Mapping of sporulation-specific functions in the yeast syntaxin gene<Emphasis Type="Italic"> SSO1</Emphasis>

The yeast Saccharomyces cerevisiae has two closely related plasma membrane syntaxins, Sso1p and Sso2p, which together provide an essential function in vegetative cells. However, Sso1p is also specifically needed during sporulation; and this function cannot be provided by Sso2p. We used fusions between SSO1 and SSO2 to map the sporulation-specific function of SSO1. We found that the two N-terminal -helices Ha and Hb of Sso1p are important for sporulation, since it is reduced 8-fold for fusions where Ha and Hb are derived from Sso2p. In contrast, the C-terminal half of Sso1p does not seem to be specifically required for sporulation. Surprisingly, we further found that the 3 untranslated region (3UTR) of SSO1 is essential for sporulation. Western blots failed to reveal a preferential expression of Sso1p in sporulating cells, indicating that effects on gene expression are unlikely to explain why the SSO1 3UTR is needed for sporulation.Communicated by S. Hohmann     

Nucleotide sequence of the coat protein genes of <Emphasis Type="Italic">Alstroemeria mosaic virus</Emphasis> and Amazon lily mosaic virus,a tentative species of genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Summary. The nucleotide sequences of the 3 terminal region of the genomes of Alstroemeria mosaic virus (AlsMV) and the Amazon lily mosaic virus (ALiMV) have been determined. These sequences contain the complete coding region of the viral coat protein (CP) gene followed by a 3-untranslated region (3-UTR). AlsMV and ALiMV share 74.9% identity in the amino acid sequence of the CP, and 55.6% identity in the nucleotide sequence of the 3-UTR. Phylogenetic analysis of these CP genes and 3-UTRs in relation to those of 79 potyvirus species revealed that AlsMV and ALiMV should be assigned to the Potato virus Y (PVY) subgroup. AlsMV and ALiMV were concluded to have arisen independently within the PVY subgroup.     

<Emphasis Type="Italic">Sarcocystis neurona</Emphasis> major surface antigen gene 1 (<Emphasis Type="Italic">SAG1</Emphasis>) shows evidence of having evolved under positive selection pressure

The major surface antigen gene 1 (SAG1) is conserved among members of Sarcocystidae and may play an important role in parasite pathogenesis. Additionally, generation and selection of different antigenic variants of SAG1 has the potential for inclusion in a subunit vaccine or in the development of a diagnostic assay. In this study, patterns of nucleotide polymorphism were used to test the hypothesis that natural selection promotes diversity in different parts of SAG1 of Sarcocystis neurona. Nucleotide and amino acid sequence analysis of SAG1 from multiple S. neurona isolates identified two alleles. Sequences were identical intra-allele and highly divergent inter-alleles. Also, phylogenetic reconstruction showed sequences clustering into two clades. Tajimas and Fu and Lis neutrality tests indicated that selection is more likely to be acting on SAG1. Moreover, a sliding window analysis based on the ratio of silent substitutions to amino acid replacements provided strong evidence that two short segments in the central and 3 domain of SAG1 have been under positive selection in the divergence of the two alleles, suggesting that it may be important for the evasion of host immune responses and would be a suitable target for vaccine development.This revised version was published online in November 2004. In the Abstract an the Results section 5 has been corrected to 3.     

The complete sequence of the genomic RNA of an isolate of <Emphasis Type="Italic">Lily virus X</Emphasis> (genus <Emphasis Type="Italic">Potexvirus</Emphasis>)

Summary. The complete sequence of the genomic RNA of an isolate of Lily virus X (LVX) has been determined for the first time. The isolate from the Netherlands was 5823 nucleotide (nt) long excluding the 3-poly(A) tail, making it the shortest reported potexvirus sequence. The 5-non-coding region begins with GGAAAA like that of Scallion virus X (ScaVX) and some isolates of Cymbidium mosaic virus (CymMV), whereas those of other sequenced potexviruses probably all begin with GAAAA. The genome organisation was similar to that of other members of the genus except that a TGBp3-like region lacked a normal AUG start codon. A phylogenetic analysis based on the entire coding sequence showed that LVX was most closely related to Strawberry mild yellow edge virus and belonged in a subgroup of the genus that also contains CymMV, Narcissus mosaic virus, ScaVX, Pepino mosaic virus, Potato aucuba mosaic virus and White clover mosaic virus.     

