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Volume 270, Number 10, Issue of March 10, 1995 pp. 5642-5648
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Sequencing of an Intronless Mouse S-Adenosylmethionine Decarboxylase Gene Coding for a Functional Enzyme Strongly Expressed in the Liver (*)

(Received for publication, October 26, 1994; and in revised form, January 4, 1995)

Kent Persson ,  Ingvar Holm ,  Olle Heby (§)

From the Department of Cellular and Developmental Biology, University of Umeå, S-901 87 Umeå, Sweden

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A genomic clone for a mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene was isolated from a cosmid library. Surprisingly, the gene proved to be intronless. With the exception of three base substitutions (changing 2 amino acids in the deduced protein), the 1002-nucleotide sequence of the open reading frame was identical to that of mouse AdoMetDC cDNA. Moreover, the gene contained a poly(dA) tract at the 3` end and was flanked by 13-base pair direct repeats. Our findings suggest that this gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the genome. By polymerase chain reaction, we positively identified the intronless gene in the mouse genome, and, by primer extension analysis, we proved the gene to be functional. Thus, its transcripts were found in many cell lines and tissues of the mouse and were particularly abundant in the liver. When the open reading frame of the intronless gene was expressed in Escherichia coli HT551, a strain with no AdoMetDC activity, it was found to encode a 38-kDa protein, corresponding to AdoMetDC proenzyme. Although the change of methionine 70 to isoleucine was close to the cleavage site at serine 68, this protein underwent proenzyme processing, generating a 31-kDa alpha subunit and an 8-kDa beta subunit. Importantly, the protein encoded by the intronless gene was functional, i.e. it catalyzed the decarboxylation of S-adenosylmethionine, and its specific activity was comparable with that of recombinant human AdoMetDC purified according to the same procedure.

INTRODUCTION

S-Adenosylmethionine decarboxylase (AdoMetDC^1; EC 4.1.1.50) is the rate-limiting enzyme in the biosynthesis of spermidine and spermine (1) . These polyamines and their diamine precursor putrescine play important roles in cell growth and differentiation(2, 3, 4, 5) . Therefore, the rate-limiting biosynthetic enzymes, ornithine decarboxylase and AdoMetDC, are useful targets for chemotherapeutic agents. Some inhibitors of these enzymes exert strong therapeutic effects in proliferative and parasitic diseases(1, 4, 5, 6) . A role of di- and polyamines in tumor cell growth is suggested by the finding that overproduction of ornithine decarboxylase is associated with neoplastic transformation(7, 8, 9) . Recently, ornithine decarboxylase has been shown to be a mediator of c-myc-induced apoptosis(10) .

The polyamines also protect cells and cell components from oxidative damage(11) . They form integral parts of many biologically important molecules such as bleomycin A(5), a cationic antibiotic from Streptomyces verticillus(12) , squalamine, an aminosterol antibiotic from the dogfish shark Squalus acanthias(13) , and the venom of the funnel-web spider Agelenopsis aperta(14) . Moreover, spermidine contributes a portion of its structure to form hypusine, an amino acid responsible for the post-translational modification of eukaryotic translation initiation factor 5A(15) .

AdoMetDC catalyzes the production of decarboxylated S-adenosylmethionine(1) . This is the aminopropyl group donor both in the conversion of putrescine to spermidine (catalyzed by spermidine synthase) and of spermidine to spermine (catalyzed by spermine synthase). Under physiological conditions, decarboxylated S-adenosylmethionine is a limiting factor in polyamine synthesis. Although ubiquitous in eukaryotic cells, AdoMetDC constitutes only a minor fraction of the intracellular proteins. This is partly due to its very short half-life and partly due to the fact that AdoMetDC expression is regulated at multiple levels, transcriptional, translational, as well as post-translational(1, 16) . Interestingly, there is evidence suggesting that the polyamines act as feedback regulators at all of these levels(1, 16) . AdoMetDC expression is induced by hormones, growth factors, tumor promoters, and other stimuli affecting growth(1, 3, 4, 5, 16) .

Cloning and sequencing of human(17) , bovine(18) , hamster(19) , rat (17) , and mouse (20, 21) AdoMetDC cDNAs have shown that the mammalian enzyme is synthesized as a 38-kDa proenzyme (333-334 amino acids) with no enzymatic activity. The proenzyme is autocatalytically cleaved into a 31-kDa alpha subunit (265-266 amino acids) and an 8-kDa beta subunit (67 amino acids), generating the pyruvate prosthetic group at the N terminus of the alpha subunit by serinolysis(22) . The mammalian enzyme contains two pairs of these nonidentical subunits (alpha(2)beta(2)) and probably two catalytic centers (1) . Both subunits seem to be necessary for catalytic activity. The amino acid sequence of the protein is highly conserved (about 90% identical) among mammalian species(17, 17, 18, 19, 20, 21) .

