Human a-Globin Gene Expression - Semantic Scholar

Vol. 261, No. : X , Issue of November 16, pp. 15327-15333.1986 Printed m [J.S.A.

THE.JOURNAL OF BlOLOClCAL CHEMISTRY (c’,1986 by The American Society of Biological Chemists, Inc

Human a-Globin Gene Expression THEDOMINANTROLE

OF THE a2-LOCUSINmRNA

AND PROTEIN SYNTHESIS (Received for publication, April 30, 1986)

Stephen A. Liebhaberts, Faith E. Cash$, and Samir K. Ballad From the $Howard Hughes Medical Institute, and the Departmentsof Human Genetics and Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania19104 and the (Cardeza Foundation for Hematologic Research, Department of Medicine, The Thomas Jefferson University, Philadelphia, Pennsylvania 19107

The two human a-globin genes,a1 and a2, are coex- 16 (3-7). Both genes are transcriptionally active (8, 9) and pressed in normal erythroid cells and encode identical encode identical protein products (10). Because the two a a-globin protein products. Based upon genetic studies, globin genes produce an identical protein product, itbeen has it has been assumed that these two adjacent and highly difficult to accurately determine their relative levels of expreshomologous genes are equally expressed. In previous sion. For this reason, such determinations have relied upon studies we have, however, demonstrated that the a2 quantitation of a-globin structural mutants. The synthesis of gene encodes a 2-%fold higher steady state level of an a-globin mutant in a heterozygous individual usually repmRNA than the crl gene. In the present study, we resents the expression of one of the four a-globin genes. Based monitor the relative levels of protein production from upon theaverage 20-25% level of expression of such structural these two loci by quantitating the synthesis of specific mutants, it has been concluded that the two a-globin genes a-globin structural mutants encoded by each a-globin are equally expressed (11).However, since a large number of gene. These values are then used to infer the relative factors can affect the levels of expression of these structural contributions of the normal a1 and a2 loci to total aglobin production. The results of eight separate stud- mutants (see “Discussion”), conclusions based upon such an ies, each based upon a different a-globin structural average determination must remain tentative. In this study,we present a detailed genetic analysis of eight mutant mapped to either the a1 or the a2 locus, are internally consistent. The data demonstrate that the unrelated individuals with distinct a-globin structural mutaa2 gene encodes 2-%fold more protein than the a1 tions. Each of these mutations is assigned to its encoding a1 gene. These results suggest that the human a-globin or a 2 locus so that the level of each mutant can beused as a gene cluster contains a major and a minor locus. The marker to monitor the expression of one of the two a-globin dominant expression of the a2 gene predicts a greater genes. We combine the modalities of DNA, mRNA,and impact of mutations at this locus, in comparison to protein analysis to arrive at a full description of a-globin gene mutations at the a1 locus, in the generation of the a- activity in these eight cases, and from this data we infer the thalassemia phenotype. relative levels of activity of the normala1 and a2 genes. These eight studies yield a consistent finding: the a2 gene is functionally the major a-globin gene in humans, encoding two to three times as much a-globin protein as the adjacent a1 gene, a-Globin is an essential subunitof the human hemoglobin This result predicts a significant difference in the impact of tetramer from the sixth week of development in utero through mutations at thetwo a-globin loci on a-globin synthesis and adult life (1).Fetal hemoglobin, a2y2, and adulthemoglobin, on the consequentseverity of a-thalassemia. a2/32, are assembled by combining two a-globin chains with two y - or P-globin chains, respectively. In a-thalassemia, a MATERIALSANDMETHODS genetic deficiency in a-globin synthesis results in the accuHemoglobin Analysis-All studies were performed on material isomulation of uncomplexed and unstable y or P chains and a lated from a single 20-100ml sample of venous bloodfrom each consequent hemolytic anemia (2). The major determinant of patient. The identities of seven of the mutant a-globin chains dephenotypicseverityineach of the specific subsets of a - scribed in this report have been previously characterized Hbs Hasthalassemia is the degree t o which a-globin synthesis is de- haron, Queens (12), and Twin Peaks (13) by the Hemoglobin Chemcreased (2). To predict the impact of a mutation in an a - istry Laboratory at theCenter for Disease Control, Atlanta, GA, and Hbs Russ, Inkster, and Winnipeg at the Comprehensive Sickle Cell globin gene upontotala-globinsynthesis,onemustfirst Center at theMedical College of Georgia, Augusta, GA. The characdefine the relative levels of expression of the two a-globin terization of the HbG-Philadelphia mutant has been published (14). loci. Prior to analysis, the blood was washed three times with ice-cold The two highly homologous a-globin genes, a1 and a2, are normal saline. To quantitate levels of mutant a-globin chainsin of chromosome circulating red cells, 200 p1 of packed cells were lysed in 1.5 volumes situated 3.4 kilobases apart on the short arm *This workwas partiallysupported by National Institutes of Health Grant 1-ROI-AM33975 (to S. A. L.) and the Dean’s Overage Research Programs of Jefferson Medical College (to S. K. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § To whom correspondence should be addressed: Dept. of Human Genetics, University of Pennsylvania,37th and Hamilton Walk, Philadelphia, PA 19104.

