scholarly journals Integration of Bombyx mori R2 Sequences into the 28S Ribosomal RNA Genes of Drosophila melanogaster

2000 ◽  
Vol 20 (1) ◽  
pp. 213-223 ◽  
Author(s):  
Danna G. Eickbush ◽  
Dongmei D. Luan ◽  
Thomas H. Eickbush
Keyword(s):  
Drosophila Melanogaster ◽  
Bombyx Mori ◽  
Dna Sequences ◽  
Reverse Transcription ◽  
Rrna Genes ◽  
Hydroxyl Group ◽  
28S Rrna ◽  
Gene Sequences ◽  
Gene Target ◽  
Integration System

ABSTRACT R2 non-long-terminal-repeat retrotransposable elements integrate into a precise location in the 28S rRNA genes of arthropods. The purified protein encoded by R2 can cleave the 28S gene target site and use the 3′ hydroxyl group generated by this cleavage to prime reverse transcription of its own RNA, a process called target-primed reverse transcription. An integration system is described here in which components from the R2 element of the silkmoth, Bombyx mori, are injected into the preblastoderm embryo ofDrosophila melanogaster. Silkmoth R2 sequences were readily detected in the 28S rRNA genes of the surviving adults as well as in the genes of their progeny. The 3′ junctions of these insertions were similar to those seen in our in vitro assays, as well as those from endogenous R2 retrotransposition events. The 5′ junctions of the insertions originally contained major deletions of both R2 and 28S gene sequences, a problem overcome by the inclusion of upstream 28S gene sequences at the 5′ end of the injected RNA. The resulting 5′ junctions suggested a recombination event between the cDNA and the upstream target sequences. This in vivo integration system should help determine the mechanism of R2 retrotransposition and be useful as a delivery system to integrate defined DNA sequences into the rRNA genes of organisms.

scholarly journals RNA-Induced Changes in the Activity of the Endonuclease Encoded by the R2 Retrotransposable Element

1998 ◽  
Vol 18 (6) ◽  
pp. 3455-3465 ◽  
Author(s):  
Jin Yang ◽  
Thomas H. Eickbush
Keyword(s):  
Reverse Transcription ◽  
Target Site ◽  
Rrna Genes ◽  
Hydroxyl Group ◽  
28S Rrna ◽  
Cleavage Reaction ◽  
Gene Target ◽  
Retrotransposable Element ◽  
Target Dna ◽  
Rnp Complex

ABSTRACT R2 is a non-long terminal repeat retrotransposable element that inserts itself site specifically in the 28S rRNA genes of arthropods. The 120-kDa protein encoded by R2 has been shown to cleave one strand of the 28S gene at the target site and to use the 3′ hydroxyl group generated from this nick to prime reverse transcription of its own RNA. This reaction has been termed target-primed reverse transcription (TPRT). Cleavage of the second DNA strand can occur in the presence or absence of reverse transcription but requires RNA. In this study, more sensitive in vitro assays have enabled further characterization of these reactions. R2 protein is capable of only a single round of TPRT because, once bound to the target DNA, it does not dissociate at physiological ionic strengths. Analysis of the role of RNA in the DNA cleavage reaction has revealed that the binding of RNA induces the R2 protein to form a multimeric complex. While larger complexes may form, the active component appears to be a dimer based on sedimentation studies and the change in stoichiometry of the cleavage reaction from a 1:1 ratio of protein subunit to target DNA in the absence of RNA to a 2:1 ratio of subunit to DNA target in the presence of RNA. Nonspecific RNA can also induce formation of this RNA-protein (RNP) complex, but the association of the protein with R2 RNA is stronger as revealed by its stability in 0.4 M NaCl. Finally, formation of the RNP complex gives rise to a 150-fold increase in the ability of the R2 endonuclease to find the target site. The specificity of this RNP complex is sufficiently great that it can find the 28S gene target site and conduct the TPRT reaction with total genomic DNA.

