scholarly journals Identification of an Active Reverse Transcriptase Enzyme Encoded by a Human Endogenous HERV-K Retrovirus

1999 ◽  
Vol 73 (3) ◽  
pp. 2365-2375 ◽  
Author(s):  
Ben Berkhout ◽  
Maarten Jebbink ◽  
Jozsef Zsíros
Keyword(s):  
Reverse Transcriptase ◽  
Human Genome ◽  
Genetic Recombination ◽  
Biochemical Properties ◽  
Rnase H ◽  
Open Reading Frame ◽  
Biologically Active ◽  
Virus Family ◽  
Reading Frame ◽  
Reverse Transcriptase Enzyme

ABSTRACT Of the numerous endogenous retroviral elements that are present in the human genome, the abundant HERV-K family is distinct because several members are transcriptionally active and coding for biologically active proteins. A detailed phylogeny of the HERV-K family based on the partial sequence of the reverse transcriptase (RT) gene revealed a high incidence of an intact RT open reading frame within the HML-2 subgroup of HERV-K elements. In this study, we report the cloning of six full-length HML-2 RT genes, of which five contain an uninterrupted open reading frame. The RT enzymes were expressed as glutathione S-transferase fusion proteins inEscherichia coli, and several HERV-K RT enzymes demonstrated polymerase as well as RNase H activity. Several biochemical properties of the RT polymerase were analyzed, including the template requirements and optimal reaction conditions (temperature, type of divalent cation). Inspection of the nucleotide sequence of the HERV-K RT genes demonstrated a mosaic structure, suggesting that a high level of genetic recombination has occurred in this virus family, which is a hallmark of replication by means of reverse transcription. The selective pressure to maintain the RT coding potential is illustrated by the sequence of a particular HERV-K isolate that contains three 1-nucleotide deletions within a small RT segment, thus maintaining the open reading frame. These combined results may suggest that these endogenous RT enzymes still have a biological function. It is possible that the RT activity was involved in the spread of this major class of retroelements by retrotransposition, and in fact it cannot be excluded that this retrovirus group is still mobile. The endogenous RT activity may also have been involved in the shaping of the human genome, e.g., by formation of pseudogenes.

scholarly journals A Group II Intron-Type Open Reading Frame from the Thermophile Bacillus (Geobacillus) stearothermophilus Encodes a Heat-Stable Reverse Transcriptase

2004 ◽  
Vol 70 (12) ◽  
pp. 7140-7147 ◽  
Author(s):  
Jaishree Vellore ◽  
Samuel E. Moretz ◽  
Bert C. Lampson
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Protein Product ◽  
Thermophilic Bacterium ◽  
Full Length ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Group Ii

ABSTRACT The production of a stable cDNA copy of an unstable RNA molecule by reverse transcription is a widely used and essential technology for many important applications, such as the construction of gene libraries, production of DNA probes, and analysis of gene expression by reverse transcriptase PCR (RT-PCR). However, the synthesis of full-length cDNAs is frequently inefficient, because the RT commonly used often produces truncated cDNAs. Synthesizing cDNA at higher temperatures, on the other hand, can provide a number of improvements. These include increasing the length of cDNA product, greater accuracy, and greater specificity during reverse transcription. Thus, an RT that remains stable and active at hot temperatures may produce better-quality cDNAs and improve the yield of full-length cDNAs. Described here is the discovery of a gene, designated trt, from the genome of the thermophilic bacterium Bacillus (Geobacillus) stearothermophilus strain 10. The gene codes for an open reading frame (ORF) similar to the ORFs encoded by group II introns found in bacteria. The gene was cloned and overexpressed in Escherichia coli, and its protein product was partially purified. Like the host organism, the Trt protein is a heat-stable protein with RT activity and can reverse transcribe RNA at temperatures as high as 75°C.

