Adult Alpha-globin Genes Research Articles

In Vivo Efficacy of Bcl11a Erythroid-Enhancer Deletion in Humanized Mouse Models of Anemia

Autologous cell therapy holds great promise for the treatment of beta thalassemia major and hemoglobinopathies like sickle cell anemia. Gene editing of a patient's own stem cells to reactivate the silenced gamma globin gene is one approach under active development. Prior to directly testing these new therapies in patients, we can answer some basic questions about their in vivo efficiency and efficacy in humanized mouse models of anemia. These models have their endogenous adult alpha and beta globin genes replaced with human alpha, gamma, and beta globin genes. These mice synthesize high level of human fetal hemoglobin during fetal life and complete their fetal-to-adult hemoglobin switch after birth. Experimental strategies designed to reactivate the silenced fetal gamma globin genes in adult erythroid cells are easily tested in vivo in these humanized hemoglobin switching mouse models. The silenced human fetal gamma globin genes can be activated by mutating the erythroid-specific enhancer of Bcl11a by gene editing. CRISPR sgRNAs, designed to target the +62 kb DNase I hypersensitive site in the second intron of Bcl11a, were microinjected along with Cas9 mRNA, into fertilized mouse embryos collected from humanized hemoglobin (Hb A) mice. The indel mutations that were generated in the founder animals were characterized and bred to homozygosity. The data demonstrates that the sgRNAs tested were successful in creating multiple unique mutations at the erythroid enhancer target sites. These mutations were transmitted through the germline allowing the effect of individual edited alleles to be analyzed. The majority of the mutations showed marginal increases in the number of F-cells over control animals. Significantly, despite having homozygous mutation of the erythroid-enhancer in all cells, fetal hemoglobin expression remains heterocellular. Importantly, the therapeutic efficacy of reactivating fetal hemoglobin with specific Bcl11a erythroid-enhancer mutations for the treatment of beta thalassemia major and sickle cell anemia was directly measured in vivo in these humanized models of disease. The reactivation of gamma globin in these humanized mouse models provides us with an opportunity to further interrogate the Bcl11a enhancer element, identify additional factors involved in hemoglobin switching and elucidate the mechanism driving pancellular vs heterocellular fetal hemoglobin expression. DisclosuresNo relevant conflicts of interest to declare.

Triiodothyronine-induced changes in the zebrafish transcriptome during the eleutheroembryonic stage: Implications for bisphenol A developmental toxicity

Thyroid disruption during early development is a current matter of concern due to its significant human health implications. We present here a transcriptome analysis of thyroid hormone-regulated genes in zebrafish during the eleutheroembryonic stage (days 2–5 post fertilization) to detect potential markers of thyroid disruption. Exposure to 3,5,3′-triiodo-l-thyroxine (T3, 50nM) induced changes in a minor portion (less than 2%) of the zebrafish transcriptome, with a significant fraction of genes involved in the haematopoietic system, eye formation, and ossification/skeletal system, including the thyroid receptor thra gene. Some of the transcriptomic changes were reflected macroscopically, as an allometric decrease of eye size and an increase on thra hybridization signal in the skeletal tissue. Using this information, changes on transcription of three genes (adult alpha globin gene si:ch211-5k11.6, embryonic globin gene hbae3, and long wavelength cone opsin gene opn1/w1) were analyzed to monitor the effect of the suspected thyroid disrupter bisphenol A (BPA) on the thyroid system during this period of development of zebrafish. BPA acted as a weak T3 agonist when tested alone, but it strongly enhanced the effect of subsaturating concentrations of T3. In thyroxine immunofluorescence quantitative disruption tests (TIQDT), BPA did not prevent the ability of thyroid follicles to synthesize thyroxine, a landmark for direct goitrogens. Our results suggest that BPA potentiates the effect of endogenous T3 in early development and demonstrate the requirement for the use of in vivo, multi-endpoint methods to evaluate thyroid disruption hazards on early developmental processes in vertebrates.

