The human episome HALF1: Structure of its genomic counterpart

A human episomal sequence (HALF1) has been identified by its ability to restore expression of hepatic functions when used to transfect a rat dedifferentiated cell line. The genomic equivalent of this human episome (gHALF1) and its flanking sequences were analyzed. HALF1 itself does not present the characteristics of a transposable element but half of its sequence corresponds to retroposons, including Alu and L1 repeats and a processed pseudogene, known to transposevia RNA intermediates. The structural characteristics of these different kinds of retroposons and their origin and evolution were analyzed.     

The Kaposi Sarcoma Herpesvirus Latency-associated Nuclear Antigen DNA Binding Domain Dorsal Positive Electrostatic Patch Facilitates DNA Replication and Episome Persistence

Kaposi sarcoma-associated herpesvirus (KSHV) has a causative role in several human malignancies. KSHV latency-associated nuclear antigen (LANA) mediates persistence of viral episomes in latently infected cells. LANA mediates KSHV DNA replication and segregates episomes to progeny nuclei. The structure of the LANA DNA binding domain was recently solved, revealing a positive electrostatic patch opposite the DNA binding surface, which is the site of BET protein binding. Here we investigate the functional role of the positive patch in LANA-mediated episome persistence. As expected, LANA mutants with alanine or glutamate substitutions in the central, peripheral, or lateral portions of the positive patch maintained the ability to bind DNA by EMSA. However, all of the substitution mutants were deficient for LANA DNA replication and episome maintenance. Mutation of the peripheral region generated the largest deficiencies. Despite these deficiencies, all positive patch mutants concentrated to dots along mitotic chromosomes in cells containing episomes, similar to LANA. The central and peripheral mutants, but not the lateral mutants, were reduced for BET protein interaction as assessed by co-immunoprecipitation. However, defects in BET protein binding were independent of episome maintenance function. Overall, the reductions in episome maintenance closely correlated with DNA replication deficiencies, suggesting that the replication defects account for the reduced episome persistence. Therefore, the electrostatic patch exerts a key role in LANA-mediated DNA replication and episome persistence and may act through a host cell partner(s) other than a BET protein or by inducing specific structures or complexes.     

Ultrastructure of Amoebophrya sp. and its changes during the course of infection

Amoebophrya is a syndinian parasite that kills harmful bloom forming algae. Previously uncharacterized ultrastructural aspects of infection and development were elucidated. The biflagellate dinospore has two mitochondria, electron-dense bodies, striated strips, trichocysts, and a nucleus with peripherally condensed chromatin. After finding an Akashiwo sanguinea host and adhering to its surface, the parasite penetrates the host surface, apparently using a microfilament based motility and electron-dense bodies within a microtubular basket in the process of parasitophorous vacuole membrane formation. After entering the host nucleus, possibly by a similar mechanism used to enter the host cell, the parasite cytosol expanded substantially prior to mitosis. From 12-36 hours mitochondria were inconspicuous but present. Chromatin condensation was variable. By 36 hours post-infection, parasites had multiple nuclei, a microtubule-supported cytopharynx, and were beginning to form a fully internal mastigocoel. By 48 hours, the characteristic "beehive" appearance was apparent with flagella projecting into a fully developed mastigocoel. The cytoplasm contained trichocysts, elongated mitochondria, and nuclei with peripherally condensed chromatin. Although Amoebophrya lacks an apical complex, its electron-dense bodies show functional similarities to apicomplexan rhoptries. Its lack of permanently condensed chromosomes, but compact dinospore chromatin, supports the idea that dinoflagellate permanently condensed chromosomes may be a remnant of a parasitic ancestor with a compact dispersal stage.     

Close sequence identity between ribosomal DNA episomes of the non-pathogenicEntamoeba dispar and pathogenicEntamoeba histolytica

Entamoeba dispar andEntamoeba histolytica are now recognized as two distinct species-the former being nonpathogenic to humans. We had earlier studied the organization of ribosomal RNA genes inE. histolytica. Here we report the analysis of ribosomal RNA genes inE. dispar. The rRNA genes ofE. dispar, like their counterpart inE. histolytica are located on a circular rDNA molecule. From restriction map analysis, the size ofE. dispar rDNA circle was estimated to be 24·4 kb. The size was also confirmed by linearizing the circle withBsaHI, and by limited DNAseI digestion. The restriction map of theE. dispar rDNA circle showed close similarity to EhR1, the rDNA circle ofE. histolytica strain HM-1:IMSS which has two rDNA units per circle. The various families of short tandem repeats found in the upstream and downstream intergenic spacers (IGS) of EhR1 were also present inE. dispar. Partial sequencing of the cloned fragments ofE. dispar rDNA and comparison with EhR1 revealed only 2·6% to 3·8% sequence divergence in the IGS. The region Tr and the adjoiningPvuI repeats in the IGS of EhR1, which are missing in thoseE. histolytica strains that have one rDNA unit per circle, were present in theE. dispar rDNA circle. Such close similarity in the overall organization and sequence of the IGS of rDNAs of two different species is uncommon. In fact the spacer sequences were only slightly more divergent than the 18S rRNA gene sequence which differs by 1·6% in the two species. The most divergent sequence betweenE. histolytica andE. dispar was the internal transcribed spacer, ITS2. Therefore, it was concluded that probes derived from the ITS1 and ITS 2 sequences would be more reliable and reproducible than probes from the IGS regions used earlier for identifying these species.     