The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase

1991 ◽  
Vol 11 (3) ◽  
pp. 1696-1706
Author(s):  
U Schulte ◽  
A M Lambowitz
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Plasmid Dna ◽  
Sequence Similarity ◽  
Polymerase Activity ◽  
Open Reading Frame ◽  
Mitochondrial Plasmid ◽  
Reading Frame ◽  
Reverse Transcriptases ◽  
Neurospora Intermedia

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.

scholarly journals Translated anti-sense product of the Na/phosphate co-transporter (NaPi-II)

1998 ◽  
Vol 332 (2) ◽  
pp. 483-489 ◽  
Author(s):  
Birgit HUELSEWEH ◽  
Beate KOHL ◽  
Hartmut HENTSCHEL ◽  
Rolf K. H. KINNE ◽  
Andreas WERNER
Keyword(s):  
Genomic Sequence ◽  
Biological Significance ◽  
Rnase H ◽  
Open Reading Frame ◽  
Regulatory Function ◽  
Female Gonad ◽  
Putative Open Reading Frame ◽  
Reading Frame ◽  
Sense Transcript ◽  
Protein Protein Interaction

The homeostasis of Pi in marine teleosts is maintained by renal Pi secretion as well as by Pi reabsorption. A Na/Pi co-transport system belonging to the NaPi-II protein family is instrumental in tightly controlled renal Pi handling in mammals and fish. We have isolated an NaPi-II related cDNA from winter flounder. It was cloned from a female gonad cDNA library and is 624 bp long. The transcript is expressed in female and male flounder gonads as well as in kidney and intestine, although at very low levels. RNase H digestion experiments revealed an opposite orientation of the transcript with regard to NaPi-II-related mRNA. The anti-sense orientation was confirmed by genomic sequence analysis and Southern blotting. Alluding to the sense transcript, the anti-sense transcript was denoted IPAN. The open reading frame of IPAN encodes a basic protein of 68 amino acid residues. Immunohistochemistry confined the anti-sense related protein, Ipan, to a submembranous compartment of immature oocytes, suggesting a role in oocyte development. In kidney and intestine Ipan is partly co-localized with the Na/Pi co-transporter, implying a regulatory function for the anti-sense protein. However, direct protein–protein interaction could not be established. The existence of a putative open reading frame in other species extends the biological significance of the novel protein.

scholarly journals The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme.

1987 ◽  
Vol 7 (6) ◽  
pp. 2221-2230 ◽  
Author(s):  
W D Burke ◽  
C C Calalang ◽  
T H Eickbush
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Transposable Elements ◽  
Reverse Transcriptase ◽  
Bombyx Mori ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Type Ii ◽  
Site Specific ◽  
Reading Frame

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

The site-specific ribosomal insertion element type II of Bombyx mori (R2Bm) contains the coding sequence for a reverse transcriptase-like enzyme

1987 ◽  
Vol 7 (6) ◽  
pp. 2221-2230
Author(s):  
W D Burke ◽  
C C Calalang ◽  
T H Eickbush
Keyword(s):  
Amino Acid ◽  
Sequence Analysis ◽  
Transposable Elements ◽  
Reverse Transcriptase ◽  
Bombyx Mori ◽  
Open Reading Frame ◽  
Nucleotide Sequence Analysis ◽  
Type Ii ◽  
Site Specific ◽  
Reading Frame

Two classes of DNA elements interrupt a fraction of the rRNA repeats of Bombyx mori. We have analyzed by genomic blotting and sequence analysis one class of these elements which we have named R2. These elements occupy approximately 9% of the rDNA units of B. mori and appear to be homologous to the type II rDNA insertions detected in Drosophila melanogaster. Approximately 25 copies of R2 exist within the B. mori genome, of which at least 20 are located at a precise location within otherwise typical rDNA units. Nucleotide sequence analysis has revealed that the 4.2-kilobase-pair R2 element has a single large open reading frame, occupying over 82% of the total length of the element. The central region of this 1,151-amino-acid open reading frame shows homology to the reverse transcriptase enzymes found in retroviruses and certain transposable elements. Amino acid homology of this region is highest to the mobile line 1 elements of mammals, followed by the mitochondrial type II introns of fungi, and the pol gene of retroviruses. Less homology exists with transposable elements of D. melanogaster and Saccharomyces cerevisiae. Two additional regions of sequence homology between L1 and R2 elements were also found outside the reverse transcriptase region. We suggest that the R2 elements are retrotransposons that are site specific in their insertion into the genome. Such mobility would enable these elements to occupy a small fraction of the rDNA units of B. mori despite their continual elimination from the rDNA locus by sequence turnover.

scholarly journals Cloning and characterizing cDNA sequences coding high-mannose n-glycan binding lectins from cultivated red algae Eucheuma denticulatum and Kappaphycus striatum

