Proc. Nat. Acad. Sci.
USA
Vol. 70, No. 6, pp. 1809-1813, June 1973
Quantitative Deficiency of Chain-Specific Globin Messenger Ribonucleic
Acids in the Thalassemia Syndromes
(RNA-DNA hybridization/RNA-dependent DNA polymerase/mouse ascites tumor cell-free system)
DAVID HOUSMAN*, BERNARD G. FORGETt, ARTHUR SKOULTCHIT, AND EDWARD J. BENZ, JR.t
*Department of Biology, Massachusetts Institute of Technology, Cambridge, Mass. 02139; and the t Division of Hematology of the
Department of Medicine, Children's Hospital Medical Center, and the Department of Pediatrics, Harvard Medical School,
Boston, Massachusetts 02115
Communicated by William B. Castle, March 29, 1973
ABSTRACT A hybridization assay procedure was de-
vised that makes possible quantitation of the ratio of
mRNA of alpha to mRNA of beta globin chains in an RNA
sample. The assay uses the radioactive synthetic DNA
copies obtained by incubation of RNA-dependent DNA
polymerase of avian myeloblastosis virus with rabbit globin
mRNA that is 80-90%4 enriched in mRNA specific for synthe-
sis of alpha or beta globin chains. The rabbit alpha-chain
mRNA is obtained from the postribosomal supernatant
of rabbit reticulocyte lysates; the rabbit beta-chain mRNA
is obtained from the largest polysomes of rabbit reticulo-
cytes treated with L-O-methylthreonine. Sufficient homol-
ogy exists between rabbit and human globin chains and
globin mRNAs that the synthetic DNA copies of chain-
specific rabbit globin mRNA hybridize with human globin
mRNA. Applied to the study of globin mRNA isolated
from reticulocytes of humans with alpha and beta thal-
assemia, the technique revealed marked quantitative
deficiency of alpha-chain mRNA relative to beta-chain
mRNA in alpha thalassemia and similar deficiency of
beta-chain mRNA relative to alpha-chain mRNA in beta
thalassemia. The thalassemia' syndromes are therefore
characterized by true quantitative deficiency of the mRNA
specific for the affected globin chain.
The thalassemia syndromes are inherited disorders of hemo-
globin (Hb) synthesis characterized by absent or decreased
synthesis of alpha (a) or beta (f) globin chains of human adult
hemoglobin (1-5). When there is some synthesis of the affected
globin chain, analysis of its peptides reveals no evidence of an
aminoacid substitution (6-8), nor is there evidence of synthesis
of incomplete globin chains where there is total absence of the
globin chain (9). There is a great deal of evidence, however,
that the genes for a and f thalassemia are closely linked to the
genes for the structural loci of the a and f globin chains,
respectively (1).
Studies on various aspects of Iolypeptide chain initiation
and translation in homozygous f thalassemia have revealed no
abnormalities (10-15). However, when globin mRNA isolated
from fi thalassemia reticulocytes is added to a heterologous
cell-free system, much less human fi chain is synthesized than
a chain (16-18). The opposite is observed in the a thalassemia
syndrome of Hb H disease (19, 20). This demonstration of a
deficienev of functional chaiin-specific mRNA in both a and f
thalassemia, however, does not rule out the possibility that
Abbreviations: cDNA, synthetic DNA copy of mRNA; Hb,
hemoglobin.
I Present address: Department of Biology, Yale University, New
Haven, Conn.
Please address reprint requests to: Dr. B. Forget, Children's
Hospital Medical Center, Boston, Maass. 02115.
1809
there exists in these conditions a normal amount of a struc-
turally and functionally abnormal mRNA. Recent studies in
,3 thalassemia of the Ferrara type, in which there is a total
absence of fl-chain synthesis (beta0 thalassemia), suggest that
strict deficiency of mRNA may not be the only factor in-
volved in that type of fl thalassemia because, in a cell-free sys-
tem, the addition of ribosome-free supernatant fraction, from
reticulocytes of nonthalassemic patients containing Hb A or
Hb S, to ribosomes of these patients with beta0 thalassemia,
leads to the synthesis of some betaA, but not betas, globin
chain(21, 22).
