Characterization of an IS element in Mycoplasma orale that is highly homologous to M. fermentans ISLE

In a previous study, using a primer set designed from Mycoplasma fermentans, we amplified a PCR fragment from Mycoplasma orale similar to the 206-bp DNA fragment amplified from M. fermentans insertion-sequence-like element (ISLE). The presence of this similar ISLE fragment has the potential to cause confusion in the PCR diagnosis of M. fermentans and M. orale, which have significantly different clinical scenarios. An ISLE from three different M. orale strains was amplified by using a primer set designed from sequence within the left and right terminal stem and loop (S&L) structures flanking the ISLE of M. fermentans. Sequence analysis showed that the M. orale ISLE is 93% identical to that of M. fermentans at the nucleotide level and codes for two open reading frames also found in the M. fermentans ISLE. This is the first finding that two different mycoplasma species harbor highly homologous IS elements. This finding has great significance in clinical diagnosis and suggests a possibility of horizontal transfer of an IS element between two different mycoplasma species.     

A <Emphasis Type="Italic">gyrB</Emphasis>-targeted PCR for Rapid Identification of <Emphasis Type="Italic">Salmonella</Emphasis>

Salmonella causes the majority of infections in humans and homeothermic animals. This article describes a specific polymerase chain reaction (PCR) method developed for a rapid identification of Salmonella. A gyrB-targeted species-specific primer pair, S-P-for (5′-GGT GGT TTC CGT AAA AGT A-3′) and S-P-rev (5′-GAA TCG CCT GGT TCT TGC-3′), was successfully designed. PCR with all the Salmonella strains produced a 366- bp DNA fragment that was absent from all the non-Salmonella strains tested. The detection limit of the PCR was 0.01 ng with genomic DNA or 3.2 cells per assay. Good specificity was also demonstrated by fecal samples, from which only the gyrB gene of Salmonella was amplified. Using the culture-PCR method, 27 isolates on Salmonella-Shigella (SS) medium were rapidly identified as Salmonella, which was confirmed by the sequencing of the gyrB gene.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">capsici</Emphasis>

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Demonstration of IS<Emphasis Type="Italic">711</Emphasis> transposition in <Emphasis Type="Italic">Brucella ovis</Emphasis> and <Emphasis Type="Italic">Brucella pinnipedialis</Emphasis>

Background  

The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.     

IS<Emphasis Type="Italic">1111</Emphasis> insertion sequences of <Emphasis Type="Italic">Coxiella burnetii</Emphasis>: characterization and use for repetitive element PCR-based differentiation of <Emphasis Type="Italic">Coxiella burnetii</Emphasis> isolates

Background  

Coxiella burnetii contains the IS1111 transposase which is present 20 times in the Nine Mile phase I (9Mi/I) genome. A single PCR primer that binds to each IS element, and primers specific to a region ~500-bp upstream of each of the 20 IS1111 elements were designed. The amplified products were characterized and used to develop a repetitive element PCR genotyping method.     

Variability in the distribution and composition of insertion sequence-like elements in strains of Mycoplasma fermentans

Mycoplasma fermentans has been reported to be pathogenic for man. All fourteen strains tested contain an insertion sequence-like element (ISLE) which may be present in multiple copies. To determine whether ISLE copies are similarly distributed in different strains of M. fermentans, restriction enzyme digest fragments of genomic DNA from 14 isolates, from a variety of sources, were separated by electrophoresis, blotted and hybridized to a biotin labelled polymerase chain reaction (PCR) amplified fragment of ISLE. A range of patterns was observed suggesting that the element has a tendency to undergo rearrangement within the genome. Analysis of ISLE sequences revealed inter- and intra-strain polymorphisms.     

IS<Emphasis Type="Italic">900</Emphasis> Restriction Fragment Length Polymorphism (RFLP) Analysis of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis>Isolates from Goats and Cattle in Norway

In Norway, paratuberculosis has been frequently diagnosed in goats, while cattle have been almost free of the infection. This difference in prevalence between goats and cattle has led to speculations about the existence of a Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis) isolate that is non-pathogenic for cattle. There is little information available on genotypic variation of M. a. paratuberculosis isolated from animals in Norway. In the present study, genotypic information on 51 isolates from goats and four isolates from cattle in Norway was obtained by use of IS 900 restriction fragment length polymorphism (RFLP) analysis. All isolates from cattle and 84% of the isolates from goats had the same RFLP pattern (B-C1). Five RFLP patterns not previously detected were found. No genotypic variation that could explain a difference in host origin was found between the isolates from cattle and the majority of the Norwegian goat isolates. This lack of difference indicates that the most common M. a. paratuberculosis isolates in Norway may infect both cattle and goats.     

