Quail (Coturnix japonica) protamine, full-length cDNA sequence, and

THEJOURNAL OF BIOLOGICAL CHEMISTRY Val. 264, No. 30, Issue of October 25, pp. 17627-1,7630, 1989 0 1989 hy The American Society for Biochemlstry and Molecular B~ology,Inc.

Communication

Printed in U.S.A.

Quail (Coturnix japonica) Protamine, Full-length cDNA Sequence, and the Function and Evolution of Vertebrate Protamines*

MATERIALS ANDMETHODS

(Received for publication, July 11, 1989) Rafael Oliva, Robyn Goren, and Gordon H. Dixon From the Departmentof Medical Biochemistry, University of Calgary, Calgary, AlbertaT2N 4N1, Canada

Using the chicken protamine gene as a probe, we have isolated and sequenced several .positive clones from a quail testis cDNA library whichrevealthe complete sequence for the quail protamine cDNA. The predicted amino acid sequence for the quail protamine contains the N-terminal tetrapeptide ARYR present in the N-terminal region of the mammalian protamines as well as several conserved motifs and arginine clusters. In addition the size of the quail protamine (56 amino acids) is closer to that of mammals (50 amino acids) than that of the chicken (61 amino acids). Altogether this data strongly suggests the existence of an avian-mammalian protamine gene line during evolution. Southern blot analysis suggests a small number of copies ( 2 ) per haploid genome (similar to that of chicken). The reported quail protamine cDNA sequence is the second avian protamine for which the amino acid sequence is available so far and provides new insights into vertebrate protamine function and evolution.

Quails (Coturnixjaponica) were obtained from Bailey’s Game Birds, Alberta, Canada. RNA and DNA were isolated as described by Oliva and Dixon (1989). Poly(A+) RNA was selected as described by Aviv and Leder (1972). cDNA was prepared as described by Okayama and Berg (1982). Transformations were performed by electroporation (Fiedler and Wirth, 1988; Taketo, 1988) using Bio-Rad equipment. Primary and secondary screenings, template DNA preparation, sequencing, double-strandedplasmidpreparation, exonuclease III/ mung bean nuclease directional deletions, and Southern blots were performed as described by Oliva and Dixon (1989). Northern blots were performed as described by Oliva et al. (1988). Dot matrix analysis was performed using the Seqaid program (Rodas and Roufa, 1987), and protaminegene and aminoacid alignments were performed with the aid of the SEQ(AL1GN) and GENALIGN (Needleman-Wunsch) programs from BIONET (1989). RESULTS

Northernblotanalysis of quail testes RNA usingthe chicken protamine cDNA probe revealed a sharp band in the 0.4 kilobase range (Fig. 1).This was the basis for quail testis cDNA library construction, screeningwith the chicken protamine cDNA probe, and sequencing the positive clones to obtain the quail protamine cDNA sequence. One colony out of every 240 turned out to be positive. Three independent positive clones were sequenced, and their sequence revealed the complete cDNA sequence forthe quail protamine (Fig. 2). The identificationof the sequenced cDNAs as corresponding to the quail protamine is based on: 1)the very high homology of these clones with the chicken protamine cDNA and amino acid sequence (Figs. 3-5); 2) the known properties of protamines (Bloch, 1969; Subirana, 1982); and 3) the consistency of the expectedsize for the quail protamine, as determined on acid gels (Chiva et al., 1987, 1988), with the size predicted from thecDNA (Fig. 2). The expression of this gene is clearly

