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Detection of identical Helicobacter DNA in the stomach and in the non-cirrhotic liver of patients with hepatocellular carcinoma.

OBJECTIVES: Studies have implied the presence of Helicobacter species in the human liver. The possible relationship between the presence of Helicobacter species in the non-cirrhotic liver of patients with hepatocellular carcinoma (HCC) and the detection of Helicobacter species in their stomach was explored. PATIENTS AND METHODS: A 16S rDNA-based polymerase chain reaction (PCR) followed by DNA sequence analysis of the obtained PCR fragments was performed on 51 surgically obtained non-cirrhotic liver specimens and 14 gastric samples. RESULTS: Analysis indicated a significant difference in the presence of Helicobacter-species-specific DNA in the liver of patients with HCC compared with controls. Sequence analysis of these PCR products obtained from HCC patients indicated that they were related most closely to the 16S rDNA sequence of Helicobacter pylori but that they always differed at the same two positions. This same aberrant Helicobacter-species-specific 16S rDNA could be isolated in gastric samples of patients with HCC. CONCLUSION: These data suggest that gastric colonization with a specific subset of Helicobacter strains is associated with the induction of HCC, either directly via colonization of the liver or indirectly, e.g. via secretion of specific toxins by Helicobacter residing in the stomach.

Carcinoma, Hepatocellular↗

Characterization of the rabbit CYP1A1 and CYP1A2 genes: developmental and dioxin-inducible expression of rabbit liver P4501A1 and P4501A2.

In adult rabbits, the CYP1A1 and CYP1A2 genes are expressed constitutively. Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) leads to elevations in both CYP1A1 and CYP1A2 gene products (S. T. Okino et al., 1985, Proc. Natl. Acad. Sci. USA 82, 5310-5314). In this report, we have characterized the rabbit CYP1A1 and CYP1A2 genes, and analyzed the pattern of expression of these genes in neonatal animals following exposure to TCDD. Genomic clones encoding the entire rabbit CYP1A1 and CYP1A2 genes were characterized. Restriction enzyme analysis and partial DNA sequence analysis identified the seven exons for the CYP1A1 and CYP1A2 genes. Primer extension analysis using mRNA from TCDD-treated neonatal rabbits helped confirm the start of transcription for the CYP1A genes. The length of the noncoding first exon of the CYP1A1 gene was 74 bases, compared to 90 and 88 bases for the human and rodent CYP1A1 genes. The length of the noncoding CYP1A2 gene first exon was 53 bases, similar to its counterpart in human and rodents. DNA sequence analysis of the 5' regulatory regions and comparison to the rodent and human CYP1 genes demonstrated that the rabbit CYP1A1 and CYP1A2 genes were most similar to their human orthologs. The 5' region of the CYP1A1 gene contained several consensus dioxin (Ah)-receptor responsive elements (XREs), while no functional XRE sequences were identified in the CYP1A2 gene. When expression of the two genes were monitored, a small amount of constitutive P4501A1 mRNA was detected in neonatal rabbits from the ages of 1 to 17 days, while P4501A2 mRNA levels could not be observed until 8-12 days postpartum. In response to TCDD treatment, P4501A1 mRNA levels were inducible at all neonatal time points, while P4501A2 mRNA levels could not be induced until the animals were 3-5 days postpartum. While the dioxin Ah-receptor most likely plays a major role in the induction of these genes by TCDD, early expression of the CYP1A1 and CYP1A2 genes is differentially regulated in a developmental fashion.

Aging↗

Isolation and functional analysis of the promoter of the rat CMP-NeuAc:GM3 alpha2,8 sialyltransferase gene 1.

A 2.1-kb 5'-flanking fragment of the rat CMP-NeuAc:GM3 alpha2,8 sialyltransferase (GD3-synthase) gene was cloned by the genomic walking procedure. The promoter activity of the fragment was assessed in F-11 cells by transient transfection and the locations for the basal and maximal promoter activities were defined. Primer extension analysis identified a transcription start site approximately 98 bp upstream of the ATG start codon. DNA sequence analysis of the promoter revealed a number of consensus binding sites for known transcription factors such as SP1, AP1, NFkappaB, C/EBP and TFIID, and a repeat GC-GT sequence motif seen for the formation of Z-type DNA. Both TATA and CCAAT boxes were not found in the promoter. Our results from deletion constructs suggested that both positive and negative cis-acting regulatory regions were present in this TATA-less promoter of the rat GD3-synthase gene.

