scholarly journals Timing of Developmentally Programmed Excision and Circularization of Paramecium Internal Eliminated Sequences

2000 ◽  
Vol 20 (5) ◽  
pp. 1553-1561 ◽  
Author(s):  
Mireille Bétermier ◽  
Sandra Duharcourt ◽  
Hervé Seitz ◽  
Eric Meyer
Keyword(s):  
Quantitative Pcr ◽  
Genomic Dna ◽  
Germ Line ◽  
Donor Site ◽  
Single Copy ◽  
Southern Blots ◽  
Macronuclear Development ◽  
Internal Eliminated Sequences ◽  
Dna Elements

ABSTRACT Paramecium internal eliminated sequences (IESs) are short AT-rich DNA elements that are precisely eliminated from the germ line genome during development of the somatic macronucleus. They are flanked by one 5′-TA-3′ dinucleotide on each side, a single copy of which remains at the donor site after excision. The timing of their excision was examined in synchronized conjugating cells by quantitative PCR. Significant amplification of the germ line genome was observed prior to IES excision, which starts 12 to 14 h after initiation of conjugation and extends over a 2- to 4-h period. Following excision, two IESs were shown to form extrachromosomal circles that can be readily detected on Southern blots of genomic DNA from cells undergoing macronuclear development. On these circular molecules, covalently joined IES ends are separated by one copy of the flanking 5′-TA-3′ repeat. The similar structures of the junctions formed on the excised and donor molecules point to a central role for this dinucleotide in IES excision.

scholarly journals Homology-Dependent Maternal Inhibition of Developmental Excision of Internal Eliminated Sequences inParamecium tetraurelia

1998 ◽  
Vol 18 (12) ◽  
pp. 7075-7085 ◽  
Author(s):  
Sandra Duharcourt ◽  
Anne-Marie Keller ◽  
Eric Meyer
Keyword(s):  
Inhibition Efficiency ◽  
Germ Line ◽  
Single Copy ◽  
Sequence Specificity ◽  
Direct Repeats ◽  
Maternal Control ◽  
Sequence Element ◽  
Conserved Sequence ◽  
Internal Eliminated Sequences ◽  
Sexual Generation

ABSTRACT Thousands of single-copy internal eliminated sequences (IESs) are excised from the germ line genome of ciliates during development of the polygenomic somatic macronucleus, following sexual events.Paramecium IESs are short, noncoding elements that frequently interrupt coding sequences. No absolutely conserved sequence element, other than flanking 5′-TA-3′ direct repeats, has been identified among sequenced IESs; the mechanisms of their specific recognition and precise elimination are unknown. Previous work has revealed the existence of an epigenetic control of excision. It was shown that the presence of one IES in the vegetative macronucleus results in a specific inhibition of the excision of the same element during the development of a new macronucleus, in the following sexual generation. We have assessed the generality and sequence specificity of this transnuclear maternal control by studying the effects of macronuclear transformation with 13 different IESs. We show that at least five of them can be maintained in the new macronuclear genome; sequence specificity is complete both between genes and between different IESs in the same gene. In all cases, the degree of excision inhibition correlates with the copy number of the maternal IES, but each IES shows a characteristic inhibition efficiency. Short internal IES-like segments were found to be excised from two of the IESs when excision between normal boundaries was inhibited. Available data suggest that the sequence specificity of these maternal effects is mediated by pairing interactions between homologous nucleic acids.

scholarly journals Absence of Replication-Competent Human-Tropic Porcine Endogenous Retroviruses in the Germ Line DNA of Inbred Miniature Swine

2004 ◽  
Vol 78 (5) ◽  
pp. 2502-2509 ◽  
Author(s):  
Linda Scobie ◽  
Samantha Taylor ◽  
James C. Wood ◽  
Kristen M. Suling ◽  
Gary Quinn ◽  
...  
Keyword(s):  
Genomic Dna ◽  
Germ Line ◽  
Human Cells ◽  
Endogenous Retroviruses ◽  
Open Reading Frames ◽  
Miniature Swine ◽  
Dna Libraries ◽  
Porcine Endogenous Retroviruses ◽  
Perv Transmission

