Serine hydroxymethyltransferase (EC 2.1.2.1, SHMT) is a pyridoxal-phosphate-binding enzyme that catalyzes the reversible reaction of serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate [1]. SHMT-catalyzed reactions are important as a primary source of one-carbon units used in folate-dependent metabolism, including purine synthesis [2]. Two isoforms of SHMT are found in eukaryotic cells: one expressed in the cytoplasm (SHMT-cyt) and one in mitochondria (SHMT-mit). The gene encoding the former is located on human chromosome 17p11.2 and the gene encoding the latter is on human chromosome 12q13.2 [3,4]. A third member of this small gene family is a processed pseudogene, SHMT-ps1, located on human chromosome 1p32.3-p33 [5,6]. Analyses of known SHMT amino acid and nucleotide sequences indicate that eukaryotic SHMT-cyt is descended from a prokaryotic glyA-like ancestor and that both SHMT-mit and SHMT-ps1 have been derived from SHMT-cyt [3,4,5,7].

PCR-based analyses of evolutionary conservation of the SHMT-ps1 nucleotide sequence suggest that the reverse transcription event giving rise to SHMT-ps1 occurred after the divergence of the order Primates from the rest of the Mammalia, thus making this locus a genetic marker unique to the primates [7]. Here, this suggestion is verified using two end-point molecular beacon assays. In addition, results presented here further refine the time frame in which the reverse transcription and retrotransposition events occurred.

End-point molecular beacon assays (see Materials and methods) were carried out on 19 primate and 12 non-primate mammalian genomic DNAs. The results of those assays confirmed the previous conclusion that SHMT-ps1 is not found outside members of the order Primates. There was, however, one notable departure from expectation among the primate DNA samples. In previous studies, genomic DNA from Lemur catta had given a 292 base pair (bp) SHMT-ps1 amplicon from which sequence had been obtained [7]. In the present study, the genomic DNA sample from the ruffed lemur, Varecia variegata, gave no evidence of the pseudogene with either molecular beacon probe, E8/E9 or E9/E10 (Figure 1). To establish which of these results was reliable, genomic DNAs from an additional 17 lemurs covering nine living species were examined by PCR using the C1/B6 primer pair under the amplification conditions given in the Materials and methods. In no case did the lemur samples yield the expected SHMT-ps1 amplicon, whereas the other primate controls succeeded each time they were used. On the basis of these new data, it now appears that the original L. catta sample did not contain pure lemur genomic DNA, and that the SHMT-ps1 locus is not present in the prosimians as previously thought.

Thermodynamic studies of molecular beacons suggest that conformationally constrained probes display enhanced hybridization specificity as a general feature [8]. The states in which a molecular beacon can exist are a random coil, the closed hairpin/loop structure, and a linearized structure bound to the target PCR product. When two or more competing molecular beacons are present, the enhanced hybridization specificity results in the ability to discriminate between target sequences that differ by as little as a single nucleotide [8,9,10,11]. In the present study, in which molecular beacon probes were used singly as end-point reporters, they were able to tolerate multiple mismatches in the target sequences. Comparative sequences homologous to the E8/E9 and E9/E10 probes (Figure 2) from several species are shown in Table 1. As can be seen, the species-specific target sequences differed from the probe sequences by as many as nine bases, with the average difference being 2.5 bases. Confidence in the ability of these highly sensitive probes to reliably detect the presence of target sequences that differ from the probe sequence by numerous bases is supported by the observation that the most divergent targets, those belonging to the Old World monkey species for the E8/E9 probe, still gave positive, albeit weakened, results. On the other hand, potential targets that were perfect matches or that differed by only one or two bases did not give any positive indications in the lemur and non-primate mammal samples (Figure 1).

The appearance of a processed pseudogene such as SHMT-ps1 is due to the reverse transcription of a processed mRNA followed by the random insertion of the derived cDNA into the genome via retrotransposition [12,13]. On the basis of the results presented here, refinement of the point at which those events giving rise to SHMT-ps1 occurred must now exclude the prosimians. Kay et al. [14] suggest that the divergence of the line leading to the monkeys and apes (Haplorrhini) from that leading to the prosimians (Strepsirrhini) occurred around the beginning of the Eocene some 55 million years ago. They also note that the divergence of the Old World monkeys and apes (Catarrhini) and the New World monkeys (Platyrrhini) probably took place before the end of the Eocene, or before 34 million years ago [14]. Given that SHMT-ps1 is found in both New World and Old World monkeys, the window in which the reverse transcription event could have taken place is further bracketed within a 12 to 15 million year period between the lower and middle Eocene. One species that has not so far been examined that would shed light on this question is, of course, the tarsier. Should the tarsier not have SHMT-ps1 in its genome, the window would be further constrained to a lower bound corresponding to the origin of the Anthropoidea, which is placed somewhat nearer the middle Eocene [14].

Finally, of interest here is the report of another primate pseudogene, a processed immunoglobulin J chain pseudogene, JΨ, whose origin is via the same process at about the same time. Max et al. [15] examined several primate species, including Old World and New World monkey species, and concluded that this locus, too, arose in the Primates some 40 to 50 million years ago. We have recently been able to confirm this conclusion by a PCR study of the same genomic DNA panel used in this paper (E.J.D. and H. Trill, unpublished observations).

Is the fact that two retroprocessed pseudogenes were produced in the order Primates at nearly the same time unusual? The answer to this is probably no. Goncalves et al. [16] suggest that retropseudogenes are a common phenomenon in mammalian genomes and estimate that the human genome contains some 23,000 to 33,000 such loci. They also note several common features among the genes giving rise to retropseudogenes: they are widely expressed; they are highly conserved; they are usually short; and they are relatively GC poor [16]. SHMT-cyt, the antecedent locus of SHMT-ps1, does, in fact, meet most of these criteria, as does the antecedent locus of JΨ. Thus, although SHMT-cyt is quite large [3,4] and neither gene is particularly GC poor [3,4,15], the appearance of these two retroprocessed pseudogenes at about the same time in the evolutionary history of the primates is probably not unusual. Both may, however, prove to be valuable molecular tools in primate phylogenetic research.