Adaptation of Puumala Hantavirus to Cell Culture Is Associated with Point Mutations in the Coding Region of the L Segment and in the Noncoding Regions of the S Segment

ABSTRACT We previously developed a model for studies on hantavirus host adaptation and initiated genetic analysis of Puumala virus variants passaged in colonized bank voles and in cultured Vero E6 cells. With the data presented in this paper, the sequence comparison of the wild-type and Vero E6-adapted variants of Puumala virus, strain Kazan, has been completed. The only amino acid substitution that distinguished the two virus variants was found in the L protein, Ser versus Phe at position 2053. Another mutation found in the L segment, the silent transition C1053U, could result from the selection of a variant with altered L RNA folding. Nucleotide substitutions observed in individual L cDNA clones, most of them A→G and U→C transitions, suggested that the population of L RNA molecules is represented by quasispecies. The mutation frequency in the L segment quasispecies appeared to be similar to the corresponding values for the S and M quasispecies. Analysis of the cDNA clones with the complete S segment sequences from passage 20 confirmed our earlier conclusion that the cell-adapted genotype of the virus is represented mostly by variants with mutated S segment noncoding regions. However, the spectrum of the S segment quasispecies appeared to be changing, suggesting that, after the initial adaptation (passages 1 to 11), the viral population is still being driven by selection for variants with higher fitness.

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Accumulation of point mutations and reassortment of genomic RNA segments are involved in the microevolution of Puumala hantavirus in a bank vole (Myodes glareolus) population

Journal of General Virology ◽

10.1099/vir.0.2008/001248-0 ◽

2008 ◽

Vol 89 (7) ◽

pp. 1649-1660 ◽

Cited By ~ 41

Author(s):

Maria Razzauti ◽

Angelina Plyusnina ◽

Heikki Henttonen ◽

Alexander Plyusnin

Keyword(s):

Genetic Diversity ◽

Bank Vole ◽

Local Population ◽

Point Mutations ◽

Myodes Glareolus ◽

Stabilizing Selection ◽

Genetic Lineage ◽

Nucleotide Substitutions ◽

Bank Voles ◽

L Segment

The genetic diversity of Puumala hantavirus (PUUV) was studied in a local population of its natural host, the bank vole (Myodes glareolus). The trapping area (2.5×2.5 km) at Konnevesi, Central Finland, included 14 trapping sites, at least 500 m apart; altogether, 147 voles were captured during May and October 2005. Partial sequences of the S, M and L viral genome segments were recovered from 40 animals. Seven, 12 and 17 variants were detected for the S, M and L sequences, respectively; these represent new wild-type PUUV strains that belong to the Finnish genetic lineage. The genetic diversity of PUUV strains from Konnevesi was 0.2–4.9 % for the S segment, 0.2–4.8 % for the M segment and 0.2–9.7 % for the L segment. Most nucleotide substitutions were synonymous and most deduced amino acid substitutions were conservative, probably due to strong stabilizing selection operating at the protein level. Based on both sequence markers and phylogenetic clustering, the S, M and L sequences could be assigned to two groups, ‘A’ and ‘B’. Notably, not all bank voles carried S, M and L sequences belonging to the same group, i.e. SAMALA or SBMBLB. A substantial proportion (8/40, 20 %) of the newly characterized PUUV strains possessed reassortant genomes such as SBMALA, SAMBLB or SBMALB. These results suggest that at least some of the PUUV reassortants are viable and can survive in the presence of their parental strains.

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THE NATURE OF NUCLEOTIDE SEQUENCE DIVERGENCE BETWEEN BARLEY AND MAIZE CHLOROPLAST DNA

Genetics ◽

10.1093/genetics/106.4.735 ◽

1984 ◽

Vol 106 (4) ◽

pp. 735-749

Author(s):

Gerard Zurawski ◽

Michael T Clegg ◽

Anthony H D Brown

Keyword(s):

