Volume 4 Number 2 February 1977

Nucleic Acids Research

Terminal deoxynucleotidyl transferase is present in athymic nude mice

J.J.Hutton* and F.J.Bollum Medical Service, Veterans Administration Hospital, Lexington, KY 40507 and Department of Biochemistry, University of Kentucky, Lexington, KY 40506, USA Received 30 December 1976

ABSTRACT Terminal deoxynucleotidyl transferase activity is normal in the bone marrow of congenitally athymic nude mice, regardless of whether transferase activity is calculated on the basis of cell number, DNA content, or activity per femur. This suggests that terminal transferase containing cells of marrow do not originate in thymus.

INTRODUCTION

Terminal deoxynucleotidyl transferase (TDT) is an unusual DNA polymerase because it does not use template information to copy deoxynucleotidyl sequences . It is normally found only in thymus and bone marrow and is assayed by measuring the addition of deoxynucleoside triphosphates onto the 3'hydroxyl ends of single stranded oligodeoxynucleotide initiators. The biological function of TDT is not known. Because of its unusual concentration in thymus and the important role of this organ in development of the immunological system, several possible mechanisms have been described in which TDT participates in the generation of immunological diversity. Baltimore has postulated that variable regions of immunoglobulins arise because single stranded gaps are made in an inherited gene which encodes these regions and that during repair of these gaps, TDT inserts the wrong base or 2 bases, causing a mutation . Alternative ways TDT could act in programming immunocompetent cells include chromosomal addition reactions and episomal variable sequence generation followed by integration into the chromosome3 Regardless of the model proposed, a role for TDT in the generation of immunological diversity in B cells is more easily envisioned than a role in generation of T cell diversity. Yet, the concentration of TDT is much higher

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Nucleic Acids Research in thymus than in bone marrow. We think it is of interest to raise the possibility that a small number of TDT containing cells is part of the normal "traffic" from thymus to marrow and that TDT has nothing whatsoever to do with B cell development or immunological diversity. If this were true, then TDT should be absent from the marrow of congenitally athymic or "nude" mice4 If TDT does play a role in B cell development and the diversification of variable regions, then TDT must be present in the bone marrow of athymic mice because these animals can make immunoglobulins and antibodies to many, but not

all, antigens 49,5 .

We have found that TDT is present in the bone marrow

of T cell deficient, congenitally athymic nude mice. Our observation proves that TDT containing cells in marrow do not arise in thymus and is consistent with a role for TDT in the differentiation of immunoglobulin producing B cells.

MATERIALS AND METHODS Nude mice on the BALB/c background were obtained from the Mammalian Genetics and Production Section, Division of Cancer Treatment, National Cancer Institute, Bethesda. Control mice were from the same stock and were of mixed genotype, some heterozygous for the nude (nu) gene and others homozygous normal. The genotypes of individual control mice were not known and are designated -/+. Animals were 6 to 8 weeks old when used in experiments. Suspensions of cells from lymph nodes and thymus were obtained by forcing tissues through wire mesh into cold RPMI 1640 tissue culture medium. Marrow was flushed from femurs using a 1 ml syringe filled with culture medium and attached to a 25 gauge needle. Cells were counted with a Coulter Counter and then pelleted by centrifugation. They were either stored at -70OC or assayed immediately. Approximately 108 cells were suspended in 0.2 ml of 0.25 M potassium phosphate buffer, pH 7.5, containing 1 mM mercaptoethanol, and sonicated 4 times in 15 second bursts with cooling. The extract was centrifuged at 100,000 x g for 60 minutes and the resulting supernatant fraction was collected. Normal rabbit serum and antiserum to TDT6 were diluted 1:10 in phosphate buffered saline. TDT reaction mixture contained 0.2 M potassium cacodylate buffer (pH 7.0), 1 mM [3H] dGTP with a specific activity of 139 cpm/pmole, 0.02 mM oligo dA50, 10 mM MgC12, and 7 1 mM mercaptoethanol . Ten p1 of supernatant fraction was incubated with 10 il of diluted serum or antiserum at O C for 60 minutes. One hundred pl of TDT reaction mixture at 350 with or without oligo dA50 was then added and the incubation was continued at 35°C. Fifty p1 aliquots were removed 458

Nucleic Acids Research at 15 and 30 minutes, placed on glass fiber disks, processed for acid insoluble radioactivity and counted. One unit of TDT equals 1 nmole of nucleotide polymerized in 1 hour. RESULTS AND DISCUSSION