Our approach to the problem has been to devise specific
assays for a- and f-chain mRNA that do not depend on the
ability of the RNA to function as messenger. It is possible to
synthesize a radioactive DNA copy of globin mRNA (cDNA)
by using the RNA dependent-DNA polymerase of avian
myeloblastosis virus (23-25). This DNA can be used in DNA-
RNA hybridization assays to identify and quantitate globin
mRNA in an RNA sample. Furthermore, it is possible to
isolate rabbit globin mRNA that is 80-90% pure for a (26) or
fi (27) globin mRNA activity when translated in a cell-free
system. DNA synthesized from such chain-specific mRNA
can be used to quantitate the ratio of a to fi mRNA in a given
preparation of rabbit globin mRNA (D. Housman, A.
Skoultchi, & G. Temple, manuscript submitted). We have ex-
tended these methods to the study of human globin mRNA.
Because there is sufficient cross-hybridization between human
globin mRNA and rabbit globin cDNA (24, 25), we have been
able to use the chain-specific rabbit globin cDNAs to quanti-
tate the ratio of a to fi globin mRNA in the reticulocyte RNA
of humans with different thalassemia syndromes.
Our results indicate that in the a thalassemia syndrome of
Hb H disease the a/fl mRNA ratio is 1: 6, whereas in homo-
zygous fl thalassemia the a/f mRNA ratio is 10: 1.
MATERIALS AND METHODS
Materials. RNA-dependent DNA polymerase of avian
myeloblastosis virus, a gift of I. Verma, D. Smoler, and D.
Baltimore, was purified as described (28, 29). Oligo(dT)12-16
was obtained from Collaborative Research Inc., Waltham,
Mlass. [3H]dGTP (specific activity 8 Ci/mmol) and [3H]
leucine (38.8 Ci/mmol) were obtained from New England
Nuclear Corp. Aspergillus oryzae SI nuclease was purified from
amylase type IV-A (Sigma Chemical Co.) as described (30, 31).
Escherichia coli tRNA was obtained from Schwarz/M\lann and
was extracted with phenol before use. Peripheral blood con-
taiiming 5-20% reticulocytes was obtained from patients with
1810 Medical Sciences: Housman et al.
1 2.5 5 to
ng RNA ADDED
FIG. 1. (A) Hybridization of rabbit a and (3 mRNAs with
[3H] DNA copy of rabbit a mRNA. Hybridization was done
for 40 hr at 650. The indicated amount of RNA added represents
total A260 units of the rabbit mRNA sample present (1 ,g =
0.025 A260 units). The cDNA input was 137 cpm per reaction
mixture over a background of 20 cpm; the digested blank reaction
mixture without mRNA contained 8 cpm above background.
Arrows indicate the points where half-saturation of hybridization
is achieved. (B) Hybridization of rabbit a and , mRNAs with
[3H]DNA copy of rabbit ,B mRNA. The conditions are the same
as in (A). The cDNA input was 350 cpm per reaction mixture
over a background of 20 cpm; the digested blank reaction mix-
ture without mRNA contained 15 cpm above background.
homozygous a thalassemia (Hb H disease), homozygous ,(
thalassemia, and a nonthalassemic patient with Hb A and
hemolytic anemia.
