THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 6, Issue of March 25. pp. 3183-3188, 1982 Printed in U S A.

The Aminoacyl-tRNA Population of Human Reticulocytes*

(Received for publication, November 23, 1981)

Dolph Hatfield+, Frederick Varricchiogl, Mary Rice+, and Bernard G. Forget11 From the *Laboratory of Molecular Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205, the §Memorial Sloan-Kettering Cancer Center, New York, New York 10021, and the IlDepartments of Internal Medicine and Human Genetics, Yale 1Jniuersity School of Medicine, New Haven, Connecticut 06510

The aminoacyl-tRNA population of human reticulo- cytes has been examined. These studies include: 1) determination of the levels of amino acid acceptance for 20 aminoacyl-tRNAs; 2) comparison of 20 amino- acyl-tRNAs from human reticulocytes to those of rabbit reticulocytes by reverse phase chromatom-aphy; 3) comparison of the levels of ne- and Leu-tRNAs in fetal and adult reticulocytes; and 4) determination of the codon recognition properties of human Ala-, Asn-, His-, Leu-, Thr-, and Val-tRNAs. These studies provide evidence that the aminoacyl-tRNA population of hu- man reticulocytes is adapted to the requirements of protein synthesis.

tors which were observed in other mammalian tissues were detected in low levels or not at all in rabbit reticulocytes (14) and the corresponding codewords were utilized infrequently or not at all in rabbit globin mRNA (10, 11). These observa- tions provide further support that the aminoacyl-tRNA pop- ulation of cells, at least those cells engaged in making large quantities of a few proteins, are adapted to the requirements of protein synthesis.

The sequences of a (15, 16), p (17,18), and y (19,20) human globin mRNAs have been determined recently. Thus, an analysis of the tRNA population of human reticulocytes would determine whether it is adapted to the requirements of globin synthesis. In the present study, the aminoacyl-tRNA popula- tion of human reticulocytes has been examined.

A significant correlation between the amino acid composi- MATERIALS AND METHODS tion of proteins and levels of the corresponding aminoacyl- tRNAs has been observed in microorganisms (1) and in plant and animal tissues which are specialized in making large quantities of a few proteins (2-6). Such observations have led Garel (3) and others (2, 4) to propose that the levels of isoacceptor aminoacyl-tRNAs in cells are adapted to the requirements of protein synthesis. Other studies, which sup- port this hypothesis, are those demonstrating that specific mRNAs are efficiently translated in vitro in the presence of tRNA isolated from cells which are engaged in synthesis of a large number of proteins, but not in presence of tRNA isolated from cells which are specialized in making large quantities of a few proteins (7 , 8).

Information generated from the sequence of mRNAs coding for proteins which are synthesized in large quantities in cells specialized for their synthesis and from an elucidation of the codon recognition properties of the isoacceptor aminoacyl- tRNAs in these cells have provided another means of exam- ining the adaptation hypothesis. For example, the codon fre- quencies in the mRNA coding for the silk protein of Bombyx mori (9) and in the mRNAs coding for the a and /? chains of rabbit hemoglobin (10, 11) have been determined. Further- more, the codon recognition properties of isoacceptor amino- acyl-tRNAs which are involved in decoding these mRNAs in the silk gland of B. mori (12, 13) and in rabbit reticulocytes (14) have been determined. Many of the relatively more abundant isoacceptor aminoacyl-tRNAs in these tissues recr ognize codons which correspond to the more frequently used codons in the mRNAs (13,14). Furthermore, several isoaccep-

* The work of B.G.F. and of F.V. was supported in part by National Institutes of Health Grants LH 20922 and CA 08748, respectively. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 Present address, National College, 200 E. Roosevelt Rd., Lorn- bard, IL 60148.