scholarly journals Transcription of Endogenous and Exogenous R2 Elements in the rRNA Gene Locus of Drosophila melanogaster

2003 ◽  
Vol 23 (11) ◽  
pp. 3825-3836 ◽  
Author(s):  
Danna G. Eickbush ◽  
Thomas H. Eickbush
Keyword(s):  
Drosophila Melanogaster ◽  
X Chromosome ◽  
Gene Locus ◽  
Rrna Genes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Integration System ◽  
Transcript Levels ◽  
Minimum Number ◽  
Different Levels

ABSTRACT R2 retrotransposons insert into the rRNA-encoding units (rDNA units) that form the nucleoli of insects. We have utilized an R2 integration system in Drosophila melanogaster to study transcription of foreign sequences integrated into the R2 target site of the 28S rRNA genes. The exogenous sequences were cotranscribed at dramatically different levels which closely paralleled the level of transcription of the endogenous R1 and R2 elements. Transcription levels were inversely correlated with the number of uninserted rDNA units, variation in this number having been brought about by the R2 integration system itself. Females with as few as 20 uninserted rDNA units per X chromosome had expression levels of endogenous and exogenous insertion sequences that were 2 orders of magnitude higher than lines that contained over 80 uninserted rDNA units per chromosome. R2 insertions only 167 bp in length exhibited this range of transcriptional regulation. Analysis of transcript levels in males suggested R2 insertions on the Y chromosome are not down-regulated to the same extent as insertions on the X chromosome. These results suggest that transcription of the rDNA units can be tightly regulated, but this regulation gradually breaks down as the cell approaches the minimum number of uninserted genes needed for survival.

scholarly journals Downstream 28S gene sequences on the RNA template affect the choice of primer and the accuracy of initiation by the R2 reverse transcriptase.

1996 ◽  
Vol 16 (9) ◽  
pp. 4726-4734 ◽  
Author(s):  
D D Luan ◽  
T H Eickbush
Keyword(s):  
Reverse Transcriptase ◽  
Long Terminal Repeat ◽  
Reverse Transcription ◽  
Terminal Repeat ◽  
Rrna Genes ◽  
28S Rrna ◽  
Gene Sequences ◽  
Cdna Synthesis ◽  
Target Dna

R2 non-long terminal repeat retrotransposable elements insert at a unique site in the 28S rRNA genes of insects. The protein encoded by the single open reading frame of R2 is capable of conducting the initial steps of its integration in vitro. The protein nicks the noncoding strand of the 28S target DNA (the strand which serves as a template for RNA synthesis) and uses the 3' hydroxyl group exposed by this nick to prime reverse transcription of the R2 RNA template. This target-primed reverse transcription (TPRT) reaction requires that the RNA template contains the 250-nucleotide 3' untranslated region of the R2 element. If this RNA template ends at the precise 3' end of the R2 element, then extra nucleotides, which we refer to as nontemplated nucleotides, are added to the target before cDNA synthesis. The presence of downstream 28S gene sequences on the RNA template reduces the total efficiency but eliminates these nontemplated additions, resulting in nearly 90% of all TPRT products reproducing the 3' junctions seen in vivo. Templates with 5 to 10 nucleotides of the 28S sequence are used most efficiently in this in vitro TPRT reaction. The requirement for downstream 28S rRNA sequences probably explains why the R2 elements of most insects differ from the majority of non-long terminal repeat retrotransposons in that they do not contain an A-rich repeat at their 3' junction with the target DNA. The presence of downstream sequences on these in vitro R2 templates also revealed that the R2 reverse transcriptase can prime cDNA synthesis by using the 3' end of another RNA molecule. This RNA-primed cDNA synthesis is not based on sequence complementarity between the RNA primer and the R2 template. The ability to use the 3' end of a noncomplementary RNA molecule has also been seen with the reverse transcriptase of the mitochondrial Mauriceville plasmid of Neurospora crassa.

scholarly journals The cytogenetic boundaries of the rDNA region within heterochromatin of the X chromosome of Drosophila melanogaster and their relation to male meiotic pairing sites

