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PubMed2002

Allozyme analysis of cryptic species in the Asplenium niduscomplex from West Java, Indonesia.

In various fern species, a large amount of rbcL sequence variation has been reported, and it is possible that these species contain several reproductively isolated cryptic species. In our previous study on Asplenium nidus L., it was suggested that the plants growing in Mt. Halimun National Park, West Java, Indonesia, consist of several cryptic species based on the results of crossing experiments among rbcL sequence types. In this study, we examined allozyme polymorphisms of five rbcL sequence types found in West Java in order to test the hypothesis that the assemblages of A. nidus delimited based on the rbcL sequences are separate Mendelian populations and gene flow is disrupted by reproductive isolation from one another. The calculated fixation indices suggested that the individuals in each rbcL type are randomly crossing at least in the investigated localities. Nevertheless, these rbcL-based assemblages were genetically differentiated in allozymes that are encoded in their nuclear genomes, and it is also suggested that gene flow is disrupted even between sympatrically distributed pairs of rbcL sequence types. Therefore, our findings support the view that the five rbcL sequence types in West Java are potential cryptic species.

Journal Article↗

PubMed2004

Identification of a novel HLA-DRB1 allele (DRB11147) by sequence-based typing in eight siblings.

We report here a novel DRB1 allele (DRB1*1147) identified by sequence-based typing. The DRB1*1147 allele was detected during routine human leukocyte antigen (HLA)-typing of a patient and his family prior to bone marrow transplantation. This allele was found in the patient and his two sisters and five brothers. The new allele is identical to DRB1*110601 at exon 2 except for a single-nucleotide substitution at codon 37 (TAC-->TCC), changing the encoded tyrosine to serine.

Alleles↗

PubMedDate not supplied

[Genetic relationships of Moscow and foreign strains of Haemophilus influenzae, serotype B].

46 Haemophilus influenzae strains of serotype b, isolated on the territory of Moscow and compared with foreign strains, were characterized by the method of multilocus sequencing-typing. Among the strains circulating in Moscow 10 variants of sequence-types were observed; of these, only one variant (CT-6) had been described earlier. The analysis of the data revealed that the strains under study were distributed in two clonal complexes; the overwhelming majority of the strains belonged to sequence-type 6. The distribution of Moscow strains in clonal complexes repeated the clonal organization detected in foreign strains. The conclusion was made that lower morbidity rate in Hib meningitis in Moscow (in comparison with that in the countries of Europe and America before the introduction of prophylactic vaccination) was not due to the peculiar genetic features of the circulating strains.

Americas↗

PubMed1998

Molecular characterization of epiphytic bacterial communities on charophycean green algae

Epiphytic bacterial communities within the sheath material of three filamentous green algae, Desmidium grevillii, Hyalotheca dissiliens, and Spondylosium pulchrum (class Charophyceae, order Zygnematales), collected from a Sphagnum bog were characterized by PCR amplification, cloning, and sequencing of 16S ribosomal DNA. A total of 20 partial sequences and nine different sequence types were obtained, and one sequence type was recovered from the bacterial communities on all three algae. By phylogenetic analysis, the cloned sequences were placed into several major lineages of the Bacteria domain: the Flexibacter/Cytophaga/Bacteroides phylum and the alpha, beta, and gamma subdivisions of the phylum Proteobacteria. Analysis at the subphylum level revealed that the majority of our sequences were not closely affiliated with those of known, cultured taxa, although the estimated evolutionary distances between our sequences and their nearest neighbors were always less than 0.1 (i.e., greater than 90% similar). This result suggests that the majority of sequences obtained in this study represent as yet phenotypically undescribed bacterial species and that the range of bacterial-algal interactions that occur in nature has not yet been fully described.

Journal Article↗

PubMed1986

Structures of glycosphingolipids isolated from human embryonal carcinoma cells. The presence of mono- and disialosyl glycolipids with blood group type 1 sequence.

Structures of glycolipids present in the human embryonal carcinoma cell PA1, were elucidated by fast atom bombardment-mass spectrometry, methylation analysis, and exo- and endoglycosidase digestion. PA1 cells contain globotriaosylceramide, sialosylgangliotriaosylceramide, sialylated and nonsialylated lacto-N-neotetraosylceramide, and the following glycolipids with a blood group type 1 sequence: (formula; see text) The two former glycolipids, lacto-N-tetraosylceramide and sialosyllacto-N-tetraosylceramide, reacted with monoclonal antibodies, K21 and K4, respectively. K21 and K4 antigens are present in many of the human embryonal carcinoma cells but not in a variety of other cell lines, suggesting that sialylated but not fucosylated blood group type 1 sequences are characteristic markers for human embryonal carcinoma cells and malignant teratocarcinomas.