31

Labeled amino acids were purchased from New England Nuclear and from Amersham Corp. with the following specific activities: 3H- amino acids (in curies per mmol), alanine (41), arginine (28.7), aspar- agine (22), aspartic acid (15.8), cysteine (1.6), glutamic acid (24.1), glutamine (20), glycine (15), histidine (60), isoleucine (80.5), leucine (57.4), lysine (61.6), methionine (15), phenylalanine (15), proline (60). serine (8.9), threonine (2.0), tryptophan (5.4), tyrosine (60.3), and valine (19); and I4C-amino acids (in millicuries per mmol), alanine (177), arginine (354), asparagine (105), aspartic acid (229), cysteine (300), glutamic acid (2921, glutamine (251.4), glycine (117), histidine (352), isoleucine (390), leucine (354), lysine (340), methionine (285), phenylalanine (513), proline (295), serine (171), threonine (210), tryp- tophan (615), tyrosine (527), and valine (295). Human reticulocyte tRNA was prepared as previously described (21) by detergent plus phenol extraction of total red cell lysates from peripheral blood of adult individuals with reticulocytosis (sickle cell anemia or other hemolytic anemias) and of newborn infants undergoing exchange transfusion from hemolytic disease of the newborn (Rh and AB0 blood group incompatibility). The tRNA was initially purified from ribosomal RNA by sucrose density gradient centrifugation (21).

Reticulocytosis was induced in white New Zealand female rabbits (3 to 4 pounds), reticulocytes subsequently obtained and tRNA and aminoacyl-tRNA synthetases prepared from rabbit reticulocytes as previously described (14). Transfer RNA preparations from human and rabbit reticulocytes were purified by Sephadex G-100 chromato- graphy. Picomoles of amino acid acceptance of human reticulocyte and rabbit reticulocyte tRNAs were determined by measuring the attachment of I4C-amino acid to tRNA under limiting tRNA condi- tions in the presence of aminoacyl-tRNA synthetases from rabbit reticulocytes (14). For co-chromatography studies, human reticulo- cyte tRNA was aminoacylated with ‘H-amino acid and rabbit retic- ulocyte tRNA with I4C-amino acid in the presence of rabbit reticulo- cyte aminoacyl-tRNA synthetases (14). The resulting labeled ami- noacyl-tRNAs were prepared for chromatography and then chromat- ographed on a reverse phase chromatographic column (designated RPC-5 (22)) as given (14). The per cent of labeled aminoacyl-tRNAs recovered from chromatographic runs were similar to those observed previously from rabbit reticulocytes and rabbit liver and from bovine liver and bovine brain and in all cases were greater than 80% with the exception of Cys-tRNA. Fifty-six per cent I4C-Cys-tRNA and 69% ,’H- Cys-tRNA were recovered from RPC-5 columns. The recoveries of labeled Pro-tRNAs were not determined. “H-1ZminoacyLtRNAs were

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3 184 Human Reticulocyte Aminoacyl-tRNAs

also prepared with human reticulocyte tRNA, fractionated on a RPC- 5 column, fractions pooled from developed columns as shown in Fig. 3 by the hatched areas and pooled fractions prepared for codon recognition studies (23). The binding of 3H-aminoacyl-tRNAs from human reticulocytes to Escherichia coli ribosomes in presence and absence of trinucleoside diphosphates was carried out by the proce- dure of Nirenberg and Leder (24) as previously given (14). A260 units of tRNA, counts/min of labeled aminoacyl-tRNA, and Mg2' levels used in ribosomal binding studies are summarized in Fig. 3. Trinu- cleoside diphosphates were the generous gift of Dr. M. Nirenberg.

Two-dimensional polyacrylamide gel electrophoresis of tRNA was carried out by the method of Varricchio and Ernst (25).

RESULTS

Aminoacylation Levels of Human and Rabbit Reticulocyte tRNA

The amino acid acceptance of each tRNA in human retic- ulocytes is given in Table I, along with the frequency of occurrence of the corresponding amino acids in human he- moglobin. Some aminoacyl-tRNAs which are more abundant in reticulocytes correspond to amino acids which are used more frequently in globin. For example, tRNA""' accepted 81 pmol/A260 unit of valine and valine occupies 62 residues in hemoglobin. On the other hand, several tRNAs which ac- cepted low levels of amino acid correspond to amino acids used infrequently or not at all in hemoglobin. For example, isoleucine does not occur in globin and only 3 pmol/A2m unit of Ile-tRNA are observed.

Smith and McNamara (26) have determined the levels of amino acid acceptance of rabbit reticulocyte tRNA and have suggested that a correlation exists between the frequency of appearance of amino acids in rabbit hemoglobin and the relative levels of the corresponding aminoacyl-tRNAs. Garel (3), using the data of Smith and McNamara (26), and Smith ( 4 ) have determined a correlation coefficient (r value) for the frequency of occurrence of amino acids in rabbit hemoglobin versus the aminoacyl-tRNA content in rabbit reticulocytes.

