Single PCR Product Research Articles

Role of Mechanical Stress-induced Glutamate Signaling-associated Molecules in Cytodifferentiation of Periodontal Ligament Cells

In this study, we analyzed the effects of tensile mechanical stress on the gene expression profile of in vitro-maintained human periodontal ligament (PDL) cells. A DNA chip analysis identified 17 up-regulated genes in human PDL cells under the mechanical stress, including HOMER1 (homer homolog 1) and GRIN3A (glutamate receptor ionotropic N-methyl-d-aspartate 3A), which are related to glutamate signaling. RT-PCR and real-time PCR analyses revealed that human PDL cells constitutively expressed glutamate signaling-associated genes and that mechanical stress increased the expression of these mRNAs, leading to release of glutamate from human PDL cells and intracellular glutamate signal transduction. Interestingly, exogenous glutamate increased the mRNAs of cytodifferentiation and mineralization-related genes as well as the ALP (alkaline phosphatase) activities during the cytodifferentiation of the PDL cells. On the other hand, the glutamate signaling inhibitors riluzole and (+)-MK801 maleate suppressed the alkaline phosphatase activities and mineralized nodule formation during the cytodifferentiation and mineralization. Riluzole inhibited the mechanical stress-induced glutamate signaling-associated gene expressions in human PDL cells. Moreover, in situ hybridization analyses showed up-regulation of glutamate signaling-associated gene expressions at tension sites in the PDL under orthodontic tooth movement in a mouse model. The present data demonstrate that the glutamate signaling induced by mechanical stress positively regulates the cytodifferentiation and mineralization of PDL cells.

The impact of PCR-generated recombination on diversity estimation of mixed viral populations by deep sequencing

Ultra-deep pyrosequencing (UDPS) of targeted amplicons allows to determine a large number of individual sequence reads from a single PCR product, and this approach is thus extremely valuable for analysis of mixed viral populations. A mixture of genetically distinct DNA templates, however, may lead to the generation of recombinant sequences during the initial PCR amplification step. In the present study, the frequency at which these misleading PCR artefacts are formed has been estimated by using artificial mixtures of genetically distinct human cytomegalovirus (HCMV) DNA templates for a given cycling profile. The presence of similar copy numbers of non-identical HCMV target sequences, combined with high levels of input HCMV DNA, as is found in some clinical samples, favored the formation of recombinant PCR products. Thus, to estimate the full natural diversity within mixed viral populations using UDPS, artificial chimeras generated during the PCR step should be taken into account as a potential artefact.

A real-time PCR assay for detection and quantification of 2-branched (1,3)-β- d–glucan producing lactic acid bacteria in cider

Ropiness in natural cider is a relatively frequent alteration, mainly found after bottling, leading to consumer rejection. It is derived from the production of exopolysaccharides (EPS) by some lactic acid bacteria most of which synthesize a 2-branched (1,3)-β-D-glucan and belong to the genera Pediococcus, Lactobacillus and Oenococcus. This polysaccharide synthesis is controlled by a single transmembrane glycosyltransferase (GTF). In this work, a method based on quantitative PCR (qPCR) and targeting the gtf gene was developed for detection and quantification of these bacteria in cider. The newly designed primers GTF3/GTF4 delimit a 151 bp fragment within the 417 bp amplicon previously designed for conventional PCR. The inclusivity and exclusivity of the qPCR assay were assessed with 33 cider isolates belonging to genus Lactobacillus, Oenoccocus and Pedioccocus, together with reference strains of 16 species and five genera including β-glucan, α-glucan and heteropolysaccharide (HePS) producing strains and non-EPS producers. The qPCR assay, followed by the melting curve analysis, confirmed the generation of a single PCR product from the β-glucan producers with a T m of 74.28 ± 0.08 and C T values (10 ng DNA) ranging between 8.46 and 16.88 (average 12.67 ± 3.5). Some EPS − LAB strains rendered C T values ranging from 28.04 to 37.75 but they were significantly higher (P(C T < 28.54) = 0.05) than those of the β-glucan producers. The assay showed a wide quantification range of 5 log units using calibrated cell suspensions of Pediococcus parvulus 2.6 and Oenococcus oeni I4. The linearity was extended over 7 log orders when calibration curves were obtained from DNA. The detection limit for β-glucan producing LAB in artificially contaminated cider was about 3 × 10 2 CFU per ml. The newly developed qPCR assay was successfully applied to monitor the cidermaking process, in 13 tanks from two cider factories, revealing a decrease in C T values derived from an increase in β-glucan producing LAB populations. In addition, 8 naturally spoiled bottled cider were tested for the quantification of these organisms using the five standard curves constructed: P. parvulus 2.6 genomic DNA and gtf amplicon (417 bp), calibrated cell suspensions of Pediococcus parvulus 2.6, Lactobacillus diolivorans G77 and Oenococcus oeni I4 and results were compared to LAB total counts on MRS. Levels obtained from the different approaches were within a log range and showed no significant differences. Therefore, the amplicon-derived standard curve is proposed for the routine estimation of gtf + populations in cider.

