tRNA Lys Research Articles

Probing the higher order structure of RNA with peroxonitrous acid

Potassium peroxinitrite (ONOOK) and [Fe(EDTA)] 2− were used to analyze the influence of chemically entirely different hydroxyl radical sources on tRNA cleavage profiles. [Fe(EDTA)] 2− gives rise to hydroxyl radicals via a Fenton-like reaction during the oxidation of chelated Fe 2+, while ONOOK generates hydroxyl radicals via its conjugate acid (ONOOK) when adding a stable alkaline solution of ONOOK in samples buffered at neutral pH. [Fe(EDTA)] 2− is known to induce oxidative strand scission at sugar moieties thought to be solvent accessible, while those residues located in the ‘inside’ of structured RNAs are protected. Although ONOOH is neutral and significantly smaller than the metal complex, both reagents generate the same protection pattern on tRNAs, suggesting that access of the commonly formed hydroxyl radical, rather than access of its source, is the determining factor when probing the higher order structure of RNA. Strong difference in reactivity is only seen at the modified 2-thiouridine S34 of tRNA Lys3 which shows hyperreactivity towards ONOOK treatment. This particular reaction may require interaction between the peroxonitrite anion and the thiocarbonyl group of the base, since hyperreactivity is not observed when probing the dethiolated tRNA Lys3.

Clinical variability associated with the mutation at nucleotide position 8344 of the mitochondrial DNA.

Myoclonic epilepsy with ragged-red fibres (MERRF) (McKusick 545000) is a maternally inherited encephalomyopathy characterized by action myoclonus, myoclonic epilepsy, ataxia, and myopathy with ragged-red fibres (RRF). Less common signs include dementia, seizures, neuropathy, hearing loss, optic atrophy and spasticity (Silvestri et al 1993). An A-to-G transition at nucleotide position 8344 in the tRNA Lys gene of the mtDNA (McKusick 590060) is a primary genetic defect causing MERRF (Shoffner et al 1990). However, both the relationship between the proportion of mutant mtDNA and clinical severity, and the extent of clinical heterogeneity in patients with the MERRF mutation are still controversial. In the present work, we looked for the MERRF mutation in a group of patients and assessed the correlations between clinical and molecular data.

Construction of a type 1 human immunodeficiency virus that maintains a primer binding site complementary to tRNA(His)

The initiation of human immunodeficiency virus type 1 reverse transcription occurs by extension of a tRNA(Lys3) primer bound near the 5' end of the viral RNA genome which is designated the primer binding site (PBS). Sequences within the viral genome upstream of the PBS which are complementary to the anticodon loop (USUU) and the T psi C loop and arm (AGGGTm psi) of tRNA(Lys3) are postulated to play a role in maintaining the selective use of tRNA(Lys3) in reverse transcription. To investigate this possibility, proviral genomes which contain a PBS complementary to the 3'-terminal 18 nucleotides of tRNA(His) [pHXB2(His)] as well as sequences upstream of this PBS which are complementary to either the anticodon loop [CCACAA; pHXB2(His-AC)] or T psi C loop [GACCGAGG; pHXB2(His-T psi C)] of tRNA(His) were constructed. Infectious virus was recovered upon transfection into COS-1 cells of pHXB2(His), pHXB2(His-AC), or pHXB2(His-T psi C). The appearance of infectious virus after cocultivation with SupT1 cells was delayed for the proviruses containing a PBS complementary to tRNA(His) compared with that obtained by transfection of the wild-type provirus [pHXB2(WT)]. However, by several passages in SupT1 cells, the mutant viruses demonstrated replication kinetics similar to those of the wild-type virus. A DNA sequence analysis of the PBS region from integrated proviruses revealed that by day 15 of culture, the PBS of viruses derived from pHXB2(His) and pHXB2(His-T psi C) reverted back to the wild-type PBS complementary to tRNA(Lys3). In contrast, viruses derived from pHXB2(His-AC) maintained a PBS complementary to tRNA(His) for over 4 months in culture encompassing 12 serial passages. This study, then, is the first report of a stable human immunodeficiency virus type 1 which utilizes an alternative tRNA primer and suggests that interactions between the primer tRNA anticodon loop and viral sequences upstream of the PBS contribute to the specificity of the tRNA primer used in reverse transcription.