AdoMetDC genes have been cloned and sequenced from Escherichia coli(23) , Saccharomyces cerevisiae(24) , rat(25, 26) , and human (27) sources. In addition to these functional AdoMetDC genes, a processed pseudogene has been identified in the rat genome(28) . The objective of the present study was to isolate a mouse AdoMetDC genomic clone from a cosmid library and to determine the primary structure of this important gene, with the ultimate goal of analyzing its transcriptional regulation. The mouse AdoMetDC gene that was cloned and sequenced (cSAMm1; EMBL Z23077) proved to be completely devoid of introns over its entire length. The presence of a poly(dA) tract at the 3` end as well as flanking direct repeats suggests that this gene has arisen by retroposition(29) , in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the mouse genome. Of particular interest is our finding that this intronless gene has acquired a functional promoter, as is evident from our identification of the mRNA specifically encoded by the intronless gene (distinguished from the putative bona fide AdoMetDC gene by primer extension analysis). Expression analysis showed that this intronless AdoMetDC gene is strongly expressed in mouse liver. When the intronless AdoMetDC gene was expressed in AdoMetDC-deficient bacteria, it was found to encode a functional enzyme, despite the fact that the coding region contained three base substitutions (as compared with the cloned cDNA(20, 21) ), causing two amino acid substitutions in the enzyme.

EXPERIMENTAL PROCEDURES

Materials

A rat AdoMetDC cDNA (pSAMr1)(17) , cloned into the Okayama-Berg vector, was maintained in E. coli strain JM101. Restriction endonucleases, T4 DNA ligase, and Tth polymerase were from Boehringer Mannheim; T4 polynucleotide kinase and avian myeloblastosis virus reverse transcriptase were from Promega; and ultrapure deoxy- and dideoxynucleotides were from Pharmacia Biotech Inc. Random primer DNA labeling system (Megaprime), Hybond-C nitrocellulose membranes, radiolabeled nucleosides, nucleotides, and [S]methionine were from Amersham Corp. Oligonucleotide primers used for DNA sequencing, PCR, and primer extension experiments were synthesized on a model 394 DNA Synthesizer (Applied Biosystems) at the Department of Cellular and Molecular Biology, University of Umeå. All chemicals, electrophoresis reagents, and media were of the highest grade commercially available.

Screening of a Mouse Genomic DNA Library for an AdoMetDC Clone

A SuperCos 1 cosmid genomic library (Mus musculus (wild type), male, 4 weeks, spleen; Stratagene) was screened by colony hybridization using a [P]dCTP random primer-labeled 1035-bp PstI-fragment of pSAMr1 (17) as hybridization probe. This fragment contains the entire coding sequence of rat AdoMetDC cDNA. Plating, replica plating and lysis of bacteria were performed according to Stratagene's recommendations. Cosmid DNA was cross-linked to Hybond-C nitrocellulose membranes using a UV Stratalinker 1800 (Stratagene). The membranes were prehybridized in a hybridization solution containing 50% deionized formamide, 0.8 M NaCl, 0.5% SDS (w/v) and 200 µg/ml tRNA in 0.02 M Pipes buffer (pH 6.5) at 42 °C for 2 h, and then hybridized to the P-labeled probe at 42 °C for 16 h in new hybridization solution. The membranes were then washed once in 1 times SSC (standard saline citrate; 20 times SSC = 3 M NaCl, 0.3 M trisodium citrate (pH 7.0)), 0.1% SDS for 5 min at room temperature, and then 3 times 15 min in 0.1 times SSC, 0.1% SDS at 60 °C. All hybridization steps were performed in a Hybaid hybridization oven. Replating of the initial, positive colonies yielded 5 purified positive cosmid clones designated cSAMm1-5. One of these clones, cSAMm1, was further characterized. After overnight culture, cosmid DNA was purified using the QIAGEN Plasmid Maxi kit.

Linear Amplification Sequencing of the Mouse AdoMetDC Gene

DNA dideoxynucleotide chain-termination sequencing (30) was performed according to the cycle sequencing method described by Murray (31) and Craxton (32) using the fmol DNA sequencing system (Promega) and a DNA thermal cycler (Perkin Elmer). The method uses Taq DNA polymerase in cycled rounds of primer extension in the presence of a double-stranded DNA template (cosmid DNA), end-labeled oligonucleotide primers, deoxy-, and dideoxynucleotides. The primers were labeled at the 5` end, using T4 polynucleotide kinase and [-P]ATP (5,000 Ci/mmol).

DNA fragments were separated according to size by high resolution denaturing gel electrophoresis using a model S2 Sequencing gel electrophoresis apparatus (Life Technologies, Inc.), and the bands were visualized by direct autoradiography. GeneWorks 2.3 (IntelliGenetics) Macintosh software was used for the DNA sequence analysis.

