Nucleotide Sequence Of Isolate Research Articles

Identification of an EcoRI restriction site for a rapid and precise determination of β-asarone-free Acorus calamus cytotypes

Calamus ( Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of β-asarone [( Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid β-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography–mass spectrometry (GC–MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of β-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-β-ocimene (3.28%), camphor (1.54%), calarene (1.42%), α-selinene (5.02%) and τ-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), β-sesquiphellandrene (3.28%), pre iso calamendiol (22.81%) and acorone (26.33%), and completely lacked of β-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3′ and 5′ ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM®-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid β-asarone-free A. calamus was also discussed.

Identification of a calicivirus isolate of unknown origin.

Chinese hamster ovary (CHO) cells manifesting striking cytopathogenic changes in culture were investigated to determine the causative agent. Electron microscopic analyses revealed viral particles of about 40 nm in diameter, displaying typical calicivirus morphology. To date, this virus, designated isolate 2117, exclusively replicates in CHO cells, achieving only moderate titres. After cloning, the coding region of 7928 nucleotides, the 3' non-coding region and the poly(A) tail were sequenced. The genome consists of three open reading frames (ORFs), with the first and second ORF having the same reading frame. The overall genomic organization as well as the nucleotide sequence of isolate 2117 is most similar to that of a recently described canine calicivirus, but also shows significant similarity to the sequences of mink calicivirus and other caliciviruses within the genus Vesivirus: In Western blots, using antibodies against the viral protease, a stable, unprocessed 3CD protein of 68 kDa was identified in homogenates of 2117-infected CHO cells. Furthermore, antibodies raised against ORF 3 reacted with the respective protein in 2117-virions, demonstrating that this predicted 9 kDa protein is a minor structural component of the virion. In addition, an RT-PCR assay was established to detect 2117 viral RNA in biological products such as foetal bovine serum, which will aid the discovery of the origin and host of the virus.

Complete Nucleotide Sequences of the Genomes of Two Brazilian Specimens of Human T Lymphotropic Virus Type 2 (HTLV-2)

We report the complete nucleotide sequences of the genomes of two human T cell lymphotropic viruses type 2 (HTLV-2) isolated from a Kayapó Indian (K96 isolate) and from an inhabitant of an urban region in the south of Brazil (RP329 isolate). The general structure of the K96 and RP329 genomes did not differ from that of other HTLV-2 genomes described in the literature. The K96 genome consisted of 8955 bp and the RP329 genome consisted of 8964 bp. The general similarity between the nucleotide sequences of the K96 and RP329 genomes was 99.4%. Comparison between the nucleotide sequences of the K96 and RP329 genomes and the nucleotide sequences of isolates considered to be HTLV-2 prototypes of subtype 2a (Mo isolate), 2b (NRA isolate), and 2d (Efe2 isolate) showed a global similarity of 98.8, 95.6, and 93.3%, respectively, for the RP329 isolate, and of 99.1, 95.6, and 93.3%, respectively, for the K96 isolate. Phylogenetic analysis permitted the classification of the K96 and RP329 isolates as HTLV-2 subtype 2a. Detailed phylogenetic analyses of the LTR, env, and Tax regions showed that the Brazilian isolates tend to form a distinct phylogenetic subgroup within subtype 2a, previously called HTLV-2c, which differs from the subtype 2a isolates found in North America, Europe, and Africa. The K96 genome is the first HTLV-2 genome obtained from a Brazilian Indian that was completely sequenced, whereas the RP329 genome represents the first specimen derived from an inhabitant of a Brazilian urban region who was not coinfected with HIV-1.

Cellulase genes from the parabasalian symbiont Pseudotrichonympha grassii in the hindgut of the wood-feeding termite Coptotermes formosanus.

