Human Α-globin Gene Research Articles

A Review of the Molecular Genetics of the Human α-Globin Gene Cluster

A Review of the Molecular Genetics of the Human α-Globin Gene Cluster

Transcriptional Promiscuity of the Human α-Globin Gene

The human alpha-globin gene displays the unusual property of transcriptional promiscuity: that is, it functions in the absence of an enhancer when transfected into nonerythroid cell lines. It is also unusual in that its promoter region lies in a hypomethylated HpaII tiny fragment (HTF) island containing multiple copies of the consensus sequence for the SP1-binding site. We have investigated whether there is a relationship between these two observations. First, we investigated the mouse alpha-globin gene since it does not lie in an HTF island. We have demonstrated that it was not transcriptionally promiscuous. Second, we studied the transcriptional activity of the human alpha-globin gene in the absence of the GC-rich region containing putative SP1-binding sites and found a small (two- to threefold) but consistent positive effect of this region on transcriptional activity in both nonerythroid and erythroid cell lines. However, this effect did not account for the promiscuous nature of the human alpha-globin gene. We found that in a nonreplicating system, the human alpha-globin gene, like that of the mouse, required a simian virus 40 enhancer in order to be transcriptionally active in nonerythroid and erythroid cell lines. Since we only observed enhancer independence of the human alpha-globin gene in a high-copy-number replicating system, we suggest that competition for trans-acting factors could explain these results. Finally, our experiments with the erythroid cell line Putko suggest that there are no tissue-specific enhancers within 1 kilobase 5' of the human alpha-globin cap site or within the gene itself.

Two BgIII RFLPs of the human α-globin gene cluster in the American sickle cell population

Two BgIII RFLPs of the human α-globin gene cluster in the American sickle cell population

Recombination at the human α-globin gene cluster: Sequence features and topological constraints

We have characterized 170 kb of DNA around the human α-globin gene cluster to enable a systematic analysis of 12 naturally occurring deletions from this region. In 8 deletions, the 3′ breakpoints lie within a 6–8 kb segment of DNA, identifying a breakpoint cluster region. Members of the Alu family of repetitive sequences are frequently found at the breakpoints and we describe a novel deletion due to homologous recombination between such repeats. In another deletion the breakpoints are separated by 131 bp of DNA, which we have shown to be transposed from a region 36 kb upstream from the 5′ breakpoint where it is present in the inverse orientation. The sizes of these deletions, the nonrandom distribution of their breakpoints, and the nature of the inversion-duplication transposition event suggest that these rearrangements are constrained by the higher-order structure of the α-globin cluster.

Long range genome structure around the human alpha-globin complex analysed by PFGE.

A map encompassing 300 kilobases (kb) in and around the human alpha-globin gene complex shows features with important implications for understanding the structure and function of the human genome. In contrast to other segments of the mammalian genome that have been analysed by pulsed field gradient electrophoresis (PFGE), this region contains an unusually high density of sites for infrequently cutting restriction enzymes that recognise GC rich motifs including the under-represented CpG doublet. This suggests that the 26 kilobase (kb) stretch of DNA containing the alpha-globin gene family, which is known from sequence analysis to be 60% GC rich, is itself embedded within a region of high GC content. This long-range structure, identified by PFGE, corresponds to a class of GC rich isochores that are thought to represent early replicating DNA present in Giemsa negative chromosomal bands. The identification of such regions by PFGE will be of value in understanding the organisation of human chromosomes and will influence the strategies used to construct a physical map of the genome.

Molecular rearrangements of the human alpha-globin gene cluster.