Preparation of Human and Rabbit Globin mRNA. Rabbit
globin mRNA was obtained as described (27). Globin mRNA
enriched for rabbit (3-chain mRNA was isolated from the
largest polysomes of reticulocytes treated with L-O-methyl-
threonine (27) and was the gift of G. Temple. Globin mRNA
enriched for rabbit a-chain mRNA was isolated from ribosome-
free, reticulocyte lysate supernatant fraction (26) and was the
gift of Mr. H. Meade and Dr. C. Baglioni. The chain-specific
rabbit mRNAs were purified by repeated sucrose gradient
centrifugations until they yielded a single RNA peak with a
sedimentation coefficient of 10 S. Translation of the mRNAs
in the cell-free system of Krebs II mouse ascites tumor
showed them to be about 90% pure for a or (3 mRNA activity.
Human reticulocyte RNA was prepared by phenol-cresol ex-
traction of membrane-free reticulocyte lysates (17), and the
total cellular RNA was fractionated by sucrose gradient
centrifugation. The RNA sedimenting between 4 and 18 S
was precipitated and served as the partially purified human
globin mRNA samples used for cell-free synthesis, hybridiza-
tion to cDNA, and synthesis of human cDNA. The amount of
1OS globin mRNA in such human mRNA samples was quanti-
tated by acrylamide gel electrophoresis of the RNA and scan-
ning of the unstained gels at A260 (17); the 10S RNA was
usually 25-30% of the total RNA in the sample, and the
amount of RNA used in the hybridization experiments shown
in Figs. 2 and 3 refer to 10S RNA, not total RNA. 1 ,ug of
RNA was considered to be equivalent to 0.025 A260 units.
Synthesis of Radioactive DNA Copy of Globin mRNA. Single-
stranded DNA copies of high specific radioactivity were ob-
Proc. Nat. Acad. Sci. USA 70 (1973)
tained by incubation of the mRNAs with partially purified
RNA-dependent DNA polymerase of avain myeloblastosis
virus, oligo(dT)12_16, [3H]dGTP, and actinomycin D (23).
The DNA product was incubated at 370 for 18 hr in 0.3 N
KOH, then neutralized with HCL. E. coli tRNA (50 ,g) was
added as carrier. The DNA was isolated by Sephadex G-150
gel filtration in 0.1 M ammonium bicarbonate buffer, lyophil-
ized, and resuspended in H20. The cDNA obtained from par-
tially purified (nonthalassemic) human globin mRNA and that
obtained from the single 1OS RNA band isolated by acrylamide
gel electrophoresis of the same human mRNA, were used as
templates for synthesis of 32P-labeled RNA by E. coli RNA
polymerase. Identical fingerprint patterns were obtained from
the [32P]RNA copies of both types of cDNA, and the nucleo-
tide sequences of most of the oligonucleotides match known
aminoacid sequences in either a or 3 globin chains (32). Fur-
thermore, the RNA transcript from the a thalassemia cDNA
used in the experiments to be presented below yields a similar
fingerprint pattern, but with definite quantitative differences
between the oligonucleotides: one very prominent set and one
very faint set of oligonucleotides. The sequences present in the
prominent oligonucleotides match (3-chain, but not a-chain,
aminoacid sequences (32).
Hybridization Assays contained in 5 ul, 0.2 M sodium phos-
phate buffer (pH 6.8), 0.5% sodium dodecyl sulfate, 100-300
cpm of [3H ]DNA copy, and 0-10 ng of mRNA. The mixture
was incubated for 40 hr at the indicated temperatures, in
ng RNA ADDED
FIG. 2. (A) Hybridization of human mRNAs with ['HI DNA
copy of rabbit a mRNA. Hybridization was done as in Fig. 1.
The indicated amount of RNA added represents total lOS RNA
added, as determined by acrylamide gel electrophoresis of the
mRNA. The input cDNA and the blank were the same as in
Fig. 1A. (B) Hybridization of human mRNAs with ['H]DNA
copy of rabbit (3 mRNA. The conditions are the same as in (A).
The cDNA input was 180 cpm per reaction mixture over a back-
ground of 20 cpm; the digested blank reaction mixture without
mRNA contained 45 cpm above background. 0, Human RNA
(nonthalassemia); X, (3-thalassemia RNA; A, a-thalassemia
RNA.