Animals↗

Genotyping with microfluidic devices.

In the past few years, electrophoresis microchips have been increasingly utilized to interrogate genetic variations in the human and other genomes. Microfluidic devices can be readily applied to speed up existing genotyping protocols, in particular the ones that require electric field-mediated separations in conjunction with restriction fragment analysis, DNA sequencing, hybridization-based techniques, allele-specific amplification, heteroduplex analysis, just to list the most important ones. As a result of recent developments, microfabricated electrophoresis devices offer several advantages over conventional slab-gel electrophoresis, such as small sample volume requirement, low reagent consumption, the option of system integration and easy multiplexing. The analysis speed of microchip electrophoresis is significantly higher than that of any other electric field-mediated separation techniques. State-of-the-art microfluidic bioanalytical devices already claim their place in most molecular biology laboratories. This review summarizes the recent developments in microchip electrophoresis methods of nucleic acids, particularly for rapid genotyping, that will most likely play a significant role in the future of clinical diagnostics.

Animals↗

Evaluation of methods for subtyping Campylobacter jejuni during an outbreak involving a food handler.

In October 1998, the Centers for Disease Control and Prevention (CDC) assisted in an investigation of an outbreak of campylobacteriosis at a school in Salina, Kansas. Twenty-two isolates were submitted from the Kansas state public health laboratory to CDC, 9 associated with the outbreak and 13 epidemiologically unrelated sporadic isolates. Pulsed-field gel electrophoresis (PFGE) using SmaI and SalI was initially used to validate the epidemiologic data. We then tested the ability of other subtyping techniques to distinguish the outbreak-associated isolates from unrelated sporadic isolates. The methods employed were somatic O serotyping, PCR-restriction fragment length polymorphism (RFLP) analysis of flaA, DNA sequence analysis of 582 bp of flaA that included the short variable region (SVR), and sequencing of the entire flaA gene. PFGE was the most discriminatory technique, yielding 11 SmaI and 10 SalI restriction profiles. All outbreak isolates were indistinguishable by PFGE, somatic O serotyping, and sequencing of the 582-bp region of the flaA gene. fla typing by PCR-RFLP grouped one sporadic isolate with the outbreak strain. Analysis of the DNA sequence of a 582-bp segment of flaA produced strain groupings similar to that generated by PCR-RFLP but further differentiated two flaA PCR-RFLP types (with a 1-bp difference in the 582-bp region). Two sporadic strains were distinct by flaA PCR-RFLP but differed only by a single base substitution in the 582-bp region. The entire flaA gene was sequenced from strains differing by a single base pair in the 582-bp region, and the data revealed that additional discrimination may in some cases be obtained by sequencing outside the SVR. PFGE was superior to all other typing methods tested for strain discrimination; it was crucial for understanding the Kansas outbreak and, when SmaI was used, provided adequate discrimination between unrelated isolates.

Bacterial Typing Techniques↗

Isolation of an osmotin-like protein gene from strawberry and analysis of the response of this gene to abiotic stresses.

A strawberry genomic clone containing an osmotin-like protein (OLP) gene, designated FaOLP2, was isolated and sequenced. FaOLP2 is predicted to encode a precursor protein of 229 amino acid residues, and its sequence shares high degrees of homology with a number of other OLPs. Genomic DNA hybridization analysis indicated that FaOLP2 represents a multi-gene family. The expression of FaOP2 in different strawberry organs was analyzed using real-time PCR. The results showed that FaOLP2 expressed at different levels in leaves, crowns, roots, green fruits and ripe red fruits. In addition, the expression of FaOLP2 under different abiotic stresses was analyzed at different time points. All of the three tested abiotic stimuli, abscisic acid, salicylic acid and mechanical wounding, triggered a significant induction of FaOLP2 within 2-6h post-treatment. Moreover, FaOLP2 was more prominently induced by salicylic acid than by abscisic acid or mechanical wounding. The positive responses of FaOLP2 to the three abiotic stimuli suggested that strawberry FaOLP2 may help to protect against osmotic-related environmental stresses and that it may also be involved in plant defense system against pathogens.

