Additional Nucleotide Substitutions Research Articles

Watanabea acidotolerans: A new trebouxiophyte lineage (Chlorophyta) inhabiting low pH environments from Europe to South America

SUMMARYHere we studied a new Watanabea strain CAUP H 8901‐CRYO isolated from an extremely acidic (pH 2.6) pit lake in Czechia. We used morphological, ultrastructural and molecular data to ascertain its taxonomic position and phylogenetic relationships within the genus Watanabea. The morphology of the studied strain matched the formal Watanabea description, including its most characteristic feature, the production of two types of autospores. The new isolate morphologically and ultrastructurally resembled the Watanabea type species, W. reniformis. However, it differed from W. borysthenica by having a pyrenoid not surrounded by starch grains. Molecular phylogenetic analyses of nuclear 18S rDNA and plastid rbcL showed that the strain CAUP H 8901‐CRYO formed an independent lineage within the genus Watanabea. Furthermore, analyses of nuclear ITS2 secondary structure demonstrated an additional side loop coiling from the helix III, the unique feature of CAUP H 8901‐CRYO, not present in the authentic strain of W. reniformis SAG 211‐9b. The ITS2 secondary structures of the two strains also differed by one compensatory base change (CBC) and a number of additional nucleotide substitutions. The new acidotolerant strain and another Watanabea isolate from a naturally acidic lake in Argentina were identical in their plastid rbcL sequence. Consequently, here we proposed a new species, Watanabea acidotolerans, to accommodate a lineage of microalgae with a possible worldwide distribution and tolerance to low pH habitats.

Genome sequencing accuracy by RCA-seq versus long PCR template cloning and sequencing in identification of human papillomavirus type 58

BackgroundGenome variations in human papillomaviruses (HPVs) are common and have been widely investigated in the past two decades. HPV genotyping depends on the finding of the viral genome variations in the L1 ORF. Other parts of the viral genome variations have also been implicated as a possible genetic factor in viral pathogenesis and/or oncogenicity.ResultsIn this study, the HPV58 genome in cervical lesions was completely sequenced both by rolling-circle amplification of total cell DNA and deep sequencing (RCA-seq) and by long PCR template cloning and sequencing. By comparison of three HPV58 genome sequences decoded from three clinical samples to reference HPV-58, we demonstrated that RCA-seq is much more accurate than long-PCR template cloning and sequencing in decoding HPV58 genome. Three HPV58 genomes decoded by RCA-seq displayed a total of 52 nucleotide substitutions from reference HPV58, which could be verified by long PCR template cloning and sequencing. However, the long PCR template cloning and sequencing led to additional nucleotide substitutions, insertions, and deletions from an authentic HPV58 genome in a clinical sample, which vary from one cloned sequence to another. Because the inherited error-prone nature of Tgo DNA polymerase used in preparation of the long PCR templates of HPV58 genome from the clinical samples, the measurable error rate in incorporation of nucleotide into an elongating DNA template was about 0.149% ±0.038% in our studies.ConclusionsSince PCR template cloning and sequencing is widely used in identification of single nucleotide polymorphism (SNP), our data indicate that a serious caution should be taken in finding of true SNPs in various genetic studies.

A deep vein thrombosis caused by 20209C>T mutation in homozygosis of the prothrombin gene in a Caucasian patient

Introduction:Additional nucleotide substitutions in the 3′-untranslated region of prothrombin gene could explain some thrombotic events and also adverse pregnancy outcomes. We describe the first case of a homozygous 20209C>T mutation as the cause of deep vein thrombosis in a Spanish patient.Case and methods:The 56-year-old male patient with a partial tear of the Achilles tendon developed calf (tibial) deep vein thrombosis after immobilization and was treated with an anticoagulant. To determine if the deep vein thrombosis was of genetic origin, a peripheral blood DNA sample was analysed for the presence of the three most frequent mutations associated with thrombotic events: factor V Leiden (1691G>A), prothrombin (20210G>A) and methylene tetrahydrofolate reductase (677C>T). The presence or absence of the normal allele of prothrombin could not be determined using the PTH-FV-MTHFR StripAssay (Vienna Lab).Results:Comprehensive analysis showed that the patient had a variant interfering with the polymerase chain reaction product, we sequenced the entire prothrombin gene and found that the patient had a homozygous C>T mutation at position 20209; this interfered with the polymerase chain reaction product, which needs a C at this position to be able to bind to the wild-type probe present in the test strip.Conclusion:The homozygous 20209C>T mutation and the presence of the mutation 677C>T in heterozygosity explained the patient’s deep vein thrombosis because the combination of mutations would increase the risk of thrombosis. Suitable genetic counselling should be provided to the patient and first-degree relatives as it important to detect prothrombin gene variants that could increase risk for thrombotic events.

