In vitro cultivation of Wolbachia pipientis in an Aedes albopictus cell line

A continuous cell line, Aa23, was established from eggs of a strain of the Asian tiger mosquito, Aedes albopictus, naturally infected with the intracellular symbiont Wolbachia pipientis. The resulting cell line was shown to be persistently infected with the bacterial endosymbiont. Treatment with antibiotics cured the cells of the infection. In the course of establishing this cell line it was noticed that RFLPs in the PCR products of two Wolbachia genes from the parental mosquitoes were fixed in the infected cell line. This indicates that the mosquito host was naturally superinfected with different Wolbachia strains, whereas the infected cell line derived from these mosquitoes only contained one of the original Wolbachia strains. The development of anin vitroculture system for this fastidious microorganism should facilitate molecular analysis of the reproduction distorting phenotypes it induces in natural arthropod hosts.

Evolutionary distinctiveness and status of the endangered Lake Eacham rainbowfish (Melanotaenia eachamensis)

The Lake Eacham rainbowfish (Melanotaenia eachamensis) was declared extinct in the wild in the late 1980s after it disappeared from its only known locality, an isolated crater lake in northeast Queensland. Doubts have been raised about whether this taxon is distinct from surrounding populations of the eastern rainbowfish (Melanotaenia splendida splendida). We examined the evolutionary distinctiveness of M. eachamensis, obtained from captive stocks, relative to M. s. splendida through analysis of variation in mtDNA sequences, nuclear microsatellites, and morphometric characters Captive M. eachamensis had mtDNAs that were highly divergent from those in most populations of M. s. splendida. A broader geographic survey using RFLPs revealed some populations initially identified as M. s. splendida, that carried eachamensis mtDNA, whereas some others had mixtures of eachamensis and splendida mtDNA. The presence of eachamensis-like mtDNA in these populations could in principle be due to (1) sorting of ancestral polymorphisms, (2) introgression of M. eachamensis mtDNA into M. s. splendida, or (3) incorrect species boundaries, such that some populations currently assigned to M. s. splendida are M. eachamensis or are mixtures of the two species. These alternatives hypotheses were evaluated through comparisons of four nuclear microsatellite loci and morphometrics and meristics. In analyses of both data sets, populations of M. s. splendida with eachamensis mtDNA were more similar to captive M. eachamensis than to M. s. splendida with splendida mtDNA, supporting hypothesis 3. These results are significant for the management of M. eachamensis in several respects. First the combined molecular and morphological evidence indicates that M. eachamensis is a distinct species and a discrete evolutionarily significant unit worthy of conservation effort. Second it appears that the species boundary between M. eachamensis and M. s. splendida has been misdiagnosed such that there are extant populations on the Atherton Tableland as well as areas where both forms coexist. Accordingly we suggest that M. eachamensis be listed as vulnerable, rather than critical (or extinct in the wild). Third, the discovery of extant but genetically divergent populations of M. eachamensis on the Atherton Tableland broadens the options for future reintroductions to Lake Eacham.

Transfer of a supernumerary chromosome between vegetatively incompatible biotypes of the fungus Colletotrichum gloeosporioides

Two biotypes (A and B) of Colletotrichum gloeosporioides infect the tropical legumes Stylosanthes spp. in Australia. These biotypes are asexual and vegetatively incompatible. However, field isolates of biotype B carrying a supernumerary 2-Mb chromosome, thought to originate from biotype A, have been reported previously. We tested the hypothesis that the 2-Mb chromosome could be transferred from biotype A to biotype B under laboratory conditions. Selectable marker genes conferring resistance to hygromycin and phleomycin were introduced into isolates of biotypes A and B, respectively. A transformant of biotype A, with the hygromycin resistance gene integrated on the 2-Mb chromosome, was cocultivated with phleomycin-resistant transformants of biotype B. Double antibiotic-resistant colonies were obtained from conidia of these mixed cultures at a frequency of approximately 10(-7). Molecular analysis using RFLPs, RAPDs, and electrophoretic karyotypes showed that these colonies contained the 2-Mb chromosome in a biotype B genetic background. In contrast, no double antibiotic colonies developed from conidia obtained from mixed cultures of phleomycin-resistant transformants of biotype B with biotype A transformants carrying the hygromycin resistance gene integrated in chromosomes >2 Mb in size. The results demonstrated that the 2-Mb chromosome was selectively transferred from biotype A to biotype B. The horizontal transfer of specific chromosomes across vegetative incompatibility barriers may explain the origin of supernumerary chromosomes in fungi.