Chicken α‐globin switching depends on autonomous silencing of the embryonic π globin gene by epigenetics mechanisms

Switching in hemoglobin gene expression is an informative paradigm for studying transcriptional regulation. Here we determined the patterns of chicken alpha-globin gene expression during development and erythroid differentiation. Previously published data suggested that the promoter regions of alpha-globin genes contain the complete information for proper developmental regulation. However, our data show a preferential trans-activation of the embryonic alpha-globin gene independent of the developmental or differentiation stage. We also found that DNA methylation and histone deacetylation play key roles in silencing the expression of the embryonic pi gene in definitive erythrocytes. However, drug-mediated reactivation of the embryonic gene during definitive erythropoiesis dramatically impaired the expression of the adult genes, suggesting gene competition or interference for enhancer elements. Our results also support a model in which the lack of open chromatin marks and localized recruitment of chicken MeCP2 contribute to autonomous gene silencing of the embryonic alpha-globin gene in a developmentally specific manner. We propose that epigenetic mechanisms are necessary for in vivo chicken alpha-globin gene switching through differential gene silencing of the embryonic alpha-globin gene in order to allow proper activation of adult alpha-globin genes.

Intermediaries of Branched-Chain Amino Acid Metabolism Can Increase Fetal Hemoglobin In Vitro and In Vivo.

Propionic acid and methylmalonic acid, which are intermediaries (BCAA-I) of branched-chain amino acid (leucine, isoleucine, valine) metabolism, can up-regulate HbF during definitive erythropoiesis [italics]in vitro[/italics] and [italics]in vivo [/italics](Little 1995). We tested ‘proximal' (prior to thiolester formation with Coenzyme A) BCAA-Is, which would be elevated in maple syrup urine disease (MSUD), and ‘distal', which would be elevated in Isovaleric Acidemia (IVA) Propionic Acidemia (PA) or Methylmalonic Acidemia (MMA), for their ability to simultaneously stimulate adult erythroid differentiation and embryonic globin gene expression in an adult erythroid cell line, murine erythroleukemia (MEL) cells after 96 hours in culture. Compounds and maximal concentrations (X) tested were: α-keto isocaproic acid, α−keto β−methylvaleric acid, and α−keto isovaleric acid, all at 50 mM, which are elevated in ‘proximal' MSUD; isobutyric acid (IB), isovaleric acid (IV), methylmalonic acid, all at 50 mM, and propionate (5mM), all of which are elevated in ‘distal' perturbations of BCAA metabolism, plus butyrate (2mM), which has been used pharmacologically to increase HbF (Atweh et al, 1999), and DMSO (adult erythroid differentiating agent in MELs). Concentration ranges were, relative to maximum, X, [X], [0.5X], [0.2X], [0.1X] and [0.01X]. RNA was analyzed by real-time PCR for murine adult alpha (α) and adult beta-major (βmaj) globin gene expression and embryonic α-type zeta (ζ) and β-type beta-H1 (βH1) and epsilon-y (εY). Only four BCAA-I compounds, plus DMSO, induced adult α−globin gene expression in MEL cells (by >100-fold, relative to uninduced). These compounds, but not DMSO, also augmented β−type embryonic globin gene expression ((βH1+εY)/(β−total))*100, as shown: propionate (5mM) 24%, butyrate (1 mM) 8% (2mM) 10%, IBA (25 mM) 5%, IVA (5 mM) 9% and (10mM) 13%. Samples from 10 individuals with ‘proximal' abnormalities in BCAA-I (n=4, median age 5.5 years) due to MSUD and ‘distal' abnormalities in BCAA-I (n=6, median age 6 years) due to IVA, PA, and MMA were examined by HPLC for % HbF. All patients were under good metabolic control at evaluation; mean Hgb (g/dL) was 13.1±0.9 in ‘proximal' (MSUD) patients, and 12.2±1.6 in patients with distal perturbations in BCAA metabolism (p=n.s.). HbF was 0.9±0.3% (one beta-thalassemia excluded) and 0.2±0.4%(p<.01) in proximal and distal BCAA-I defects, respectively. Gamma (γ)-globin gene composition was examined by HPLC-mass spectromety in 3 patients with proximal BCAA-I and 5 with distal BCAA-I. No γ chains were detectable in patients with MSUD, and only Gγ chains, at >5% of total β−type chains, were present in 3 of 5 patients with distal BCAA-I (p<.05). Our data show that, as predicted by MEL cells [italics]in vitro[/italics], a subset of metabolic intermediaries can up-regulate fetal globin gene expression in non-anemic patients with endogenous elevations in BCAA-I due to inborn metabolic errors. Gγ, which predominates during normal fetal life, may be most affected by these compounds.