Chloroplast Dna ◽

Dna Sequences ◽

Large Subunit ◽

Leader Region ◽

Coding Region ◽

Nucleotide Substitutions ◽

Bisphosphate Carboxylase ◽

Noncoding Regions ◽

The Third ◽

Maize Chloroplast

ABSTRACT Analysis of a 2175-base pair (bp) SmaI-HindIII fragment of barley chloroplast DNA revealed that rbcL (the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase) and atpB (the gene for the β subunit of ATPase) are transcribed divergently and are separated by an untranscribed region of 155-166 bp. The rbcL mRNA has a 320-residue untranslated leader region, whereas the atpB mRNA has a 296- to 309-residue leader region. The sequence of these regions, together with the initial 113 bp of the atpB-coding region and the initial 1279 bp of the rbcL-coding region, is compared with the analogous maize chloroplast DNA sequences. Two classes of nucleotide differences are present, substitutions and insertions/deletions. Nucleotide substitutions show a 1.9-fold bias toward transitions in the rbcL-coding region and a 1.5-fold bias toward transitions in the noncoding region. The level of nucleotide substitutions between the barley and maize sequences is about 0.065/bp. Seventy-one percent of the substitutions in the rbcL-coding region are at the third codon position, and 95% of these are synonymous changes. Insertion/deletion events, which are confined to the noncoding regions, are not randomly distributed in these regions and are often associated with short repeated sequences. The extent of change for the noncoding regions (about 0.093 events/bp) is less than the extent of change at the third codon positions in the rbcL-coding region (about 0.135 events/bp), including insertion/delection events. Limited sequence analysis of the analogous DNA from a wild line (Hordeum spontaneum) and a primitive Iranian barley (H. vulgare) suggested a low rate of chloroplast DNA evolution. Compared to spinach chloroplast DNA, the barley rbcL-atpB untranslated region is extremely diverged, with only the putative rbcL promoters and ribosome-binding site being extensively conserved.

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Association of genetic variants of the prolactin gene with milk production traits in Russian Red Pied cattle

Proceedings of the British Society of Animal Science ◽

10.1017/s1752756200020597 ◽

2007 ◽

Vol 2007 ◽

pp. 156-156 ◽

Cited By ~ 3

Author(s):

Masoud Alipanah ◽

Lobov Kalashnikova ◽

Genadi Rodionov

Keyword(s):

Genetic Marker ◽

Regulatory Function ◽

Cdna Clones ◽

Production Traits ◽

Coding Region ◽

Nucleotide Substitutions ◽

Prolactin Gene ◽

Pcr Rflp ◽

Rsai Polymorphism

Prolactin plays an important regulatory function in mammary gland development, milk secretion, and expression of milk protein genes. Hence the PRL gene is a potential genetic marker of production traits in dairy cattle. The gene was mapped on chromosome 23 by Hallerman et al. (1988). It consists of 5 exons and four introns (Camper et al. 1984) encoding the 199-amino-acid mature protein (Wallis 1974). On the basis of sequence analysis of four different cDNA clones, seven possible nucleotide substitutions were described by Sasavage et al. (1982). One of them, recognized by RsaI endonuclease, has become a popular genetic marker used for genetic characterization of cattle populations by means of PCR-RFLP (Mitra et al., 1995). Two allelic variants (B and b) have been distinguished at the DNA level, based on RsaI polymorphism in the third exon of the coding region. It has been suggested that prolactin alleles correlate with milk yield (Lewin et al., 1992).

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Boundaries of somatic mutation in rearranged immunoglobulin genes: 5' boundary is near the promoter, and 3' boundary is approximately 1 kb from V(D)J gene.

Journal of Experimental Medicine ◽

10.1084/jem.172.6.1717 ◽

1990 ◽

Vol 172 (6) ◽

pp. 1717-1727 ◽

Cited By ~ 222

Author(s):

S G Lebecque ◽

P J Gearhart

Keyword(s):

Structural Information ◽

Gene Segment ◽

Point Mutations ◽

Sequence Motif ◽

Immunoglobulin Gene ◽

Coding Region ◽

Nucleotide Substitutions ◽

Coding Regions ◽

Dna Strands ◽

Transcriptional State

To investigate why somatic mutations are spatially restricted to a region around the rearranged V(D)J immunoglobulin gene, we compared the distribution of mutations flanking murine V gene segments that had rearranged next to either proximal or distal J gene segments. 124 nucleotide substitutions, nine deletions, and two insertions were identified in 32,481 bp of DNA flanking the coding regions from 17 heavy and kappa light chain genes. Most of the mutations occurred within a 2-kb region centered around the V(D)J gene, regardless of which J gene segment was used, suggesting that the structural information for mutation is located in sequences around and within the V(D)J gene, and not in sequences downstream of the J gene segments. The majority of mutations were found within 300 bp of DNA flanking the 5' side of the V(D)J gene and 850 bp flanking the 3' side at a frequency of 0.8%, which was similar to the frequency in the coding region. The frequency of flanking mutations decreased as a function of distance from the gene. There was no evidence for hot spots in that every mutation was unique and occurred at a different position. No mutations were found upstream of the promoter region, suggesting that the promoter delimits a 5' boundary, which provides strong evidence that transcription is necessary to generate mutation. The 3' boundary was approximately 1 kb from the V(D)J gene and was not associated with a DNA sequence motif. Occasional mutations were located in the nuclear matrix association and enhancer regions. The pattern of substitutions suggests that there is discrimination between the two DNA strands during mutation, in that the four bases were mutated with different frequencies on each strand. The high frequency of mutations in the 3' flanking region and the uniqueness of each mutation argues against templated gene conversion as a mechanism for generating somatic diversity in murine V(D)J genes. Rather, the data support a model for random point mutations where the mechanism is linked to the transcriptional state of the gene.

