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Prothymosin α and parathymosin: mRNA and polypeptide levels in rodent tissues
ARCHIVES OF BIOCHEMISTRY ANT) BIOPHYSICS Vol. 289, No. 1, February 15, pp. 266-263,1989
Prothymosin
cy and Parathymosin: mRNA and Polypeptide in Rodent Tissues
M. CLINTON, Department
of Biochemistry,
Levels
M. FRANGOU-LAZARIDIS,l C. PANNEERSELVAM,’ AND B. L. HORECKER3 Cornell University
Medical College,
1300
York Avenue, New York, New Ymk 10021
Received August 10,1988, and in revised form October 10,198X
Blot hybridization analyses have established the presence of mRNAs for prothymosin (Y(ProTa) and for parathymosin (ParaT) in rat and mouse lung, liver, kidney, and brain, confirming the biosynthesis of these peptides in nonlymphoid tissues. In these tissues the levels of mRNAs paralleled the content of the polypeptides, determined with specific radioimmunoassays. The mRNA levels also confirmed the reciprocal relation between the two polypeptides; ProTa and its mRNA were found in highest concentrations in spleen and thy&us, followed by lung, kidney, and brain, with lowest concentrations in liver. On the other hand, liver contained highest concentrations of ParaT and the mRNA for ParaT, with lowest levels present in spleen and thymus. In comparison to tissues from young (6-8 week) mice, older (18 month) mice contained lower concentrations (2040%) of both polypeptides, with qualitatively similar decreases in mRNA content. 019~9 Academic
Press, Inc.
The polypeptides prothymosin 01 (ProTLu)4 and parathymosin (ParaT) were first isolated from rat thymus (1, 2) but have since been shown to be present in many other mammalian tissues (2-4). Rat ProT~v and rat ParaT are similar in size, containing 111 and 101 amino acid residues, respectively (5), and share an unusual amino acid composition characterized by a high content of glutamyl and aspartyl residues and the complete absence of aromatic and sulfur-containing amino acids. Despite these similarities in 1 Current address: Laboratory of Biological Chemistry, University of Ioannina Medical School, GR45332, Ioannina, Greece. ’ On leave from the Department of Biochemistry, University of Madras, Madras, 600113, India. 3 To whom correspondence should be addressed. ’ Abbreviations used: ProTa, prothymosin a; ParaT, parathymosin; RIA, radioimmunoassay; Mops, 4-morpholinepropanesulfonic acid; SDS, sodium dodecyl sulfate.
0003-9861/89 $3.00 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
256
size and composition, they show sequence homologies in only limited regions in the NHz-terminal portion (5). The almost ubiquitous distribution of ProTa and ParaT in mammalian tissues raises interesting questions regarding their origin in these tissues. Based on the structure of the cloned cDNAs for both rat and human ProTors (5, 6), which contain initiator codons preceding the codons for the first amino acid residue and terminator codons immediately following the codon for the COOH-terminal residues, their synthesis via a larger precursor is excluded. The absence of a hydrophobic signal peptide makes it unlikely that ProTcv is exported by the usual mechanism for secretory proteins. However, this lack of a leader sequence is shared by a number of secreted peptides, including interleukin 1 (7, 8) and endothelial growth factor (9). The release of interleukin 1 from a membrane-bound form (10) is thought to be mediated by extracellular proteinases (111,
RODENT
TISSUE
PROTHYMOSIN
but this mechanism is unlikely to apply to ProTa and ParaT which lack hydrophobic sequences. The present studies were undertaken to evaluate the presence of mRNAs for prothymosin o( and parathymosin in rat and mouse tissues, as evidence for their biosynthesis in these tissues, using the cloned cDNAs (5) for blot hybridization analysis. We have also compared the levels of the mRNAs with the concentrations of the respective polypeptides determined with the recently developed specific RIAs (4,12). We find that all tissues examined contain mRNAs for both ProTcy and ParaT and that the relative levels of these mRNAs parallel the levels of polypeptides. The results also confirm that most active synthesis of ProTa occurs in thymus and spleen, in contrast to ParaT, which is most actively synthesized in nonlymphoid tissues such as liver and kidney. Comparison of the levels of mRNAs and polypeptides in tissues from young and old mice indicates that aging is accompanied by small but consistent decreases in the synthesis and tissue content of both polypeptides. EXPERIMENTAL
PROCEDURES
Materials. Synthetic thymosin 01~and the synthetic prothymosin LYfragments 23-32 and 26-35 were generously provided by Dr. E. P. Heimer, Roche Research Center, Nutley, New Jersey. Prothymosin cyand parathymosin were isolated as described (13) from rat thymus and rat liver, respectively. The content of these peptides in solution was determined by amino acid analysis carried out with aliquots hydrolyzed in 6.0 N HCI at 150°C for 1 h, using a Waters Model PicoTag analyzer, as described by Heinrikson and Meredith (14). Reagents and solvents were analytical or chromatography grade commercial preparations. Solvents were redistilled as required. Collection oftissue. Male Charles River CD rats, 56 weeks old, were sacrificed by decapitation and the tissues removed immediately and frozen in liquid nitrogen. Larger organs, such as liver, brain, lung, and kidney, were minced with scissors before freezing. Frozen tissues from 10 rats were pooled and stored at -70°C. The same pools of frozen tissues were sampled for RNA and for polypeptide analyses. For the experiments with mice, young (6-8 weeks) or “old” (18 months) mice, strain C57BL/6, were employed. For
cy AND
PARATHYMOSIN
257
both mRNA and peptide analyses tissues from six mice in each group were collected as described for rat tissues and pooled after freezing. RNA e&r&ion and analyses. Tissue samples (l-2 g) were taken from each frozen tissue pool and total RNA was isolated by the guanidine isothiocyanatecesium chloride method (15). After two ethanol precipitations from 0.3 M Na acetate, the last precipitate was stored at -70°C as a suspension in ethanol. For electrophoretic analysis, aliquots of the RNA suspension (15 wg), as determined by uv absorption analysis, were removed, the RNA was pelleted, washed with ethanol, and dried on a Speed-Vat concentrator, and the residues were suspended in RNA sample buffer containing 1X running buffer (1X = 20 mM Mops, pH 7.0, 5 mM Na acetate, and 1 mM EDTA), 50% formamide, and 2.2 M formaldehyde. The solutions were heated to 65°C for 5 min and applied to horizontal 0.8% agarose gels containing 1X running buffer and 2.2 M formaldehyde. After electrophoresis at 90 V for 2.5 h, the gels were stained for 30 min in 1X running buffer containing 10 fig/ml of ethidium bromide, destained twice for 15 min in 1X running buffer, and transferred by blotting overnight to nylon membranes (Hybond-N, Amersham) with 10X SSC buffer (1X SSC buffer contains 15 mM trisodium citrate and 0.15 M NaCl). The membranes were baked at 80°C for 2 h in a vacuum oven, immersed briefly in 6X SSC at room temperature, and prehybridized for 4 h at 42°C in 1X Denhardt’s solution (0.2% Ficoll, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin) plus 6X SSC buffer, 0.2% SDS, 50 mM NaH2P04, pH 7.0, 0.1 mg/ml denatured salmon sperm DNA, and 50% formamide. The cDNA probes employed for detection of prothymosin 01 and parathymosin mRNAs were from clones pRSpro-187 and pRSpara-5187 (5), respectively. The probes were labeled with [“‘P]dCTP by random priming (16) (l-2 X lo9 cpm/pg) or by nick translation (17) (2-4 X lo* cpm/pg) as indicated. Hybridization with these probes was at 42°C for 18-20 h in a solution containing 1X Denhardt’s solution (see above), plus 6X SSC buffer, 0.2% SDS, 50 mM NaH2P04, pH 7.0,15 mM EDTA, 0.1 mg/ml denatured salmon sperm DNA, and 50% formamide, with 5 X lo6 cpm/ml of labeled probe. Final washing of the membranes was in 1X SSC, 0.1% SDS at 55°C for both probes. After being washed, the membranes were exposed to Kodak XAR film for 2-12 h using DuPont Codex intensifying screens. For hybridization with cDNA probes on slot blots aliquots of the RNA suspension (125 Kg) were centrifuged, washed, and dried, as described above. The RNA was dissolved in Ha0 containing 1 mg/ml of yeast tRNA (Sigma) and seven serial twofold dilutions were tested for each RNA sample, covering a range of 15 fig to 110 ng per slot. Immediately before
258
CLINTON
ET AL.