Gene and genotypic diversity of Phytophthora cinnamomi in South Africa and Australia revealed by DNA polymorphisms

Phytophthora cinnamomi isolates from South Africa and Australia were compared to assess genetic differentiation between the two populations. These two populations were analysed for levels of phenotypic diversity using random amplified polymorphic DNAs (RAPDs) and gene and genotypic diversity using restriction fragment length polymorphisms (RFLPs). Sixteen RAPD markers from four decanucleotide Operon primers and 34 RFLP alleles from 15 putative loci were used. A few isolates from Papua New Guinea known to posses alleles different from Australian isolates were also included for comparative purposes. South African and Australian P. cinnamomi populations were almost identical with an extremely low level of genetic distance between them (D-m = 0.003). Common features for the two populations include shared alleles, low levels of phenotypic/genotypic diversity, high clonality, and low observed and expected levels of heterozygosity. Furthermore, relatively high levels of genetic differentiation between mating type populations (D-m South Africa = 0.020 and D-m Australia = 0.025 respectively), negative fixation indices, and significant deviations from Hardy-Weinberg equilibrium, all provided evidence for the lack of frequent sexual reproduction in both populations. The data strongly suggest that both the South African and Australian P. cinnamomi populations are introduced.

Investigation of IGF2/ApaI and H19/RsaI polymorphisms in patients with cutaneous melanoma

Objective: The etiology of cutaneous melanoma is complex, involving both heterogeneous genetic and environmental components. The aim of our study was to verify if single polymorphic sites within IGF2 and H19 genes and their consequent haplotypes influence risk and/or prognosis of familial melanoma. Design: Twenty one patients with clinical criteria of hereditary melanoma (early onset, presence of multiple primary melanoma, and/or one or more affected first- or second-degree relatives) and previously screened for CDKN2A mutations were genotyped by IGF2/ApaI and H19/RsaI PCR-RFLPs. Data were compared between patients and a control group (100 healthy young individuals) using Chi-square and Fisher`s exact tests. We also investigated if these polymorphic sites could be microRNAs potential targets, using RegRNA software. Results: Although the IGF2 and HI9 genotypes/haplotypes were not significantly associated with melanoma, two of the most severe cases (very early onset or multiple melanomas) showed to be heterozygous for both genes. We found an overlap between IGF2/ApaI and miR-615-5p, and between H19/RsaI and miR-574-3p. Conclusions: Some studies have shown H19, and IGF2 genes (or related genes or protein, for example, IGF2R and IMP-3) differential expression in melanoma. However, no study has attempted to examine markers across this cluster in relation to melanoma until now. Since the base change may impair the pairing of microRNA and its binding site, our results suggest a new window for future studies of IGF2 and H19 genetic variability and posttranscriptional regulation. Due to the importance and based on the present results, we suggest that the genotype/haplotype analysis of IGF2 and H19 polymorphisms should be better investigated in large populations with cutaneous melanoma, attempting to tie the association with progression of the disease. (C) 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different-colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were samplcd. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating inde- pendently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once.