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Polymorphisms in the goat β-lactoglobulin gene

Journal of Dairy Research ◽

10.1017/s0022029905000981 ◽

2005 ◽

Vol 72 (3) ◽

pp. 379-384 ◽

Cited By ~ 12

Author(s):

Maria Ballester ◽

Armand Sánchez ◽

Josep M Folch

Keyword(s):

Genetic Variants ◽

Promoter Region ◽

Point Mutations ◽

Proximal Promoter ◽

Coding Region ◽

Nucleotide Substitutions ◽

Single Nucleotide ◽

Β Lactoglobulin ◽

Pyrosequencing Technology ◽

Frequent Haplotype

β-lactoglobulin polymorphisms have been reported in the milk of different goat breeds, although no genetic variants affecting the protein have been characterized. In the present study, we amplified and sequenced the proximal promoter and the first six exons containing the entire coding region for the β-lactoglobulin gene in eleven goat breeds from Spain, France, Italy, Switzerland, Senegal and Asia to identify genetic variants. Fifteen polymorphisms were detected, nine in the promoter region and six in the exons of the β-lactoglobulin gene. All polymorphisms were single nucleotide substitutions with the exception of one deletion/insertion in the promoter region. The polymorphisms in the coding region did not produce any amino acid change. In addition, pyrosequencing technology was used to genotype four polymorphisms in the promoter region in 200 goats belonging to eleven breeds. Differences in allelic frequencies for these polymorphisms between breeds are described and a specific polymorphism for the Italian populations was identified. Finally, the analysis of association between these four promoter point mutations was investigated resulting in five haplotypes, GCGC being the most frequent haplotype in all breeds analysed.

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Isolation and characterization of calmodulin genes from Xenopus laevis

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.507-513.1984 ◽

1984 ◽

Vol 4 (3) ◽

pp. 507-513

Author(s):

Y H Chien ◽

I B Dawid

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Complete Analysis ◽

Cdna Clones ◽

Coding Region ◽

Nucleotide Substitutions ◽

Protein Coding ◽

Isolation And Characterization ◽

Electric Eel ◽

Complete Protein

Two cDNAs derived from Xenopus laevis calmodulin mRNA have been cloned. Both cDNAs contain the complete protein-coding region and various lengths of untranslated segments. The two cDNAs encode an identical protein but differ from each other by 5% nucleotide substitutions. The 5' and 3' untranslated regions, to the extent available, are highly homologous between the two cDNAs. The predicted sequence of X. laevis calmodulin is identical to that of vertebrate calmodulins from mammals and chickens and shows one substitution compared with electric eel calmodulin. Genomic DNA sequences homologous to each of the two cDNA clones have been isolated and were shown to account for the major calmodulin-coding DNA sequences in X. laevis. These data suggest that X. laevis carries two active, nonallelic calmodulin genes. Although no complete analysis has been carried out, it appears that the X. laevis calmodulin genes are interrupted by at least four introns. The relative concentrations of calmodulin mRNA have been estimated in different embryonic stages and adult tissues and found to vary by up to a factor of 10. The highest levels of calmodulin mRNA were found in ovaries, testes, and brains. In these three tissues, the two calmodulin genes appear to be expressed at approximately equal levels.

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Characterisation of 96 mutations in 128 unrelated severe haemophilia A patients from France

Thrombosis and Haemostasis ◽

10.1160/th05-05-0379 ◽

2006 ◽

Vol 95 (04) ◽

pp. 593-599 ◽

Cited By ~ 11

Author(s):

Christine Vinciguerra ◽

Christophe Zawadzki ◽

Yesim Dargaud ◽

Gilles Pernod ◽

Claire Berger ◽

...

Keyword(s):

Factor Viii ◽

Direct Sequencing ◽

Point Mutations ◽

Haemophilia A ◽

Missense Mutations ◽

Severe Haemophilia ◽

Coding Region ◽

Nucleotide Substitutions ◽

Large Deletions ◽

Fviii Inhibitor

SummaryDirect sequencing of the coding region of factor VIII (F8) gene was used to determine the mutations responsible for severe haemophilia A (FVIII<1%) in 128 unrelated haemophiliacs A, negative for intron 22 and intron 1 inversions. A mutation was found in 122/128 patients (95%). Ninety-six distinct mutations were identified in this cohort, 62 of these are novel. They consisted of deletions (7 large and 24 small deletions), insertions (n=9), associations of insertion/deletion (n=2), association of deletion/substitution (n=1), and single nucleotide substitutions (53 point mutations consisting of 31 missense, 20 nonsense, and 2 splicing mutations). Twenty-two patients had developed inhibitors, and among this subgroup 3 large deletions, 6 frameshift, 9 nonsense and 4 missense mutations were detected. For6 patients, among which one developed an anti-FVIII inhibitor, no mutations were detected in the coding and splicing regions of factor VIII gene. Different approaches of molecular modelling were performed in addition to familial linkage analysis to determine the pathophysiological responsibility of these novel missense mutations.