Fungal species identification from avian lung specimens by single hypha laser microdissection and PCR product sequencing

Accurate species diagnosis in cases of fungal pneumonia may be hampered by environmental contamination and colonization resulting in false-positive results. Our novel approach for fungal species diagnostics combines fluorescent staining of mounted cryosections with the optical brightener Blankophor, laser capture microdissection and PCR amplification with subsequent sequencing of the first internal transcribed spacer region (ITS-1). Using clinical specimens from infected birds, we show that the procedure is suitable for species identification from single hyphae of intralesional filamentous fungi. Our data also suggest that multiple Aspergillus fumigatus strain infections may occur frequently in pulmonary aspergillosis of birds.

A PCR-Based Assay for Detection of Fusarium oxysporum f. sp. lactucae in Lettuce Seed.

A nested polymerase chain reaction-based (nPCR) assay was developed and evaluated for the rapid detection of Fusarium oxysporum f. sp. lactucae in seed of lettuce. Three primers were designed from sequences of the intergenic spacer region of the rDNA and were used in the PCR amplifications. The first amplification employed the primer pair GYCF1 and GYCR4C and produced a product of 2,270 bp. The second amplification employed the forward primer GYCF1 and the nested primer R943 and produced a single 936-bp PCR product. The nPCR protocol developed successfully detected the target sequence in genomic DNA at 1 fg/μl. A seed assay was tested that included a 4-day incubation step in which seed were maintained under high humidity conditions to increase fungal biomass for DNA extraction. In seed lots prepared by mixing known amounts of F. oxysporum f. sp. lactucae-infested seed with noninfested seed, this assay permitted the detection of the pathogen from lots with infestation rates as low as 0.1%. Samples of lettuce seed obtained from 88 commercial lettuce seed lots were assayed for the pathogen by direct plating and by using the nPCR assay. The pathogen was not detected by either diagnostic method, suggesting the seed lots were pathogen free or the level was below detection limits.

Discovery of single nucleotide polymorphism in Capsicum and SNP markers for cultivar identification

Molecular markers based on single nucleotide polymorphisms (SNPs) are abundant and evenly distributed in a whole genome enough to distinguish individuals in a population. In recent years, sets of SNP markers have been designed and applied for cultivar identification in various crop species. This paper is the first to report the development of a panel of SNP markers for variety identification in peppers. We used conserved ortholog set II (COSII) markers developed from conserved unigenes between tomato and Arabidopsis to identify SNPs in peppers. We tested 438 COSII primer sets amplified as single PCR products out of a total 600 COSII primer sets. Among the 438 COSII primers, 170 primer sets (38.8%) showed polymorphisms between Capsicum annuum ‘RNaky (RN)’and C. chinense ‘PI 159234 (234)’. In contrast, only 48 primer sets (11.0%) out of 438 primers sets were polymorphic between C. annuum ‘Perennial (PER), and ‘Dempsey (DEMP)’. The average frequency of SNPs plus InDels between C. annuum and C. chinense was 1/189 bp and between C. annuum spp. was 1/948 bp. Primer sets showing SNP between C. annuum PER and DEMP were re-designed to Allele Specific PCR (AS-PCR) primers and we finally selected a total of 40 SNP markers for cultivar identification. As the result, we were able to discriminate 97.5% of the 81 commercial hot cultivars and 100% of the 17 sweet pepper cultivars. We conclude the paper by discussing the use of the SNP marker set for cultivar identification and other applications.

