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.     

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     

Novel marker for recombination in the 3′-untranslated region of members of the species Human enterovirus A

Human enterovirus A (HEV-A) is a species in the genus Enterovirus. Viruses belonging to this species are often responsible for hand, foot and mouth disease and associated acute neurological disease. Studies of the 3′ untranslated region (UTR) of human enterovirus 71 (HEV71) revealed a possible role in virus replication. We compared the 3′-UTRs of all members of HEV-A and confirmed the presence of a secondary structure comprising three stem-loop domains (SLDs). SLD-Z is situated closest to the stop codon and has been shown previously to affect plaque morphology. The prototype strains of coxsackieviruses A4 (CVA4), CVA14, and CVA16 carried the longer group I SLD-Z, whilst other CVAs and HEV71 carried the shorter group II SLD-Z. We demonstrate the importance of SLD-Z as a marker for the emergence of newer strains of HEV71 and CVA16 through inter-typic recombination and propose that SLD-Z is a novel evolutionary marker for recombination in HEV-A.     

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.     

Promoter activity in the 5′ flanking regions of the Bohle iridovirus ICP 18, ICP 46 and major capsid protein genes

Summary. Bohle iridovirus (BIV) belongs to the genus Ranavirus, of which Frog virus 3 (FV-3) is the type species. We are developing BIV as a recombinant viral delivery vector, and as a first step we located specific BIV promoter sequences to drive foreign gene expression in the recombinant virus. By comparison with FV-3 sequences, the genes encoding ICP 18 and ICP 46 in BIV were identified and sequenced. Putative promoter regions of these two early genes and of the major capsid protein (MCP) gene were identified, cloned into pSFM21, and luciferase production was then used to assess the promoter activity of these regions.     

A point mutation in the 5′-untranslated leader that affects translational activation of the mitochondrial COX 3 mRNA

The 613-base 5-untranslated leader (5-UTL) of the Saccharomyces cerevisiae mitochondrial COX 3 mRNA contains the target of an mRNA-specific translational activator complex composed of at least three nuclearly encoded proteins. We have genetically mapped a collection of cox 3 point mutations, using a set of defined COX 3 deletions, and found one to be located in the region coding the 5-UTL. The strain carrying this allele was specifically defective in translation of the COX 3 mRNA. Nucleotide-sequence analysis showed that the allele was in fact a double mutation comprised of a single-base insertion in the 5-UTL (T inserted between bases-428 and-427 with respect to the start of translation) and a G to A substitution at+3 that changed the ATG initiation codon to ATA. Both mutations were required to block translation completely. The effects of the ATG to ATA mutation alone (cox 3-1) had previously been analyzed in this laboratory: it reduces, but does not eliminate, translation, causing a slow respiratory growth phenotype. The T insertion in the 5-UTL had no detectable respiratory growth phenotype as a single mutation. However, the 5-UTL insertion mutation enhanced the respiratory defective phenotype of missense mutations in pet 54, one of the COX 3-specific translational-activator genes. This phenotypic enhancement suggests that the-400 region of the 5-UTL, where the mutation is located, is important for Pet54p-COX 3 mRNA interaction.     

Age-related expression analysis of mouse liver nuclear protein binding to 3′-untranslated region of <Emphasis Type="Italic">Period2</Emphasis> gene

In mammals, both circadian rhythm and aging play important roles in regulating time-dependent homeostasis. We previously discovered an age-related increase element binding protein, hnRNP A3, which binds to the 3′-untranslated region (UTR) of blood coagulation factor IX (FIX). Here, we describe other members of this protein family, hnRNP C and hnRNP H, which bind to the 3′-UTR of the mouse circadian clock gene Period 2 (mPer2). RNA electrophoretic mobility shift assays using a ³²P-labeled Per2 RNA probe coupled with two-dimensional gel electrophoresis followed by MALDI-TOF/MS peptide mass fingerprint analysis was used to analyze these proteins. Western blotting suggested that the total expression of these proteins in mouse liver cell nuclei does not increase with age. Two-dimensional gel electrophoresis analysis of age-related protein expression showed that many isoforms of these proteins exist in the liver and that each protein exhibits a complex age-related expression pattern. These results suggest that many isoforms of proteins are regulated by different aging systems and that many age regulation systems function in the liver.     

Transforming growth factor β mRNA and protein expression in the ovary of the chicken embryo

The expression pattern of transforming growth factor beta (TGFβ) isoforms in chicken embryo gonads was studied at 6–10 days of incubation. TGFβ2 mRNA was expressed predominantly in the cortex of the left ovary from day 8 of incubation onwards. TGFβ3 mRNA was not detected at any of the stages studied. Similarly, immunofluorescence for the TGFβ protein revealed that at day 9 it was located throughout the cortex of the left ovary and in the medulla of both the left and right ovaries. The presence of phosphorylated Smad2 in the nuclei of these regions suggests that TGFβ signaling is most likely active at this developmental stage. Culturing the left ovary in a TGFβ1-supplemented medium induced a shift of cortical structures toward the medulla, suggesting a role for TGFβ in the morphogenesis of the female gonad in chickens.     