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.     

2-micron circle plasmids do not reduce yeast life span

Extrachromosomal rDNA circles (ERCs) and recombinant origin-containing plasmids (ARS-plasmids) are thought to reduce replicative life span in the budding yeast Saccharomyces cerevisiae due to their accumulation in yeast cells by an asymmetric inheritance process known as mother cell bias. Most commonly used laboratory yeast strains contain the naturally occurring, high copy number 2-micron circle plasmid. 2-micron plasmids are known to exhibit stable mitotic inheritance, unlike ARS-plasmids and ERCs, but the fidelity of inheritance during replicative aging and cell senescence has not been studied. This raises the question: do 2-micron circles reduce replicative life span? To address this question we have used a convenient method to cure laboratory yeast strains of the 2-micron plasmid. We find no difference in the replicative life spans of otherwise isogenic cir⁺ and cir⁰ strains, with and without the 2-micron plasmid. Consistent with this, we find that 2-micron circles do not accumulate in old yeast cells. These findings indicate that naturally occurring levels of 2-micron plasmids do not adversely affect life span, and that accumulation due to asymmetric inheritance is required for reduction of replicative life span by DNA episomes.     

Transient transgene transmission to piglets by intrauterine insemination of spermatozoa incubated with DNA fragments

An efficient and low-cost production of transgenic pigs has significant applications to the pig industry and biomedical science. Generation of transgenic pig by sperm-mediated gene transfer (SMGT) was inexpensive and convenient, and reported with high efficiency. To test the method of SMGT in pigs, we employed deep post-cervical intrauterine insemination of incubated spermatozoa in this study. A test of sperm motility of semen from nine Landrace boars after incubation with radioactively labeled DNA construct indicated that DNA uptake of the sperm was highly correlated with sperm motility at the time of collection. DNA concentration of 50 and 300 microg per one billion sperm was incubated with washed high-motility sperm at 17 degrees C for 2 hr. Twenty one hybrid gilts and sows of Meishan crossed with Large White were inseminated with transgene-incubated sperm and produced 156 piglets. Transgene DNA sequences were identified in 31 piglets by PCR amplification of genomic DNA isolated from piglet ears at the age of 3 days. The deep intrauterine insemination had a higher rate of positive transgenic piglets than regular insemination (29.6% of 98 piglets vs. 3.4% of 58 piglets). However, the exogenous transgene DNA was not detected in any piglets at the age of 70-100 days. Therefore, the results further demonstrated that transgene through incubation with spermatozoa was mostly transiently transmitted to the offspring at early growing stage and lost in adulthood, which may result from episomal DNA replications during cell divisions only at the early stage of development.     

State of Human Papilloma Virus DNA in Cervical Carcinomas

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.     

Expression of a transgene encoded on a non-viral episomal vector is not subject to epigenetic silencing by cytosine methylation

Currently available vectors for mammalian cells suffer from a number of limitations which make them only partially useful for genetic modification of eukaryotic cells and organisms and for gene therapy. While integration of a vector can lead to unpredictable interactions with the host genome and silencing of the integrated transgene, most non-integrating vectors mediate only transient expression of a transgene. All available vector types can lead to transformation of the recipient cell and many of them can cause serious immunological side effects in the organism. The ideal vector has to be free of these side effects and should allow long-term expression of a transgene in the absence of selection. In this report we describe a novel non-viral episomal expression system fulfilling these criteria. The gene encoding the truncated rat NGF-receptor gene under the control of the CMV-promoter was inserted into a vector construct containing a scaffold/matrix attached region (S/MAR). This vector was then transfected into CHO cells and human HaCat cells. We show that this vector replicates episomally in these cells and is mitotically stable in the abscence of selection over more than 100 generations. Moreover, we provide the first experimental data that the CMV-promoter in an episome is not subject to silencing by cytosine methylation, thus allowing long-term expression of the transgene in the absence of selection.     

Circular YAC vectors containing a small mammalian origin sequence can associate with the nuclear matrix

Three different mammalian origins of DNA replication, 343, S3, and X24, have been cloned into a 15.8 kb circular yeast vector pYACneo. Subsequent transfection into HeLa cells resulted in the isolation of several stably maintained clones. Two cell lines, C343e2 and CS3e1, were found to have sequences maintained as episomes in long-term culture with a stability per generation of approximately 80%. Both episomes also contain matrix attachment region (MAR) sequences which mediate the binding of DNA to the nuclear skeleton and are thought to play a role in DNA replication. Using high salt extraction of the nucleus and fluorescent in situ hybridization, we were able to demonstrate an association of the 343 episome with the nuclear matrix, most probably through functional MAR sequences that allow an association with the nuclear matrix and associated regions containing essential replication proteins. The presence of functional MARs in small episomal sequences may facilitate the replication and maintenance of transfected DNA as an episome and improve their utility as small episomal constructs, potential microchromosomes. J. Cell. Biochem. 67:439–450, 1997. © 1997 Wiley-Liss, Inc.