 Proc. Nat. Acad. Sci. USA 70 (1978) mRNA Deficiency in Thalassemia 1811

sealed capillary tubes. The amount of labeled DNA hybrid-

ized was determined by diluting the reaction mixture into 2
ml of a solution containing 0.1 M sodium acetate (pH 4.5),
1 mM ZnSO4, 10 Ag/ml of calf-thymus DNA (heat-denatured),
and sufficient Aspergillus oryzae S1 nuclease to digest de-
natured 3H-labeled DNA of bacteriophage P22 so that less
than 5% of its cpm remained acid-precipitable, while no more
than 5% acid-soluble counts were released from the native
(double-stranded) phage DNA; incubation was for 30 min at
45°. The remaining nuclease-resistant trichloroacetic acid-
precipitable counts (less the counts in a similarly processed
blank containing cDNA but no RNA) were taken to represent
the specific hybrid; percent hybridization was calculated in
reference to the acid-precipitable counts in a similarly in-
cubated but nondigested blank reaction mixture containing
cDNA but no RNA.
RESULTS
Hybridization of Rabbit Globin mRNA to Rabbit cDNA. Our
hybridization experiments measure the ratio of a-chain mRNA
to f-chain mRNA in a given sample. We perform a saturation
curve for each RNA preparation against ['H ]a-chain and ['H ]
f-chain cDNA of the same specific activity. The ratio of the
amount of RNA required to reach a saturation value in
hybridization with f cDNA to the amount required to reach a 4 --I

saturation value of hybridization with a cDNA gives a direct

measurement of the ratio of a-chain mRNA sequences to (-
chain mRNA sequences in the sample. The validity of this
measurement depends on two considerations: (i) The cDNAs
are composed predominantly of sequences complementary to
a- and (-chain mRNAs; (ii) cross-hybridization between a-
chain mRNA and f-chain cDNA and between (-chain mRNA
and a-chain cDNA does not take place.
Detailed experimental support for these propositions will be
presented elsewhere (Housman, Skoultchi, and Temple, manu-
script submitted). In brief, these results are as follows:
(i) Fingerprint analysis of the RNA transcript of human
cDNA strongly indicates that only sequences complementary
to globin mRNA are present in the cDNA preparations.
(ii) Hybridization was performed under conditions that
should favor duplex formation between f-chain cDNA and
a-chain mRNA by incubation of a cDNA with a large excess
of an RNA preparation containing a and ( mRNAs in a ratio
greater than 5:1 [a mRNA (26)]. No hybrids between a
mRNA and (3 cDNA sequences were detected, as judged by
analysis of the melting temperature of the hybrids formed.
(iii) The results of hybridization of rabbit a mRNA and fl
mRNA to cDNA synthesized from unfractionated rabbit
globin mRNA (a/f ratio = 1) indicate that this cDNA is a
representation of a and (3 mRNA sequences in about the same
proportions as in the template RNA and that no additional
sequences are present in the cDNA in amounts greater than
10%.
Analysis of the saturation curves for rabbit a mRNA and
rabbit ( mRNA (Fig. 1) gives an a mRNA to (3 mRNA con-
tent of about 8/1 for the rabbit a mRNA preparation and 1/6
for the rabbit (3 mRNA preparation. These values are deter-
mined by dividing the amount of RNA required to give half
of the saturation value obtained in hybridization of this RNA
against rabbit ( cDNA by the amount of this same RNA re-
quired to give half of the saturation value obtained in hy-
bridization of this RNA against rabbit a cDNA. For the rabbit
ng RNA ADDED
FIG. 3. Hybridization of human mRNAs with [3H] DNA copy
of human a-thalassemia mRNA (human (3 eDNA). (Symbols
are the same as in Fig. 2.) (A) Hybridization was done at 650
for 40 hr. The cDNA input was 232 cpm per reaction mixture
over a background of 20 cpm; the digested blank reaction mixture
without mRNA contained 18 cpm above background. The amount
of human 10S mRNA added was determined as in Fig. 2. (B)
The conditions were the same in (A), except that the temperature
was 78°. The cDNA input was 211 cpm per reaction mixture
over a background of 19 cpm; the digested blank reaction mixture
without mRNA contained 0 cpm above background.
