Anthony V. Furano

NGF stimulates incorporation of fucose or glucosamine into an external glycoprotein in cultured rat PC12 pheochromocytoma cells

Abstract Rat PC12 pheochromocytoma cells respond to Nerve Growth Factor (NGF) by ceasing to undergo mitosis and acquiring neuronal characteristics, including outgrowth of neurites and electrical excitability. We have found that NGF treatment results in no consistent qualitative and only a few minor quantitative changes in the two-dimensional electrophoresis pattern of 14 C-amino acid-labeled proteins synthesized by the cells. On the other hand, NGF stimulates the incorporation of radiolabeled fucose or glucosamine into several components, including one of apparent molecular weight 230,000 daltons on one- and two-dimensional SDS-polyacrylamide gels. This component is removed by mild trypsinization of intact cells and therefore appears to be a glycoprotein (named here NILE) at least partially exposed on the cell surface. Stimulation of NILE glycoprotein labeling can first be detected after 2 days of exposure to NGF and increases progressively with time of treatment. This change is not solely a consequence of the cessation of cell division caused by NGF, since it does not occur in nondividing PC12 populations prepared by cytosine arabinoside treatment. NGF stimulates labeling of NILE glycoprotein even when attachment and process outgrowth are prevented by growing the cells in spinner suspension cultures. The relative rate of labeling attained under these conditions is less, however, than in NGF-treated, substrate-attached cells. Stimulation of NILE glycoprotein labeling by NGF is selectively blocked (as is neurite outgrowth) by camptothecin, an RNA synthesis inhibitor, and thus may require transcription. Despite their similarities in apparent size and exposure on cell surfaces, the NILE and LETS glycoproteins are shown to be immunologically distinct.

Fruit flies and humans respond differently to retrotransposons.

Retrotransposable element insertions are 20 times more numerous per unit length of DNA in the large human genome compared to the small Drosophila genome. Whereas all Drosophila elements are subject to constant turnover (recent insertion and elimination by selection), this has not generally been the case for human retrotransposons. We suggest that a difference in recombination adopted by these organisms in response to the deleterious effects of interspersed repeated DNA can explain in part this fundamental difference between the evolutionary dynamics of fruit fly and human retrotransposons.

The evolution of long interspersed repeated DNA (L1, LINE 1) as revealed by the analysis of an ancient rodent L1 DNA family

SummaryAll modern mammals contain a distinctive, highly repeated (⩾50,000 members) family of long interspersed repeated DNA called the L1 (LINE 1) family. While the modern L1 families were derived from a common ancestor that predated the mammalian radiation ∼80 million years ago, most of the members of these families were generated within the last 5 million years. However, recently we demonstrated that modern murine (Old World rats and mice) genomes share an older long interspersed repeated DNA family that we called Lx. Here we report our analysis of the DNA sequence of Lx family members and the relationship of this family to the modern L1 families in mouse and rat. The extent of DNA sequence divergence between Lx members indicates that the Lx amplification occurred about 12 million years ago, around the time of the murine radiation. Parsimony analysis revealed that Lx elements were ancestral to both the modern rat and mouse L1 families. However, we found that few if any of the evolutionary intermediates between the Lx and the modern L1 families were extensively amplified. Because the modern L1 families have evolved under selective pressure, the evolutionary intermediates must have been capable of replication. Therefore, replicationcompetent L1 elements can reside in genomes without undergoing extensive amplification. We discuss the bearing of our findings on the evolution of L1 DNA elements and the mammalian genome.

CpG dinucleotides and the mutation rate of non-CpG DNA.

The neutral mutation rate is equal to the base substitution rate when the latter is not affected by natural selection. Differences between these rates may reveal that factors such as natural selection, linkage, or a mutator locus are affecting a given sequence. We examined the neutral base substitution rate by measuring the sequence divergence of approximately 30,000 pairs of inactive orthologous L1 retrotransposon sequences interspersed throughout the human and chimpanzee genomes. In contrast to other studies, we related ortholog divergence to the time (age) that the L1 sequences resided in the genome prior to the chimpanzee and human speciation. As expected, the younger orthologs contained more hypermutable CpGs than the older ones because of their conversion to TpGs (and CpAs). Consequently, the younger orthologs accumulated more CpG mutations than the older ones during the approximately 5 million years since the human and chimpanzee lineages separated. But during this same time, the younger orthologs also accumulated more non-CpG mutations than the older ones. In fact, non-CpG and CpG mutations showed an almost perfect (R2 = 0.98) correlation for approximately 97% of the ortholog pairs. The correlation is independent of G + C content, recombination rate, and chromosomal location. Therefore, it likely reflects an intrinsic effect of CpGs, or mutations thereof, on non-CpG DNA rather than the joint manifestation of the chromosomal environment. The CpG effect is not uniform for all regions of non-CpG DNA. Therefore, the mutation rate of non-CpG DNA is contingent to varying extents on local CpG content. Aside from their implications for mutational mechanisms, these results indicate that a precise determination of a uniform genome-wide neutral mutation rate may not be attainable.