Antigens↗

PubMed1999

Sequencing based typing for genetic polymorphisms in exons, 2, 3 and 4 of the MICA gene.

We have established a sequencing based typing (SBT) method for detection of genetic polymorphism in the exon 2 to 4 domains of the major histocompatibility complex (MHC) class I chain-related gene A (MICA) and applied it to allele typing of 130 healthy Japanese individuals. A 2.2-kb segment including exons 2, 3 and 4 of the MICA gene was amplified by a pair of generic primers followed by cycle sequencing using exon-specific nested primers. In total, 8 alleles were observed in a Japanese population and the most frequent allele was MICA008 with the gene frequency of 30.8%. MICA009 was the second most frequent (16.5%), while the rarest one was MICA007 (1.2%). MICA alleles displayed strong linkage equilibria with HLA-B antigens (i.e. MICA008 with B7, B48, B60 and B61; MICA009 with B51 and B52; MICA002 with B35, B39, B58 and B67; MICA004 with B44, MICA007 with B13 and B27; MICA010 with B46, B62 and B48, MICA012 with B54, B55, B56 and B59; MICA019 and B70, B71 and B62). Recently, the B48 haplotype has been reported to lack the entire MICA gene by a large-scale deletion in a Japanese population. Among 8 serologically B48 homozygous individuals, 4 were found to represent this MICA null allele as assessed by no polymerase chain reaction (PCR) amplification using MICA-specific primers, while the remaining four possessed the intact MICA gene with MICA008 or MICA010.

Asian People↗

PubMed1988

Recognition sequences of type II restriction systems are constrained by the G + C content of host genomes.

I show that the recognition sequences of Type II restriction systems are correlated with the G + C content of the host bacterial DNA. Almost all restriction systems with G + C rich tetranucleotide recognition sequences are found in species with A + T rich genomes, whereas G + C rich hexanucleotide and octanucleotide recognition sequences are found almost exclusively in species with G + C rich genomes. Most hexanucleotide recognition sequences found in species with A + T rich genomes are A + T rich. This distribution eliminates a substantial proportion of the potential variance in the frequency of restriction recognition sequences in the host genomes. As a consequence, almost all restriction recognition sequences, including those eight base pairs in length (Not I and Sfi I), are predicted to occur with a frequency ranging from once every 300 to once every 5,000 base pairs in the host genome. Since the G + C content of bacteriophage DNA and of the host genome are also correlated, the data presented is evidence that most Type II "restriction systems" are indeed involved in phage restriction.

Bacteriophages↗

PubMed2002

Novel HLA-DRB1 alleles revealed by sequencing based typing, DRB104053 and DRB11143.

We report the discovery of two HLA-DRB1 alleles by sequencing based typing (SBT). DRB1*04053 differs from previously reported DRB1 alleles by a single synonymous nucleotide substitution, resulting in a unique polymorphism at codon 93. DRB1*1143 differs from previously identified DRB1 alleles by a single non-synonymous nucleotide substitution, resulting in a polymorphism observed in other DRB1 and DRB3 alleles1.

Alleles↗

PubMed2006

Rapid detection of the "highly virulent" group B Streptococcus ST-17 clone.

Group B streptococcus (GBS) is a leading cause of neonatal morbidity and mortality. Multilocus sequence typing (MLST) revealed that the sequence type ST-17 defines a "highly virulent" serotype III clone strongly associated with neonatal invasive infections. Our aim was to identify a target sequence enabling rapid, simple, and specific detection of this clone by a real-time PCR assay. Conventional methods for DNA manipulation and gene analyses were used to characterize the gbs2018 gene variant specific for ST-17 clone and to design ST-17- and GBS-specific primers. Conventional and real-time PCR assays were developed to detect GBS and ST-17 clones in bacterial cultures and directly on clinical samples. One hundred and fifty-six French GBS strains from various geographical areas in France isolated between 1990 and 2005 were screened by PCR with ST-17-specific primers. Forty strains were positive, and all were validated by MLST as ST-17. A representative sampling of 49 ST-17-PCR-negative strains was confirmed by MLST as non-ST-17. Real-time PCR was further used to directly test 85 vaginal samples. Among these, 13 were GBS-positive, and one was identified as ST-17. The association between strain invasiveness and ST-17 lineage in neonates with late onset disease was highly significant: 78% (P<0.0001) of strains isolated were ST-17. In conclusion, an ST-17-specific gbs2018 allele was identified and used to develop a sensitive and specific rapid-screening molecular assay for identifying ST-17 "highly virulent" GBS. Using this technique, accurate identification of women and neonates colonized by ST-17 can be readily achieved within less than 2 h.

