A newly discovered human alpha-globin gene

Abstract

A previously undefined transcript with significant homology to the pseudo-alpha2 region of the alpha-globin locus on human chromosome 16 was detected as part of an effort to better define the transcriptional profiles of human reticulocytes. Cloning and sequencing of that transcript (GenBank AY698022; named mu-globin) revealed an insert with a 423-nucleotide open reading frame. BLASTP and ClustalW and phylogenetic analyses of the predicted protein demonstrated a high level of homology with the avian alpha-D globin. In addition, the heme- and globin-binding amino acids of mu-globin and avian alpha-D globin are largely conserved. Using quantitative real-time polymerase chain reaction (PCR), mu-globin was detected at a level of approximately 0.1% that measured for alpha-globin in erythroid tissues. Erythroid-specific expression was detected by Northern blot analysis, and maximal expression during the erythroblast terminal differentiation was also detected. Despite this highly regulated pattern of mu-globin gene transcription, mu-globin protein was not detected by mass spectrometry. These results suggest the human genome encodes a previously unrecognized globin member of the avian alpha-D family that is transcribed in a highly regulated pattern in erythroid cells.

Figures

Figure 1.

Unsupervised hierarchic clustering analysis. Microarray data for 14 cord blood and 14 adult blood reticulocyte samples probed by 9 globin genes and the 240336_AT (μ-globin) probe set. Data were clustered (unsupervised), and the signal scale is based on the raw intensities for each probe set.

Figure 2.

Bioinformatics analyses of μ-globin. (A) Alignment of the μ-globin mRNA and the ORF-predicted protein with α-globin cluster on chromosome 16p13. Locations (arrowheads) of the Kozak (CGCCAUGC), stop (UGA), and polyA signal (pA) relative to the three μ-globin exons are shown. The protein is annotated according to globin helical domains, and the residues involved in heme binding and α-β-globin contact are indicated under the gray bar (conserved residues between human α-globin and μ-globin are denoted with colored boxes1). (B) Transcription factor binding site comparison of 200-bp promoter regions of the selected globin genes. Locations of erythroid transcription factor binding motifs are indicated in red, hypoxia-inducible factor motif in green, and general transcription factor binding motifs in blue; other binding motifs are shown as open boxes. AP2 indicates activator protein 2; EKLF, erythroid Kruppel-like factor; GATA1, GATA binding factor 1; HIF, hypoxia-inducible factor; Muscle-INI, muscle initiation factor; MZF1, myeloid zinc finger protein 1; NRSE, neural restrictive silencer element; and SRF, serum response factor.

Figure 3.

Northern blot analyses of μ and α-globin expression. The hybridizations of erythroid tissues with α-globin (A) and μ-globin (B) probes are shown with molecular weight positions on the right. CB indicates cord blood; AB, adult blood; FL, fetal liver; BM, adult bone marrow. (C) Multitissue Northern blot hybridized with α-globin (top) and μ-globin (bottom) probes.

Figure 4.

Quantitative PCR analyses. Total RNAs from erythroid tissues were amplified for detection of μ-globin (A) and α-globin (B). One million copies of DNA encoding α-globin (α clone) and μ-globin (μ clone) were included as negative controls to demonstrate specificity. Also shown is a comparison of μ-globin (C) and α-globin (D) expression levels in differentiating erythroblasts during culture (14 days) of CD34+ cells in erythropoietin (culture day on x-axis). All studies were performed in triplicate. Copy numbers were calculated by standard curve comparison. Mean values and standard deviation bars are shown. Asterisks indicate a significant change (t test; P < .001) compared with no significant (N.S.) change between fetal and adult erythroid tissues. For abbreviations, see the legend to Figure 3.

Figure 5.

SELDI-TOF-MS analyses of red blood cell lysates. Globin proteins detected in erythrocytes from 3 cord blood (A) and adult blood (B) samples are displayed in the 14 000-Da to 17 000-Da mass range (top of each panel). Arrows indicate the α-, β-, and γ-globin proteins. Dotted lines indicate the expected location of μ-globin if it were present.

Figure 6.

Phylogenetic tree constructed by MP algorithm method. Two hundred ninety-one α-like globin proteins and the predicted μ-globin protein were clustered, as described in “Results.” General locations of the human α-like globin chains (open boxes) and a general description of the predominant globin type within each cluster are provided.