Cellulase genes of Pseudotrichonympha grassii (Hypermastigida: Eucomonymphidae), the symbiotic flagellate in the hindgut of the wood-feeding termite Coptotermes formosanus, were isolated and characterized. The nucleotide sequences of the major cellulase component in the hindgut of C. formosanus were determined based on its N-terminal amino acid sequence. The five isolated nucleotide sequences (PgCBH-homos) had an open reading frame of 1350 bp showing similarity to catalytic domains of glycoside hydrolase family (GHF) 7 members, and primary structure comparison with GHF7 members whose tertiary structures are well-characterized revealed the overall similarity between PgCBH-homo and the catalytic domain of a processive cellulase Cel7A (formerly CBHI) from the aerobic fungus Trichoderma reesei. Functional expression of PgCBH-homos in Escherichia coli, using the carboxymethylcellulose-Congo red assay, demonstrated the actual cellulolytic activity of PgCBH-homo. RT-PCR showed that PgCBH-homos were expressed, from the three flagellates in the hindgut, specifically in P. grassii.

First report of Beet virus Q in Belgium.

Beet necrotic yellow vein virus (BNYVV), the causal agent of rhizomania disease on sugar beet, has been reported in Belgium for more than 16 years. Other soilborne viruses belonging to the genus Pomovirus, such as Beet soilborne virus (BSBV) (3) and Beet virus Q (BVQ) (1), are suspected pathogens of sugar beets grown in Belgium. During the 2000 growing season, more than 20 fields showing rhizomania-like and yellowing symptoms on sugar beet leaves were investigated for the presence of BVQ, BNYVV, and BSBV. All samples were checked by enzyme-linked immunosorbent assay (ELISA) using commercial BNYVV (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France) and BSBV/BVQ (DSMZ, Braunschweig, Germany - AS-0576 polyclonal, AS-0576/2 MAb) antisera. RNA was extracted from sugar beet rootlets using an RNeasy extraction kit (Qiagen, Hilden, Germany), before performing a reverse transcription-polymerase chain reaction (RT-PCR) using primers (5'-GCTGGAGTATATCACCGATGAC-3' and 5'-AAAATC TCGGATAGCATCCAAC-3') designed to specifically amplify a 510-bp region of BVQ RNA-1. The presence of BSBV and BNYVV was also checked by RT-PCR using previously described primers (1,2). The BVQ-derived PCR product was sequenced and proved to be more than 99% identical to the Wierthe BVQ isolate nucleotide sequence. Soil transmission of BVQ was demonstrated through a bioassay using soil dilutions with quartz and sugar beet cv. Cadyx as bait. After 6 weeks, BVQ was detected by RT-PCR in bait plants. The putative vector, Polymyxa betae, was identified by lactophenol-cotton blue staining of the roots followed by microscopic examination. BVQ produces irregularly shaped local lesions that appear ≈5 days after mechanical inoculation and tend to spread along veins. BVQ was detected in six fields located in the Polders Region and Brabant Province of Belgium. BVQ was always found in sugar beet samples coinfected with BNYVV and BSBV. The economic significance of BVQ and its interaction with other viruses is not known.

Identification and toxicity of Alternaria brassicicola, the causal agent of dark leaf spot disease of Brassica species grown in Thailand

Fungi, with spores characteristic of the genus Alternaria, were isolated from necrotic lesions on leaves of cabbage, cauliflower, Chinese kale and choi‐sum growing in Thailand, and were proved by Koch's postulates to be the causal agents of a disease known as dark leaf spot. All isolates corresponded in morphology to descriptions of Alternaria brassicicola and the identification was confirmed by analysis of the ITS1, 5.8S gene and ITS2 regions of rDNA, the nucleotide sequences of isolates from all four plants being identical to each other and to the published sequence of a known isolate of A. brassicicola. Culture filtrates of isolates of the fungus from each host, grown on a defined medium consisting of Czapek–Dox nutrients supplemented with cations, were toxic to cells isolated from the four host plants. Taking the data overall, filtrates from the cauliflower isolate were significantly less toxic than those from the other isolates. Although the filtrate from the cabbage isolate was most toxic to cabbage cells and that of the choi‐sum isolate most toxic to choi‐sum cells, filtrates of the Chinese kale and cauliflower isolates were most toxic to cells of plants other than those from which they were isolated.

Molecular diversity of 5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis.

