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[beta]-Thalassemia pathology is due not only to loss of [beta]-globin (HBB), but also to erythrotoxic accumulation and aggregation of the [beta]-globin-binding partner, [alpha]-globin (HBA1/2). Here we describe a Cas9/AAV6-mediated genome editing strategy that can replace the entire HBA1 gene with a full-length HBB transgene in [beta]-thalassemia-derived hematopoietic stem and progenitor cells (HSPCs), which is sufficient to normalize [beta]-globin:[alpha]-globin messenger RNA and protein ratios and restore functional adult hemoglobin tetramers in patient-derived red blood cells. Edited HSPCs were capable of long-term and bilineage hematopoietic reconstitution in mice, establishing proof of concept for replacement of HBA1 with HBB as a novel therapeutic strategy for curing [beta]-thalassemia. A new genome editing strategy can normalize the [beta]-globin:[alpha]-globin balance in human hematopoietic stem cells from patients with [beta]-thalassemia and restore functional adult hemoglobin tetramers in patient-derived red blood cells., Author(s): M. Kyle Cromer [sup.1] , Joab Camarena [sup.1] , Renata M. Martin [sup.1] , Benjamin J. Lesch [sup.1] , Christopher A. Vakulskas [sup.2] , Nicole M. Bode [sup.2] , [...]

Electrophoretic surveys of hemoglobin (Hb) polymorphism in house mice from South Asia and the Middle East have revealed that two alternative [beta]-globin haplotypes, [Hbb.sup.d] and [Hbb.sup.p], are often present at intermediate frequencies in geographically disparate populations. Both haplotypes harbor two functionally distinct [beta]-globin paralogs, HBB-T1 (which encodes the [beta]-chain subunits of the major Hb isoform) and HBB-T2 (which encodes the [beta]-chains of the minor Hb isoform). The [Hbb.sup.d] and [Hbb.sup.p] haplotypes share identical HBB-T1 alleles, but products of the alternative HBB-T2 alleles ([d.sub.minor] and [p.sub.minor] ) are distinguished by two amino acid substitutions. To investigate the possible adaptive significance of the [Hbb.sup.d]/[Hbb.sup.p] polymorphism we conducted a population genetic analysis of the duplicated [beta]-globin genes of Indian house mice (Mus castaneus) in conjunction with experimental studies of Hb function in inbred strains of mice that carry the alternative [Hbb.sup.d] and [Hbb.sup.p] haplotypes. The main objectives of this study were (i) to characterize patterns of nucleotide polymorphism and linkage disequilibrium in the duplicated [beta]-globin genes of M. castaneus, (ii) to test the hypothesis that the [Hbb.sup.d] and [Hbb.sup.p] haplotypes are maintained as a balanced polymorphism, and (iii) to assess whether allelic differences in the alternative minor Hb isoforms ( [d.sub.minor] and [p.sub.minor] ) are associated with different [O.sub.2]- binding properties. A multilocus analysis of polymorphism and divergence revealed that levels of diversity at the HBB-T2 gene exceeded neutral expectations, and reconstructed haplotype networks for both [beta]-globin paralogs revealed extensive allele sharing with several other closely related species of Mus. However, despite this suggestive evidence for balancing selection, [O.sub.2]-equilibrium curves revealed no discernible functional differences between red cell lysates containing the [d.sub.minor] and [p.sub.minor] Hb isoforms. If the [d.sub.minor] and [p.sub.minor] alleles are maintained as a balanced polymorphism, our results indicate that the associated fitness variance is not directly related to respiratory functions of Hb.

In this work, we report that flavohemoglobin contributes to the azole susceptibility of Staphylococcus aureus. We first observed that deletion of the flavohemoglobin gene leads to an increase in the viability of imidazole-treated S. aureus cells and that reversion to the wild-type phenotype occurs upon expression of flavohemoglobin from a multicopy plasmid. Further spectroscopic analyses showed that miconazole, the most efficient azole antibiotic against S. aureus, ligates to heme of both oxidized and reduced flavohemoglobin. The binding of miconazole to oxidized flavohemoglobin, with an association constant of 1.7 x [10.sup.6] [M.sup.-1], typical of a tight, specific binding equilibrium, results in augmentation of the superoxide production by the enzyme. These results are corroborated by in vivo studies showing that imidazole-treated S. aureus cells expressing flavohemoglobin contain a larger amount of reactive oxygen species. Moreover, it was observed that the survival of miconazole-treated S. aureus internalized by murine macrophages is higher for cells lacking flavohemoglobin. Altogether, the present data revealed that in S. aureus, flavohemoglobin enhances the antimicrobial activity of imidazoles via an increase of intracellular oxidative stress. doi: 10.1128/JB.01378-09