Identification of the Intronless AdoMetDC Gene in the Mouse Genome by PCR Analysis

Genomic mouse DNA was isolated from the tails of two inbred mouse strains (129/SvJ and DBA/2J) and from 3 cell lines (Ehrlich ascites tumor cells, F9 teratocarcinoma stem cells, and L1210 lymphoid leukemia cells). The DNA was extracted using the Nucleon II DNA extraction kit (ScotLab). The oligonucleotides used for PCR were 5`-TCCAATAGTGCAAGTGGCACGTAT-3` (Primer 1; see Fig. 3) corresponding to a sequence (-49 to -26) in the putative promoter and 5`-CAAAATGAAACGTCTCTTGGAGAC-3` (Primer 2; see Fig. 3) complementary to a sequence (567 to 544) in the open reading frame (ORF) of the intronless AdoMetDC gene.

Figure 3: Positive identification of the intronless AdoMetDC gene in the genome of various mouse strains and cell lines by PCR. The experimental design is shown in A, and the result is shown in B. A, Primer 1 corresponds to a sequence in the putative promoter of the intronless gene, and Primer 2 is complementary to a sequence in the ORF. In the presence of the intronless gene, PCR results in amplification of a unique 615-bp product. B, genomic DNA, isolated from inbred mouse strains and from mouse cell lines, was subjected to PCR, and the amplified material was separated by electrophoresis in a 1.5% agarose gel and visualized by UV light. Lane1, 129/SvJ mouse; lane2, DBA/2J mouse; lane3, Ehrlich ascites tumor cells; lane4, F9 teratocarcinoma stem cells; lane5, L1210 lymphoid leukemia cells; lane6, control PCR with no DNA added; lane7, 100-bp DNA ladder (Pharmacia).


PCR reactions were carried out in a total volume of 100 µl with 0.5 µg of genomic mouse DNA, 0.25 µM final concentration of each of the two oligonucleotide primers, 2.0 units of Tth polymerase, 200 µM final concentration of each of dGTP, dATP, dCTP, and dTTP in the buffer (10 mM Tris-HCl (pH 8.9), 0.1 M KCl and 0.15 mM MgCl(2)) supplied with the Tth polymerase (Boehringer Mannheim). All PCRs were carried out in a DNA thermal cycler under the following conditions: 2 min of denaturation at 95 °C and 30 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 40 s, and extension at 72 °C for 1 min completed by a final extension at 72 °C for 7 min. An aliquot of the amplified material was loaded on a 1.5% agarose gel containing ethidium bromide. Amplified material was visualized by UV light.

Identification of Transcripts from the Intronless Mouse AdoMetDC Gene by Primer Extension Analysis

Total RNA from various tissues of a 5-week-old 129/SvJ male mouse was isolated using a guanidinium isothiocyanate extraction protocol(33) . The oligonucleotide used for primer extension was 5`-GAATATGCCCTAGAAAGTGGA-3` (Primer Tsn4; see Fig. 4) complementary to a sequence (nt +1811 to +1791) in the 3` untranslated region (UTR) of the intronless AdoMetDC gene.

Figure 4: Identification of the specific transcripts for the intronless and the bona fide mouse AdoMetDC genes by dideoxy-ATP-terminated primer extension analysis. The experimental design is shown in A, and the results are shown in B. The primer (Primer Tsn4) was designed such that the extension product would be 25 nt long in the presence of a transcript from the intronless gene and 31 nt long in the presence of a transcript from the bona fide gene (corresponding to the published mouse cDNAs(20, 21) ). Thus the primer was complementary to a sequence (1811-1791) in the 3`-UTR. Primer extension reactions were carried out in the presence of P-labeled primer and dideoxy-ATP using total RNA from the liver (lane1), lung (lane2), spleen (lane3), kidney (lane4), and testis (lane5) of 129/SvJ mice and from F9 teratocarcinoma stem cells (lane6), Ehrlich ascites tumor cells (lane7), and L1210 lymphoid leukemia cells (lane8). The extension products were fractionated by PAGE and detected on autoradiographic film using an amplifying screen.


For the analysis of cSAMm1 expression by primer extension, 50 µg of total RNA and 1.25 times 10^5 cpm of Primer Tsn4 (5` end-labeled with [-P]ATP; 3,000 Ci/mmol) were used per reaction(34) . For termination by dideoxynucleotides, dATP was replaced by dideoxy-ATP. In each reaction, 16 units of avian myeloblastosis virus reverse transcriptase were used. Extension products were treated with RNase A (20 mg/ml) for 30 min, extracted with phenol/chloroform, and finally ethanolprecipitated and fractionated by PAGE (15% gel containing 8 M urea). The gel was dried, and the radioactivity was detected on autoradiographic film using amplifying screens.

Production of Antiserum to Recombinant Human AdoMetDC

Rabbits were injected subcutaneously with 100 µg of recombinant human AdoMetDC (22) in complete Freund's adjuvant. Booster injections with 100 µg of AdoMetDC in incomplete Freund's adjuvant were given 1 and 2 months after the initial injection. Rabbits were bled 10 days after each injection to determine the titer of the antiserum. After the second booster injection, two rabbits produced antiserum of good quality, and these were bled regularly over a period of 6 weeks. When the titer started to decrease (4 months after the initial injection) a final booster injection was given as above, and 3 weeks later both animals were exsanguinated.