Structural variation among retroviral primer-DNA junctions: solution structure of the HIV-1 (-)-strand Okazaki fragment r(gcca)d(CTGC).d(GCAGTGGC).

The three-dimensional solution structure of the hybrid-chimeric duplex r(gcca)d(CTGC).d(GCAGTGGC) has been determined by two-dimensional NMR, restrained molecular dynamics (rMD), and NOE back-calculation methods. This chimera, consisting of a chimeric RNA-DNA strand and its complementary DNA strand, is formed after priming (-)-strand DNA synthesis by tRNA(Lys3) and subsequent (+)-strand DNA synthesis by reverse transcriptase and is an obligatory intermediate in the formation of double-stranded DNA prior to HIV-1 retrovirus integration. The duplex consists of two different types of double helix: a hybrid form (H-form) and a B-form structure connected by a junction. It is chemically similar to several other Okazaki fragments whose structures have been previously determined in our laboratory. However, some structural parameters are not the same and were found to be sequence dependent. In particular, the sugar conformations at the DNA base pair proximal to the hybrid segment vary from O4'-endo to C2'-endo depending on the base composition. The position of the transition from the relatively wide groove of H-form to the narrow groove of B-form is also sequence dependent, occurring either exactly at the RNA-DNA junction or within the purely DNA segment of the chimera-as is the case in the structure of the present HIV-1 (-)-strand primer. This structural change produces a kink at the DNA-DNA step adjacent to the RNA-DNA junction in the HIV-1 (-)-strand primer. The sequence dependence of structures of RNA-DNA chimeric duplexes may be responsible for the variable cleavage pattern of different Okazaki fragments by reverse transcriptase RNase H.

MERRF syndrome with overwhelming lactic acidosis

Myoclonic epilepsy with ragged-red fiber syndrome has been associated with a mitochondrial DNA base substitution at nucleotide 8344 in the mitochondrial tRNA Lys gene. In several reported series, adult patients with these mutations have mild to moderate symptoms that progress slowly over many years. We describe a girl with MERRF syndrome who had an unusually rapid and severe clinical course, with onset of symptoms at age 7 years and death by age 14 years of overwhelming lactic acidosis. Postmortem tissue biopsy revealed variable but generally high percentages of mutant mitochondrial genomes in multiple organ systems. Twenty-one other members of her family were tested for the mutation and had varying percentages in leukocytes.

Essential Amino Acids Regulate Fatty Acid Synthase Expression through an Uncharged Transfer RNA-dependent Mechanism

To better understand the regulation of gene expression by amino acids, we studied the effects of these macronutrients on fatty acid synthase (FAS), an enzyme crucial for energy storage. When HepG2 cells were fed serum-free media selectively deficient in each amino acid, the omission of any single classic essential amino acid as well as Arg or His (essential in some rapidly growing cells) resulted in FAS mRNA levels that were about half of those in complete medium. Control message levels were unaffected and omission of nonessential amino acids did not alter FAS expression. FAS mRNA levels peaked 12-16 h after feeding complete and Ser (nonessential)-deficient media but did not increase in cells fed Lys (essential)-deficient medium. With Lys, FAS mRNA increased over the physiologic concentration range of 15-150 microM, and low concentrations of lysine decreased FAS but not apoB protein mass. Transcription inhibitors mimicked treatment with Lys-deficient media, and nuclear run-off assays showed that Lys-deficient media abolished FAS but not apoB transcription. After treatment with Lys-deficient media, the intracellular Lys pool was rapidly depleted in association with an increase of uncharged (deacylated) tRNA Lys from < 1 to 64% of available tRNA Lys. Even in the presence of the essential amino acid His, increasing the level of uncharged tRNA His with histidinol, a competitive inhibitor of the histidinyl-tRNA synthetase, blocked FAS expression. Tyrosinol treatment did not alter FAS mRNA levels. These results suggest that essential amino acids regulate FAS expression by altering uncharged tRNA levels, a novel mechanism for nutrient control of gene expression in mammalian cells.

