Biochimica et Biophysica Acta, 950 (1988) 215-220 215 Elsevier

BBA 91825

Nucleotide sequence of a c D N A clone encoding mouse transition protein 1

Kenneth C. Kleene a, Adel Borzorgzadeh a, James F. Flynn a, Pamela C. Yelick b and Norman B. Hecht b

a Department of Biology, University of Massachusetts at Boston, Boston, MA and b Department of Biology, Tufts University, Medford, MA (U.S.A.)

(Received 22 December 1987)

Key words: Transition protein 1; Nuclear protein; cDNA clone; (Mouse)

We have determined the nucleotide sequence of cDNA clones encoding mouse transition protein 1 (TP1), a basic nuclear protein involved in nuclear condensation during spermiogenesis. The nucleotide sequence predicts that transition protein 1 in rats and mice differs by only one amino acid. The rate of substitution of nucleotides in the coding region of mouse and rat transition protein 1 mRNA is close to the average of many proteins in rats and mice, and the usage of degenerate codons is typical of the mouse. The identification of this cDNA clone, in conjunction with previous work (Kleene et al. (1983) Dev. Biol. 98, 455-464; Hecht et al. (1986) Exp. Cell Res. 164, 183-190), demonstrates that the mRNA for mouse transition protein 1 accumulates during the haploid phase of spermatogenesis.

Introduction

During the haploid (spermatid) phase of spermatogenesis in many vertebrates, the histones are replaced by protamines [1-12], low molecular weight proteins, 28-65 amino acids long, which typically contain about 60% arginine grouped in several clusters, little or no lysine, and an ex- tremely limited amino-acid composition [7,8,13- 17]. The replacement of histones by protamines causes a marked reduction in the volume of the sperm nucleus [18], a cellular specialization which enhances sperm motility, and leads to the com- plete cessation of transcription [19]. In trout, the histones are replaced directly by protamines [1], whereas in dogfish [6] and mammals [4,5,9-12],

Abbreviation: TP1, transition protein 1.

Correspondence: K.C. Kleene, Department of Biology, Univer- sity of Massachusetts at Boston, Boston, MA 02125, U.S.A.

the histones are replaced initially by two or more spermatid-specific basic nuclear proteins, known as transition proteins. The transition proteins ap- parently function as intermediates while chro- matin condensation is in progress, because they do not appear in the nuclei of mature sperm [2,4-6,9-12].

We became interested in the transition proteins and protarnines when we isolated three clones from a mouse testis cDNA library that hybridized to small mRNAs which are detectable only in haploid spermatogenic cells [20]. Two of these clones have been sequenced previously, demon- strafing that they encode protamine 1 [13] and the precursor for protamine 2 [17].

We report here the sequence of the third clone demonstrating that it, too, encodes a spermatidal basic nuclear protein, transition protein 1 (TP1). TP1 was first isolated from the testes of rats by Kistler et al. [10]. Subsequently, Kistler and his colleagues determined the sequence of the protein directly [21] and indirectly from a cDNA clone

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

216

[22]. Rat TP1 is 54 amino acids long, contains about 50% basic amino adds, and differs from mammalian protamines because it is soluble in 5% trichloroacetic acid [10], contains about 19% lysine and lacks cysteine and clusters of arginines. Ho- mologues of TP1, which sometimes contain cy- steine [4,23], have been found in nuclei of con- densing spermatids of mice, rams and many other mammals [3,5,9,11,23]. Unexpectedly, considering its very basic composition, rat TP1 is able to lower the thermal stability of double-stranded DNA [24], an activity which is mediated by intercalation of tyrosine side-chains [24]. We report here the pre- dicted amino-acid sequence of mouse TP1. The identification of this clone also clarifies the inter- pretation of previous Northern blots using uniden- tiffed probes (previously designated HSAR 700 [20] and 11A [26]) and supports the findings of several laboratories that the synthesis of the tran- sition proteins and protamines in mammals is regulated at both the transcriptional and transla- tional levels during spermiogenesis [20,25-30].