ABSTRACT The potential transmission of porcine endogenous retroviruses (PERVs) has raised concern in the development of porcine xenotransplantation products. Our previous studies have resulted in the identification of animals within a research herd of inbred miniature swine that lack the capacity to transmit PERV to human cells in vitro. In contrast, other animals were capable of PERV transmission. The PERVs that were transmitted to human cells are recombinants between PERV-A and PERV-C in the post-VRA region of the envelope (B. A. Oldmixon, J. C. Wood, T. A. Ericsson, C. A. Wilson, M. E. White-Scharf, G. Andersson, J. L. Greenstein, H. J. Schuurman, and C. Patience, J. Virol. 76:3045-3048, 2002); these viruses we term PERV-A/C. This observation prompted us to determine whether these human-tropic replication-competent (HTRC) PERV-A/C recombinants were present in the genomic DNA of these miniature swine. Genomic DNA libraries were generated from one miniature swine that transmitted HTRC PERV as well as from one miniature swine that did not transmit HTRC PERV. HTRC PERV-A/C proviruses were not identified in the germ line DNAs of these pigs by using genomic mapping. Similarly, although PERV-A loci were identified in both libraries that possessed long env open reading frames, the Env proteins encoded by these loci were nonfunctional according to pseudotype assays. In the absence of a germ line source for HTRC PERV, further studies are warranted to assess the mechanisms by which HTRC PERV can be generated. Once identified, it may prove possible to generate animals with further reduced potential to produce HTRC PERV.

scholarly journals Focal Distribution of Baculovirus IE1 Triggered by Its Binding to the hr DNA Elements

2005 ◽  
Vol 79 (1) ◽  
pp. 39-46 ◽  
Author(s):  
Toshihiro Nagamine ◽  
Yu Kawasaki ◽  
Tetsutaro Iizuka ◽  
Shogo Matsumoto
Keyword(s):  
Fluorescent Protein ◽  
Direct Repeat ◽  
Single Copy ◽  
Targeted Mutagenesis ◽  
Homologous Region ◽  
Focus Formation ◽  
Primary Determinant ◽  
Dna Elements ◽  
Species Specific ◽  
Green Fluorescent

ABSTRACT In BmN cells infected with the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), IE1, a principal transcriptional activator, localizes to sites of viral DNA replication. IE1 initially displays focal distribution in BmNPV-infected cells prior to DNA synthesis, whereas the protein expressed by transfection with the ie1 gene is distributed throughout the nucleoplasm instead of localized to discrete subnuclear structures. To identify the inducer of focus formation for IE1, we conducted transfection experiments with an IE1-GFP construct and found that cotransfection with genomic DNA fragments bearing the homologous region (hr) sequences caused the formation of IE1-green fluorescent protein (GFP) foci. The transfection of insect cells with a single plasmid containing exclusively the hr3 sequence and the IE1-GFP gene was sufficient to form IE1-GFP foci. These results suggest that hr elements are a primary determinant of the focal distribution of IE1. An analysis of a series of hr3 deletion mutants showed that a single copy of the direct repeat could induce the formation of IE1 foci. Targeted mutagenesis within the hr-binding domain of IE1-GFP caused impairment of the hr-dependent IE1 localization, suggesting that binding of IE1 to the hr elements is essential for the onset of IE1 focus formation. The observation of BmNPV IE1 foci in non-BmNPV-susceptible cells suggests that no species-specific factors are required for hr-dependent IE1 focus formation.

scholarly journals A Novel Chromodomain Protein, Pdd3p, Associates with Internal Eliminated Sequences during Macronuclear Development inTetrahymena thermophila

2000 ◽  
Vol 20 (11) ◽  
pp. 4128-4134 ◽  
Author(s):  
Mikhail A. Nikiforov ◽  
Martin A. Gorovsky ◽  
C. David Allis
Keyword(s):  
De Novo ◽  
Germ Line ◽  
Chromosome Breakage ◽  
Specific Protein ◽  
Developmentally Regulated ◽  
Dna Elimination ◽  
Internal Eliminated Sequences ◽  
Subsequent Elimination ◽  
Rearrangement Processes