Expression and S Labeling of the Intronless Mouse AdoMetDC Gene in E. coli for Analysis of Proenzyme Processing

The coding sequence of cSAMm1 was inserted in place of the human AdoMetDC cDNA fragment in the pCQV2A construct (22) derived from the pCQV2 expression vector(35) . Thus, the pCQV2A construct was digested with Csp 45 I (Promega) and XbaI, and the larger fragment was purified on a 1% agarose gel. A 1048-bp fragment of cSAMm1 containing the coding sequence was similarly isolated by digestion with Csp 45 I and XbaI and purification on a 1% agarose gel. This fragment was ligated into the Csp 45 I-XbaI-digested fragment of the pCQV2A construct according to standard procedures(36) . The resulting construct was designated pCQcSAM. Cells of E. coli strain HT551, which have no AdoMetDC activity because of a deletion in the speED operon(37) , were transformed with pCQcSAM using the CaCl(2) procedure(36) . Plasmid DNA from transformed colonies was partially sequenced to ensure that it contained the 1048-bp cSAMm1 fragment.

E. coli strain HT551 carrying the appropriate expression vector construct (pCQV2A or pCQcSAM) were grown overnight at 32 °C in M9 minimal medium. They were then diluted 50-fold, and 4 h later the temperature was raised to 42 °C to induce expression. After 2 h, the cells were exposed to [S]methionine (50 µCi/ml) for 5 min. Then (time = 0) incorporation was stopped by adding cold methionine (final concentration, 100 µg/ml), and the cells were kept growing. Samples were taken at 0, 5, 10, 15, 20, 30, 45, and 60 min. Cells were sonicated in 10 mM Tris-HCl (pH 7.5) containing 0.1 mM EDTA, 0.5 mM dithiothreitol, 0.1% bovine serum albumin, 0.1% Triton X-100, 0.1% SDS, and 0.1% Tween 80. After centrifugation for 30 min at 30,000 times g, 4 °C, the supernatant was incubated for 30 min at room temperature with antiserum to recombinant human AdoMetDC (see above), whereupon protein A was added and the incubation continued for a further 30 min. After thorough washing with the above buffer, immunoprecipitated proteins were solubilized in SDS-PAGE loading buffer and subjected to SDS-PAGE (12.5% gel). Gels were incubated in Amplify (Amersham Corp.), and proteins were visualized by fluorography.

Expression of the Intronless Mouse AdoMetDC Gene in E. coli and Purification of the Resulting Protein

E. coli HT551 cells containing the pCQcSAM fragment were grown overnight at 32 °C. They were then diluted 100-fold and grown for 4 h at 32 °C and for 4 h at 42 °C in order to induce cSAMm1 protein expression. The processed recombinant protein was purified by (NH(4))(2)SO(4) precipitation and affinity chromatography on methylglyoxal-bis(guanylhydrazone)-Sepharose, essentially as described for human AdoMetDC (22) with the exception that the Mono Q column step was omitted. The migration of the purified protein was analyzed by SDS-PAGE and compared with molecular weight standards. Proteins were visualized by Coomassie Brilliant Blue G-250 staining.

Assay for AdoMetDC Activity of the Protein Encoded by the Intronless Mouse AdoMetDC Gene

AdoMetDC activity was assayed by measuring the release of ^14CO(2) from S-adenosyl-L-[carboxyl-^14C]methionine as described by Pegg and Williams-Ashman(38) .

RESULTS AND DISCUSSION

Isolation and Sequencing of a Mouse AdoMetDC Gene

With the ultimate goal to understand the control of mouse AdoMetDC gene expression and its involvement in cell growth and differentiation, we isolated a genomic clone from a M. musculus (wild type) genomic DNA library and determined its primary structure. Many surprising results came out of this study. The size of the gene was less than one-tenth of that expected, and its sequence was colinear with that of mouse AdoMetDC cDNA (20, 21) (Fig. 1). This implies that the cloned gene is completely devoid of introns over its entire length. With the exception of 3 point mutations of the base substitution type, which change 2 amino acids (Met-70 Ile and Ala-139 Val) in the deduced protein, the 1002-nt sequence of the ORF is identical to that of mouse AdoMetDC cDNA (Fig. 1). The base substitutions at positions 540 (GA) and 746 (CT) are transition mutations, whereas the substitution at position 858 (AC) is a transversion mutation. The protein-coding nucleotide sequence of the intronless AdoMetDC gene was consolidated by sequencing of the complementary strand.