Phylogenetic reconstructions of the [beta]-globin gene family in vertebrates have revealed that developmentally regulated systems of hemoglobin synthesis have been reinvented multiple times in independent lineages. For example, the functional differentiation of embryonic and adult [beta]-like globin genes occurred independently in birds and mammals. In both taxa, the embryonic [beta]-globin gene is exclusively expressed in primitive erythroid cells derived from the yolk sac. However, the '[epsilon]-globin'' gene in birds is not orthologous to the [epsilon]-globin gene in mammals, because they are independently derived from lineage-specific duplications of a proto [beta]-globin gene. Here, we report evidence that the early and late expressed [beta]-like globin genes in monotremes and therian mammals (marsupials and placental mammals) are the products of independent duplications of a proto [beta]-globin gene in each of these two lineages. Results of our analysis of genomic sequence data from a large number of vertebrate taxa, including sequence from the recently completed platypus genome, reveal that the e- and [beta]-globin genes of therian mammals arose via duplication of a proto [beta]-globin gene after the therian/monotreme split. Our analysis of genomic sequence from the platypus also revealed the presence of a duplicate pair of [beta]-like globin genes that originated via duplication of a proto [beta]-globin gene in the monotreme lineage. This discovery provides evidence that, in different lineages of mammals, descendent copies of the same proto [beta]-globin gene may have been independently neofunctionalized to perform physiological tasks associated with oxygen uptake and storage during embryonic development. [beta]-globin gene family | platypus | hemoglobin | gene family evolution | gene duplication

The relationship between nuclear localization and transcriptional activity of the endogenous murine beta-globin locus during erythroid differentiation was examined. The results suggested that the locus control region (LCR)-dependent association of the beta-globin locus with transcriptionally engaged polymerase II foci provided the driving force for relocalization of the locus toward the nuclear interior during erythroid maturation.

Experiments and studies of the locus control region (LCR) implied that a direct functional interaction between the LCR and globin gene was necessary for high-level transcription. A report is presented which shows that the LCR directs the nuclear relocation of the globin locus during erythroid differentiation and directs its colocalization to specific nuclear subcomponents, providing further insight into the mechanisms of long-range transcriptional regulation and LCR function.

Phylogenetic inferences drawn from comparative data on mammalian [beta]-globin gene clusters indicate that the ancestral primate cluster contained a locus control region (LCR) and five paralogously related [beta]-type globin loci (5'-LCR-[epsilon]-[gamma]-[psi][eta]-[delta]-[beta]-3'), with [epsilon] and [gamma]a] expressed solely during embryonic life. A [gamma] locus tandem duplication (5'-[[gamma].sup.1]-[[gamma].sup.2]-3') triggered [gamma]'s evolution toward fetal expression but by a different trajectory in platyrrhines (New World monkeys) than in catarrhines (Old World monkeys and apes, including humans). In platyrrhine (e.g., Cebus) fetuses, [[gamma].sup.1] at the ancestral distance from [epsilon] is down-regulated, whereas [[gamma].sup.2] at increased distance is up-regulated. Catarrhine [[gamma].sup.1] and [[gamma].sup.2] acquired longer distances from [epsilon] (14 and 19 kb, respectively), and both are upregulated throughout fetal life with [[gamma].sup.1]'s expression predominating over [[gamma].sup.2]'s. On enlarging the platyrrhine expression data, we find Aotus [gamma] is embryonic, Alouatta [gamma] is inactive at term, and in Callithrix, [[gamma].sup.1] is down-regulated fetally, whereas [[gamma].sup.2] is up-regulated. Of eight mammalian taxa now represented per taxon by embryonic, fetal, and postnatal [beta]-type globin gene expression data, four taxa are primates, and data for three of these primates are from this laboratory. Our results support a model in which a short distance (10 kb) impedes embryonic activation of the downstream gene. embryonic activation | gene duplication | gene expression | hemoglobin

The organization of genes within the nucleus may influence transcription. We have analyzed the nuclear positioning of the coordinately regulated [alpha]- and [beta]-globin genes and show that the gene-dense chromatin surrounding the human [alpha]-globin genes is frequently decondensed, independent of transcription. Against this background, we show the frequent juxtaposition of active [alpha]- and [beta]-globin genes and of homologous [alpha]-globin loci that occurs at nuclear speckles and correlates with transcription. However, we did not see increased colocalization of signals, which would be expected with direct physical interaction. The same degree of proximity does not occur between human [beta]-globin genes or between murine globin genes, which are more constrained to their chromosome territories. Our findings suggest that the distribution of globin genes within erythroblast nuclei is the result of a self-organizing process, involving transcriptional status, diffusional ability of chromatin, and physical interactions with nuclear proteins, rather than a directed form of higher-order control.