The human α-globin gene complex on chromosome 16 consists of two adult α genes (α1 and α2) separated from the embryonic α-like (ζ) gene by two pseudogenes (ψα and ψζ) arranged in the order 5'-ζ-ψζ-ψα-α2-α1-3'. The globin chains derived from the two α genes are identical and they are produced in roughly equal amounts. Disorders that result in reduced α-chain synthesis (α-thalassemia) are common and result in either diminished (α+-thalassemia) or absent (α°-thalassemia) α-chain production from the affected chromosome. Molecular analysis has shown that α-thalassemia is caused by a large variety of deletion and nondeletion lesions, which are reviewed by Higgs and Weatherall. It is now clear that the α-thalassemias in contrast to the β-thalassemias, are most frequently due to large (> 3 kb) deletions from within the gene cluster. It is possible that this reflects an inherently high rate of recombination within the α-gene complex that is dependent on its overall structure. Therefore, investigation of the α-thalassemia mutants and the normal variants that result from these recombination events may give some insight into the way in which the present-day human α-gene cluster has been molded during evolution. In this paper, we will summarize our current knowledge of the common molecular rearrangements of this multigene family and attempts to relate some of them to the normal structure of the α complex.

Amplification and expression of human alpha-globin genes in Chinese hamster ovary cells.

We studied the effects of gene amplification on human globin gene expression in Chinese hamster ovary cells. The normal human alpha-globin gene (N alpha 2) and a hybrid gene (M alpha G) containing the 5' promoter-regulator region of the mouse metallothionein gene and the human structural alpha 2-globin gene were linked to a modular SV2-cDNA dihydrofolate reductase (DHFR) gene. The recombinant DNA molecules were introduced into Chinese hamster ovary cells by calcium phosphate precipitation. After initial selection to retain the DHFR and linked sequences, the cells were cultured in increasing concentrations of methotrexate up to 0.2 mM. Southern blot analysis of total cellular DNA showed an approximately 500- to 1,000-fold increase in the number of copies of DHFR and human alpha-globin genes. The transcription of the alpha-globin and DHFR genes increased as their copy number within the cells increased. The transcription of the amplified hybrid M alpha G gene was also inducible with cadmium treatments. Both mature mRNA and "read-through" transcripts were observed. DHFR constituted approximately 10% of pulse-labeled cellular proteins in these cells, but no human alpha-globin was detected. In vitro translation of polyadenylated RNA from these cells showed that alpha-globin mRNA transcribed from the amplified alpha-globin genes was functional and directed alpha-globin chain synthesis. In situ hybridization of 3H-labeled alpha-globin and DHFR DNA probes in chromosome preparations from the two cell lines indicated that both genes were coamplified in the same chromosomal locations in each cell type. These results indicate that gene amplification enhances human globin gene expression in cultured Chinese hamster ovary cells.

Differences in human α-, β- and δ-globin gene expression in monkey kidney cells

We have compared the function of the human α-, β- and δ-globin genes using various plasmid expression vectors derived from pBR322. Amplification of recombinants occurred after their introduction, by calcium-phosphate-mediated DNA transfer, into monkey kidney cells that constitutively produce T antigen (COS cells). The human α-globin gene promoter functioned independently, but the β-globin gene promoter was nearly totally dependent on the enhancing activity of the 72 bp direct repeats from the SV40 genome. Furthermore, when the human α- and β-globin genes were linked in the same vector, the a promoter was active but the β promoter was not. Function of the δ-globin gene promoter also depended on the enhancer element. In vectors containing the 72 bp repeats and the β- or δ-globin gene, the activity of the β-globin gene was approximately 50 times greater than that of the β-globin gene, approximating the ratio of β and δ mRNA observed in normal human bone marrow cells.

Localization of the human α-globin gene cluster to the short arm of chromosome 16 (16p12–16pter) by hybridization in situ

The human α-globin gene cluster has been assigned previously to chromosome 16 using a molecular hybridization assay of somatic cell hybrids. We have carried out a regional assignment to the upper half of the short arm of chromosome 16 in the region 16p12-pter by hybridization in situ of α-globin-specific probes to fixed metaphase chromosomes. Suitable hybridization probes, of sufficient length to give a detectable signal, were prepared free from highly repetitive sequences, by the isolation of restriction enzyme fragments from genomic recombinant clones covering the ζ, ψα and α-globin gene regions of the chromosome. A total of 5.2 × 10 3 bases of hybridized sequence was used to detect the α-globin gene cluster. The criteria of chromosome specificity, overall efficiency of hybridization and regional localization were used to establish the localization. These criteria have been applied previously to the mapping of the β-globin gene cluster.

