Characterization of an X-chromosome PCR-RFLP marker associated with fat deposition and growth in the pig

The X-chromosome, highly conserved within mammals, has been shown to contain major quantitative trait loci (QTL) for growth and fat deposition in the pig. We have discovered a BamHI polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) marker that was assigned to the porcine X-chromosome by two-point and multi-point linkage analysis following genotyping of a three-generation Berkshire by Yorkshire reference family. The marker was positioned 9 cM telomeric to SW2126 and 15.6 cM centromeric to SW1943. Sequence flanking the marker was found to have high similarity to existing database porcine DNA repeat elements. Association analyses of the BamHI marker for growth and meat quality traits in the reference family revealed significant association with marbling (P < 0.03), 10th rib back fat (P < 0.09) and total lipid percentage (P < 0.05), as well as with loin eye area (P < 0.04), average glycolytic potential (P < 0.03) and average lactate content (P < 0.04). Further studies are required to determine the X-chromosome functional gene affecting fat deposition and growth in the pig.     

Characterization of Two Perkinsus spp. from the Softshell Clam, Mya arenaria Using the Small Subunit Ribosomal RNA Gene

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.     

Taenia saginata: differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Molecular Characterization of Human Rotavirus VP4 Genes by Polymerase Chain Reaction and Restriction Fragment Length Polymorphism Assay

A restriction fragment length polymorphism (RFLP) assay was developed to examine the genetic variability and similarity of the VP4 genes of human rotaviruses. The VP4 genes of 14 human rotavirus strains, including VP4 serotype P1A strains (Wa, P, VA70), serotype P1B strain (DS-1), serotype P2 strains (M37, 1076, McN, ST3) and serotype P3 strains (AU-1, AU228, K8, PA151, PCP5, MZ58), and those of 2 feline strains (FRV-1 and Cat2) were reverse-transcribed and amplified by the polymerase chain reaction (PCR). The amplified VP4 cDNAs were then digested with a panel of restriction endonucleases (HindIII, NruI, HaeIII, and EcoRI), resulting in the identification of at least one enzyme with which digestion produced an RFLP profile specific for a particular P serotype. Of interest was the presence of two distinct RFLP patterns within the serotype P3 VP4 genes: one corresponding to the VP4 gene carried by the members of the AU-1 genogroup and the other corresponding to the VP4 genes carried by naturally-occurring reassortants between members of the AU-1 and other genogroups.     

Detection and Identification of Aster Yellows (16SrI) Phytoplasma in Peach Trees in Jordan by RFLP Analysis of PCR-Amplified Products (16S rDNAs)

Aster yellows phytoplasma were detected, for the first time, in peach trees in Al‐Jubiha and Homret Al‐Sahen area. Leaves of infected trees showed yellow or reddish, irregular water‐soaked blotches. Discoloured areas become dry and brittle and the dead tissues dropped out. Under severe infections, leaves fall down and fruits dropped prematurely. Phytoplasmas were detected from all symptomatic peach trees by polymerase chain reaction (PCR) using universal phytoplasmas primers P1/P7 followed by R16F2/R2. No amplification products were obtained from templates of asymptomatic peaches. PCR products (1.2 kb) used for restriction fragment length polymorphism analysis (RFLP) after digestion with endonuclease AluI, HpaII, KpnI and RsaI produced the same restriction profiles for all samples, and they were identical with those of American aster yellows (16SrI) phytoplasma strain. This paper is the first report on aster yellows phytoplasma affecting peach trees in Jordan.     

都柏林念珠菌的PCR-RFLP鉴定

目的建立一种准确、可靠的鉴定都柏林念珠菌基因型的方法。方法临床念珠菌分离自临床生殖器念珠菌病患者,45℃温度试验时几乎不生长,且其他表型实验结果也符合都柏林念珠菌特征。对41例临床念珠菌和1例白念珠菌标准株、1例都柏林念珠菌标准株rDNA内部转录间隔区的基因进行聚合酶链反应（PCR）扩增,HpyF10Ⅵ酶切后观察PAGE图谱。结果聚合酶链反应-限制性片段长度多态性（PCR-RFLP）后,39例临床株鉴定为白念珠菌。2例临床菌株带型特殊,测序后行BLAST比对分析,1例鉴定为白念珠菌,另1例尚不能肯定为都柏林念珠菌,还需要进一步以其他分子生物学方法鉴定。结论PCR-RFLP方法酶切后两种念珠菌带型区分明显,可以鉴别大部分临床菌株。基因测序是该方法有意义的补充。     