Erythroid Development in the Absence of Hemoglobin.

The mammalian erythrocyte is a highly specialized blood cell that differentiates via an orderly series of committed progenitors in the bone marrow in a process termed erythropoiesis. Homeostasis of the erythron is carefully maintained by balancing the proliferation and destruction of early and late erythroid progenitors. In mature red blood cells over ninety-five percent of the protein is hemoglobin (Hb). What happens to committed erythroid cells in the absence of hemoglobin? To answer this question we have derived a novel line of embryonic stem (ES) cells from mouse embryos that have all eight adult alpha and beta globin genes knocked out. These “Null” Hb ES cells were injected into wild-type blastocysts to examine their in vivo potential to contribute to the tissues of developing chimeric mice. Examination of the peripheral blood and bone marrow of these chimeras by flow cytometry revealed that the “Null” Hb ES cells were able to produce normal levels of each type of white blood cell analyzed. However, “Null” erythrocytes were absent from the circulation and only early committed progenitors were found in the bone marrow. Very few “Null” erythroid cells matured beyond the proerythoblast to the basophilic erythroblast stage (Ter119low, CD71hi). To study this maturational block in more detail, an erythroid culture system was established by in vitro differentiation of the “Null” Hb ES cells. These pure erythroid progenitor (EP) cultures support and amplify the proerythroblast stage of development. Interestingly, EP cells could be derived from “Null” Hb ES cells demonstrating that Hb is not required for the development of proerythroblasts. “Null” derived EP cells express erythroid lineage markers (EKLF, GATA1, GypA, EpoR, Tal1), but express no adult globins or markers of other hematopoietic lineages (Mpl, GATA3, IL7R, PAX5, CEBPα, CD41b). Upon terminal differentiation most “Null” derived EP cells undergo apoptosis by 48 hours (7AAD−, Annexin V+) and are dead (7AAD+) by 72 hours. These “Null” Hb ES cells provide a novel experimental system to elucidate the role of hemoglobin during erythroid differentiation, maturation, and homeostasis.

The α-Globin Gene Family of an Australian Marsupial, Macropus eugenii: The Long Evolutionary History of the θ-Globin Gene and Its Functional Status in Mammals

Comparative evolutionary analyses of gene families among divergent lineages can provide information on the order and timing of major gene duplication events and evolution of gene function. Here we investigate the evolutionary history of the alpha-globin gene family in mammals by isolating and characterizing alpha-like globin genes from an Australian marsupial, the tammar wallaby, Macropus eugenii. Sequence and phylogenetic analyses indicate that the tammar alpha-globin family consists of at least four genes including a single adult-expressed gene (alpha), two embryonic/neonatally expressed genes (zeta and zeta'), and theta-globin, each orthologous to the respective alpha-, zeta-, and theta-globin genes of eutherian mammals. The results suggest that the theta-globin lineage arose by duplication of an ancestral adult alpha-globin gene and had already evolved an unusual promoter region, atypical of all known alpha-globin gene promoters, prior to the divergence of the marsupial and eutherian lineages. Evolutionary analyses, using a maximum likelihood approach, indicate that theta-globin, has evolved under strong selective constraints in both marsupials and the lineage leading to human theta-globin, suggesting a long-term functional status. Overall, our results indicate that at least a four-gene cluster consisting of three alpha-like and one beta-like globin genes linked in the order 5'-zeta-alpha-theta-omega-3' existed in the common ancestor of marsupials and eutherians. However, results are inconclusive as to whether the two tammar zeta-globin genes arose by duplication prior to the radiation of the marsupial and eutherian lineages, with maintenance of exon sequences by gene conversion, or more recently within marsupials.

