Molecular identification and phylogeny of parasitic wasp species (Hymenoptera: Trichogrammatidae) by mitochondrial DNA RFLP and RAPD markers

Mitochondrial DNA (mtDNA) restriction fragment length polymorphism (RFLP) and random amplified polymorphic DNA (RAPD) were tested for their ability to discriminate between seven species of minute parasitic wasps belonging to the genus Trichogramma. They proved to be reliable species-diagnostic molecular markers. Pairwise comparisons of the mtDNA restriction maps revealed considerable differentiation among the seven species. Percentage of common restriction sites ranged from 30% to 83%. Phylogenetic analyses performed either on the mtDNA nucleotide distance matrix or on the matrix of the restriction site-state generated a tree congruent with those based on allozymes and morphology.
RAPD procedures also revealed species-specific banding patterns and seem promising for a rapid and easy identification of Trichogramma species. Moreover, for some Trichogramma species, RAPD banding patterns might be informative of the phylogenetic related ness.     

Attempts to molecularly distinguish cryptic taxa in Anopheles gambiae s.s

Analyses of inversions in polytene chromosomes indicate that, in West Africa, Anopheles gambiae (sensu stricto) may be a complex of more than a single taxonomic unit, and these units have been called chromosomal forms. In order to determine whether this genetic discontinuity extends to the rest of the genome, as would be expected if reproductive isolation exists, we have sequenced several regions of both the nuclear and mitochondrial genomes. With one exception, we were unable to identify any nucleotide sites that differentiate the chromosomal forms. The exception was the internal transcribed spacer (ITS) of the ribosomal DNA (rDNA). Three sites in this region distinguish Mopti chromosomal form from Savanna and Bamako in Mali and Burkina Faso. However, outside these two countries, the association between chromosomal form and rDNA type does not always hold. Together with the variants in the rDNA intergenic spacer (IGS) described in the accompanying papers ( della Torre et al., 2001 ; Favia et al., 2001 ), we can recognize two major types of rDNA, Type I and Type II (corresponding to molecular forms S and M in della Torre et al., 2001 ). Type I is widespread in West Africa and is the only type found outside of West Africa (i.e. Tanzania and Madagascar). Type II is confined to West Africa. We were unable to detect any heterozygosity for the ITS types even in five collections containing both types. A sample from the island of São Tomé could not be classified into either Type I or Type II as the rDNA had characteristics of both. In general, our results confirm that An. gambiae is not a single pan‐mictic unit, but exactly how to define any new taxa remains problematic. Finally, we have found minor variants of the major rDNA types fixed in local populations; contrary to most previous studies, this suggests restricted gene flow among populations of this species.     

Typing of hepatitis C virus by a new method based on restriction fragment length polymorphism.

A new restriction fragment length polymorphism (RFLP) analysis has been developed for hepatitis C virus (HCV) typing in the viral 5' non-coding region and contiguous core region. These genomic sequences were chosen for the relative nucleotide homology among different genotypes and for the presence of polymorphic sites. By employing two endonucleases (AccI and MboI) and, in some instances, a third one (EcoRII), we can unambiguously and reproducibly distinguish between genotypes and subtypes 1a, 1b, 1c, 2a, 2c, 2b, 3a, 3b, 4a, 5a and 6a. The method was applied for diagnosing two Italian groups of HCV-infected individuals reflecting a randomly collected population and a group of intravenous drug users. The accuracy of this method has been validated by comparison with INNOLiPA and by sequencing. Our approach represents an improvement over previous RFLP methods, since typing is accurate and simpler.     

Variation in the ribosomal RNA cistron among host-adapted races of an aphid (Schizaphis graminum)

The greenbug, Schizaphis graminum, is an aphid species that consists of races that can be separated based on morphology, life histories, cytogenetics, mitochondrial DNA RFLPs and virulence to plant culti-vars. Variation in the greenbug rDNA multigene family was studied to determine the extent to which rDNA cistrons have diverged among and within races. A restriction map of the rDNA cistron was constructed. Probing DNA from different races with subclones from rDNA coding regions and internal spacers identified little variation. However, probing with subclones of the intergenic spacer (IGS) identified continuous length variation within and among races. Race specific patterns were identified. Within a race, almost continuous variation in total IGS length was detected and asexual lineages possessed distinct patterns useful in genetic fingerprinting studies.     