Of the three groups of haemoglobins identified in micro-organisms (single-domain globins, flavohaemoglobins and truncated globins), the last group is the least well understood. The function of the truncated haemoglobin (Ctb) encoded by Cj0465c in the microaerophilic food-borne bacterial pathogen Campylobacter jejuni was investigated by constructing a ctb mutant and characterizing its phenotype. The effects of the ctb mutation on the kinetics of terminal oxidase function in C. jejuni were investigated using oxyleghaemoglobin and oxymyoglobin as sensitive reporters of [O.sub.2] consumption. The [V.sub.max] of ctb mutant cells for [O.sub.2], calculated using either globin, was greater than that of wild-type cells at extracellular [O.sub.2] concentrations up to ~ 1 [micro]M, suggesting a role for Ctb in moderating [O.sub.2] supply for reduction by high-affinity terminal oxidases. However, cells mutated in ctb were disadvantaged when grown under conditions of high aeration, as revealed by measurements of growth yields and rates in batch culture. Furthermore, the rate at which ctb mutant cells consumed [O.sub.2] in an [O.sub.2] electrode (10-200 [micro]M [O.sub.2]) was approximately half the rate displayed by wild-type cells, reflecting a role for Ctb in respiration at physiologically relevant external [O.sub.2] concentrations. However, a lack of sensitivity of the mutant to paraquat or [H.sub.2][O.sub.2] indicated that increased oxidative stress under such conditions was not the cause of these phenotypes. [O.sub.2] affinities of cells (Kin values of approximately 40 nM and 1 [micro]M) were unaffected by mutation of either Ctb or the full-length C. jejuni globin, Cgb. Although the gene encoding Ctb was found to be upregulated by S-nitrosoglutathione (GSNO) and the NO-donating compound S-nitroso-N-acetylpenicillamine (SNAP), a ctb mutant did not display sensitivity to a number of nitrosative stress-generating compounds. The authors conclude that Ctb is involved in moderating [O.sub.2] flux within C. jejuni.

A study to verify if previous results on the haplotype frequencies of normal individuals are confirmed for beta(super 0)39-thalassemia carriers and to analyze the phylogenetic position of Sardinians among the Mediterranean population using the haplotype frequencies associated with mutations in the beta-globin genes is conducted. It concluded that the haplotype frequencies of the globin cluster in heterozygotes carrying the beta(super 0) mutation have underlined the genetic distinctiveness.

Although most globins, including the N-terminal domains within chimeric proteins such as flavohemoglobins and globin-coupled sensors, exhibit a 3/3 helical sandwich structure, many bacterial, plant, and ciliate globins have a 2/2 helical sandwich structure. We carried out a comprehensive survey of globins in the genomes from the three kingdoms of life. Bayesian phylogenetic trees based on manually aligned sequences indicate the possibility of past horizontal globin gene transfers from bacteria to eukaryotes. BLASTP searches revealed the presence of 3/3 single-domain globins related to the globin domains of the bacterial and fungal flavohemoglobins in many bacteria, a red alga, and a diatom. Iterated PSI-BLAST searches based on groups of globin sequences found that only the single-domain globins and flavohemoglobins recognize the eukaryote 3/3 globins, including vertebrate neuroglobins, [alpha]- and [beta]-globins, and cytoglobins. The 2/2 globins recognize the flavohemoglobins, as do the globin coupled sensors and the closely related single-domain protoglobins. However, the 2/2 globins and the globin-coupled sensors do not recognize each other. Thus, all globins appear to be distributed among three lineages: (i) the 3/3 plant and metazoan globins, single-domain globins, and flavohemoglobins; (ii) the bacterial 3/3 globin-coupled sensors and protoglobins; and (iii) the bacterial, plant, and ciliate 2/2 globins. The three lineages may have evolved from an ancestral 3/3 or 2/2 globin. Furthermore, it appears likely that the predominant functions of globins are enzymatic and that oxygen transport is a specialized development that accompanied the evolution of metazoans. evolution | sequences

Three-dimensional organization of a gene locus is important for its regulation, as recently demonstrated for the beta-globin locus. It is reported that erythroid Kruppel-like transcription factor (EKLF), an erythroid transcription factor required for adult beta-globin gene transcription is also required for active chromatin Hub (ACH).

