A novel Sac I RFLP in the 3′ untranslated region of the myotonin protein kinase gene

We found a novel Sac I polymorphism downstream of CTG repeats in the 3′ untranslated region of the myotonin protein kinase (MT-PK) gene. A C to G transition at nucleotide 13,590 in the gene was revealed by Southern blotting and confirmed by sequencing analyses. The allelic frequency of the C : G polymorphism in 63 unrelated Japanese individuals was estimated to be 0.98: 0.02. When Southern blotting is employed in the analysis of the CTG repeat length in the MT-PK gene, this Sac I polymorphism should be taken into consideration. Received: 15 October, 1998 / Accepted: 31 October, 1998     

Comparative analysis of the large fragment of the 5′ untranslated region (LF-5′ UTR) of serotype A foot-and-mouth disease virus field isolates from India

India is endemic for foot-and-mouth disease (FMD) and in recent years a unique group within serotype A, carrying a codon deletion at an antigenically critical site in capsid protein VP3 has emerged (VP3⁵⁹-deletion group). This tempted us to analyze the noncoding region, which is an under represented area, though critically associated with virus biology and pathogenesis. Analysis of the large fragment of 5′ untranslated region (LF-5′ UTR) of type A FMD virus revealed discrepancy in the overall tree topology between LF-5′ UTR and 1D region possibly due to independent evolution of coding and noncoding regions. The VP3⁵⁹-deletion group maintained its phylogenetic distinctness even at the LF-5′ UTR. Eighteen lineage specific signatures detected here support independent evolutionary paths for the lineages. Extensive deletions of 45 and 89 nucleotides corresponding to the pseudoknot region were noticed. Conservation pattern in the ‘A₂₅₃AACA’ motif in the cre/bus stem-loop indicates the importance of first three ‘A’ residues in VPg uridylylation. Of the three polypyrimidine tract binding protein (PTB) binding sites mapped on the internal ribosome entry site (IRES), the pyrimidine tract (Py tract) in the loop of domain 2 was found to be maximally conserved and it might be the major PTB binding site. Strikingly, a deletion group lineage specific transversion was noticed in the Py tract at the 3′ end of IRES without significantly affecting its in vitro infectious titer. Hence, we presume that for efficient cap-independent viral translation, either a minimum number of pyrimidine residues rather than a complete Py tract or a Py tract tolerating transversions only at specific locations and a core motif ‘CUUU’ within the Py tract is essential.     

The nucleotide sequence of the coat protein gene and 3′ untranslated region of papaya ringspot virus type W (Aust)

Summary The nucleotide sequence of the 3 terminal region of the Australian isolate of papaya ringspot virus type W [PRSV-W (Aust)] was determined. An open reading frame (864 bp), encoding the putative coat protein gene, occurs upstream of the putative 3 untranslated region (206 bp) and poly(A) tail. A 23 amino acid sequence was obtained from N-terminal analysis of the coat protein from purified virions. This sequence has 100% homology with a region of the amino acid sequence inferred from the nucleic acid sequence of the coat protein gene. However, this region is 13 amino acids downstream from the N terminus predicted for two American isolates of PRSV. The coat protein gene of PRSV-W (Aust) was shown to have 96.8% and 96.4% nucleotide sequence similarity with American isolates of PRSV-W and PRSV-P, respectively.     

Molecular characterization of a foot-and-mouth disease virus containing a 57-nucleotide insertion in the 3′untranslated region

A foot-and-mouth disease virus containing a 57-nucleotide (nt) insertion in the 3′untranslated region (3′UTR) was generated by transposon (tn)-mediated mutagenesis. Characterization of the mutant virus (A24-3′UTR8110) revealed no significant differences in virus growth, translation efficiency or virulence in cattle compared to the A24 wild-type virus. RNA modeling showed that the structures predicted in the 3′UTR were not affected by the tn insertion. These results revealed that the 3′UTR can tolerate foreign sequences that do not disrupt essential signals required for virus replication.     

