JOURNAL OF CELLULAR PHYSIOLOGY 144:287-294 (1990)

Patterns of Spectrin Transcripts in Erythroid and Non-Erythroid Cells JOSEF 1. PRCHAL,* THALIA PAPAYANNOPOULOU, AND SA-HYUN Y O O N Division of Hematology and Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama 35294 0.7 ./?, S.-H.Y.); Division of Hematology, University of Washington, Seattle, Washington 98 195 (T.P.) Spectrin is the major protein of the membrane erythrocyte skeleton. More recently, homologous but non-identical spectrins (fodrins) were also found in various non-erythroid tissues. Spectrin mRNA in erythroid and various non-erythroid cells was examined by direct hybridization with human a-spectrin, p-spectrin (erythroid spectrins), and a-fodrin (non-erythroid spectrin) cDNA probes. Northern blot analysis of poly (A)' RNA revealed a distinct pattern of expression in erythroid vs. non-erythroid cells. Erythroid cells from early erythroblasts to reticulocyte stage expressed two mRNA species of P-spectrin, whereas they expressed only a single species of a-spectrin, and no a-fodrin mRNA. In contrast, nonerythroid cells (platelets, myeloid cells, liver, muscle, heart, cerebellum, and eye lens) expressed either no a-spectrin mRNA or a different molecular weight transcript(s) of this gene, and a single species of a-fodrin mRNA. Additionally, they also expressed from none to multiple species of P-spectrin, and these were of different molecular size(s) from that found in erythroid cells (with the exception of platelets). Transcripts of non-erythroid spectrin, a-fodrin, were found as a single copy only in non-erythroid tissues. Human and murine erythroleukemia cells expressed both erythroid spectrin transcripts in addition to a-fodrin and raise the possibility that erythroid progenitors may have the potential to express both erythroid and non-erythroid species. These data indicated that several mRNA species of P-spectrin could be detected in both erythroid and some non-erythroid cells. Whether multiple spectrin peptides could also be found with functional heterogeneity is unclear. However, in each case, the pattern combination observed appeared to be tissue-specific

Spectrin is the major protein of the membrane skeleton which underlies the internal side of the erythrocyte membrane (3,12,35,41).Erythroid spectrin is the fibre-like protein composed of two 240,000 and 220,000 dalton homologous but non-identical polypeptides, a and p, which are twisted along each other into a heterodimer. At their head region, spectrin heterodimers are self-associated to form tetramers and higher-degree oligomers (32,54).At their distal end, these tetramers are interconnected into a two-dimensional network by their linkage to oligomers of actin. This interaction is greatly strengthened by protein 4.1, which binds to the distal ends of the spectrin tetramers and also t o the integral glycoproteins (1,4,12). At a site about 20 nm from the distal end of p-spectrin is a binding region for ankyrin which connects the p-spectrin chain to the major transmembrane protein band 3-the anion channel protein (3,431.Thus, the cytoskeleton is attached to the membrane and renders durable flexibility and stability to the cell. Spectrin was initially thought to be unique to erythrocytes (7,35,36) but recently, similar but non-identical spectrin-like peptides (fodrins) have been identified in various non-erythroid cells, suggesting the existence of a family of spectrin genes. Biochemical and functional comparisons of these immunologically cross-reacting proteins have defined them as 0 1990 WILEY-LISS, INC

isoforms of spectrin (5,8,16,19,38,45).The spectrin isoforms show tissue-specific distribution and differential expression during development (5,19,20,28,29,39).Interestingly, another cytoskeletal protein, protein 4.1, also has isoforms (2,13,14,40,51). The number and structure of the genes encoding the spectrin peptides still remain to be fully elucidated. Partial information a t both cDNA and genomic levels has been recently presented (6,15,30,31,37,44). However, information about spectrin transcripts from erythroid purified nucleated precursors has not been presented before. We have examined erythroid and non-erythroid spectrin gene transcripts in various human erythroid and non-erythroid tissues. We have identified multiple transcripts of p-spectrin and a single size message of a-spectrin in human erythroid precursor cells but no a-fodrin transcript. Although p- and a-spectrin transcripts were found in some non-erythroid cells, they were of different size than those found in erythroid precursors cells. Transcripts of non-erythroid spectrin, a-fodrin, were found as a single copy

Received December 13, 1989; accepted April 10, 1990. *To whom reprint requestskorrespondence should be addressed.