a mRNA preparation, for example, this half-saturation value
corresponds to 2.5 ng in hybridization against rabbit fi cDNA,
and 0.3 ng in hybridization against rabbit a cDNA; for the
rabbit (3 mRNA preparation, these values are 0.5 ng and 3 ng,
respectively. The ratio of these values is a direct measure of
the ratio of a to ( mRNA sequences in these RNA prepara-
tions, and indicate that the a mRNA has (3 mRNA in it to the
extent of 12% and that the (3 mRNA has a mRNA in it to the
extent of 16%. This procedure is used for determining the ratio
of a to (3 mRNA sequences in each RNA preparation con-
sidered below.
Hybridization of Human Globin mRNA to Chain-Specific
Rabbit cDNA. Hybridization of total (or mixed) human globin
mRNA to rabbit a- and f-chain-specific cDNA is shown in
Fig. 2. With both cDNAs, half-saturation of hybridization is
obtained with about the same amount of RNA (0.2-0.25 ng).
This result indicates that the relative amounts of a and (3
mRNA in the human RNA are about equal. The maximum
amount of hydridization or percent hybridization of human
mRNA is higher with rabbit a cDNA (70-80%) than with
rabbit (3 cDNA (40%). This difference in percent hybridiza-
tion of human RNA with the different rabbit cDNAs could
be due to several factors. One possible explanation is that
 1812 Medical Sciences: Housman et al.
differences in the third or "wobble" position of each codon
may be much more frequent in 3 mRNA sequences between
human and rabbit than they are in the a mRNA sequences.
Hybridization of a-Thalassemia mRNA with Chain-Specific
Rabbit cDNA. The results of hybridization obtained with
reticulocyte mRNA from a patient with a thalassemia (Hb
H disease) are shown in Fig. 2. The ratio of a/f mRNA
sequences determined by half-saturation values of the RNA
against fi cDNA (0.5 ng) and a cDNA (2.9 ng) is roughly 1/6
and indicates a marked deficiency of a mRNA relative to fi
mRNA in the a-thalassemia RNA. Since the sample includes
RNA from a whole-cell lysate rather than a polysome prepara-
tion, these results rule out the presence of a significant amount
of functionally deficient a-chain mRNA that is unable to
enter polysomes.
Although the a//3 globin chain synthetic ratio of this pa-
tient's intact reticulocytes was 1/3 (data not shown), the
mRNA isolated from these reticulocytes showed a much more
profound deficiency of a mRNA activity, when translated in
the cell-free system of Krebs II mouse ascites tumor (19):
the a/f globin chain synthetic ratio in the cell-free system was
1/25 (data not shown). The a/fl mRNA ratio obtained by
hybridization gives an intermediate value between these two
extremes. In a second unrelated patient with Hb H disease,
similar hybridization anid intact-cell and cell-free globin syn-
thesis results were obtained. Discrepancy between intact-cell
and cell-free mRNA-directed protein synthesis in a thalassemia
has also been reported by others (20). All of these findings may
indicate the presence, in the a-thalassemia reticulocyte, of
interchain protein synthesis regulation, and translational de-
pression of f-chain synthesis or release, due to the absence of
a chains. A role of a chains in the regulation of f-chain syn-
thesis has been suggested (33-37).
Hybridization of f-Thalassemia mRNA with Chain-Specific
Rabbit cDNA. The fi-thalassemia mRNA shows the same
pattern of hybridization with rabbit a cDNA as the normal
human mRNA (Fig. 2A): half-saturation of hybridization is
achieved with 0.25 ng of RNA. Hybridization of the ,B-thal-
assemia mRNA with rabbit fi cDNA revealed half-saturation
of hydridization achieved with 0.5 ng of RNA (Fig. 2B).