An AciI polymorphism in the 3′ untranslated region of the human phosphomannomutase 2 (PMM2) gene

We found an AciI polymorphism in the 3′ untranslated region of the phosphomannomutase 2 (PMM2) gene located at 16p13. A G-to-C transition at nucleotide position 96 bp downstream from the PMM2 stop codon was detected in polymerase chain reaction (PCR) products after AciI digestion. The heterozygosity of the polymorphic alleles was 0.375 in a Japanese population. This polymorphism is useful for genetic analysis in patients with carbohydrate-deficient glycoprotein syndromes, of which there are four subtypes. Received: March 17, 1999 / Accepted: April 3, 1999     

Genetic diversity of 3′ region of glycoprotein D gene of bovine herpesvirus 1 and 5

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) are closely related alphaherpesviruses of cattle. While BoHV-1 is mainly associated with respiratory/genital disease and rarely associated with neurological disease, BoHV-5 is the primary agent of meningoencephalitis in cattle. The envelope glycoprotein D of alphaherpesviruses (BoHV-1/gD1 and BoHV-5/gD5) is involved in the early steps of virus infection and may influence virus tropism and neuropathogenesis. This study performed a sequence analysis of the 3′ region of gD gene (gD3′) of BoHV-1 isolates recovered from respiratory/genital disease (n = 6 and reference strain Cooper) or from neurological disease (n = 7); and from seven typical neurological BoHV-5 isolates. After PCR amplification, nucleotide (nt) sequencing, and aminoacid (aa) sequence prediction; gD3′ sequences were compared, identity levels were calculated, and selective pressure was analyzed. The phylogenetic reconstruction based on nt and aa sequences allowed for a clear differentiation of BoHV-1 (n = 14) and BoHV-5 (n = 7) clusters. The seven BoHV-1 isolates from neurological disease are grouped within the BoHV-1 branch. A consistent alignment of 346 nt revealed a high similarity within each viral species (gD1 = 98.3 % nt and aa; gD5 = 97.8 % nt and 85.8 % aa) and an expected lower similarity between gD1 and gD5 (73.7 and 64.1 %, nt and aa, respectively). The analysis of molecular evolution revealed an average negative selection at gD3′. Thus, the phylogeny and similarity levels allowed for differentiation of BoHV-1 and BoHV-5 species, but not further division in subspecies. Sequence analysis did not allow for the identification of genetic differences in gD3′ potentially associated with the respective clinical/pathological phenotypes, yet revealed a lower level of gD3′ conservation than previously reported.     

Genetic characterization of vaccine and field strains of serotype A foot-and-mouth disease virus from India

Extreme antigenic and genetic heterogeneity of serotype A foot-and-mouth disease virus (FMDV) population has resulted in change of vaccine strains in India twice in the last decade. In such a situation, complete characterization of the vaccine strains is imperative. With regard to the frequent outbreaks of this disease, FMDV field strains are also of interest. Therefore three vaccine strains and two field strains of type A FMDV from India were completely sequenced and the obtained sequences were subjected to sequence and phylogenetic analyses. Based on the complete coding region, all the Indian strains clustered in the Asia topotype and exhibited a more than 11% nt divergence from the other Asian strains. The 5'-UTR of some Indian strains revealed block deletions of 43 and 86 nt corresponding to the pseudoknot region. Amino acids S44 in VP2 and F164 in VP1 were found to be the exclusive signatures for the Asia topotype. The vaccine strains differed at 65 aa positions in the capsid region, 13 of them antigenically critical. Variability at such positions is likely to affect the antigenic profile of these strains. Complete genome sequences of the vaccine strains presented here could serve as the reference for any comparative genomics in future.     