The r value for these studies was 0.82. However, neither Garel nor Smith included the levels of Leu-tRNA and Met-tRNA,,, in their determinations. We determined the levels of amino acid acceptance of rabbit reticulocyte tRNA (see Table I) in order to compare them with those of human tRNA. The r value, which includes the levels of all 20 rabbit reticulocyte aminoacyl-tRNAs in Table I, is 0.78. The r value for the corresponding data from human reticulocytes is 0.71. The p value for each of these studies is less than 0.001.

The levels of aminoacylation of tRNAL"" in both human and rabbit reticulocytes are lower than the frequency of appearance of leucine in the hemoglobin from the respective mammalian tissues. Further consideration of this observation is given under "Discussion."

Chromatography of Human and Rabbit Reticulocyte Aminoacyl-tRNAs

Twenty aminoacyl-tRNAs from human reticulocytes la- beled with 3H-amino acids are compared by RPC-5 chroma- tography to the corresponding aminoacyl-tRNAs from rabbit reticulocytes labeled with 14C-amino acids (see Fig. 1). The elution profiies of Gly- and Met-tRNAs from both tissues are very similar. Slight variations are observed in the profiies of human and rabbit Arg-, Cys-, Glu-, Gln-, Ile-, Lys-, Phe-, Pro-, Trp-, and Val-tRNAs. Variations are also present in the elution profiles of Ala-, Leu-, Ser-, and Thr-tRNAs. The most pronounced differences are observed in Asn-, Asp-, His-, and

Ile-:Leu-tRNA Levels in Adult versus Neonatal Reticulocytes

Tv-tRNAs.

Human (Y and /3 (adult) globin chains lack isoleucine whereas the y chain of fetal hemoglobin contains 4 isoleucine residues. The ratio of isoleucine to leucine acceptance was, therefore, determined in unfractionated reticulocyte tRNA samples from a number of different adults and newborn in- fants. The results of these studies are summarized in Table 11.

TABLE I Aminoacylation of human and rabbit reticulocyte tRNA

Levels of acceptance Hemoglobin"

Amino acid Human Rabbit Human Rabbit

Acceptance Sioh Acceptance % * pmol/&m

unit'

No. resi- dues ? b h

No. resi- % b

dues

pmo1/A2m unit'

Alanine 74 8.7 86 10.0 72 12.5 56 9.8 Arginine 60 7.1 47 5.5 12 2.1 12 2.1 Asparagine 60 7.1 44 5.1 20 3.5 24 4.2 Aspartic acid 40 4.7 26 3.0 30 5.2 22 3.8 Cysteine 10 1.2 18 2.1 6 1.1 4 0.7 Glutamic acid 47 5.5 30 3.5 24 4.2 34 5.9 Glutamine 19 2.2 17 2.0 8 1.4 10 1.7 Glycine 95 11.2 98 11.4 40 7.0 40 7.0 Histidine 49 5.8 50 5.8 38 6.6 40 7.0 Isoleucine 3 0.4 14 1.6 0 0 8 1.4 Leucine 43 5.1 56 6.5 72 12.5 70 12.2 Lysine 55 6.5 50 5.8 44 7.7 48 8.4 Methionine 9" 1.1 7" 0.8 6 1.1 4 0.7 Phenylalanine 43 5.1 34 4.0 30 5.2 32 5.6 Proline 47 5.5 43 5.0 28 4.9 22 3.8 Serine 41 4.8 58 6.8 32 5.6 42 7.3 Threonine 43 5.1 46 5.4 32 5.6 32 5.6 Tryptophan 11 1.3 21 2.5 6 1.1 6 1.1 Tyrosine 18 2.1 16 1.9 12 2.1 12 2.1 Valine 81 10.0 97 11.3 62 10.8 56 9.8

" a&-tetramer; amino acid composition of hemoglobin is taken is 1 cm. The human tRNA preparation was derived from a pool of six from Ref. 15 to 18 (human) and 10 and 11 (rabbit). different individuals, in order to correct for any bias based on individ-

Per cent of total. ual variations. e One Azm unit is that amount of material when dissolved in 1 ml " Level corresponds only to internal Met-tRNA.

has an absorbance at 260 nm of 1 A unit in a cell whose path length

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FRACTION NUMBER

FIG. 1. Comparison of human and rabbit reticulocyte ami- noacyl-tRNAs (AA-tR.NA) by reverse phase chromatography. “H-Aminoacyl-tRNAs were prepared with human reticulocyte tRNA and “C-aminoacyl-tRNAs with rabbit reticulocyte tRNA and were co-chromatographed on a RPC-5 column as given under “Materials and Methods.”