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under stringent conditions as described elsewhere (34); specifically, the hybridization was performed with 50% formamide a t 42"C, and the final washing conditions of the filter were 0.1 x SSC buffer at 65°C. The hybridizaMATERIALS AND METHODS tion probes were 32P-labeled by nick translation by using a kit from Bethesda Research Laboratories (GaithPreparation of RNA ersburg, Maryland). The same filter (filter a) was then Subjects undergoing phlebotomies for hemochroma- used originally with a human p-spectrin cDNA probe, tosis served as a source of normal reticulocyte RNA. followed by a n a-fodrin cDNA probe, and then a n aBlood from a n ankyrin-deficient patient also undergo- spectrin cDNA probe. The second Northern blot filter, ing phlebotomies for secondary hemochromatosis (11) containing different tissue mRNAs (filter b), was also and from sickle cell patients under a n exchange trans- used sequentially: first, with a human p-spectrin fusion program was also used as a source of human cDNA probe, followed by a n a-fodrin cDNA probe, and reticulocyte RNA. Total RNA was isolated by the then with a n a-spectrin cDNA probe. method of Goossens and Kan (22) and modified by adSlot blotting and quantitation of ditional purification of the acid RNA precipitate by the spectrin transcripts guanidine HC1-phenol method (10). The normal human bone marrow was prepared from a resected human rib RNA samples were applied on nitrocellulose paper of a patient undergoing exploratory thoracotomy. In by using a slot blot system (Schleicher & Schuell, this particular bone marrow preparation, about 50% of Keene, New Hampshire). The cloned, authentic human its nucleated cells were recognizable erythroid precur- p-spectrin cDNA, and human a-spectrin cDNA (see besors. Human erythroblasts were purified from buffy low) were used as internal standards. The quantitation coat marrow aspirates, or from fetal liver suspension of mRNA by slot blot was performed by using a Helena cells, by direct panning on Ep-I antibody-coated plates densitometer (Helena Laboratories, Beaumont, Texas). (56). These erythroblasts represented a spectrum of The relative amounts of the two species of p-spectrin cells ranging from basophilic erythroblasts to polychro- transcript present in reticulocyte RNA were determatophilic erythroblasts to late orthochromic erythro- mined by the autoradiography of Northern blots by usblasts with polychromatophilic erythroblasts being ing a n LKB 2400 Ultrascan XL laser densitometer predominant. They also included CFU-e (approximate (LKB Produkter, Broma, Sweden). The hybridization Human platelets were obtained and washing conditions were the same a s described abundancy about 1%). from a patient undergoing therapeutic platelet apher- above. Autoradiograms were scanned by densitometer esis for symptomatic essential thrombocythemia. The and the amount of message was calculated by comparapheresis was performed by using a n IBM 2977 Cell ison of the relative intensity of hybridization of the Separator under conditions securing maximum purity sample with that of a known amount of cloned p-specof platelets following the instruction of the manufac- trin cDNA. turer. The usual contamination quoted by the manuPreparation of P-spectrin, a-spectrin, and facturer is 1 leukocyte/lOs platelets. The residual a-fodrin cDNA probes erythrocytes were removed by osmotic lysis. Direct microscopic examination of this platelet preparation reThe human p-spectrin cDNA probe was isolated from vealed less than one erythrocyte and leukocyte per the plasmid pGemp3.7 previously characterized in our 10,000 pheresed platelets. Human liver, left cardiac laboratory (44). A human a-fodrin cDNA probe (37) ventricle, skeletal muscle (biceps), and apheresed T- was made from the plasmid pHaFod2.7A (kindly prolymphocytes (from a patient with atypical chronic lym- vided by Dr. Randall T. Moon, University of Washingphocytic leukemia undergoing therapeutic leukaphe- ton, Seattle, Washington). Taq I polymerase, Sp6, and resis) were also used as a source of RNA. The human T7 promoter primers (Promega Biotec, Madison, Wisand murine erythroleukemia cell lines, HEL (HEL-92 consin) were used to amplify the cDNA insert by polyand HEL-R), K 562, line OCI M2, mouse erythroleuke- merase chain reaction (18,46). The polymerase chain mia cell line MEL (grown with and without agents fa- reaction was cycled 30 times, annealing was performed voring erythroid induction (42), the human myeloid at 37°C for 2 min, denaturing was performed at 94°C cell lines, KG 1 (25), PLB-985 (53), and B1086 (27) for 2 min, and extension a t 72°C was performed for 6 were also used a s a source of RNA and were main- min. The 2.7 kb a-fodrin cDNA band was eluted from tained as previously described. Total RNA from these the agarose preparative gel by electroelution (34). The tissues was prepared by the guanidine HC1-phenol human a-spectrin cDNA probe (15) was prepared from method (10). Poly(A)+ RNA was isolated by oligo(dT)- the plasmid pHcusp5 (kindly provided by Dr. Peter Curcellulose chromatography (34), and by one to three pas- tis) and made by polymerase chain reaction with two sages through oligo(dT) resin. All human tissues were primers of the flanking PstI site of pBR 322. The opobtained from the Tissue Procurement Facility of the erating conditions were similar to the a-fodrin probe University of Alabama at Birmingham (UAB) and pro- preparation (see above). tocols previously approved by the UAB Human Use RESULTS Committee were used. The fact that the abundance of spectrin gene prodNorthern blot hybridization ucts in many of the tissues is extremely low and their Poly(A) + RNA was fractionated by formaldehyde size is large is compounded by limited availability of agarose gel electrophoresis (34) and transferred to ni- some tissues of interest (i.e., purified erythroblasts, trocellulose filters ( 5 2 ) . Hybridization was performed etc.). This required that only the least degraded RNA

only in non-erythroid tissues. Additionally, both erythroid- and non-erythroid-type spectrin transcripts were found in erythroleukemia cells.

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preparations were used for these analyses and several different Northern blots were performed, of which only two are shown here. When only a limited quantity of high-quality RNA was available, all was used for the analysis. Each Northern blot was exposed for variable time intervals in order to characterize optimally the message in a given tissue. Thus, only informative data are shown. For the purposes of size comparison among individual tissues, data obtained from one gel are kept in a separate figure.