Abscisic Acid↗

Characterization of a novel D1S80 pseudoallele.

During a D1S80 population study conducted for databasing purposes in the New York City Ashkenazi Jewish population, eight out of 96 samples were typed with a band corresponding to the position of a #15 allele. In seven of the eight samples, three bands appeared. Further investigation was needed to explain the high frequency of an allele considered so rare that it is not included in the commercially provided allelic ladder. After extraction of the putative D1S80 15-repeat amplicon band from the 6% polyacrylamide genotyping gel, the amplicon bands were reamplified with D1S80 primers. After retyping as putative 15 alleles, these samples underwent Southern hybridization with a D1S80 locus-specific probe followed by DNA sequence analysis. Sequence analysis revealed that these bands did not arise from true D1S80 15 alleles. However, the PCR product was of a size that fell within the allelic ladder region corresponding to the 15 band and contained end sequences with strong homology to the D1S80 primers. An alignment search of the sequenced product revealed that a portion of the amplicons contained 72% identity to a known gene. These results emphasize the importance of sequencing analysis when questions arise about the authenticity of an allele.

Alleles↗

Phylogeographical disjunction in abundant high-dispersal littoral gastropods.

Abstract Phylogeographical disjunctions in high-dispersal marine taxa are variously ascribed to palaeogeographical conditions or contemporary ecological factors. Associated biogeographical studies, however, seldom incorporate the sampling design required to confidently discriminate among such competing hypotheses. In the current study, over 7800 gastropod specimens were examined for operculum colour, and 129 specimens genetically, to test ecological and historical biogeographical hypotheses relating to biogeographical disjunction in the Southern Hemisphere, and to southern Australia in particular. Mitochondrial DNA sequence analysis of the high-dispersal intertidal gastropod Nerita atramentosa in southern Australia (88 specimens; 18 localities) revealed an east-west phylogeographical split involving two highly divergent clades (26.0 +/- 1.9%) exhibiting minimal geographical overlap in the southeast. The eastern clade of Nerita atramentosa is also widespread in northern New Zealand (43 specimens, 10 localities), but no significant genetic differentiation is explained by the Tasman Sea, a 2000-km-wide oceanic barrier. Spatial genetic structure was not detected within either clade, consistent with the species' dispersive planktotrophic phase lasting for 5-6 months. Digital analysis of operculum colouration revealed substantial differences between eastern (tan) and western (black) specimens. Genetic analysis and visual inspection of 88 Australian specimens revealed a completely nonrandom association between mtDNA data and operculum colouration. Independent examination of a further 7822 specimens from 14 sites in southern Australia revealed both colour morphs at all localities, but reinforced the phylogeographical data by indicating a marked turnover in colour morph abundance associated with a palaeogeographical barrier: Wilsons Promontory. This sharp biogeographical disjunction is in marked contrast to the species' high dispersal abilities. The genetic similarity of Nerita morio (Easter Island) and the eastern Australian + New Zealand lineage (1.1 +/- 0.3%) provides further evidence of long-distance dispersal in southern Nerita. Phylogenetic relationships of nine species (four genera) of Neritidae, an almost exclusively tropical gastropod family, are consistent with the hypothesis that southern temperate black nerites comprise a monophyletic radiation.

Analysis of Variance↗

Molecular basis of the LOCR (Rh55) antigen.