Positive selection signals of hepatitis B virus and their association with disease stages and viral genotypes

The hepatitis B virus (HBV) is a global health problem that causes different types of liver diseases. The high mutation rate of HBV, which results from a lack of proofreading activity of the viral polymerase, leads to the actively adaptive evolution of mutant strains under various selection pressures. This study focuses on the positive selection signals in the whole HBV genome and the association of these selection signals with the disease stages and/or viral genotypes. A total of 486 complete HBV genomes from HBV-infected individuals of different illness categories (i.e., acute, chronic, and severe hepatitis) were analyzed. To obtain a panoramic view of the selection signals, codon-based maximum likelihood analysis, three-dimensional (3D) mapping, and allele frequency comparison were conducted on genotypes B and C HBV from subjects with different stages of hepatitis. A total of 95 selected codons were resolved, and a significantly higher number of positive selection signatures were found in the chronic and severe hepatitis groups compared with the acute groups. Many of the selected codons were associated with either a unique disease stage or a specific genotype. The conservation analysis of the selection signals in the viral core protein (HBcAg) illustrated the occurrence of selected codons in the highly diversified regions. The allele-frequency-based analysis identified eight additional nucleotide substitutions, and the frequencies of these mutations were found to increase with disease progression. Moreover, we found that three substitutions, including A1762T, G1764A, and A2739C, were nearly fixed. The mapping of all of the selected codons and nucleotide substitutions to the functional domains of the viral proteins suggested that more than 60% of the mutations were subject to selection forces from host immune surveillance, antiviral therapy, and replication fitness.

Rapid genotyping of the human renin (REN) gene by the LightCycler® instrument: identification of unexpected nucleotide substitutions within the selected hybridization probe area.

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) in REN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

Rapid Genotyping of the Human Renin (REN) Gene by the LightCycler® Instrument: Identification of Unexpected Nucleotide Substitutions within the Selected Hybridization Probe Area

Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) inREN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.

Optimization of the doxycycline-dependent simian immunodeficiency virus through in vitro evolution

BackgroundVaccination of macaques with live attenuated simian immunodeficiency virus (SIV) provides significant protection against the wild-type virus. The use of a live attenuated human immunodeficiency virus (HIV) as AIDS vaccine in humans is however considered unsafe because of the risk that the attenuated virus may accumulate genetic changes during persistence and evolve to a pathogenic variant. We earlier presented a conditionally live HIV-1 variant that replicates exclusively in the presence of doxycycline (dox). Replication of this vaccine strain can be limited to the time that is needed to provide full protection through transient dox administration. Since the effectiveness and safety of such a conditionally live virus vaccine should be tested in macaques, we constructed a similar dox-dependent SIV variant. The Tat-TAR transcription control mechanism in this virus was inactivated through mutation and functionally replaced by the dox-inducible Tet-On regulatory system. This SIV-rtTA variant replicated in a dox-dependent manner in T cell lines, but not as efficiently as the parental SIVmac239 strain. Since macaque studies will likely require an efficiently replicating variant, we set out to optimize SIV-rtTA through in vitro viral evolution.ResultsUpon long-term culturing of SIV-rtTA, additional nucleotide substitutions were observed in TAR that affect the structure of this RNA element but that do not restore Tat binding. We demonstrate that the bulge and loop mutations that we had introduced in the TAR element of SIV-rtTA to inactivate the Tat-TAR mechanism, shifted the equilibrium between two alternative conformations of TAR. The additional TAR mutations observed in the evolved variants partially or completely restored this equilibrium, which suggests that the balance between the two TAR conformations is important for efficient viral replication. Moreover, SIV-rtTA acquired mutations in the U3 promoter region. We demonstrate that these TAR and U3 changes improve viral replication in T-cell lines and macaque peripheral blood mononuclear cells (PBMC) but do not affect dox-control.ConclusionThe dox-dependent SIV-rtTA variant was optimized by viral evolution, yielding variants that can be used to test the conditionally live virus vaccine approach and as a tool in SIV biology studies and vaccine research.