of sterile water, clarified by centrifugation at 15,000 X g, and analyzed by one of three methods: carboxymethyl cellulose (CMC’) chromatography ( X ) , Triton-acid urea (TAU) gel electrophoresis (16,17),or isoelectric focusing (IEF) (18). Each of these procedures was carried out in the presence of 8 M urea so that individual globin chains could be identified and quantitated. The CMC chromatography and TAU The abbreviations used are: CMC, carboxylmethyl cellulose chromatography; TAU, Triton-acidurea; IEF, isoelectric focusing; Temed, N,N,N‘,N’-tetramethylethylenediamine; a, a-globin; am,mutant aglobin.

15327

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Human a-Globin GeneExpression

electrophoresiswere performed as published with no significant mod- total erythrocyte a-globin ( a ) was quantitated by one of the ifications. The IEF procedure was modified from the published pro- three techniques described under “Materials and Methods,” tocol. The final concentrations of constituents in the gel are 8 M urea, depending upon which method gave the best resolution. Hb 6% acrylamide (acrylamide: bis-acrylamide, 300.91, 3% Nonidet P40, 1.8% Pharmalyte (Pharmacia), pH 6.5-9, 0.18% Pharmalyte, pH J-Oxford andHb Queens were analyzed by CMC chromatogby TAU electrophoresis, 3-10, and 0.02% TEMED. Dried samples taken up in 8 M urea, 3% raphy, Hb Twin Peaks and Hasharan byelectrophoresis. The results Nonidet P-40,1.8% Pharmalyte, pH 6.5-9,0.18%Pharmalyte, pH 3- and the remaining samplesIEF 10, and 10% 0-mercaptoethanol were applied to the gel in precast of these analyses are shown in TableI (am/&, Hemolysate). wells (application of samples using loading pads or plastictemplates In four cases where fresh samples were available (Hbs Jresulted in significant artifacts). After electrophoresis, Nonidet P-40 Oxford, Queens, Hasharon and Twin Peaks), whole bloodwas and ampholines were removed fromthe gel by soaking intwo changes incubated in the presence of [3H]leucine to measure relative of 10%trichloroacetic acid, 30% ethanol, then fixed in 7% acetic acid, 30% methanol.Gels with radioactive samples were impregnated with proportions of mutant a-globin in newly synthesized reticuEN”HANCE(New England Nuclear) and dried prior to autoradiog- locyte a-globin. The results of these studies are displayed in raphy on Kodak XAR film at -70 “Cin the presence of an intensifying Table I (amla,Labeled Retics). screen (Du Pont, Lightening Plus). The TAU gels were treated in a a-Globin Gene Mapping andmRNA Analysis-The a-globin similar fashion except that the initial soak in 10% trichloroacetic, genotype was directly determined in sevenof the eight cases 30% ethanol was eliminated. The intensity of autoradiograph bands by Southern mapping using an a-globin cDNA probe. In all was quantitated by densitometric analysis usinga Zeineh Soft Laser a1 Scanning densitometer (Biomed Instruments Inc., Fullerton, CA). seven cases the mapping demonstrated normally arranged The Coomassie Blue-stained protein bands on the IEF, and TAU and a2 loci as evidence by a single 23-kilobase EcoRI band, a slab gelswere also quantitated by densitometry. The amount of single 14-kilobase BamHI band, and twoBglII bands of 12.7 protein loaded on the gel and the intensity of the autoradiograph was and 7.0 kilobases (data not shown). The DNA isolated from always adjustedso that the scanning was done in the linear range of the Hb Twin Peaks sample was not of sufficient quality for detection. CMC column fractions were analyzed for protein content gene mapping and further samples could not be obtained for by UV absorbance at 280 nm, and radioactivity was quantitated by repeat studies. scintillation counting to an error of less than 3%. The relative levels of a l - a n d a2-globin mRNA in total The relative rates of synthesis of globin chains in the intact by S1 nuclease reticulocytes of each individual were determined by incubating pe- reticulocyte RNAwas measured in each sample a1 and a2 cDNA ripheral blood for 2 h at 37 “C in the presence of [3H]leucine or[“C] mapping (9) using probes isolated from both leucine and 2 mg/ml glucose as previously detailed(19).The red cells clones(22). A representativestudydisplayingthea2:al were then washed, lysed, and analyzed as described above. mRNA