Isolation and Sequencing of Two α-Globin Genes αAand αDin Pigeon and Evidence for Embryo-Specific Expression of the αD-Globin Gene1

By screening a pigeon genomic DNA library, we isolated a recombinant phage clone containing the alpha(A)-globin gene. The DNA sequence of the approximately 6kbp-long insert fragment of the phage clone was determined. The sequence suggested the existence of pigeon alpha(D)-globin gene located 3.1 kbp upstream from the alpha(A)-globin gene. The expression of the alpha(D)-globin in late embryo was also shown by the N-terminal amino-acid sequence of the intact globin chain. These results show that two adult alpha-globin genes, alpha(A) and alpha(D), exist in the pigeon genome, and the alpha(D)-globin is expressed at the late embryo stage. The stage-specific expression suggests the existence of regulatory elements and factors interacting to inhibit transcription at the adult stage.

Inactivation of Mouse α-Globin Gene by Homologous Recombination: Mouse Model of Hemoglobin H Disease

We have disrupted the 5' locus of the duplicated adult alpha-globin genes by gene targeting in the mouse embryonic stem cells and created mice with alpha-thalassemia syndromes. The heterozygous knockout mice (.alpha/alpha alpha) are asymptomatic like the silent carriers in humans whereas the homozygous knockout mice (.alpha/.alpha) show hemolytic anemia. Mice with three dysfunctional alpha-globin genes generated by breeding the 5' alpha-globin knockouts (.alpha/alpha alpha) and the deletion type alpha-thalassemia mice (../alpha alpha) produce severe hemoglobin H disease and they die in utero. These results indicate that the 5' alpha-globin gene is the predominant locus in mice, and suggest that it is even more dominant than its human homologue.

Inactivation of mouse alpha-globin gene by homologous recombination: mouse model of hemoglobin H disease

We have disrupted the 5′ locus of the duplicated adult alpha-globin genes by gene targeting in the mouse embryonic stem cells and created mice with alpha-thalassemia syndromes. The heterozygous knockout mice (.alpha/alpha alpha) are asymptomatic like the silent carriers in humans whereas the homozygous knockout mice (.alpha/.alpha) show hemolytic anemia. Mice with three dysfunctional alpha-globin genes generated by breeding the 5′ alpha-globin knockouts (.alpha/alpha alpha) and the deletion type alpha-thalassemia mice (../alpha alpha) produce severe hemoglobin H disease and they die in utero. These results indicate that the 5′ alpha-globin gene is the predominant locus in mice, and suggest that it is even more dominant than its human homologue.

Lethal α–thalassaemia created by gene targeting in mice and its genetic rescue

Mutations at the alpha-globin locus are the most common class of mutations in humans, with deletion of all four adult alpha-globin genes resulting in the perinatal lethal condition haemoglobin Barts hydrops fetalis. Using gene targeting in mice, we have deleted a 16 kilobase region encompassing both adult alpha-globin genes. Animals homozygous for this deletion become hydropic and die late in gestation mimicking humans with hydrops fetalis. Introduction of a human alpha-globin transgene rescued these animals from perinatal death thus demonstrating the utility of this murine model in the development of cellular and gene based approaches for treating this human genetic disease.

Transcriptional activation of human adult alpha-globin genes by hypersensitive site-40 enhancer: function of nuclear factor-binding motifs occupied in erythroid cells.