A sex determination protocol for the Iberian desman (<Emphasis Type="Italic">Galemys pyrenaicus</Emphasis>) based on a three primer amplification of <Emphasis Type="Italic">DBX</Emphasis> and <Emphasis Type="Italic">DBY</Emphasis> fragments with non-invasive samples

We have sequenced partial fragments of DBX and DBY genes of the endangered Iberian desman (Galemys pyrenaicus). The sequences were used to design a sex determination protocol for non-invasive samples based on a PCR reaction, using only three primers. This protocol allows the simultaneous amplification of two fragments, one corresponding to the DBX gene and the other to the DBY gene, both differing in size. To increase sensitivity on the detection of positive amplifications and on the determination of fragment size we use a fluorescently labelled primer. The protocol has been tested in DNA samples from hair and stool, revealing major difficulties in sexing faecal samples, but unambiguous sexing of hair samples.     

Development of expressed sequence tag-derived microsatellite markers for <Emphasis Type="Italic">Saccharina</Emphasis> (<Emphasis Type="Italic">Laminaria</Emphasis>) <Emphasis Type="Italic">japonica</Emphasis>

Expressed sequence tag-derived microsatellite markers (EST-SSR) were generated and characterized in Laminaria japonica using data mining from updated public EST databases and polymorphism testing. Fifty-eight of 578 ESTs (10.0%) containing various repeat motifs were used to design polymerase chain reaction (PCR) amplification primers. A total of 12 pairs of primer were generated and used in the PCR amplification. Alleles per locus ranged from two to ten (average of 5.7). The observed heterozygosities and expected heterozygosities were from 0.045 to 0.543 and from 0.056 to 0.814, respectively. All loci were in Hardy–Weinberg equilibrium and no linkage disequilibrium was detected. These robust, informative, and potentially transferable polymorphic markers appear suitable for population, genetic, parentage, and mapping studies of L. japonica.     

<Emphasis Type="Italic">Mycoplasma alkalescens</Emphasis> demonstrated in bronchoalveolar lavage of cattle in Denmark

Mycoplasma alkalescens is an arginine-metabolizing mycoplasma, which has been found in association with mastitis and arthritis in cattle. Routine bacteriological examination of 17 bronchoalveolar lavage samples from calves with pneumonia in a single herd in Denmark, identified M. alkalescens in eight samples. The organism was found as a sole bacterilogical findings in five of the samples as well as in combination with Mannheimia haemolytica, Haemophilus somni and Salmonella Dublin. This is the first report of isolation of M. alkalescens in Denmark.     

Genetic stability, <Emphasis Type="Italic">ex vitro</Emphasis> rooting and gene expression studies in <Emphasis Type="Italic">Hagenia abyssinica</Emphasis>

Randomly amplified polymorphic DNA (RAPD) markers were used to assess genetic stability of 80 micropropagated Hagenia abyssinica plants, 40 of axillary origin and 40 of adventitious origin. The shoots were isolated from the same mother tree and micropropagated for over two years. Among the 83 RAPD primers screened, 16 gave reproducible band patterns. These 16 primers produced 115 bands for each plant. One plant from axillary origin showed two unique bands with primer OPC-11. All other plants showed identical band patterns. Generally, there was no significant difference in the shoot multiplication rate between shoots of axillary and adventitious origin. Indole-3-acetic acid (IAA) resulted in better ex vitro rooting compared to indole-3-butyric acid (IBA) and α-naphthaleneacetic acid (NAA). Non-micropropagated plants that were grown in the greenhouse for about one year were better in ex vitro rooting compared to those of juvenile material and mature tree derived micropropagated plants of the same treatment. Adventitious rooting related oxygenase gene (ARRO-1) isolated from apple (Malus domestica) was not expressed in H. abyssinica using a complementary DNA representational difference analysis fragment (cDNA RDA14) as a probe.     