Methods

cDNA clones encoding mouse TP1 were identi- fied in a mouse male germ cell lambda gtl0 cDNA [25] library by plaque hybridization using the in- sert from the plasmid, pCE3 [20], as a probe. The phage cDNA inserts were subcloned and se- quenced by primer extension [25].

Results and Discussion

Nucleotide sequence of cDNA clones encoding mouse TP1

The nucleotide sequence of a 405 base cDNA insert encoding mouse TP1 is shown in Fig. 1 together with the sequence of a cDNA fragment encoding rat TP1 [22]. The AUG codon at base + 1 contains an A in the - 3 position, so it is expected to be used efficiently as a translation start site [31]. The reading frame following this initiation codon encodes a polypeptide 55 amino acids long which differs by one non-conservative substitution from the revised sequence of rat TP1 [22], the substitution of a valine for a serine at amino acid 36. The calculated molecular weight is 6280, as-

suming that the first methionine is removed post- translationally and that serine is the amino- terminus of the mature protein, as in the rat [21].

The sequence of the TP1 cDNA clone demon- strates that mouse TP1 contains three leucines and no cysteines, whereas mouse protamine 1 contain nine cysteines and no leucines [13]. Thus, the sequences of these cDNA clones validate the use of radiolabeled cysteine and leucine to distinguish between translation products for protamine 1 and TP1 in the mouse [9]. The sequences containing the two tyrosines, which are thought to be crucial for the helix-destabilizing activity of TP1 [24], are identical in the mouse and rat. A search through the April 1, 1987, National Biomedical Research Foundation Protein Data Base with the FASTP program [32] revealed no proteins with sequences similar to rat or mouse TP1. In other words, there is no obvious similarity to other proteins with related functions, such as condensation of sperm nuclei (protamines and transition protein 2), packaging of DNA (histones) or prokaryotic DNA helix-destabilizing proteins [33].

The cDNA clone shown in Fig. 1 includes nine bases of 5' non-coding, and its 3' end terminates in an eight-base fragment of the poly(A) tail. The 3' termini of three other cDNA inserts appear to be incomplete, since they end in tracts of 3 or 4 A's, corresponding to the internal oligo(A) tracts at bases + 365-368 and + 383-388. The truncated 3' ends of these cDNA inserts probably result from annealing of the oligo(dT) primer used to construct the library to these internal oligo(A) sequences. Mouse TP I -m RN A lacks the 16-base hyphenated repeat signal for processing the 3' ends of the mRNAs for the class of histones whose synthesis is coupled to DNA replication [34], and the 17-base sequence found adjacent to the polyadenylation signal of the mRNAs for pro- tamine 1 and the precursor for protamine 2 in the mouse [17]. A canonical polyadenylation signal, AAUAAA, and a rare variant, AAUAAC [35], appear 12 and 30 bases upstream from the poly(A) tract. Every recombinant DNA for a transition protein in mammals or a protamine in dogfish, trout and mammals that has been sequenced to date is polyadenylated, contains a polyadenylation signal, and lacks the conserved stem-loop of repli- cative histone mRNAs [13-17,25,30,36,37].

217

1 Met Ser Thr Ser Arg Lys Leu Lys Thr

Mouse GAAAGTACC ATG TCG ACC AGC CGC AAG CTA AAG ACT Rat ATTTTGGC A .... T---A ................. A .........

-9 1

i0 20 His Gly Met Arg Arg Gly Lys Asn Arg Ala Pro His Lys Gly Val CAT GGC ATG AGG AGA GGC AAG AAC CGA GCT CCT CAC AAG GGC GTC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30 60

30 Lys Arg GIy Sly Ser Lys Arg Lys Tyr Arg Lys Ser Val Leu Lys AAG AGA GGT GGA AGC AAG AGA h~ TAC CGG hAG AGC GTC CTG AAA ........ A ........................... AG ...... G

90 Ser

40 50 Ser Arg Lys Arg Gly Asp Asp Ala Ser Arg Asn Tyr Arg Ser His AGT AGG AAA CGG GGC GAT GAT GCA AGT CGC AAT TAC CGA TCC CAC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

