Globin Family Research Articles

Developmental regulation of globin gene expression.

We have used the globin family of genes in chicken to study developmental regulation of gene expression, both at the level of individual interaction of trans-acting factors with local promoters and enhancers, and at the level of chromatin structure. Regulation of all members of the alpha- and beta-globin clusters is affected by the erythroid regulatory factor GATA-1. Separate mechanisms exist for regulation of individual members of the family. As an example, we describe the control mechanisms that play a role in the expression of the rho-globin gene, which is expressed only in primitive lineage erythroid cells. In addressing the involvement of chromatin structure in gene activation, we have examined the role of locus control elements, and also considered the way in which RNA polymerase molecules might accommodate to the presence of nucleosomes on transcribed genes.

Was the loss of the D helix in α globin a functionally neutral mutation?

Proteins in the globin family are found in a variety of species from bacteria to man. From the many globin sequences known, evolutionary trees have been constructed showing that alpha and beta globins diverged from a common ancestor between 425 and 500 million years ago, after vertebrate species had appeared and roughly when sharks and bony vertebrates diverged. The alpha and beta globins assemble to form tetrameric haemoglobin, alpha 2 beta 2, which can switch between quaternary states having high and low oxygen affinity. This allows the protein to bind oxygen cooperatively and therefore efficiently transport oxygen from the lungs to respiring tissues. The alpha and beta globins have closely related tertiary structures, being alpha-helical proteins with similar haem-binding sites. Most globins consist of eight helices, designated A to H from the N terminus, connected by short nonhelical segments, but all known vertebrate alpha globins lack a D helix. Because the loss of this helix by alpha globin occurred shortly before tetrameric haemoglobin appeared, it might be a functionally important mutation required for a tetramer assembly or allostery. We have now tested this idea by engineering human haemoglobins containing beta subunits without a D helix and alpha subunits with a D helix. Both of these mutations have little effect on the oxygen-binding properties of the molecule. Thus it is possible that deletion of the D helix in the alpha subunit was caused by a neutral mutation.

Unique sequence organization and erythroid cell-specific nuclear factor-binding of mammalian Θ I globin promoters

The theta 1 globin gene is an alpha globin-like gene, and started to diverge from the other members of the alpha globin family 260 million years ago. DNA sequencing and transcriptional analysis indicated that it is functional in erythroid cells of the higher primates, but not in prosimians and rabbit. The theta 1 promoter region of higher primates including man consists of GC-rich sequences characteristic of housekeeping gene promoters, and CCAAT and TATA boxes located further upstream. It is shown here that the housekeeping gene promoter-like region of human theta 1 contains two tandemly arranged, GC-rich motifs (GC-I and GC-II). Of these, GC-II interacts with nuclear factor(s) present in the globin-expressing, erythroleukemia cell line K562, before and after hemin induction. GC-I, however, interacts with nuclear factor(s) only present in hemin-induced K562 cells. These factors are different from previously reported erythroid cell-specific factors, and are not detectable in non-erythroid Hela cells. Furthermore, the sequence of the motif GC-I and its location relative to ATG codon have been conserved among all known mammalian theta 1 globin genes. Finally, and most interestingly, the CCAAT box of theta 1 is contained within a 38 bp internal segment of Alu repeat sequence. Immediately upstream from this CCAAT box-containing Alu repeat segment is a 241 bp Alu repeat pointing in the opposite direction. The conservation of this novel arrangement among the higher primates suggests that an inserted Alu family repeat and its flanking genomic sequence have co-evolved, for at least 30 million years, to provide the canonical CCAAT and TATA promoter elements of the theta 1 globin genes in higher primates.

Minimal energy foldings of eukaryotic mRNAs form a separate leader domain

We have investigated the minimal energy foldings of 38 mature mRNAs, including the globin family, the insulins, the growth hormones and interleukin-2, and have compared these foldings with those of fully and partly randomised sequences. The mRNAs differ from the random sequences in that they form a separate leader hairpin of 40-60 nucleotides, with the initiation codon typically located downstream of this hairpin, followed by a main fold in which a region flanking the initiation codon is basepaired with the trailer: resulting in a close proximity of the 5' and 3' end of the mRNA. The formation of this conformation depends not only--or primarily--on the structure of the leader, but on both the leader and trailer sequence and their interaction with the coding sequence. Thus if, as the frequent occurrence of this pattern suggests, the secondary structure of the leader regions plays a role in the initiation of translation, possibly accounting for the specificity of initiation and the different translational efficiencies of various mRNAs, we expect that these features may be influenced both by leader and trailer mutants.

Dual evolutionary modes in the bovine globin locus.

Five bovine globin pseudogenes were subjected to sequence analysis. These genes include the three pseudogenes in the beta-type globin gene cluster as well as two allelic forms. Comparison of the sequences with those of the adult and fetal bovine globin genes shows that together they form a multigene family that was created by large-scale duplication. The structures are explained by invoking sequence exchange mediated by gene conversion. After their creation these genes evolved in a concerted fashion, exchanging sequence freely by intrachromosomal gene conversion. Subsequently, one by one, the genes were uncoupled from this exchange. This was accomplished by the creation of nonhomologies that formed barriers to gene conversion. These nonhomologies were several hundred bases in length and were formed by either deletion or by insertion of short repetitive sequences within the gene structures. In this way the genes made the transition from a rapid, coupled mode to a slow, solitary mode of evolution. Allelic gene polymorphisms were distributed inhomogeneously in the bovine globin family. It is proposed that this was due to interruption of interchromosomal gene conversion by a recent pseudogene duplication in the fetal globin gene cluster.