TABLE
I
RELATIVE CONTENT OF mRNAs FOR PROTHYMOSIN 01AND PARATHYMOSIN ESTIMATED FROM SLOT BLOT HYBRIDIZATION EXPERIMENTS’ Prothymosin
Thymus Spleen Lung Brain Kidney Liver
1
2
3
1.00 0.77 0.68 0.45 0.43 0.08
1.00 0.78 0.63 0.43 0.42 0.07
1.00 0.75 0.71 0.50 0.50 0.07
o(
Parathymosin Average 1.00 0.76 0.67 0.46 0.45 0.07
1
2
3
0.40 0.27 0.54 0.83 0.51 1.00
0.47 0.30 0.58 0.86 0.55 1.00
0.47 0.27 0.65 0.83 0.65 1.00
Average 0.45 0.28 0.59 0.84 0.57 1.00
a Slot blot hybridization analyses were carried out with RNA samples prepared from aliquots of the pooled frozen tissues from 10 rats (see Experimental Procedures). The values represent the areas under the peak of each densitometer scan, with the values obtained for ProTcv mRNA from thymus and ParaT mRNA from liver taken as 1.0. Only densitometer scan areas in the linear range of the seven serial dilutions were used. Three independent slot blot hybridization analyses were carried out for each sample.
application, each sample was adjusted to 2.2 M formaldehyde, 10X SSC and incubated at 65°C for 5 min. Immobilization of the RNA was performed on a Minifold II slot-blot apparatus (Schleicher & Schuell) with BA85 nitrocellulose membranes (Schleicher & Schuell) which had been prewetted with water, then 10X SSC, and after application each individual well was washed with 500 pl 10~ SSC. The membranes were then air-dried and baked under vacuum at 80°C for 2 h. Prehybridization, hybridization, and washings were as described earlier for the nylon membranes. Multiple exposures of each film were prepared to ensure a linear response of the film. Densitometric measurements were with a Quick Scan, Jr. (Helene Laboratories). Radioimmunoassays. Extracts for the radioimmunoassays were prepared as described by Komiyama et al. (13). Briefly, aliquots (2 g) were taken from each frozen tissue pool and pulverized under Nz in a chilled mortar and pestle. The frozen powders were then quickly dispersed into 20 ml of boiling water and boiled continually for 5 min. After being cooled in ice, each suspension was homogenized with a Polytron homogenizer (Brinkman Type PT 10/35) and centrifuged. Aliquots from each clear supernatant solution were taken for radioimmunoassays, carried out in duplicate or triplicate in Eppendorf microfuge tubes essentially as described for prothymosin o( by Haritos and Horecker (12) and for parathymosin by Panneerselvam et al. (4). For ProTa, the radiolabeled ligand was 3H-methylated thymosin 01~,and synthetic thymosin 01~was employed to standardize the assay. With the anti-thymosin o(~ antibody employed, displacement of thymosin 01~required five times as much
ProTa on a molar basis (12). We have previously shown (1, 18) that with this extraction method the cross-reacting material is ProTa, with less than detectable amounts of thymosin at present. For ParaT the radioimmunoassay employed an antibody raised against ParaT, with 3H-methylated ParaT as the competing radioligand. The epitope recognized by this antibody includes residues 26-32 of ParaT (4). This specificity was confirmed using synthetic peptides corresponding to residues 23-32 and 26-35 of ParaT (see Materials). With these synthetic peptides, the displacement curves followed closely the curve described previously (4) for ParaT 1-42 (data not shown). RESULTS
Levels of mRNAs fin- ProTa and ParaT in rat tissues. Blot hybridization
analysis confirmed the presence of mRNAs for both ProTa and ParaT in a variety of rat tissues. Quantitative analysis on slot blots showed highest quantities of ProTa mRNA in thymus, spleen, and lung, with intermediate levels in brain and kidney, and low, but definitely detectable, quantities in liver. For parathymosin, on the other hand, the levels of mRNA were highest in liver and brain, with relatively low levels in spleen and thymus (Table I). Sizes of the mRNAs for ProTa and ParaT were estimated by blot hybridiza-
RODENT
Prothymosin
CI
TISSUE
PROTHYMOSIN
Porothymosin
FIG. 1. Sizes and distribution in rat tissues of mRNAs for ProTo and ParaT. Total RNA was isolated as under Experimental Procedures. Aliquots containing 15 pg of RNA were applied to each lane and electrophoresis, transfer, hybridization, and autoradiography carried out as described under Experimental Procedures, with cDNA probes labeled by random priming. The positions of 18 and 28 S ribosomal RNA were determined from the ethidiumstained gels, as indicated.