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     

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.     

Nucleotide sequence of the coat protein gene of a strain of clover yellow vein virus from New Zealand: conservation of a stem-loop structure in the 3′ region of potyviruses

Summary The sequence of the 3-terminal 1492 nucleotides of the genome of a New Zealand isolate of clover yellow vein potyvirus (CYVV) has been determined. This sequence encodes a large open reading frame of 1314 nucleotides, the start of which was not identified, but which encodes a putative 272 amino acid coat protein. Downstream of the coat protein coding region is a 177 nucleotide untranslated sequence terminated by a polyadenylate tract. Comparison of the deduced CYVV-NZ coat protein amino acid sequence with two other strains of CYVV showed 86–93% similarity, suggesting CYVV-NZ should be regarded as a separate CYVV strain. CYVV-NZ shares with other CYVV strains a direct repeat of 14–16 nucleotides that is capable of forming a stem-loop structure. Examination of 35 strains of 15 other potyviruses showed a similar stem-loop structure conserved in all cases. A possible role in replication is hypothesized for the structure.     

Relationship between single nucleotide polymorphisms in the 3’-untranslated region of the vascular endothelial growth factor gene and susceptibility to diabetic peripheral neuropathy in China

Introduction

This study is to elucidate the relationship between a 936C/T mutation at the 3’-untranslated region of the human vascular endothelial growth factor (VEGF) gene and diabetic peripheral neuropathy (DPN).

Material and methods

All subjects recruited in this study were divided into DM (diabetes without neuropathy, retinopathy or nephropathy), DPN (diabetes with peripheral neuropathy only) and healthy control groups. The gene polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism, as well as other clinical methods including serum VEGF by ELISA.

Results

The C allele frequency and CC genotype frequency in the DPN group were higher than those in the NC group and DM group. The T allele frequency and CT+TT genotype (carrying the T allele) frequency in the DPN group were lower than those in the NC group (χ² = 19.051 and 18.533, both p < 0.001) and DM group (χ² = 11.117 and 11.156, both p = 0.001). However, there was no statistically significant difference in the three genotype (CC/CT+TT) frequencies and allele (C/T) frequencies between the DM group and the NC group. The multivariate logistic regression analysis showed that the levels of glycated hemoglobin (HbA_1c), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C) and plasma VEGF positively correlated with DPN, while the 936C/T gene polymorphism of VEGF negatively correlated with DPN.

Conclusions

Allele 936C of VEGF may serve as a genetic marker susceptible to DPN, while allele 936T may be a protective genetic marker of DPN.     

Characterization of VP6 genes from rotavirus strains collected in the United States from 1996–2002

We sequenced 22 VP6 genes from common rotavirus strains P[8], G1; P[4], G2; P[8], G3; P[8], G4 and P[8], G9 and uncommon type P[6], G9 collected in the US over a 6-year period. All strains defined as members of VP6 antigenic subgroup (SG) I according to reactivity patterns with monoclonal antibodies formed a genetic cluster (Genogroup I) with SG I reference strains. Similarly, all strains in antigenic SGII formed a group (Genogroup II) with corresponding standard strains of the same SG. Most US strains of each genogroup had diverged by 10–15% from the VP6 gene sequence of reference strains collected >20 years earlier and some recent isolates from other countries. Evolutionary analysis demonstrated that recently isolated US strains of both genogroups have diverged into 2–3 related clusters consistent with other recent findings. Unexpectedly, some recent isolates from other countries have diverged greatly from both older reference isolates and from the recent US isolates characterized here. This finding suggests that genetic diversity in human rotavirus VP6 genes may be greater than previously recognized. These sequences will help in the construction of a VP6 gene database to aid in the development of broadly reactive molecular assays and permit identification of regions where primers and probes for existing assays may need to be redesigned. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of CDC.     

A mitochondrial intron sequence in the 5′-flanking region of a plant nuclear lectin gene

Summary A sequence fragment from the cis-splicing intron between exons a and b of the NADH-dehydrogenase subunit 5 gene (nad5) in plant mitochondria is also present in one of two closely related nuclear-encoded lectin genes of Dolichos biflorus. This sequence of 116 nucleotides is the major difference in the 5-flanking region of two recently described lectin genes (Harada et al. 1990). The stem and leaf lectin DB58 does not contain the insert, while the otherwise more than 90% identical 5-flanking region of the seed lectin is interrupted by this mitochondrial intron sequence.