The developmental stage- and erythroid lineage-specific activation of the human embryonic zeta- and fetal/adult alpha-globin genes is controlled by an upstream regulatory element [hypersensitive site (HS)-40] with locus control region properties, a process mediated by multiple nuclear factor-DNA complexes. In vitro DNase I protection experiments of the two G+C-rich, adult alpha-globin promoters have revealed a number of binding sites for nuclear factors that are common to HeLa and K-562 extracts. However, genomic footprinting analysis has demonstrated that only a subset of these sites, clustered between -130 and +1, is occupied in an erythroid tissue-specific manner. The function of these in vivo-occupied motifs of the alpha-globin promoters, as well as those previously mapped in the HS-40 region, is assayed by site-directed mutagenesis and transient expression in embryonic/fetal erythroid K-562 cells. These studies, together with our expression data on the human embryonic zeta-globin promoter, provide a comprehensive view of the functional roles of individual nuclear factor-DNA complexes in the final stages of transcriptional activation of the human alpha-like globin promoters by the HS-40 element.

Recombination and Genetic Polymorphism at the Mouse α-Globin Locus

A recent recombination event within the mouse alpha-globin gene region was discovered in the recombinant inbred mouse strain AKXL-7. Polymorphisms (RFLPs) that distinguish the AKR and C57L strains were used to determine the parental origins of segments of the AKXL-7 Chromosome 11. Southern blot analysis revealed that the left and right AKXL-7 flanking regions originated from different parental chromosomes, indicating that a recombination event had occurred within the locus complex during the generation of this RI strain. Further analysis using an intergenic region probe narrowed the region of crossover to approximately 5.2 kb, located between the tandem adult alpha-globin genes. Additional mouse strains were studied concurrently to identify RFLPs that may be informative for defining evolutionary relationships and serve as markers to discriminate among different inbred strains as well as different mouse species.

Homology of a 130-kb region enclosing the ?-globin gene cluster, the ?-locus controlling region, and two non-globin genes in human and mouse

The human alpha-globin gene cluster (30 kb) is embedded in a GC-rich isochore very close to the telomere of Chromosome (Chr) 16p. The alpha-Locus Controlling Region (alpha-LCR) is located upstream of the adult alpha-globin genes and has been shown to be essential for their expression. In this study we have been looking for expressed genes in the region upstream of the alpha-globin cluster to understand the role of the LCR-like element in the expression and replication timing of flanking gene clusters. We show that the upstream alpha-globin region is conserved over a 75-kb range and includes at least two oppositely transcribed non-globin genes, here referred to as Mid1 and Dist1. Complementary DNA sequences of 250 bp and 2.5 kb from Mid1 (coordinate -68) and Dist1 (coordinate -90 to -99), respectively, were isolated from human and mouse. The deduced partial amino acid sequences of these cDNAs are 81% and 95% identical for the Mid1 and Dist1 gene respectively. We have cloned a mouse cosmid "contig" which includes Dist1, Mid1, and the entire murine alpha-globin cluster. The murine homolog of the alpha-LCR was mapped upstream of the mouse globin genes at approximately the same position as in the human locus. Our results indicate that, in mouse and human, the alpha-globin loci and their flanking sequences are homologous over a range of at least 130 kb. The structural homology of this region in both mammals suggests also a functional one and indicates the mouse as a potential model for studying the role of the alpha-LCR controlling element in the regulation of expression and replication timing of the flanking gene clusters.

