Molecular evolution of two lineages of L1 (LINE-1) retrotransposons in the california mouse, Peromyscus californicus.

The large number of L1 [long interspersed elements (LINE)-1] sequences found in the genome is due to the insertion of copies of the retrotransposon over evolutionary time. The majority of copies appear to be replicates of a few active, or "master" templates. A continual replacement of master templates over time gives rise to lineages distinguishable by their own unique set of shared-sequence variants. A previous analysis of L1 sequences in deer mice, Peromyscus maniculatus and P. leucopus, revealed two active L1 lineages, marked by different rates of evolution, whose most recent common ancestor predates the expansion of the Peromyscus species. Here we exploit lineage-specific, shared-sequence variants to reveal a paucity of Lineage 2 sequences in at least one species, P. californicus. The dearth of Lineage 2 copies in P. californicus suggests that Lineage 2 may have been unproductive until after the most recent common ancestor of P. californicus and P. maniculatus. We also show that Lineage 1 appears to have a higher rate of evolution in P. maniculatus relative to either P. californicus or P. leucopus. As a phylogenetic tool, L1 lineage-specific variants support a close affinity between P. californicus and P. eremicus relative to the other species examined.

Total protein content and protein synthesis within pre-elongation stage bovine embryos.

Protein content was measured in zona-free bovine oocytes and pre-elongation stage embryos, following in vitro maturation, fertilisation, and then culture in Synthetic Oviduct Fluid medium supplemented with amino acids and 8 mg ml(-1) bovine serum albumin (BSA). Values (ng embryo(-1)) of 122 +/- 7.8, 137 +/- 8.6, 111 +/- 8.8, 115 +/- 10.4, 139 +/- 9.0 and 152 +/- 10.1 were obtained for zona-free mature oocytes, 2-cell (day 2), 8-cell (day 3), compact morula (day 6), blastocyst (day 7), and expanded blastocyst (day 8) stage embryos, respectively. The protein content of day 7 zona-enclosed blastocysts was 337 +/- 58.0 ng embryo(-1). These values suggest that prior to compaction and blastulation, the early cleavage stage bovine embryo has a higher rate of protein degradation than that of synthesis. Net growth is observed only after initiation of compaction. The protein content of day 7 blastocysts was measured in embryos following in vitro production and culture in the same media supplemented with either 0.5% w/v polyvinyl alcohol (PVA), 8 mg ml(-1) BSA, 8 mg ml(-1) BSA and further supplemented with 10% fetal calf serum (FCS) from the beginning of culture (FCS-D1), 8 mg ml(-1) BSA and 10% FCS from the fourth day of culture (day 5 of development) or from in vivo-derived day 7 blastocysts. Protein content was significantly (P < 0.05) lower in PVA-cultured embryos than other treatments. To determine if this difference in PVA-cultured embryos was due to a difference in the rate of protein synthesis, comparisons were made between day 7 embryos derived from BSA-culture and either PVA-culture, FCS-D1 culture or in vivo-derived embryos. Despite differences in diameter, no significant difference was observed in the incorporation of L-[2,3,4,5,6-3H]-phenylalanine into the TCA-precipitable fraction in any of the three comparisons made. However, incubation in the presence of FITC-abelled BSA or beta-casein and examination under either fluorescence or confocal microscopy revealed that protein in the extra-embryonic environment was actively taken up by the trophectoderm of day 7 blastocysts, most likely by endocytosis. These results suggest that exogenous protein is an important nutritive source, probably maintaining intracellular amino acid pools. Results obtained from the production of em bryos in protein-free medium should be viewed with the knowledge that such embryos differ metabolically from those embryos grown in the presence of protein, including in vivo-derived embryos.

Two persistent LINE-1 lineages in Peromyscus have unequal rates of evolution.

LINE-1, the major family of long, interspersed repeats in the mammalian genome, moves via an RNA intermediate and encodes its own reverse transcriptase. Comparative sequence analysis was used to reconstruct the phylogenetic history of LINE-1 dynamics in the deer mouse, Peromyscus. As is the case in Mus and Rattus, a very small number of active templates produce the majority of LINE-1 copies in Peromyscus. However, in contrast to the single LINE-1 lineage seen in the muroid rodents, Peromyscus has at least two LINE-1 lineages whose most recent common ancestor probably existed before the peromyscine radiation. Species-specific variants of Lineage 1, and intact open reading frames in the youngest elements of both Lineages 1 and 2, suggest that both lineages have remained active within the same genome. The higher number of shared-sequence variants in Lineage 1 relative to Lineage 2 suggests that Lineage 1 has replaced its master template much more frequently than Lineage 2 or that the reverse transcriptase Lineage 1 is more error prone. The implications of the method used to acquire LINE-1 sequences for analysis are discussed.