ratio in the Hb J-Oxford sample is shown in the first RNA Analysis-Isolation of mRNA from acid-precipitated reticu- two lanes of Fig. 1. In lane 1 the RNA ishybridized to thea2locyte polysomes was carried out as previously described (20). The 2 the RNA is hybridized t o t h e a l leukocyte pellets from this procedure were saved for subsequent DNA derived probe and in lane derived probe. A diagram of the assay using the two reciprocal isolation. Total RNA extracted from the polysomes was used for in uitro translations, quantitation of the relative a l - and a2-globin probes is shownbelow the autoradiograph. The average result mRNA levels and the purification of a1- and a2-globin mRNAby of each assay is shown in the last column of Table I. In all hybrid selection. Hybrid selection of a l - and &globin mRNA was cases, this ratio obtained using both of the probeswas within carried out as previously described (21), and the relative content of the previously defined normal limits (22). This suggests that the two mRNAs in unfractionated and hybrid-selected sampleswas in all eight individuals, the a-globin genes encode normal determined by S1 nucleasemappingaspreviouslydetailedusing steady state levels of mRNA. probes isolated from botha l - and a2-globin genes (22). Assignment of Each Structural Mutation to Its Encoding In Vitro Translations-In vitro translations were all carried out in of the a-globin a micrococcal nuclease-treated rabbit reticulocyte lysate. The lysate Locus by Hybrid-selected Translation-Each was prepared according to standard protocol (23), and the in vitro mutants was mapped to the a l - or a2-globin locus by the translations were carried out in 15-pl reaction volumes as previously technique of hybrid-selected translation aspreviously detailed described (24). When the translation was to be analyzed by IEF, the (21). The a1 and a2 mRNAs were purified by selective hytranslation was labeled with [3H]leucine(~-[4,5-~H]leucine 130 Ci/ mmol, Amersham) rather than [35S]methionine(~-[~‘S]methionine bridization to recombinant plasmids containing cDNAs representing the divergent 3”noncoding regions of a1 and the 1400 Ci/mmol, Amersham). DNAAnalysis-DNAwas isolatedfrom a leukocytepellet as a2 mRNAs, respectively. The purity of the hybrid-selected mRNA was assessed in eachcase by S1 nuclease mapping. A previously described (25). The structure of the a-globin genes was analyzed by Southern blotting (26) after single enzyme digests with representative example in the case of Hb J-Oxford is shown the restriction endonucleases EcoRI, BglII,and BamHI, all as previ- in lanes3-6 in Fig. 1. Hybrid-selected a1 mRNA was mapped ously detailed (22). with both an a2 and a1 anprobe (lanes3 and 4, respectively). The analysis demonstrates that the al-globin mRNA is unRESULTS contaminated with a2-globin mRNA. The analysis of hybridThe Level of a-Globin Mutant Chains inCirculating Eryth- selected &?-globin mRNA (lanes 5 and 6 ) demonstrates a rocytes and in Newly Synthesized Globin Chains-Peripheral blood samples were obtained from eight unrelated individuals, TABLEI each heterozygous for a different a-globin structural mutant. Genetic profileof eight individuals with a-globinstructural mutants Four of the samples, J-Oxford, Queens, Hasharon, and Twin am/a Peaks, were processed immediately after venipuncture while Geno- a2:al Mutant HemolLabeled type mRNA Gthe remaining four samples, Russ, Winnipeg, Inkster, and ysate retics Philadelphia were processed after overnight delivery on wet 40 47 aa/aa 2.8 J-Oxford (15 Gly-Asp) ice. In each case, except thatof Hb J-Oxford, the identity of 14 14 aa/aa 2.6 (34 Leu-Arg) Queens themutantchainhadbeen previously characterized(see 34 35 aa/aa 3.0 Hasharon (47 Asp-His) “Materials and Methods” section). The identityof the Hb J 17 ND” aa/aa 2.3 Russ (51 Gly-Arg) Oxford mutation was established by isolation of an abnormal 26 ND aa/aa 2.6 G-Philadelphia (68 Asn-Lys) 11 ND aa/aa 2.7 a-globin tryptic peptideby CMC chromatographyfollowed by (75 Asp-Tyr) Winnipeg aa/aa 2.6 Inkster (85 Asp-Val)ND 24 amino acid analysisof this peptide ona Beckman 118 amino 23 17 2.8 ND Twin Peaks (113 Leu-His) acid analyzer (data not shown). a ND, not determined. The level of mutant a-globin ( a m )chain as a percentage of