The mutational spectrum of non-CpG DNA varies with CpG content

The accumulation of base substitutions (mutations) not subject to natural selection is the neutral mutation rate. Because this rate reflects the in vivo processes involved in maintaining the integrity of genetic information, the factors that affect the neutral mutation rate are of considerable interest. Mammals exhibit two dramatically different neutral mutation rates: the CpG mutation rate, wherein the C of most CpGs (i.e., methyl-CpG) mutate at 10-50 times that of C in any other context or of any other base. The latter mutations constitute the non-CpG rate. The high CpG rate results from the spontaneous deamination of methyl-C to T and incomplete restoration of the ensuing T:G mismatches to C:Gs. Here, we determined the neutral non-CpG mutation rate as a function of CpG content by comparing sequence divergence of thousands of pairs of neutrally evolving chimpanzee and human orthologs that differ primarily in CpG content. Both the mutation rate and the mutational spectrum (transition/transversion ratio) of non-CpG residues change in parallel as sigmoidal (logistic) functions of CpG content. As different mechanisms generate transitions and transversions, these results indicate that both mutation rate and mutational processes are contingent on the local CpG content. We consider several possible mechanisms that might explain how CpG exerts these effects.

DNA FOSSILS AND PHYLOGENETIC ANALYSIS : USING L1 (LINE-1, LONG INTERSPERSED REPEATED) DNA TO DETERMINE THE EVOLUTIONARY HISTORY OF MAMMALS

The L1 element (LINE-1, long interspersed repeated DNA) is the mammalian version of the non-long terminal repeat class of transposable elements that replicate via an RNA intermediate (retrotransposons) (1). Every modern mammalian species studied to date contains a distinctive L1 family consisting of tens of thousands of members, which are interspersed throughout the genome. Despite their distinctiveness, all full-length mammalian L1 elements share the same organization: a 59-UTR, which includes a regulatory sequence; ORF I, which encodes a protein of unknown function; ORF II, which encodes an RT (2); and a 39-UTR that contains a G-rich polypurine:polypyrimidine tract and terminates in an A-rich sequence (Fig. 1). Each of the modern L1 families evolved independently in the various mammalian lineages from a common ancestral L1 element that dates back to sometime before the mammalian radiation ;100 million years ago (3–5). Being capable of prodigious amplification, the modern L1 elements and their evolutionary antecedents (see below) now account for at least 30% of the mass of mammalian DNA. In addition, L1 elements are active in present day species and are a frequent cause of genetic polymorphisms including a number of non-inherited genetic defects in humans (6–8). It is also possible that the L1 RT catalyzed the retrotransposition of elements that do not encode their own RT such as the mammalian SINE families (e.g. Alu in primates, B1, B2, ID, etc., in rodents) (5, 9–11). Since these families can reach copy numbers as high as 1 3 10 and alone contribute up to 5% of mammalian DNA (e.g. Alu (9)), L1 elements quite likely have had, and continue to have, a profound effect on the structure, function, and evolution of mammalian genomes. In spite of their prominence, most of the biochemical and molecular details of L1 regulation, replication, and transposition remain unknown. To a large extent, what is known has been derived from evolutionary studies, and these have yielded two kinds of information. The first is derived from comparisons between different mammalian L1 families or between L1 elements and their counterparts in other organisms. This comparative biochemical approach identified and assigned possible functional significance to different features of non-long terminal repeat retrotransposons. The second type of information, generated by the analytical techniques of evolutionary biology, revealed the evolutionary dynamics of L1 families. These studies suggest that L1 evolution is a paradigm for a novel, but as yet incompletely understood, evolutionary process that is taking place within the “ecosystem” of the mammalian genome and that L1 evolution is quite dynamic, with novel L1 variants continually emerging over relatively short periods of time. As a consequence, L1 evolution has generated a rather complex family structure, and it has become apparent that this feature of L1 evolution can be exploited to examine the evolutionary (phylogenetic) history of the mammalian hosts that harbor these elements (12–16). It is this last aspect of L1 biology that will be the focus of this review. By way of introduction, we will briefly summarize some results derived from the comparative biochemical analysis and the evolutionary studies of L1 families.