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Highly Reliable Heterologous System for Evaluating Resistance of Clinical Herpes Simplex Virus Isolates to Nucleoside Analogues

Journal of Virology ◽

10.1128/jvi.75.7.3105-3110.2001 ◽

2001 ◽

Vol 75 (7) ◽

pp. 3105-3110 ◽

Cited By ~ 44

Author(s):

Julie Bestman-Smith ◽

Isabelle Schmit ◽

Barbara Papadopoulou ◽

Guy Boivin

Keyword(s):

Herpes Simplex Virus ◽

Herpes Simplex ◽

Point Mutations ◽

Protozoan Parasite ◽

Nucleoside Analogues ◽

Coding Region ◽

Nucleotide Substitutions ◽

Heterologous System ◽

Clinical Resistance ◽

Simplex Virus

ABSTRACT Clinical resistance of herpes simplex virus (HSV) types 1 and 2 to acyclovir (ACV) is usually caused by the presence of point mutations within the coding region of the viral thymidine kinase (TK) gene. The distinction between viral TK mutations involved in ACV resistance or part of viral polymorphism can be difficult to evaluate with current methodologies based on transfection and homologous recombination. We have developed and validated a new heterologous system based on the expression of the viral TK gene by the protozoan parasiteLeishmania, normally devoid of TK activity. The viral TK genes from 5 ACV-susceptible and 13 ACV-resistant clinical HSV isolates and from the reference strains MS2 (type 2) and KOS (type 1) were transfected as part of an episomal expression vector inLeishmania. The susceptibility of TK-recombinant parasites to ganciclovir (GCV), a closely related nucleoside analogue, was evaluated by a simple measurement of the absorbance ofLeishmania cultures grown in the presence of the drug. Expression of the TK gene from ACV-susceptible clinical isolates resulted in Leishmania susceptibility to GCV, whereas expression of a TK gene with frameshift mutations or nucleotide substitutions from ACV-resistant isolates gave rise to parasites with high levels of GCV resistance. The expression of the HSV TK gene inLeishmania provides an easy, reliable, and sensitive assay for evaluating HSV susceptibility to nucleoside analogues and for assessing the role of specific viral TK mutations.

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Isolation and characterization of calmodulin genes from Xenopus laevis.

Molecular and Cellular Biology ◽

10.1128/mcb.4.3.507 ◽

1984 ◽

Vol 4 (3) ◽

pp. 507-513 ◽

Cited By ~ 105

Author(s):

Y H Chien ◽

I B Dawid

Keyword(s):

Xenopus Laevis ◽

Dna Sequences ◽

Complete Analysis ◽

Cdna Clones ◽

Coding Region ◽

Nucleotide Substitutions ◽

Protein Coding ◽

Isolation And Characterization ◽

Electric Eel ◽

Complete Protein

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Two isoforms of the kidney androgen-regulated protein are encoded by two alleles of a single gene in OFl mice

Genetics Research ◽

10.1017/s0016672300030329 ◽

1992 ◽

Vol 59 (2) ◽

pp. 117-124 ◽

Cited By ~ 2

Author(s):

E. Melanitou ◽

D. Tronik ◽

F. Rougeon

Keyword(s):

Amino Acid ◽

Mouse Genome ◽

Single Gene ◽

Point Mutations ◽

Apparent Molecular Weight ◽

Analysis Data ◽

Size Difference ◽

Cdna Clones ◽

Coding Region

SummaryTwo cDNA clones coding for two forms of the mouse kidney androgen-regulated protein (KAP) distinguished by their electrophoretic mobilities on SDS gel electrophoresis have been isolated from libraries prepared from strains of mice having one (BALB/c) or two (OFl) forms of the KAP protein. The corresponding mRNAs have identical sizes, as well as identical sequences in their 5' non-translated regions. The size difference observed between the two proteins is due to two point mutations in the coding region of the KAP mRNA, leading to two amino-acid changes one of which resulted in the substitution of a glycine for a glutamic acid. As shown by in vitro transcription/translation experiments, these two amino-acid differences are responsible for the shift in the apparent molecular weight of the protein on SDS gels. Both forms of the protein are more abundant in males than in females.In vitro translation of kidney RNAs isolated from six different strains and species of mice revealed the presence of other forms of the KAP protein, characterized by small variations of their molecular weights. Southern blot analysis data are consistent with the presence of only one kap gene in the mouse genome. A restriction fragment length polymorphism has been observed, which does not correlate with the protein polymorphism, indicating the presence of another allele in the OF1 mouse genome.

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