o( AND
259
PARATHYMOSIN
fold higher for ProTa (cf. Table II and Table III of Ref (2)) and one-third lower for ParaT (cf. Table III and Table I of Ref. (4)). The reasons for these differences are not immediately apparent but may reflect variations in content from one group of animals to another. The reciprocal relation between the concentrations of both mRNAs and polypeptides in the tissues examined is best illustrated by comparing the content in each tissue to the corresponding content in thymus (Fig. 2). The values for both mRNA and polypeptide were highest for ProTa in lymphoid tissues, whereas for ParaT the concentrations of both mRNA and peptide were highest in nonlymphoid tissues. The results also suggest that regulation of the
TABLE
II
PROTHYMOSIN 01IN RAT Trssuss
tion after electrophoresis on agarose gels, which also confirmed the relative quantities of these mRNAs in these tissues (Fig. 1). The mRNA for rat ProTa was estimated to contain approximately 1.3 kb, similar to the value, 1.4 kb, reported for the mRNA isolated from a human fibroblast cell line (19). The mRNA for rat ParaT was considerably larger, containing approximately 1.8 kb (Fig. 1). For each polypeptide only a single RNA species hybridizing to the respective DNA probe was detected, although the presence of several transcripts of similar size was not excluded. Tissue levels of ProTa! and ParaT. Preliminary values for the content of these polypeptides, employing the recently developed radioimmunoassays, have been reported (Z-4). In order to relate the tissue content of the polypeptides to the mRNA levels reported in the previous section, we analyzed extracts prepared from the same rat tissue pools used for the RNA analyses. For all of the tissues tested, the relative quantities of the two polypeptides (Tables II and III) were similar to those reported previously (2-4), although the absolute values were different, approximately two-
Thymosin 01,equivalents* (nmol/g wet tissue) Tissue” Thymus Spleen Lung Kidney Brain Liver Muscle
Extract
I
13.9 -t 2.2 8.5 + 0.3 3.4 2 0.19 3.1 kO.11 1.4 + 0.09 1.78 +_0.03 0.5 iz 0.02
Extract 15.9 9.5 3.5 3.1 1.8 1.5 0.37
II
* 1.5 k 0.8 t 0.1
* 0.1 -+ 0.2 -+ 0.1 t 0.05
ProTo” (cLg/g wet tissue) 916 554 216 195 100 110 27
a Tissues from 10 rats were collected as described under Experimental Procedures and stored at -70°C. “Two separate extracts were prepared from 2 g samples of each frozen tissue pool (see Experimental Procedures). Each extract was analyzed in triplicate (Extract I) or duplicate (Extract II) for cross-reacting material, and the results expressed as thymosin 01~equivalents/g wet tissue. The numbers shown represent the arithmetic means k the maximum deviation. “To convert thymosin 0~~ equivalents to micrograms of ProTcu, the arithmetic mean values for thymosin a1 equivalents were multiplied by a factor of 5 to account for the Iower reactivity of ProTo in the RIA for thymosin 01~(see Ref. 12) and converted to micrograms using the molecular weight of 12,282 for rat prothymosin LYestablished from the eDNA sequence (5).
260
CLINTON TABLE
III
PARATHYMOSININRATTISSUES Parathymosin nmol/g Tissue
Extract
I
Thymus Spleen Lung Kidney Brain Liver Muscle
7.1 + 4.8 F 5.5 f 13.8 f 8.3 f 19.2 f 1.4 *
0.4 0.3 0.6 0.6 0.4 0.9 0.2
tissue” Extract
II
tissue*
6.6 + 0.3 5.5 + 0.4 8.5 ‘-c 0.4 11.3 +- 0.9 7.4 i 0.4 18.7 + 0.8 1.0 f 0.2
78 59 80 144 90 217 14
a Aliquots of the extracts prepared for analysis of ProTa (see Table 11) were also analyzed for ParaT. Each extract was analyzed in duplicate and the arithmetic means and maximum deviations are reported. bFor conversion of nanomoles to micrograms the molecular weight was taken to be 11,470, calculated from the amino acid sequence deduced from the cloned cDNA (5).
ET AL.
same pooled tissues (Fig. 3). The content of mRNA for ProTa was distinctly lower in all four tissues from old, as compared to young, animals. This was also true for the content of mRNA for ParaT, except for kidney where the levels did not appear to be significantly different. We were unable to compare the content of the mRNAs in young and old thymus because thymus tissue could not be recovered from the 18month-old animals. The mRNAs for ParaT were similar in size, -1.8 kb, in the rat and the mouse. For ProTa, however, important species differences were observed, -1.9 kb for mouse ProTo mRNA compared to 1.3 kb for this mRNA in rat tissues and 1.4 kb reported by Eschenfeldt and Berger (19) for human fibroblasts. DISCUSSION
The presence of mRNAs for ProTa and ParaT in nonlymphoid tissues supports PROTHYMOSIN
tissue content for both polypeptides is primarily at the level of transcription, because in general the relative content of the mRNA paralleled the relative content of the polypeptide. Efect of aging on levels of ProTa and
Cl
060.4 02-
ParaT and their mRNAs in mouse tissue.
The effects of aging were studied in tissues from C57BL/6 mice, since this strain is currently employed in other laboratories at this institution for studies on the effects of aging on the immune system. Radioimmunoassays for ProTcv and ParaT were carried out on extracts from tissues of 6- to Sweek-old and l&month-old mice (Tables IV and V). The values obtained for both peptides in tissues from young mice were very similar to those reported in Tables II and III for 5- to 6-week-old rats, but the values for ProTa in tissues of 18-monthold mice were 25-40% lower. For ParaT the differences in content in old and young animals were smaller, only 20-30%. Qualitatively similar differences were observed in the levels of mRNAs analyzed in brain, spleen, liver, and kidney from the
30
U 0
RNA PeptIde
PARATHYMOSIN n
20-
1 o-
FIG. 2. Comparison of ProTa and ParaT mRNA and polypeptide levels in rat tissues. The data from Table I and Tables II and III are represented as ratios to the values for the mRNAs and polypeptides in thymus. The values for polypeptide content for ProTa and ParaT in thymus were 916 and 78 @g/g tissue, respectively, taken from the data in Tables II and III.