The adult α-globin locus of old world monkeys: An abrupt breakdown of sequence similarity to human is defined by an Alu family repeat insertion site

The haploid genomes of all known primates have two or more adult alpha-globin genes contained within tandemly arranged duplication units. Although the tandem duplication event generating these alpha-globin loci is believed to occur prior to the divergence of primates, a number of length polymorphisms exist within the loci among different primate species. In order to understand the molecular basis of these length polymorphisms, we have cloned and determined the nucleotide sequence of a major portion of the rhesus monkey adult alpha-globin locus. Sequence comparison to human suggests that the length difference between the adult alpha-globin loci of human and Old World monkey is the result of one or more DNA recombination processes, all of which appeared to be related to the transposition of Alu family repeats. First, the finding of a monomeric Alu family repeat at the junction between nonhomology block I and homology block Y of the alpha 2 gene-containing unit in rhesus macaque suggests that the dimeric Alu family repeat, Alu 3, at the orthologous position in human was generated by insertion of a monomeric Alu family repeat into the 3' end of another preexisting Alu family repeat. Second, two Alu family repeats, Alu 1 and Alu 2, exist in human at the 3' end of each of the two X homology blocks, respectively. However, this pair of paralogous Alu family repeats is absent at the corresponding positions in rhesus macaques. This raises interesting questions regarding the evolutionary origin of Alu 1 and Alu 2. Finally, DNA sequences immediately downstream from the insertion site of Alu 2 are completely different between human and rhesus macaque.(ABSTRACT TRUNCATED AT 250 WORDS)

Human alpha-globin genes demonstrate autonomous developmental regulation in transgenic mice.

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Human α-Globin Genes Demonstrate Autonomous Developmental Regulation in Transgenic Mice

Recent studies have demonstrated that transcriptional activation of the human adult beta-globin transgene in mice by coinsertion of the beta-globin cluster locus control region (beta-LCR) results in loss of its adult restricted pattern of expression. Normal developmental control is reestablished by coinsertion of the fetal gamma-globin transgene in cis to the adult beta-globin gene. To test the generality of this interdependence of two globin genes for their proper developmental control, we generated transgenic mice in which the human adult alpha-globin genes are transcriptionally activated by the beta-LCR either alone or in cis to their corresponding embryonic zeta-globin gene. In both cases, the human globin transgenes were expressed at the appropriate developmental period. In contrast to the beta-globin gene, developmental control of the human adult alpha-globin transgenes appears to be autonomous and maintained even when activated by an adjacent locus control region.

Adult chcken α-globin gene expression in transfected QT6 quail cells: evidence for a negative regulatory element in the αD gene region

The chicken adult alpha-globin genes, alpha A and alpha D, are closely linked in chromosomal DNA and are coordinately expressed in vivo in an approximate 3:1 ratio, respectively. When subcloned DNAs containing one or the other gene are stably transfected into QT6 quail fibroblasts, the alpha A-globin gene is expressed at measurable RNA levels, but the alpha D gene is not. The alpha A gene expression can be considerably increased by the presence of a linked Rous sarcoma virus long terminal repeat enhancer, but that of the alpha D gene remains undetectable. Transfection with subclones containing both genes, either in cis or in trans, leads to considerably reduced alpha A RNA levels and still no observable alpha D gene expression. Transfection with deleted subclones suggests that maximal expression levels in this system require the alpha A-globin gene promoter, as opposed to that of the alpha D gene, but that such expression is greatly reduced by one or more DNA sequences which lie approximately 2,000 base pairs upstream of the alpha A gene, within the body of the alpha D gene.