These results indicate the presence of only twice as much a as
non-a mRNA in the fi-thalassemia mRNA. In the cell-free sys-
tem this same mRNA promotes the synthesis of only 1/10 as
much ,B chain as a chain, but it also promotes the synthesis of
significant amounts of the y chain of fetal hemoglobin
which is usually increased in fi thalassemia. There is about half
as much -y- as a-chain synthesis in the cell-free system,
whereas there is 1/3 as much y- as a-chain synthesis in
the patient's intact reticulocytes (data not shown). Because
there is a good deal of homology between the human y
and ,B globin chains (39 amino acids different out of 146), the
human -y mRNA present in the sample could be hy-
bridizing with the rabbit ,B cDNA and giving a falsely high
value for fi mRNA content in the sample. For this reason a
more specific test for quantitation of human fi mRNA was
devised.
Hybridization of Human mRNA with Human f cDNA. Two
major variables were altered to permit quantitation of ,B
mRNA (relative to a mRNA) in the presence of an excess of
human -y-chain mRNA. First, human fl-chain [3H ]cDNA
was synthesized and replaced rabbit fl-chain cDNA in the
Proc. Nat. Acad. Sci. USA 70 (1973)
hybridization mixture. Secondly, the hybridization tempera-
ture was increased from 650 to 780 in order to render less stable
any -y mRNA-,B cDNA hybrids.
To obtain cDNA containing predominantly sequences com-
plementary to human fl-chain mRNA, we took advantage of
our previous demonstration of marked a-chain mRNA de-
ficiency in a-thalassemia mRNA. Fingerprint analysis of the
RNA copy synthesized from this cDNA bears out the conten-
tion that this cDNA contains predominantly fl-chain se-
quences.
Initial hybridization experiments were done at 650. The
results are shown in Fig. 3A. With the normal (nonthal-
assemic) human mRNA, half-saturation of hybridization is
achieved with the same amount of RNA (0.25 ng) as required
to achieve half-saturation of hybridization against the rabbit a
cDNA (Fig. 2A); this result again indicates about equal
amounts of and B mRNA in normal human RNA.
a
The a/fl mRNA ratio obtained, by the same calculation, for
the f-thalassemia mRNA in this experiment is 4.8/1 (1.2 ng
against human cDNA/0.25 ng against rabbit a cDNA).
However, the plateau value for fi-thalassemia mRNA hy-
bridized to human cDNA (% hybrid) was lower (50%) than
that for the normal human mRNA (80%). This observation
suggested the possibility that some of the hybrids formed at
650 were duplexes between -y mRNA and cDNA.
Analysis of the melting profile of the hybrids formed at 650
confirmed that a significant fraction (30%) of the fi-thal-
assemia mRNA-human cDNA hybrids had a lower melting
temperature (800) than the hybrids between normal human
mRNA and human cDNA (930) (data not shown). To elim-
inate this low-melting component, which we believe to be due
to hybrids between y-chain mRNA and fl-chain cDNA,
we increased the temperature of hybridization to 780. The re-
sults of the 780 hybridization are shown in Fig. 3B: no change
occurred in the saturation curve of normal (human) mRNA
compared to the results obtained at 650. However, the amount
of fi-thalassemia mRNA required to reach half-saturation of
hybridization was much greater than at 650 (5 ng instead of
1.2 ng). The ratio of a-chain mRNA to f-chain mRNA in the
B-thalassemia RNA is therefore about 10:1 (5 ng against
human cDNA/0.5 ng against rabbit a cDNA). Again, as the
ratio of a to mRNA in total cellular RNA rather than poly-
somal RNA has been determined, it seems very unlikely that
a significant amount of fl-chain mRNA sequences that cannot
be translated is present in these cells.