Byline: L. V. Basova (1), E. I. Tiktopulo (1), I. A. Kashparov (1), V. E. Bychkova (1) Keywords: apomyoglobin; phospholipid vesicles; protein denaturation; intermediate state Abstract: The conformational state of sperm whale apomyoglobin (apoMb) was studied at neutral pH in the presence of negatively charged vesicles using near and far UV circular dichroism, tryptophan fluorescence, differential scanning microcalorimetry, and fast performance liquid chromatography. Under these conditions, the apoMb structure undergoes transition from its native to an intermediate state. In this state the protein loses its rigid native structure but retains its secondary structure. However, the environment of tryptophan residues remains rather hydrophobic. This intermediate state of apoMb shows properties similar to those of its molten globule state in solution. It is shown that apoMb can bind to negatively charged phospholipid vesicles even at neutral pH. A possible functional role of this intermediate state is discussed. Author Affiliation: (1) Institute of Protein Research, Russian Academy of Sciences, Pushchino, Moscow Region, 142290, Russia Article History: Registration Date: 18/10/2004

Byline: S. E. Dmitriev (1), I. M. Terenin (1), M. P. Rubtsova (1), I. N. Shatsky (1) Keywords: translation initiation; [beta]-globin mRNA; mRNA secondary structure; 5'-untranslated region; initiation factor eIF2; reconstruction of the 48S initiation complex; mammals Abstract: Nucleotide sequence changes increasing the number of paired bases without producing stable secondary structure in the 5'-untranslated region (5'-UTR) of the [beta]-globin mRNA had a slight effect on its translation in rabbit reticulocyte lysate at low mRNA concentration and dramatically decreased translation efficiency at a high concentration. The removal of paired regions restored translation. Addition of purified eIF2 to the lysate resulted in equal translation efficiencies of templates differing in structure of 5'-UTR. A similar effect was observed for p50, a major mRNP protein. Other mRNA-binding initiation factors, eIF4F and eIF4B, had no effect on the dependence of translation efficiency on mRNA concentration. Analysis of the assembly of the 48S initiation complex from its purified components showed that less eIF2 is required for translation initiation on the [beta]-globin mRNA than on its derivative containing minor secondary structure elements in 5'-UTR. According to a model proposed, eIF2 not only delivers Met-tRNA, but it also stabilizes the interaction of the 40S ribosome subunit with 5'-UTR, which is of particular importance for translation initiation on templates with structured 5'-UTR. Author Affiliation: (1) Belozerskii Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia Article History: Registration Date: 12/10/2004

We have previously reported the structure of a chromatin remodeling complex (PYR complex) with Ikaros as its DNA binding subunit that is specifically present in adult murine and human hematopoietic cells. We now show that homozygous Ikaros 'knockout' (null) mice lack the PYR complex, demonstrating the requirement for Ikaros in the formation of the complex on DNA. Heterozygous Ikaros null mice have about half as much PYR complex, indicating a dosage effect for both Ikaros and PYR complex. We also show that Ikaros null mice have multiple hematopoietic cell defects including anemia and megakaryocytic abnormalities, in addition to previously reported lymphoid and stem cell defects. The null mice also have a delay in murine embryonic to adult [beta]-globin switching and a delay in human [gamma] to [beta] switching, consistent with a previously suggested role for PYR complex in this process. Lastly, cDNA array analyses indicate that several hematopoietic cell-specific genes in all blood lineages are either up- or down-regulated in 14-day embryos from Ikaros null as compared with wild-type mice. These results indicate that Ikaros and PYR complex function together in vivo at many adult hematopoietic cell-specific genes and at intergenic sites, affecting their expression and leading to pleiotropic hematopoietic defects.

Research on the locus control region (LCR) of the embryonic epsilon-globin gene is discussed. The LCR has little influence on chromatin sensitivity, but chromatin structure can affect nonglobin transcription within LCR.