Transcripts of p-spectrin P-spectrin mRNA species in reticulocytes. Reticulocytes from subjects with hemochromatosis, sickle cell disease, and a n ankyrin deficiency (11)were the source of RNA for our study. We showed that human reticulocytes do have p-spectrin low-abundance mRNA (Figs. la,b). In addition, P-spectrin mRNA was present as two distinct species. The major species, about 7.8 kb, was approximately seven times more abundant than that of the minor, larger transcript measuring about 9.5 kb. These same two distinct transcripts, in similar relative proportions, were present in comparable low abundance in reticulocytes from one normal, one ankyrin-deficient, and one sickle cell patient (data not shown). P-spectrin RNA species in erythroblasts. Since reticulocytes are enucleated cells representing terminal stages of erythroid cell maturation, we tested whether earlier nucleated precursors expressed the same or additional species of P-spectrin transcripts. Thus, we have repeated the Northern blot analysis with inclusion of bone marrow cells and purified fetal and adult erythroblasts. In these tissues, the qualitatively identical but more abundant transcripts, when compared to reticulocytes, were easily detected, although the total RNA and not the poly (A)' RNA was used (Fig. lb, lanes 3-5). P-spectrin RNA species in erythroleukemia lines. Similar to reticulocytes, human erythroleukemia lines (HEL, K562, OCI M2) contained the major 7.8 kb transcript, but, in contrast, they also contained two other distinct, low molecular weight transcripts estimated to be approximately 4.4and 3.4 kb (see Fig. l a , lane 3). These low molecular weight P-spectrin transcripts were seen on repeated evaluations of erythroleukemia RNAs obtained from three independent batches of cultured cells over a period of about a year. Two higher molecular weight species 19 kb and 10 kb) were observed repeatedly in HEL human erythroleukemia line (see Fig. l a , lane 3). Interestingly, MEL cells were also found to have three distinct mRNA species (7.2, 8.5, and 10 kb) in approximately the same proportion, but in different sizes compared to human erythroid cells (Fig. l b , lane 6). P-spectrinRNA species in human non-erythroid cells. No transcripts of P-spectrin were detected in human poly(A)' RNA preparations from human leukemic lines without erythroid differentiation including KG 1 (251, PLB (53), and B1086 (27). Similarly, no P-spectrin transcripts were detected in adult liver or T-lymphocytes (data not shown). In contrast, a single P-spectrin mRNA of the same size as the major erythroid transcript was detected in human platelets (Fig. l b , lane 9). In order to assure that the P-spectrin

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Fig. 1. a: Representative Northern blot analysis of different erythroid cells analyzed with (Il-spectrinprobe. Lane #1: 25 pg of poly(A)+ RNA from normal reticulocytes. Lane #2: 25 pg of poly(A)+ RNA from a patient with ankyrin deficiency. Lane #3: 30 pg of poly(A)+ RNA from a n erythroleukemia cell line, HEL-92. The filter was hybridized at 42°C with nick-translated 738 bp Hinf I fragment (44) that served as a P-spectrin cDNA probe and washed at 65°C with lowsalt-concentration solution. RNA ladder (Bethesda Research Laboratories, Gaithersburg, Maryland) served as a molecular weight marker. Size markers are in kb. The film was developed after 10 days of exposure at -70°C with intensifying screens. b: Representative Northern blot analysis of selected erythroid and non-erythroid cells analyzed with P-spectrin probe performed at a different time than in a. Lane #1: 30 pg of poly(A) RNA from normal reticulocytes. Lane #2: 32 pg of poly(A) ' RNA from a n erythroleukemia cell line HEL92. Lane #3: 60 pg of total RNA from bone marrow. Lane #4: 24 pg of total RNA from adult erythroblasts. Lane # 5 40 pg of total RNA from fetal erythroblasts. Lane # 6 20 pg of poly(A) RNA from MEL cells. Lane # 7 35 pg of poly(A)+ RNA from left cardiac ventricle. Lane # 8 30 pg of poly(A) RNA from skeletal muscle. Lane #9 30 pg of poly(A) RNA from platelets. Lanes 7' and 8' represent a short exposure (3 hours of lanes 7 and 8.The filter was hybridized at 42°C with nick-translated 738 bp-Hinf I fragment (441,that served as a P-spectrin cDNA probe and washed at 65°C with lowsalt-concentration solution. RNA ladder (Bethesda Research Laboratories, Gaithersburg, Maryland) served as a molecular weight marker. Size markers are in kb. The film was developed after 24 hour exposure (except 7' and 8') at -70°C with intensifying screens. '

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mRNA seen in the platelet poly(A) RNA preparation was not the result of a reticulocyte contamination, the same platelet and reticulocyte RNA as examined in Figure l b was blotted on the nitrocellulose paper and hybridized with a,-globin cDNA (kindly provided by Dr. Steve Liebhaber). No visible globin signal was seen in the platelet preparation, while abundant globin mRNA was present as expected in the reticulocyte RNA. Similarly, to rule out a possible signal from a small amount of contamination by granulocytes, the Northern filter, containing 2 pg of granulocyte poly(A) RNA preparation (kindly provided by Dr. +

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Fig. 2. Representative Northern blot analysis of erythroid and nonerythroid cells hybridized to the human a-spectrin cDNA probe. The filters used in Figure l a (lane #1)and l b (lanes #s 2-6) were stripped of the previously used a-fodrin probe thoroughly and hybridized to the human a-spectrin cDNA probe. Time of exposure indicated in parenthesis. Lane #1: 30 pg of poly(A) t RNA from an erythroleukemia cell line, HEL-92 (7 days). Lane #2: Adult erythroblasts-total RNA (3 days). Lane #3: Fetal erythroblasts-total RNA ( 3 days). Lane # 4 Left cardiac ventricle poly(A)+ RNA ( 3 days). Lane # 5 Skeletal muscle poly(A)+ RNA ( 3 days). Lane # 6 30 pg of poly(A)+ RNA from platelets ( 3 days). The hybridization and washing condition were the same as those used in Figure lb.