Figure 1: Nucleotide sequence of the ORF of the intronless mouse AdoMetDC gene and the deduced amino acid sequence of the corresponding protein as compared with the mouse AdoMetDC cDNA (dash, same nucleotide) and amino acid sequences (no mark, same amino acid)(20, 21) . Numbering of the nucleotides begins with the putative transcription start site (+1), and numbering of the amino acids begins with the initiating amino acid (Met-1). Out of the 1002 nucleotides specifying amino acids, there are three substitutions (boxes) resulting in two amino acid replacements. The point mutations affecting nucleotide 540 (GA) and nucleotide 746 (CT) result in Met-70 Ile and Ala-139 Val substitutions, respectively. The point mutation at nucleotide 858 (AC), however, is in the third position of a codon and does not specify a different amino acid. The 334-amino acid sequence corresponds to the 38-kDa AdoMetDC proenzyme, which is processed to a 31-kDa alpha subunit and an 8-kDa beta subunit(1) . The bond split is likely to occur between glutamic acid 67 and serine 68 (*). In this process, the serine is converted to pyruvate, which becomes the N terminus of the large alpha subunit and acts as the prosthetic group of the enzyme. The glutamic acid becomes the C terminus of the small beta subunit. cDNA*, sequence from(20) .


The start site for AdoMetDC mRNA transcription has been identified in the human (27) and rat (26) genomes. It is localized to the same G residue (within the 5`-CTCGCTT-3` context) in both species, although the 5`-UTR of the rat mRNA is 5 nt longer (325 nt as compared with 320). When comparing the intronless mouse AdoMetDC gene sequence with that of the human and rat AdoMetDC genes, the nucleotide sequence from the putative transcription start site and downstream is identical for at least 40 nt. Therefore, it is very likely that the transcription of the intronless gene starts at the same G residue, which is consequently numbered +1. On this assumption, the 5`-UTR of the mRNA encoded by the intronless AdoMetDC gene is 330 nt long (Fig. 1).

In addition to the ORF coding for AdoMetDC, there is a small ORF in the 5`-UTR of the intronless gene. This ORF is also present in other mammalian AdoMetDC genes(39) . It codes for a hexapeptide (MAGDIS or Met-Ala-Gly-Asp-Ile-Ser), which appears to suppress translation of AdoMetDC mRNA in a cell-specific manner(39, 40, 41) . The 5`-UTR of the intronless AdoMetDC gene also has a high G+C content, implying that it may have stable secondary structures affecting its translation(42, 43) .

Downstream of the termination codon for the intronless AdoMetDC gene, there are at least two sets of potential polyadenylation signals (Fig. 2). Their positions correspond to those found in human (27) and rat (26) AdoMetDC genes and in mouse AdoMetDC cDNA(20, 21) . The most upstream signal (AATTAAA) at position 1869, yields a 3`-UTR of >540 nt; the actual length depending on the site and extent of polyadenylation. The second signal, at position 3173, is identical to the typical polyadenylation signal AATAAA(44) , yielding a 3`-UTR of >1843 nt; the actual length again depending on the site and extent of polyadenylation. The numbers of nucleotides between the putative transcription start site and the polyadenylation sites are 1872 and 3175, respectively. When taking into account that a poly(A) tail is added, these figures are in agreement with the 2.1 and 3.4-kilobase AdoMetDC mRNA species found in mouse tissues and cell lines(17) . Therefore, utilization of both polyadenylation signals in the intronless gene will contribute to the formation of transcripts, which are likely to be indistinguishable from the transcripts derived from the bona fide AdoMetDC gene.

Figure 2: Comparison of the human (A) (27) and rat (B) (25, 26) AdoMetDC genes with the intronless AdoMetDC gene of the mouse (C). The exons (E1-E9) and the corresponding regions of the intronless gene are depicted by boxes, with the openboxes referring to the protein-coding region, and the closedboxes referring to the 5`- and 3`-UTRs. The boundaries of the intronless gene are defined by a 13-bp direct repeat (AAGAAACATTCTA). All three genes contain two major polyadenylation/termination sequences in their 3`-UTR, the one most upstream being AATTAAA and the other AATAAA(25, 26, 27) . The 5`-flanking region of the human and rat genes possess TATA boxes, and in the corresponding region of the intronless mouse gene there is a TATA-like box (TATTAAT) at -28 (the number refers to the first nucleotide in the sequence). The mouse AdoMetDC gene shares with long interspersed elements (46) their four canonical structural features: (a) it does not contain introns, (b) it represents a full-length copy of the processed transcript from the functional gene, (c) it contains a poly(dA) tract (dA) at the 3` end, and (d) it is flanked by target site duplications, i.e. the cellular DNAs adjacent to the retrotransposed structure display direct repeats.


The bona fide mouse AdoMetDC gene(s) has not yet been isolated and sequenced, but the corresponding rat genes are known to be interrupted by seven introns(26) , and the human AdoMetDC gene has one additional intron (27) (Fig. 2). Exon-intron junctions in these genes are in identical positions except that intron 6 of the human gene is missing in the rat gene (Fig. 2, A and B)(27) .