In the nine different adult reticulocyte tRNA samples, the isoleucine:leucine ratio was consistently low. The values ranged between 0.04 and 0.21 with all but one of the samples giving ratios of 0.13 or less. The neonatal reticulocyte tRNA sample yielded a wider range of values: two of the six samples gave values in the adult range, three samples gave markedly higher values (0.71 to 0.76), and one sample yielded an inter-

mediate value of 0.52. There is, therefore, a tendency for neonatal reticulocyte tRNA samples to possess a relatively higher content of Ile-tRNA than adult tRNA, as would be expected from the isoleucine content. of y globin chains, but this phenomenon was not uniformly observed.

A comparison of adult and fetal human reticulocyte tRNAs by two-dimensional polyacrylamide gel electrophoresis (Fig. 2) showed only minor differences between the two samples.

Fractionation of Human Reticulocyte Aminoacyl-tRNAs for Coding Studies

The fractionation of four aminoacyl-tRNAs from human reticulocytes for coding studies is shown in Fig. 3. Fractions were pooled from the developed columns and designated with a Roman numeral in the order of elution from the column. Each fraction was assayed for its codon recognition properties and responses to codons are shown in the figures. The results of these studies are:

Ala-tRNA-The binding of the initial eluting shoulder of Ala-tRNA (Fraction I) to ribosomes is stimulated in the presence of GCU, GCC, GCA, and GCG. The large eluting peak (Fraction II) responds to GCU, GCC, and GCA. The terminal eluting peak (Fraction III) responds to GCU, GCC, GCA, and GCG. Additional fractionation would be required to determine if the terminal eluting peak consists of an isoac- ceptor recognizing GCU, GCC, and GCA, and an isoacceptor recognizing GCG or a single isoacceptor which recognizes all four codons. The initial eluting shoulder of Ala-tRNA most certainly consists of an isoacceptor that recognizes only GCG by analogy to rabbit reticulocyte Ala-tRNA (14). Rabbit re- ticulocyte Ala-tRNA contains two minor peaks which elute before the larger peak and appear to be more abundant in rabbit than human reticulocyte Ala-tRNA (see Fig. 1). These peaks in rabbit reticulocytes recognize only GCG (14). The initial eluting shoulder of human reticulocyte Ala-tRNA is

TABLE II Aminoacylation of neonatal versus adult human reticulocyte tRNA

The tRNAs used in these experiments were purified by sucrose density gradient centrifugation only without further purification by Sephadex G-100 gel filtration. The variability between different sam- ples in the amount of charging (picomoles per A& may be related to this lack of further purification and the presence in the samples of variable amounts of AZ60 absorbing material that is not tRNA, or the presence of variable amounts of different substances that can inhibit the charging enzymes. The aminoacylation assays were performed with different isotopes and enzymes than those reported in Table I. Specific activity of isotopes were: [“‘Clleucine, 283 mCi/mmol, and [‘%]isoleucine, 306 mCi/mmol. The results are expressed as pico- moles of amino acid acceptance per AZM) unit of tRNA sample. Adult sample 4 and neonatal samples 1.4, and 6 were also analyzed by two- dimensional acrylamide gel electrophoresis (Fig. 2).

tRNA Amino acid acceptance

sample Ile LISI

pmol/Azso unit Adult 1 3.8 37.5

2 4.8 72.9 3 5.6 50.3 4 2.0 50.8 5 4.8 66.5 6 15.3 54.5 7 2.3 66.1 8 3.5 33.2 9 2.5 18.1

Neonatal 1 29.2 56.1 2 8.1 11.9 3 27.2 38.1 4 8.4 11.0 5 6.1 44.7 6 4.5 68.2

Ile/Leu

0.10 0.07 0.11 0.04 0.07 0.21 0.03 0.11 0.13 0.52 0.71 0.72 0.76 0.14 0.07

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3186 Human Reticulocyte Aminoacyl-tRNAs