Beimu (bulbs of Fritillaria) is an important traditional Chinese herbal medicine commonly used as an antitussive and expectorant. There are about 25 species and varieties of Fritillaria that carry the name Beimu on commercial markets. The price for each Beimu may differ by more than 100-fold. However, the identification of the origin of a particular species on the market is difficult. Here, a molecular method was used to identify various species of Fritillaria regardless of their geographical origin. The 5S-rRNA coding sequence is highly conserved in higher eukaryotes, but the spacer sequence of the 5S-rRNA gene is variable among different species. Total genomic DNA from fresh leaves and bulbs of Fritillaria cirrhosa, F. puqiensis, F. anhuiensis, and F. thunbergii was extracted. The 5S-rRNA spacer region of the extracted DNAs was amplified by PCR with a pair of primers located within the conserved coding region. The isolated cDNA clones (approximately 600 bp) covering the 5S-rRNA spacer domain were sequenced. By aligning the isolated nucleotide sequences of the four Fritillaria species, sequence diversity was found in the spacer region. Furthermore, a unique EcoR I site was used for the rapid identification of different species of Fritillaria. To our knowledge, this is the first report on the detection of 5S-rRNA spacer domain sequences of Fritillaria and their use to identify species.

Human papillomavirus‐5b DNA integrated in a metastatic tumor: cloning, nucleotide sequence and genomic organization

Human papillomavirus‐5b DNA integrated in a metastatic tumor: cloning, nucleotide sequence and genomic organization

5389515 Isolated nucleotide sequences for identifying Neisseria Gonorrhoeae : Chemlo Rich; Foltz Lisa Indianapolis, IN, United States Assigned to Boehringer Mannheim Corporation

5389515 Isolated nucleotide sequences for identifying Neisseria Gonorrhoeae : Chemlo Rich; Foltz Lisa Indianapolis, IN, United States Assigned to Boehringer Mannheim Corporation

5389515 Isolated nucleotide sequences for identifying Neisseria Gonorrhoeae : Chmelo Rich; Foltz Lisa Indianapolis, IN, United States Assigned to Boehringer Mannheim Corporation

5389515 Isolated nucleotide sequences for identifying Neisseria Gonorrhoeae : Chmelo Rich; Foltz Lisa Indianapolis, IN, United States Assigned to Boehringer Mannheim Corporation

5177179 Isolated nucleotide sequence expressing human transforming growth factor-beta1-binding protein

5177179 Isolated nucleotide sequence expressing human transforming growth factor-beta1-binding protein

5177197 Isolated nucleotide sequence expressing human transforming growth factor- betal-binding protein: Tetsuto Kanzaki, Anders Olofsson, Anita Moren, Christer Wernstedt, Ulf Hellman, Kohei Miyazono, Lena Claesson-Welsh, Carl-Henrik Heldin, Chiba, Japan assigned to Ludwig Institute for Cancer Research

5177197 Isolated nucleotide sequence expressing human transforming growth factor- betal-binding protein: Tetsuto Kanzaki, Anders Olofsson, Anita Moren, Christer Wernstedt, Ulf Hellman, Kohei Miyazono, Lena Claesson-Welsh, Carl-Henrik Heldin, Chiba, Japan assigned to Ludwig Institute for Cancer Research

5177197 Isolated nucleotide sequence expressing human transforming growth factor- beta1-binding protein: Tetsuto Kanzaki, Anders Olofsson, Anita Moren, Christer Wernstedt, Ulf Hellman, Kohei Miyazono, Lena Claesson-Welsh, Carl-Henrik Heldin, Chiba, Japan assigned to Ludwig Institute for Cancer Research

5177197 Isolated nucleotide sequence expressing human transforming growth factor- beta1-binding protein: Tetsuto Kanzaki, Anders Olofsson, Anita Moren, Christer Wernstedt, Ulf Hellman, Kohei Miyazono, Lena Claesson-Welsh, Carl-Henrik Heldin, Chiba, Japan assigned to Ludwig Institute for Cancer Research

Two distinct subtypes of hepatitis C virus defined by antibodies directed to the putative core protein.