120 150

Leu End TTG TGA TGCGGCAATG AGCTCTGCCC TGGTGGTCTT CAAACAACAC GGGGCAGGAG .... A . . . . . . . AG ................. T .... CA ..... C-A

180 210

CATGAGGACA TCAGAGGGGG ACTGCCAAAG AGATCTGAAG TTAGACCAAA --**-* .... 240

AGCCAAAGAT CCTATCAGAG TGGGTAAATG CCAGTCGTGA CGAAATTCGG 270 300

AATGTATATG TTGGCTGTTT CTCCCCAACA TCTCAATAAC ATTTTGAAAA 330 360

CAAATAAAAT TGTGAAAAAC AAAAAAAA 390

Fig. 1. Nucleotide sequence of mouse transition protein 1 c D N A inserts and predicted amino-acid sequence. The sequences of four c D N A subclones for mouse TP1 were determined on one strand us ing primer extension with dideoxynucleotides by sequencing in from the T7 and SP6 promoters. The nueleotide sequence of one of these inserts was determined on both s trands using subclones generated using the BgllI site at base 249. The predicted amino-acid sequence of mouse TP1 is listed above the nucleotide sequence. The nucleotides are numbered below the nucleotide sequence and the amino acids are numbered above the amino-acid sequence. Base number 1 has been assigned to the A of the A T G codon and amino acid number 1 is assumed to be serine as in rat TP1 [21]. The initiation codon and the putative polyadenylation signal in the nucleotide sequence have been underlined. The nucleotide sequence of a c D N A fragment containing the coding region of rat TP1 m R N A and part of the 3 ' -nontranslated leader [22] is shown below mouse TP1. Bases which are the same in rat and mouse have been indicated by -; bases which are different are shown as A, C, G, or T; and bases which are absent in the rat are shown by *. The single amino-acid difference between rat and mouse TP1 at amino

acid 36 is also shown.

Nucleotide substitution rate of rat and mouse TP1 It is widely recognized that the protamines and

transition proteins are erratically distributed in the animal kingdom [8]. Accurate measurements of the rates of nucleotide substitution in the pro- tein-coding and regulatory elements of the genes for the transition proteins and protamines should help to elucidate the origins and eccentric distri-

bution of these proteins. We used the method of Li et al. [38] to determine the rate of nucleotide substitution of rat and mouse TP1. This method classifies nucleotide sites as completely non-de- generate, completely degenerate, or partly degen- erate and non-degenerate. For example, the third site in the codon for lysine, AAA, is scored as 1 /3 degenerate and 2 /3 non-degenerate because tran-

218

s i t i o n a l s u b s t i t u t i o n s a r e s y n o n y m o u s ( ly s ine ) a n d

t r a n s v e r s i o n a l s u b s t i t u t i o n s a re n o n - s y n o n y m o u s

( a s p a r a g i n e ) . T h e a n a l y s i s o f s u b s t i t u t i o n r a t e o f

r a t a n d m o u s e T P 1 is s t r a i g h t f o r w a r d b e c a u s e t h e

o p t i m a l a l i g n m e n t o f c o d i n g s e q u e n c e s d o e s n o t

i n c l u d e gaps . T h e c o d i n g s e q u e n c e s o f r a t a n d

m o u s e T P 1 i n Fig. 1 i n c l u d e t w o n o n - s y n o n y m o u s

s u b s t i t u t i o n s a t a t o t a l o f 36.67 n o n - d e g e n e r a t e

s i tes a n d t h r e e s y n o n y m o u s s u b s t i t u t i o n s a t a t o t a l

o f 125.33 d e g e n e r a t e si tes. T h e r a t e o f n u c l e o t i d e

s u b s t i t u t i o n is c a l c u l a t e d by :

substitution rate number of substitutions

number of sites × divergence time × 2

A l t h o u g h i t is c o n t r o v e r s i a l w h e t h e r r a t s a n d m i c e