The confirmation of three repeated sequence elements in the 3′ untranslated region of Chikungunya virus

In this study, the complete genomic nucleotide sequence of Chikungunya virus (CHIKV) strain S27 African prototype was determined and three 21 nucleotides repeated sequence elements (RSEs) at positions 11398–11418, 11533–11553, and 11620–11640 in the 3′ untranslated region (3′UTR) were confirmed. In addition, the 3′UTRs of all CHIKV strains deposited in GenBank were analyzed. The results displayed that the majority of the CHIKV strains consisted of the three 21 nucleotides RSEs in the 3′UTRs, and the third RSE was the most conservative. The conservation of the three RSEs of 21 nucleotides within the 3′UTR of CHIKV genome may play an important role on the virus replication cycle.     

Molecular cloning and sequence analysis of the 3′-terminal region of iris severe mosaic virus RNA

Summary Sequence analysis of iris severe mosaic potyvirus genomic RNA revealed an unusual E/G cleavage site between the deduced large nuclear-inclusion protein and coat protein sequences. The latter showed an N-terminus of only 15 amino acids. The 3 non-translated region of the viral RNA was 340 nucleotides long.     

HLA-G 3′ untranslated region gene variants are promising prognostic factors for BK polyomavirus replication and acute rejection after living-donor kidney transplant

The immunosuppressive non-classical human leukocyte antigen-G (HLA-G) promotes transplant tolerance as well as viral immune escape. HLA-G expression is associated with regulatory elements targeting certain single nucleotide polymorphisms (SNPs) in the HLA-G 3′ untranslated region (UTR). Thus, we evaluated the impact of HLA-G 3′UTR polymorphisms as surrogate markers for BK polyomavirus (BKPyV) replication or nephropathy (PyVAN) and acute cellular and antibody mediated rejection (ACR/AMR) in 251 living-donor kidney-transplant recipient pairs. After sequencing of the HLA-G 3′UTR, fourteen SNPs between +2960 and +3227 and the 14 bp insertion/deletion polymorphism, which arrange as UTR haplotypes, were identified. The UTR-4 haplotype in donors and recipients was associated with occurrence of BKPyV/PyVAN compared to the other UTR haplotypes. While the UTR-4 recipient haplotype provided protection against AMR, the UTR-2 donor haplotype was deleteriously associated with ACR/AMR. Deduction of the UTR-2/4 haplotypes to specific SNPs revealed that the +3003C variant (unique for UTR-4) in donors as well as in recipients is responsible for BKPyV/PyVAN and also provides protection against AMR; whereas the +3196G variant (unique for UTR-2) promotes allograft rejection. Thus, HLA-G 3′UTR variants are promising genetic predisposition markers both in donors and recipients that may help to predict susceptibility to either viral infectious complication of BKPyV or allograft rejection.     

Analysis of the 3′-variable region of the cagA gene from Helicobacter pylori strains infecting patients at New York City hospitals

Helicobacter pylori infects the gastric mucosa in humans and is a causative agent for peptic ulcer disease (PUD) and gastric cancer (GC). CagA is produced by H. pylori and is associated with more severe outcomes. cagA genes vary at the 3′-region with respect to phosphorylation motifs (EPIYA-A, -B, -C, or -D) and CagA multimerization motifs (CM). This variability may be associated with the clinical outcomes. We examined the variable region of cagA genes expressed in H. pylori-infected patients treated at three NYC Hospitals. DNA was isolated from gastric biopsies of patients undergoing upper endoscopy. Most H. pylori-infected patients were Black or Hispanic. The cagA 3′-region of CagA-positive samples was amplified by PCR, purified and sequenced. The patterns of EPIYA and CM motifs were examined and related to clinical outcomes. We obtained 42 CagA sequences from our sample collection. The EPIYA phosphorylation motif pattern was ABC in 81.0% of our samples. Western (W) and Eastern (E) CM motifs have also been defined. CagA proteins lacking an Eastern CM motif and possessing one or two Western CM motifs were observed more frequently in patients with PUD and GC when compared with non-ulcer gastritis (50.0% vs 11.8%, respectively), suggesting that these CM motif patterns are more virulent than those containing at least one Eastern CM motif. We conclude that In H. pylori-infected patients treated at NYC Hospitals, CM motif patterns in the CagA 30-variable region may be more significant than EPIYA motif patterns with respect to clinical outcomes.     