ABSTRACT Conversion of the germ line micronuclear genome into the genome of a somatic macronucleus in Tetrahymena thermophila requires several DNA rearrangement processes. These include (i) excision and subsequent elimination of several thousand internal eliminated sequences (IESs) scattered throughout the micronuclear genome and (ii) breakage of the micronuclear chromosomes into hundreds of DNA fragments, followed by de novo telomere addition to their ends. Chromosome breakage sequences (Cbs) that determine the sites of breakage and short regions of DNA adjacent to them are also eliminated. Both processes occur concomitantly in the developing macronucleus. Two stage-specific protein factors involved in germ line DNA elimination have been described previously. Pdd1p and Pdd2p (for programmed DNA degradation) physically associate with internal eliminated sequences in transient electron-dense structures in the developing macronucleus. Here, we report the purification, sequence analysis, and characterization of Pdd3p, a novel developmentally regulated, chromodomain-containing polypeptide. Pdd3p colocalizes with Pdd1p in the peripheral regions of DNA elimination structures, but is also found more internally. DNA cross-linked and immunoprecipitated with Pdd1p- or Pdd3p-specific antibodies is enriched in IESs, but not Cbs, suggesting that different protein factors are involved in elimination of these two groups of sequences.

A rapid procedure for the isolation of C0t-1 DNA from plants

Genome ◽  
1997 ◽  
Vol 40 (1) ◽  
pp. 138-142 ◽  
Author(s):  
Michael S. Zwick ◽  
Robert E. Hanson ◽  
M. Nurul Islam-Faridi ◽  
David M. Stelly ◽  
Rod A. Wing ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Repetitive Dna ◽  
Copy Number ◽  
Genomic Dna ◽  
Mammalian Species ◽  
Repetitive Dnas ◽  
Rapid Procedure ◽  
Dna Elements ◽  
Repetitive Dna Elements

In situ hybridization (ISH) for the detection of single- or low-copy sequences, particularly large DNA fragments cloned into YAC or BAC vectors, generally requires the suppression or "blocking" of highly-repetitive DNAs. C0t-1 DNA is enriched for repetitive DNA elements, high or moderate in copy number, and can therefore be used more effectively than total genomic DNA to prehybridize and competitively hybridize repetitive elements that would otherwise cause nonspecific hybridization. C0t-1 DNAs from several mammalian species are commercially available, however, none is currently available for plants to the best of our knowledge. We have developed a simple 1-day procedure to generate C0t-1 DNA without the use of specialized equipment.Key words: C0t-1 DNA, in situ hybridization, BACs, plants.

scholarly journals Evidence supporting somatic assembly of the DNA segments (minigenes), coding for the framework, and complementarity-determining segments of immunoglobulin variable regions.

1979 ◽  
Vol 149 (6) ◽  
pp. 1299-1313 ◽  
Author(s):  
E A Kabat ◽  
T T Wu ◽  
H Bilofsky
Keyword(s):  
Adult Mouse ◽  
Germ Line ◽  
Variable Region ◽  
Single Copy ◽  
Variable Regions ◽  
X Ray ◽  
V Genes ◽  
V Region ◽  
Complementarity Determining Regions ◽  
The One

Two sets of apparently conflicting data on the genes coding for the variable region are being accumulated. One suggests that the sets of nucleotides coding for the framework segments of immunoglobulin light and heavy (VL and VH) chains assort independently and are therefore germ-line minigenes which, together with sets of nucleotides coding for the complementarity-determining regions (CDR) or segments assemble to form complete variable (V)-region genes (15, 16, 33). The other, based on the findings with clones from 12-d-old embryo and adult mouse coding for V-regions, infer that the first three frameworks and the three complementarity-determining segments are already assembled as germ-line V-genes (17-21). It is now generally accepted that the J segment, which in the one instance sequenced (21) is made up of nucleotides coding for framework (FR)4 plus two residues of CDR3, is a minigene. An examination of sequences of human, mouse, and rabbit V-regions, assuming the latter hypothesis, indicates that individual framework sets would have to be present in many copies. The FR2 segment found in one human, 20 mice, and 13 rabbits would have to be present in at least 10/14 copies in the NZB, and 5/6 in the BALB/c mouse, and 12/13 in the rabbit. The X-ray crystallographic data show this region to be a loop, projecting out from the V-domain, capable of accommodating many substiutions and 12 and 8 alternative sequences for this FR2 segment have been found in mouse and rabbit VK chains with substitutions possible at 13 of the 15 positions. These alternative sequences occur much less frequently than the preserved FR2 segment. Thus, there is no basis in the protein structure to account for evolutionary stability of this FR2 segment if it occurs in so many copies in germ-line genes coding for residues 1-96, but its stability is easily explained if it were coded for by a separate germ-line minigene present as a single copy; the alternative forms could then have arisen by duplication and mutation of this minigene. Somatic assembly of the minigene segments for the three framework and three complementarity-determining segments during differentiation would account completely for our assortment data from which FR4 was inferred to be a minigene.