The functional consequences of the the organization of human beta-type-globin genes are investigated with regard to temporal regulation of the individual genes. The position of the epsilon-globin gene adjacent to the locus control region (LCR) is mandatory for expression during yolk-sac stage fo erythropoiesis. Simple inversion of the human genes or the LCR thus alters the transcription of beta-type globin genes.

SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures. Little is known, however, about how these complexes target specific genes for activation. We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine [Beta]-globin loci. PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent. Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170. PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult [Beta]-globin genes. A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice. Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of [Beta]-globin transcriptional activators.

A study was conducted to analyze the molecular basis of feline adult beta-globin polymorphism reflected in restriction fragment length patterns. Southern blot analysis was carried out on the beta-globin gene region from 25 cats with 13 different high-performance liquid chromatography patterns. Results indicated an autosomal codominant mode of inheritance of the beta-globin chains with seven haplotypes.

The function of the erythroid Kruppel-like factor (EKLF) was investigated in relation to stage-specific expression of the human beta-globin gene. EKLF deficiency in transgenic mice resulted to death caused by anemia during the developmental stage. Also, the function of the chromatin remodeling complexes such as the SWI/SNF contributed to the transcription of a tissue-specific regulated gene. Therefore, chromatin remodeling and transcriptional activity depended upon the coactivation of EKLF and the SWI/SNF complex, E-RC1.

The human [Beta]-globin locus is activated transcriptionally by a complex series of events that culminate in appropriate temporal and tissue-specific control over five separate genes during embryonic and early postnatal development. One cis-regulatory element in the locus, originally identified as an enhancer 3[prime] to the A[Gamma]-globin gene, more recently has been suggested to harbor alternative or additional properties, including stage-specific silencer, insulator, nuclear matrix, or chromosome scaffold attachment activities. We have re-evaluated the activity during erythropoiesis that is conferred by this element by deleting it from a yeast artificial chromosome (YAC) containing the entire human [Beta]-globin locus and then assaying for the expression of each gene at each developmental stage after incorporation of the mutant YAC into the mouse germline. The data show that loss of the A[Gamma]-globin 3[prime] element confers no phenotype in six independent lines of intact YAC mutant transgenic mice, thus demonstrating (minimally) that any activities attributable to this element are fully compensated by other DNA sequences within the [Beta]-globin locus.

The human [Beta]-globin locus control region (LCR), which consists of four erythroid-specific DNase I hypersensitive sites (HS1-HS4), functions over a long distance to control the transcription, chromatin structure, and replication of the [Beta]-globin genes. We have used stable transfection assays to show that activation of the mitogen-activated protein (MAP) kinase pathway by low concentrations of the phorbol ester phorbol 12-tetradecanoate 13-acetate (TPA) induces enhancer activity of the LCR subregion HS2, but not HS3. Although HS2 enhancer activity is diminished with increasing distance from the promoter, the relative level of induction by TPA is independent of HS2-promoter distance. Mutation of cis-elements within HS2 reveals that the tandem-binding sites for the hematopoietic-specific transcription factor NF-E2 are required for induction by TPA, and induction is conferred by expressing NF-E2 in an NF-E2-null cell line. These results show that MAP kinases target factors functioning through the NF-E2 sites to enhance long-range transactivation by the LCR.

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human [Beta]-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the N[H.sub.2]-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous [Beta]- or [Alpha]-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TA[F.sub.II]130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Analysis of bleomycin (BLM) activity in the beta-globin gene cluster indicates that intensity of BLM damage sites differs in purified DNA and intact cells. The chromatin structure probably affects BLM activity at the beta-globin promoter sequence. BLM activity is low at the site of protein-DNA interactions in intact cells. It is affected at nuclear factor binding sites in the beta-globin locus control region hypersensitive site 2 of intact cells. The activity of BLM is lowest in K562 cells with a ten-fold reduction in damage at the tandem nuclear factor-erythroid 2/AP1 DNA sequence element.

We have isolated a new hemoglobin gene from soybean. It is expressed in cotyledons, stems of seedlings, roots, young leaves, and in some cells in the nodules that are associated with the nitrogen-fixing Bradyrhizobium symbiont. This contrasts with the expression of the leghemoglobins, which are active only in the infected cells of the nodules. The deduced protein sequence of the new gene shows only 58% similarity to one of the soybean leghemoglobins, but 85-87% similarity to hemoglobins from the nonlegumes Parasponia, Casuarina, and barley. The pattern of expression and the gene sequence indicate that this new gene is a nonsymbiotic legume hemoglobin. The finding of this gene in legumes and similar genes in other species strengthens our previous suggestion that genomes of all plants contain hemoglobin genes. The specialized leghemoglobin gene family may have arisen from a preexisting nonsymbiotic hemoglobin by gene duplication.