a-GlobinHuman

Gene Expression

15329

similar degree of purity, In all cases but one, the mRNAs are similarly pure; in the case of Hb Queens, the a1 mRNA was "contaminated with residual 02 mRNA and, due to limited 1 2 3 4 5 6 material, the hybrid selection could not be repeated. Unfractionated RNA and aliquots of the hybrid-selected mRNAs from each individual were translated in vitro, and the labeled - 263 translation products were fractionated and quantitated by CMC chromatography,TAU electrophoresis, or IEF. The results are displayed in Figs. 2 and 3. (The results of these studies on Hb Hasharon have been presented previously (21).) - 175 In each case, the position of the mutant a-globin (a"')is well separated from the normal a-globin (aA). Visual inspection of the fractionation patterns allows an unambiguous locus assignment in each case. The results of the locus assignment a2 probe a1 probe are tabulated in Table 11. a Queens, Russ, Winnipeg, and (lanes 1,3,51 (lanes 2.4.61 AUG Hind Ill UAA Twin Peaks are encoded at the a1 locus while (Y J-Oxford, AUG Hind 111 UAA a1 41 ,f Hasharon, G-Philadelphia, and Inkster areencoded at thea2 ( o 52 locus. a1 probe a; probe (JW1011 (pRP91 In thefour cases where the resultsof the in uitro translation can be compared to thoseof the in uiuo reticulocyte synthesis 1 S' 1 S' (J-Oxford, Queens, Hasharon, and Twin Peaks), the results 175 nt 263nt a1 fragment odemonstrate a close correspondence (compare Table I, a"'/a, *- 175 nt 52 fragment o 263nt Labeled retics,with Table11, in vitrotranslation #/a).There FIG. 1. S I nuclease mapping of relative a l - and a2-globin is also a close correspondence between the levels of mutant mRNA content in total reticulocyte RNA and in hybrid-se- synthesized in uitro and the levels of mutant present in the lected a l - and a2-globin mRNA. Total reticulocyte RNA (lunes circulating erythrocytes (compare TableI, am/aHemolysate, 1 and 2), hybrid-selected a1 mRNA (lanes 3 and 4 ) , and hybridselected a2 mRNA (lunes 5 and 6 ) were hybridized to a single strand, with Table 11, In uitro translation a"'/a). The one exception 32P end-labeled probe isolated from either the al-globin cDNA clone is in the case of Twin Peaks. In this case, the relative levels pJWlOl (40) in lunes 2,4, and 6 or the a2-globin cDNA clone pRP9 of mutant chains synthesizedin uitro (11%) and in the intact (41)in lanes 1,3,and 5. Hybrids were digested with S1 nuclease and reticulocytes (17%) are lower than thatfound in the hemoglothe products were analyzed on a 5% acrylamide, 8 M urea gel, all as bin of the circulating redcells (23%). This may reflect a previously described (9, 22). The assay is shown schematically below higher affinity of the mutant a-globinfor B chains compared the gel autoradiograph. The larger S1 resistant fragment of 263 nucleotides (nt ) is generated by the hybridization of the probe to its to that of the normal @-globin;the basis of this difference is homologous a-globin mRNA (e.g. a1 probe to the a1 mRNA) while not further explored in the presentstudy. T o directly determine whether theexpression of a mutant the smaller S1 resistant fragment of 175 nt is generated when the probe hybridizes to the nonhomologous a-globin mRNA species (e.g. a-globin encoded a t a particular locus accurately represents a1 probe to the a2 mRNA). The two probes give reciprocal results the level of expression of the normal a-globin allele at the and are used for mutual confirmation. The sizes of the fragments, same locus, we compared the level of each a1 mutant (al"') indicated next to the gel, are in nucleotides and the position of the initiation codon (AUC),termination codon ( U A A ) ,and the mRNA to its normal alA counterpart and each a2 mutant (a2"') to its normal aZAcounterpart. Thiswas accomplished by detercap site (0)are noted in the diagram. Hybrid selected