RODENT
TISSUE
PROTHYMOSIN TABLE
LY AND
261
PARATHYMOSIN
IV
LEVELS OF ProTol IN TISSUES OF YOUNG AND OLD MICE Age 18 months
Age 6-8 weeks
Tissue
Thymosin (pi equivalents” (nmol/g tissue)
Prothymosin 01 (pg/g tissue) *
Thymus Spleen Lung Kidney Brain Liver Muscle
10.3 + 5.2 f 3.2 + 2.4 c 1.2 * 1.7 k 0.4 It
638 320 196 150 74 106 22
1.5 1.0 0.5 0.4 0.2 0.1 0.1
Thymosin 01~equivalents” (nmol/g tissue)
Prothymosin CY (fig/g tissue) NA 238 (0.74)d 122 (0.62) 112 (0.75) 42 (0.57) 64 (0.60) 16 (0.73)
NA” 3.8 + 0.4 2.0 f 0.3 1.8 f 0.1 0.7 -t 0.1 1.0 f 0.1 0.3 f 0.1
’ Tissues from six mice were collected and frozen, and the frozen tissues pooled (see Experimental Procedures). Aliquots (0.5-2.0 g) of the pooled tissues were extracted and the extracts analyzed in triplicate as described for rat tissues (see Table II), and the arithmetic means and maximum deviations reported. *The values for thymosin 01~equivalents were converted to micrograms of ProTo as described in the footnotes to Table II. ‘No thymus was obtained from the old mice. ‘The numbers in parentheses represent the ratio of the content in tissues from 18-month-old mice to the content in tissues in 6- to 8-week-old mice.
the concept that these polypeptides are synthesized in these tissues. It is thus unlikely that either ProTcx or ParaT functions as a thymic hormone, as has been proposed by others for thymosin LYE,the NHa-terminal fragment of ProTLv (20). This is particularly true for ParaT; for this
polypeptide the quantities of mRNA recovered from nonlymphoid tissues, especially liver, were substantially larger than those recovered from thymus and spleen (see Fig. 2). In general, the tissue content of mRNAs for both ProToc and ParaT paralleled the
TABLE
V
LEVELS OF ParaT IN TISSUES OF YOUNG AND OLD MICE Young mice
Old mice
Tissue
nmol/g tissue”
pg/g tissue
nmol/g tissue”
pg/g tissue
Thymus Spleen Lung Kidney Brain Liver Muscle
6.6 ?I 0.5 5.8 + 0.5 12.2 * 1.1 15.2 + 1.1 7.7 -t 0.8 17.3 t 1.2 1.1 I? 0.2
76 66 140 174 88 198 13
NA* 4.3 YE0.3 8.6 f 0.9 11.2 IL 0.6 6.1 + 0.4 14.0 k 1.0 0.9 570.1
NA* 49 (0.74)’ 99 (0.71) 129 (0.74) 70 (0.79) 161(0.81) 10 (0.77)
a Aliquots of the extracts described in Table IV were taken for analysis of ParaT. Each assay was performed in triplicate and the arithmetic means and maximum deviations reported. b No thymus was obtained from the old mice. ’ Values in parentheses are the ratios of content in old versus young mice.
262
CLINTON
ET AL. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes on Aging (NP-534) and the National Institutes of Allergy and Infectious Diseases (AI 22901). Maria Frangou-Lazaridis was the recipient of a Fellowship from the Fogarty International Center, National Institutes of Health (1 F05 TW03770). REFERENCES
Prothymosin
Q
Parothymosin
FIG. 3. Sizes and distribution in mouse tissue of mRNA for ProTa and ParaT. The procedures were as described for Fig. 1. The cDNA probes were labeled by nick-translation (see Experimental Procedures).
content of the polypeptides. For lung, kidney, and brain, the relative quantity of ProTa mRNA was greater than the relative quantity of polypeptide, suggesting either more rapid turnover of the polypeptide in these tissues or regulation of synthesis at the translational level. This was also true for ParaT in brain and possibly also in lung. Only for ParaT in kidney did we observe a higher content of polypeptide relative to the content of mRNA. With respect to the comparison of the content of ProTol and ParaT in tissues of old mice, we had hoped to find larger changes in the levels of these polypeptides, which might be related to differences in immune responses in young and old animals (for a review see (21)). The small differences observed appear to be consistent from tissue to tissue, but additional studies are required to confirm these observations and to evaluate their significance. Marginal differences in serum thymosin 01~levels in young and aged mice have also been reported by Weindruch et aZ. (22).
While ProTa and ParaT have not yet been isolated and characterized from mouse tissue, it is of interest that the mRNA for ProTa in the mouse contains approximately 500-600 additional bases, compared to the mRNAs in rat and man. It is also not clear why these messages,which code for only 100-110 amino acid residues, should contain such large noncoding regions.
1. HARITOS, A. A., GOODALL, G. J., AND HORECKER, B. L. (1984) P~oc. N&l. Acad Sci. USA 81,10081011. 2. HARITOS, A. A., SALVIN, S. B., BLACHER, R., STEIN, S., AND HORECKER, B. L. (1985) Pra. Natl. Acad. Sci. USA 82,1050-1053. 3. HARITOS, A. A., TSOLAS, O., AND HORECKER, B. L. (1984) Proc. Nat1 Ad. Sci. USA 81,1391-1393. 4. PANNEERSELVAM, C., CALDARELLA, J., AND HORECKER, B. L. (1987) J. Immunol Methods 104, 131-136. 5. FRANGOU-LAZARIDIS, M., CLINTON, M., GOODALL, G. J., AND HORECKER, B. L. (1988) Arch. Bitthem. Biophys. 263,305-310. 6. GOODALL, G. J., DOMINGUEZ, F., AND HORECKER, B. L. (1986) Proc. N&l. Aend. Sci. USA 83,89268928. 7. LOMEDICO, P. T., GUBLER, U., HELLMANN, C. P., DUKOVICH, M., GIRI, J. G., PAN, Y.-C. E., COLLIER, K., SEMIONOW, R., CHUA, A. O., AND MIZEL, S. B. (1984) Nature (Lmdon) 312, 458462. 8. FURUTANI, Y., NOTAKE, M., YAMAYOSHI, M., YAMAGISHI, J., NOMURA, H., OHUE, M., FURUTA, R., FUKUI, T., YAMADA, M., AND NAKAMURA, S. (1985) Nucleic Acids Res. 13,5869-5882. 9. JAYE, M., HOWK, R., BURGESS, W., RICCA, G. A., CHILJ, I.-M., RAVERA, M. W., O’BRIEN, S. J., MODI, W. S., MACIAG, T., AND DROHAN, W. N. (1986) Science 233,541-545. 10. KURT-JONES, E. A., BELLER, D. I., MIZEL, S. B., AND UNANUE, E. R. (1985) Proc. Natl. Acad. Scil USA 82,1204-1208. 11. MATSUSHIMA, K., TAGUCHI, M., KOVACS, E. J., YOUNG, H. A., AND OPPENHEIM, J. J. (1986) J. Immunol. 136,2883-2891. 12. HARITOS, A. A., AND HORECKER, B. L. (1985) J. Immunol. Methods Sl, lSS-205. 13. KOMIYAMA, T., PAN, L.-X., HARITOS, A. A., WIDEMAN, J. W., PAN, Y.-C. E., CHANG, M., ROGERS, I., AND HORECKER, B. L. (1986) Proc. Natl. Acad Sci. USA 83,1242-1245. 14. HEINRIKSON, R. L., AND MEREDITH, S. C. (1984) Anal. Biochem. 136,65-74. 15. CHIRGWIN, J. M., PRZYBYLA, A. E., MACDONALD, R. J., AND RUTTER, W. J. (1979) Biochemistry 18.5294-5299.