Development of genome-specific primers for homoeologous genes in allopolyploid species: the waxy and starch synthase II genes in allohexaploid wheat (Triticum aestivum L.) as examples

BackgroundIn allopolypoid crops, homoeologous genes in different genomes exhibit a very high sequence similarity, especially in the coding regions of genes. This makes it difficult to design genome-specific primers to amplify individual genes from different genomes. Development of genome-specific primers for agronomically important genes in allopolypoid crops is very important and useful not only for the study of sequence diversity and association mapping of genes in natural populations, but also for the development of gene-based functional markers for marker-assisted breeding. Here we report on a useful approach for the development of genome-specific primers in allohexaploid wheat.FindingsIn the present study, three genome-specific primer sets for the waxy (Wx) genes and four genome-specific primer sets for the starch synthase II (SSII) genes were developed mainly from single nucleotide polymorphisms (SNPs) and/or insertions or deletions (Indels) in introns and intron-exon junctions. The size of a single PCR product ranged from 750 bp to 1657 bp. The total length of amplified PCR products by these genome-specific primer sets accounted for 72.6%-87.0% of the Wx genes and 59.5%-61.6% of the SSII genes. Five genome-specific primer sets for the Wx genes (one for Wx-7A, three for Wx-4A and one for Wx-7D) could distinguish the wild type wheat and partial waxy wheat lines. These genome-specific primer sets for the Wx and SSII genes produced amplifications in hexaploid wheat, cultivated durum wheat, and Aegilops tauschii accessions, but failed to generate amplification in the majority of wild diploid and tetraploid accessions.ConclusionsFor the first time, we report on the development of genome-specific primers from three homoeologous Wx and SSII genes covering the majority of the genes in allohexaploid wheat. These genome-specific primers are being used for the study of sequence diversity and association mapping of the three homoeologous Wx and SSII genes in natural populations of both hexaploid wheat and cultivated tetraploid wheat. The strategies used in this paper can be used to develop genome-specific primers for homoeologous genes in any allopolypoid species. They may be also suitable for (i) the development of gene-specific primers for duplicated paralogous genes in any diploid species, and (ii) the development of allele-specific primers at the same gene locus.

Detection of Wolbachia DNA in blood from dogs infected with Dirofilaria immitis

Dirofilaria immitis is the causative agent of heartworm disease in canines and felines, and pulmonary dirofilariasis in man . It harbors a symbiotic intracellular bacterium from the genus Wolbachia that plays an important role in its biology and contributes to the inflammatory pathology of the heartworm. This endosymbiont is sensitive to the tetracycline family of antibiotics prompting its use in the treatment of filariasis. To track Wolbachia during treatment, primers were designed based on the FtsZ gene from Wolbachia. These primers amplify a single PCR product with the expected size from DNA samples derived from various species of worms that harbor Wolbachia ( D. immitis, Brugia malayi and Brugia pahangy). The detection limit of Wolbachia DNA in the assay was 80 pg of D. immitis DNA. Furthermore, the primer set successfully amplified the expected PCR product using blood samples from dogs harboring the heartworm and circulating microfilariae.