Total

a1

a2

(e

FIG.2. In vitro translation of total reticulocyte mRNA and hybridselected a l - and a2-globin mRNAs. Unfractionated reticulocyte RNA and hybrid-selected a l - and a2-globin mRNA from the indicated individuals were translatedinrabbit reticulocyte lysate in the presence of [3H]leucineor ['"Slmethionine. The samples were analyzed by CMC column chromatography. Prior to CMC column chromatography, 50 mg of autologous erythrocyte carrier hemoglobin wasadded to theproducts of the in oitro translation in order to mark the positions of the normal and mutant a-globin radioactive peaks: normal p(BA), p26c"4L''"(BE), normal a ( a A ) , a Q l e n s (aQ), and aJ-oxford (aJ). The OD280 profile (- - -) of the CMC column represents the relative levels of the globin chains in the circulating erythrocytes repwhile the radioactive profile (-) resents the corresponding in oitro synthesized globin chains of unfractionated RNA (Total),or hybrid-selected a1 and a 2 mRNA.

/c

1. ,d;

,2A

0

0

10

.

.

20

30

40

50

60

Fraction number

10

20

30

40

50

0

60

15330

Human a-Globin Gene Expression

G - Phila. Winnipeg lnkster

Russ

a a1 a”’ aA

a2

a a1

...

a2

Twin Peaks

a a1

a2 aA

L.

~.

am

I

I

Total

FIG. 3. In vitro translation of total reticulocyte mRNA and hybrid selected a l - and a2-globin mRNAs. Results of experiments as described in Fig. 2 except that samples are analyzed by IEF or TAU (for analysis of Twin Peaks) electrophoresis. Theautoradiograph of each gel is shown at the top and the position of the normal a-globin ( a A )andthemutanta-globin (aM)is noted. The relative level of normal and mutant a-globin inthetranslation of unfractionatedRNA (lanes marked a and scam marked Total) or the hybrid selected a1 and a2 mRNAs (lanes and scam marked a1 and 0 2 ) is quantitated by the densitometric scans shownbelow eachautoradiograph. Thedatafrom these studies is summarized in Table 11.

d

I

a2

I

I

/ \ mining the relative levels of in vitro synthesis of am and aA encoded by hybrid-selected al-or a2-globin mRNA (from a1 mutants and a2 mutants, respectively). The column and gel analyses of these translations are shown in Figs. 2 and 3, respectively, and the quantitative data is summarized in the last column of Table 11. In the caseof Hb Queens, the in vitro translation of a1 mRNA could not be used for this quantitative comparison due to contamination with a 2 mRNA (see above). Relative Contribution of the Two Normal a-Globin Loci to a-Globin Synthesis-The results of the gene mapping studies (Table I) and thein vitro translational analyses (Figs. 2 and 3 and Table 11) are compiled for each mutant in Fig. 4. A normal a-globingenotype is assigned in each casebased upon the Southern analysis. In the caseof Hb Twin Peaks, South-