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PROTHYMOSIN
16. FEINBERG, A. P., AND VOGELSTEIN, B. (1984) Anal. Biochem. 137,6-13,266-267. 17. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 18. HARITOS, A. A., CALDARELLA, J., AND HORECKER, B. L. (1985) Anal. Rio&em. 144,436-440. 19. ESCHENFELDT, W. H., AND BERGER, S. L. (1986) Proc. Natl. Acad. Sci. USA 83,9403-9407.
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20. Low, T. L. K., THURMAN, G. B., MCADOO, M., McCLURE, J., ROSSIO, J. L., NAYLOR, P. H., AND GOLDSTEIN, A. L. (1979) J. Biol. Chem. 254,981986. 21. SISKIND, G. W., AND WEKSLER, M. E. (1982) in Annual Review of Gerontology and Geriatrics (Eisdorfer, C., Ed.), pp. 3-26, Springer Press, New York. 22. WEINDRUCH, R.,NAYLOR, P. H., GOLDSTEIN, A. L., AND WALFORD, R. L. (1988) J. Germtol. 43, B40-42.
Prothymosin
cy and Parathymosin: mRNA and Polypeptide in Rodent Tissues
M. CLINTON, Department
of Biochemistry,
Levels
M. FRANGOU-LAZARIDIS,l C. PANNEERSELVAM,’ AND B. L. HORECKER3 Cornell University
Medical College,
1300
York Avenue, New York, New Ymk 10021
Received August 10,1988, and in revised form October 10,198X
Blot hybridization analyses have established the presence of mRNAs for prothymosin (Y(ProTa) and for parathymosin (ParaT) in rat and mouse lung, liver, kidney, and brain, confirming the biosynthesis of these peptides in nonlymphoid tissues. In these tissues the levels of mRNAs paralleled the content of the polypeptides, determined with specific radioimmunoassays. The mRNA levels also confirmed the reciprocal relation between the two polypeptides; ProTa and its mRNA were found in highest concentrations in spleen and thy&us, followed by lung, kidney, and brain, with lowest concentrations in liver. On the other hand, liver contained highest concentrations of ParaT and the mRNA for ParaT, with lowest levels present in spleen and thymus. In comparison to tissues from young (6-8 week) mice, older (18 month) mice contained lower concentrations (2040%) of both polypeptides, with qualitatively similar decreases in mRNA content. 019~9 Academic
Press, Inc.
The polypeptides prothymosin 01 (ProTLu)4 and parathymosin (ParaT) were first isolated from rat thymus (1, 2) but have since been shown to be present in many other mammalian tissues (2-4). Rat ProT~v and rat ParaT are similar in size, containing 111 and 101 amino acid residues, respectively (5), and share an unusual amino acid composition characterized by a high content of glutamyl and aspartyl residues and the complete absence of aromatic and sulfur-containing amino acids. Despite these similarities in 1 Current address: Laboratory of Biological Chemistry, University of Ioannina Medical School, GR45332, Ioannina, Greece. ’ On leave from the Department of Biochemistry, University of Madras, Madras, 600113, India. 3 To whom correspondence should be addressed. ’ Abbreviations used: ProTa, prothymosin a; ParaT, parathymosin; RIA, radioimmunoassay; Mops, 4-morpholinepropanesulfonic acid; SDS, sodium dodecyl sulfate.
0003-9861/89 $3.00 Copyright All rights
0 1989 by Academic Press, Inc. of reproduction in any form reserved.
256
size and composition, they show sequence homologies in only limited regions in the NHz-terminal portion (5). The almost ubiquitous distribution of ProTa and ParaT in mammalian tissues raises interesting questions regarding their origin in these tissues. Based on the structure of the cloned cDNAs for both rat and human ProTors (5, 6), which contain initiator codons preceding the codons for the first amino acid residue and terminator codons immediately following the codon for the COOH-terminal residues, their synthesis via a larger precursor is excluded. The absence of a hydrophobic signal peptide makes it unlikely that ProTcv is exported by the usual mechanism for secretory proteins. However, this lack of a leader sequence is shared by a number of secreted peptides, including interleukin 1 (7, 8) and endothelial growth factor (9). The release of interleukin 1 from a membrane-bound form (10) is thought to be mediated by extracellular proteinases (111,
RODENT
TISSUE
PROTHYMOSIN
but this mechanism is unlikely to apply to ProTa and ParaT which lack hydrophobic sequences. The present studies were undertaken to evaluate the presence of mRNAs for prothymosin o( and parathymosin in rat and mouse tissues, as evidence for their biosynthesis in these tissues, using the cloned cDNAs (5) for blot hybridization analysis. We have also compared the levels of the mRNAs with the concentrations of the respective polypeptides determined with the recently developed specific RIAs (4,12). We find that all tissues examined contain mRNAs for both ProTcy and ParaT and that the relative levels of these mRNAs parallel the levels of polypeptides. The results also confirm that most active synthesis of ProTa occurs in thymus and spleen, in contrast to ParaT, which is most actively synthesized in nonlymphoid tissues such as liver and kidney. Comparison of the levels of mRNAs and polypeptides in tissues from young and old mice indicates that aging is accompanied by small but consistent decreases in the synthesis and tissue content of both polypeptides. EXPERIMENTAL
PROCEDURES
Materials. Synthetic thymosin 01~and the synthetic prothymosin LYfragments 23-32 and 26-35 were generously provided by Dr. E. P. Heimer, Roche Research Center, Nutley, New Jersey. Prothymosin cyand parathymosin were isolated as described (13) from rat thymus and rat liver, respectively. The content of these peptides in solution was determined by amino acid analysis carried out with aliquots hydrolyzed in 6.0 N HCI at 150°C for 1 h, using a Waters Model PicoTag analyzer, as described by Heinrikson and Meredith (14). Reagents and solvents were analytical or chromatography grade commercial preparations. Solvents were redistilled as required. Collection oftissue. Male Charles River CD rats, 56 weeks old, were sacrificed by decapitation and the tissues removed immediately and frozen in liquid nitrogen. Larger organs, such as liver, brain, lung, and kidney, were minced with scissors before freezing. Frozen tissues from 10 rats were pooled and stored at -70°C. The same pools of frozen tissues were sampled for RNA and for polypeptide analyses. For the experiments with mice, young (6-8 weeks) or “old” (18 months) mice, strain C57BL/6, were employed. For
cy AND
PARATHYMOSIN
257