Identification of human <Emphasis Type="Italic">Clock</Emphasis> gene variants by denaturing high-performance liquid chromatography

The human Clock gene (hClock) encodes the CLOCK protein essential for the function of the circadian system. We have screened the entire coding region, including the 5 and 3 untranslated regions (UTRs) and the flanking intronic regions, of the hClock gene for sequence variations in 70 unrelated Chinese Singaporeans with denaturing high-performance liquid chromatography (dHPLC). A total of 15 sequence variations were detected, five of which were novel. All involved single base changes. There were 12 substitutions and three insertions/deletions. All except one were found in the introns or the UTRs. Frequencies of the minor allele for all 15 polymorphisms ranged from 0.7% to almost 50%. For the eight sites whose minor allele frequency was found to be at least 10%, pair-wise comparisons revealed that all except one were in almost complete linkage disequilibrium. Our identification of additional single nucleotide polymorphisms in the hClock gene would provide markers whose frequencies could be established in the selected population and used for further analysis of the phenotypic effects. Our results would also be useful for better planning in the selection of polymorphisms for future genetic association studies involving the hClock gene.     

Characterization of the small subgenomic RNA of <Emphasis Type="Italic">Soybean dwarf virus</Emphasis>

Summary. We have characterized a small subgenomic RNA of Japanese strains of Soybean dwarf virus (SbDV). Northern blot analyses of SbDV-infected plants showed that the small RNA contained the 3 terminal sequence of the genome and was detected in four typical Japanese SbDV strains, YS, YP, DS and DP. In the case of SbDV-DS, the RNA was 220 nucleotides in length and was transcribed from the 3 terminal region of the genome. This RNA appeared at a similar time to genomic RNA and a large sgRNA, and thereafter persisted in the infected plant. Since no conserved open reading frame (ORF) among the strains was postulated in the 3 terminal region, the small subgenomic RNA may have some regulatory roles in SbDV infections.Received December 12, 2002; accepted April 16, 2003 Published online July 2, 2003     

Association analyses of DNA methyltransferase-1 (<Emphasis Type="Italic">DNMT1</Emphasis>) polymorphisms with systemic lupus erythematosus

The etiology of systemic lupus erythematosus (SLE) is very complex, and genetic factors appear to play a significant role in susceptibility to SLE, in determining the disease expression, and in the autoantibody profiles of individuals with SLE. DNA methyltransferase-1 (DNMT1) is a major enzyme that determines genomic methylation patterns and both maintains methyltransferase and exhibits de novo DNA methylation activity in vivo. In order to clarify the association of DNMT1 polymorphisms with SLE, we scrutinized the genetic polymorphisms in exons and their boundaries of DNMT1, including the –1,500 bp promoter region, by direct sequencing in 24 Korean individuals. Twenty-nine sequence variants were identified: two in 5UTR, six in exons, and 21 in introns. Eight of these polymorphisms were selected for a larger-scale genotyping (n=680) by considering their allele frequencies, haplotype-tagging status, and linkage disequilibrium coefficiencies (LDs) among polymorphisms. The associations between DNMT1 polymorphisms and the clinical profiles of SLE were analyzed. No significant associations with the risk of SLE were detected. However, further analyses of association with autoantibody production among SLE patients revealed that one nonsynonymous SNP, +14463G>C (V120L) in exon 4, was weakly associated with an increased risk of anti-La antibody production (P=0.04), although the significance could not be retained after correction of multiple tests. The DNMT1 variations and haplotypes clarified in this study would provide valuable information for future genetic studies of other autoimmune diseases.     