Thomas Rado), was hybridized with the same P-spectrin probe and again no detectable signal was seen. Multiple cross-hybridizing signals, clearly different from those seen in erythroid cells, were seen in human heart and skeletal muscle (6.5 kb, 8 kb, and 10 kb), albeit in varying relative proportions (Fig. lb, lanes 7, 8 and 7') 8'). The relative abundance of these signals was particularly high. The molecular sizes of these transcripts contrast with yet different sizes of P-spectrin mRNAs we had previously reported in cerebellum (11 kb) and eye lens (8.6 and 7.4 kbj. In the latter tissue, the abundance of this transcript was unusually high (57-59). Relative amount of P-spectrin transcripts. Accounting for the different amounts of RNA, the different lengths of exposure, and whether the total RNA or poly(A)+ RNA has been used, we estimated that the relative amount of p-spectrin message in the sources examined was as follows (in descending order): 1)highest in bone marrow and fetal erythroblasts. It is estimated that there is 50 times as much 6-spectrin transcript in bone marrow and erythroblasts as in reticulocytes, 2) followed by muscle, heart; 3) HEL and reitculocytes; 4)MEL; and 5 ) the least amount of message was found in platelets. a-spectrin mRNA transcripts In contrast to the finding of multiple transcripts of P-spectrin gene, only a single 9.2 kb a-spectrin mRNA was detected in erythroid tissues-including bone marrow cells, purified adult and fetal erythroblasts, and MEL cells (Fig. 2, lanes 2 , 3 and data not shown). The HEL cells had an additional higher molecular weight of approximately 10 kb a-spectrin transcripts as well as

a low molecular weight of approximately 4.3 kb transcript (see Fig. 2, lane 1). In contrast, unusually high molecular weight transcripts were seen in heart and skeletal muscle of estimated sizes of over 20 kb and 15 kb (Fig. 2, lanes 4,5). The only other tissues where we detected a-spectrin transcript mRNA were cerebellum (about 11 kb) and eye lens (7.5 kb) where we had previously reported (57,591 the a-spectrin transcript distinct from those seen in erythroid cells. No detectable transcripts of a-spectrin were present in T-lymphocytes, platelets (Fig. 2, lane 61, or adult liver, or in human poly(A) RNA preparations from human leukemic lines without erythroid differentiation-KG 1, PLB-985, and B1086. Relative amount of a-spectrin transcripts in different tissues. The abundance of a-spectrin message in the tissues examined is in the following descending order: 1)it is the greatest in bone marrow, fetal erythroblasts, and adult erythroblasts; 2) heart; 3) skeletal muscle; 4) HEL and MEL; and 5) reticulocyte. While there were qualitative differences of a-spectrin transcripts in erythroid, as compared to non-erythroid cells, the relative abundancy of the a-spectrin mRNA does not distinguish erythroid and non-erythroid tissues. +

a-fodrin RNA transcripts In order to examine the presence of the non-erythroid spectrin transcripts, the Northern blots used in the studies described above were hybridized with a human a-fodrin cDNA probe: a distinct band was seen in the erythroleukemia lines at a molecular weight of about 8 kb (Fig. 3a, lane 3 and Fig. 3b, lane 2). The molecular weight of this band differed from that of the P-spectrin transcripts and the a-spectrin transcript. No bands were seen in any of the human reticulocyte preparations (Fig. 3a, lanes 1, 2 and Fig. 3b, lane l),nor in purified fetal and adult erythroblast (Fig. 3b, lanes 4, 5). However, in bone marrow cells (Fig. 3b, lane 31, and in human poly(A)+ RNA preparations from human leukemic lines without erythroid differentiation (B1086-Fig. 3b, lane 6, and KG 1,PLB-985-data not shown), T-lymphocytes, skeletal muscle, MEL cells (data not shown), and platelets (Fig. 3b, lane 8)-the same single transcript of a-fodrin as seen in the HEL cells was identified. In the human heart, the relative amounts of a-fodrin transcript were estimated to be about l o x more than in the skeletal muscle (Fig. 3b, lane 7), and its relative abundancy was greater than those found in any other tissues examined in this report. To summarize these data, no a-fodrin transcript was seen in purified primarily erythroid cells, i.e., reticulocytes, adult or fetal erythroblasts-in contrast to nonerythroid cells and erythroleukemia lines.

DISCUSSION We have found distinct patterns of expression for several spectrin mRNA species in erythroid vs. nonerythroid cells. Two distinct transcripts of P-spectrin, a single distinct transcript of a-spectrin and no expression of a-fodrin, appeared to be characteristic for erythroid precursors from erythroblasts to the reiculocyte stage. By contrast, non-erythroid cells were characterized by the presence of a-fodrin and either the absence

ERYTHROID AND NON-ERYTHROID SPECTRIN TRANSCRIPTS

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4.42.41.4Fig. 3. a: Northern blot hybridization of different erythroid cells analyzed with a-fodrin cDNA probe. The same filter used in Figure l a lanes 1-3 was used after stripping the previously used probe. Lane #1: Normal reticulocytes. Lane #2: Ankyrin deficient patient. Lane #3: Erythroleukemia cell line, HEL-92. The representative samples, together with experimental conditions, were identical to those described in Figure la-except that Ihe film was developed after 3 days at -70°C. b: Representative Northern blot analysis of selected erythroid and non-erythroid cells analyzed with a-fodrin cDNA probe performed a t a different time than in a using the same filter used as in Figures l b and 2. Lane #1: Normal reticulocytes. Lane #2: An erythroleukemia cell line, HEL-92. Lane #3: Bone marrow cells. Lane # 4 Adult erythroblasts. Lane # 5 Fetal erythroblasts. Lane # 6 B1086 cells. Lane # 7 Heart. Lane #8: Platelets. The film was developed after 8 days exposure at -70°C with intensifying screens.