both mRNA and peptide analyses tissues from six mice in each group were collected as described for rat tissues and pooled after freezing. RNA e&r&ion and analyses. Tissue samples (l-2 g) were taken from each frozen tissue pool and total RNA was isolated by the guanidine isothiocyanatecesium chloride method (15). After two ethanol precipitations from 0.3 M Na acetate, the last precipitate was stored at -70°C as a suspension in ethanol. For electrophoretic analysis, aliquots of the RNA suspension (15 wg), as determined by uv absorption analysis, were removed, the RNA was pelleted, washed with ethanol, and dried on a Speed-Vat concentrator, and the residues were suspended in RNA sample buffer containing 1X running buffer (1X = 20 mM Mops, pH 7.0, 5 mM Na acetate, and 1 mM EDTA), 50% formamide, and 2.2 M formaldehyde. The solutions were heated to 65°C for 5 min and applied to horizontal 0.8% agarose gels containing 1X running buffer and 2.2 M formaldehyde. After electrophoresis at 90 V for 2.5 h, the gels were stained for 30 min in 1X running buffer containing 10 fig/ml of ethidium bromide, destained twice for 15 min in 1X running buffer, and transferred by blotting overnight to nylon membranes (Hybond-N, Amersham) with 10X SSC buffer (1X SSC buffer contains 15 mM trisodium citrate and 0.15 M NaCl). The membranes were baked at 80°C for 2 h in a vacuum oven, immersed briefly in 6X SSC at room temperature, and prehybridized for 4 h at 42°C in 1X Denhardt’s solution (0.2% Ficoll, 0.2% polyvinylpyrrolidone, and 0.2% bovine serum albumin) plus 6X SSC buffer, 0.2% SDS, 50 mM NaH2P04, pH 7.0, 0.1 mg/ml denatured salmon sperm DNA, and 50% formamide. The cDNA probes employed for detection of prothymosin 01 and parathymosin mRNAs were from clones pRSpro-187 and pRSpara-5187 (5), respectively. The probes were labeled with [“‘P]dCTP by random priming (16) (l-2 X lo9 cpm/pg) or by nick translation (17) (2-4 X lo* cpm/pg) as indicated. Hybridization with these probes was at 42°C for 18-20 h in a solution containing 1X Denhardt’s solution (see above), plus 6X SSC buffer, 0.2% SDS, 50 mM NaH2P04, pH 7.0,15 mM EDTA, 0.1 mg/ml denatured salmon sperm DNA, and 50% formamide, with 5 X lo6 cpm/ml of labeled probe. Final washing of the membranes was in 1X SSC, 0.1% SDS at 55°C for both probes. After being washed, the membranes were exposed to Kodak XAR film for 2-12 h using DuPont Codex intensifying screens. For hybridization with cDNA probes on slot blots aliquots of the RNA suspension (125 Kg) were centrifuged, washed, and dried, as described above. The RNA was dissolved in Ha0 containing 1 mg/ml of yeast tRNA (Sigma) and seven serial twofold dilutions were tested for each RNA sample, covering a range of 15 fig to 110 ng per slot. Immediately before
258
CLINTON
ET AL.
TABLE
I
RELATIVE CONTENT OF mRNAs FOR PROTHYMOSIN 01AND PARATHYMOSIN ESTIMATED FROM SLOT BLOT HYBRIDIZATION EXPERIMENTS’ Prothymosin
Thymus Spleen Lung Brain Kidney Liver
1
2
3
1.00 0.77 0.68 0.45 0.43 0.08
1.00 0.78 0.63 0.43 0.42 0.07
1.00 0.75 0.71 0.50 0.50 0.07
o(
Parathymosin Average 1.00 0.76 0.67 0.46 0.45 0.07
1
2
3
0.40 0.27 0.54 0.83 0.51 1.00
0.47 0.30 0.58 0.86 0.55 1.00
0.47 0.27 0.65 0.83 0.65 1.00
Average 0.45 0.28 0.59 0.84 0.57 1.00
a Slot blot hybridization analyses were carried out with RNA samples prepared from aliquots of the pooled frozen tissues from 10 rats (see Experimental Procedures). The values represent the areas under the peak of each densitometer scan, with the values obtained for ProTcv mRNA from thymus and ParaT mRNA from liver taken as 1.0. Only densitometer scan areas in the linear range of the seven serial dilutions were used. Three independent slot blot hybridization analyses were carried out for each sample.
application, each sample was adjusted to 2.2 M formaldehyde, 10X SSC and incubated at 65°C for 5 min. Immobilization of the RNA was performed on a Minifold II slot-blot apparatus (Schleicher & Schuell) with BA85 nitrocellulose membranes (Schleicher & Schuell) which had been prewetted with water, then 10X SSC, and after application each individual well was washed with 500 pl 10~ SSC. The membranes were then air-dried and baked under vacuum at 80°C for 2 h. Prehybridization, hybridization, and washings were as described earlier for the nylon membranes. Multiple exposures of each film were prepared to ensure a linear response of the film. Densitometric measurements were with a Quick Scan, Jr. (Helene Laboratories). Radioimmunoassays. Extracts for the radioimmunoassays were prepared as described by Komiyama et al. (13). Briefly, aliquots (2 g) were taken from each frozen tissue pool and pulverized under Nz in a chilled mortar and pestle. The frozen powders were then quickly dispersed into 20 ml of boiling water and boiled continually for 5 min. After being cooled in ice, each suspension was homogenized with a Polytron homogenizer (Brinkman Type PT 10/35) and centrifuged. Aliquots from each clear supernatant solution were taken for radioimmunoassays, carried out in duplicate or triplicate in Eppendorf microfuge tubes essentially as described for prothymosin o( by Haritos and Horecker (12) and for parathymosin by Panneerselvam et al. (4). For ProTa, the radiolabeled ligand was 3H-methylated thymosin 01~,and synthetic thymosin 01~was employed to standardize the assay. With the anti-thymosin o(~ antibody employed, displacement of thymosin 01~required five times as much
ProTa on a molar basis (12). We have previously shown (1, 18) that with this extraction method the cross-reacting material is ProTa, with less than detectable amounts of thymosin at present. For ParaT the radioimmunoassay employed an antibody raised against ParaT, with 3H-methylated ParaT as the competing radioligand. The epitope recognized by this antibody includes residues 26-32 of ParaT (4). This specificity was confirmed using synthetic peptides corresponding to residues 23-32 and 26-35 of ParaT (see Materials). With these synthetic peptides, the displacement curves followed closely the curve described previously (4) for ParaT 1-42 (data not shown). RESULTS
Levels of mRNAs fin- ProTa and ParaT in rat tissues. Blot hybridization
analysis confirmed the presence of mRNAs for both ProTa and ParaT in a variety of rat tissues. Quantitative analysis on slot blots showed highest quantities of ProTa mRNA in thymus, spleen, and lung, with intermediate levels in brain and kidney, and low, but definitely detectable, quantities in liver. For parathymosin, on the other hand, the levels of mRNA were highest in liver and brain, with relatively low levels in spleen and thymus (Table I). Sizes of the mRNAs for ProTa and ParaT were estimated by blot hybridiza-
RODENT
Prothymosin
CI
TISSUE
PROTHYMOSIN
Porothymosin
FIG. 1. Sizes and distribution in rat tissues of mRNAs for ProTo and ParaT. Total RNA was isolated as under Experimental Procedures. Aliquots containing 15 pg of RNA were applied to each lane and electrophoresis, transfer, hybridization, and autoradiography carried out as described under Experimental Procedures, with cDNA probes labeled by random priming. The positions of 18 and 28 S ribosomal RNA were determined from the ethidiumstained gels, as indicated.