Development of AFLP and STS markers linked to a waterlogging tolerance in Korean soybean landraces

Among the 400 soybean (Glycine max) landraces, we selected 3 tolerant (KAS150-9, KAS160-15, and KAS170-9) and 3 susceptible lines (KAS160-14, KAS160-20, and KAS201-6-1) by the survival percentage and injury scores. Susceptible lines showed decrease in chlorophyll content and increase in glucose and malondialdehyde (MDA) contents under waterlogging stress, while tolerant lines did not change significantly. For AFLP analysis, 8 EcoRI (+3) and 8 MseI (+3) primers used in 32 primer combinations generated a total of 2 566 bands with a mean of 80 bands per primer combination, of which 1 117 (43.5 %) were clearly polymorphic between the tolerant and susceptible lines. A genetic similarity coefficient, based on cluster analysis using an unweighted pair grouping method of average (UPGMA), was 0.79 for the tolerant group, while the susceptible landraces were genetically less related, with a genetic similarity coefficient of 0.17. The 10 reproducible polymorphic PCR products present in the 3 tolerant or susceptible lines were sequenced and converted into sequence tagged site (STS) markers. These STS primer sets were designated GmWT01-GmWT06 and GmWS01-GmWS04. Two STS primer sets, GmWT06 and GmWS02, generated a single monomorphic PCR product identical in size to the original AFLP fragments. For the broad application of these STS markers in marker-assisted selection (MAS) for soybean genotypes tolerant to waterlogging stress, two developed STS markers are being evaluated with putative waterlogging tolerant mutant lines induced by &gamma;-radiation in soybean mutation breeding programs.

Testing avian, squamate, and mammalian nuclear markers for cross amplification in turtles

We used PCR amplifications to assess 120 previously described nuclear markers for phylogeographic and phylogenetic analysis in turtles. Twenty-seven of 120 markers amplified a single PCR product for both the western pond turtle (Emys marmorata) and the West African mud turtle (Pelusios castaneus), 71 amplified a single product in either E. marmorata or P. castaneus, and a subset of eight markers amplified single products across a test panel of 11 additional turtle species representing a broad sample of turtle diversity. Our ongoing research shows that nuclear markers developed for birds, squamates, and other vertebrate taxa can be useful for analyses of turtles, suggesting that many primers are transferable across large phylogenetic distances.

An Accurate and Rapid PCR-Based Zygosity Testing Method for Genetically Modified Maize

Using genetically modified NK603 maize as an example, an accurate and cost-effective PCR-based procedure was described for genetically modified maize zygosity testing. The method involved a simple PCR reaction with four primers. The primers were designed from the inserted transgene at 5' and 3' ends (5'TP and 3'TP), the 5' flanking genomic DNA (5'GP), and the 3' flanking genomic DNA (3'GP), respectively. If the plant was a null (wild type), genomic DNA between 5'GP and 3'GP were amplified and a single PCR product was produced; If the plant was a homozygote, DNA between 5'GP and 5'TP, 3'TP and 3'GP were amplified and two PCR products were produced; If the plant was a hemizygote, DNA between 5'GP and 5'TP, 3'TP and 3'GP, 5GP and 3'GP were all amplified, producing three PCR products. PCR products with different sizes were separated on an agarose gel, and one, two and three bands were easily scored, which represents null, homozygote and hemizygote, respectively. Compared to expensive quantitative PCR and time-consuming bioassay, this method coupled with high throughput genomic DNA extraction is of great assistance to corn breeders for zygosity selection. Hundreds of leaf discs from individual plants can be punched with eppendorf tubes in field and plant zygosity result is readily available. Similar strategy can be applied to develop event-specific zygosity testings for other maize genetically modified events such as Bt11, Event176, GA21, MON810, MON863 and TC1507 provided that their sequences at both insertion junctions are available.