Structural Requirements for the Binding of tRNA3Lys to Reverse Transcriptase of the Human Immunodeficiency Virus Type 1

Reverse transcription of the human immunodeficiency virus type 1 (HIV-1) RNA genome is primed by the cellular tRNA Lys3 molecule. Packaging of this tRNA primer during virion assembly is thought to be mediated by specific interactions with the reverse transcriptase (RT) protein. Portions of the tRNA molecule that are required for interaction with the RT protein remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified RT to mutant forms of tRNA Lys3. The anticodon loop could be mutated without eliminating RT recognition. However, mutations in the T psi C stem were found to partially interfere with RT binding, and D arm mutants were completely inactive in RT binding. Interestingly, binding of the RT protein to tRNA Lys3 facilitates the subsequent annealing of template strand to the 3'-terminus of the tRNA molecule. Consistent with this finding, we demonstrate that mutant HIV-1 virions lacking the RT protein do contain a viral RNA genome without an associated tRNA Lys3 primer. We also found that a preformed primer tRNA-template complex is efficiently recognized by RT protein in vitro. Extension of the template molecule over the T psi C loop did result in complete inhibition of RT binding, suggesting the presence of additional recognition elements in the T psi C loop. These results, combined with a comparative sequence analysis of tRNA species present in HIV-1 virions and RNA motifs selected in vitro for high affinity RT binding, suggest that RT recognizes the central domain of the tRNA tertiary structure, which is formed by interaction of the D and T psi C loops.

Analysis of Primer Extension and the First Template Switch during Human Immunodeficiency Virus Reverse Transcription.

Reverse transcription by human immunodeficiency virus (HIV) reverse transcriptase (RT) entails several distinct, early steps in the synthesis of double-stranded DNA from viral RNA templates. These steps include tRNA(lys3) priming of RNA-dependent DNA polymerization and template switching for synthesis of near full-length (-) DNA. However, HIV RT lacks the fluent processivity of other viral reverse transcriptases or cellular polymerases. Previous studies in our laboratory showed that RT reactions primed with tRNA(lys3) had higher efficiencies of template switching than those primed by synthetic oligonucleotides. To further study primer extension, pausing, and template switching, we utilized an in vitro reconstituted reverse transcription/template switching reaction consisting of HIV RNA templates, recombinant HIV RT and primer tRNA(lys3). We observed initial pause sites within the first five nucleotides of the primer terminus; these were extended efficiently to full-length (-) strong-stop DNA with prolonged incubation. Other pause sites occurred in the R/U5/PBS template adjacent to homopolymeric sequences and regions thought to be involved in secondary structure. This assay provides a sensitive means of assessing template switching at times between 10 s and 3 h. Copyright 1995 S. Karger AG, Basel

Complementation and segregation behavior of disease-causing mitochondrial DNA mutations in cellular model systems

The recent development of cellular models of mitochondrial DNA-linked diseases by transfer of patient-derived mitochondria into human mtDNA-less (ϱ°) cells has provided a valualble tool for investigating the complementation and segregation of mtDNA mutations. In transformants carrying in heteroplasmic form the mitochondrial tRNA Lys gene 8344 mutation of tRNA Leu(UUR) gene 3243 mutation associated, respectively, with the MERRF or the MELAS encephalomyopathy, full protection of the cells against the protein synthesis and respiration defects caused by the mutations was observed when the wild-type mtDNA exceeded 10% of the total complement. In the MERRF transformants, the protective effect of wild-type mtDNA was shown to involve interactions of the mutant and wild-type gene products, probably coexisting within the same organelle from the time of the mutation event. In striking contrast, in experiments in which two mtDNAs carrying either the MERRF or the MELAS mutation were sequentially introduced within distinct organelles into the same ϱ° cells, no evidence of cooperation between their products was observed. These results pointed to the phenotypic independence of the two genomes. A similar conclusion was reached in experiments in which a chloramphenicol (CAP) resistance-conferring mtDNA mutation was introduced into CAP-sensitive cells. In the area of segregation of mtDNA mutations, in unstable heteroplasmic MELAS transformants, observations were made which pointed to a replicative advantage of mutant molecules, leading to a rapid shift of the genome towards the mutant type. These results are consistent with a model in which the mitochondrion, rather than the mtDNA molecule, is the segregating unit.