Protamines are small highly basic proteins which act by compacting the DNA in the sperm nuclei of many species (Bloch, 1969; Dixon, 1972; Subirana, 1975; Mezquitaand Teng, 1977a, 1977b; Oliva et al., 1982; Oliva and Mezquita, 1982, 1986; Christensen and Dixon, 1982; Oliva et al., 1987). Because of the great variabilityof sequence in these proteins, the determination of the structure of the nucleoprotamine, thepathways of vertebrateprotamineevolution,andthe function of protamines have remained elusive (Coelingh et al., 1972; Dixon et al., 1975; Warrant and Kim, 1978; Mezquita, 1985a, 1985b; Poccia, 1986; Risley, 1988). It is, therefore, necessaryto compareclosely related sequences or species in order to answer many of these questions (Dixon et al., 1985; Kasinsky et al., 1987; Kasinsky, 1989). In this paper, the determination of the sequence of quail protaminecDNA coupled with knowledge of the domestic rooster protamine gene suggests the existence of an avian-mammalian protamine gene line in evolution. *This work was supported by a term operating grant from the Medical Research Council of Canada (to G. H. D.) and an Alberta Heritage Foundation for Medical Research Post-Doctoral Fellowship (to R. 0.).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.

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FIG. 1. Northern blot analysis of testis RNA from different species using a chicken protamine probe.

17627

17628

Quail cDNA Sequence -1 GCCGTCCCAC CGCAGCCCGG C

FIG. 2. Quail protamine cDNAsequence. Translation start codon (ATG), termination codon (TGA), polyadenylation signal (AATAAA),and poly(A) tail site are underlined.

-

ATG GCC M A

CGC TACCGG CGC ACG AGG ACC CGC R Y R R T R T R

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FIG. 4. Detailedalignments of the protaminecDNA sequence from quail with that from chicken and their corresponding predicted amino acid sequences. Nucleotide positions are indicated for the quail cDNA using the ATG start codon as a reference. Neutral mismatches are indicated by an asterisk, whereas the changes leading to a different amino acid sequence are indicated by a black dot. a and b designate two alternative alignments for the region shown in brackets. Amino acid differences between quail and chicken are underlined.

DOGFISH

FIG. 3. Dot matrix analysis of all the protamine gene sequences presently available. 20 matches or more in a 32-nucleotide window are indicated as a dot. The arrow at the x or y axis indicates the scale size of the transcriptional unit. The shaded area indicates the coding region. The positions of known introns is indicated; however, intron sequences have not been included in the comparison. The quail protamine sequence is from Fig. 2. The other sequences are from the following sources: chicken (Oliva and Dixon, 1989); bull P1 (Krawetz et al., 1987, 1988; Lee et al., 1987a), mouse P1 (Kleene et al., 1985), mouse P2 (Johnson et al., 1988), human P1 (Lee et al., 1987b), human P2 (Domenjoud et al., 1988), boar P1 (Maier et aL, 1988), trout plOl(States et al., 1982), and dogfish (Berlot-Picard et al., 1986).

established from its poly(A+) mRNA origin from quail testis. The differences between the chicken and quail sequences (Figs. 4 and 5) rule outthe possibility of artifactual (by contamination) subcloning and resequencing the cDNA corresponding to the chicken (Fig. 6).

Southern blot analysis indicates a reduced complexity of the quail protamine genes, comparable to the chicken genes (Oliva and Dixon, 1989) and suggests a low copy number, perhaps of two, based on the existence of two bands on the Southern blots (Fig. 6) and on a preliminary intensity comparison with known standards (notshown). Dot matrix analysis (Fig. 3) shows the relationships between the various protamine genes for which the nucleotide sequence is so far available. The similarity between the two members of the avian family, among the members of the mammalian P1 protamine family, and among the members of the mammalian P2 family can be clearly seenin Fig. 3. Detailed alignments of the mammalian and avian amino acid protamine sequences are shown in Fig. 5, in which the homologous nature of this group of protamines is apparent. DISCUSSION

We have cloned and sequenced the cDNA corresponding to the quail protamine, the second avian protamine for which

Quail cDNA Sequence

17629

FIG.5. Detailed alignment of the amino acid sequences corresponding to the avian and mammalian P1 protamine sequences. Protamine amino acid sequences arefromquail (Fig. 2), chicken (Oliva and Dixon, 1989). bull (Mazrimas et al., 1986), hoar (Tohita et al., 1983), goat (Ammer and Henschen, 1988h), stallion (Belaiche et al., 1987;Ammer and Henschen, 1987), ram (SautiPre et al., 1984), mouse P1 (Bellvi. et al., 1988), rat (Ammer and Henschen, 1988h), rabhit (Ammer and Henschen, 1988a, 1988h), and human P1 (McKay et al., 1985, 1986; Ammer et al., 198% Gusseet al., 1986).