DISCUSSION
A large amount of experimental evidence has accumulated that
indirectly suggests that there must exist a quantitative defi-
ciency in mRNA for one or another globin chain in the various
thalassemia syndromes. Even the demonstration that mRNA
isolated from thalassemic reticulocytes reproduces, in heterol-
ogous cell-free systems, the imbalance of globin-chain syn-
thesis characteristic of intact thalassemic reticulocytes, does
not rule out the possibility of a structurally and functionally
abnormal mRNA for the affected chain, present in normal
amounts within the thalassemic reticulocytes. The results of
the hybridization experiments described in this report provide
direct evidence for the suspected quantitative deficiency of
chain-specific globin mRNA in both a- and fi-thalassemia
syndromes. This direct demonstration is provided not by a
functional test of the mRNA but by a quantitative chemical
 Proc. Nat. Acad. Sci. USA 70 (1973)
assay: a DNA-RNA hybridization assay capable of detecting
the presence of nanogram amounts of a specific mRNA mole-
cule. Previous attempts-to demonstrate deficiency of mRNA
in thalassemia by similar hybridization techniques (18) have
not given clear-cut results because of the use, as hybridization
probes, of DNA copies of total globin mRNA containing copies
of both a and mRNA in presumably equal amounts. In the
current study, we have used the DNA copies of chain-specific
mRNA for rabbit a and globin chain. By comparing the
point at which a standard set of dilutions of a mRNA prepara-
tion achieves half-maximum hybridization with and then
with a globin cDNA, one obtains a quantitative ratio of a to
mRNA content of the given RNA sample. Nonthalassemic
samples give a ratio of 1 to 1; on the contrary, in a thalassemia
the a to ,B mRNA ratio is roughly 1 to 6, and in B thalassemia
this ratio is roughly 10 to 1. These findings provide strong
evidence against the hypothesis that there exists, in the usual
types of thalassemia, a mRNA that is functionally inactive
but present in normal amounts. Such a mRNA would still be
expected to have sufficient sequence homology with the nor-
mal mRNA to be detected in the hybridization assay.
Demonstration of a quantitative deficiency in a or mRNA
in thalassemia leaves open the question of the molecular basis
for this defect. One possibility is that the deficient mRNA
sequence is not transcribed as efficiently or processed and
transported from nucleus to cytoplasm as effectively as are
normal mRNA sequences. Alternatively, a quantitative de-
ficiency could result from a less-stable mRNA due to an altera-
tion in its primary or secondary structure. Although in homo-
zygous and heterozygous thalassemia there is more /3-chain
synthesis relative to a-chain synthesis in the bone-marrow
cells than in the peripheral blood reticulocytes (38, 39), it is
not known whether this discrepancy is due to differences in
the /3/a mRNA content present in marrow cells and reticulo-
cytes. Comparison of the /3/a mRNA content in thalassemic
marrow RNA to that found in corresponding reticulocyte
RNA should provide some further insight into the problem.
Finally, application of the hybridization technique to the
study of Ferrara-type thalassemia should help resolve the
issue of whether or not /3-chain mRNA is present in that syn-
drome in normal amounts.
We thank Drs. D. Baltimore, D. G. Nathan, J. Huberman, and
P. Gross for generous support and encouragement; Dr. G. Tem-
ple, H. Meade, and Dr. C. Baglioni for use of their chain-specific
rabbit globin mRNAs; Dr. I. Verma, D. Smoler, and Dr. D.
Baltimore for the purified avian myeloblastosis virus RNA-
dependent DNA polymerase. This work was supported in part
by the following grants from the National Institutes of Health:
CA-13472, AM-15929, and AM-05581. D.H. and A.S. are fellows
of the Jane Coffin Childs Memorial Fund for Medical Research.
B.G.F. is the recipient of a Public Health Service Research Career
Development Award, AM-70234.
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