Four distinct genotypes of hepatitis C virus types I, II, III and IV have been identified by comparison of nucleotide sequences of isolates from different areas of the world. We examined the possibility that hepatitis C virus may have serologically definable subtypes. Enzyme-linked immunosorbent assay systems were prepared by use of two synthetic peptides deduced from the putative core protein of hepatitis C virus. The following are the two peptides that were used: (a) IPKARRPEGRTWAQPGY (subtype-1) conserved in hepatitis C virus isolates with type I and type II genotypes; and (b) IPKDRRSTGKSWGKPGY (subtype-2) conserved in type III and type IV genotypes. With the enzyme-linked immunosorbent assays, the subtype-1 antibodies were detected in 26 (68%) of 38 subjects whose hepatitis C virus RNA had been genotyped as type I or type II, whereas subtype-2 antibodies were not detected. Inversely, the subtype-2 antibodies were detected in 10 (56%) of 18 subjects with hepatitis C virus RNA genotypes III or IV, whereas subtype-1 antibodies were detected in none of them. These results suggest that hepatitis C virus has two serologically distinguishable core antigen subtypes, corresponding to either genotype I/II or genotype III/IV. Subtyping of HCV by serological methods would contribute to tracking transmission routes of the virus, especially in cases where serum samples were not stored under conditions to preserve RNA or in infected hosts who have cleared the virus and therefore have only antibodies remaining to identify the infection.

Phylogenetically different strains of a variant of coxsackievirus A 24 were repeatedly introduced but discontinued circulating in Japan.

Variations in the nucleotide sequence of 3 C proteinase of coxsackievirus A 24 variant (CA 24 v) were analyzed to define the route of transmission and spread of the virus which was introduced to Japan on three separate occasions, 1985-86, 1988, and 1989. The nucleotide sequences of isolates from the same year's outbreak in Japan were identical or closely related, while the isolates from different outbreaks were less closely related to one another than to those from other countries in the same year. All Japanese isolates from Okinawa and other prefectures in 1985 and 1986 were closely related to the Taiwan strains in those same years, indicating common-source outbreaks. Two 1988 isolates from Chiba Prefecture, Japan, were closely related to those from Singapore in 1987, China in 1988 and Hong Kong in 1988. All seven Japanese isolates from Chiba Prefecture in 1989 comprised a group together with the Taiwan and Singapore strains in 1988. The results indicate that CA 24 v was introduced into Japan on each occasion from the outside. Furthermore, in contrast to the explosive epidemics in Okinawa Prefecture in 1985 and 1986, the virus which was repeatedly introduced to other areas in Japan did not circulate endemically, and disappeared within a short time.

Genetic heterogeneity among human papillomaviruses (HPV) associated with epidermodysplasia verruciformis: evidence for multiple allelic forms of HPV5 and HPV8 E6 genes

In order to get some insight into modifications of human papillomavirus (HPV) genomes which could play a role in tumor progression in epidermodysplasia verruciformis (EV), we studied three EV patients infected by HPV5 and one by HPV8, with cancers containing mostly or only episomal viral genomes with a deletion. The mutants were compared with the full-length genomes present in the benign lesions of each patient. Deletions affected the L1 and/or L2 open reading frames (ORFs), and extended in the 5′ end of the long control region in two cancers. The isolates studied showed a polymorphism of restriction endonuclease cleavage sites and variations in the nucleotide sequence of the E6 ORF and the regions flanking the deletions. However, except for one patient infected by two distinct HPV5 variants, no difference was observed in the nucleotide sequence of isolates cloned from the benign lesions and the cancer of the same patient. This may suggest that point mutations are not involved in tumor progression. Comparison of nucleotide sequence data revealed an unexpectedly high number of nucleotide substitutions among the four HPV5 variants and the HPV8 variant, as compared with HPV5 and HPV8 published sequences. Changes involved 49 of the 457 nucleotides of HPV5 E6 ORF and 14 of the 465 nucleotides of HPV8 E6 ORF. This corresponds to amino acid substitutions affecting 17 of the 157 amino acids of HPV5 E6 proteins and 7 of the 155 amino acids of HPV8 E6 proteins. Half of the substitutions represent nonconservative changes. The variants showing the highest degree of sequence variation were detected in additional EV patients by PCR. This points to the existence of a set of HPV5 and HPV8 stable variants, encoding for multiple allelic forms of the transforming E6 gene.