Detection of Phytoplasma Infection in Rose, with Degeneration Symptoms

In 1998 a severe disease was observed on rose cvs. 'Patina', 'Papillon' and 'Mercedes' cultivated in a commercial greenhouse in Poland. The symptoms included stunted growth, bud proliferation, leaf malformation and deficiency of flower buds. Sporadically some plants yielded flower buds transformed into big-bud structures and degenerated flowers. The presence of phytoplasma in roses with severe symptoms as well as in recovered plants and Catharanthus roseus experimentally infected by grafting and via dodder was demonstrated by nested polymerase chain reaction assay with primers pair R16F2/R2 or R16F1/R0 and R16(I)F1/R1 amplifying phytoplasma 16S rDNA fragment. The polymerase chain reaction products (1.1 kb) used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes Alu I and Mse I produced the same restriction profiles for all samples. The restriction profiles of phytoplasma DNA from these plants corresponded to those of an aster yellows phytoplasma reference strain. Electron microscope examination of the ultra-thin sections of the stem showed wall thickenings of many sieve tubes of the diseased roses and single phytoplasma cells within a sieve element of the phloem of experimentally infected periwinkles. This paper is the first report on aster yellows phytoplasma in rose identified at a molecular level.     

Functional polymorphisms of caveolin-1 variants as potential biomarkers of esophageal squamous cell carcinoma

Abstract

Objective: To investigate the association of caveolin-1 (CAV1) genetic variants (C239A (rs1997623), G14713A (rs3807987), G21985A (rs12672038), T29107A (rs7804372)) with esophageal squamous cell carcinoma (ESCC) susceptibility.

Methods: A total of 427 patients with ESCC and 427 healthy controls were genotyped using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method.

Results: There were significant differences between patients and controls in distributions of their genotypes and allelic frequencies in G14713A and T29107A polymorphisms. Furthermore, haplotype analysis revealed that haplotypes CAAT and CAGT were associated with high risk for ESCC, while haplotype CGGA was protective against ESCC. Stratified analysis showed the associations between the SNPs (G14713A and T29107A) and ESCC risk were noteworthy among female patients and patients who never smoke or drank alcohol.

Conclusions: Genetic polymorphisms of CAV1 G14713A and T29107A might affect an individual’s susceptibility in developing ESCC, making them efficient potential genetic biomarkers for early detection of ESCC.     

Identification of phytoplasmas associated with a decline of European hackberry (Celtis australis)

The presence of phytoplasmas in declining trees of European hackberry was demonstrated for the first time using polymerase chain reaction assays with primers amplifying phytoplasma 16S rDNA regions. Restriction fragment length polymorphism analysis of these DNA fragments together with PCR, employing primers specific for particular phylogenetic groups of phytoplasmas, made it possible to detect the presence of aster yellows group (16SrI) related phytoplasmas. These were classified into two different subgroups (I-B and I-C) and were present in both symptomatic and asymptomatic hackberry plants. Aster yellows-related phytoplasmas were found in all the root samples collected during the winter. In addition, phytoplasmas from the peach X disease group (16SrIH) were found in four out of 10 root samples; in five root samples phytoplasmas of the elm yellows group (16SrV) were also present.     

Characterisation of phytoplasma (16SrVI) associated with little leaf disease of Portulaca grandiflora – An in silico analysis

A new severe little leaf disease was observed on P. grandiflora, popular as Moss-rose Purslane, widely grown in temperate zones. Characteristic symptoms, ultrastructural studies, antibiotic response and amplification of 16S ribosomal DNA fragments (about 1.5 kb) by polymerase chain reaction (PCR) from infected samples, suspect the involvement of phytoplasma as a pathogen. Nested PCR product, 1.2 kb, with primer pairs R16F2n/R16R2 used for cloning and sequencing. Comparision of the 16S rRNA gene sequences showed that the causal, PLL phytoplasma, is very close (98%) to Indian brinjal little leaf (EF186820) and “Candidatus Phytoplasma trifolii” (AY390261), 16SrVI group phytoplasmas, previously reported from India and Canada respectively. Here, the status of PLL (EF651786) is verified by computer-simulated restriction fragment length polymorphism analysis of 16S rRNA genes of the F2n/R2 sequences of closely related strains of the 16SrVI group using 17 restriction enzymes.     

Cryptic species in the Pyropia yezoensis complex (Bangiales,Rhodophyta): Sympatric occurrence of two cryptic species even on same rocks

In a previous study on wild populations of Pyropia, the occurrence of two possible new species (Pyropia sp. 2 and Pyropia sp. 3) which are closely related to the two commercially important Pyropia species, P. yezoensis and P. tenera, was confirmed as the result of molecular phylogenetic analyses. To characterize the morphological features of the two wild Pyropia species, we collected Pyropia blades in a natural population in which Pyropia sp. 3 was known to occur, and carried out molecular identification before detailed morphological observations. Through the molecular identification we found, unexpectedly, that Pyropia sp. 2 blades grew sympatrically in the same site. Therefore, after molecular identification, we examined in detail the external morphology and anatomy of the two wild Pyropia species using more than 10 blades each. As a result, it is concluded that all of the blades of the two species are morphologically identical to P. yezoensis, but distinct from P. tenera. It is therefore considered that both of the two wild Pyropia species are cryptic species within the P. yezoensis complex. Furthermore, this study revealed that the two cryptic species grew sympatrically, even on the same rocks within the natural habitat.     