Phylogenetic relationships among fruit flies, Bactrocera (Diptera, Tephritidae), based on the mitochondrial rDNA sequences

Nucleotide sequences of a 1.6 kb long portion of the mitochondrial DNA containing the majority of the 16S rRNA gene, the tRNAval gene, and the 5' half-region of the 12S rRNA gene were determined for forty-eight individuals of nineteen Bactrocera species and one other tephritid taxon, Anastrepha ludens. Phylogenetic analyses were performed using the consistently aligned 1.5 kb long sequences, excluding seventeen portions that could not be aligned unambiguously and were aligned inconsistently among analyses using different programs and parameters. Results of phylogenetic analyses were highly congruent among distance, unweighted parsimony, and weighted parsimony methods in terms of topology of resulting trees. Bootstrap analyses supported many of the phylogenetic relationships resolved by these analyses. Although aligned sequences showed apparent biases in nucleotide content and substitutions that differed among sites within the stem/loop structure of rRNA molecules, compensation for such factors did not greatly affect the topology. The results are discussed in relation to the taxonomic positions of species used in this study and in relation to the phylogenetic and diagnostic utility of the mitochondrial rDNA fragment.     

Detection and characterization of thefimAgene ofEscherichia coliO157:H7

An expected 850-bp DNA fragment containingfimA, the structural gene for type 1 fimbriae, and flanking sequences was amplified from 39 (of 46) pathogenic and commensal strains ofEscherichia coliusing the polymerase chain reaction (PCR). Restriction fragment length polymorphism (RFLP) analysis of the amplified products showed 13HinPI and fourSau96I restriction profiles among these 39E. colistrains, revealing the polymorphic nature of this allele. A unique RFLP pattern was shared byE. coliO157:H7, O157:H^- and a few O55 serotype strains. DNA sequence analysis of thefimAregion demonstrated thatE. coliO157:H7 strain 933 and O157:H⁻strain E32511 contained identical DNA sequences that were distinct from otherE. colistrains, especially a 16-bp sequence 5′ tofimAthat was conspicuously absent only inE. coliO157 strains. Exploiting these differences, a PCR assay was developed that amplifies a 936-bp fragment from allE. coliO157:H7 strains examined to date. This PCR assay offers a simple, rapid, and reliable means to detectE. colistrains of the O157:H7 serotype.     

Heterogeneity of the internal transcribed spacer (ITS-2) region within individual deer ticks

To determine whether nuclear rDNA sequences provide a useful means for assessing the structure of populations of Ixodes ticks, we compared variability among copies of an internal transcribed spacer (ITS-2) sequence within individual ticks to the variability between ticks. At least 4% of the nucleotides comprising this sequence vary among the copies present within individual ticks. ITS-2 diversity in each of two ticks is nearly half as great as that reported between ticks from geographically disparate populations. Because individual ticks retain ancestral polymorphism, ITS-2 variation does not accurately reflect descent relationships among these ticks. Sequencing single copies of PCR-amplified ITS-2 therefore does not permit assessment of the phylogenetic relationships among the I. ricinus-like ticks in eastern North America. We recommend caution in future analyses, and emphasize the importance of procedures designed to ensure that the many paralogous coples of the rDNA cistron have been sufficiently homogenized by concerted evolutionary processes. Such precautionary measures will make certain that phylogenetic trees based on these gene sequences reflect the phyletic relatedness of the biological species.     

Mutagenic primer‐based PCR‐RFLP assay for genotyping IRGM gene promoter variant rs4958843 (C/T)

Background

Single‐nucleotide polymorphisms play an important role in the susceptibility of many diseases, evolutionary studies, and genetic mapping. The rs4958843 in IRGM promoter is associated with tuberculosis and Crohn's disease. As this SNP is not present in any of the restriction sites, PCR‐RFLP is not possible. Therefore, we have developed artificial‐RFLP method to genotype this SNP.

Methods

We designed forward primer with mismatches that resulted in the creation of a restriction site for enzyme NheI in the amplicon. Control samples of known genotypes were obtained by sequencing. The amplified product for SNP rs4958843 was digested with NheI restriction enzyme and resolved on an agarose gel to know the genotypes of the samples.

Results

Results of sequencing and A‐RFLP were concordant. The developed method was applied to genotype this polymorphism in 100 samples from healthy individuals. The allelic frequencies of SNP rs4958843 were C (0.16) and T (0.84), while corresponding genotypic distribution was CC (2), CT (29), and TT (69).