Gene expression pattern of chicken erythrocyte nuclei in heterokaryons

Expression of erythro-specific chick genes was studied in heterokaryons prepared by fusing chick erythrocytes (CE) with rat myoblasts. In this type of heterokaryon the inactive erythrocyte nucleus takes up nuclear proteins of myoblast origin and undergoes transcriptional reactivation. In order to study the stability of the genetic programming of the reactivated CE nucleus, chick gene expression was examined by analysis of RNA from the heterokaryons. Probes for several erythro- and chick-specific genes were used. The heterokaryons showed strong expression of the chick histone H5 and adult alpha-globin genes, while other genes, e.g. the transcription factor Eryf1 gene, normally expressed during erythroid differentiation, were not transcribed. Although the CE used were of the definitive lineage, the heterokaryons showed activation of the chick embryonic beta-globin gene, i.e. a gene normally expressed only in CE of the primitive lineage. We conclude that the reactivation of the mature CE nucleus in a rat cytoplasm resulted in a more immature erythroid gene expression pattern. The activation of the embryonic beta-globin gene indicated a switch of the lineage-specific gene expression pattern. This switch occurred in the absence of DNA replication. The strong expression of the globin and H5 genes in heterokaryons, in the absence of expression of the regulatory factor Eryf1, suggested the existence of Eryf1-independent regulatory mechanisms for erythroid gene expression in these cells.

The chicken alpha-globin gene domain is transcribed into a 17-kilobase polycistronic RNA.

The 5' start sites and the 3' ends of giant transcripts of the approximately 20-kilobase (kb)-long chicken alpha-globin gene domain were identified by reverse transcription with specific primers and by nuclease S1 mapping using cloned and sequenced restriction fragments of the domain. A transcriptional unit of approximately 17 kb was found that includes all three embryonic and adult genes of the cluster. The largest transcript initiates 8 kb upstream of the gene, within a cluster of A + T-rich sequences placed upstream of a matrix attachment point, at one of several CAA(A)T boxes framing a cluster of four TATA boxes. The 5' ends of a group of 2.5-, 5-, and 12-kb globin transcripts accumulating in avian erythroblastosis virus-transformed cells, which transcribe globin genes abortively, map to the sequence ATATATAATAA 1 kb upstream of the embryonic pi-globin gene. This sequence might correspond to a site of RNA processing or of alternative transcription initiation. Transcription of the domain ends about 2 kb downstream of the last gene of the cluster, downstream of an enhancer and immediately upstream of a CR1 repetitive element in an A + T-rich sequence that includes a matrix attachment site. These data indicate that full-domain transcripts including embryonic as well as adult alpha-globin genes exist, and that the region transcribed is framed by A + T-rich linkers and matrix attachment points.

The rabbit alpha-like globin gene cluster is polymorphic both in the sizes of BamHI fragments and in the numbers of duplicated sets of genes.

The alpha-like globin gene cluster in rabbits contains embryonic zeta-globin genes, an adult alpha-globin gene, and theta-globin genes of undetermined function. The basic arrangement of genes, deduced from analysis of cloned DNA fragments, is 5'-zeta 0-zeta 1-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3'. However, the pattern of restriction fragments containing zeta- and theta-globin genes varies among individual rabbits. Analysis of BamHI fragments of genomic DNA from 24 New Zealand white rabbits revealed eight different patterns of fragments containing zeta-globin genes. The large BamHI fragments containing genes zeta 0 and zeta 1 are polymorphic in length, whereas a 1.9-kb fragment containing the zeta 2 gene and the 3.5-kb fragment containing the zeta 3 gene do not vary in size. In contrast to this constancy in the size of the restriction fragments, the copy number of the zeta 2 and zeta 3 genes does vary among different rabbits. No length polymorphism was detected in the BamHI fragments containing the theta-globin genes, but again the copy number varies for restriction fragments containing the theta 2 gene. The alpha 1- and theta 1-globin genes are located in a nonpolymorphic 7.2-kb BamHI fragment. The combined data from hybridization with both zeta and theta probes shows that the BamHI cleavage pattern does not vary within the region 5'-alpha 1-theta 1-zeta 2-zeta 3-theta 2-3', but the pattern genomic blot-hybridization patterns for the progeny of parental rabbits with different zeta-globin gene patterns shows that the polymorphic patterns are inherited in a Mendelian fashion. Two different haplotypes have been mapped based on the genomic blot-hybridization data. The variation in the alpha-like globin gene cluster in the rabbit population results both from differences in the copy number of the duplication block containing the zeta-zeta-theta gene set and from the presence or absence of polymorphic BamHI sites.