Two unrelated double-stranded RNA molecule patterns in <Emphasis Type="Italic">Gremmeniella abietina</Emphasis> type A code for putative viruses of the families <Emphasis Type="Italic">Totiviridae</Emphasis> and <Emphasis Type="Italic">Partitiviridae</Emphasis>

Summary. Two double stranded (ds) RNA molecule patterns, probably of viral origin, were sequenced from Gremmeniella abietina var. abietina type A. The genome of Gremmeniella abietina RNA virus L1 (GaRV-L1) from isolate HR2 was 5133bp and contained two open reading frames (ORFs). The 5-proximal ORF coded for a putative coat protein (CP) and the 3-proximal ORF encoded putative RNA-dependent RNA polymerase (RdRp). GaRV-L1 had sequence similarities with a previously described totivirus (Helminthosporium victoriae 190S virus) and two unclassified members of family Totiviridae (Sphaeropsis sapinea RNA virus 1 and Sphaeropsis sapinea RNA virus 2). The genome of Gremmeniella abietina RNA virus MS1 (GaRV-MS1) from isolate C5 was composed of three dsRNA molecules coding for a putative RdRp (dsRNA1), a putative CP (dsRNA2) and protein of unknown function (dsRNA3). The lengths of these dsRNA molecules were 1782, 1586 and 1181bp, respectively. Sequence comparisons indicated that the GaRV-MS1 dsRNA pattern comprises a putative virus that is highly similar to Discula destructiva virus 1, Discula destructiva virus 2 and Fusarium solani virus 1 of the family Partitiviridae.     

Analysis of genetic variability of Indian isolates of <Emphasis Type="Italic">Hepatitis C virus</Emphasis>

Summary. The degree of genetic variability among Hepatitis C virus strains circulating in India is currently unknown. In order to get insight into this matter, sequence data obtained from the 5 non coding region from 8 patients from New Delhi were compared with sequences from 16 HCV isolates from different geographic locations of India. The phylogenetic analysis of most prevalent genotypes revealed the presence of novel HCV variants in type 1 strains.     

Expression of exogenous genes in<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis>: improving vectors and electroporation protocols

To improve transfection efficiency in Trypanosoma cruzi, we developed a new electroporation protocol and expression vectors which use luciferase and green and red fluorescent proteins as reporter genes. In transient transfections, the electroporation conditions reported here resulted in luciferase expression 100 times higher than the levels obtained with previously described protocols. To verify whether sequences containing different trans-splicing signals influence reporter gene expression, we compared DNA fragments corresponding to 5 untranslated plus intergenic (5 UTR plus Ig) regions from GAPDH, TcP2, - and -tubulin and amastin genes. Vectors containing sequences derived from the first four genes presented similar efficiencies and resulted in luciferase expression in transiently transfected epimastigotes that was up to 10 times higher than that for a control vector. In contrast, the amastin 5 UTR plus Ig resulted in lower levels of reporter gene expression. We also constructed a vector containing an expression cassette designed to be targeted to the tubulin locus of the parasite.     

Strains of <Emphasis Type="Italic">Peru tomato virus</Emphasis> infecting cocona (<Emphasis Type="Italic">Solanum sessiliflorum</Emphasis>), tomato and pepper in Peru with reference to genome evolution in genus <Emphasis Type="Italic">Potyvirus</Emphasis>

Summary. Two isolates (SL1 and SL6) of Peru tomato virus (PTV, genus Potyvirus) were obtained from cocona plants (Solanum sessiliflorum) growing in Tingo María, the jungle of the Amazon basin in Peru. One PTV isolate (TM) was isolated from a tomato plant (Lycopersicon esculentum) growing in Huaral at the Peruvian coast. The three PTV isolates were readily transmissible by Myzus persicae. Isolate SL1, but not SL6, caused chlorotic lesions in inoculated leaves of Chenopodium amaranticolor and C. quinoa. Isolate TM differed from SL1 and SL6 in causing more severe mosaic symptoms in tomato, and vein necrosis in the leaves of cocona. Pepper cv. Avelar (Capsicum annuum) showed resistance to the PTV isolates SL1 and SL6 but not TM. The 5- and 3-proximal sequences of the three PTV isolates were cloned, sequenced and compared to the corresponding sequences of four PTV isolates from pepper, the only host from which PTV isolates have been previously characterised at the molecular level. Phylogenetic analyses on the P1 protein and coat protein amino acid sequences indicated, in accordance with the phenotypic data from indicator hosts, that the PTV isolates from cocona represented a distinguishable strain. In contrast, the PTV isolates from tomato and pepper were not grouped according to the host. Inclusion of the sequence data from the three PTV isolates of this study in a phylogenetic analysis with other PTV isolates and other potyviruses strengthen the membership of PTV in the so-called PVY subgroup of Potyvirus. This subgroup of closely related potyvirus species was also distinguishable from other potyviruses by their more uniform sizes of the protein-encoding regions within the polyprotein.The first two authors contributed equally.     