2016 ◽  
Vol 14 (2) ◽  
pp. 327-336
Author(s):  
Le Dinh Hung ◽  
Makoto Hirayama ◽  
Kanji Hori
Keyword(s):  
Amino Acids ◽  
Red Algae ◽  
Untranslated Region ◽  
Biochemical Properties ◽  
Amino Acid Sequences ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Eucheuma Denticulatum ◽  
Cdna Sequences ◽  
High Mannose

The red algae, Eucheuma denticulatum and Kappaphycus striatum have been widely cultivated in Vietnam as a source of carrageenophytes for industry. In the past, biochemical properties of lectins isolated from these algae has been characterized  and  evaluated extensively. However, gene coding for such lectins isn’t studied yet. In this study, their full length cDNA is amfplified using cDNA ends (RACE) methods. Sequence analysis revealed that cDNA of EDA-2 from E. denticulatum consisted of 1,158 bp containing 103 bp of a 5'untranslated region, 248 bp of 3'untranslated region, and 807 bp of an open reading frame; and cDNA of KSA-2 from K. striatum consisted of 1174 bp containing 94 bp of the 5'-untranslated region, 273 bp of 3'untranslated region and 807 bp of the open reading frame. The cDNA of both EDA-2 and KSA-2 encoded for a polypeptide of 269 amino acids including an initiating methionine, but differed in sequences and molecular masses. The deduced amino acid sequences of EDA-2 and KSA-2 composed of four tandem repeated domains with about 67 amino acids each. The primary structure of EDA-2 and KSA-2 is highly similar to those of the high mannose N-glycan specific lectins including OAA from cyanobacterium, BOA, MBHA and PFA from bacteria, and ESA-2, KAA-1, KAA-2 from macro red algae, which showed strong anti-HIV and anti-influenza virus activities. These results indicate that these cultivated algae are becoming promising materials for production of anti-virus reagent or functional food that can prevent virus infection in future.

scholarly journals The LaBelle mitochondrial plasmid of Neurospora intermedia encodes a novel DNA polymerase that may be derived from a reverse transcriptase.

1991 ◽  
Vol 11 (3) ◽  
pp. 1696-1706 ◽  
Author(s):  
U Schulte ◽  
A M Lambowitz
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Plasmid Dna ◽  
Sequence Similarity ◽  
Polymerase Activity ◽  
Open Reading Frame ◽  
Mitochondrial Plasmid ◽  
Reading Frame ◽  
Reverse Transcriptases ◽  
Neurospora Intermedia

The LaBelle-1b strain of Neurospora intermedia contains a 4.1-kb closed-circular mitochondrial plasmid DNA, which encodes a single long open reading frame of 1,151 amino acids reported to have sequence similarity to reverse transcriptases. Here, we show that the LaBelle strain contains a novel DNA polymerase activity that is highly specific for the endogenous LaBelle plasmid DNA in nucleoprotein particles and can be distinguished from the mitochondrial DNA polymerase by several characteristics. Photolabeling experiments indicate that the LaBelle-specific DNA polymerase activity is associated with a polypeptide of 120 kDa, which is in good agreement with the size predicted for the protein encoded by the LaBelle plasmid open reading frame (132 kDa). This 120-kDa polypeptide is found only in the LaBelle strain that contains the mitochondrial plasmid, and it cosegregates with mitochondria in sexual crosses, suggesting that it is encoded by the plasmid. The LaBelle-specific DNA polymerase efficiently uses the artificial DNA substrates, poly(dA)-oligo(dT) and poly(dC)-oligo(dG), but despite its reported sequence similarity to reverse transcriptases, it has very low activity with analogous RNA substrates, poly(rA)-oligo(dT), poly(rC)-oligo(dG), or poly(rCm)-oligo(dG). Considered together with the previous sequence comparisons, our results suggest that the LaBelle plasmid encodes a novel DNA polymerase, which was derived from a protein that was at one time a reverse transcriptase but lost its ability to use RNA templates. This DNA polymerase now presumably functions in replication of the plasmid. Our results constitute the first biochemical evidence for a DNA polymerase activity associated with a mitochondrial plasmid. Further, they may provide insight into the evolution of DNA polymerases from reverse transcriptases, as presumably occurred in the course of evolution following the transition from the so-called RNA world to the present DNA world.

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