ern analysis is not available, and a normal genotype is assumed based upon the normal relative levels of a1 and a2 mRNAs (22). The level of expression of each of the four genes is represented as a percentage, with all four genes adding up to 100%. In eachcase, the level of a-globin synthesis assigned to the mutant locus is a directly determined value obtained from thein vitro translation of total reticulocyte RNA (Table 11). In eachof the mutants(excluding Hb Queens, see above), the value for expression of the normalallele at thehomologous normal locus on the second chromosome is calculated from the in vitro translation of the appropriate a1 or a2 hybridselected mRNA (Table 11, alm/alAor a2”’/a2*). The remaining percentage of a-globin synthesis is equally divided between the two remaining(a1or a2)genes. The relative levels of protein expression of the a1 and a2 loci on the normal

Expression Human Gene a-Globin TABLE I1

15331

level of expression at that locus, it cannot be assumed that these two values are exactly equal. One of the most important factors which can affect the apparent level of expression of In vitro translation an a-globin structural mutant is the coexistance in the geMutant Locus nome of an a-thalassemia mutation. Such a mutation, affecting the expression of one or more of the three remaining aglobin genes, would decrease the expression of normal aa2 40.0 1.15 J-Oxford globin chains and increase the apparent level of expression of a1 14.0 ND" Queens a2 35.0 0.90 Hasharon the mutant. We have studied each of the presently reported a1 13.0 0.75 Russ individuals to rule out such mutations. Single gene deletions 29.5 0.79 a2 G-Philadelphia (a-thalassemia-2 mutations) can be detected by any of the a1 12.5 0.85 Winnipeg three different restriction digests used in the present study, a2 24.0 0.65 Inkster and deletion of both a-globin genes on a single chromosome Twin Peaks a1 11.0 0.68 (a-thalassemia-1 mutations) is ruled out in the individuals ND, not determin id. presently studied by the in uitro translation of hybrid-selected a-globin mRNAs which demonstrates eithertwo a1 or two a 2 Relative Expression 01 Each 01 the a-Globin Loci alleles in each individual. Nondeletion a-thalassemias usually a2 a1 a2 a1 result in a reduced steady state level of mRNA from the affected locus and can therefore be detected by alterations in H -&-" Queens J-Oxford the relative levels of a1 and a2 mRNAs (8, 27-29). The one 12 5 2 - " 8.1 documented exception to this generalization is a missense ( m R N A = 2 6 1) (rnRNA=28.1] mutation which encodes a highly unstable protein product "-&" " I (30). However such mutations can be detected by in uitro translational analysis (20). Therefore, based upon the DNA, RNA, and translationalanalyses detailed in the present study, we conclude with reasonable certainty that the genomes of the eight individuals do not contain coexisting thalassemic mutations. In addition to the above noted influence of coexisting athalassemia upon the level of mutant expression, a number of ' e other factors can significantly alter relative levels of expresPeaks Twin 36 5 lnksler 37 sion of the mutant chain. Such variables, some theoretical lrnRNA=26 rnRNA-281) 1) and some documented in the thalassemia syndromes, include FIG. 4. Relative expression of the four a-globin genes in secondary effects of the missense mutation upon the mRNA each of the eight individuals studied. The name of each a-globin functions of splicing (32), transport, stability (8), and transmutant is placed to theleft of its respective genome. Each normal aglobin gene