1982 ◽  
Vol 39 (2) ◽  
pp. 149-156 ◽  
Author(s):  
R. Appels ◽  
A. J. Hilliker
Keyword(s):  
Drosophila Melanogaster ◽  
Rrna Genes ◽  
Type I ◽  
28S Rrna ◽  
Meiotic Pairing ◽  
Coding Region ◽  
Intervening Sequences ◽  
Single Region ◽  
Rrna Sequences

SummaryThe proximal breakpoints of the inversion chromosomes In(1)ωm4 and In(1)m51b were shown, by in situ hybridization, to define the boundaries of the ribosomal DNA region located within the X chromosome heterochromatin (Xh). We estimate that at least 95% of the rDNA is located between the In(1)ωm4 and In(1)ωm51b proximal breakpoints. In contrast only 60–70% of the Type I intervening sequences located in Xh are located between these breakpoints. The Type I intervening sequences in the rDNA region occur as inserts in the 28S rRNA sequences while the remainder of the sequences are distal to the In(1)ωm4 breakpoint and not associated with rRNA genes.The regions of Xh which contain rDNA and Type I intervening sequences were related to regions shown by Cooper (1964) to contribute to meiotic pairing between the X and Y chromosomes in male Drosophila. We demonstrate that the rRNA coding region contributes to X / Y pairing. However, no single region of Xh is required for fidelity of male meiotic pairing of the sex chromosomes.

Copro-PCR based detection of bovine schistosome infection in India

2015 ◽  
Vol 90 (1) ◽  
pp. 102-107 ◽  
Author(s):  
B. Lakshmanan ◽  
K. Devada ◽  
S. Joseph ◽  
T.V. Aravindakshan ◽  
L. Sabu
Keyword(s):  
Mitochondrial Dna ◽  
Ribosomal Rna ◽  
Dna Sequences ◽  
Rrna Genes ◽  
12S Rrna ◽  
Gene Sequences ◽  
Schistosome Infection ◽  
Faecal Samples ◽  
Polymerase Chain ◽  
Trematode Infections

AbstractSchistosomosis and amphistomosis are the two economically important and widely prevalent snail-borne trematode infections in grazing cattle of southern India. Acute infections are symptomatically similar and difficult to detect by routine microscopy for eggs. The present study was directed towards the development of a copro-polymerase chain reaction (copro-PCR) for detection of bovine schistosome species, using custom-designed primers targeting 18S and 28S ribosomal RNA as well as mitochondrial DNA. The study demonstrated the enhanced diagnostic specificity of mitochondrial DNA markers over ribosomal RNA genes as genus-specific probes to detect schistosomes. We developed a sensitive PCR assay using primers designed from mitochondrial DNA sequences targeting the partialrrnl(16S rRNA), tCys (transfer RNA for cysteine) and partialrrnS(12S rRNA) genes ofSchistosoma spindaleto specifically detect schistosome infection from faecal samples of naturally infected bovines. The salient findings of the work also throw light on to the high similarity of the ribosomal RNA gene sequences of schistosomes with those ofGastrothylax crumeniferandFischoederius elongatus,the most prevalent pouched amphistomes of the region. Further investigation has to be directed towards unravelling the complete gene sequences of 18S and 28S ribosomal RNA as well as mitochondrial DNA sequences of amphistome isolates from India.

