Organization of non-vertebrate globin genes.

The organization of non-vertebrate globin genes exhibits substantially more variability than the three-exon, two-intron structure of the vertebrate globin genes. (1) The structures of genes of the single-domain globin chains of the annelid Lumbricus and the mollusc Anadara, and the globin gene coding for the two-domain chains of the clam Barbatia, are similar to the vertebrate plan. (2) Genes for single-domain chains exist in bacteria and protozoa. Although the globin gene is highly expressed in the bacterium Vitreoscilla, the putative globin gene hmp in E. coli, which codes for a chimeric protein whose N-terminal moiety of 139 residues contains 67 residues identical to the Vitreoscilla globin, may be either unexpressed or expressed at very low levels, despite the presence of normal regulatory sequences. The DNA sequence of the globin gene of the protozoan Paramecium, determined recently by Yamauchi and collaborators, appears to consist of two exons separated by a short intron. (3) Among the lower eukaryotes, the yeasts Saccharomyces and Candida have chimeric proteins consisting of N-terminal globin and C-terminal flavoprotein moieties of about the same size. The structure of the gene for the chimeric protein of Saccharomyces exhibits no introns. According to Riggs, the presence of chimeric proteins in E. coli and other prokaryotes, such as Alcaligenes and Rhizobium, as well as in yeasts, suggests a previously unrecognized evolutionary pathway for hemoglobin, namely that of a multipurpose heme-binding domain attached to a variety of unrelated proteins with diverse functions. (4) The published globin gene sequences of the insect larva Chironomus have an intron-less structure and are present as clusters of multiple copies; the expression of the globin genes is tissue and developmental stage-specific. Furthermore, the expression of many of these genes has not yet been demonstrated despite the presence of apparently normal regulatory sequences in the two flanking regions. Unexpectedly, Bergtrom and collaborators have recently shown that at least three Ctt globin II beta genes contain putative introns. (5) Pohajdak and collaborators have found a seven-exon and six-intron structure for the globin gene of the nematode Pseudoterranova which codes for a two-domain globin chain. Although the second and fourth introns of the N-terminal domain correspond to the two introns found in vertebrate globin genes, the position of the third intron is close to that of the central intron in plant hemoglobins.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chicken globin gene number.

Using complementary DNA prepared from adult chicken globin messenger RNA, we show the existence of 2-3 non-cross-hybridising globin sequences in the chicken genome, each of which is present in only one copy per haploid genome. This was done by solution hybridization to total DNA under conditions of cDNA excess. The data agreses with results of RNA-driven hybridisation of globin complementary DNA, and with protein data.     

Hydrophobic interaction between globin helices.

The interhelical interfaces have been examined in seven high-resolution globin chains. The profiles of hydrophobic contact, as measured by the residue solvent-accessible area loss upon folding, have been calculated. The seven globins studied differ in their overall loss of solvent-accessible area upon packing of their helices, the order being 1MBD greater than 1LH1 greater than 1ECD greater than 2MHBB greater than 2HHBB greater than 2HHBA greater than 2MHBA, which gives a measure of the difference in stability due to the hydrophobic interaction. The five helix-pair packings (AH, BE, BG, FH and GH) examined in detail have qualitative similarities. There are, however, substantial quantitative differences both at the equivalent residue level and at the level of overall helix-helix contact, which has significance in some models of folding. The AH pair has the most uniform area loss over the seven globins and the largest variation in accessible area loss on packing among the five helix pairs is the GH pair. The set of residues required to produce the globin fold has been deduced from the residue area losses.     

Organization of sequences of avian globin mRNA.

Formamide polyacrylamide gel electrophoresis shows that chicken globin mRNA contains about 6.50 nucleotides, and since only 435 of these code for globin, a further 215 are not translated, and their function and position are not known. This work has produced the following conclusions. 1. 45-50 of these untranslated nucleotides are present as poly (A) at the 3' terminus. 2. The 3' untranslated region of chicken globin mRNA is at least 90 nucleotides in length. This minimal estimate is based on data derived from hybridization of defined lenghts of chicken globin cDNA to rabbit globin mRNA. The percentage of avian globin cDNA sequences which hybridize to rabbit globin mRNA is directly proportional to the length of the cDNA in each case. This relationship holds for lengths of cDNA from 115 up to 620 nucleotides. The low percentage homology for short cDNA molecules is not due to their being short per se. In homologous mRNA excess hybridizations (chicken cDNA/chicken mRNA), all cDNA preparations were completely protected from S1 nuclease digestion. 3. It is probable that there is greater evolutionary divergence in the 3' untranslated region of chicken and rabbit globin mRNA when compared with the coding regions of these molecules; The combined data is sued to formulate a regional map of chicken globin mRNA,     

Design and synthesis of a globin fold.

We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.     

Increase in globin chains and globin mRNA in erythroleukemia cells in response to hemin.

Addition of 50 μm hemin to mouse erythroleukemia cells cultured in 0.5% dimethyl-sulfoxide (DMSO) resulted in >10-fold stimulation of globin chain synthesis as a percentage of acid precipitable protein. In cultures fully induced with 1.5% DMSO, addition of 15 mm 3-amino-1,2,4-triazole (AT), an inhibitor of heme synthesis, reduced globin chain synthesis to uninduced levels and reduced globin mRNA levels to less than 20% of induced values. The inhibition of AT was prevented by simultaneous addition of 25 μm hemin to the cultures. Using RNA-DNA hybridization analysis, the amount of globin mRNA sequences as a fraction of total cytoplasmic RNA was also increased by addition of 50 μm hemin to cultures with 0.5% DMSO. The results suggest that exogenous hemin can promote globin chain synthesis, that endogenously synthesized heme can be required for globin chain synthesis, and that hemin directly or indirectly also alters the appearance or degradation of globin mRNA sequences in the cytoplasm.     

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.     

Unexpected intron location in non-vertebrate globin genes.

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.     

Age-dependent silencing of globin transgenes in the mouse.

Variegation of transgene expression, a heterocellular or mosaic pattern of expression seen in all mice in a given transgenic line, is a frequently observed but unexplained phenomenon. We have encountered variegation with globin transgenes; when lacZ expression is driven by globin control elements a proportion of erythrocytes express beta-galactosidase (beta-gal), while the remaining erythrocytes express none. The percentage of expressing cells is constant within each line (at any particular developmental stage), but varies between lines. Such variation may account for much of the line-to-line variability which has been reported in the expression of a transgene construct. We have now extended these observations by studying expression of several globin/lacZ transgenes with increasing age. Expression of beta-gal is variegated in all lines in adult mice, including those made with a beta-globin promoter and locus control region driving lacZ. The extent of variegation differs widely between lines, but in all lines there is a marked decline in the number of erythrocytes expressing beta-gal with increasing age. Progression of silencing continues long past the point at which globin switching is complete, suggesting that it is not related to this process. We observe that age-dependent silencing is most severe in high copy number animals. Increasing variegation of transgene expression with ageing of mice is likely to complicate interpretation of the developmental regulation of transgenes. We speculate that it reflects a general mechanism of epigenetic regulation.