Molecular diagnosis of Candida albicans using real-time polymerase chain reaction of a CaYST1 gene

Candida albicans is an opportunistic pathogen that causes infections in immuno-compromised individuals. Virulence of this organism is a function of a multiplicity of factors working jointly to overwhelm the host defenses. From a previous study, the in vitro adherence capabilities of seven isolates (collected from patients suffering vaginitis) to vaginal epithelial cells were tested. Isolate No. 2 recorded the highest adherence ability. In this part of work, Real-time PCR assay was applied for the identification of isolate No. 2. This isolate could be discriminated by species-specific primers, deduced from the intron nucleotide sequence of the C. albicans CaYST1 gene. To confirm that the isolated strain is a fungal strain, the universal fungal primers (ITS5 and ITS4) were used to amplify the complete internal transcribed spacer (ITS) region including the 5.8S ribosomal gene that is present in all fungi. The ITS region was successfully amplified from the tested yeast, provided a single PCR product of approximately 520 bp. In another run, a single pair of specific primers [Intron Nucleotide Sequence (INT1 and INT2)] was then used to amplify the intron nucleotide sequence of the CaYST1 gene, which present only in C. albicans. The generated amplified product gave the expected single band of 310 bp. The melting points of all PCR products were routinely determined. The results showed that the melting temperatures for the tested isolate and the reference strain of C. albicans (ATCC 10231) using ITS5 and ITS4 primers were 80.09°C and 83.99°C, respectively, while they were 80.78°C and 80.10°C using INT1 and INT2 when assayed individually. Based on their very close melting profiles, the tested isolate proved to be identical to C. albicans reference strain. These results supported the idea of using genes containing intron sequences to design species-specific primers for the identification of fungal strains by real time PCR.

Nuclear Receptor-Induced Chromosomal Proximity and DNA Breaks Underlie Specific Translocations in Cancer

Chromosomal translocations are a hallmark of leukemia/lymphoma and also appear in solid tumors, but the underlying mechanism remains elusive. By establishing a cellular model that mimics the relative frequency of authentic translocation events without proliferation selection, we report mechanisms of nuclear receptor-dependent tumor translocations. Intronic binding of liganded androgen receptor (AR) first juxtaposes translocation loci by triggering intra- and interchromosomal interactions. AR then promotes site-specific DNA double-stranded breaks (DSBs) at translocation loci by recruiting two types of enzymatic activities induced by genotoxic stress and liganded AR, including activation-induced cytidine deaminase and the LINE-1 repeat-encoded ORF2 endonuclease. These enzymes synergistically generate site-selective DSBs at juxtaposed translocation loci that are ligated by nonhomologous end joining pathway for specific translocations. Our data suggest that the confluence of two parallel pathways initiated by liganded nuclear receptor and genotoxic stress underlies nonrandom tumor translocations, which may function in many types of tumors and pathological processes.

Multiplex PCR system for identifying the carnivore origins of faeces for an epidemiological study on Echinococcus multilocularis in Hokkaido, Japan

A multiplex PCR system was developed to identify the carnivore origins of faeces collected in Hokkaido, Japan, for epidemiological studies on Echinococcus multilocularis. Primers were designed against the D-loop region of mitochondrial DNA. Two separate primer mixtures (mix 1, specific forward primers to fox, raccoon dog and dog, and a universal reverse primer [prH]; and mix 2, specific forward primers to cat, raccoon and weasels and prH) were used so that the PCR products (160 bp, fox and cat; 240 bp, raccoon dog and raccoon; and 330 bp, dog and weasel) were distinguished by size. The multiplex PCR exhibited no cross-reactivity between carnivore species and did not amplify DNA from rodent prey. When 270 field-collected faeces were examined, 250 showed single PCR products belonging to specific target sizes, suggesting successful carnivore identification for 92.6% of samples. Taeniid eggs were detected in 11.1% of samples and coproantigen in 30.4%; whereas the prevalences of taeniid eggs and coproantigen were 12.9% and 34.0% in fox faeces, and 0% and 26.3% in cat faeces, respectively. These results suggest that the prevalence in different target animals can be evaluated individually and precisely using multiplex PCR system.