scholarly journals In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells

2007 ◽  
Vol 88 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Daniel Doucet ◽  
Anic Levasseur ◽  
Catherine Béliveau ◽  
Renée Lapointe ◽  
Don Stoltz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Virus Replication ◽  
Genomic Dna ◽  
Cultured Cells ◽  
Germ Line ◽  
Choristoneura Fumiferana ◽  
Genome Segment ◽  
Host Cells ◽  
Dsdna Viruses

Polydnaviruses (PDVs) are dsDNA viruses transmitted by ichneumonid and braconid endoparasitoids to their lepidopteran hosts during oviposition. Wasp carriers are asymptomatic and transmit the virus to their progeny through the germ line; replication is confined to the calyx region of the wasp ovary, where the virus accumulates in the fluid bathing the eggs. In the lepidopteran host, however, no virus replication takes place, but PDV gene expression is essential for successful parasitism. Sustained gene expression in the absence of virus replication thus requires that the circular PDV genome segments persist for days within host cells. Available evidence suggests that most genome segments persist as episomes, but recent studies have indicated that some genome segments may undergo integration within lepidopteran genomic DNA, at least in vitro. In the present study, an integrated form of a Tranosema rostrale ichnovirus (TrIV) genome segment was cloned from genomic DNA extracted from infected Choristoneura fumiferana CF-124T cells and junction regions on either side of the viral DNA sequence were sequenced. This is the first proven example of integration of an ichnovirus genome segment in infected lepidopteran cells. Interestingly, circular forms of this genome segment do not appear to persist in these cells; none the less, a gene (TrFrep1) carried by this genome segment displays long-term transcription in infected cultured cells.

scholarly journals GAPDH Pseudogenes and the Quantification of Feline Genomic DNA Equivalents

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
A. Katrin Helfer-Hungerbuehler ◽  
Stefan Widmer ◽  
Regina Hofmann-Lehmann
Keyword(s):  
Reference Gene ◽  
Copy Number ◽  
Genomic Dna ◽  
Single Copy ◽  
Variable Number ◽  
Suitable Reference Gene ◽  
Suitable Alternative ◽  
Future Studies ◽  
Gene Assay ◽  
Suitable Reference

Quantitative real-time PCR (qPCR) is broadly used to detect and quantify nucleic acid targets. In order to determine cell copy number and genome equivalents, a suitable reference gene that is present in a defined number in the genome is needed, preferably as a single copy gene. For most organisms, a variable number of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) pseudogenes have been reported. However, it has been suggested that a single-copy of the GAPDH pseudogene is present in the feline genome and that a GAPDH assay can therefore be used to quantify feline genomic DNA (gDNA). The aim of this study was to determine whether one or more GAPDH pseudogenes are present in the feline genome and to provide a suitable alternative qPCR system for the quantification of feline cell copy number and genome equivalents. Bioinformatics and sequencing results revealed that not just one but several closely related GAPDH-like sequences were present in the cat genome. We thus identified, developed, optimized, and validated an alternative reference gene assay using feline albumin (fALB). Our data emphasize the need for an alternative reference gene, apart from the GAPDH pseudogene, for the normalization of gDNA levels. We recommend using the fALB qPCR assay for future studies.

scholarly journals Proof of Concept for Recombinant Cellular Controls in Quantitative Molecular Pathogen Detection

2011 ◽  
Vol 77 (7) ◽  
pp. 2531-2533 ◽  
Author(s):  
Peter Rossmanith ◽  
Patrick Mester ◽  
Karin Frühwirth ◽  
Sabine Fuchs ◽  
Martin Wagner
Keyword(s):  
Listeria Monocytogenes ◽  
Sample Preparation ◽  
Quantitative Pcr ◽  
Pathogen Detection ◽  
Single Copy ◽  
Deletion Strain ◽  
Proof Of Concept ◽  
Targeted Deletion ◽  
Process Controls

ABSTRACTIn this study, we present the concept of internal sample process controls (ISPCs) to monitor the efficiency of an analytical chain using sample preparation and quantitative PCR (qPCR). A recombinantListeria monocytogenesΔprfA(targeted deletion) strain containing a competitive artificial single-copy genomic target was applied to naturally contaminated samples to demonstrate its analytical suitability as an ISPC.

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