Identification of a Naturally Occurring Recombinant Isolate of <Emphasis Type="Italic">Sugarcane Mosaic Virus</Emphasis> Causing Maize Dwarf Mosaic Disease

The complete nucleotide sequence of a potyvirus causing severe maize dwarf mosaic disease in Shaanxi province, northwestern China was determined (GenBank accession No. AY569692). The full genome is 9596 nucleotides in length excluding the 3 -terminal poly (A) sequence. It contains a large open reading frame (ORF) flanked by a 149 nt 5-untranslated region (UTR) and a 255 nt 3-UTR. The putative polyprotein encoded by this large ORF comprises of 3063 amino acid residues. Sequence comparisons and phylogenetic analyses showed that this potyvirus is an isolate of Sugarcane mosaic virus (SCMV). The entire sequences shared identities of 89.6–97.6 % and 79.3–93.3% with 9 sequenced SCMV isolates at the nucleotide and deduced amino acid levels, respectively. But it showed much lower identities with Maize dwarf mosaic virus (MDMV), Sorghum mosaic virus (SrMV) and Johnsongrass mosaic virus (JGMV) isolates. The putative coat protein sequence is identical to that of a Chinese maize isolate SCMV-HZ. However, partition comparisons and phylogenetic profile analyses of the viral nucleotide sequences indicated that it is a recombinant isolate of SCMV. The recombination sites are located within the 6K1 and CI coding regions.     

Endogenous retrovirus HC2 <Emphasis Type="Italic">pol</Emphasis> fragments in the squirrel monkey: expression,evolution, and phylogeny

Summary. Human endogenous retrovirus HC2 is an incomplete provirus containing the entire gag and pol genes and a 3 LTR, whereas the 5 LTR and env gene are missing. We investigated expression of the HC2 pol gene in the squirrel monkey (Saimiri sciureus) by RT-PCR. The pol gene was expressed in cerebellum, liver, lung, and spleen of the squirrel monkey, but not in six other tissues tested. RT-PCR products were cloned and sequenced resulting in seven sequences that were analyzed. These sequences showed 73.7–89.2% sequence similarity to HC2 pol genes present in the human genome. No frameshifts or termination codons caused by deletion/insertion or point mutation were found in clones SM-HC27-1 and SM-HC27-4 isolated from squirrel monkey lung tissues. Phylogenetic analysis showed that HC2 pol elements from the squirrel monkey were randomly clustered with those in human genome and the genomes of other nonhuman primates, indicating that substantial evolution of the HC2 elements occurred prior to primate speciation with additional evolution of the elements, independent of each other, after speciation.Received January 8, 2003; accepted June 11, 2003 Published online August 7, 2003     

Complete nucleotide sequence of the RNA 1 of a grapevine isolate of <Emphasis Type="Italic">Arabis mosaic virus</Emphasis>

Summary. The complete nucleotide sequence of the genomic RNA 1 of the grapevine isolate NW (Neustadt an der Weinstrasse) of Arabis mosaic virus (ArMV) was determined. It is 7334 nucleotides long excluding the poly(A) tail, and contains one long open reading frame encoding a polypeptide of 2284 amino acids. The 5 and 3 non-coding regions were 227 and 252 nucleotides long respectively, and showed stretches of high identity with the corresponding 5 and 3 non-coding regions of ArMV-NW RNA 2. The analysis of the amino acid sequence of the polyprotein encoded by the RNA 1 of the ArMV-NW showed that the conserved amino acid motifs, characteristic for the viral protease co-factor, the NTP-binding protein, the cystein protease, and the RdRp core domains, were all present. Amino acid sequence comparisons between the polyproteins encoded by the RNAs 1 of ArMV-NW and other nepoviruses showed 75% identity with the GFLV-F13, and up to 36% with other nepoviruses.     