d i v e r g e d 10 o r 30 m i l l i o n y e a r s ago, 15 m i l l i o n

y e a r s is c o n s i d e r e d a r e a s o n a b l e e s t i m a t e [39]. W e

e s t i m a t e t h a t t h e s u b s t i t u t i o n r a t e a n d s t a n d a r d

e r r o r a t s y n o n y m o u s a n d n o n s y n o n y m o u s s i tes is

(2 .9 + 1 .7 ) . 10 - 9 a n d (0.5 + 0 . 4 ) . 10 -9 s u b s t i t u -

t i o n s / s i t e p e r year . T h e s e r a t e s o f b a s e s u b s t i t u -

t i o n a t s y n o n y m o u s a n d n o n - s y n o n y m o u s s i t es o f

T P 1 a re r e s p e c t i v e l y a b o u t o n e - h a l f t he a v e r a g e o f

a v a r i e t y o f g e n e s in m o u s e a n d ra t , 6.5 • 10 - 9 a n d

1.1 • 10 - 9 s u b s t i t u t i o n s / s i t e p e r y e a r [39].

Codon usage in mouse transition protein 1

T a b l e I s h o w s t h e c o d o n u s a g e d e t e r m i n e d f r o m

TABLE I

CODON CHOICE IN MOUSE TP1

Maruyama et al. [41] presented their compilation of codon usage as the number of each codon per thousand amino acids. We have converted their data to the percentage of each degenerate codon for each amino acid. In performing the X 2 test, the expected numbers of degenerate codons were calculated from the percentage of each codon for each amino acid in the mouse and the total number of times each amino acid appears in mouse TP1. We assumed that there were 1, 2, 3 and 5 degrees of freedom for amino acids with 2, 3, 4 and 6 degenerate codons.

AA MTP1 Mouse AA MTP1 Mouse AA MTP1 mouse codon No. (%) codon No. (%) codon No. (%)

Arg Pro Gin CGA 2 10.0 CCA 0 28.0 CAA 0 23.8 CGC 2 20.9 CCC 0 32.0 CAG 0 76.2 CGG 2 15.9 CCG 0 10.2 His CGU 0 6.8 CCU 1 29.7 CAC 2 62.7 AGA 3 22.6 Ala CAU 1 37.3 AGG 2 23.7 GCA 1 18.5 Glu

Leu GCC 0 40.3 GAA 0 34.2 CUA 1 6.3 GCG 0 10.0 GAG 0 65.8 CUC 0 23.5 GCU 1 31.1 Asp CUG 1 44.0 Gly GAC 0 59.8 CUU 0 11.5 GGA 1 25.2 GAU 2 40.2 UUA 1 3.7 GGC 4 35.1 Tyr UUG 1 11.1 G G G 0 21.3 UAC 2 64.0

Ser GGU 1 18.3 UAU 0 36.0 UCA 0 11.8 Val Cys UCC 1 25.5 GUA 0 6.9 UGC 0 57.5 UCG 1 4.3 GUC 2 28.6 UGU 0 42.5 UCU 0 18.5 G U G 0 49.7 Ile AGC 3 28.5 GUU 0 14.7 AUA 0 12.7 AGU 2 11.2 Lys AUC 0 56.1

Thr AAA 3 35.6 AUU 0 31.1 ACA 0 26.5 AAG 7 64.4 Ter 0 ACC 1 39.4 Asn UAA 0 32.3 ACG 0 11.2 AAC 1 62.1 UAG 0 14.7 ACU 1 22.9 AAU 1 37.9 UGA 1 52.9

Phe Met UUC 0 64.6 AUG 2 100.0 UUU 0 35.3

219

the nucleotide sequence of mouse TP1. Since there are phylogenetic differences in codon usage [40,41], it seems appropriate to ask whether the selection of degenerate codons in mouse TP1 is characteris- tic of the mouse. We accordingly used the chi- square test to determine whether the observed number of codons for each amino acid in TP1 differs from the expected number based on the codon usage in the coding regions of 66 mouse genes compiled by Maruyama et al. [41]. The choice of codons for every amino acid in mouse TP1 is similar to the normal mouse usage judging from chi-square probabilities greater than 0.05. In addition, the third positions of degenerate codons of mouse TP1 contain similar proportions of G and C, 60.3%, and purines, 47.1%, as the mouse, 62.1% and 44.5% [41]. The finding that codon choice in TP1 is typical of mouse proteins implies that the coding sequence of mouse TPI-mRNA is insensitive to the selective pressures which in- fluence codon usage in histone genes [42,43].