Adult↗

PubMed2006

The identification of three novel MICA alleles by sequence-based typing.

During a study of MICA frequency in a healthy population and a cohort of patients suffering with inflammatory bowel disease, three DNA samples produced unusual reactivity patterns using polymerase chain reaction sequence-specific primers (PCR-SSP). These samples were subsequently characterized by sequence-based typing (SBT). Here, we report the sequence of these three novel MICA alleles.

Alleles↗

PubMed1993

Pro-alpha 2(V) collagen gene; pairwise analysis of the amino-propeptide coding domain, and cross-species comparison of the promoter sequence.

Type V collagen is a minor represented and poorly characterized fibrillar collagen type. Previous cDNA cloning experiments showed that the amino-propeptide of the pro-alpha 2(V) chain shares structural features in common with pro-alpha 1(I), pro alpha 1(II) and pro-alpha 1(III) collagens. In the present paper, this analysis was extended to the gene level. Accordingly the exon/intron arrangement of the amino-propeptide coding domain was compared among pro-alpha 1(I) (COL1A1), pro alpha 1(II) (COL2A1), pro-alpha 1(III) (CO13A1) and pro-alpha 2(V) (COL5A2) collagen genes. This revealed that COL3A1 is the most closely related gene to COL5A2. Based on the assumption that critical regulatory elements might be phylogenetically conserved, we also compared the promoter sequences of the mouse and human COL5A2 genes. This revealed the highest level of sequence homology (97%) in a 52-bp segment which was previously shown to be essential in conferring cell type specificity to the human promoter.

Amino Acid Sequence↗

PubMed2002

Two new HLA class I alleles recognised by PCR sequence-specific primer and sequencing based typing: B3805 and Cw0408.

Two new HLA class I alleles have been recognised by molecular-based typing. B*3805 was initially identified by polymerase chain reaction using sequence-specific primers (PCR-SSP) and afterwards confirmed by sequencing based typing (SBT) studies in a Spanish Caucasian blood cord unit. A unique nucleotide change throughout exons 2, 3 and 4, leading to the amino acid replacement Ser11Ala, differentiates B*3801 and *3805. This position behaves as a dimorphic residue in HLA-B and -C loci, and seems to be structurally unrelated to peptide and TcR recognition. Cw*0408 was first detected by SBT in two African American bone marrow donors in combination with its most structurally related allele, Cw*04011. The single amino acid change found between Cw*04011 and Cw*0408 was Thr163Leu, a residue involved in pocket A of the peptide-binding cleft. This new allele could be the result of a gene conversion event between Cw*04011 and any of the Cw*03 alleles.

Alleles↗

PubMed1999

Forensic mtDNA hair analysis excludes a dog from having caused a traffic accident.

A dog was suspected of having caused a traffic accident. Three hair fragments were recovered from the damaged car and subjected to DNA sequence analysis of the canine mitochondrial D-loop control region. The results were compared to saliva and hair samples from the alleged dog, as well as to control hair samples from four unrelated dogs of different breeds. Two sequence types exhibiting five nucleotide differences in a 377 bp fragment were identified among the four controls. Whereas the evidence hair fragment was identical to the type 1 control sequence, the alleged dog shared the type 2 control sequence except for one position. Thus the dog could be excluded as the origin of the hair fragment. As canine mtDNA appears to exhibit only limited polymorphism, mitochondrial D-loop sequence comparison is currently only suitable for exclusions.

Accidents, Traffic↗

PubMed2001

High-resolution sequence-based typing strategy for HLA-DQA1 using SSP-PCR and subsequent genotyping analysis with novel spreadsheet program.