chicken allelic variants much more unlikely. Dot matrix analysis of all protamine gene sequences available so far clearly indicates their relationships (Fig. 3). This K b. is seen as a diagonal succession of dots at theintersection of the corresponding genes. The similarities between the two avian protamine genes, among the four mammalian P1 prot23.1 amine family genes, and within the two mammalian P2 family 9.4genes are clearly apparent (Fig. 3). Although less clear at this 6.5 level of nucleotide comparison, the avian protamine genes 4.3 also display significant similarity (in their coding regions) to W the mammalian P1 protamine family genes (indicated as several dots in the dot matrix corresponding to the coding 2.3 regions). The similarities among avian and mammalian P1 20 protamines are also clearly evident by a comparison of their amino acid sequences (Fig. 5). Such similaritiesare sufficient to postulate a homologous relationship and hence a common avian-mammalian protamine gene line during evolution. Particularly conserved are the N-terminal tetrapeptides ARYR and SRSRfollowed bycluster of 5-7 arginines (Fig. 5). These sequences are also present in the marsupials opossum and 0.5 wallaby (Balhorn et aZ., 1989). In addition, in this region at I position 8, either a serine or a theonine occurs, both polar uncharged amino acids being susceptible to phosphorylation. The similarities at the C terminus are less marked than at FIG.6. Southern blot analysis of quail genomic DNA using the N terminus (Fig. 5); nevertheless, the arginine clustering, the quail protaminecDNA as a probe. Lane I , EcoRI digest; lane the presence of the valine a t position 44 (allowing for gaps in 2, &lII digest; and lane 3, BarnHI digest. kb, kilobase. the alignment), the tyrosines at position 52, andthe Cterminal tyrosine follow a clearly conserved pattern. Among the differences between the avian and mammalian the nucleotide and amino acid sequence is now known (Fig. protamines, the main one is the absence of cysteines in bird 2). The predicted amino acid sequence for the quail protamine 5). This implies that either cysteines were protamines (Fig. shows 11 differences and is shorter by 5 amino acids than chicken protamine (Figs. 2, 4, and 5), resulting in a total lost in birds or gained in mammals. Several mechanisms for length of 56 amino acids. The homologous relationship be- the appearance or loss of cysteines are possible. If cysteine tween the quail and the chicken protamine is even more codons appeared in mammalian protamine genes, a potential marked when the corresponding nucleotide sequences are mechanism would be their origin by mutation of arginine compared (Figs. 3 and 4). These similarities between chicken codons CGT/C to a TGT/C cysteine codon. This pathway and quail protamines clarify to some extent theorigin of the might be greatly facilitated as a result of a previous cytosine differences between the amino acid sequence for the chicken methylation in thearginine codon, since 5mC is thought tobe protamine determined by Nakano et al. (1976) and the pre- evolutionarily unstable and tends to mutate toT by deamidicted amino acid sequence from the genome or the redeter- nation a t position 5 (Coulondre et al., 1978). Despite many conserved motifs between mammalian and mined amino acid sequence for galline (Oliva and Dixon, bird protamines,the presence of several cysteines in mammals 1989). These differences raised the question of whether different protamineallelic variants existed among chickens, with leads to anessentially different mechanism for condensation the possibility that Nakano et al. (1976) had sequenced one of the nucleoprotamine through the formation of disulfide of those, and Oliva and Dixon (1989) a differentone. The fact bonds in the sperm nuclei (Balhorn, 1982; Balhorn et al., that the unusual amino acids (such as threonine andvaline) 1984). The fact that cysteines are not conserved while other are present in the same position in the quail and chicken motifs are (such as the N-terminal tetrapeptide ARYR) may protamines (two different avian species) as well as in many indicate arelatively less essential functionof the cysteines as mammals (Fig. 5), only if the recently redetermined sequence compared to theconserved amino acid clusters. Therefore, we (Oliva and Dixon, 1989) is considered, makes the hypothesis think that theconsensus sequences between avian and mamof the differences in sequence corresponding to different malian protamines (Fig. 5) will provide very useful insights