The Intronless Mouse AdoMetDC Gene Is a Retroposon

The lack of introns and the presence of a poly(dA) tract downstream of the second polyadenylation signal and flanking 13-bp direct repeats (AAGAAACATTCTA) (Fig. 2) suggest that the cloned gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a cDNA copy and inserted into the genome(29, 45, 46) . There was a 26-nt span between the end of the second polyadenylation signal (at position 3178) and the beginning of the poly(dA) tract (at position 3205). Direct repeats are frequently found bordering processed genes and are thought to result from the repair of a staggered break at an A-rich sequence where the cDNA (or DNA-RNA hybrid) has been inserted into the genome(45) . Processed genes are believed to be vertically transmitted(47) . Consequently, their formation should take place in germ cells. Since little or no cellular differentiation occurs at this stage of development, germ cells contain relatively few transcripts encoding tissue-specific proteins (only those transcripts that are specific for oocytes and early development). Therefore, the majority of the processed genes will be of the housekeeping type. Indeed, the AdoMetDC genes are considered to belong to this category, although tissue-specific expression of the protein may be achieved by translational regulation(39, 40, 41) .

The Intronless AdoMetDC Gene Acquired a Functional Promoter

The nucleotide sequence of the 5`-flanking region of the intronless mouse AdoMetDC gene has been only partially sequenced. It does not contain a conventional TATA box but a sequence (TATTAAT), which bears close resemblance to the consensus TATA(A/T)A sequence (48) found in eukaryotic promoters. The distance between this TATA-like sequence (at position -28) and the putative transcription start site (+1) is within the range (-34 to -26) observed for other eukaryotic genes(48) . Further upstream of the intronless AdoMetDC gene, there are several potential promoter and enhancer elements, which could serve as recognition sites for a variety of transcription factors. It remains to be determined, by direct footprinting and transcriptional analysis, whether these sites are indeed recognized by transcription factors and whether they function in vivo as cis-regulatory elements.

In the rat genome, two distinct but closely related AdoMetDC loci have been found, both located on chromosome 20(26) . Despite some differences between their exon sequences, the genes code for identical proteins. The 5`-flanking regions upstream of nt -63 are totally different. Both promoters appear to be efficient but controlled by different sets of transcription factors. In view of their structures and chromosomal location, it has be suggested that these AdoMetDC genes have arisen from a recent duplication event in the rat genome(26) . In the mouse genome, a functional duplication of an AdoMetDC gene instead seems to have arisen through retroposon recruitment. The fact that the intronless gene has acquired a unique 5`-flanking region and has lost introns that, in the corresponding rat gene, contain potential promoter and enhancer elements(26) , suggests that the transcriptional regulation of the intronless AdoMetDC gene is completely different from that of the bona fide gene. These changes may also imply that the transcription of the intronless AdoMetDC gene is not subject to the same feedback regulation by polyamines (1, 16) as the bona fide gene.

Identification of the Intronless AdoMetDC Gene in the Mouse Genome

A PCR experiment was designed as outlined in Fig. 3A, with primers specifying a unique 615-bp gene fragment from the intronless gene. Only the intronless gene could result in this fragment because Primer 1 is located upstream of the direct repeat in the putative promoter, which together with the intronless AdoMetDC gene must be considered a unique sequence. Assuming that the intron structure of the bona fide mouse AdoMetDC gene is similar to those of the rat and human AdoMetDC genes(25, 26, 27) , the amplified fragment from the bona fide gene would include two introns and thus would be very much larger than the 615-bp fragment. When using genomic mouse DNA as the template, the predicted 615-bp fragment was found to be amplified (Fig. 3B), thus demonstrating that the intronless AdoMetDC gene is indeed present in the genome of the mouse. This is not the only AdoMetDC gene in the mouse genome, however, because Southern blot analysis clearly shows that this intronless gene is part of a gene family. Evidence for an AdoMetDC gene family in the mouse genome, as well as in the rat genome, also comes from other studies(26) .

AdoMetDC gene sequences have been mapped to human chromosomes 6 and X with the use of a panel of human-mouse somatic cell hybrids(49) . In agreement, Maricet al.(27) found AdoMetDC gene-related sequences in DNA libraries specific for human chromosomes 6 and X. They found the gene on chromosome 6 to be active. Since partial nucleotide sequencing revealed that the gene on the X chromosome lacked introns, it was suggested that this locus represents a processed AdoMetDC pseudogene(27) . Whether this human AdoMetDC-related gene sequence also represents a functional intronless gene or is merely a nonfunctional pseudogene remains to be determined.

The Intronless AdoMetDC Gene Is Functional

Very importantly, primer extension experiments show that the intronless AdoMetDC gene is functional and not merely a processed pseudogene from which no transcripts are generated. Thus, the intronless gene encodes a transcript that can be specifically identified by a primer extension reaction designed as shown in Fig. 4. This transcript is found in many cell lines and tissues of the mouse, and is particularly abundant in the liver (Fig. 4). It also appears as if the transcript from the intronless AdoMetDC gene is more abundant than that from the bona fide gene, at least in those tissues and cell lines studied.

