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A major non-LTR retrotransposon of Bombyx mori, L1Bm.

Repetitive sequences with oligo A tails were observed in Dra1 fragments of Bombyx mori genomic DNA. The full sequence of the element, an abundant non-LTR retrotransposon of B. mori, was determined by assembling inner restriction fragments. This element, designated L1Bm, contained two ORFs encoding a gag-like protein and reverse transcriptase (RT), respectively. An endonuclease domain was identified at the N-terminus of the RT sequence. The homology search of the amino acid sequences revealed that L1Bm belongs evolutionally to the same family as various other non-LTR retrotransposons of Drosophila and Mosquito. On the other hand, L1Bm resembles the L1 element of human genome in its high copy number and its frequent truncation at the 5'-side. Some units of the histone gene repeat in B. mori possess complete L1Bm elements in a 3'-flanking region of H2b.

Amino Acid Sequence↗

The Evolution of SINEs and LINEs in the genus Chironomus (Diptera).

Genomic DNA amplification from 51 species of the family Chironomidae shows that most contain relatives of NLRCth1 LINE and CTRT1 SINE retrotransposons first found in Chironomus thummi. More than 300 cloned PCR products were sequenced. The amplified region of the reverse transcriptase gene in the LINEs is intact and highly conserved, suggesting active elements. The SINEs are less conserved, consistent with minimal/no selection after transposition. A mitochondrial gene phylogeny resolves the Chironomus genus into six lineages (Guryev et al. 2001). LINE and SINE phylogenies resolve five of these lineages, indicating their monophyletic origin and vertical inheritance. However, both the LINE and the SINE tree topologies differ from the species phylogeny, resolving the elements into "clusters I-IV" and "cluster V" families. The data suggest a descent of all LINE and SINE subfamilies from two major families. Based on the species phylogeny, a few LINEs and a larger number of SINEs are cladisitically misplaced. Most misbranch with LINEs or SINEs from species with the same families of elements. From sequence comparisons, cladistically misplaced LINEs and several misplaced SINEs arose by convergent base substitutions. More diverged SINEs result from early transposition and some are derived from multiple source SINEs in the same species. SINEs from two species (C. dorsalis, C. pallidivittatus), expected to belong to the clusters I-IV family, branch instead with cluster V family SINEs; apparently both families predate separation of cluster V from clusters I-IV species. Correlation of the distribution of active SINEs and LINEs, as well as similar 3' sequence motifs in CTRT1 and NLRCth1, suggests coevolving retrotransposon pairs in which CTRT1 transposition depends on enzymes active during NLRCth1 LINE mobility.

Animals↗

Influence of substrate structure on cleavage by hammerhead ribozyme.

We compared the cleavage by a hammerhead ribozyme of a wild-type precursor tRNA (pre-tRNA leu(3)) and a structurally altered mutant form. We also analyzed the cleavage reactions of these tRNAs catalyzed by a ribozyme variant that was designed to complement the mutant precursor tRNA. Kinetic analyses reveal that the kcat values are nearly the same for the wild-type and the mutant substrate RNAs. However, the Km values differ considerably, being higher for the wild-type substrate. Thus, the formation of the ribozyme-substrate complex, but not the chemical cleavage step, is affected by these changes. Time course studies were performed, at different temperatures, to estimate the efficiency of the cleavage reactions and the effect of temperature. The cleavage of mutant precursor tRNA is generally faster than the wild-type at all temperatures analyzed. These results suggest that substrate structures can limit ribozyme efficiency, presumably by hindering the hybridization step.

Base Composition↗

DNA-binding fingers encoded by a trypanosome retroposon.

A Trypanosoma brucei repeated DNA element (TRS-1, for trypanosome repeated sequence), which seems transposable, may encode a 1651 amino acid polypeptide showing homology with reverse transcriptase. This polypeptide would also carry a DNA-binding domain, as suggested by the presence of five DNA-binding "fingers" homologous to those of the transcription factor TFIIIA of Xenopus laevis and retroviral DNA-binding proteins.

Animals↗

Identification of a repetitive element in the snail Biomphalaria glabrata: relationship to the reverse transcriptase-encoding sequence in LINE-1 transposons.

BamHI-digested Biomphalaria glabrata DNA contains a repetitive 2.0-kb fragment which is readily discernible by ethidium bromide staining. We present evidence that this repetitive element is related at both the nucleotide and amino acid levels to long interspersed nuclear element (LINE)-like transposons. Although comparable elements have been described in several invertebrates, this is the first report of a molluscan homologue. In common with LINE transposons, an open reading frame in the B. glabrata element shows significant homology to reverse transcriptase--a feature believed to allow the dissemination of these elements in the eukaryotic genome.