o( AND
259
PARATHYMOSIN
fold higher for ProTa (cf. Table II and Table III of Ref (2)) and one-third lower for ParaT (cf. Table III and Table I of Ref. (4)). The reasons for these differences are not immediately apparent but may reflect variations in content from one group of animals to another. The reciprocal relation between the concentrations of both mRNAs and polypeptides in the tissues examined is best illustrated by comparing the content in each tissue to the corresponding content in thymus (Fig. 2). The values for both mRNA and polypeptide were highest for ProTa in lymphoid tissues, whereas for ParaT the concentrations of both mRNA and peptide were highest in nonlymphoid tissues. The results also suggest that regulation of the
TABLE
II
PROTHYMOSIN 01IN RAT Trssuss
tion after electrophoresis on agarose gels, which also confirmed the relative quantities of these mRNAs in these tissues (Fig. 1). The mRNA for rat ProTa was estimated to contain approximately 1.3 kb, similar to the value, 1.4 kb, reported for the mRNA isolated from a human fibroblast cell line (19). The mRNA for rat ParaT was considerably larger, containing approximately 1.8 kb (Fig. 1). For each polypeptide only a single RNA species hybridizing to the respective DNA probe was detected, although the presence of several transcripts of similar size was not excluded. Tissue levels of ProTa! and ParaT. Preliminary values for the content of these polypeptides, employing the recently developed radioimmunoassays, have been reported (Z-4). In order to relate the tissue content of the polypeptides to the mRNA levels reported in the previous section, we analyzed extracts prepared from the same rat tissue pools used for the RNA analyses. For all of the tissues tested, the relative quantities of the two polypeptides (Tables II and III) were similar to those reported previously (2-4), although the absolute values were different, approximately two-
Thymosin 01,equivalents* (nmol/g wet tissue) Tissue” Thymus Spleen Lung Kidney Brain Liver Muscle
Extract
I
13.9 -t 2.2 8.5 + 0.3 3.4 2 0.19 3.1 kO.11 1.4 + 0.09 1.78 +_0.03 0.5 iz 0.02
Extract 15.9 9.5 3.5 3.1 1.8 1.5 0.37
II
* 1.5 k 0.8 t 0.1
* 0.1 -+ 0.2 -+ 0.1 t 0.05
ProTo” (cLg/g wet tissue) 916 554 216 195 100 110 27
a Tissues from 10 rats were collected as described under Experimental Procedures and stored at -70°C. “Two separate extracts were prepared from 2 g samples of each frozen tissue pool (see Experimental Procedures). Each extract was analyzed in triplicate (Extract I) or duplicate (Extract II) for cross-reacting material, and the results expressed as thymosin 01~equivalents/g wet tissue. The numbers shown represent the arithmetic means k the maximum deviation. “To convert thymosin 0~~ equivalents to micrograms of ProTcu, the arithmetic mean values for thymosin a1 equivalents were multiplied by a factor of 5 to account for the Iower reactivity of ProTo in the RIA for thymosin 01~(see Ref. 12) and converted to micrograms using the molecular weight of 12,282 for rat prothymosin LYestablished from the eDNA sequence (5).
260
CLINTON TABLE
III
PARATHYMOSININRATTISSUES Parathymosin nmol/g Tissue
Extract
I
Thymus Spleen Lung Kidney Brain Liver Muscle
7.1 + 4.8 F 5.5 f 13.8 f 8.3 f 19.2 f 1.4 *
0.4 0.3 0.6 0.6 0.4 0.9 0.2
tissue” Extract
II
tissue*
6.6 + 0.3 5.5 + 0.4 8.5 ‘-c 0.4 11.3 +- 0.9 7.4 i 0.4 18.7 + 0.8 1.0 f 0.2
78 59 80 144 90 217 14
a Aliquots of the extracts prepared for analysis of ProTa (see Table 11) were also analyzed for ParaT. Each extract was analyzed in duplicate and the arithmetic means and maximum deviations are reported. bFor conversion of nanomoles to micrograms the molecular weight was taken to be 11,470, calculated from the amino acid sequence deduced from the cloned cDNA (5).
ET AL.
same pooled tissues (Fig. 3). The content of mRNA for ProTa was distinctly lower in all four tissues from old, as compared to young, animals. This was also true for the content of mRNA for ParaT, except for kidney where the levels did not appear to be significantly different. We were unable to compare the content of the mRNAs in young and old thymus because thymus tissue could not be recovered from the 18month-old animals. The mRNAs for ParaT were similar in size, -1.8 kb, in the rat and the mouse. For ProTa, however, important species differences were observed, -1.9 kb for mouse ProTo mRNA compared to 1.3 kb for this mRNA in rat tissues and 1.4 kb reported by Eschenfeldt and Berger (19) for human fibroblasts. DISCUSSION
The presence of mRNAs for ProTa and ParaT in nonlymphoid tissues supports PROTHYMOSIN
tissue content for both polypeptides is primarily at the level of transcription, because in general the relative content of the mRNA paralleled the relative content of the polypeptide. Efect of aging on levels of ProTa and
Cl
060.4 02-
ParaT and their mRNAs in mouse tissue.