Conclusions

Four cDNA clones encoding major spermatidal basic nuclear proteins in the mouse have now been sequenced, protamine 1 [13], the precursor for protamine 2 [17], TP1 and TP2 [25]. Together, these four cDNA clones account for most, and possibly all, of the 7-9 polypeptides found in sonication-resistant mouse spermatids which migrate faster than histone H4 on acid-urea poly- acrylamide gels [2,9,11]. The number of poly- peptides is greater than the number of clones, because the mRNA for protamine 2 is translated as a precursor which is processed proteolyticaUy generating 5 polypeptides [17], and the mRNA for mouse TP2 has two in-phase initiation codons so it may be translated as two polypeptides which are identical except for a difference in length at the amino-terminus [25]. Whether these cDNA clones account for all the basic nuclear proteins which replace the histories in late spermatids in mice cannot be answered until the proteins have been further characterized.

The sequence reported here also demonstrates that a small mRNA, about 600 bases long, which is found only in haploid spermatogenic cells (pre- viously referred to as HSART00 [20] and l l A [26])

encodes mouse TP1. Studies using recombinant DNA clones for protamine 1 [20,26,27,30], pro- tamine 2 [20,26], transition protein 1 [20,26] and transition protein 2 [25] in the mouse, protamine 1 in bull [14,29] and transition protein 1 in the rat [28] concur that all these mRNAs accumulate in a translationaUy inactive state in early haploid cells and the mRNAs are translationally activated in late haploid cells.

Acknowledgements

This work was supported by NSF Grant DCB- 8510350 (K.C.K.) and NIH Grant GM-29224 (N.B.H.). The authors are grateful to Dr. Wen- Hsiung Li and the Molecular Biology Computer Research Resource for programs used in sequence analysis.

References

1 Christensen, M.E. and Dixon, G.H. (1982) Dev. Biol. 93, 404-415.

2 Balhorn, R., Weston, S., Thomas, C. and Wyrobek, A.J. (1984) Exp. Cell Res. 150, 298-308.

3 Bellvt, A.R., Anderson, E. and Hanley-Bowdoin, L. (1975) Dev. Biol. 47, 349-365.

4 Dupressoir, T., Sautiere P., Lanneau, M. and Loir, M. (1985) Exp. Cell Res. 161, 63-74.

5 Grimes, S.R., Jr., Meistrich, M.L., Platz, R.D. and Hnilica, L.S. (1977) Exp. Cell Res. 110, 31-39.

6 Gusse, M. and ChevaiUier, P. (1981) Exp. Cell Res. 136, 391-397.

7 Balhorn, R. (1982) J. Cell Biol. 93, 298-305. 8 Dixon, G.H., Aiken, J.M., Jankowski, J.M., McKenzie,

D.I., Moir, R., and States, J.C. (1986) in Chromosomal Proteins and Gene Expression (Reeck, G.R., Goodman, G.A. and Puigdomenench, P., eds.), pp. 287-314, Plenum, New York.

9 Kleene, K.C. and Flynn, J. (1987) Dev. Biol. 123, 125-135. 10 Kistler, W.S., Geroch, M.E. and Williams-Ashman, H.G.

(1973) J. Biol. Chem. 248, 4532-4543. 11 Mayer, J.F., Chang, T.S.K. and Zirkin, B.R. (1981) Biol.