Interaction of primer tRNA Lys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

In the interaction between HIV-1 RT and tRNA Lys3 each subunit of the heterodimer interacts with tRNA showing a different affinity: K d (p66) = 23 nM, K d (p51) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the K d value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, lacking one, two or three nucleotides from the 3′-end, or ribo- and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 RT than natural tRNA. A possible mechanism of RT p66/p51 activation by tRNA and its analogs, mediated through the p51 subunit, is discussed.

HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA.

Efficient extension of a misaligned tRNA-primer during replication of the HIV-1 retrovirus.

The human immunodeficiency virus (HIV) and other retroviruses show extensive genomic variation, which is primarily due to error-prone replication by the viral reverse transcriptase (RT) enzymes. RT errors include misincorporation with subsequent extension of the mismatched terminal base, and extension of realigned primer-template duplexes. Whereas both RT-mediated mechanisms have been extensively studied in vitro, almost no in vivo experiments have been performed. In this work, we analyzed the ability of HIV-1 RT to extend a misaligned tRNA(Lys3) primer in vivo. This tRNA binds with its 3'-terminal 18 nt to a complementary sequence in the viral genome, referred to as the primer-binding site (PBS). We constructed a series of mutant viral genomes with small insertions or deletions in the PBS sequence, resulting in misalignment of the tRNA primer. Extension of the misaligned primer did occur with reasonable efficiency for some of the mutants, resulting in reversion to the wild-type viral sequence. The infectivity and reversion frequency of the PBS mutants is therefore a measure of the efficiency of extending a misaligned primer in vivo. Using virion-derived primer-template complexes, we also measured the tRNA-priming efficiency in vitro. The combined results show that HIV-1 RT can elongate a misaligned primer and that the efficiency of primer extension is determined by the extent of the mismatch.

Incorporation of excess wild-type and mutant tRNA(3Lys) into human immunodeficiency virus type 1.

Human immunodeficiency virus (HIV) particles produced in COS-7 cells transfected with HIV type 1 (HIV-1) proviral DNA contain 8 molecules of tRNA(3Lys) per 2 molecules of genomic RNA and 12 molecules of tRNA1,2Lys per 2 molecules of genomic RNA. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a human tRNA3Lys gene, there is a large increase in the amount of cytoplasmic tRNA3Lys per microgram of total cellular RNA, and the tRNA3Lys content in the virus increases from 8 to 17 molecules per 2 molecules of genomic RNA. However, the total number of tRNALys molecules per 2 molecules of genomic RNA remains constant at 20; i.e., the viral tRNA1,2Lys content decreases from 12 to 3 molecules per 2 molecules of genomic RNA. All detectable tRNA3Lys is aminoacylated in the cytoplasm of infected cells and deacylated in the virus. When COS-7 cells are transfected with a plasmid containing both HIV-1 proviral DNA and a mutant amber suppressor tRNA3Lys gene (in which the anticodon is changed from TTT to CTA), there is also a large increase in the relative concentration of cytoplasmic tRNA3Lys, and the tRNA3Lys content in the virus increases from 8 to 15 molecules per 2 molecules of genomic RNA, with a decrease in viral tRNA1,2Lys from 12 to 5 molecules per 2 molecules of genomic RNA. Thus, the total number of molecules of tRNALys in the virion remains at 20. The alteration of the anticodon has little effect on the viral packaging of this mutant tRNA in spite of the fact that it no longer contains the modified base mcm 5s2U at position 34, and its ability to be aminoacylated is significantly impaired compared with that of wild-type tRNA3Lys. Viral particles which have incorporated either excess wild-type tRNA3Lys or mutant suppressor tRNA3Lys show no differences in viral infectivity compared with wild-type HIV-1.