Nucleotide sequence of the 3′ terminal region of the genome of four Lettuce mosaic virus isolates from Greece and Yemen

Summary.  Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3′ terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3′ non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1 ¹ and mo1 ² and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR. Received July 1, 1998 Accepted March 25, 1999     

Molecular cloning and nucleotide sequencing of the 3′-terminal region of a South African isolate of ryegrass mosaic virus RNA and in vitro expression of the coat protein gene

Summary The 2094 nucleotides at the 3-terminus of a South African isolate of ryegrass mosaic virus (RGMV) was cloned and sequenced. Two putative poly-protein cleavage sites were found: Q/L and E/A, both of which are novel in thePotyviridae. The RGMV-SA cDNA was cloned into an expression vector, pUEX, and a fusion protein of 185 kDa was obtained which reacted strongly to anti-RGMV-SA antiserum. Alignment of the predicted amino acid sequence of RGMV-SA with those of otherPotyviridae members showed limited identity, indicating that RGMV-SA is a definite and distinct virus.     

HLA-E coding and 3′ untranslated region variability determined by next-generation sequencing in two West-African population samples

HLA-E is a non-classical Human Leucocyte Antigen class I gene with immunomodulatory properties. Whereas HLA-E expression usually occurs at low levels, it is widely distributed amongst human tissues, has the ability to bind self and non-self antigens and to interact with NK cells and T lymphocytes, being important for immunosurveillance and also for fighting against infections. HLA-E is usually the most conserved locus among all class I genes. However, most of the previous studies evaluating HLA-E variability sequenced only a few exons or genotyped known polymorphisms. Here we report a strategy to evaluate HLA-E variability by next-generation sequencing (NGS) that might be used to other HLA loci and present the HLA-E haplotype diversity considering the segment encoding the entire HLA-E mRNA (including 5′UTR, introns and the 3′UTR) in two African population samples, Susu from Guinea–Conakry and Lobi from Burkina Faso. Our results indicate that (a) the HLA-E gene is indeed conserved, encoding mainly two different protein molecules; (b) Africans do present several unknown HLA-E alleles presenting synonymous mutations; (c) the HLA-E 3′UTR is quite polymorphic and (d) haplotypes in the HLA-E 3′UTR are in close association with HLA-E coding alleles. NGS has proved to be an important tool on data generation for future studies evaluating variability in non-classical MHC genes.     

Polymorphism in the 3′-untranslated region of the thymidylate synthase gene and sensitivity of stomach cancer to fluoropyrimidine-based chemotherapy

To investigate the relationship between polymorphism in the 3-untranslated region (3-UTR) of the thymidylate synthase (TS) gene and sensitivity of gastric cancer to 5-fluorouracil (5-FU)-based chemotherapy, 106 cases of advanced gastric cancer were analyzed. All patients were treated with 5-FU-based chemotherapy; DNA from peripheral blood leukocytes was obtained before therapy. TS 3-UTR genotypes were detected by PCR-RFLP. Polymorphism in the TS 3-UTR can be classified into three groups according to the presence or absence of a 6 bp nucleotide fragment: the –6/–6 bp, –6/+6 bp and +6/+6 bp groups. The response rate of the –6/–6 bp and –6/+6 bp groups was found to be significantly higher than the +6/+6 bp group. These results show that the presence of the TS 3-UTR 6 bp nucleotide fragment can be correlated with the sensitivity of gastric cancer to 5-FU-based chemotherapy, and that the TS 3-UTR polymorphism profile can be used to guide the choice of 5-FU-based chemotherapy in advanced gastric cancer.