Amino Acid Sequence↗

The lymphoproliferative disease virus of turkeys represents a distinct class of avian type-C retrovirus.

The lymphoproliferative disease virus of turkeys (LPDV) is the etiological agent of a rapidly developing lymphoproliferative process in turkeys. To better understand the genetic relationships of LPDV to other retroviruses we determined the nucleotide sequence of its pol gene. Comparative computer analyses of the deduced amino acid sequences of the reverse transcriptase and integrase domains within pol established that LPDV represents a distinct class of avian retroviruses that is most closely related to the avian leukemia-sarcoma viruses.

Amino Acid Sequence↗

Natural resistance of human immunodeficiency virus type 2 to zidovudine.

Zidovudine (AZT) is a reverse transcriptase (RT) inhibitor widely used to treat persons infected with HIV-1 and HIV-2. Recent data on treated patients suggest differences in the antiviral activity of AZT between HIV-1 and HIV-2. We evaluated the antiviral activity of AZT on HIV-2 by using multiple approaches including in vitro selection experiments, analysis of growth kinetics with AZT, and phenotypic testing. A total of 5 wild-type (WT) HIV-2 viruses were used in the analysis. For comparison, 4 control WT HIV-1 strains and one HIV-1 mutant carrying the 215S mutation were evaluated in parallel. All 5 HIV-1 isolates acquired AZT resistance mutations after 3-6 passages with AZT or a 4- to 32-fold increase in AZT concentration. Among these viruses, the fastest selection of resistance was seen in HIV-1(S215), which acquired S215Y (1-nucleotide change only) at passage 3 after only 17 days in culture. In contrast, none of the 5 HIV-2 viruses that naturally have S215 acquired S215Y/F or any other RT mutation during 10 passages with AZT (1025-fold increase in AZT concentration). In the presence of AZT + didanosine (ddI), 3 of the 5 HIV-1 isolates acquired AZT or ddI resistance mutations, while only ddI resistance mutations were seen in HIV-2 (4 of 5 isolates). All HIV-2 viruses replicated efficiently in high AZT concentrations and were about 200-fold less sensitive to AZT than HIV-1. In contrast, HIV-2 and HIV-1 were equally susceptible to ddI, a finding consistent with the selection of HIV-2 mutants with AZT + ddI. Our results demonstrate that the activity of AZT on HIV-2 is lower than previously thought, and emphasize the need for novel antiretroviral drugs specific for HIV-2.

Amino Acid Sequence↗

Protection against feline immunodeficiency virus using replication defective proviral DNA vaccines with feline interleukin-12 and -18.

A molecular clone of the Glasgow-8 isolate of FIV (FIVGL8) was rendered replication defective by an in-frame deletion in either reverse transcriptase (deltaRT) or integrase (deltaIN) genes for use as DNA vaccines. To test the ability of these multi-gene vaccines to protect against two feline immunodeficiency virus (FIV) isolates of differing virulence, cats were immunized using either DNA vaccine alone or co-administered with interleukin-12 (IL-12) and/or interleukin-18 (IL-18) cytokine DNA. Animals were challenged sequentially with FIV-Petaluma (FIVPET) an FIV isolate of relatively low virulence and subsequently with the more virulent FIVGL8. A proportion of vaccinates (5/18 deltaIN and 2/12 deltaRT) were protected against primary challenge with FIV(PET). Five of the vaccinated-protected cats were re-challenged with FIV(PET); four (all deltaIN) remained free of viraemia whilst all naive controls became viraemic. Following subsequent challenge with the more virulent FIVGL8 these four vaccinated-protected animals all became viraemic but showed lower proviral loads than naive cats. This study suggests that while our current DNA vaccines may not produce sterilizing immunity against more virulent isolates of FIV, they may nevertheless significantly reduce the impact of infection.

Amino Acid Sequence↗

Molecular and phylogenetic analysis of SRS 19-6 murine leukemia virus.

The complete nucleotide sequence of the genome of Solid-type Reticulum cell Sarcoma 19-6 murine leukemia virus (SRS 19-6 MuLV) was determined. This virus was isolated in mainland China from laboratory mice that had been separated from western mice since the 1930s. The genome is 8,256 nucleotides in length and exhibits a genetic organization characteristic of replication competent MuLVs. Phylogenies constructed from reverse transcriptase (RT) domains showed that SRS 19-6 MuLV is closely related to other MuLV-related retroviruses; however, it has clearly diverged from previously isolated MuLVs. Comparative sequence analysis of the env sequences indicated that SRS 19-6 MuLV encodes a surface (SU) glycoprotein that is related to other ecotropic MuLVs in the VR-A and VR-B variable regions. However, SRS 19-6 MuLV env glycoprotein was distinct from all other MuLVs (ecotropic and non-ecotropic) in the proline-rich hypervariable region. No evidence for recombination with endogenous MuLV env sequences in generation of SRS 19-6 MuLV was observed. Comparisons of long terminal repeat (LTR) sequences revealed that the GV 1.4 molecular clone of Graffi MuLV contained 96% sequence identity to SRS 19-6 MuLV's LTR with 99% identity when comparisons were restricted to the U3 regions of the two viruses. The consensus enhancer binding motifs contained in the U3 regions of the two viruses were nearly identical. Nevertheless the two viruses have previously been shown to induce distinct patterns of disease. Comparisons between 196 and Graffi GV1.4 MuLVs may provide insights into the mechanisms of disease specificity induced by MuLVs.