FIG. 2. Two-dimensional polyacrylamide gel electrophore- sis of human adult and fetal reticulocyte tRNA. 80 pg of neonatal reticulocyte tRNA samples I and 4 in Table I1 (see A and B, respectively, in the figure), 75 pg of neonatal sample 6 in Table I1 ( 0 , and 150 pg of adult sample 4 in Table I1 (D) were separated by two-

0 1 0 30 h, 60 90

FRACTION NUMBER

FIG. 3. Fractionation and coding responses of human retic- ulocyte aminoacyl-tRNAs. ["HIAla-, Asn-, His-, and Leu-tRNAs were prepared, chromatographed on a RI'C-5 column, the resulting "H-aminoacyl-tRNA fractions pooled as designated by the hatched areas and prepared for coding studies, and coding studies carried out as given under "Materials and Methods." The results of the coding studies are shown in the figure. Codons are given in the first column to the left. The order in which the counts/min are listed by columns corresponds to the binding obtained with each fraction in response to codons in the order of fraction elution (designated by Roman numer- als) from HPC-5 columns. The counts/min were obtained by subtract- ing the number of counts/min bound in the absence of codon from that bound in the presence of codon. Counts/min obtained in the absence of codon are given in parentheses and listed as None. Total counts/min, AZwl units, and Mi'' levels used in the assay are also shown.

better resolved in the co-chromatography studies with rabbit reticulocyte Ala-tRNA (see Fig. 1).

Am-tRNA-The binding of both peaks of Asn-tRNA (Frac- tions I and 11) to ribosomes is stimulated in the presence of AAU and AAC. The response of both peaks is significantly greater in the presence of AAC than AAU at 0.02 M Mg2+. Similar codon recognition properties were observed with rab- bit reticulocyte Asn-tRNA (27).

His-tRNA-Three peaks of His-tRNA (Fractions I to 111)

dimensional polyacrylamide gel electrophoresis. The pH 8.3, 7 M urea first dimension and pH 8.3 second dimension system (23) was used. Separation in the first dimension was from right to left and in the second dimension from top to bottom.

respond to CAU and CAC. The response of each peak was greater in the presence of CAU than CAC. Similar codon recognition properties were observed with rabbit reticulocyte His-tRNA (27).

Leu-tRNA-The initial eluting peak of Leu-tRNA (Frac- tion I) responds to CUG. The shoulder which elutes from the column just after the initial peak (Fraction 11) also recognizes CUG. Fraction I11 binds tightly to ribosomes in the absence of codons and its binding was not stimulated in the presence of any of the leucine codons. Therefore, a codon assignment was not made to this Leu-tRNA. However, Pirtle et al. (28) have determined the primary sequence of the corresponding Leu-tRNA from bovine liver and have shown that it contains I in the wobble position of the anticodon. Thus, Fraction 111 can tentatively be assigned the leucine codons CUU, CUC, and CUA. The large initial eluting peak in rabbit reticulocytes also recognizes CUG (14). Rabbit reticulocytes also contain a minor peak, which elutes between the initial and third eluting peaks, that recognizes UUG (14). This peak is not detected in human reticulocyte tRNA.

The coding properties of human reticulocyte Thr- and Val- tRNAs were also examined (data not shown). Responses of the Thr-tRNA fractions to ACU, ACC, and ACA were ob- served. The initial eluting peak of Val-tRNA responded to GUG and other Val-tRNA fractions responded to GUU, GUC, GUA, and GUG. These observations were similar to those observed with Thr-tRNA (14) and Val-tRNA (14, 29) from other mammalian tissues.

DISCUSSION

The amino acid acceptance of all 20 aminoacyl-tRNAs in human reticulocytes was determined. A linear correlation coefficient of 0.71 was obtained for the aminoacyl-tRNA con- tent in human reticulocytes uer.w.9 the amino acid content in human hemoglobin. This value indicates a significant, but not exact, correlation between the two parameters. Indeed, for some amino acids, such as arginine and glycine, the relative levels of aminoacylation of tRNA are greater than the fre- quency of appearance of that amino acid in globin. For other amino acids, such as leucine, the levels of aminoacylation of tRNA'^" are lower than the frequency of appearance of leucine in globin. The concentration of Leu-tRNA is also lower in rabbit reticulocytes than would be predicted from the leucine content in rabbit globin (4). The latter observation has led to the speculation that Leu-tRNA may be limiting in rabbit