Molecular phylogeny of several species of Hoplolaimina (Nematoda: Tylenchida) associated with turfgrass in Korea, with comments on their morphology

Nematology ◽  
2020 ◽  
pp. 1-18
Author(s):  
Abraham Okki Mwamula ◽  
Gayeong Lee ◽  
Yeong Ho Kim ◽  
Young Ho Kim ◽  
Kwang-Soo Lee ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Integrative Taxonomy ◽  
Rrna Genes ◽  
28S Rrna ◽  
Gene Sequences ◽  
Meloidogyne Graminicola ◽  
Molecular Phylogenetic ◽  
Its Rrna ◽  
First Time ◽  
Pratylenchus Zeae

Summary Seven species belonging to Suborder Hoplolaimina are characterised using integrative taxonomy, considering both morphological and molecular phylogenetic analyses of the 28S-rRNA, ITS-rRNA and COI gene sequences. It is evident that, as more populations of Pratylenchus zeae are continuously characterised, the species continues to display an ever-increasing intraspecific genetic variation within the 28S-rRNA and ITS-rRNA genes. However, the COI gene sequences exhibit minimum intraspecific variation and thus might be the most powerful DNA barcoding marker for the precise identification of P. zeae and should therefore be recommended as a complementary technique in the identification process of the species. Pratylenchus zeae, Meloidogyne graminicola and Heterodera pratensis are characterised herein for the first time in Korea, while the presence in Korea of P. penetrans, P. scribneri, H. avenae, and M. marylandi, is molecularly confirmed.

scholarly journals Chromosomal distribution of the major insert in Drosophila melanogaster 28S rRNA genes

1981 ◽  
Vol 37 (2) ◽  
pp. 209-214 ◽  
Author(s):  
W. J. Peacock ◽  
R. Appels ◽  
S. Endow ◽  
D. Glover
Keyword(s):  
Drosophila Melanogaster ◽  
Polytene Chromosomes ◽  
Ribosomal Gene ◽  
Rrna Genes ◽  
Major Type ◽  
Type I ◽  
28S Rrna ◽  
Mitotic Chromosomes ◽  
Chromosomal Distribution

SUMMARYThe major type I insert sequence for the 28S rRNA genes of Drosophila melanogaster has been mapped within the chromosomes using a probe synthesized from a cloned sequence containing the entire 5·4 kb segment. The genomic distribution was shown to be complex in that the insert sequence occurred next to many different types of sequences, in addition to occurring as an insert in the 28S rRNA genes of the X chromosome. In situ hybridization of mitotic chromosomes showed most of the insert units not contained in the ribosomal genes to be located near the ribosomal gene cluster on the X chromosome. Additional sites were detected in polytene chromosomes in region 102C, 8–12 and in the hetero-chromatin of the autosomes.

scholarly journals From 18S to 28S rRNA Gene: An Improved Targeted Sarcocystidae PCR Amplification, Species Identification with Long DNA Sequences

2021 ◽  
Vol 104 (4) ◽  
pp. 1388-1393
Author(s):  
Florence C. H. Lee ◽  
Vickneshwaran Muthu
Keyword(s):  
Species Identification ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Environmental Samples ◽  
Pcr Amplification ◽  
Rrna Genes ◽  
Rrna Gene ◽  
28S Rrna ◽  
Index Test ◽  
Identity Matching

ABSTRACTSarcocystosis outbreaks in Tioman and Pangkor islands of Malaysia between 2011 and 2014 have raised the need to improve Sarcocystis species detection from environmental samples. In-house works found that published primers amplifying the 18S rRNA gene of Sarcocystis either could not produce the target from environmental samples or produced Sarcocystis DNA sequence that was insufficient for species identification. Using the primer pair of 18S S5 F (published) and 28S R6 R (new), this study improved the PCR amplification of Sarcocystidae to overcome these two difficulties. The PCR product spanned from the 18S to 28S rRNA genes, providing more information for species identification. The long DNA sequence allowed comparison between the “Ident” and “Query Cover” sorting in GenBank identity matching. This revealed the ambiguity in identity matching caused by different lengths of reference DNA sequences, which is seldom discussed in the literature. Using the disparity index test, a measurement of homogeneity in nucleotide substitution pattern, it is shown that the internal transcribed spacer (ITS)1-5.8S-ITS2 and 28S genes are better than the 18S gene in indicating nucleotide variations, implying better potentials for species identification. The example given by the handful of Sarcocystidae long DNA sequences reported herein calls for the need to report DNA sequence from the 18S to the 28S rRNA genes for species identification, especially among emerging pathogens. DNA sequence reporting should include the hypervariable 5.8S and ITS2 regions where applicable, and not be limited to single gene, per the current general trend.