is represented by an open box and the a-globin gene lational efficiency, as well as protein instability (20, 33) and encoding the missense mutation is represented as a closed box. The altered efficiency in hemoglobin tetramer formation. Finally, relative expression of each of the four a-globin genes is represented the level of the mutant may be unexpectedly high in rare above the respective box as a percentage of total a-globin synthesis. situations in which the same mutation is encoded at both of The derivation of these values is detailed in the text. The ratio of the a-globin loci on the same chromosome (34) or in which protein expression encoded by the two a-globin loci on the normal an individual is homozygous for a structural mutant(35). For (bottom) chromosome is shown to the right of the arrow. For direct must be comparison, the ratioof a 2 to a1 mRNA expression in each individual these reasons, each case of astructuralmutant studied in detail before conclusions can be made based upon (see Table I) is shown in parenthesis below the normal chromosome. its level of expression. To detect minor alterations in mRNA metabolism and/or (bottom) chromosome is then calculated. For direct compartranslational efficiency whichmight affect the level of expresative purposes, the relative levels of the a2 and a1 mRNAs sion of the mutanta-globin, we directly compare the synthesis (from Table I) are also shown in this figure. of the mutant andnormal a-globin by in uitro translation of DISCUSSION hybrid-selected mRNA. Without this measurement, it is not In the present study we attempt to quantitate the relative possible to accurately determine the degree to which the level expression of the two a-globin genes, a1 and 0 2 . Since these of expression of a structural mutant at an a-globin locus two genes encode identical a-globin proteins, theirindividual represents the normal level of expression at that locus. For levels of expression can only be distinguished when one of example, in two of the four a 2 mutants, Hb Inkster and Hb them encodes a structural mutant. Eachof the mutants stud- G-Philadelphia, the am:aAratio encoded by a2 mRNA is ied in this report contains a single amino acid substitution significantly less than 1. This ratio suggests that themRNAs which alters the physical properties of the protein sufficiently encoding these two mutant proteins may have significantly for it to be separated from the normal a-globin by chromato- lower translational efficiencies thanthe normal a-globin graphic or electrophoretic techniques. In thisway, the relative mRNA. Regardless of the mechanism, if this difference went levels of mutant and normal a-globin chains can be quanti- undetected, the level of expression of the normal a2 loci in tated. In addition,each mutant can be assigned to either the these two individuals would be underestimated. The deviations of this ratio from unity in the other individuals studied a1 or a 2 locus by hybrid-selected translation. With this information, each of these mutants can be used to specifically (see the last column of Table 11) may also reflect alterations monitor the activity of a single a-globin gene at a known inthe stability or translational efficiency of themutant locus. mRNA. Each of these values was included in calculating the Whereas the level of expression of a mutant a-globin en- levels of gene expression shown in Fig. 4. coded at a particular locus can be used to infer the normal A final concern in interpreting dataon the a-globin mutants Locus assignment and in vitro translationof a-globin structural mutants