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Human Reticulocyte Aminoacyl-tRNAs 3187

globin synthesis (4). However, a more appropriate correlation would be to determine the frequency of codon usage in globin mRNA and the relative amounts of isoacceptor aminoacyl- tRNAs which recognize specific codons. Most of the leucine residues in human (15-20) and rabbit (10, 11) hemoglobin are coded by CUG codons and the corresponding isoacceptor which recognizes CUG is clearly the most abundant Leu- tRNA in rabbit (14) and human reticulocytes. The possibility, therefore, that the Leu-tRNA which recognizes CUG may be present in maximum levels in reticulocytes for translating CUG codons in globin mRNA should also be considered.

The aminoacyl-tRNA population of human reticulocytes was compared to that of rabbit reticulocytes by reverse phase chromatography. Variations were observed in the elution pro- fides of several aminoacyl-tRNAs. The most pronounced dif- ferences occurred in the elution profiles of Asn-, Asp-, and His-tRNAs. Each of these aminoacyl-tRNAs contain the hy- permodified base, Q, in the 3”position of the anticodon (30). The more abundant, later eluting peaks of Asn-, Asp-, and His-tRNAs in human reticulocytes are consistent with ami- noacyl-tRNAs deficient in Q base (30, 31). The possible role of this base in protein synthesis is not clear. McNamara and Smith (32) have observed that His-tRNA with Q base and without Q base read CAU and CAC codons with the same efficiency. Therefore, it does not appear that Q is involved in restricting wobble (33) such that isoacceptors containing this highly modified base preferentially read codons with a 3‘- undine or codons with a 3”cytidine. It is of interest to note, however, that some of the major differences in frequency of codon usage between human and rabbit globin mRNAs occur with the pyrimidine selected in the 3’ position of asparagine, aspartic acid, and histidine codons (IO, 11, 15-18).

The possibility that the variations observed in the elution profiles between human and rabbit reticulocytes (Fig. I) may be due to factors other than isoacceptor levels in the respective tRNA populations should be considered. For example, both tRNA populations were aminoacylated with a preparation of rabbit reticulocyte synthetases. It might be considered that rabbit reticulocyte synthetases may selectively aminoacylate some human tRNA isoacceptors. Although we have not ex- amined the aminoacylation of human tRNA in the presence of human reticulocyte synthetases, the fact that the present studies were carried out with limiting tRNA levels and excess enzymes for a longer incubation period than required for complete attachment of amino acid (see “Materials and Meth- ods” and Ref. 14) should minimize this possibility. Addition- ally, some of the observed differences in elution profides may be due to selective deacylation of isoacceptors from one tRNA population during chromatography. However, the high recov- ery of most labeled aminoacyl-tRNAs from RPC-5 columns would tend to rule out this possibility with the exception of Cys-tRNA. The lowest recovery from columns occurred with Cys-tRNAs (see “Materials and Methods”).

Reticulocytes are enucleated cells which are at the end of their protein synthetic life cycle. Therefore, a question may be raised as to how representative the tRNA populations observed in the present study are to the actual populations in cells actively synthesizing globin proteins. Burka (34) and Zehavi-WiUner and Danor (35) have observed the turnover of rRNA, mRNA, and tRNA in maturing reticulocytes and have found that tRNA turnover is much slower than that of rRNA or mRNA. Smith and collaborators (26,27,36) have examined the tRNA population of rabbit reticulocytes prepared by similar procedures as those used in the present study and have concluded that it is representative of the population involved in making globin. In an additional study, we have examined the levels of isoacceptors which have been assigned

codons in human and rabbit reticulocytes and compared these levels to the frequency of their cognate codons in the respec- tive a and P globin mRNAs.’ A highly significant correlation was observed between these two parameters. These results provide evidence that the tRNA populations in the present study are representative of those in cells actively synthesizing globin and further that the isoacceptor levels in these cells are largely adapted to the requirements of protein synthesis.