Agarose-encapsulated, metabolically active, permeabilized nuclei for human hematopoietic cell lines were analyzed for Z-DNA formation in the beta-globin gene cluster. Biotinylated monoclonal antibodies against Z-DNA were diffused into the nuclei and cross-linked to DNA. After digestion, the fragments that had formed Z-DNA were isolated. Five of the seventeen regions with Z-DNA motifs in the 73-kb region were found in the Z formation.

Positioned nucleosomes contribute to both the structure and the function of the chromatin fiber and can play a decisive role in controlling gene expression. We have mapped, at high resolution, the translational positions adopted by limiting amounts of core histone octamers reconstituted onto 4.4 kb of DNA comprising the entire chicken adult [beta]-globin gene, its enhancer, and flanking sequences. The octamer displays extensive variation in its affinity for different positioning sites, the range exhibited being about 2 orders of magnitude greater than that of the initial binding of the octamer. Strong positioning sites are located 5' and 3' of the globin gene and in the second intron but are absent from the coding regions. These sites exhibit a periodicity ([nearly equal to]200 bp) similar to the average spacing of nucleosomes on the inactive [beta]-globin gene in vivo, which could indicate their involvement in packaging the gene into higher-order chromatin structure. Overlapping, alternative octamer positioning sites commonly exhibit spacings of 20 and 40 bp, but not of 10 bp. These short-range periodicities could reflect features of the core particle structure contributing to the pronounced sequence-dependent manner in which the core histone octamer interacts with DNA.

Erythyroid Kruppel-like factor- (EKLF) deficient mice were created via gene targeting to determine EKLF's function in vivo. The embryos, which succumb to anemia during erythropoiesis of the fetal liver, were found to possess the molecular and hematological features of beta-globin deficiency, which is found in beta-thalassemia. Yolk sac erythropoiesis, erythroid commitment or the expression of other potential target genes do not require EKLF even if it is expressed at all stages. The stage-specific and beta-globin-gene specific requirements hint at the ability of EKLF to make the fetal-to-adult switch easier in humans.

Nuclease S1 facilitated the excision of the entire chicken alpha-globin gene domain proximal to matrix attachment regions. Isolated chicken erythroid cell nuclei when treated with nuclease S1 enabled separation of a 20 kilobase genomic DNA fragment that contained the entire alpha-globin gene cluster.

Locus control region blockade by the domain insulating 5' constitutive hypersensitive site of chicken beta globin domain insulator was studied. Chromatin structural alteration was found to be linked to this mechanism, which is directional, while acting at transcriptional levels. The insulating action did not display any unique activator or blockading impact when not assayed in human erythroid cell line K562. The insulator also behaves as a protective agent for white minigenes in transgenic Drosophila.

The mutations in the beta-globin poly(A) signals in patients suffering from beta-thalassemia were identified and characterized. The mutations were identified as either point mutations or deletions. Point mutations in the poly(A) tail resulted in expression of 12-34% of normal beta-globin mRNA. The deletions did not produce any normal transcripts, although the patient homozygous for this mutation had a beta/alpha chain ratio of 0.10. These results suggest that the poly(A) signal is not an absolute requirement for mRNA cleavage and the extended RNA is translated in vivo. Further analysis indicates that the primary beta-globin transcriptional unit is > 5 kb long.

The effect of a nonsense mutation at codon 39 (beta-39) ofthe beta-globin chain on mRNA production was investigated in transfected AF8 cells. Results show that, in addition to reducing protein translation of beta- globin, the beta-39 mutation also decreases beta-39 mRNA production. This ability of a beta-39 nonsense mutation to influence both cytoplasmic translation and nuclear transcription suggests that a communication pathway exists between the nucleus and the cytoplasm that allows nuclear detection of cytoplasmic events.

The locus control region (LCR) provides an erythroid-specific active chromatin territory and grants high-level, proper expression on linked globin genes. The hypersensitive site (HS) components of the globin LCR exhibit extreme reaction to DNase I. This susceptibility intensifies DNase I cleavage of specific base pairs. Eryhtroid-specific and protein-dependent interaction is necessary for HS formation.