We present a new sequence-based typing (SBT) strategy for the polymorphic HLA-DQA1 locus that is based on sequence-specific primer - polymerase chain reaction (SSP-PCR) amplification from genomic DNA. This method allows high-resolution genotyping in the second exon of the DQA1 gene. This gene presents a unique situation in which half of the known alleles contain an inframe three base pair deletion of codon 56. This deletion confounds direct SBT methodologies of heterozygous individuals containing both a deletion and nondeletion allele. The primary HLA haplotype associated with type 1 diabetes susceptibility is DR3/DR4. The DQA1 genotype for these two haplotypes are DQA1 *0501, a non-deletion allele and *0301, a deletion allele, thus creating a situation that cannot be resolved using a direct sequencing approach. Our group-specific SBT strategy isolates the deletion alleles from the nondeletion alleles, allowing them to be resolved by direct sequencing. Additionally, we present a novel spreadsheet program that accurately assigns the genotype of both homozygous and heterozygous persons.

Alleles↗

PubMed2003

Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences.

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

Afipia↗

PubMed1992

Evolutionary origin of a Kunitz-type trypsin inhibitor domain inserted in the amyloid beta precursor protein of Alzheimer's disease.

The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid beta precursor protein (beta APP). It is known that beta APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhibitors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present beta APP gene diverged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in beta APP have an evolutionarily close relationship with the inter-alpha-trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membrane-bound serine protease in human T4+ lymphocytes.

Alzheimer Disease↗

PubMed1994

Preferential usage of the bone-type leader sequence for the transcripts of liver/bone/kidney-type alkaline phosphatase gene in neutrophilic granulocytes.

Alkaline phosphatase in neutrophils (NAP) is a product of the liver/bone/kidney-type alkaline phosphatase gene, the chromosomal structure of which has recently been analyzed. The gene has two alternative leader sequences resulting in the two types of mRNA (liver-type and bone-type mRNAs), which suggests that the expression of the gene is regulated by independent promoters. To determine the mechanism underlying NAP induction, it is essential to know which type of the mRNAs is dominant in neutrophils. We adopted quantitative polymerase chain reaction method to determine the relative amount of bone-type mRNA in neutrophils. The bone-type mRNA was found to be at least 5 times more than the liver-type mRNA. mRNA of NAP is known to be induced by in vitro treatment of the cells with granulocyte colony-stimulating factor (G-CSF), which is enhanced by a simultaneous addition of retinoic acid. In neutrophils treated with G-CSF, the bone-type mRNA was at least 6 times more than the liver-type mRNA. In neutrophils treated by both G-CSF and retinoic acid, the bone-type mRNA was at least 22 times more than the liver-type mRNA. The results show that the bone-type mRNA is predominantly transcribed in peripheral neutrophils and in neutrophils cultured in vitro.

Alkaline Phosphatase↗

PubMed2002

Obligate bacterial endosymbionts of Acanthamoeba spp. related to the beta-Proteobacteria: proposal of 'Candidatus Procabacter acanthamoebae' gen. nov., sp. nov.

All obligate bacterial endosymbionts of free-living amoebae currently described are affiliated with the alpha-Proteobacteria, the Chlamydiales or the phylum Cytophaga-Flavobacterium-Bacteroides. Here, six rod-shaped gram-negative obligate bacterial endosymbionts of clinical and environmental isolates of Acanthamoeba spp. from the USA and Malaysia are reported. Comparative 16S rDNA sequence analysis demonstrated that these endosymbionts form a novel, monophyletic lineage within the beta-Proteobacteria, showing less than 90% sequence similarity to all other recognized members of this subclass. 23S rDNA sequence analysis of two symbionts confirmed this affiliation and revealed the presence of uncommon putative intervening sequences of 146 bp within helix-25 that shared no sequence homology to any other bacterial rDNA. In addition, the 23S rRNA of these endosymbionts displayed one polymorphism at the target site of oligonucleotide probe BET42a that is conserved in all other sequenced beta-Proteobacteria. Intra-cytoplasmatic localization of the endosymbionts within the amoebal host cells was confirmed by electron microscopy and fluorescence in situ hybridization with a specific 16S rRNA-targeted oligonucleotide probe. Based on these findings, the provisional name 'Candidatus Procabacter acanthamoebae' is proposed for classification of a representative of the six endosymbionts of Acanthamoeba spp. studied in this report. Comparative 18S rDNA sequence analysis of the Acanthamoeba host cells revealed their membership with either Acanthamoeba 18S rDNA sequence type T5 (Acanthamoeba lenticulata) or sequence type T4, which comprises the majority of all Acanthamoeba isolates.

Acanthamoeba↗