The comparison of Ile-/Leu-tRNA ratios in adult uersus neonatal human reticulocytes suggests that reticulocyte tRNA levels can change in response to need. The finding of increased Ile-/Leu-tRNA ratios in neonatal reticulocytes was variable, however, and levels similar to those found in adult reticulo- cytes were observed in two of six samples. The variability of the Ile/Leu ratio in different neonatal samples could be related to the fact that the neonates were of different ages (some premature, others full term) and, therefore, at different stages of the fetal to adult switch. It may also be that increased levels of Ile-tRNA are not necessarily associated with high levels of y gene expression in human erythroid cells. In fact, one of the adult samples (No. 3) was from an individual with homozygous P-thalassemia whose reticulocytes synthesized substantial amounts of y globin chains, and the Ile-/Leu- tRNA ratio in this sample was similar to that found in reticulocyte tRNA samples from other adults who did not manifest increased fetal hemoglobin synthesis. It is certainly’ possible that the Ile-tRNA genes are preferentially expressed in fetal erythroid cells as part of an overall fetal erythroid cell program of gene expression that differs from that of adult erythroid cells in many components other than only y globin gene expression. Although a and ,8 globin chains totally lack isoleucine, adult erythroid cells nevertheless must have a requirement for Ile-tRNA in order to synthesize a number of nonglobin proteins during the early phases of their prolifera- tion and differentiation. Because the stability of this Ile-tRNA should permit it to persist to the reticulocyte stage of cell maturation, one would not expect absent or exceptionally low levels of Ile-tRNA in human adult reticulocytes.

The codon recognition properties of six aminoacyl-tRNAs fractionated from human reticulocytes were determined. These included Leu-, Ala-, Asn-, His-, Thr-, and Val-tRNAs. No response of Leu-tRNA to the leucine codon UUG was detected. UUG codons do not occur in human a or /3 globin mRNA (15-18). There is one UUG codon for leucine in rabbit globin mRNA (IO, 11) and only a minor leucine isoacceptor which recognizes UUG was detected in rabbit reticulocytes (14). Minor Ala-tRNA isoacceptors which recognize only GCG have been detected in rabbit reticulocytes (14) and 3 of 28 alanine codons in rabbit globin mRNA are coded by GCG codons (IO, 11). On the other hand, 7 of 36 alanine codons are coded by GCG in human adult globin mRNA. However, an alanine isoacceptor which recognizes only GCG was not de- tected in human reticulocytes. Co-chromatography of human and rabbit reticulocyte Ala-tRNAs suggest that human Ala- tRNA contains one of the minor isoacceptors which recognizes GCG (Fig. 1). This isoacceptor which is proportionally less of the total Ala-tRNA in human than in rabbit reticulocytes was not resolved from other alanine isoacceptors in the coding studies (Fig. 3). The terminal eluting peak of human reticu- locyte Ala-tRNA shows a strong response to GCG relative to the other alanine codons. Most certainly, this Ala-tRNA par- ticipates in translating alanine GCG codons. Human Asn-, His-, Thr-, and Val-tRNAs manifested similar codon recog- nition properties as the corresponding aminoacyl-tRNAs de- scribed in other mammalian tissues (14, 27, 29).

The studies carried out with human reticulocyte tRNA ’ D. Hatfield, M. Rice, and B. Forget, manuscript in preparation.

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3 188 Human Reticulocyte Aminoacyl-tRNAs

provide further support that the tRNA populations of cells, at least those specialized in making large quantities of a few proteins, are adapted to the requirements of protein synthesis (3).

Acknowledgment-We express our appreciation to Miss Sandra Lane for her assistance in developing the RPC-5 columns.

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3. Garel, J.-P. (1974) J. Theor. Biol. 43, 211-225 4. Smith, D. W. E. (1975) Science 190,529-535 5. Hentzen, D. (1976) Cancer Res. 36,3082-3085 6. Viotti, A,, Balducci, C., and Weil, J. H. (1978) Biochim. Biophys.

7. Sharma, 0. K., Beezley, D. N., and Roberts, W. K. (1976) Bio-

8. LeMeur, M., Gerlinger, P., and Ebel, J. P. (1976) Eur. J. Biochem.

9. Suzuki, Y., and Brown, D. (1972) J. Mol. Biol. 63,409-429

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chemistry 15,4313-4318

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D Hatfield, F Varricchio, M Rice and B G ForgetThe aminoacyl-tRNA population of human reticulocytes.

1982, 257:3183-3188.J. Biol. Chem. 

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