Hemoglobins, found in members of almost all invertebrate phyla, display an extraordinary diversity of form and function. Although some are intracellular with chains and assemblies similar in size to those of vertebrates, others are giant extracellular proteins with masses as large as 8,000 kilodaltons. Two very different strategies have evolved for the stabilization of these large molecules. The first is the formation of both intra- and inter- chain disulfide bonds that effectively immobilize segments of the protein, and the second is the evolution by gene duplication of multi-domain chains with from two to eighteen myoglobin-like, heme-containing domains in a single polypeptide that may be as large as 260 kilodaltons. The genes for vertebrate globins have a characteristic two-intron, three-exon structure. The gene encoding chain c of the hemoglobin of the earthworm Lumbricus terrestris has precisely the same organization and splice junction positions. This shows that these positions have been conserved for at least 600 million years, the estimated time of divergence of annelids and the ancestor to chordates. The gene encoding a hemoglobin (leghemoglobin) of higher plants shows exactly the same splice junctions as in the globin genes of vertebrates except that the middle exon is split by an additional intron which is believed to have been lost early in animal evolution. The occurrence of hemoglobins in diverse higher plants suggests that they might be present in all plants albeit at very low concentrations and perhaps serving an enzymatic function. Hemoglobin, broadly defined as a heme-containing protein capable of reversible combination with oxygen, also occurs in bacteria and fungi. The discovery of a bacterial hemoglobin that is 26% identical with lupin leghemoglobin indicates a procarvotic origin. The possibility that hemoglobin may have evolved from a cytochrome is suggested by the presence of hemoglobins in the yeast, Candida, and the bacterium, Alcaligenes, that contain both heme and flavin. They must therefore have evolved by the fusion of the genes for two different proteins. However, the possible homology of the heme domains with plant or animal hemoglobins remains to be determined. The lactic dehydrogenase of yeast, cytochrome [b.sub.5], is also a soluble flavoheme protein with a heme domain that is homologous with mammalian cytochrome [b.sub.5]. Globin may have evolved in part from a member of the cytochrome [b.sub.5] family, but if so, the event must have occurred so early that only a borderline perhaps random correspondence of amino acid sequences remains.

A study was conducted to examine the detoxifying enzymatic activity of the first heme-containing globin isolated from Amphitrite ornata dehaloperoxidase (DHP) which exhibit the ability to catalyze the [H.sub.2][O.sub.2]-dependent dehalogenation of halophenols. The study observations provide insight into the dual physiological link between the [O.sub.2] carrier role of the ferrous protein and the peroxidase activity of the ferric enzyme in the bifunctional protein DHP.

Flash photolysis studies of ([alpha]-cp[beta] ) were performed to understand the biphasic CO and O2 kinetics for a circularly permuted human [beta]-globin (cp[beta]) coexpressed with human [alpha]-globin in bacterial cells which form [alpha]-cp[beta] hemoglobin in solution. The equilibrium oxygen binding measurements obtained for [alpha]-cp[beta] supported the generated intermediate, higher affinity, deoxyHb quaternary state by the deoxygenated permutated [beta] subunits.

A multilocus analysis European rabbit was conducted by using a set of 25 polymorphic allozyme loci and a set of 15 population samples from the Iberian Peninsula. The results obtained provide insight into the contrasting levels of spatial differentiation at these two globin genes and the variation of patterns in the [alpha]- and [beta]-chain subunits of rabbit haemoglobin that are shaped by different modes of selection.

The mechanisms by which expression of the Β-like globin genes are developmentally regulated are under intense investigation. The temporal control of human embryonic (ζ) globin expression was analyzed. A 3.7-kilobase [...]

Several extended molecular dynamics simulations are employed to analyze the dynamical regulation of ligand migration by a gate-opening molecular switch in the truncated hemoglobin-N from the Mycobacterium tuberculosis. The described molecular mechanism ensures access of NO to the heme cavity in the oxygenated form of the protein, which then confirms the survival of different microorganisms under stress conditions.

The consequences of heme binding and post-translational heme modification on individual structural elements in the cyanobacterium Synechoccous sp. PCC 7002 (S7002 rHb) are investigated. The [super 1]H/[super 2]H exchange data has indicated that the B, G, and H helices formed a robust core regardless of the presence of the heme or cross-link, which encompasses the early folding nucleus of two-on-two globins.