Reprod. 25, 1041-1051. 12 Loir, M. and Lanneau, M. (1978) Exp. Cell Res. 115,

231-243. 13 Kleene, K.C., Distel, R.J. and Hecht, N.B. (1985) Biochem-

istry 24, 719-722. 14 Krawetz, S.A., Connor, W. and Dixon, G.H. (1987) DNA

6, 47-57. 15 Lee, C-H., Mansouri, A., Hecht, W., Hecht, N.B. and

Engel, W. (1987) Biochem. Hoppe-Seyler 368, 131-135. 16 States, J.C., Connor, W., Wosnick, M.A., Aiken, J.M.,

Gedamu, L. and Dixon, G.H. (1982) Nucleic Acids Res. 10, 4551-4563.

220

17 Yelick, P.C., Balhorn, R., Johnson, P.A., Corzett, M., Mazrimas, J., Kleene, K.C. and Hecht, N.B. (1987) Mol. Cell Biol. 7, 2173-2179.

18 Pogany, G., Corzett, M., Weston, S. and Balhorn, R. (1981) Exp. Cell Res. 136, 127-136.

19 Kierszenbaum, A.L. and Tres, L.L. (1975) J. Cell Biol. 65, 258-270.

20 Kleene, K.C., Distel, R.J. and Hecht, N.B. (1983) Dev. Biol. 98, 455-464.

21 Kistler, W.S., Noyes, C., Hsu, R. and Heinrickson, R.L. (1975) J. Biol. Chem. 250, 1847-1853.

22 Heidaran, M.A. and Kistler, W.S. (1987) Gene 54, 281-284. 23 Lanneau, M. and Loir, M. (1982) J. Repro& Fertil. 65,

163-170. 24 Singh, J. and Rao, M.R.S. (1987) J. Biol. Chem. 262,

734-740. 25 Kleene, K.C. and Flynn, J.F. (1987) J. Biol. Chem. 262,

17272-17277. 26 Hecht, N.B., Bower, P.A., Waters, S.H., Yelick, P.C. and

Distel, R.J, (1986) Exp. Cell Res. 164, 183-190. 27 Kleene, K.C., Distel, R.J. and Hecht, N.B. (1984) Dev.

Biol. 105, 71-79. 28 Heidaran, M.A. and Kistler, W.S. (1987) J. Biol. Chem.

262, 13309-13315. 29 Lee, C-H., Bartels, I. and Engel, W. (1987) Biochem.

Hoppe-Seyler 368, 807-811.

30 Peschon, J.L., Behringer, R.R., Brinster, R.L. and Palmiter, R.D. (1987) Proc. Natl. Acad. So. USA 84, 5316-5319.

31 Kozak, M. (1984) Nucleic Acids Res. 12, 857-872. 32 Lipman, D.J. and Pearson, W.R. (1985) Science 227,

1435-1441. 33 Prasad, B.V. and Chiu, W. (1987) J. Mol. Biol. 193, 579-584. 34 Birchmeier, C., Folk, W. and Birnstiel, M. (1983) Cell 35,

433-440. 35 Wickens, M. and Stephenson, P.A. (1984) Science 226,

1045-1051. 36 Berlot-Picard, F., Vodjani, G. and Doly, J. (1986) Eur. J.

Biochem. 160, 305-310. 37 Berlot-Picard, F., Vodjani, G. and Doly, J. (1987) Eur. J.

Biochem. 165, 553-557. 38 Li, W-H., Wu, C-I. and Luo, C-C. (1985) Mol. Biol. Evol. 2,

150-174. 39 Li, W.-H., Tanimura, M., Sharp. P.M. (1987) J. Mol. Evol.

25, 330-342. 40 Grantham, R., Gautier, C., Gouy, M., Mercier, R. and

Pave, A. (1980) Nucleic Acids Res. 8, r49-62. 41 Maruyama, T., Gojobori, T., Aota, S. and Ikemura, T.

(1985) Nucleic Acids Res. 14, r151-197. 42 Taylor, J.D., Wellman, S.E. and Marzluff, W.F. (1986) J.

Mol. Evol. 23, 242-249. 43 Wells, D., Bains, W. and Kedes, L. (1986) J. Mol. Evol. 23,

224-241.