or presence of different size transcripts of a- and Pspectrins.

p-spectrin transcripts Erythroid p-spectrin gene transcripts were detected in normal human erythroid precursors in two distinct transcripts of different relative proportions. In our earlier studies (44), we were unable to detect the small amounts of P-spectrin message in reticulocyte RNA from a sickle cell patient. This was probably a result of our previous inability to obtain a large amount of intact, high molecular weight poly(A)+ RNA from these cells. I n fact, in those experiments, we used only 1.5 kg of poly(A)+ RNA (441, compared to the 25 pg of poly(A) RNA used in these present experiments. +

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With refinement of our techniques, and the availability of larger amounts of blood (one unit or more) as a starting material for RNA preparation, we have now concluded that human reticulocytes do have p-spectrin low-abundance mRNA. These data indicate that the abundance of a- and P-spectrin message decreases from erythroblasts to reticulocytes. It is of interest that the only other hemopoietic cells containing the P-spectrin mRNA were platelets. In these a n mRNA of the same size as that of a major erythroid transcript was seen. Interestingly, protein immunologically cross-reacting with red cell spectrin was reported in platelets (16). In that report, the cross-reacting platelet-derived spectrin contained immunoreactive material at the position of a-spectrin but not P-spectrin; however, a faintly staining additional band was seen that may have represented a P-spectrin species. We could not detect a-spectrin mRNA on the same filter. The reason for the discrepancy between our observations and the above report is not immediately obvious, but may be due to the differences in the sensitivity of different techniques. In contrast, in other hemopoietic cells such as myeloid precursors and T-lymphocytes, the product of the P-spectrin gene was not detected. The finding of erythroid P-spectrin in platelets may have physiologic relevance. Several erythroid markers have been found in megakaryocytes (Epo-R, GF-1) and majority of erythroleukemia lines were reported to have both erythroid and megakaryocytic markers. On the basis of these results it has been speculated that bi-potential, erythroid-megakaryocytic, progenitor may normally exist in bone marrow. The significance of finding two P-spectrin transcripts in human erythroid precursors is not clear. Although there is no evidence of heterogeneity of P-spectrin a t the protein level, the presence of a minor peptide isoform cannot be entirely excluded. An alternative explanation for our findings may be one of the following: alternate site of transcription initiation (24), alternate processing of introns (47-49), use of a different poly(A)+ signal (491, or possibly other newly described types of mRNA processing, such a s RNA editing (50). Interestingly, in birds and humans another cytoskeletal protein, protein 4.1, has been found to have differential processing of RNA from a single gene (23,40,51) and translational control-giving rise to multiple polypeptides of different molecular weights during maturation. In some non-hemopoietic tissues, the P-spectrin gene also appeared to be utilized, although processed differently, in a n apparent tissue-specific manner. p-spectrin transcripts were also present in the human heart and skeletal muscle, wherein three different transcripts, although in different relative proportions, were seen. A recent preliminary description of the differential processing of the 3' region of erythroid p-spectrin mRNA in skeletal muscle (55) indicated that the observed difference between P-spectrin mRNAs of muscle and erythroid cells should be about 300 bp, while our data suggests greater differences in molecular weight. The molecular sizes of these transcripts contrast with yet different sizes of P-spectrin mRNAs we had previously reported in cerebellum and eye lens. In the latter tissue, the abundance of this transcript was unusually high (57,591. Preliminary evidence has recently been