BACKGROUND: In 1994 during the investigation of a case of hemolytic disease of the newborn, a new low-incidence red cell (RBC) antigen, LOCR, was described. Although the presence of LOCR was associated with altered expression of Rh antigens, its formal assignment to the Rh blood group system did not occur until haplotype and linkage analysis conducted in 2003 provided the necessary proof. The current study was undertaken in an attempt to define the underlying RH mutation in LOCR+ individuals. STUDY DESIGN AND METHODS: Genomic DNA from five unrelated LOCR+ individuals and three Rh-matched control individuals was amplified by polymerase chain reaction with intronic primers flanking all 10 exons of RH. Amplified products were separated on 1 percent agarose gels and isolated for DNA sequence analysis in both the forward and the reverse directions. RESULTS: DNA sequence analysis of the three LOCR+ D- individuals revealed a single heterozygous 286G>A nucleotide substitution resulting in a predicted Gly>Ser substitution at amino acid 96. DNA sequence analysis from the two LOCR+ D+ individuals revealed the identical mutation, as well as all of the changes associated with the common RHD gene. CONCLUSIONS: Based on our results, a Gly96Ser substitution in the Rhce polypeptide defines the low-incidence RBC antigen known as LOCR. This same amino acid change has previously been shown to be involved in the Rh:-26 phenotype, which suggests that LOCR and Rh26 are antithetical. Serologic investigations with various Rh:-26 cells and serum samples, however, reveal that only some c+ Rh:-26 phenotypes are LOCR+.

Adult↗

Natural simian virus 40 strains are present in human choroid plexus and ependymoma tumors.

Simian virus 40 (SV40) sequences for large tumor antigen (T-ag) were recently detected in a significant fraction of certain human brain tumors of early childhood (Bergsagel et al., N. Engl. J. Med. 326, 988-993, 1992). In the current study, we sought to determine whether authentic SV40 was present in the choroid plexus and ependymoma tumors previously examined. Polymerase chain reaction and DNA sequence analysis revealed authentic SV40 regulatory region and major capsid (VP1) sequences in 14 of 17 tumors tested. Only one 72-basepair element was detected in the SV40 enhancer region of positive tumor samples, an arrangement designated as "archetypal." The C terminus of the T-ag gene was detected in the same 14 tumors and was sequenced from 5 tumors; some nucleotide changes were found that would result in amino acid changes in T-ag. Infectious SV40 was isolated from one sample after lipofection of tumor DNA into monkey kidney cells. Sequence analysis of the rescued virus SVCPC revealed (i) an archetypal regulatory region, (ii) nucleotide changes in the C terminus of the T-ag gene that distinguished it from SV40 laboratory strains 776 and SV40-B2 and from human isolate SVPML-1, and (iii) identity with previous human brain tumor isolate SVMEN in the three genomic regions sequenced. No human-isolate-specific distinguishing features were detected among the viral sequences analyzed. Thus, authentic SV40 is present in humans and associated with two tumor types known to be induced experimentally by the virus.

Amino Acid Sequence↗

PCR primers that allow intergeneric differentiation of ascomycetes and their application to Verticillium spp.

A pair of conserved PCR primers, designated NMS1 and NMS2, that amplify a region in the mitochondrial small rRNA gene region were designed for fungi belonging to the class Ascomycetes. These primers were tested with members of eight fungal genera (Aspergillus, Fusarium, Magnaporthe, Mycospharella, Neurospora, Saccharomyces, Sclerotinia, Verticillium) and 10 Verticillium species (Verticillium albo-atrum, Verticillium chlamydosporium, Verticillium cinnebarium, Verticillium dahliae, Verticillium fungicola, Verticillium lecanii, Verticillium lateritium, Verticillium nigrescens, Verticillium psaliotae, and Verticillium tricorpus). The primers were also tested with 35 isolates of V. dahliae obtained from diverse geographic areas and diverse hosts. The results of a restriction fragment length polymorphism analysis of the region amplified by the primers differentiated the genera examined and the results of a DNA sequence analysis of the amplified region differentiated the Verticillium species. Two Fusarium species were also differentiated by the results of the restriction fragment length polymorphism analysis. On the basis of the nucleotide sequences of the amplified regions, we obtained a pair of PCR primers that could be used to differentiate V. dahliae from the other fungal isolates tested, including V. albo-atrum, a closely related plant-pathogenic species. The V. dahliae-specific PCR primer may aid in more rapid and specific detection of the pathogen directly in plant and/or soil samples. PCR primers NMS1 and NMS2 may be used as potential mitochondrial markers for studying fungal cytoplasmic inheritance of ascomycetes and for identifying DNA probes that are informative at or below the genus level.

Aspergillus↗

Analysis of the polymorphic structure of short tandem repeats locus D18S555.