Amino Acid Sequence↗

Retroelements, transposons and methylation status in the genome of oil palm (Elaeis guineensis) and the relationship to somaclonal variation.

We isolated and characterized different classes of transposable DNA elements in oil palm (Elaeis guineensis) plants grown from seed, and plants regenerated from tissue culture that show mantling, an abnormality leading to flower abortion. Using PCR assays, reverse transcriptase fragments belonging to LINE-like and gypsy-like retroelements and transposase fragments of En/Spm transposons were cloned. Sequence analysis revealed the presence of a major family of LINEs in oil palm, with other diverged copies. Gypsy-like retrotransposons form a single homologous group, whereas En/Spm transposons are present in several diverged families. Southern analysis revealed their presence in low (LINEs) to medium (gypsy and En/Spm) copy numbers in oil palm, and in situ hybridization showed a limited number of distinct loci for each class of transposable element. No differences in the genomic organization of the different classes of transposable DNA elements between ortet palm (parent) and regenerated palm trees with mantled phenotype were detected, but different levels of sequence methylation were observed. During tissue culture, McrBC digestion revealed the genome-wide reduction in DNA methylation, which was restored to near-normal levels in regenerated trees. HPLC analysis showed that methylation levels were slightly lower in the regenerated trees compared to the ortet parent. The genomic organization of the transposable DNA elements in different oil palm species, accessions and individual regenerated trees was investigated revealing only minor differences. The results suggest that the mantled phenotype is not caused by major rearrangements of transposable elements but may relate to changes in the methylation pattern of other genomic components.

Amino Acid Sequence↗

Many human L1 elements are capable of retrotransposition.

Using a selective screening strategy to enrich for active L1 elements, we isolated 13 full-length elements from a human genomic library. We tested these and two previously-isolated L1s (L1.3 and L1.4) for reverse transcriptase (RT) activity and the ability to retrotranspose in HeLa cells. Of the 13 newly-isolated L1s, eight had RT activity and three were able to retrotranspose. L1.3 and L1.4 possessed RT activity and retrotransposed at remarkably high frequencies. These studies bring the number of characterized active human L1 elements to seven. Based on these and other data, we estimate that 30-60 active L1 elements reside in the average diploid genome.

Animals↗

Reverse transcriptase activity innate to DNA polymerase I and DNA topoisomerase I proteins of Streptomyces telomere complex.

Replication of Streptomyces linear chromosomes and plasmids proceeds bidirectionally from a central origin, leaving recessed 5' termini that are extended by a telomere binding complex. This complex contains both a telomere-protecting terminal protein (Tpg) and a telomere-associated protein that interacts with Tpg and the DNA ends of linear Streptomyces replicons. By using histidine-tagged telomere-associated protein (Tap) as a scaffold, we identified DNA polymerase (PolA) and topoisomerase I (TopA) proteins as other components of the Streptomyces telomere complex. Biochemical characterization of these proteins indicated that both PolA and TopA exhibit highly efficient reverse transcriptase (RT) activity in addition to their predicted functions. Although RT activity innate to other DNA-dependent DNA polymerases has been observed previously, its occurrence in a topoisomerase is unprecedented. Deletion mapping and sequence analysis showed that the RT activity of Streptomcyces TopA resides in a peptide region containing motifs that are absent from most bacterial topoisomerases but are highly conserved in a novel subfamily of eubacterial topoisomerases found largely in Actinobacteria. Within one of these motifs, and essential to the RT function of Streptomyces TopA, is an Asp-Asp doublet sequence required also for the RT activities of human immunodeficiency virus and eukaryotic cell telomerases.

Amino Acid Sequence↗

Retroviral-like element in a marine invertebrate.