Amplification of the ancient murine Lx family of long interspersed repeated DNA occurred during the murine radiation

We identified and characterized the relics of an ancient rodent Ll family, referred to as Lx, which was extensively amplified at the time of the murine radiation about 12 million years ago, and which we showed was ancestral to the modern L1 families in rat and mouse. Here we have extended our analysis of the Lx amplification by examining more murine and nonmurine species for Lx sequences using both blot hybridization and the polymerase chain reaction for a total of 36 species. In addition we have determined the relative copy number and sequence divergence, or age, of Lx elements in representative murine genera. Our results show that while Lx sequences are confined to murine genera, the extent of the amplification was different in the different murine lineages, indicating that the amplification of Lx did not precede, but was coincident with, the murine radiation. The implications of our findings for the evolutionary dynamics of L1 families and the utility of ancestral amplification events for systematics are discussed.

DETERMINATION OF THE EVOLUTIONARY RELATIONSHIPS IN RATTUS SENSU LATO (RODENTIA : MURIDAE) USING L1 (LINE-1) AMPLIFICATION EVENTS

We determined ∼215 bp of DNA sequence from the 3′-untranslated region (UTR) of 240 cloned L1 (LINE-1) elements isolated from 22 species of Rattus sensu lato and Rattus sensu stricto murine rodents. The sequences were sorted into different L1 subfamilies, and oligonucleotides cognate to them were hybridized to genomic DNA of various taxa. From the distribution of the L1 subfamilies in the various species, we inferred the partial phylogeny of Rattus sensu lato. The four Maxomys species comprise a well-defined clade separate from a monophyletic cluster that contains the two Leopoldamys and four Niviventer species. The Niviventer/ Leopoldamys clade, in turn, shares a node with the clade that contains Berylmys, Sundamys, Bandicota, and Rattus sensu stricto. The evolutionary relationships that we deduced agree with and significantly extend the phylogeny of Rattus sensu lato established by other molecular criteria. Furthermore, the L1 amplification events scored here produced a unique phylogenetic tree, that is, in no case did a character (a given L1 amplification event) appear on more than one branch. The lack of homoplasy found in this study supports the robustness of L1 amplification events as phylogenetic markers for the study of mammalian evolution.

Repair of naturally occurring mismatches can induce mutations in flanking DNA

‘Normal’ genomic DNA contains hundreds of mismatches that are generated daily by the spontaneous deamination of C (U/G) and methyl-C (T/G). Thus, a mutagenic effect of their repair could constitute a serious genetic burden. We show here that while mismatches introduced into human cells on an SV40-based episome were invariably repaired, this process induced mutations in flanking DNA at a significantly higher rate than no mismatch controls. Most mutations involved the C of TpC, the substrate of some single strand-specific APOBEC cytidine deaminases, similar to the mutations that can typify the ‘mutator phenotype’ of numerous tumors. siRNA knockdowns and chromatin immunoprecipitation showed that TpC preferring APOBECs mediate the mutagenesis, and siRNA knockdowns showed that both the base excision and mismatch repair pathways are involved. That naturally occurring mispairs can be converted to mutators, represents an heretofore unsuspected source of genetic changes that could underlie disease, aging, and evolutionary change. DOI: http://dx.doi.org/10.7554/eLife.02001.001

Rapid evolution of a young L1 (LINE-1) clade in recently speciated rattus taxa

L1 elements are retrotransposons that have been replicating and evolving in mammalian genomes since before the mammalian radiation. Rattus norvegicus shares the young L1mlvi2 clade only with its sister taxon, Rattus cf moluccarius. Here we compared the L1mlvi2 clade in these recently diverged species and found that it evolved rapidly into closely related but distinct clades: the L1mlvi2-rm clade (or subfamily), characterized here from R. cf moluccarius, and the L1mlvi2-rn clade, originally described in R. norvegicus. In addition to other differences, these clades are distinguished by a cluster of amino acid replacement substitutions in ORF I. Both rat species contain the L1mlvi2-rm clade, but the L1mlvi2-rn clade is restricted to R. norvegicus. Therefore, the L1mlvi2-rm clade arose prior to the divergence of R. norvegicus and R. cf moluccarius, and the L1mlvi2-rn clade amplified after their divergence. The total number of L1mlvi2-rm elements in R. cf moluccarius is about the same as the sum of the L1mlvi2-rm and L1mlvi2-rn elements in R. norvegicus. The possibility that L1 amplification is in some way limited so that the two clades compete for replicative supremacy as well as the implications of the other distinguishing characteristic of the L1mlvi2-rn and L1mlvi2-rm clades are discussed.