Emergence of a Novel Lamivudine-Resistant Hepatitis B Virus Variant with a Substitution Outside the YMDD Motif

Lamivudine is a major drug approved for treatment of chronic hepatitis B virus (HBV) infection. Emergence of drug-resistant mutants with amino acid substitutions in the YMDD motif is a well-documented problem during long-term lamivudine therapy. Here we report a novel lamivudine-resistant strain of HBV with an intact YMDD motif, which included an amino acid substitution, rtA181T, in the reverse transcriptase (RT) domain of HBV polymerase. The substitution also induced a unique amino acid substitution (W172L) in the overlapping hepatitis B surface (HBs) protein. The YMDD mutant strains were not detected even by using the sensitive peptide nucleic acid-mediated PCR clamping method. The detected nucleotide substitution was accompanied by the emergence of an additional nucleotide substitution that induced amino acid change (S331C) in the spacer domain. The rtA181T mutant strain displayed a threefold decrease in susceptibility to lamivudine in in vitro experiments in comparison with the wild type. In vivo analysis using human hepatocyte-chimeric mice confirmed the resistance of this mutant strain to lamivudine. We developed a method to detect this novel rtA181T mutation and a previously reported rtA181T mutation with the HBs stop codon using restriction fragment length polymorphism PCR and identified one patient with the latter pattern among 40 patients with lamivudine resistance. In conclusion, although the incidence is not high, we have to be careful regarding the emergence of lamivudine-resistant mutant strains with intact YMDD motif.

Prothrombin Gene Variants in Non-Caucasians with Fetal Loss and Intrauterine Growth Retardation

Thrombotic predisposition may affect pregnancy outcome, but in non-Caucasians the contributing genetic factors are poorly characterized. Two recently identified prothrombin gene mutations (20209C>T and 20221C>T) have been observed in non-Caucasian patients with thrombosis. The mutations are located near the commonly identified variant 20210G>A and have not been reported in Caucasian patients. The authors report a novel connection with pregnancy complications. The identification of sequence variants other than 20210G>A in the 3'-untranslated region of the prothrombin gene suggests that additional nucleotide substitutions may contribute to the development of thrombotic events and adverse pregnancy outcomes, especially in less well-characterized populations.

Nucleotide substitutions within U5 are critical for efficient reverse transcription of human immunodeficiency virus type 1 with a primer binding site complementary to tRNA(His).

Sequence analysis of integrated proviruses of human immunodeficiency virus type 1 (HIV-1) which utilize tRNA(His) to initiate reverse transcription [virus derived from pHXB2(His-AC-TGT)] revealed five additional nucleotide substitutions in the U5 and primer binding site (PBS) regions (ATGAC for CCTGT at nucleotides 152, 160, 174, 181, and 200, respectively) (Z. Zhang et al., Virology 226:306-317, 1996). We constructed a mutant proviral genome [pHXB2(His-AC-GAC)] which contained the ATGAC substitutions to test if they represented a necessary adaptation by the virus for use of tRNA(His) to initiate reverse transcription. Viruses from pHXB2(His-AC-TGT) and pHXB2(His-AC-GAC) were infectious. Sequence analysis of the U5 and PBS regions of integrated provirus from a cell culture infected with virus derived from pHXB2(His-AC-TGT) revealed a G-to-A change in CCTGT at nucleotide 181 after limited in vitro culture, suggesting that this nucleotide change represented an adaptation by the virus to efficiently utilize tRNA(His) to initiate reverse transcription. To further address this possibility, we used a specific mutation in reverse transcriptase (RT), a methionine-to-valine change in the highly conserved YMDD amino acid motif of HIV-1 RT (M184V), which has been shown in previous studies to influence the fidelity and activity of the enzyme. The M184V RT mutation was cloned into pHXB2(His-AC-GAC) and pHXB2(His-AC-TGT). Virus derived from pHXB2(His-AC-GAC) with M184V RT had slightly delayed replication compared to the virus from pHXB2(His-AC-GAC) with wild-type RT; in contrast, virus from pHXB2(His-AC-TGT) with M184V RT was severely compromised in replication. Using an endogenous reverse transcription-PCR assay to analyze the reverse transcription of viruses obtained after transfection, we found that viruses derived from pHXB2(His-AC-GAC) with the wildtype RT were slightly faster in the initiation of reverse transcription than viruses with M184V RT. The initiation of reverse transcription was delayed in viruses derived from pHXB2(His-AC-TGT) with wild-type RT and M184V RT compared to viruses derived from pHXB2(His-AC-GAC). Finally, sequence analysis of U5 and PBS regions of proviruses from pHXB2(His-AC-GAC) with wild-type RT revealed considerably more nucleotide substitutions than in viruses derived from pHXB2(His-AC-GAC) containing the M184V mutation in RT after extended in vitro culture. Our studies point to a role for these additional nucleotide substitutions in U5 as an adaptation by the virus to utilize an alternative tRNA to initiate reverse transcription.