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spectrin (220,000 daltons), the mRNA should be longer than 6 kb; the two shorter P-spectrin transcripts are not likely t o have coding capabilities for spectrin peptides as we know them. Based on several lines of evidence we have established that the smaller molecular weight bands do not represent non-specific hybridization with ribosomal RNA. These observed differences between human erythroleukemias vs. human erythroblasts and reticulocytes may represent a) an earlier differentiation stage of erythroleukemia cells, b) could a-spectrin transcripts be present by virtue of pluri- o r oligopotency of these In contrast to multiple transcripts of P-spectrin seen cells, since they can express other non-erythroid markin erythroid tissues, we detected only a single, 9.2 kb ers such as megakaryocyte-platelet-specific lineage mRNA of a-spectrin in erythroid tissues. The tran- proteins (42), or c) could be features of “tumor” nature script size of 9.2 kb appears to be erythroid tissue spe- of these cells. However, these differences cannot be excific. The relative abundance of a-spectrin in erythroid plained by the presence of nuclei since the same tranprecursors in relation to their mutation stage appears scripts were observed in purified human erythroblasts to be analogous to that of the p-spectrin, i.e., decreas- and bone marrow cells. Thus, the nature of these multiple smaller and larger transcripts of spectrins reing toward the reticulocyte stage. In non-hemopoietic cells, unusually large, cross-hy- mains puzzling. One has to consider that the lower mobridizing bands were seen in Northern blot analyses of lecular weight bands seen on Northern blots could be heart and skeletal muscle tissues. In our hands the due to cross-hybridization of our probes with yet-unpreparation of high-quality RNA from these tissues characterized gene products for smaller species, or may was particularly difficult due to high levels of nuclease represent intermediates in the RNA degradation proactivity and the fact that these autopsy tissues were cess. (Similar faint bands were also seen with the aavailable only several hours after death. These factors spectrin and a-fodrin probes.) Conversely, higher momay account for some observed smearing. However, lecular weight species seen in erythroleukemia cells since these bands on Northern blots were seen in sev- may represent incompletely processed spectrin gene eral blots of RNA from these tissues, and were not seen transcripts. However, since we have seen these smaller in concomitantly handled RNAs prepared from differ- and higher molecular weight species consistently in ent tissues, we believe the cross-hybridizing bands do multiple preparations prepared from several isolates not represent degraded RNA or high molecular weight over period of 1 year we consider these possibilities aggregates (incompletely denatured RNA). Their sur- unlikely. The presence of a-fodrin transcript in erythprisingly large size raises the possibility that our probe roleukemia cells is not surprising since it has been predetected the transcripts of other genes. Interestingly, viously suggested by Glenney and Glenney (1984) and the homology of a-spectrin, a-actinin (17), and dystro- by Mangeat and Burridge (1984) that even precursors phin (26) has been recently reported. Even though the committed solely to erythroid differentiation contain dystrophin gene and its cDNA are very large, it would fodrin. Specifically, Friend erythroleukemia cells were appear that even the reported transcript size of dystro- found to contain fodrin in addition to spectrin (21). A phin (12 kb) is not large enough to account for our microinjection of an affinity-purified polyclonal antiobservations. The only other non-erythroid tissues in fodrin antibody t o various cultured mouse and human which we could detect a-spectrin transcripts were cer- cells was reported to cause distortion and condensation ebellum and eye lens, wherein we detected 11 kb and of intermediate filaments (33). Our data corroborate 7.5 kb transcripts (57,59)-clearly differing from those these observations and underscore the hazard of isolatfound in erythroid tissues. ing and cloning the erythroid spectrin message from these sources. Presence of erythroid- and non-eryrr-fodrin transcripts throid-type spectrins in erythroleukemia cell lines may We also report that the normal human erythroid indicate the bi-potential nature of these cells or be a cells do not contain a detectable a-fodrin gene tran- consequence of leukemic transformation. However, the script. By contrast, more primitive, possibly pluripo- finding of a-fodrin in MEL cells, which are only erytent nucleated hematopoietic cells with a potential for throid cells, raises the possibility that erythroid proerythroid differentiation, such as erythroleukemia genitors (BFUe, CFUe) may have the potential t o exlines, contain a-fodrin transcripts. In all non-erythroid press some a-fodrin, but this potential is extinguished tissues examined, a single 8 kb a-fodrin gene transcript with further erythroid differentiation. was detected, although its relative abundance had varThe nature of the multiple P-spectrin transcripts and ied, being more abundant in the human heart than in their significance needs to be elucidated by further any other tissues examined in this report. Interest- studies. The fact that in some tissues both erythroid ingly, the only other tissue with a distinctly different and non-erythroid genes are transcribed raises the possize of a-fodrin mRNA was eye lens wherein this tran- sibility of the presence of spectrin peptides composed of script was found to be particularly abundant (57,59). mixed heterodimers (of both erythroid and non-erythroid subunits). This report, our previous studies of Spectrin transcripts in erythroleukemia erythroid spectrin transcripts in human lens and cereThe presence of additional and multiple a- and p- bellum (57,59), and a recent demonstration by Winkelspectrin transcripts in erythroleukemia lines has also mann and co-workers (1989) on the differential probeen unexpected. Based on the molecular weight of p- cessing of the 3‘ region of erythroid p-spectrin mRNA

presented for the existence of a separate gene for nonerythroid spectrin-P-fodrin (9). The homology between these genes apparently ranges from 61%to 76%. It is unclear whether the transcript of this gene may be detected by our probe and whether this accounts for some of the mRNAs we describe here. However, the transcript of P-fodrin was detected in human lymphocytes (9),while our p-spectrin probe failed t o react with any mRNA in lymphocyte tissue.