We analyzed the polymorphic structure of the short tandem repeats (STR) (RRGG) locus D18S555 by DNA sequencing, and examined the distribution of alleles in DNA samples from Japanese and Chinese individuals. DNA sequence analysis showed that the alleles at this locus consisted of deletions or insertions of (gaaagaaagaat), (ggaa), (ggaaggaaggag), (ggaaggagggaaggaaggag), (gggaggaa), and (ggga) 5, with each allele having its own DNA sequence. Analysis of 200 unrelated Japanese and 169 unrelated Chinese subjects identified 11 alleles, of which alleles 4, 6, and 13 occurred with a high frequency. There was no difference in allele frequency between the two ethnic groups. These results indicate that the genotyping of D18S555 alleles requires no sequence analysis, and making the locus applicable to forensic identity.

Asian People↗

Eight hundred-base sequencing in a microfabricated electrophoretic device.

The human genome will be sequenced using capillary array electrophoresis technology. Although currently achieving only 550 base reads per run, capillary arrays have increased the efficiency and lowered the cost of sequencing by eliminating gel plate preparation, reducing sample volumes, and offering automation and speed. However, much higher throughput and greater cost reductions are needed. The next major advancement in sequencing technology is expected from the development of arrays of microfabricated channels in a plate or "chip" format. For de novo sequencing, the practical utility of the microdevice approach has been limited by device length to a read of 500-600 bases per run. We demonstrate a significant milestone for a microfabricated device by obtaining an average read length of 800 bases in 80 min (98% accuracy) for either M13 standards or DNA sequencing samples from the Whitehead Institute Center for Genomic Research (WICGR) production line. This result is achieved in 40-cm-long channels using a new class of large-scale microfabricated devices. Both microfabrication of extended structures and achievement of long reads are essential steps toward a 384-lane very-large-scale microfluidic (VLSMF) system for ultrahigh-throughput DNA sequencing analysis, currently under construction in our laboratory.

Base Sequence↗

Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA.

Eukaryotic DNA topoisomerase I introduces transient single-stranded breaks on double-stranded DNA and spontaneously breaks down single-stranded DNA. The cleavage sites on both single and double-stranded SV40 DNA have been determined by DNA sequencing. Consistent with other reports, the eukaryotic enzymes, in contrast to prokaryotic type I topoisomerases, links to the 3'-end of the cleaved DNA and generates a free 5'-hydroxyl end on the other half of the broken DNA strand. Both human and calf enzymes cleave SV40 DNA at the identical and specific sites. From 827 nucleotides sequenced, 68 cleavage sites were mapped. The majority of the cleavage sites were present on both double and single-stranded DNA at exactly the same nucleotide positions, suggesting that the DNA sequence is essential for enzyme recognition. By analyzing all the cleavage sequences, certain nucleotides are found to be less favored at the cleavage sites. There is a high probability to exclude G from positions -4, -2, -1 and +1, T from position -3, and A from position -1. These five positions (-4 to +1 oriented in the 5' to 3' direction) around the cleavage sites must interact intimately with topo I and thus are essential for enzyme recognition. One topo I cleavage site which shows atypical cleavage sequence maps in the middle of a palindromic sequence near the origin of SV40 DNA replication. It occurs only on single-stranded SV40 DNA, suggesting that the DNA hairpin can alter the cleavage specificity. The strongest cleavage site maps near the origin of SV40 DNA replication at nucleotide 31-32 and has a pentanucleotide sequence of 5'-TGACT-3'.

Animals↗

Lateral flagellar gene system of Vibrio parahaemolyticus.