Deoxynucleotide sequence of an insect cDNA codes for an unreported member of the Chironomus thummi globin family

Synthetic oligonucleotides served as probes to isolate insect globin clones from a Chironomus thummi cDNA bank. The cDNA insert of one clone (pC-S9) was completely sequenced by the dideoxy termination procedure. Beginning at the 5′ end of the coding region, the 584 base pair sequence encodes most of an N-terminal hydrophobic signal sequence and the complete sequence for a mature secreted globin, and contains a polyadenylation recognition site 3′ to an appropriate stop codon. The inferred amino acid sequence is that of an unreported variant of hemoglobin VIIB. Based on the number of differences between Hb VIIB chains, the pC-S9 gene has been evolutionarily independent longer than the other (two) members of the globin VIIB subfamily.

Relation between sequence similarity and structural similarity in proteins. Role of important properties of amino acids

In a previous paper we obtained ten (orthogonal) factors, linear combinations of which can express the properties of the 20 naturally occurring amino acids. In this paper, we assume that the most important properties (linear combinations of these ten factors) that determine the three-dimensional structure of a protein are conserved properties, i.e., are those that have been conserved during evolution. Two definitions of a conserved property are presented: (1) a conserved property for an average protein is defined as that linear combination of the ten factors that optimally expresses the similarity of one amino acid to another (hence, little change during evolution), as given by the relatedness odds matrix of Dayhoff et al.; (2) a conserved property for each position in the amino acid sequence (locus) of a specific family of homologous proteins (the cytochromec family or the globin family) is defined as that linear combination of the ten factors that is common among a set of amino acids at a given locus when the sequences are properly aligned. When the specificity at each locus is averaged over all loci, the same features are observed for three expressions of these two definitions, namely the conserved property for an average protein, the average conserved property for the cytochromec family, and the average conserved property for the globin family; we find that bulk and hydrophobicity (information about packing and long-range interactions) are more important than other properties, such as the preference for adopting a specific backbone structure (information about short-range interactions). We also demonstrate that the sequence profile of a conserved property, defined for each locus of a protein family (definition 2), corresponds uniquely to the three-dimensional structure, while the conserved property for an average protein (definition 1) is not useful for the prediction of protein structure. The amino acid sequences of numerous proteins are searched to find those that are similar, in terms of the conserved properties (definition 2), to sequences of the same size from one of the homologous families (cytochromec and globin, respectively) for whose loci the conserved properties were defined. Many similar sequences are found, the number of similarities decreasing with increasing size of the segment. However, the segments must be rather long (≥15 residues) before the comparisons become meaningful. As an example, one sufficiently large sequence (20 residues) from a protein of known structure (apo-liver alcohol dehydrogenase that is not a member of either family) is found to be similar in the conserved properties to a particular sequence of a member of the family of human hemoglobin α chains, and the two sequences have similar structures. This means that, since conserved properties are expected to be structure determinants, we can use the conserved properties to predict an initial protein structure for subsequent energy minimization for a protein for which the conserved properties are similar to those of a family of proteins with a sufficiently large number of homologous amino acid sequences; such a large number of homologous sequences is required to define a conserved property for each locus of the homologous protein family.

Hemoglobin — A Working Molecule

The characteristic structure of the globin family has been maintained with little variation for at least 540 million years,1 , 2 evolution being witness to the necessity of maintaining a precise st...

The evolution of the globin family genes: Concordance of stochastic and augmented maximum parsimony genetic distances for α hemoglobin, β hemoglobin and myoglobin phylogenies

We compare the amino acid sequences of 70 globing, representing the following families: (a) α hemoglobin chains; (b) β hemoglobin chains; (c) myoglobins; (d) two lamprey, a mollusc, and two plant globins. The comparisons show a convergence of maximal and minimal estimates of genetic differences as calculated respectively by the stochastic and maximum parsimony procedures, thus demonstrating for the first time the logical consistency and complementarity of the two procedures. Evolutionary rates are non-constant, varying over a range of 1 to 75 nucleotide replacements per 100 codons per 10 8 years. These rate differentials are resolved into two components (a) due to change in the number of codon sites free to fix mutations during the period of divergence of the species involved; (b) due to change in fixation intensity at each site. These two components also show non-uniformity along different lineages. Positive Darwinian natural selection can bring about an increase in either component, and negative or stabilizing selection in protein evolution can lead to decreases. Accelerated rates of globin evolution were found in lineages of cold-blooded vertebrates, some marsupials, and early placental mammals, while slower rates were found in warm-blooded vertebrates, especially higher primates. One manifestation of negative selection in the globins is that minimal 3-base type amino acid replacements occur less frequently than would be expected if base replacements had occurred and were accepted at random. The selection against these replacements is not due to atypical behavior with respect to the change in electrical charge involved in the replacements. Interestingly, the globins from the lamprey, sea hare and the legumes are as distant from one another as are α-hemoglobin and β-hemoglobin from myoglobin.