Identification of DNA sequences required for transcription of the human α1-globin gene in a new SV40 host-vector system

We have developed a rapid and simple method for studying the transcription of cloned eucaryotic genes, which involves transfecting SV40-transformed monkey cell lines (COS cells) with derivatives of the plasmid pBR322 that contain the SV40 viral replication origin but lack regions necessary for viral transcription (SV-ORI vectors). Because COS cells produce SV40 T antigen and are permissive for SV40 viral replication, transfected SV-ORI plasmids replicate to a high copy number. SV-ORI plasmids carrying a human α-globin gene are also replicated in COS cells. Moreover, the α-globin gene is faithfully transcribed to produce high levels of RNA, which is accurately processed to produce authentic α-globin mRNA. We have used this transcription system to demonstrate that a sequence located between 55 and 87 base pairs upstream from the mRNA capping site is required for efficient transcription of the α-globin gene in COS cells.

Regional assignment of genes for human α-globin and phosphoglycollate phosphatase to the short arm of chromosome 16

The human alpha-globin and phosphoglycollate phosphatase (EC 3.1.3.18) genes have been regionally localized to the short arm of human chromosome 16 (HC16). This was accomplished by fusing mouse fibroblasts (A9) to human fibroblasts that contain a reciprocal translocation between the long arms of chromosomes 16 and 11. The murine A9 cells are deficient in adenine phosphoribosyltransferase (APRT), an enzyme present on the long arm of HC16 (HC16q). Hybrid cells were grown in selection culture medium that required the cells to retain human APRT. Therefore, the hybrids exhibited stable retention of the entire HC16 or the rearranged chromosome containing HC16q. We isolated five independent primary and secondary hybrid cell lines which retained either HC16 or HC16q at a high frequency. The presence of human alpha-globin genes in the various clones was established directly by DNA extraction and hybridization to a cDNA probe for human alpha-globin genes. Autoradiographs showed that hybrid cells containing the long arm, but not the short arm, of HC16 showed only the background mouse bands. Hybrid cells that retained the entire HC16 demonstrated the band(s) containing the human alpha-globin genes. Hybrid cells that contained HC16 with its alpha-globin genes were then placed in culture medium that contained diaminopurine, which is lethal for cells containing APRT. These counter-selected hybrid cells had lost HC16 and also lost the human alpha-globin genes as determined by blot hybridization. The presence of alpha-globin gene sequences in the hybrid clones was concordant with HC16 only and not with any other human chromosome. These results confirm the assignment of alpha-globin genes to HC16 and localize the genes to the short arm. We also assign the locus for phosphoglycollate to the short arm of HC16.

The duplicated human α-globin genes: Their relative expression as measured by RNA analysis

The human α-globin genes are duplicated and encode identical polypeptides. Recently we detected in cloned genomic DNAs characteristic sequence differences between the 3′ untranslated regions of the 5′ (α2) and 3′ (α1) genes, not previously recognized by direct analysis of mRNA and cDNA transcripts. Based on these untranslated region differences, we have now used S1 nuclease mapping of RNA to detect and quantitate the two predicted α-mRNA species. With this assay we have examined the relative expression of the α-globin genes during normal development and in α-thalassemia syndromes. In normal adult reticulocytes, α2 RNA is slightly more abundant than the α1 species (ratio 60:40). This relative abundance of the a RNAs was consistently observed in fetal blood and liver RNA samples from 10 weeks of gestation to birth. In both deletion and nondeletion forms of α thalassemia, only the α1 RNA species was present in erythroid RNA. These studies resolve prior conflicts regarding the heterogeneity of α RNA and establish the normal pattern of relative α-gene expression during development independent of protein variants. RNA analysis also permits for the first time identification of the mutant genes in nondeletion forms of a thalassemia.