Rapid pathotyping of <Emphasis Type="Italic">Newcastle disease virus</Emphasis> from allantoic fluid and organs of experimentally infected chickens using two novel probes

Summary. A system including RNA isolation, primers and two novel oligonucleotide probes (NC22 and VC22) was established to rapidly pathotype Newcastle disease virus (NDV) from infected allantoic fluid; The sequence of the probes is: VC22, 5-AAGGAGGCAGAAACGCTTTATA-3; NC22, 5-GGGGAGACAGG GGCGCCTTATA-3. Application efficacy of the probes was verified by differentiating NDV that derived from experimentally infected organs. NC22 and VC22 both were labeled with digoxigenin (DIG) and were successful in differentiating NDV strains from the infected allantoic fluid and organs of experimentally infected SPF chickens. The RT-PCR products of NDV isolates and strains were dotted onto nylon membrane and then hybridized with two specific probes separately. The VC22 probe is specific to virulent strains, and the NC22 probe is specific to avirulent strains. The results of hybridization coincide with viral intracerebral pathologenicity index (ICPI). The specificity of two probes and sensitivity of NC22 probe were evaluated. NC22 could detect down to 10^–8 dilution from 10^7.5 EID₅₀/ml or 800fg of viral RNA. The system could be completed within 1 day to conduct experiment from clinical sample to the result of assay, and its potential practical application in clinical assay was discussed basing on specificity, sensitivity and rapidness.     

Determination of complete nucleotide sequence of <Emphasis Type="Italic">Hibiscus latent Singapore virus</Emphasis>: Evidence for the presence of an internal poly(A) tract

Summary. We have sequenced the complete genome of a hibiscus-infecting tobamovirus, Hibiscus latent Singapore virus (HLSV). The experimental host range of HLSV is similar to that of another distinct species of hibiscus infecting tobamovirus, Hibiscus latent Fort Pierce virus (HLFPV). The genomic structure of HLSV is similar to other tobamoviruses in general. It consists of a 5 untranslated region (UTR), followed by ORFs encoding for a 128kDa protein and a 186kDa readthrough protein, a 30kDa movement protein (MP), 18kDa coat protein (CP) and a 3 UTR. The unique feature of HLSV is the presence of a poly(A) tract within its 3 UTR. In our previous work, we have reported MP and CP sequences of HLSV and its phylogenetic analysis. Here we report the complete nucleotide sequence of HLSV, phylogenetic analysis of the nucleotide and amino acid sequences of 128/186kDa ORFs and the presence of a uniquely located poly(A) tract within the 3 UTR.The GenBank accession numbers of the sequences reported in this paper are AF400156, AF400157 and AY497578, respectively.     

Molecular characterization of a partitivirus from the plant pathogenic ascomycete <Emphasis Type="Italic">Rosellinia necatrix</Emphasis>

Summary. The W8 isolate of the phytopathogenic fungus, Rosellinia necatrix that causes white root rot, contained three segments of double-stranded (ds) RNA, namely L1, L2 and M. Purified viral particles of about 25nm in diameter contained an RNA segment with almost the same mobility as M-dsRNA, but the band was sensitive to S1 nuclease. Molecular analysis revealed that M-dsRNA consisted of two (RNA 1 and RNA 2) similarly sized species of 2299 and 2279bp excluding an interrupted poly (A or U) tail of 16–51bp. The predicted largest open reading frame in RNA 1 and RNA 2 was similar to those of RNA dependent RNA polymerase (RdRp) and coat protein (CP), respectively, encoded by the family Partitiviridae. The non-coding regions (NCR) of the two segments were similar (approximately 70% base identity) at the 5 end, but different at the 3 end. The NCR at the 3 end contained adenosine-uracil rich elements (AREs) in both segments. Northern analyses revealed RNA 1 and RNA 2 in mycelial and viral particle fractions. We coined the name Rosellinia necatrix partitivirus 1-W8 (RnPV1-W8) for M-dsRNA based on viral particle morphology and sequence information.     