The Protein Encoded by the Intronless AdoMetDC Gene Is Functional

To determine whether the intronless mouse AdoMetDC gene encoded a functional protein, we expressed the protein in E. coli HT551, a strain with no AdoMetDC activity(37) . The ORF was found to encode a 38-kDa protein corresponding to AdoMetDC proenzyme, which underwent proenzyme processing generating a 31-kDa alpha subunit and an 8-kDa beta subunit (Fig. 5), even though the change of methionine 70 to isoleucine was close to the cleavage site at serine 68. Processing of the proenzyme is essential for catalytic activity, because changing serine 68 to alanine by site-directed mutagenesis completely prevents the processing and abolishes AdoMetDC activity(22) . The fact that the processing of the proenzyme encoded by the intronless AdoMetDC gene occurred readily in the bacterial cell suggests that the cleavage is autocatalytic, as has previously been concluded for the bona fide AdoMetDC gene product(22) .

Figure 5: Analysis of the autocatalytic processing of the protein encoded by the intronless mouse AdoMetDC gene. Human and mouse AdoMetDC proteins were pulse-labeled with [S]methionine for 5 min while overexpressed in E. coli strain HT551(37). At 0, 5, 10, 15, 20, 30, 45, and 60 min after radiolabeling, extracts were immunoprecipitated with a recombinant human AdoMetDC antibody, and the precipitates were analyzed by SDS-PAGE. Radiolabeled proteins were visualized by fluorography. Migration of [^14C]methylated protein standards of the indicated molecular masses (kDa) is shown on the left. The protein encoded by the intronless mouse AdoMetDC gene is equivalent to the 38-kDa proenzyme of the human AdoMetDC and is shown to be processed to a 31-kDa alpha subunit (and an 8-kDa beta subunit).


The protein purified and migrated in accordance with the 31-kDa alpha subunit of human AdoMetDC (Fig. 6) and exhibited catalytic activity characteristic of AdoMetDC (see the legend to Fig. 6) despite the two amino acid substitutions. The specific activity of the purified mouse enzyme was 236 units/mg of protein, which is comparable with that of the human enzyme (530 units/mg of protein) purified according to the same procedure. One unit of enzyme activity is defined as releasing 1 nmol of CO(2)/min. The essentially normal behavior of the protein encoded by the intronless AdoMetDC gene is consistent with the fact that the two amino acid substitutions (methionine 70 to isoleucine and alanine 139 to valine) do not involve amino acids known to be essential for AdoMetDC activity or proenzyme processing, as determined by site-directed mutagenesis(1) , nor do they change the net charge of the protein.

Figure 6: Electrophoretic mobility of the protein encoded by the intronless mouse AdoMetDC gene. Human (B) and mouse (C) AdoMetDC proteins, expressed in E. coli strain HT551(37), were purified on a methylglyoxal-bis(guanylhydrazone)-Sepharose affinity column, separated by SDS-PAGE (12.5% gel) and stained with Coomassie Brilliant Blue. Migration of protein standards of the indicated molecular masses (kDa) is shown in A. The protein encoded by the intronless mouse AdoMetDC gene purified and migrated in accordance with the 31-kDa alpha subunit of the human AdoMetDC and its specific activity (236 units/mg of protein) was comparable with that of the human enzyme (530 units/mg of protein) purified according to the same procedure. One unit of enzyme activity is defined as releasing 1 nmol of CO(2)/min.


Except for the two amino acid substitutions mentioned above, the primary structure of the protein encoded by the intronless AdoMetDC gene is identical to the human and rat AdoMetDC proteins ( Fig. 1and 2). Thus, structurally important domains of the AdoMetDC protein are unaffected by the mutations, including the only conserved region between eukaryotic and prokaryotic AdoMetDCs (1, 23) (amino acids 81-91 of the intronless gene), and the PEST region (50) (amino acids 243-269 of the intronless gene), which may be important for the rapid turnover of the enzyme.

It is interesting to note that multiple forms of AdoMetDC have been observed in the rat(51) . Whether a gene corresponding to the intronless AdoMetDC gene of the mouse, which is strongly expressed in the liver, can account for the alternate form observed in the rat liver remains to be determined. Irrespectively, mouse tissues may have two types of AdoMetDC homodimers, those containing two alpha subunits encoded by the intronless gene and those containing two alpha subunits encoded by the bona fide gene as well as a heterodimer containing one alpha subunit of each.