Accumulation of Somatic Nucleotide Substitutions in Mitochondrial DNA Associated with the 3243 A-to-G tRNALeu(UUR)Mutation in Encephalomyopathy and Cardiomyopathy

To understand the pathogenesis of mitochondrial encephalomyopathy and cardiomyopathy, we analyzed the sequence heterogeneity of the skeletal muscle mitochondrial DNA from a patient with Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS). A mtDNA segment of 347 bp amplified from the total DNA was cloned into a vector. Analysis of 60 independent clones (20,800 bp in total) revealed the 3243 A→G transition in all the sequenced clones and additional nucleotide substitutions at 5 sites in 10 clones. The frequency of mutant clones (10/60) in the MELAS patient was significantly higher [χ2= 10.909,P< 0.05] than that in an age-matched skeletal muscle control (0/60) as well as in a normal placenta (2/60). These results supports our hypothesis that secondary somatic mtDNA mutations can be initiated by the 3243 A→G mutation and that the accumulation of somatic mutation in individuals with deleterious inherited mitochondrial genotype can contribute to the progressive mitochondrial dysfunction in MELAS.

Isolation and characterization of mumps virus strains in a mumps outbreak with a high incidence of aseptic meningitis.

In 1993, mumps with a high incidence of aseptic meningitis became prevalent in Akita prefecture, Japan. Three mumps virus isolates obtained from the nonvaccine-associated cases lacked the BamHI restriction cleavage site of the P gene, like the Urabe strain (Yamada, A. et al, Vaccine 8: 553-557). However, four additional nucleotide substitutions were found in the determined region of 157 bp. Fourteen of 19 cases from which mumps virus showing the Urabe-like RFLP profile was detected were complicated with symptomatic meningitis, whereas there were only four cases of meningitis among 23 individuals infected with the wild type showing no Urabe-like RFLP profile (non-"Urabe-like" wild-type). The incidence of meningitis was over 70% among patients infected with the "Urabe-like" wild-type virus. The "Urabe-like" wild-type. disappeared after February 1994 in the epidemic area and was replaced by the non-"Urabe-like" wild-type. Patients infected with the "Urabe-like" wild-type lived in a closed colony, in which there were two instances of transmission between siblings. Thus this outbreak was transient and narrowly localized.

Permutation of a pair of tertiary nucleotides in a transfer RNA.

The tertiary nucleotides at positions 15 and 48 in a tRNA establish non-Watson-Crick hydrogen interactions that connect the dihydrouridine (D) loop with the variable loop and stabilize the "L"-shaped tRNA structure. Although the majority of tRNAs have G15.C48 or A15.U48, all of the 16 possible nucleotide pairs at positions 15 and 48 can be found in the existing cytoplasmic and mitochondrial tRNA sequences. Because tRNAs contain a variety of slightly different sets of tertiary nucleotides, this complexity raises the question of whether a given tRNA sequence framework can accommodate all of the 16 compositions at positions 15 and 48. In this work, G15 and C48 in an Escherichia coli alanine amber suppressor tRNA were permuted, and variants were tested for biological activity in vivo. All but an A15.A48 variant were functional, indicating substantial flexibility at positions 15 and 48 to accommodate nucleotide variations. Analysis of the A15.A48 variant with chemical probes showed that this mutant harbors a defect that specifically changes the conformation of the anticodon sequence. Interestingly, human tRNA(Ala) has A15.A48. Additional nucleotide substitutions in E. coli A15.A48 tRNA(Ala) that recreate the D loop sequence of human tRNA(Ala) restored the biological activity to this tRNA by reestablishing the wild-type conformation of the anticodon sequence. The results suggest a distal relationship between the D and the anticodon loops in a tRNA and delineate covariation of specific nucleotides in the evolution of tRNA(Ala) from E. coli to human.

Evidence for a base-paired region of hepatitis B virus pregenome encapsidation signal which influences the patterns of precore mutations abolishing HBe protein expression.

In two natural HBe-minus hepatitis B virus mutants, expression of HBe protein was abrogated by a nonsense mutation at precore codon 28 and a frameshift mutation at codon 29, respectively. Both mutants contained an additional nucleotide substitution(s) which was found by transfection experiments to be required for efficient packaging of pregenomic RNA. The observed mutational profiles were consistent with the presence of a base-paired region of the pregenome encapsidation signal overlapping the HBe-coding sequence. Results obtained with artificial mutants with significant changes in the primary sequence suggested that base pairing is required but insufficient for efficient pregenome packaging. However, the predicted first four base pairs of the stem are dispensable.