Conclusion

The newly developed method is simple, easy, and cost‐effective which could be used to genotype IRGM polymorphism −1161 C/T (rs4958843) in various populations in the replication studies and has its applicability in the clinical settings. The developed method was applied for genotyping samples from healthy individuals from North India. For the first time, we report the frequency of this polymorphism from this region.

     

A facile PCR‐RFLP method for genotyping of ITPA rs1127354 and rs7270101 polymorphisms

Background

Inosine triphosphate pyrophosphatase (ITPA) gene single nucleotide polymorphisms (SNPs), rs1127354 and rs7270101, may cause a functional impairment in ITPase enzyme, resulting anemia protection in patients with chronic hepatitis C virus (HCV) infection undergoing ribavirin (RBV)‐dependent regimens. The main purpose of this study was to provide and validate a simple, rapid, and inexpensive polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) technique for genotyping of ITPA rs1127354 and rs7270101 polymorphisms in chronic HCV‐infected patients.

Methods

In the current study, 100 Iranian patients with chronic hepatitis C were examined and genotyped for ITPA rs1127354 and rs7270101 gene polymorphisms. To genotype rs1127354 and rs7270101 polymorphisms, PCR‐RFLP technique and sequencing technique were performed on these samples. To validate the PCR‐RFLP method, the PCR‐RFLP genotyping results should be 100% concordant with the PCR‐sequencing results.

Results

The rs1127354 and rs7270101 polymorphisms of ITPA gene were genotyped by PCR‐RFLP technique and sequencing simultaneously, and the results of both techniques were 100% concordant in all 100 patients. Both PCR‐RFLP and sequencing techniques indicated that the genotypic frequency of rs7270101 was 80% AA, 19% AC and 1% CC, and for rs1127354 was 79% CC, 20% CA and 1% AA, respectively.

Conclusion

We developed and validated a rapid and inexpensive PCR‐RFLP technique for the detection of ITPA rs1127354 and rs7270101 gene polymorphisms.

     

A mutation in the 65,000 Dalton heat shock protein gene, commonly used for molecular identification of non-tuberculous mycobacteria, leads to the misidentification of Mycobacterium malmoense as Mycobacterium marinum

We describe a case of a Mycobacterium isolated from a patient with cervical lymphadenitis which was initially identified by hsp65 RFLP as Mycobacterium marinum. Sequence analysis of the hsp65 DNA fragment and the 16S rDNA signature sequence, however, led to the identification of Mycobacterium malmoense. A point mutation in one of the restriction sites had shifted the M. malmoense typical RFLP pattern to the M. marinum specific RFLP pattern. As a consequence, care should be taken when identifying mycobacteria with the use of one molecular technique, only.     

Genetic diversity of Flavobacterium columnare examined by restriction fragment length polymorphism and sequencing of the 16S ribosomal RNA gene and the 16S-23S rDNA spacer

Genetic variability among strains of Flavobacterium columnare, isolated in the United States, was characterized by restriction fragment length polymorphism (RFLP) and phylogenetic analysis based on the sequence of the 16S rRNA gene. Twenty-seven isolates of F. columnare were differentiated into three genotypes. The isolates within the genotypes were further grouped based on RFLP of the 16S-23S rDNA spacer. The first genotype had five strains that were further divided into group A (4 strains) and B (1 strain) while the second genotype had 10 strains that were also further divided into group A (4 strains) and B (6 stains). The third genotype had 12 isolates with no differences in the RFLP patterns of the 16S-23S rDNA spacers. The 16S rRNA gene sequences representing the three identified genotypes were compared to the different published sequences by phylogenetic analysis and the results showed the American genotypes 1, 2 and 3 corresponding to genomovar 1, 2, and 3, respectively, reported by Triyanto and Wakabayashi [Triyanto, Wakabayashi H. Genotyping of strains of Flavobacterium columnare from diseased fishes. Fish Pathol 1999; 34: 65-71]. The study demonstrates a method for RFLP and sequencing of the 16S rRNA gene and the 16-23S rDNA spacer as a useful tool in epidemiological studies of F. columnare.     