Retroviral-like elements (RL elements) include retroviruses and long terminal repeat (LTR)-containing retrotransposons. We report the presence of sea urchin RL elements (termed SURL) in eight species of sea urchins and find that these RL elements belong to several subfamilies. The complete DNA sequence of one SURL element in Tripneustes gratilla is 5266 base pairs long, including 254-nucleotide-long identical long terminal repeats (LTRs). It contains a single open reading frame nearly 4 kilobases long including the gag and pol genes. Comparison of conserved DNA sequences of RL elements from different sea urchin species indicates that active elements have been inserting copies into echinoid genomes for at least 200 million years.

Animals↗

In vitro bypass replication of the cisplatin-d(GpG) lesion by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase is highly mutagenic.

Eukaryotic DNA polymerase beta and the reverse transcriptases are the most inaccurate of the known DNA polymerases. We report here mutagenic replication in vitro past intrastrand N(7)G-N(7)G chelates of the cis-diamminedichloroplatinum(II), the major DNA adduct of the antitumor agent cisplatin by calf thymus DNA polymerase beta and human immunodeficiency virus type I reverse transcriptase (42% and 26% mutations, respectively). The most frequent modifications generated by both enzymes were one-base frameshift deletions. Only one mutational hot spot opposite the platinated guanines was observed with human immunodeficiency virus type I reverse transcriptase, while two hot spots were generated by DNA polymerase beta, one at the base situated 5' to the lesion and the other situated 4-6 nucleotides 5' to the adduct. An unusual mutagenic event, tandem replication of a 12-base pair sequence, was observed with DNA polymerase beta. The mutational spectra of the two DNA polymerases suggest that template slippage occurred with higher frequency in the presence of the more distributive DNA polymerase beta.

Animals↗

Identification of a simian immunodeficiency virus reverse transcriptase variant with enhanced replicational fidelity in the late stage of viral infection.

Genomic hypermutation of human and simian immunodeficiency viruses (HIV and SIV) enables these viruses to adapt and escape from various types of anti-viral selection by altering the molecular properties of viral gene products. In this study, we examined whether the biochemical and catalytic properties of SIV DNA polymerases (reverse transcriptases; RT) can change during the course of viral infection. For this test, we analyzed RTs obtained from two SIV clones, SIVMNE CL8 and SIVMNE 170. SIVMNE 170 was isolated during the late symptomatic phase of infection with the parental strain, SIVMNE CL8. We found these two RTs have identical DNA polymerase specific activities and kinetics with three different DNA and RNA templates. In addition, the processivity of these two SIV RT proteins were also similar. However, as demonstrated by a misincorporation assay, the SIVMNE 170 RT showed much higher fidelity than SIVMNE CL8. The fidelity difference between these two SIV RTs was also confirmed by a steady state kinetic fidelity assay. These findings suggest that the fidelity of lentiviral RTs may change during the course of viral infection, possibly in response to alterations of host anti-viral immune capability. In addition, our sequence analysis of these two RT genes proposes possible structural strategies that the virus may employ to alter RT fidelity.

Amino Acid Sequence↗

ProMSED: protein multiple sequence editor for Windows 3.11/95.

MOTIVATION: Most protein sequence alignment algorithms give similar results on closely related proteins, while manual intervention may be needed for distantly related molecules. To correct the alignment, it is often necessary to repeat calculations on selected parts of the alignments and edit the alignment manually. Software implementing such interactive alignment procedures is of significance. RESULTS: This paper presents a new MS Windows application called ProMSED for both automatic and manual protein sequence alignment. The program reads main sequence formats and has a user-friendly interface. ProMSED performs automatic (ClustalV algorithm) alignments, alignment visualization and editing, and it allows sequences to be aligned interactively leaving previously aligned regions unchanged. Manual alignment and sequence analysis are facilitated by colouring schemes reflecting amino acid similarity of mutational and physicochemical properties. The interactive alignment of a diverged set of reverse transcriptases has located four out of six known conserved motifs. AVAILABILITY: ProMSED is available on request from the authors. DEMO is available from ftp://ftp.ebi.ac.uk/pub/ software/dos/promsed/ or ftp://iubio.bio.indiana.edu/molbio/ ibmpc/.

Algorithms↗

Potential for insect transmission of HIV: experimental exposure of Cimex hemipterus and Toxorhynchites amboinensis to human immunodeficiency virus.

Human immunodeficiency virus (HIV) was detected in bedbugs (Cimex hemipterus) up to 8 d after oral exposure to highly concentrated virus in blood meals, but no virus replication was observed. HIV did not replicate in either intraabdominally inoculated bedbugs or intrathoracically inoculated mosquitoes (Toxorhynchites amboinensis). The virus was not detected in bedbug feces. Mechanical transmission of HIV by bedbugs could not be demonstrated in an in vitro model. The persistence of HIV in an insect or on its mouthparts is one of many factors necessary for mechanical transmission in nature. The risk of insect transmission of HIV appears to be extremely low or nonexistent.

Animals↗