-

Expression Human Gene a-Globin

15332

TABLEI11 Locus assignment of a-globin point mutations Mutations Locus

Structural J-Oxford

I

(a15 Gly-Asp)

Refs

a2

34 (a34Leu-Arg) Queens Hasharon a2 21 (a47AsWHis) Russ a1 (a51Gly-Arg) a2 (a68Asn-Lys) G-Philadelphia a1 (a75A s p T y r ) Winnipeg Jnkster a2 (a85A s p V a l ) Twin Peaks ff1 (a113Leu-His) Combined structuralthalassemic 30,31a2 (a125Leu-Pro) Quong Sze a2 b (a138UCC4JC-) Wayne Constant Spring a2 b (a142UAA-CAA) Icara a2 b (a142UAA-AAA) Kaya Dora a2 b (a142UAA4JCA) Seal Rock a2 b (a142UAA-LJCA) Thalassemic Initiation codon (AUG-ACG) a2 28 IVS 1 donor site a2 29 deletion Poly(A) addition sig- (AAUAAA+AAUAAG)/ a2 / 27 nal/Codon 14 frame shift (TGG-+-GG) a1 Determined in the present report. bDeduced from the amino acid sequence of the readthroughC terminus. (a16 Lys-Glu)

a2 + a 1 a1

(I

with a deletion of both a-globin genes from one chromosome and an a2 structural mutation ( a Quong Sze; a125Leu-Pra) on the other chromosomes. While the a2:al mRNA ratio in this sample demonstrated the normal excess of a2 mRNA (2.6:1), in vitro translation of this mRNA produced equal levels of mutant (a2) andnormal ( a l ) globin. This result is clearly at variance with the present results which are based upon eight different mutations. The basis for this discrepancy may be suggested by the Hb Inkster and Hb G-Philadelphia studies which demonstrate that the expression of this mutantprotein may be suppressed by a secondary effect of the encoded missense mutation, possibly related to an alteration in the translational efficiency of the mutant mRNA. The direct comparison of expression of the mutant allele to the homologous normal allele ( ~ 2 ~ : a 2as * )carried out in the present study was not possible in the case of a2 Quong Sze because the normal a2 gene on the sister chromosome is deleted. Therefore, the explanation of the Hb Quong Sze result must remain tentative pending further studies. The existence of major and minor a-globin loci in the human genome i s consistent with existing data on a-globin expression in non-human species. It is possible to directly assess the relative expression of the two a-globin loci in a number of species in which the a-globin encoded at the two loci differ by one or two amino acids. In the 15 species so studied, the two a-globin genes are expressed unequally. The relative expression of the major and minor loci varies from ratios of 1.4 to 3.8 with an average value of 2.3 (38), a ratio remarkably close to thatreported in the presentstudy. The results of the present study are also consistent with the patternof point mutations (andsmall deletions) detected in the human a-globin gene cluster. Those mutations which have been assigned to one of the two a-globin loci in intact (two a-globin gene) chromosomes arelistedinTable 111. Structural mutationswhich are phenotypically silent (do not cause significant loss of hemoglobin synthesis) are equally distributed between the two a-globin loci. This suggests that the rate of mutation at the two loci is approximately equal. In contrast, the distribution of point mutations which adversely affect gene function (combined structural-thalassemic and thalassemic in Table111)is clearly asymmetric. If consideration is limited to chromosomes on which there is a single, nondeletion a-thalassemia mutationon the chromosome, eight of eight mutations are located at the a2 locus. As it appears that themutation rates of the two genes are equal, it is reasonable to speculate that anequal number of thalassemic mutations also exist at thea1 locus. One possible reason why these mutations have not been detected is that the loss of an a1 gene would not result in an appreciable loss of a-globin synthetic capacity. According to thepresent study, the loss of a single a1 gene would result in the loss of only 13% of total a-globin synthesis. In contrast, the loss of an a 2 gene would result in a more significant (37%) loss of a-globin synthesis. a1 mutations would, therefore, not result in appreciable thalassemic phenotypes (unless present in a genome with additional a-thalassemia mutations (27, 39)) and would not be detected. The major role of the a 2 gene, therefore predicts, that most functionally significant nondeletion a-thalassemia mutations will occur within the a2-globin locus.

is whether or not the stability of the mutant protein is the general same as thatof normal a-globin.It is often assumed in discussions of a-globin mutants thatthose mutants which are expressed a t significantly less than the average 20-25% level are unstable. While instabilitycan certainly havean effect on the level of a mutant chain (see for example the recent reexamination of the relative expression of Hbs J-Buda andGPest (36)), the data in the present study suggest that low levels arenot always due to proteininstability. We have specifically ruled out instability of each of thesemutant proteins by demonstrating a close correlation between the levels of the mutant in hemolysates of circulating red cells and the levels of newly synthesized a-globin chains synthesized either inintact reticulocytes or in an in vitro translation. The low level of expression of the a-globin mutants Queens, Russ, Winnipeg, and Twin Peaks appears to reflect their positioning at thea1 locus rather thanprotein instability. The conclusion that the a2 gene is the major human aglobin gene is consistent with previous studies which demonstrate both a 2- to %fold excess of a 2 mRNA in normal fetal and adult erythroid cells (8, 9, 22) and an equivalent translational activity of the two a-globin mRNA species.' The eight individuals in the present study have an average 2.8fold excess of a 2 to a1 mRNA which parallels the relative excess of a-globin encoded by the a2-globin gene (summarized in Fig. 4 ) . It should be noted that the ratiosof a2:al mRNA levels and a221 protein synthesis are determined by independent experimentalapproaches and calculations. These studies suggest that the basis of the a2 locus predominance in a-globin protein production is based upon its synthesis of a higher level of steady state a-globin mRNA. In a previous report, we suggested that thecontributions of Acknowledgments-Weacknowledge the generosity ofDr. Titus the a1 and a 2 loci tototal a-globin synthesis might be balanced by a higher translational efficiency of the less abun- Huisman and Dr. Donald Rucknagel in contributing blood samples dant a1 mRNA (37). This conclusion was based upon in vitro from several of their patients for this study. translation studies of reticulocyte mRNA from an individual REFERENCES

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