1 2 3

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Quail cDNA Sequence

17630

in understanding the key elements in nucleoprotamine formation, structure, and function(Oliva et al., 1987; Nakano et al., 1989; Tobita et al., 1988). In a similar way, comparisons of the conserved nucleotide sequences at the 3’ or 5’ of the quail and chicken genes should contribute to an understanding of the important regulatory and structural elements controllingprotamine gene expression. For example, thereis strong conservation of the 5’ region immediately preceding the initiation codon as well as the 3’ region from position +240 to the polyadenylation site at +290 (onlytwomismatches). In contrast, theregion immediately 3‘ to the TGA termination codon (+174 to +240) is much less conserved (13 mismatches aswell as 2 deletionsin the chicken). was It noted (Oliva et ai., 1988; Oliva and Dixon, 1989) that this region in the chicken represented an in-frame duplication of the Cterminal coding region; however, such a region is absentfrom the quail cDNA. One explanation would be that the chicken has undergone a partial duplication of its C-terminal coding region with the possible evolutionary result of a lengthening of the protamine polypeptide in the event that the present day TGA stop codon were to mutate to, for example, CGA (Arg). It hasbeen established that longer protamines such as the present day galline are much more effective in replacing histones anddisassembling nucleosomesthan the shorter fish protamines, iridine and salmine (Oliva et al., 1987). Thus, such a hypothetical evolutionary event could lead to an improvement in the “fitness” of the protamine molecule in the replacement reaction and condensation of nucleoprotamine in the sperm nucleus in the chickenline. The divergence of quail and chicken appears tohave taken place 25-36 X lo6years ago in the Oligocene era (Olson, 1985). Based on this data and the known 11amino acid changes and a deleted cluster (12 events) between quail and chicken protamines, it can be calculated that the rate of mutation for of speciation bethese protamines since the establishment tween quail and chicken is 5.5-8 mutations/lO million years/ 100 peptide bonds. This mutation rate is much faster than the average in proteins; 1.2 mutations in 10million years/100 bonds (McLaughlin andDayhoff, 1972). REFERENCES Ammer, H., and Henschen, A. (1987) Biol. Chem. Hoppe-Seyler 3 6 8 , 16191626 Ammer H., and Henschen, A. (1988a) FEBS Lett. 242,111-116 Ammer.. H... and Henschen. A. (198813) Biol. Chem. HoDDe-Seykr .. - 369, 13011306 Ammer, H., Henschen, A., and Lee, C. H. (1986) Biol. Chem. Hoppe-Seyler 3 6 7 , 515-522 Aviv, H., and Leder, P. (1972) Proc. Nati. Acad. Sci. U. S. A. 6 9 , 1408-1412 Balhorn, R. (1982) J. Cell Biol. 93,298-305 Balhorn. R.. Weston. , , , S.., Thomas. C.. and Wvrobek. A. J. (1984) EXD.Cell Res. 1 5 0 , 2981308 Balhorn, R., Mazrimas, J. A,, Corzett, M., Cumming, J., and Fadem, B. (1989) J. Cell Biol. 1 0 7 , 950 (Abstr. 167) Belaiche. D., Loir. M.. K r u d e ,. W., and Sautiere P. (1987) Biochim. Biophys. A C ~ U 9’13,’145-i49 Bellve, A. R., McKay, D. J., Renaux, B. S., andDixon, G. H. (1988) Biochemistry 27,2890-2897 Berlot-Picard, F., Vodjdani, G., and Doly, J. (1986) Eur. J . Biochem. 160,305~

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