Studies on the phylogenetic position of the Ctenodactylidae (Rodentia)

More than half of all mammalian species belong to the order of the rodents. Although many phylogenetic relationships among rodents have been established beyond doubt, others are not yet established. The phylogenetic position of the Ctenodactylidae (gundis) was discussed in several contributions to a symposium in Paris in 1984 (Luckett and Hartenberger 1985). This is a small rodent family with only four species living in the border regions of the Sahara Desert in Africa. This taxon has been grouped either with the hystricognathous rodents or as a separate branch, as a very early o&hoot from the rodent stem: Luckett ( 1985), Bugge (1985), and George ( 1985) concluded, from different sets of data, that the Ctenodactylidae share with the hystricognathous rodents a number of derived traits and that the Ctenodactylidae should be considered as an early offshoot of the latter. Wood ( 1985) discussed the phylogenetic position of several sciurognathous hystricomorph families, including the Ctenodactylidae. These families are also restricted to Africa and have often been associated with the hystricognathous rodents. However, in Wood’s opinion there are no indications for these relationships, the similarities being the result of conservation of ancestral features or of convergence. Hartenberger ( 1985) concluded from the fossil evidence that the Ctenodactylidae are one of the oldest recognized rodent families, diverging from all other rodent taxa in the Lower Eocene. With additional molecular data, it may be possible to shed more light on this controversy and to learn more about the origin of the rodents. No molecular evidence is yet available on the phylogenetic position of the Ctenodactylidae. Therefore, several tissues and blood of gundis were collected, and a number of proteins were isolated and investigated. Gundi myoglobin was isolated, and its amino acid sequence was determined (Beintema et al. 1990). Only four other rodent myoglobin sequences have been determined so far: mouse (A4z.o musculus) (Harris et al. 1985), casiragua (Proechimys guairue) (Harris et al. 1985 ), viscacha (Lugostomus muximus) (Gumett et al. 1984)) and mole rate (Spulux ehrenbergi) (Gumett et al. 1984). The gundi amino acid myoglobin sequence was compared with the 73 other known vertebrate myoglobin amino acid sequences by using computer procedures, described by Czelusniak et al. ( 1990), that search for the tree or set of trees with shortest nucleotide substitution length. The shortest trees for the 74 myoglobin sequences found in this search failed to represent rodents, primates, and artiodactyls as monophyletic orders. In these shortest trees, the gundi joins the hystricognath (casiragua/viscacha) branch, which is rather widely separated from the mouse/mole rat branch. If the four other rodents are forced together, which requires four additional nucleotide substitutions, the branch to the gundi still joins the two hystricognaths, or, for the same score, the gundi branches off before the divergence of the other four rodent sequences. The amino acid sequences of the a and p hemoglobin chains of gundi were

Nucleotide sequence analysis of the C.AKR Lyt-2a gene: structural polymorphism in alleles encoding the Lyt-2.1 T-cell surface alloantigen.

The Lyt-2a allele of the C.AKR strain of mice (genotype Lyt-2a, Lyt-3a) was cloned, and its complete nucleotide sequence as well as that of 2 kb of 5' flanking DNA was determined. The sequence was compared with the partial sequence of the Lyt-2a allele of DBA/2 (genotype Lyt-2a, Lyt-3b) and the nearly complete sequence of the B10.CAS2 Lyt-2b allele reported by Liaw and coworkers (1986). The coding regions of the two Lyt-2a alleles differ from each other by two nucleotide substitutions in the three exons over which they could be compared, resulting in two amino acid substitutions in the leader and transmembrane segments. The coding region of the C.AKR Lyt-2a allele differs from the Lyt-2b allele by two nucleotide substitutions in the extracellular V-like domain, one of which is silent and the second of which leads to substitution of valine for methionine at amino acid position 78 giving rise to the Lyt-2.1 allotypic specificity. The coding region of the DBA/2 Lyt-2a allele shares with C.AKR the allotypic substitution at position 78 and differs from Lyt-2b by three additional nucleotide substitutions in the coding regions, two of which lead to amino acid substitutions in the leader and transmembrane segments. It would therefore appear that the Lyt-2 alleles of the three strains analyzed are distinct, and the nomenclature Lyt-2a1 and Lyt-2a2 is suggested to distinguish the alleles of C.AKR and DBA/2, respectively. These alleles share a common difference from the Lyt-2b gene product at position 78, and since the amino acid substitutions which distinguish them from each other are in the leader and transmembrane segments, their mature Lyt-2 gene products appear antigenically identical.