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tein 4.1 of avian erythrocytes is composed of multiple variants that exhibit tissue-specific expression. Cell, 37:595-607. 24. Jahn, C.L., Hutchison, C.A. 111, Phillips, S.J., Weaver, S., Haigwood, N.L., Voliva, C.F., and Edgell, M.H. (1980) DNA sequence organization of the p-globin complex in the BLABic mouse. Cell, 21: 159-168. 25. Koefler, H.P., and Golde, D.W. (1978) Acute myelogenous leukemia. Science, 200t1153-1154. ACKNOWLEDGMENTS 26. Koenig, M., Monaco, A.P., and Kunkel, L.M. (1988) The complete sequence of dystrophin predicts a rod-shaped cytoskeletal protein. This work was supported in part as follows: NIH Cell, 53:219-228. grants HL 37462 and DK-3085, RPB grant of Dpt. of 27. Krauss, R., Lilly, M.B., Prasthofer, E., and Rado, T.A. (1988) Ophthalmology, and VA grant #FICAP/2237. Structure and expression of myeloid and lymphoid genes in a spontaneously arising B-cell line (B1086). Blood Suppl. 72:210a. LITERATURE CITED 28. Lazarides. E.. and Nelson, W.J. (1983) Erythrocyte form of spectrin in cerebellum: Appearance a t a specif& stage in the terminal 1. Anderson, R.A., and Lovrien, R.E. (1984) Glycophorin is linked by differentiation of neurons. Science, 222:931-933. band 4.1 protein to the human erythrocyte membrane skeleton. 29. Lazarides, E., Nelson, W.J., and Kasamatsu, T. (1984) SegregaNature, 307:655-658. tion of two spectrin forms in the chicken optic system: A mecha2. Baines, A.J., and Bennett, V. (1985) Synapsin I is a spectrinnism for establishing restricted membrane-cytoskeletal domains binding protein immunologically related t o erythrocyte protein in neurons. Cell, 36t269-278. 4.1. Nature, 315:410-416. 3. Bennett, V. (1982) The molecular basis for membrane-cytoskel- 30. Leto, T.L., Fortugno-Erikson, D., Barton, D., Yang-Feng, T.L., E’rancke, U., Harris, AS., Morrow, J., Marchesi, V.T., and Benz, eton association in human erythrocytes. J . Cell. Biochem., 18: E.J., J r . (1988) Comparison of nonerythroid alpha-spectrin genes 49-65. reveals strict homology among diverse species. Mol. Cell. Biol., 4. Bennett,, V. (1985) The membrane skeleton of human erythro8tl-9. cytes and its implications for more complex cells. Annu. Rev. 31. Idnnenbach, A.J., Speicher, D.W., Marchesi, V.T., and Forget, Biochem., 54.273-304. B.G. (1986) Cloning of a portion of the chromosomal gene for 5. Bennett, V., Davis, V., and Fowler, W.E. (1982) Brain spectrin, a human erythrocyte alpha-spectrin by using a synthetic gene fragmembrane-associated protein related in structure and function to ment. Proc. Natl. Acad. Sci. USA, 83:2397-2401. erythrocyte spectrin. Nature, 299t126-131. 6. Birkenmeier, C.S., Bodine, D.M., Repasky, E.A., Helfman, D.M., 32. Liu, S.C., Windisch, P., Kim, S., and Palek, J . (1984) Oligomeric states of spectrin in normal erythrocyte membranes: Biochemical Hughes, S.H., and Barker, J.E. (1985) Remarkable homology and electron microscopic studies. Cell, 37587-594. among the internal repeats of erythroid and nonerythroid spec33. Mangeat, P.H., and Burridge, K. (1984) Immunoprecipitation of trin. Proc. Natl. Acad. Sci. USA, 82:5671-5675. nonervthrocvtic soectrin within live cells following microiniec7. Branton, D., Cohen, C.M., and Tyler, J. 11981) Interaction of cytion o? speccrin aAtibodies: Relation to cytoskeletalkucture“. J. toskeletal proteins on the human erythrocyte membrane. Cell, Cell Biol., 98t1363-1377. 24:24-32. 34. Maniatis, T., Fritsch, E.F., and Sambrook, J. (1982) Molecular 8. Burridge, K., Kelly, T., and Mangeat, P. (1982) Nonerythrocyte Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, spectrins: Actin-membrane attachment proteins occurring in Cold Spring Harbor, N.Y. many cell types. J. Cell Biol., 95:478-486. 9. Chang, J.G., Sahr, K.E., and Forget, B.G. (1988)Characterization 35. Marchesi, V.T. (1983) The red cell membrane skeleton: Recent progress. Blood, 61:l-11. of P-fodrin in human lymphoid cells. Blood [Suppl.] 72545. 10. Chromczynski, P., and Sacchi, N. (1987) Single-step method of 36. Marchesi, V.T., and Steers, E., Jr. (1968) Selective solubilization of a protein component of the red cell membrane. Science, 159: RNA isolation by acid gluanidinium thiocyanate-phenol-chloro203-204. form extraction. Anal. Biochem., 162r156-159. 11. Coetzer, T.L., Lawler, J., Liu, S.C., Prchal, J.T., Gualtieri, R.T., 37. McMahon, A.P., Giebelhaus, D.H., Champion, J.E., Bailes, J.A., Lacey, S., Carritt, B., Henchman, H.K., and Moon, R.T. (1987) Brain, M.C., Dacie, Sir J.V., and Palek, J. (1988) Partial ankyrin cDNA cloning, sequencing and chromosome mapping of a nonand spectrin deficiency in severe, atypical hereditary spherocytoerythroid spectrin, human a-fodrin. Differentiation, 34r68-78. sis. N. Engl. J. Med., 318:230-234. 38. Nelson, W.J., and Lazarides, E. (1983)Expression ofthe p subunit 12. Cohen, C.M. (1983) The molecular organization of the red cell of spectrin in nonerythroid cells. Proc. Natl. Acad. Sci. USA, 80: membrane skeleton. Semin. Hematol., 20r141-156. 363-367. 13. Cohen, C.M., Foley, S.M., and Korgsen, C. (1982) A protein im39. Nelson, W.J., and Lazarides, E. (1983) Switching of subunit communologically related to erythrocyte band 4.1 is found on stress position of muscle spectrin during myogenesis in vitro. Nature, fibers of non-erythroid cells. Nature, 299:648-650. 304t363-371. 14. Conboy, J., and Kan, Y.W. (1986) Molecular cloning of protein 40. Ngai, J., Stack, J.H., Moon, R.T., and Lazarides, E. (1987) Regu4.1, a major structural element of the human erythrocyte memlated expression of multiple chicken erythroid membrane skeletal brane skeleton. Proc. Natl. Acad. Sci. USA, 83:9512-9516. protein 4.1 variants is governed by differential RNA processing 15. Curtis, P.J., Palumbo, A., Ming, J., Fraser, P., Cioe, L., Meo, P., and translational control. Proc. Natl. Acad. Sci. USA, 84t4432Shane, S., and Rovera, G. (1985) Sequence comparison of human 4436. and murine erythrocyte alpha-spectrin cDNA. Gene, 36t357-362. 16. Davies, G.E., and Cohen, C.M. (1985) Platelets contain proteins 41. Palek, J . (1987) Hereditary elliptocytosis, spherocytosis and related disorders: Consequences of a deficiency or a mutation of immunologically related to red cell spectrin and protein 4.1. membrane skeletal proteins. Blood, 1.147-168. Blood, 65.52-59. 17. Davison, M.D., and Critchley, D.R. (1988) a-Actinins and the 42. Papayannopoulou, T.H., Nakamoto, B., Kurachi, S., and Nelson. DMD protein contain spedrin-like repeats. Cell, 52r159-160. R. (1987)Analysis of the erythroid phenotype of HEL cells: Clonal variations and the effect of inducers. Blood, 70:1764-1772. 18. Erlich, H.A., Gelfand, D.H., and Saiki, R.K. (1988) Specific DNA amplification. Nature, 331,461-462. 43. Pasternack, G.R., Anderson, R.A., Leto, T.A., and Marchesi, V.T. (1985) Interactions between protein 4.1 and band 3. J . Biol. 19. Glenney, J.R., Jr., Glenney, P., and Weber, K. (1982) Erythroid spectrin, brain fodrin, and intestinal brush border proteins (TWChem., 260:3676-3683. 2601240) are related molecules containing a common calmodulin- 44. Prchal, J.T., Morley, B.J., Yoon, S.H., Coetzer, T., Palek, J., Conbinding subunit bound to a variant cell type-specific subunit. boy, J . , and Kan, Y.W. (1987) Isolation and characterization of cDNA clones for human erythrocyte P-spectrin. Proc. Natl. Acad. Proc. Natl. Acad. Sci. USA, 79t4002-4005. 20. Glenney, J.R., Jr., and Glenney, P. (1983) Fodrin is the general Sci. USA, 84:7468-7472. 45. Repasky, E.A., Granger, B.L., and Lazarides, E. (1982) Widespectrin-like protein found in most cells whereas spectrin and the spread occurrence of avian spectrin in nonerythroid cells. Cell, TW protein have a restricted distribution. Cell, 34:503-512. 21. Glenney, J.R., Jr., and Glenney, P. (1984) Co-expression of spec29:821-833. trin and fodrin in Friend erythroleukernic cells treated with 46. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., DMSO. Exp. Cell Res., I52r15-21. Horn, G.T., Mullis, K.B., and Erlich, H.A. (1988) Primer-directed 22. Goossens, M., and Kan, Y.W. (1981) DNA analysis in the diagenzymatic amplification of DNA with a thermostable DNA polynosis of hemoglobin disorders. Methods Enzymol., 76r805-817. merase. Science, 239r487-494. 23. Granger, B.L., and Lazarides, E. (1984) Membrane skeletal pro47. Schmelzer, C., and Muller, M.W. (1987) Self-splicing of group I1