scholarly journals R2 Target-Primed Reverse Transcription: Ordered Cleavage and Polymerization Steps by Protein Subunits Asymmetrically Bound to the Target DNA

2005 ◽  
Vol 25 (15) ◽  
pp. 6617-6628 ◽  
Author(s):  
Shawn M. Christensen ◽  
Thomas H. Eickbush
Keyword(s):  
Dna Binding ◽  
Reverse Transcription ◽  
Insertion Site ◽  
Rrna Genes ◽  
28S Rrna ◽  
Protein Subunit ◽  
Binding Motifs ◽  
Dna Binding Motifs ◽  
Target Dna ◽  
Dna Strand

ABSTRACT R2 elements are non-long terminal repeat retrotransposons that specifically insert into 28S rRNA genes of many animal groups. These elements encode a single protein with reverse transcriptase and endonuclease activities as well as specific DNA and RNA binding properties. In this report, gel shift experiments were conducted to investigate the stoichiometry of the DNA, RNA, and protein components of the integration reaction. The enzymatic functions associated with each of the protein complexes were also determined, and DNase I digests were used to footprint the protein onto the target DNA. Additionally, a short polypeptide containing the N-terminal putative DNA-binding motifs was footprinted on the DNA target site. These combined findings revealed that one protein subunit binds the R2 RNA template and the DNA 10 to 40 bp upstream of the insertion site. This subunit cleaves the first DNA strand and uses that cleavage to prime reverse transcription of the R2 RNA transcript. Another protein subunit(s) uses the N-terminal DNA binding motifs to bind to the 18 bp of target DNA downstream of the insertion site and is responsible for cleavage of the second DNA strand. A complete model for the R2 integration reaction is presented, which with minor modifications is adaptable to other non-LTR retrotransposons.

scholarly journals RNA template requirements for target DNA-primed reverse transcription by the R2 retrotransposable element.

1995 ◽  
Vol 15 (7) ◽  
pp. 3882-3891 ◽  
Author(s):  
D D Luan ◽  
T H Eickbush
Keyword(s):  
Reverse Transcriptase ◽  
Long Terminal Repeat ◽  
Reverse Transcription ◽  
Transcription Initiation ◽  
Terminal Repeat ◽  
Hydroxyl Group ◽  
Rrna Gene ◽  
28S Rrna ◽  
Retrotransposable Element ◽  
Target Dna

R2 is a non-long terminal repeat-retrotransposable element that inserts specifically in the 28S rRNA gene of most insects. The single protein encoded by R2 has been shown to contain both site-specific endonuclease and reverse transcriptase activities. Integration of the element involves cleavage of one strand of the 28S target DNA and the utilization of the exposed 3' hydroxyl group to prime the reverse transcription of the R2 RNA transcript. We have characterized the RNA requirement of this target DNA-primed reverse transcription reaction and found that the 250 nucleotides corresponding to the 3' untranslated region of the R2 transcript were necessary and sufficient for the reaction. To investigate the sequence requirements at the site of reverse transcription initiation, a series of RNA templates that contained substitutions and deletions at the extreme 3' end of the RNA were tested. The R2 templates used most efficiently had 3' ends which corresponded to the precise boundary of the R2 element with the 28S gene found in vivo. Transcripts containing short polyadenylated tails (8 nucleotides) were not utilized efficiently. R2 RNAs that were truncated at their 3' ends by 3 to 6 nucleotides were used less efficiently as templates and then only after the R2 reverse transcriptase had added extra, apparently nontemplated, nucleotides to the target DNA. The ability of the reverse transcriptase to add additional nucleotides to the target DNA before engaging the RNA template might be a mechanism for the generation of poly(A) or simple repeat sequences found at the 3' end of most non-long terminal repeat-retrotransposable elements.

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