Simplex and Duplex Event-Specific Analytical Methods for Functional Biotech Maize

Analytical methods are very important in the control of genetically modified organism (GMO) labeling systems or living modified organism (LMO) management for biotech crops. Event-specific primers and probes were developed for qualitative and quantitative analysis for biotech maize event 3272 and LY 038 on the basis of the 3' flanking regions, respectively. The qualitative primers confirmed the specificity by a single PCR product and sensitivity to 0.05% as a limit of detection (LOD). Simplex and duplex quantitative methods were also developed using TaqMan real-time PCR. One synthetic plasmid was constructed from two taxon-specific DNA sequences of maize and two event-specific 3' flanking DNA sequences of event 3272 and LY 038 as reference molecules. In-house validation of the quantitative methods was performed using six levels of mixing samples, from 0.1 to 10.0%. As a result, the biases from the true value and the relative deviations were all within the range of +/-30%. Limits of quantitation (LOQs) of the quantitative methods were all 0.1% for simplex real-time PCRs of event 3272 and LY 038 and 0.5% for duplex real-time PCR of LY 038. This study reports that event-specific analytical methods were applicable for qualitative and quantitative analysis for biotech maize event 3272 and LY 038.

Applicability of the 16S–23S rDNA internal spacer for PCR detection of the phytostimulatory PGPR inoculant Azospirillum lipoferum CRT1 in field soil

To assess the applicability of the 16S-23S rDNA internal spacer regions (ISR) as targets for PCR detection of Azospirillum ssp. and the phytostimulatory plant growth-promoting rhizobacteria seed inoculant Azospirillum lipoferum CRT1 in soil. Primer sets were designed after sequence analysis of the ISR of A. lipoferum CRT1 and Azospirillum brasilense Sp245. The primers fAZO/rAZO targeting the Azospirillum genus successfully yielded PCR amplicons (400-550 bp) from Azospirillum strains but also from certain non-Azospirillum strains in vitro, therefore they were not appropriate to monitor indigenous Azospirillum soil populations. The primers fCRT1/rCRT1 targeting A. lipoferum CRT1 generated a single 249-bp PCR product but could also amplify other strains from the same species. However, with DNA extracts from the rhizosphere of field-grown maize, both fAZO/rAZO and fCRT1/rCRT1 primer sets could be used to evidence strain CRT1 in inoculated plants by nested PCR, after a first ISR amplification with universal ribosomal primers. In soil, a 7-log dynamic range of detection (10(2)-10(8) CFU g(-1) soil) was obtained. The PCR primers targeting 16S-23S rDNA ISR sequences enabled detection of the inoculant A. lipoferum CRT1 in field soil. Convenient methods to monitor Azospirillum phytostimulators in the soil are lacking. The PCR protocols designed based on ISR sequences will be useful for detection of the crop inoculant A. lipoferum CRT1 under field conditions.

Identification of a phytoplasma from the aster yellows group infecting purple coneflower (Echinacea purpurea) in Slovenia

Purple coneflower (Echinacea purpurea) (Asteraceae family), is a perennial flowering plant native to eastern North America which is widely grown as an ornamental and medicinal plant. Since 2002, phytoplasma-like symptoms have been observed in several gardens in the Savinja valley in Slovenia. Disease incidence (5–30%) has varied between years and increases with plant age. The symptoms seen include plant weakness, leaf yellowing, and floral malformations particularly virescence and phyllody. For molecular identification of the pathogen, the total DNA was isolated from samples of several parts of an infected plant and from a plant without symptoms using a CTAB protocol (Kump & Javornik, 1996). PCR was carried out using three universal 16S rDNA-specific primers P1/P7, Fu5/Ru3 and R16F2n/R16R2. All three primer pairs gave a single PCR products from tissue with symptoms, with the expected lengths of ~1·8, 0·9, and 1·2 kbp, but no products from samples from plants without symptoms. All PCR products were TA cloned (Promega T-easy), sequenced and assembled into a single sequence of a total length of 1840 bp, which was deposited in GenBank (Accession No. EU416172). A blast search revealed 100% sequence identity for the entire contig with poa stunt phytoplasma (DQ640502) and clover phyllody phytoplasma strain CPh (AF222066) sequences, both belonging to the 16SrI-C subgroup of the aster yellows group (AY). Other matches with high sequence similarities (98–99%) also belonged to aster yellows phytoplasma members, e.g. onion yellows phytoplasma (AP006628.2), epilobium phyllody phytoplasma (AY101386), silene virescence phytoplasma (AY744070) and to the Czech strain of purple coneflower phyllody phytoplasma (EF546778). Phytoplasmas belonging to the AY group have been previously identified in purple coneflower in the USA, Canada (Stanosz & Heimann, 1997; Lee et al., 2008) and in the Czech Republic. This is the first report of detection of an isolate of ‘Candidatus Phytoplasma asteris’ (subgroup 16SrI-C) infecting purple coneflower in Slovenia.