in skeletal muscle (55) indicate the arbitrariness ofthe concept Of “erythroid” and “non-erythroid” spectrin genes. However, for purposes Of continuity, it may be practical to retain the erythroid and non-erythroid spectrin gene terminology.

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introns in uztro: Lariat formation and 3' splice site selection in mutant RNAs. Cell, 51r753-762. Schwarzbauer, J.E., Tamkun, J.W., Lemischka, I.R., and Hynes, R.O. (1983) Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell, 35:421-431. Sharp, P.A. (1987) Splicing of messenger RNA precursors. Science, 235:766-771. Shaw, J.M., Feagin, J.E., Stuart, K., and Simpson, L. (1988) Editing of kinetoplastid mitochondria1 mRNAs by uridine addition and deletion generates conserved amino acid sequences and AUG initiation codons. Cell, 53:401-411. Tang, T.K., Leto, T.L., Correas, I., Alonso, M.A., Marchesi, V.T., and Benz, E.J., J r . (1988) Selective expression of a n erythroidspecific isoform of protein 4.1. Proc. Natl. Acad. Sci. USA, 85: 3713-3717. Thomas, P.S. (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA, 77t5201-5205. Tucker, K.A., Killy, M.B., Heck, L., and &do, T.A. (1987) Characterization of a new human diploid myeloid luekemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity. Blood, 70(2):372-378.

54. Ungewickell, E., and Gratzer, W. (1978) Self-association of human spectrin: A thermodynamic and kinetic study. Eur. J . Biochem., 88t379-385. 55. Winkelmann, J.C., Costa, F.F., and Forget, B.G. (1989) Expression of the red erythroid p spectrin gene% nonerythroid tissues: Evidence for tissue-specific differential processing of 3' p spectrin pre-mRNA. J . Cell. Biochem. [Suppl.], 13B;221. 56. Yokochi, T., Brice, M., Rabinovitch, P.S., Papayannopoulou, T., and Stamatoyanuopoulos, G. (1984) Monoclonal antibodies detecting antigenic determinants with restricted expression on erythroid cells from the erythroid committed progenitor level to the mature erythroblast. Blood, 63:1376-1384. 57. Yoon, S.-H., Skalka, H., and Prchal, J.T. (1988) Presence of erythroid and non-ervthroid sDectrin transcriDts in human lens and cerebellum. Blood ISuppl.1: 72(5):35a. 58. Yoon, S.-H., Papayannopoulou, T., and Prchal, J.T. (1988) Characterization and quantitation of a-spectrln mRNA in erythroid precursors, Blood iSuppl.1, 72151.106a. 59. Yoon, S.-H., Skalka, H., and Prchal, J.T. (1989) Presence of erythroid and nonerythroid spectrin transcripts in human lens and cerebellum. J . Invest. Ophthalmol., 30t1860-1866.