Ribosomal spacer length variability in the large raspberry aphid, Amphorophora idaei (Aphidinae: Macrosiphini)

The large raspberry aphid, Amphorophora idaei , has several biotypes described by their abilities to overcome plant resistance genes. Bioassays of field populations showed a strong shift towards A, resistance-breaking biotypes since the 1960s. RFLP analysis of the rDNA cistron was used to study variation found within and between standard clones of three A. idaei biotypes and twenty-nine field populations collected over 3 years. Probing genomic DNA with the ribosomal DNA probe pBG 35 produced consistent differences in RFLPs between standard clones of biotypes. However, analysis of field populations gave more complex RFLP patterns that were not biotype-specific, unlike characteristic intergenic spacer (IGS) patterns reported for Schizaphis graminum biotypes. All but one sample collected from separate fields showed considerable genetic diversity within populations, attributed to alate migrations of parthenogenetic females in summer and males in autumn.     

Molecular basis of polymorphisms of human complement component C3

C3 exhibits two common allotypic variants that may be separated by gel electrophoresis and are called C3 fast (C3 F) and C3 slow (C3 S). C3 F, the less common variant, occurs at appreciable frequencies only in Caucasoid populations (gene frequency = 0.20). An increased prevalence of the C3 F allele has been reported in patients with partial lipodystrophy, IgA nephropathy, and Indian childhood hepatic cirrhosis. Studies of the genomic organization of the human C3 gene led to the identification of a single change (C to G) between C3 S and C3 F at nucleotide 364 in exon 3. This leads, at the translation level, to the substitution of an arginine residue (positively charged) in C3 S for a glycine residue (neutral) in C3 F. This substitution results in a polymorphic restriction site for the enzyme HhaI. The resulting restriction fragment length polymorphism (RFLP) was investigated using genomic DNA, amplified using the polymerase chain reaction; there was absolute concordance between the genomic polymorphism and the distribution of C3 S and C3 F in 50 normal subjects. The molecular basis of a second structural polymorphism, defined by the monoclonal antibody HAV 4-1, was also characterized. The polymorphic determinant was identified at codon 314 in the exon 9 of the beta chain where a leucine residue (HAV 4-1+) is substituted for a proline residue (HAV 4-1-). Identification of the amino acid sequences of these polymorphic variants will facilitate characterization of possible functional differences between different allotypes of C3. Three RFLPs (BamHI, EcoRI, and SstI) were located to introns in the C3 gene. There was no allelic association between these three RFLPs, or between the RFLPs and the C3 F/S polymorphic site. Genetic equilibration of these polymorphisms has occurred within a gene of 41 kb.     

Molecular taxonomy using single-strand conformation polymorphism (SSCP) analysis of mitochondrial ribosomal DNA genes

Single-strand conformation polymorphism (SSCP) analysis detects single point mutations in DNA molecules. We demonstrate that SSCP analysis of mitochondrial ribosomal DNA (rDNA) genes is a sensitive taxonomic tool because these genes often differ at numerous sites among closely related species. Using conserved primers, portions of the 12S or 16S rDNA genes were amplified using the polymerase chain reaction (PCR) in congeneric species of ticks, leaf hoppers, mosquitoes, and closely related endoparasitic wasps. SSCP was performed and products were visualized with silver staining. Species-specific patterns were observed in all taxa. Intraspecific variation at the level of single nucleotide substitutions was detected. SSCP diagnostics are less expensive and time consuming to develop than PCR with species-specific primers, and, unlike PCR with arbitrary primers, there is minimal concern with DNA contamination from non-target organisms.     

DNA variation in the genes of the Na,K-adenosine triphosphatase and its relation with resting metabolic rate, respiratory quotient, and body fat.

The aim of this study was to investigate in 261 subjects from 58 families the association between DNA variation at the genes coding for the Na,K-ATPase peptides and resting metabolic rate (RMR), respiratory quotient (RQ), and percent body fat (%FAT). Five restriction fragment length polymorphisms (RFLP) at three Na,K-ATPase genes were determined: one at the alpha 1 locus (BglII), and two at the beta locus (beta MspI and beta PvuII). Haplotypes were determined from the two variable sites of the alpha 2 gene (alpha 2 haplotypes) and the beta gene (beta haplotypes). There was a strong trend for %FAT to be related to the RFLP generated by BglII at the alpha 2 exons 21-22 in males (P = 0.06) and females (P = 0.05). RQ was (a) associated with the BglII RFLP at the alpha 2 exon 1 (P = 0.02) and with the alpha 2 8.0 kb/4.3 kb haplotype (P = 0.04) and (b) linked with the beta gene MspI marker (P = 0.04) and with the beta 5.3 kb/5.1 kb haplotype (P = 0.008) based on sib-pair analysis. The present study suggests that the genes encoding Na,K-ATPase may be associated or linked with RQ and perhaps with %FAT but not with RMR.     