On the Occurrence of Intronless Genes in Higher Eukaryotes

Our finding that a gene for AdoMetDC is not only intronless and functional but produces a functional protein at that is intriguing. It seems to be a rather uncommon phenomenon, whereas inactive retropseudogenes are quite abundant. Thus, only a small number of functional intronless genes have been discovered, notably the gene families coding for histones(52) , protamines(53) , and interferons (54, 55) . Also, individual genes such as those encoding a beta(2)-adrenergic receptor(56) , an atrial M(2) muscarinic acetylcholine receptor(57) , a serotonin 1D receptor variant (58) , a D(1) dopamine receptor subtype(59) , a thrombomodulin (60) , a JUN protooncogene(61) , an N-myc-2 protooncogene(62) , a leukosialin(63) , a SCIP transcription factor (64) , a nuclear pore glycoprotein p62(65) , a testisspecific isoform of phosphoglycerate kinase 2(66) , the major gastrointestinal tumor-associated antigen, GA733-1(67) , an insulinoma-associated protein(68) , a calmodulin-like protein(69, 70, 71) , a glutamine synthetase(72) , a protein kinase A anchor protein(73) , and a casein kinase II-alpha subunit (74) have been discovered. Even though many of these genes are functional in the sense that they are transcribed, i.e. have a functional promoter, it has not been unequivocally demonstrated that they all encode a functional protein. Some of the genes above have introns in their 5`- and 3`-flanking regions (57, 63) or have truncated UTRs(61, 67) , and some, e.g. the histones, do not exhibit the hallmarks of retroposons. Although it has not been established for all intronless genes, it appears that at least some of them have intron-containing counterparts in the genome where they are found(53, 62, 66, 69, 70, 71, 72, 74) .

Assuming that primordial genes evolved with introns(75) , the lack of introns in genes of higher eukaryotes can be due to (a) intron loss or (b) reinsertion into the genome of genetic material copied from mRNA by reverse transcription. The existence of an intron in the 5`-UTR of the human leukosialin gene, a gene that is devoid of introns in its coding region(63) , argues for intron elimination. On the other hand, our finding that the cloned mouse AdoMetDC gene is intronless over its entire length argues for a mechanism involving reverse transcription.

What is the biological significance of the multiplicity of AdoMetDC genes? Is it simply an evolutionary accident that brings no selective advantage to the organism, or have the distinct genes evolved to exercise specific functions? Probably, accidental gene amplification and fixation of gene families can occur as an essentially neutral event. Our finding, that the intronless AdoMetDC gene in mice exhibits a quantitatively distinct pattern of expression, suggests that it may have acquired a novel role in cell stimulatory activities. The fact that the AdoMetDC genes are not co-ordinately turned on in response to induction but that different genes are turned on to varying extents in different tissues and cells suggests that the individual AdoMetDC species may have further, as yet unrecognized, activities of importance in physiological growth control and differentiation.

Although the protein encoded by the intronless AdoMetDC gene is functional, we cannot exclude the possibility that mutations at the sites observed could lead to inadequate expression of the gene by impairing transcription, translation, and/or post-translational events. Because the formation of mRNA from the intronless AdoMetDC gene does not require splicing, less time may elapse between transcription of the gene and appearance of the mature message in the cytoplasm and its translation into functional AdoMetDC protein. Thus, a signal that leads to increased transcription of this gene may be more rapidly translated into increased levels of AdoMetDC protein than a signal stimulating the bona fide gene, which would produce a nonprocessed message that would have to be spliced.

Conclusions

A surprising and remarkable sequence of evolutionary events has been revealed in the present study. The mouse genome is shown to contain an intronless AdoMetDC gene, which displays the hallmarks of a retroposon generated from a fully processed RNA message, i.e. lack of introns, presence of a poly(dA) tract at the 3` end, and a short directly repeated sequence (AAGAAACATTCTA) at both ends. The most surprising and improbable event is that by insertion, the gene has acquired a novel and functional promoter. This is evident from primer extension analyses, which permitted unequivocal identification of specific transcripts from the intronless gene in mouse tissues and cell lines.

FOOTNOTES

*
This work was supported by the Swedish Natural Science Research Council (B-BU-4086), the Swedish Cancer Society, the Royal Swedish Academy of Sciences (the Hierta-Retzius Foundation), and the J. C. Kempe Foundation. A preliminary report on these findings was presented at a conference on TheOrganizationandExpressionoftheGenome, Lorne, Victoria, Australia, February 14-18, 1994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z23077[GenBank].

§
To whom correspondence and reprint requests should be addressed.

(^1)
The abbreviations used are: AdoMetDC, S-adenosylmethionine decarboxylase; PCR, polymerase chain reaction; bp, base pair; Pipes, 1,4-piperazinediethanesulfonic acid; UTR, untranslated region; ORF, open reading frame; nt, nucleotide(s).

ACKNOWLEDGEMENTS

We thank Dr. Antti Pajunen, Department of Biochemistry, University of Oulu, Finland, for providing a rat AdoMetDC cDNA clone (pSAMr1) and for sharing unpublished data regarding rat AdoMetDC genes; Drs. Celia and Herbert Tabor, NIDDK, National Institutes of Health, Bethesda, Maryland, for kindly providing the E. coli HT551 strain; and Dr. Franklin G. Berger and Karen W. Barbour, Department of Biological Sciences, University of South Carolina, Columbia for generously sharing experience in primer extension analysis. We also thank Astrid Brorsson and Birgitta Grahn for technical assistance.

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