionation is based predominantly on differences in charge. The monomer heterogeneity persists even when the nucleosomes are trimmed down to 145 bp with Exo III or when H1 and H5 are removed. The slowest migrating monomers are associated with HMG 14 and 17; however, we do not believe that these proteins are entirely responsible for the altered mobility since the nucleosome heterogeneity persists even after removal of HMG 14 and 17. The DNA associated with the HMG 14 and 17 containing nucleosomes is shown to be enriched in actively transcribed globin sequences.

Amino Acid Sequence of the a-Chain of the Tetrameric Haemoglobin of the Bivalve Mollusc Anadara Trapezia

The amino acid sequence of the IX-chain of the tetrameric haemoglobin of the bivalve mollusc A. trapezia shows 153 amino acid residues, longer than invertebrate globins previously sequenced. The amino terminal residue is acetylated as shown by nuclear magnetic resonance and mass spectrographic analysis of an N-terminal peptide. There are additional residues at the amino terminus, similar to the IX-globin of the shark and lamprey globin. In common with other invertebrate globin sequences, when compared with vertebrate globins it is necessary to insert extra gaps in interhelical regions of the vertebrate alignment to preserve homology.

The isolation and partial characterization of recombinant DNA containing genomic globin sequences from the goat.

Total genomic DNA from the spleen of a juvenile goat was partially digested with the restriction endonuclease Eco RI, and the 12 to 22 kilobase pair size class isolated by sucrose density gradient centrifugation. The two internal Eco RI fragments of X bacteriophage Charon 4A DNA were removed and the goat DNA ligated to the remaining phage DNA. The recombinant DNAs were packaged into infectious phage particles in vitro and amplified by growth of plaques on the Escherichiu coli host DP50 sup F. Twelve recombinants containing globin gene sequences were identified by their ability to hybridize to globin Q’P]cDNA and four of these were selected for further study. DNA prepared from these clones was digested with various restriction endonucleases and the products analyzed by agarose and polyacrylamide gel electrophoresis. Restriction fragments containing the globin genes were identified by hybridization following transfer to nitrocellulose filters. Clones 1,2, and 4 appear to contain overlapping sequences in the inserted goat DNA while clone 3 exhibits a unique pattern of restriction enzyme fragments. Orientation experiments show that the globin gene coding sequences are located in the (I) strand of clones 1,2, and 3, and in the (r) strand of clone 4. Cell-free translation of goat globin mRNAs which hybridize to the recombinant clones show that clones 1, 2, and 4 contain yor fi-globin sequences but not those for a-globin. The inserted DNA of the clones range from 13.4 to 16.7 kilobase pairs, making it possible to characterize, in addition to the globin genes, extensive regions surrounding these genes.

Fitting the Gene Lineage into its Species Lineage, a Parsimony Strategy Illustrated by Cladograms Constructed from Globin Sequences

activating regulator mutations. Analysis of amino acid sequence data from the alpha and beta hemoglobins and myoglobins suggests that the gene lineages deviate relatively little from established species relationships, and that most contemporary gene pairs in this phylogenetic system may be regarded as orthologous (split at the time of speciation) rather than paralogous (split prior to speciation). It also appears that as the statistical sample of orthologous sequence sites becomes greatly enlarged by tandem alignment of enough different types of protein chains, the parsimony reconstruction based strictly on minimal nucleotide replacement length will itself reveal the correct cladogram for the set of contemporary species. [Phylogeny reconstruction; parsimony methods; globin sequences; gene phylogeny; species phylogeny.]

Fitting the Gene Lineage into its Species Lineage, a Parsimony Strategy Illustrated by Cladograms Constructed from Globin Sequences

Fitting the Gene Lineage into its Species Lineage, a Parsimony Strategy Illustrated by Cladograms Constructed from Globin Sequences

Specificity of chromatin transcription in vitro Anomalies due to RNA-dependent RNA synthesis

In the presence of Mn 2+, globin mRNA can be transcribed into a partial RNA copy by Escherichia coli RNA polymerase. This process also occurs when the mRNA is transcribed together with chromatin. A fraction, at least, of the newly synthesized RNA copy (anti-globin RNA) can serve as a template for the synthesis of globin sequences of the same polarity as the original mRNA. This process is sufficient to explain the specific synthesis of a subset of the globin RNA on mouse foetal liver chromatin. It also accounts for the synthesis of double-stranded RNA sequence by E. coli RNA polymerase, on chromatin as well as on pure mRNA. Results are presented suggesting that the poly(A) tract of the mRNA could be preferentially transcribed. In the presence of Mg 2+, the RNA-dependent transcription is strongly inhibited, as well as the synthesis of double-stranded RNA. Under these conditions, the transcription on chromatin appears to be largely DNA dependent, and the synthesis of globin sequences is completely asymmetric. Spermine (0.3 mM) seems to improve the specificity of transcription. The transcription of chromatin in vitro is thus largely dependent on the nature of the divalent cation present in the incubation mixture.