A Taql polymorphism in the human interleukin-1β (IL-1β) gene correlates with IL-1β secretion in vitro

In the present study we searched for restriction fragment length polymorphisms (RFLP) in the human interleukin-1 beta (IL-1 beta) gene and for correlations to monocyte (Mo) function in non-related healthy donors and insulin-dependent diabetic patients. We demonstrated a diallelic polymorphism with the restriction enzyme TaqI consisting of fragments of 9.4 kb and 13.4 kb. No differences in allele or genotype frequencies of this RFLP were observed between randomly selected controls and randomly selected patients with insulin-dependent diabetes mellitus (IDDM). However, when analysing IDDM patients negative for HLA-DR3 and -DR4, our data demonstrate that the 13.4 kb allele is more frequent in this group compared to a matched control group. The functional impact of this RFLP was studied by analysing in vitro stimulated Mo IL-1 beta response. An IL-1 beta allele dosage effect on secretory capacity was observed after LPS-stimulation: 13.4/13.4 kb homozygous individuals secreted significantly more IL-1 beta than 9.4/13.4 kb heterozygous individuals, who secreted significantly more than 9.4/9.4 kb homozygous individuals. Analyses of supernatants from LPS-stimulated Mo cultures from individuals with each TaqI IL-1 beta genotype revealed no differences in the mouse thymocyte co-stimulatory assay when compared on a molar basis, indicating that the TaqI polymorphism gave rise only to quantitative differences in expression levels and probably not to a mutant IL-1 beta.(ABSTRACT TRUNCATED AT 250 WORDS)     

高脂血症患儿载脂蛋白AI－CIII基因区限制性片段长度多态性研究

利用２．２ｋｂ人载脂蛋白ＡＩ基因探针，检测５７例高脂血症患儿及３８例正常血脂儿童的载脂蛋白（ａｐｏ）ＡＩ－ＣＩＩＩ基因区限制性片段长度多态性。发现中国上海汉族儿童人群ａｐｏＡＩ－ＣＩＩＩ基因区存在Ｓｓ　ｔＩ多态位点，其等位基因频率Ｓ＿１为０．９８，Ｓ＿２为０．１２，明显不同于其他种族。并发现该多态位点与高胆固醇血症、高甘油三酯血症、高胆固醇合并高甘油三酯血症，均无密切关系，不能作为该地区高脂血症患儿的遗传标记。     

A PCR-RFLP assay for identification of Anisakis simplex from different geographical regions

Anisakis simplex, a nematode from the family Anisakidae, is a parasite of fish and mammals. It is a casual agent of a human disease called anisakiosis. We found that the assay based on PCR amplification of the ITS-1–5·8 S–ITS-2 fragment of rDNA and subsequent restriction fragment length polymorphism, previously described on the basis of A. simplex isolated solely from one geographical region, can be used as a general test for identification of this worm species. The restriction patterns analysed for four restriction enzymes were found to be identical in the case of allA. simplex individuals isolated from as different geographical regions as Baltic Sea, Norwegian Sea, Bering Sea and Sea of Okchotsk. Moreover, our results support the previously proposed hypothesis, based on the studies of isoenzymes, that there is a remarkable genetic homogeneity within A. simplex from different geographical regions.     

Genetic similarity among pheromone and voltinism races of Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae)

The genetic variability of seven European corn borer populations, Ostrinia nubilalis, from North America and Europe was assessed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis and DNA sequencing. The nuclear ribosomal internal transcribed spacer 1 (ITS-1) region (≈ 500 base pair [bp]) and four mitochondrial (mtDNA) regions (1550 bp total) were examined. The smartweed borer, Ostrinia obumbratalis, and south-Western corn borer, Diatraea grandiosella, were used for comparisons. Of 106 restriction sites identified (80 in mtDNA and 26 in ITS-1), none differentiated geographical populations, pheromone races, or voltine ecotypes of the European corn borer. The lack of variation in the ITS-1 of European corn borer was confirmed by DNA sequence analysis. The genetic similarity of European corn borer populations, despite their wide geographical range and physiological differences, may be explained by a relatively recent origin for the voltinism and pheromone races, gene flow among races, and/or expansion from genetic bottlenecks.