The complete nucleotide sequence,genome organization,and specific detection of <Emphasis Type="Italic">Beet mosaic virus</Emphasis>

Summary. Beet mosaic virus (BtMV) was identified almost five decades ago but has not been fully characterized at the molecular level. In this study, we have determined for the first time the complete nucleotide sequence of BtMV genomic RNA and have developed a specific molecular means for its diagnosis. The viral genome of BtMV comprises 9591 nucleotides, excluding the 3 terminal poly (A) sequence, and contains a single open reading frame (ORF) that begins at nt 166 and terminates at nt 9423, encoding a single polyprotein of 3086 amino acid residues. A 3 untranslated region of 168 nucleotides follows the ORF. The deduced genome organization is typical for a member of the family Potyviridae and includes 10 proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, NIa-Pro, NIb and coat protein (CP). Nine putative protease cleavage sites were predicted computationally and by analogy with genome arrangements of other potyviruses. Conserved sequence motifs of homologous proteins of other potyviruses were found in corresponding positions of BtMV. BtMV is a distinct species of the genus Potyvirus with the most closely related species being Peanut mottle virus (55% amino acid identity). Based on the nucleotide sequence obtained, we have developed a virus-specific RT-PCR assay for accurate diagnosis and differentiation of BtMV.     

A novel<Emphasis Type="Italic"> Cryptosporidium parvum</Emphasis> antigen,CP2, preferentially associates with membranous structures

The present study addresses the cloning and characterization of a Cryptosporidium parvum antigen, CP2. Sequencing of cDNA and genomic clones revealed a novel gene capable of coding a message of 2,136 nucleotides flanked by 28 and 140 nucleotides of the 5- and 3-noncoding regions, respectively. The deduced amino acid sequence suggests that CP2 is a secreted and/or membrane protein. Immunofluorescence microscopy detected CP2 enrichment in sporozoites that subsequently appeared to encase type I meronts in infected HCT-8 cells. Immunogold electron microscopy revealed that CP2 consistently localized to membranous structures throughout development. In addition, progression from macrogametocyte to sporulated oocyst revealed CP2 initially at the periphery of amylopectin-like granules, in the cytoplasm and discrete vesicles, the parasitophorous vacuole, on the surface of sporozoites, and finally on the parasitophorous vacuole membrane (PVM). The observed expression pattern suggests that CP2 may be involved in the invasion process and/or PVM integrity.     

Characterization and expression of the<Emphasis Type="Italic"> Neurospora crassa nmt-1</Emphasis> gene

The Neurospora crassa homologue of the yeast no message in thiamine (nmt-1) gene was characterized. The deduced 342-amino-acid gene product has more than 60% identity with other fungal homologues and 42% similarity to a putative bacterial permease. In addition to three introns disrupting the coding sequence, a differentially spliced intron in the 5 untranslated region was also detected. Unlike other fungi, the N. crassa nmt-1 gene is repressed only 6- to 8-fold by exogenous thiamine concentrations above 0.5 µM; and a high basal level of nmt-1 mRNA persists even at 5 µM thiamine. Immuno-blotting with purified antibodies detected two variants of NMT-1 which differ in size and charge. The more abundant 39-kDa form is more strongly repressed by thiamine than the 37-kDa protein. NMT-1 abundance modulates slowly in response to changes in the concentration of exogenous thiamine, suggesting that N. crassa maintains thiamine reserves in excess of immediate needs. Disruption of the nmt-1 gene demonstrated that it is essential for growth in the absence of exogenous thiamine. NMT-1-deficient strains had a growth rate and colony density which was about 70% of the wild type, despite supplementation with a wide range of exogenous thiamine. These results suggest that the nmt-1 gene plays some other role in addition to thiamine biosynthesis.Communicated by U. Kück