Characterization of the highly variable <Emphasis Type="Italic">cry</Emphasis> gene regions of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strain ly4a3 by PCR-SSCP profiling and sequencing

Characterization of cry gene contents can help to predict the insecticidal activities of Bacillus thuringiensis isolates and in the searching of new cry genes. PCR-Single-strand conformation polymorphism (SSCP) profiling and sequencing of the highly variable cry gene regions were used to characterize cry gene content of B. thuringiensis strain ly4a3. The highly variable regions with about 1100 bp in sizes were amplified using a degenerate primer pair for cry genes, OL2(d) and OL5(r). A library of the PCR product was constructed, and all white colonies were subjected to PCR using another degenerate primer pair for cry genes, OL3(d) and OL5(r), with products about 250 bp in sizes. Two different profiles were observed based on SSCP profiling for the PCR products. The cry genes in the two corresponding colonies were sequenced and their deduced amino acids showed high identities to Cry1Ab (84.5%∼98.4%) and Cry1I (88.78%∼98.4%), respectively. This method allows the quick characterization of cry gene content of B. thuringiensis isolates and the detection of new cry genes.     

Mycoplasma adaptation to stress factors: Nucleotide sequences absent in vegetative forms of <Emphasis Type="Italic">Mycoplasma gallisepticum</Emphasis> S6 cells are found in nonculturable forms

The adaptation of Mycoplasma gallisepticum S6 to adverse environmental conditions is associated with the transformation of vegetative cell forms to viable but nonculturable (VBNC) forms. The vegetative and VBNC forms proved to differ in the spectrum of PCR products amplified from pvpA-gene, which codes for the phase-variable cytoadhesion protein. The vegetative forms displayed only one amplicon, which contained one open reading frame (1086 bp) with a high homology (97%) to pvpA-gene of M. gallisepticum R and Pendik. The VBNC forms of M. gallisepticum S6 had additional amplicons, whose open reading frames were absent from the complete database sequence of the mycoplasma genome. A high nucleotide sequence homology (54–55%) between pvpA-gene and the additional pvpA-gene amplicons made it possible to suggest that pvpA-gene provided a source for the formation of new regions within the mycoplasma genome during adaptation to adverse environmental conditions.     

Molecular identification of isolates of <Emphasis Type="Italic">Peronosclerospora sorghi</Emphasis> from maize using PCR-based SCAR marker

The fungus Peronosclerospora sorghi [Weston and Uppal (Shaw)] infects both sorghum and maize and incites downy mildew disease. Pathogenic and molecular variability among isolates of P. sorghi from sorghum and maize has been reported. In the present study we developed a DNA sequence characterized amplified region (SCAR) marker for identification of isolates of P. sorghi from maize by using polymerase chain reaction (PCR). The random amplified polymorphic DNA (RAPD) primer OPB15 consistently amplified a 1,000 base pairs (bp) product in PCR only from DNA of P. sorghi isolates from maize and not from isolates of sorghum. The PCR-amplified 1,000-bp product was cloned and sequenced. The sequence of the SCAR marker was used for designing specific primers for identification of maize isolates of P. sorghi. The SCAR primers amplified a 800 bp fragment only from genomic DNA of maize isolates of P. sorghi. The SCAR primers developed in this study are highly specific and reproducible, and proved to be powerful tool for identification of P. sorghi isolates from maize.     

Molecular detection of <Emphasis Type="Italic">Ehrlichia ruminantium</Emphasis> infection in <Emphasis Type="Italic">Amblyomma variegatum</Emphasis> ticks in The Gambia

In West Africa, losses due to heartwater disease are not known because the incidence/prevalence has not been well studied or documented. To develop a diagnostic tool for molecular epidemiology, three PCR-based diagnostic assays, a nested pCS20 PCR, a nested map1 PCR and a nested reverse line blot (RLB) hybridization assay, were evaluated to determine their ability to detect infection in vector ticks, by applying them simultaneously to A. variegatum field ticks to detect Ehrlichia ruminantium, the causative agent of heartwater. The nested pCS20 PCR assay which amplified the pCS20 gene fragment showed the highest detection performance with a detection rate of 16.6%; the nested map1 PCR, which amplified the gene encoding the major antigenic protein1 (map1 gene) showed a detection rate of 11% and the RLB, based on the 16S rDNA sequence of anaplasma and ehrlichial species, detected 6.2%. The RLB, in addition, demonstrated molecular evidence of Ehrlichia ovina, Anaplasma marginale and Anaplasma ovis infections in The Gambia. Subsequently, the pCS20 assay was applied to study the prevalence and distribution of E. ruminantium tick infection rates at different sites in five divisions of The Gambia. The rates of infection in the country ranged from 1.6% to 15.1% with higher prevalences detected at sites in the westerly divisions (Western, Lower River and North Bank; range 8.3–15.1%) than in the easterly divisions (Central River and Upper River; range 1.6–7.5%). This study demonstrated a gradient in the distribution of heartwater disease risk for susceptible livestock in The Gambia which factor must be considered in the overall design of future upgrading programmes.     