Candidate New Species ofKobuvirusin Porcine Hosts

Candidate New Species of<i>Kobuvirus</i>in Porcine Hosts

Molecular Detection of Colletotrichum lindemuthianum by Duplex PCR

AbstractA duplex PCR technique was developed to detect the pathogenic fungus Colletotrichum lindemuthianum infection in the tissues of common bean. Based on the differences of 24 internal transcribed spacer, DNA sequences of Colletotrichum spp. retrieved from GeneBank database, one pair of specific primers of CY1/CY2 (CY1: 5′‐CTT TGT GAA CAT ACC TAA CC‐3′; CY2: 5′‐GGT TTT ACG GCA GGA GTG‐3′), was designed. The CY1/CY2 primers amplified a single PCR product of 442 bp only from C. lindemuthianum and Colletotrichum orbiculare, not from any other tested species. By using random amplification of polymorphic DNA technique, a product closely associated with C. lindemuthianum was generated. This product was cloned, sequenced and used for designing a species‐specific primers of CD1/CD2 (CD1: 5′‐ACC TGG ACA CAT AAG TCA AAG‐3′; CD2: 5′‐CAA CAA TGC CAG TAT CAG AG‐3′). The CD1/CD2 primers could distinguish C. lindemuthianum from C. orbiculare by a 638 bp PCR band. A duplex PCR method, combining both primers of CY1/CY2 and CD1/CD2, was used to detect C. lindemuthianum infection. The sensitivity of the detection with this PCR method was 1 pg of pure genomic DNA from the pathogen. Therefore, the PCR‐based methods could be used for accurate and rapid detection of C. lindemuthianum from common bean.

The Hairless Phenotype of the Hirosaki Hairless Rat Is Due to the Deletion of an 80-kb Genomic DNA Containing Five Basic Keratin Genes

Most models of hereditary hypotrichosis are due to alterations in growth factors and transcription factors, and the examples of causative mutations in hair keratin genes are limited. The Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDRs). In this study, the locus of the responsible gene was examined by linkage analysis and mapped on chromosome 7q36. Because many basic keratin genes are clustered on 7q36, their expression was examined. Reverse transcription-PCR and genomic PCR indicated that the Kb21 (Krt81), -23 (Krt83), and -26 (Krt86) genes encoding basic hair keratins were not expressed and were deleted. Furthermore, 80-kb genomic DNA ranging from exon 9 of Kb25 (Krt85) to exon 9 of Krt2-25 was deleted. The breakpoints of these genes were within a 95-bp portion shared by the two genes, suggesting that deletion due to non-allelic homologous recombination occurred. Proteins identified as Kb21, Kb23, and Krt2-25 in SDR hairs by mass spectrometry were not detected in HHR. Instead, the product of a fusion gene became dominant in HHR. Because fusion occurred between the exons of the two genes with the same sequences, the product was identical to the wild-type Kb25 protein. By using immunohistochemistry, Kb21 was not detected in HHR hair follicles. Kb25 was expressed in the cortex in HHRs, whereas it was in the medulla in SDRs. This study clearly illustrates the importance of hair keratin genes in hair growth.