Two novel CTNS mutations in cystinosis patients in Thailand

Cystinosis is an autosomal recessive disorder characterized by defective transport of cystine across the lysosomal membrane and resulting in renal, ophthalmic, and other organ abnormalities. Mutations in the CTNS gene cause a deficiency of the transport protein, cystinosin. We performed mutation analysis of CTNS in six cystinosis patients from four families in Thailand. Using PCR sequencing of the entire coding regions, we identified all eight mutant alleles, including two mutations, p.G309D and p.Q284X, that have not been previously reported. This study expands the mutational and population spectrum of nephropathic cystinosis.     

Detection and Molecular Characterization of ‘Candidatus Phytoplasma asteris’ in European Hazel (Corylus avellana) in Poland

Stunted European hazel (Corylus avellana L.) plants showing leaf yellowing were observed in south‐eastern Poland. Phytoplasma‐specific primers P1/P7 and R16F2n/R16R2, as well as primers specific for aster yellows (16SrI), X‐disease (16SrIII) and apple proliferation (16SrX) groups were singly used in nested polymerase chain reaction (PCR) to amplify the 16S rDNA from 22 symptomatic and asymptomatic hazel plants. Restriction fragment length polymorphism with MseI, HhaI, RsaI and BfaI enzymes of the 16S rRNA gene fragments amplified with the primers R16F2n/R16R2 from three symptomatic hazel plants of cvs Katalonski, Webba and Halle revealed patterns identical to those from the AY1 strain related to ‘Candidatus Phytoplasma asteris’. The nucleotide sequence analysis confirmed this result. This is the first report of the natural occurrence of ‘Ca. P. asteris’ in European hazel in Poland.     

Association of cytotoxic T lymphocyte-associated antigen 4 gene single nucleotide polymorphism with type 1 diabetes mellitus in Madurai population of Southern India

Type 1 diabetes mellitus formerly called juvenile diabetes, is an organ specific T-cell mediated autoimmune disease characterized by the progressive loss of function of the insulin producing beta-cells of the islets of Langerhans. Cytotoxic T lymphocyte-associated antigen 4 gene (CTLA-4) has been proposed as a candidate gene for conferring susceptibility to autoimmunity. Association of CTLA-4 gene polymorphism is well established in autoimmune endocrinopathies across world population. The present study was conducted to investigate the association of CTLA-4 exon 1 49A/G polymorphism with TIDM in Madurai, a city in Southern India. Fifty three clinically proven T1DM patients and 53 control subjects with no history of autoimmune disease were recruited for the study. Genomic DNA was extracted from peripheral blood. CTLA-4 exon 1 49 A/G polymorphism was assessed using PCR-RFLP methods. Our findings revealed a significant association of CTLA-4 exon 1 49 A/G polymorphism with T1DM in Madurai population.     

Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

Genetic Diversity in Ralstonia solanacearum Strains from Mauritius using Restriction Fragment Length Polymorphisms

Race 1, biovar III of Ralstonia (synonym Pseudomonas ) solanacearum , causal organism of bacterial wilt, has been reported in Mauritius on several crops and plant species. The genetic relationship among 38 strains isolated from potato, tomato, bean and anthurium was determined by restriction fragment length polymorphisms (RFLPs). After hybridization with probe 5a67, five RFLP patterns could be distinguished. Types V and I were most commonly encountered. A common band of approximately 6.5 kb was found in 35 strains. Type I pattern consisted of only this band and was observed in 12 out of 16 anthurium strains tested. Type V was associated with 12 out of 16 potato strains and consisted of a band of approximately 3.3 kb in addition to the one observed in type I. RFLP patterns II, III and IV were less frequently encountered. The RFLP analysis showed that genetic diversity was present in race 1, biovar III strains. The relationship between the host and RFLP pattern is discussed.     

Primers for sequence characterization and polymorphism detection in the Atlantic salmon (Salmo salar) growth hormone 1 (GH1) gene

We report the identification of intraspecific sequence variation in the Atlantic salmon (Salmo salar) growth hormone 1 gene. Rapid and inexpensive assays for polymorphism detection were developed for 10 sites. Five of the assays detected single nucleotide polymorphisms (SNPs) using polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses, and five were indel polymorphisms, detected using fragment length analyses. The average within population frequency of the most common allele varied from 0.52 to 0.90, and the average within population heterozygosity varied from 0.02 to 0.37 in seven European salmon populations.