Generality of the tRNA Origin of Short Interspersed Repetitive Elements (SINEs): Characterization of Three Different tRNA-derived Retroposons in the Octopus

Transcription in vitro of total genomic DNA of Octopus vulgaris resulted in PolIII transcripts of 200 to 500 nucleotides in length. These transcripts were used as probes and, as a result, three different kinds of short interspersed element (SINE) were isolated and characterized. Two SINEs, designated the octopus OR1 and OR2 families, seem to have been derived from tRNA Arg. The other SINE, designated the octopus OK family, have originated from tRNA, but the parental tRNA species cannot be identified due to sequence divergence from the original tRNA sequence. The OR1 and OR2 families exhibit considerable similarity to one another, in the 5′ region of the tRNA-unrelated region as well as in the tRNA Arg-unrelated region, an observation that suggests that these two families may have had the same origin in evolution. The three SINEs together constitute at least 6% of the genome of O. vulgaris . Results of a dot hybridization experiment suggest that the OR1 and OR2 families are present in fewer species than the OK family and that the OK family exists in many species of octopodid. The present observations indicate that SINEs have generally been derived from tRNAs in invertebrates, as well as in vertebrates, and that retroposition is widely involved in the genetic and structural variability of invertebrate genomes. It has been demonstrated that tRNA Arg and tRNA Lys appear to be tRNA species found as progenitors of vertebrate SINEs, and the same preference for progenitor species of tRNAs seems also to be a feature of invertebrate SINEs.

Mutating the “primer grip” of p66 HIV-1 reverse transcriptase implicates tryptophan-229 in template-primer utilization

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides were reconstituted in vitro with wild type p51 to generate the "selectively mutated" heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp229 toward architecture of the p66 primer grip.

Detection of novel transcripts in the human mitochondrial DNA region coding for ATPase8—ATPase6 subunits

We have analyzed the tRNA Lys, ATPase8, ATPase6, COIII region of mitochondrial DNA in several human tissues. Beside the mature tRNA Lys, ATPase8 and ATPase6 common mRNA, and COIII mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNA Lys plus the ATPase8 and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.

Comparison of deoxyoligonucleotide and tRNA(Lys-3) as primers in an endogenous human immunodeficiency virus-1 in vitro reverse transcription/template-switching reaction.

We developed an endogenous in vitro reverse transcription assay to study the properties of priming and template switching during human immunodeficiency virus (HIV) replication. Reactions were primed with HIV reverse transcriptase (RT) and either a deoxyoligonucleotide primer (dPR) or tRNA(Lys-3), the natural primer for reverse transcription. The RNA templates utilized were the actual HIV sequences involved in the first template switch, namely a primer binding sequence (PBS)/U5/R RNA donor template and a R/U3 RNA acceptor template. Reverse transcription reactions using the latter templates and dPR or tRNA(Lys-3) as primers yielded four major products: (-)-strong-stop DNA, a partial template-switched DNA, full template-switched DNA, and a pseudo-PBS-primed product. Use of dPR resulted in three times less template switching than was obtained with tRNA(Lys-3). When reactions were primed with either dPR or tRNA(Lys-3), increases in acceptor:donor template ratios resulted in augmented template switching. Increasing the concentration of RT resulted in increased priming from the PBS but had no effect on the efficiency of template switching. Decreasing the extent of R region overlap resulted in a drop in efficiency of template switching. Decreases in the R region on the donor template also caused a drop in initiation of transcription that was primed by tRNA(Lys-3) from the PBS. In contrast, a corresponding reduction of the R region on the acceptor template had no effect on priming. We conclude that a transcriptional complex of tRNA(Lys-3) and RT may be associated not only with the PBS but also with other cis RNA sequences and secondary structures in a manner essential for efficient priming and template switching.

