Predicting mammalian SINE subfamily activity from A-tail length

Based on previous observations that newly inserted LINEs and SINEs have particularly long 3' A-tails, which shorten rapidly during evolutionary time, we have analyzed the rat and mouse genomes for evidence of recently inserted SINEs and LINEs. We find that the youngest predicted subfamilies of rodent identifier (ID) elements, a rodent-specific SINE derived from tRNA(Ala), are preferentially associated with A-tails over 50 bases in the rat genome, as predicted. Furthermore, these studies detected a subfamily of ID elements that has made over 15,000 copies that is younger than any previously reported ID subfamily. We use PCR analysis of genomic loci to demonstrate that all subfamily members tested inserted after the divergence of Rattus norvegicus from Rattus rattus. We also found evidence that the rodent B1 family of elements is much more active currently in mouse than in rat. These data provide useful estimates of recent activity from all of the mammalian retrotransposons, as well as allowing identification of the most recent insertions for use as population and speciation markers in those species. Both the current rat ID and mouse B1 elements that are active have small, specific interruptions in their 3' A-tail sequences. We suggest that these interruptions stabilize the length of the A-tails and contribute to the activity of these subfamilies. We present a model in which the dynamics of the 3' A-tail may be a central controlling factor in SINE activity.     

Not born equal: increased rate asymmetry in relocated and retrotransposed rodent gene duplicates

Duplicated genes frequently evolve at different rates. This asymmetry is evidence of natural selection's ability to discriminate between the 2 copies, subjecting them to different levels of purifying selection or even permitting adaptive evolution of one or both copies. However, if gene duplication creates pairs of protein-coding sequences that are initially identical, this raises the question of how selection tells the 2 copies apart. Here, we investigated asymmetric sequence divergence of recently duplicated genes in rodents and related this to 2 possible sources of such asymmetry: gene relocation as a consequence of duplication and retrotransposition as a mechanism of gene duplication. We found that most young rodent duplicates that have been relocated were created by retrotransposition. The degree of rate asymmetry in gene pairs where one copy has been relocated (either by retrotransposition or DNA-based duplication) is greater than in pairs formed by local DNA-based duplication events. Furthermore, by considering the direction of transposition for distant duplicates, we found a consistent tendency for retrogenes to undergo accelerated protein evolution relative to their static paralogs, whereas DNA-based transpositions showed no such tendency. Finally, we demonstrate that the faster sequence evolution of retrogenes correlates with the profound alteration of their expression pattern that is precipitated by retrotransposition.     

An S/MAR-based L1 retrotransposition cassette mediates sustained levels of insertional mutagenesis without suffering from epigenetic silencing of DNA methylation

L1 is an insertional mutagen that is capable of mediating permanent gene disruption in mammalian genomes. However, currently available L1 retrotransposition vectors exhibit low or unstable transgene expression when expressed in somatic cells and tissues. This restriction limits their potential utility in long-term screening procedures or somatic mutagenesis applications. In this study, we addressed this problem by developing a minicircle, nonviral L1 retrotransposition vector using a scaffold/matrix attachment region (S/MAR) in the vector backbone and evaluated its utility in human cell lines. The S/MAR-based L1 retrotransposition vector provides stable, elevated levels of L1 expression compared to the currently used EBNA1-based L1 vector. In addition, the S/MAR elements effectively mediate sustained levels of L1 retrotransposition in prolonged cell culturing without suffering from epigenetic silencing by DNA methylation or from vector integration problems even in the absence of selection pressure. These findings indicate that the simple inclusion of S/MAR in the vector backbone increased levels of L1 expression and retrotransposition that can be used as an effective tool to generate insertional mutagenesis in large-scale somatic mutagenesis applications in mammalian cells.     

ALU-ring elements in the primate genomes

Elucidation of complete nucleotide sequence of the human has revealed that coding sequences that store the information needed to synthesize functional proteins, occupy only 2% of the genomic region. The remaining 98%, barring few regulatory sequences, has been referred to as non-functional or junk DNA and consists of many kinds of repeat elements. In fact, human genome is the most repeat rich genome sequenced so far, in which more than half of the region is occupied by such sequences. Determination of significance of these repeats in the human genome has become the focus of many studies all over the world, especially after genome sequencing did not reveal any significant difference in coding regions between lower eukaryotes and human. In this article, we have focused on Alu repeats that are primate specific elements with many interesting biological properties. Moreover, these are the repeats with highest copy number in the human genome. We have highlighted different facets of their interaction with the genome and changing paradigms regarding their role in genome organization.     

High copy number in human endogenous retrovirus families is associated with copying mechanisms in addition to reinfection

There are at least 31 families of human endogenous retroviruses (HERVs), each derived from an independent infection by an exogenous virus. Using evidence of purifying selection on HERV genes, we have shown previously that reinfection by replication-competent elements was the predominant mechanism of copying in some families. Here we analyze the evolution of 17 HERV families using d(N)/d(S) ratios and find a positive relationship between copy number and the use of additional copying mechanisms. All families with more than 200 elements have also used one or more of the following mechanisms: (1) complementation in trans (elements copied by other elements of the same family; HERV-H and ERV-9), (2) retrotransposition in cis (elements copying themselves) within germ-line cells (HERV-K(HML3)), and (3) being copied by non-HERV machinery (HERV-W). We discuss why these other mechanisms are rare in most families and suggest why complementation in trans is significant only in the larger families.     

Development of hybrid viral vectors for gene therapy

Adenoviral, retroviral/lentiviral, adeno-associated viral, and herpesviral vectors are the major viral vectors used in gene therapy. Compared with non-viral methods, viruses are highly-evolved, natural delivery agents for genetic materials. Despite their remarkable transduction efficiency, both clinical trials and laboratory experiments have suggested that viral vectors have inherent shortcomings for gene therapy, including limited loading capacity, immunogenicity, genotoxicity, and failure to support long-term adequate transgenic expression. One of the key issues in viral gene therapy is the state of the delivered genetic material in transduced cells. To address genotoxicity and improve the therapeutic transgene expression profile, construction of hybrid vectors have recently been developed. By adding new abilities or replacing certain undesirable elements, novel hybrid viral vectors are expected to outperform their conventional counterparts with improved safety and enhanced therapeutic efficacy. This review provides a comprehensive summary of current achievements in hybrid viral vector development and their impact on the field of gene therapy.