The 3′ untranslated regions of the duplicated human α-globin genes are unexpectedly divergent

DNA sequence analysis of a cloned partially deleted human α-thalassemia globin gene revealed a novel 3′ untranslated region displaying at least nineteen differences when compared with previously published α mRNA sequences. Restriction enzyme mapping established the origin of the α-thalassemia gene as the more 3′ of the normal, duplicated α genes (α1). DNA sequencing of a previously isolated α1 gene revealed a 3′ untranslated region identical to that of the α-thalassemia gene. The sequence of the corresponding region of the more 5′ α gene (α2) was consistent with published mRNA sequences except in three probably polymorphic positions. Therefore the 3′ untranslated regions of the highly homologous α-globin genes differ significantly. The recognition that the duplicate α genes differ in a region expressed in mature mRNA should now permit direct assessment of relative gene output in various normal and pathologic states. The divergence of the α gene 3′ untranslated regions in the face of minimal coding sequence differences must be reconciled with current models for matching homologous gene sequences by recombination events.

Expression of human alpha-globin genes in hybrid mouse erythroleukaemia cells depends on differentiated state of human donor cell.

We have developed a system which can be used to study the mechanisms that may govern the expression of human alpha-globin genes in human erythroid and non-erythroid haematopoietic cells. Human chromosome 16, which has been shown to bear the human alpha-globin genes, is introduced by cell fusion into mouse erythroleukemia (MEL) cells to generate continuously proliferating cell lines that retain permanently the human alpha-globin genes. We have shown that hybrid diploid MEL cells with human alpha-globin genes from erythroid donor cells express these genes fully through globin chain synthesis, while hybrid diploid MEL cells containing human alpha-globin genes from non-erythroid human haematopoietic donor cells contain very low levels of human alpha-globin mRNA and no detectable human alpha-globin chains. The levels of human alpha-globin mRNA in these hybrid cells were found to depend on factors present in the MEL recipient cell as well as on the differentiated state of the human donor cell, suggesting that this system may be suitable for characterisation of mechanisms governing haematopoietic differentiation in man.

Human α-globin gene expression following chromosomal dependent gene transfer into mouse erythroleukemia cells

We have used the genetic marker, adenine phosphoribosyl transferase (APRT), an enzyme known to be on human chromosome 16, to establish a method for the transfer of human α-globin genes into mouse erythroleukemia cells. Mouse erythroleukemia cells devoid of detectable levels of APRT were fused with fractions of human marrow enriched in human erythroid cells. The hybrid cells arising from this fusion were isolated in medium supplemented with aminopterin and thymidine, and used adenine as the sole purine source. This population of hybrid cells was dominated by cells (80%) in which human chromosome 16 was present. Human chromosomes 4, 5 and 6 were also found in these cells. The hybrid cells were then placed in medium supplemented with diaminopurine (DAP), which is lethal for cells containing APRT. Greater than 95% of the DAP-selected hybrid cells lacked human chromosome 16. Cytoplasmic RNA was extracted from the two hybrid cell populations and assayed by molecular hybridization for sequences coding for human α-globin. Carboxymethyl cellulose chromatography was used to study the level of synthesis of human a-globin in the hybrids. The original hybrid cell, which contained a high frequency of human chromosome 16, also contained high levels of human a-globin mRNA and human α-globin chains. Hybrid cells counter-selected in DAP and thus lacking human chromosome 16 were devoid of detectable levels of human APRT, human α-globin mRNA and human α-globin chains. This work shows that transfer of human chromosome 16 into the MEL cell is possible using a chromosomedependent, APRT-mediated method of gene transfer. Using this system in which expression of the human α-globin gene occurs, we were also able to confirm our earlier assignment of the human α-globin gene to human chromosome 16. This system may be of further use in identifying genetic elements governing expression of the human α-globin gene which can be carried with human chromosome 16 as it is donated to the mouse erythroleukemia cell by donor cells of different epigenotypes.

Localization of the human α-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.