Preferential incorporation of nucleoside analogs after template switching during human immunodeficiency virus reverse transcription

We assessed the effects of 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC) on reverse transcription in CD4-positive cells by isolating truncated human immunodeficiency virus (HIV) DNA fragments. Jurkat cells were treated with AZT (2 microM), ddI (200 microM), or 3TC (50 microM) prior to infection with HIV. Low-molecular-weight DNA was isolated and amplified by PCR with primer pairs which identify different segments of HIV proviral DNA. We found that the HIV DNA fragments generated from drug-treated, HIV-exposed Jurkat cells were truncated at a ratio of 15:1 [i.e., (-) strong-stop DNA to HIV DNA generated after the first template switch]. Full-length DNA was observed in the case of untreated, HIV-infected cultures. Following nucleoside analog treatment of HIV-exposed Jurkat cells, reverse transcription was terminated only after the synthesis of (-) strong-stop DNA. The nucleoside analogs tested, i.e., AZT, ddI, and 3TC, preferentially chain terminated viral DNA synthesis immediately following the first template switch. The (-) strong-stop HIV DNA was present in AZT-treated and untreated cultures for at least 6 days. We also carried out cell-free reverse transcription/template-switching reactions involving tRNA(Lys3) or a deoxyoligonucleotide as a primer, as a means of studying the selective incorporation of AZT triphosphate into proviral DNA. When reactions were primed with tRNA(Lys3), we found that AZT triphosphate was preferentially incorporated after template switching.

A Rapid and Sensitive PCR Screening Method for Point Mutations Associated with Mitochondrial Encephalomyopathies

Alterations of the mitochondrial DNA, encoding important parts of the cellular energy-generating system (oxidative phosphorylation, OXPHOS), are often associated with the occurence of degenerative neuromuscular diseases. Especially point mutations in the mitochondrial tRNA genes, which cannot be complemented by the nuclear encoded tRNAs, are candidates for severe defects of the OXPHOS system. An A to G transition at nt 8344 in the tRNA Lys gene has been associated with MERRF disease whereas an A to G substitution at nt 3243 in the tRNA Leu gene has been linked to the MELAS syndrome. These two mtDNA alterations as well as point mutations in protein-coding genes can be detected simultaneously by an allele-specific amplification of the altered mtDNA. This assay allows the reliable detection of heteroplasmic point-mutations, even if the mutated DNA appears to a small extent of less than 1%.

Human immunodeficiency virus uses tRNA Lys,3 as primer for reverse transcription in HeLa-CD4 + cells

Significant amounts of different tRNA molecules are present in retroviral particles, but one specific tRNA species functions as primer in reverse transcription. It is generally believed that the HIV-1 virus uses the tRNA Lys,3 molecule as primer. This is based on sequence complementarity between the 3' end of tRNA Lys,3 and the primer-binding site (PBS) on HIV-1 genomic RNA. Recent biochemical analyses indicated that tRNA Lys,3 is indeed incorporated into viral particles. Interestingly, tRNA Lys,3 could not be detected in virions produced by HeLa-CD4 + cells [(1992) Biochem. Biophys. Res. Commun. 185, 1105-1115]. In order to test whether alternative tRNA molecules can function as primer in HIV replication, we performed a series of experiments based on the observation that tRNA primer sequences are inherited by the viral progeny. We cultured HIV-1 for prolonged periods of time in HeLa-CD4 + cells, but did not detect sequence changes in the PBS region. Furthermore, we found PBS-mutants to be replication incompetent, again suggesting that HIV-1 solely uses tRNA Lys,3 as primer. Most importantly, we obtained revertants of one such PBS-mutant, which had restored a wild-type PBS sequence. This tRNA Lys,3-mediated repair demonstrates a general requirement for this primer in HIV-1 reverse transcription.