Vibrio parahaemolyticus possesses dual flagellar systems adapted for movement under different circumstances. A single polar flagellum propels the bacterium in liquid (i.e., swimming) with a motor that is powered by the sodium motive force. Multiple proton-driven lateral flagella enable translocation over surfaces (i.e., swarming). The polar flagellum is produced continuously, while production of lateral flagella is induced when the organism is grown on surfaces. This work describes the isolation of mutants with insertions in the structural and regulatory laf genes. A Tn5-based lux transcriptional reporter transposon was constructed and used for mutagenesis and subsequent transcriptional analysis of the laf regulon. Twenty-nine independent insertions were distributed within 16 laf genes. DNA sequence analysis identified 38 laf genes in two loci. Among the mutants isolated, 11 contained surface-induced lux fusions. A hierarchy of laf gene expression was established following characterization of the laf::lux transcriptional fusion strains and by mutational and primer extension analyses of the laf regulon. The laf system is like many enteric systems in that it is a proton-driven, peritrichous flagellar system; however, laf regulation was different from the Salmonella-Escherichia coli paradigm. There is no apparent flhDC counterpart that encodes master regulators known to control flagellar biosynthesis and swarming in many enteric bacteria. A potential sigma(54)-dependent regulator, LafK, was demonstrated to control expression of early genes, and a lateral-specific sigma(28) factor controls late flagellar gene expression. Another notable feature was the discovery of a gene encoding a MotY-like product, which previously had been associated only with the architecture of sodium-type polar flagellar motors.

Bacterial Outer Membrane Proteins↗

[Detection of Mycoplasma genitalium in throat by nested polymerase chain reaction and analysis of DNA sequencing in pediatric patients with acute upper respiratory tract infections].

OBJECTIVE: To explore the relation between Mycoplasma genitalium (Mg) and acute upper respiratory tract infection in pediatric patients by using nested polymerase chain reaction(nPCR) and analysis of DNA sequencing techniques. METHODS: The specimens of throat swab were obtained in 62 acute upper respiratory tract infections children and 80 health children. The two kinds of Mg species-specific, in which the sequences of I-III and V-VII variable regions of Mg 16Sr RNA gene were detected by using nPCR technique. DNA sequences of positive product were detected and analysed. RESULTS: The Positive rate of health children is 5% (4/80), while the acute upper respiratory tract infection children is 21% (13/62). The difference is significant (P < 0.001). The DNA sequences of positive product shows that: there are two point mutation(T-->C) in the sequences of V-VII variable regions of throat swab of one pediatric patient, other sequences are same as those Mg type strain(G-37T). CONCLUSIONS: Acute upper respiratory tract infection of pediatric patients have relation with Mg. There are Mg mutant strain in China.

Adhesins, Bacterial↗

Molecular properties and activity of a carboxyl-terminal truncated form of xylanase 3 from Aeromonas caviae W-61.

Aeromonas caviae W-61 produces five species of xylanases, xylanases 1, 2, 3, 4, and 5 [Nguyen, V.D. et al., Biosci. Biotechnol. Biochem., 56, 1708-1712 (1993) and Appl. Environ. Microbiol., 57, 445-449 (1991)]. While preserving a purified xylanase 3 preparation from A. caviae in solution at 4 degrees C, the xylanase 3 was found to be proteolyzed to give a truncated form with a smaller molecular mass than that of the intact one. It appears likely that the truncated form of xylanase 3 was produced in this particular purification experiment by the action of a contaminating protease. We isolated the truncated form of xylanase 3 (Xyn3tr), of which the C-terminal 102-residue segment is missing. By the chemical analysis of the N- and C-terminal amino acid residues of Xyn3tr and the DNA sequencing analysis of the xylanase 3 gene (xyn3), the N-terminal 398th proline residue of xylanase 3 was found to be the C-terminus of Xyn3tr. Xyn3tr had the activity to form xylotriose (X3), xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) as main final products from oat spelt xylan. In contrast, intact xylanase 3 released X6 and higher xylo-oligosaccharides as main products. Xylanase 3 hydrolysed X4 through X6. However, Xyn3tr had no activity towards X4 and X5. The recombinant Xyn3tr and recombinant xylanase 3 (XYN3) were purified homogeneously from the periplasmic space of E. coli harboring the plasmids pXYN3 and pXYN3tr, which include xyn3 and xyn3tr genes, respectively, and their enzymatic activities were measured. The cleavage patterns of oat spelt and xylo-oligosaccharides by XYN3tr were identical with that by intact Xyn3tr. Thus, we conclude that the C-terminal region comprising a 102-residue segment in xylanase 3 is involved in governing the molecular size of xylo-oligosaccharides cleaved from beta-1,4-xylan by the enzyme and in the hydrolytic activity towards X4 and X5.

Aeromonas↗