Terminal deoxynucleotidyl transferase activities in bone marrow and lymph nodes from normal and athymic nude mice are given in Table 1. Thymus from control mice was also assayed. Criteria for the presence of TDT included inhibition of transferase activity by antiserum to purified calf thymus TDT6 and stimulation of activity by the initiator, oligo dA50. Terminal deoxynucleotidyl transferase activity was easily demonstrated in bone marrow from athymic nude mice at a level comparable to controls. Marrow from both groups of animals contained 0.39 units of TDT/10 cells compared to a level of 8.9 units/108 cells in thymus. In the absence of the initiator, oligo dA5, activities of approximately 0.1 units/108 cells were present. This may represent TDT activity in the presence of an endogenous initiator or it may represent some other type of nucleotide binding in the crude extracts assayed. In all experiments TDT activity in marrow and thymus was clearly inhibited by antiserum to TDT. The inhibition of activity by antiserum was not complete, but is consistent with earlier neutralization tests on mouse enzyme 6. Terminal deoxynucleotidyl transferase activity was not detectable in lymph nodes. The yield of nucleated cells from marrow and lymph nodes was approxiTable 1.

Transferase activity in tissues or normal and athymic nude mice Each value of TDT activity is the mean of 3 separate determinations,

on 3 different samples of tissue.

Standard errors are reported for marrow

from nude mice, and were similar for other tissues with similar mean values. Each sample of bone marrow and lymph nodes represented pools of tissue from 10 mice.

Thymus was from individual animals.

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Nucleic Acids Research mately 30% less per nude mouse than per control animal. For example, average of 2.2 x 107 nucleated cells was obtained from the two femurs

an

of

a

The an average of 2.8 x 107 cells per control animal. of DNA per 108 cells was 0.8 mg in marrow of nude mice compared to 0.6 mg in the same tissue from controls. Regardless of whether TDT activities are calculated on the basis of cell count, DNA content, or number of femurs, there is no evidence that TDT values differ significantly in nude

nude

mouse

compared to

average amount

control mice. The homozygous nude mouse lacks a thymus 4 . There is an absolute lymphopenia in the peripheral blood with a deficiency of T cells. Levels of circulating immunoglobulin are usually reduced, but the nude mouse can mount an antibody response to many, but not all, antigens4'5. Lymphoid stem cells of both T and B lymphocytes are present in nude mice because nude bone marrow transplanted to irradiated normal recipients can repopulate the thymus and 8 give rise to both T and B cells8. Failure of T cell development in nude mice is not due to an absence of lymphoid precursor cells, but to the absence of the epithelial elements of normal thymus. The presence of TDT in marrow of athymic mice is consistent with the presence of the enzyme in lymphoid progenitor cells that can mature in marrow independent of thymic influence. A role for TDT in the generation of immunological diversity in both T and B cells cannot be excluded.

versus

supported by the Veterans Administration and by research grants from the National Institutes of Health. John Meier and James Donofrio provided expert technical assistance. This work

*

was

To whom correspondence should be sent.

REFERENCES 1 Bollum, F.J. (1974) in The Enzymes. Vol. X, pp. 145-171. Academic Press. New York 2 Baltimore, D. (1974) Nature 248, 409-411 Bollum, F.J. (1975) Karl August Forster Lectures. Vol. 14, pp. 1-47. 3 Franz Steiner VerlaA. Wiesbaden 4 Rygaard, J. (1973) Thymus and Self, Inmunobiology of the Mouse Mutant Nude. 193 pp. F.A.D.L., Copenhagen 5 Crewther, P. and Warner, N.L. (1972) Aust, J. Exp. Biol. Med. Sci. 50, 625-635 6 Bollum, F.J. (1975) Proc. Nat. Acad. Sci. USA 72, 4119-4122 7 Coleman, M.S., Greenwood, M.F., Hutton, J.J., Bollum, F.J., Lampkin, B. and Holland, P. (1976) Cancer Res. 36, 120-127 8 Pritchard, H. and Micklem, H.S. (1973) Clin. Exp. Immunol. 14, 597-607

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Terminal deoxynucleotidyl transferase is present in athymic nude mice.

Volume 4 Number 2 February 1977 Nucleic Acids Research Terminal deoxynucleotidyl transferase is present in athymic nude mice J.J.Hutton* and F.J.Bo...
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