The effects of aging were studied in tissues from C57BL/6 mice, since this strain is currently employed in other laboratories at this institution for studies on the effects of aging on the immune system. Radioimmunoassays for ProTcv and ParaT were carried out on extracts from tissues of 6- to Sweek-old and l&month-old mice (Tables IV and V). The values obtained for both peptides in tissues from young mice were very similar to those reported in Tables II and III for 5- to 6-week-old rats, but the values for ProTa in tissues of 18-monthold mice were 25-40% lower. For ParaT the differences in content in old and young animals were smaller, only 20-30%. Qualitatively similar differences were observed in the levels of mRNAs analyzed in brain, spleen, liver, and kidney from the
30
U 0
RNA PeptIde
PARATHYMOSIN n
20-
1 o-
FIG. 2. Comparison of ProTa and ParaT mRNA and polypeptide levels in rat tissues. The data from Table I and Tables II and III are represented as ratios to the values for the mRNAs and polypeptides in thymus. The values for polypeptide content for ProTa and ParaT in thymus were 916 and 78 @g/g tissue, respectively, taken from the data in Tables II and III.
RODENT
TISSUE
PROTHYMOSIN TABLE
LY AND
261
PARATHYMOSIN
IV
LEVELS OF ProTol IN TISSUES OF YOUNG AND OLD MICE Age 18 months
Age 6-8 weeks
Tissue
Thymosin (pi equivalents” (nmol/g tissue)
Prothymosin 01 (pg/g tissue) *
Thymus Spleen Lung Kidney Brain Liver Muscle
10.3 + 5.2 f 3.2 + 2.4 c 1.2 * 1.7 k 0.4 It
638 320 196 150 74 106 22
1.5 1.0 0.5 0.4 0.2 0.1 0.1
Thymosin 01~equivalents” (nmol/g tissue)
Prothymosin CY (fig/g tissue) NA 238 (0.74)d 122 (0.62) 112 (0.75) 42 (0.57) 64 (0.60) 16 (0.73)
NA” 3.8 + 0.4 2.0 f 0.3 1.8 f 0.1 0.7 -t 0.1 1.0 f 0.1 0.3 f 0.1
’ Tissues from six mice were collected and frozen, and the frozen tissues pooled (see Experimental Procedures). Aliquots (0.5-2.0 g) of the pooled tissues were extracted and the extracts analyzed in triplicate as described for rat tissues (see Table II), and the arithmetic means and maximum deviations reported. *The values for thymosin 01~equivalents were converted to micrograms of ProTo as described in the footnotes to Table II. ‘No thymus was obtained from the old mice. ‘The numbers in parentheses represent the ratio of the content in tissues from 18-month-old mice to the content in tissues in 6- to 8-week-old mice.
the concept that these polypeptides are synthesized in these tissues. It is thus unlikely that either ProTcx or ParaT functions as a thymic hormone, as has been proposed by others for thymosin LYE,the NHa-terminal fragment of ProTLv (20). This is particularly true for ParaT; for this
polypeptide the quantities of mRNA recovered from nonlymphoid tissues, especially liver, were substantially larger than those recovered from thymus and spleen (see Fig. 2). In general, the tissue content of mRNAs for both ProToc and ParaT paralleled the
TABLE
V
LEVELS OF ParaT IN TISSUES OF YOUNG AND OLD MICE Young mice
Old mice
Tissue
nmol/g tissue”
pg/g tissue
nmol/g tissue”
pg/g tissue
Thymus Spleen Lung Kidney Brain Liver Muscle
6.6 ?I 0.5 5.8 + 0.5 12.2 * 1.1 15.2 + 1.1 7.7 -t 0.8 17.3 t 1.2 1.1 I? 0.2
76 66 140 174 88 198 13
NA* 4.3 YE0.3 8.6 f 0.9 11.2 IL 0.6 6.1 + 0.4 14.0 k 1.0 0.9 570.1
NA* 49 (0.74)’ 99 (0.71) 129 (0.74) 70 (0.79) 161(0.81) 10 (0.77)
a Aliquots of the extracts described in Table IV were taken for analysis of ParaT. Each assay was performed in triplicate and the arithmetic means and maximum deviations reported. b No thymus was obtained from the old mice. ’ Values in parentheses are the ratios of content in old versus young mice.
262
CLINTON
ET AL. ACKNOWLEDGMENTS This work was supported by grants from the National Institutes on Aging (NP-534) and the National Institutes of Allergy and Infectious Diseases (AI 22901). Maria Frangou-Lazaridis was the recipient of a Fellowship from the Fogarty International Center, National Institutes of Health (1 F05 TW03770). REFERENCES
Prothymosin
Q
Parothymosin
FIG. 3. Sizes and distribution in mouse tissue of mRNA for ProTa and ParaT. The procedures were as described for Fig. 1. The cDNA probes were labeled by nick-translation (see Experimental Procedures).
content of the polypeptides. For lung, kidney, and brain, the relative quantity of ProTa mRNA was greater than the relative quantity of polypeptide, suggesting either more rapid turnover of the polypeptide in these tissues or regulation of synthesis at the translational level. This was also true for ParaT in brain and possibly also in lung. Only for ParaT in kidney did we observe a higher content of polypeptide relative to the content of mRNA. With respect to the comparison of the content of ProTol and ParaT in tissues of old mice, we had hoped to find larger changes in the levels of these polypeptides, which might be related to differences in immune responses in young and old animals (for a review see (21)). The small differences observed appear to be consistent from tissue to tissue, but additional studies are required to confirm these observations and to evaluate their significance. Marginal differences in serum thymosin 01~levels in young and aged mice have also been reported by Weindruch et aZ. (22).
While ProTa and ParaT have not yet been isolated and characterized from mouse tissue, it is of interest that the mRNA for ProTa in the mouse contains approximately 500-600 additional bases, compared to the mRNAs in rat and man. It is also not clear why these messages,which code for only 100-110 amino acid residues, should contain such large noncoding regions.
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TISSUE
PROTHYMOSIN
16. FEINBERG, A. P., AND VOGELSTEIN, B. (1984) Anal. Biochem. 137,6-13,266-267. 17. MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 18. HARITOS, A. A., CALDARELLA, J., AND HORECKER, B. L. (1985) Anal. Rio&em. 144,436-440. 19. ESCHENFELDT, W. H., AND BERGER, S. L. (1986) Proc. Natl. Acad. Sci. USA 83,9403-9407.
a AND
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20. Low, T. L. K., THURMAN, G. B., MCADOO, M., McCLURE, J., ROSSIO, J. L., NAYLOR, P. H., AND GOLDSTEIN, A. L. (1979) J. Biol. Chem. 254,981986. 21. SISKIND, G. W., AND WEKSLER, M. E. (1982) in Annual Review of Gerontology and Geriatrics (Eisdorfer, C., Ed.), pp. 3-26, Springer Press, New York. 22. WEINDRUCH, R.,NAYLOR, P. H., GOLDSTEIN, A. L., AND WALFORD, R. L. (1988) J. Germtol. 43, B40-42.