Molecular cloning and analysis of the UDP-Glucose Pyrophosphorylase in <Emphasis Type="Italic">Streptococcus equi</Emphasis> subsp<Emphasis Type="Italic">. zooepidemicus</Emphasis>

UDP-Glucose Pyrophosphorylase (EC 2.7.7.9, UGPase) plays an important role in Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) cell envelope Hyaluronic acid (HA) biosynthesis and it is also recognized as a virulence determinant in several bacterial species. HA is valuable biopolymer used in the pharmaceutical and cosmetic industry. In addition, encapsulation by HA is considered an important virulence factor in other streptococci. Research UGPase will contribute to the vaccine development of S. zooepidemicus and the production of HA. In this study, The UGPase gene fragment (789 bp) obtained from previous research was amplified using PCR, and located by Genome walking technology (Genebank No.GQ423507). The UGPase was expressed, purified and identified using UGPase antibody. The enzyme kinetic parameters were determined, the temperature and pH of the highest activity for the cloned UGPase were 37°C, pH 7.5. The K _m and K _cat value against UTP and G-1-P was 8.5 μM, 69.05 s⁻¹ and 36.41 μM, 48.81 s⁻¹, respectively. The homology-modeling was operated. Overexpression of the UGPase in S. zooepidemicus, its virulence was slightly affected, and HA yield reduced. Real-time PCR was carried out to determine the UGPase expression levels of both SEZp and SEZugp in different grow period, the level is high in logarithmic phase and low in Decline phase.     

Simple and efficient methods for <Emphasis Type="Italic">S</Emphasis> genotyping and <Emphasis Type="Italic">S</Emphasis> screening in genus <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis

In F₁ hybrid breeding of Brassica vegetables utilizing the self-incompatibility system, identification of S genotypes in breeding lines is required. In the present study, we developed S-tester lines of 87 S haplotypes, i.e., 42 S haplotypes in B. rapa and 45 S haplotypes in B. oleracea. With these materials, we established a simple, efficient, and reliable dot-blot technique for S genotyping for 40 S haplotypes of B. rapa and and 33 of B. oleracea using allele-specific oligonucleotide probes and allele-specific primer pairs designed from sequences of each SP11 allele. In this method, DNA fragments amplified using multiplex primer pairs with digoxigenin-dUTP were hybridized with dot-blotted allele-specific oligonucleotide probes with distinct signals. In addition, we developed a screening method for identification of plants harboring a particular S haplotype using a labeled allele-specific oligonucleotide probe. This method is considered to be useful for purity testing of F₁ hybrid seeds.     

Amplification and detection of polymorphic sequence-tagged sites in<Emphasis Type="Italic">Lathyrus sativus</Emphasis>

A simple procedure was developed to convertLathyrus sativus defence-related expressed sequence tags (ESTs) into mappable genetic markers by using PCR. Twenty-nine STS primer pairs were generated on the basis of sequence information from anL. sativus cDNA library. These primers were used to screen for polymorphisms between 2L. sativus accessions, ATC 80878 and ATC 80407, resistant and susceptible, respectively, toMycosphaerella pinodes infection. All 29 primer pairs amplified PCR products in both accessions, 11 of which amplified multiple RAPD-like products. The remaining 18 primer pairs amplified single monomorphic products. Following cloning, sequencing, and database searches, 17 of 18 PCR products were confirmed to have amplified the targeted genome region. Ten of these 17 STS primer pairs revealed polymorphisms between ATC 80878 and ATC 80407 when PCR products were digested with a range of restriction endonucleases. These results suggest that the STS-based PCR analysis will be useful for generating informative molecular markers inL. sativus for future genome mapping experiments.