Genetic relationships of Corynebacterium diphtheriae strains isolated from a diphtheria case and carriers by restriction fragment length polymorphism of rRNA genes

In the present study we report the results of an analysis, based on ribotyping of Corynebacterium diphtheriae intermedius strains isolated from a 9 years old child with clinical diphtheria and his 5 contacts. Quantitative analysis of RFLPs of rRNA was used to determine relatedness of these 7 C.diphtheriae strains providing support data in the diphtheria epidemiology. We have also tested those strains for toxigenicity in vitro by using the Elek's gel diffusion method and in vivo by using cell culture method on cultured monkey kidney cell (VERO cells). The hybridization results revealed that the 5 C.diphtheriae strains isolated from contacts and one isolated from the clinical case (nose case strain) had identical RFLP patterns with all 4 restriction endonucleases used, ribotype B. The genetic distance from this ribotype and ribotype A (throat case strain), that we initially assumed to be responsible for the illness of the patient, was of 0.450 showing poor genetic correlation among these two ribotypes. We found no significant differences concerned to the toxin production by using the cell culture method. In conclusion, the use of RFLPs of rRNA gene was successful in detecting minor differences in closely related toxigenic C.diphtheriae intermedius strains and providing information about genetic relationships among them.

Meningococcal Disease Caused by Neisseria meningitidis Serogroup B Serotype 4 in São Paulo, Brazil, 1990 to 1996

A large epidemic of serogroup B meningococcal disease (MD), has been occurring in greater São Paulo, Brazil, since 1988.21 A Cuban-produced vaccine, based on outer-membrane-protein (OMP) from serogroup B: serotype 4: serosubtype P1.15 (B:4:P1.15) Neisseria meningitidis, was given to about 2.4 million children aged from 3 months to 6 years during 1989 and 1990. The administration of vaccine had little or no measurable effects on this outbreak. In order to detect clonal changes that could explain the continued increase in the incidence of disease after the vaccination, we serotyped isolates recovered between 1990 and 1996 from 834 patients with systemic disease. Strains B:4:P1.15, which was detected in the area as early as 1977, has been the most prevalent phenotype since 1988. These strains are still prevalent in the area and were responsible for about 68% of 834 serogroup B cases in the last 7 years. We analyzed 438 (52%) of these strains by restriction fragment length polymorphism (RFLPs) of rRNA genes (ribotyping). The most frequent pattern obtained was referred to as Rb1 (68%). We concluded that the same clone of B:4:P1.15-Rb1 strains was the most prevalent strain and responsible for the continued increase of incidence of serogroup B MD cases in greater São Paulo during the last 7 years in spite of the vaccination trial.

Molecular characterization of Torque teno virus and SEN virus co-infection with HIV in patients from Southern Iran

Introduction Torque teno virus (TTV) and SEN virus are circular single-stranded DNA viruses that cause blood-borne infections. The SEN virus (SEN-V) was originally detected in the serum of an injection drug user infected with human immunodeficiency virus (HIV). Recently TTV was discovered as a potential causative agent of non-A-E hepatitis. The aim of this study was to investigate the prevalence of the SEN-V-D/H and TTV in HIV patients and healthy blood donors in Iran. Methods One hundred and fifty HIV patients with a mean age of 50.46 ± 18.46 years and 150 healthy blood donors with a mean age of 48.16 ± 13.73 years were included in this study. TTV and SEN-V were detected by the PCR and were quantitatively assayed by competitive PCR (nested and semi-nested PCR). Restriction fragment length polymorphisms (RFLPs) were used to determine the heterogeneity of TTV. Results TTV and SEN-V were detected 96 (64%) and 84 (56%) of 150 HIV patients respectively. These rates were 34% (n=51) and 37.33% (n=56) in healthy blood donors (significant, p<0.05). PCR detected SEN-V/TTV DNA from 32 of the healthy blood donors (21.33%), while 65 (43.33%) of HIV patients were positive for SEN-V/TTV DNA. Of 150 HIV patients, 32.66% and 23.33% were positive for SEN-V-H and SEN-V-D, respectively and 18.66% (n=28) were co-infected with SEN-V-D/H. Conclusions The prevalence of SEN-VD/H and TTV is higher in HIV patients than in healthy blood donors in Southern Iran. Our results suggest that TTV and SEN-V might play a role in the development of liver disease in patients with immunodeficiency diseases.

Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil

Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB) polymerase chain reaction (PCR) was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs). We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human), respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia) braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.

Development and validation of PCR-based assays for diagnosis of American cutaneous leishmaniasis and identificatio nof the parasite species

In this study, PCR assays targeting different Leishmania heat-shock protein 70 gene (hsp70) regions, producing fragments ranging in size from 230-390 bp were developed and evaluated to determine their potential as a tool for the specific molecular diagnosis of cutaneous leishmaniasis (CL). A total of 70 Leishmania strains were analysed, including seven reference strains (RS) and 63 previously typed strains. Analysis of the RS indicated a specific region of 234 bp in the hsp70 gene as a valid target that was highly sensitive for detection of Leishmania species DNA with capacity of distinguishing all analyzed species, after polymerase chain reaction-restriction fragment length polymorfism (PCR-RFLP). This PCR assay was compared with other PCR targets used for the molecular diagnosis of leishmaniasis: hsp70 (1400-bp region), internal transcribed spacer (ITS)1 and glucose-6-phosphate dehydrogenase (G6pd). A good agreement among the methods was observed concerning the Leishmania species identification. Moreover, to evaluate the potential for molecular diagnosis, we compared the PCR targets hsp70-234 bp, ITS1, G6pd and mkDNA using a panel of 99 DNA samples from tissue fragments collected from patients with confirmed CL. Both PCR-hsp70-234 bp and PCR-ITS1 detected Leishmania DNA in more than 70% of the samples. However, using hsp70-234 bp PCR-RFLP, identification of all of the Leishmania species associated with CL in Brazil can be achieved employing a simpler and cheaper electrophoresis protocol.

Genetic structure of a linear population of Beta vulgaris ssp maritima (sea beet) revealed by isozyme and RFLP analysis

Knowledge of the genetic structure of plant populations is necessary for the understanding of the dynamics of major ecological processes. It also has applications in conservation biology and risk assessment for genetically modified crops. This paper reports the genetic structure of a linear population of sea beet, Beta vulgaris ssp. maritima (the wild relative of sugar beet), on Furzey Island, Poole Harbour. The relative spatial positions of the plants were accurately mapped and the plants were scored for variation at isozyme and RFLP loci. Structure was analysed by repeated subdivision of the population to find the average size of a randomly mating group. Estimates of F-ST between randomly mating units were then made, and gave patterns consistent with the structure of the population being determined largely by founder effects. The implications of these results for the monitoring of transgene spread in wild sea beet populations are discussed.

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

A search for RFLP markers to identify genes for aluminum tolerance in maize

The objective of this study was to identify restriction fragment length polymorphism (RFLP) markers linked to QTLs that control aluminum (Al) tolerance in maize. The strategy used was bulked segregant analysis (BSA) and the genetic material utilized was an F2 population derived from a cross between the Al-susceptible inbred line L53 and Al-tolerant inbred line L1327. Both lines were developed at the National Maize and Sorghum Research Center - CNPMS/EMBRAPA. The F2 population of 1554 individuals was evaluated in a nutrient solution containing a toxic concentration of Al and relative seminal root length (RSRL) was used as a phenotypic measure of tolerance. The RSRL frequency distribution was continuous, but skewed towards Al-susceptible individuals. Seedlings of the F2 population which scored the highest and the lowest RSRL values were transplanted to the field and subsequently selfed to obtain F3 families. Thirty F3 families (15 Al-susceptible and 15 Al-tolerant) were evaluated in nutrient solution, using an incomplete block design, to identify those with the smallest variances for aluminum tolerance and susceptibility. Six Al-susceptible and five Al-tolerant F3 families were chosen to construct one pool of Al-susceptible individuals, and another of Al-tolerant, herein referred as "bulks", based on average values of RSRL and genetic variance. One hundred and thirteen probes were selected, with an average interval of 30 cM, covering the 10 maize chromosomes. These were tested for their ability to discriminate the parental lines. Fifty-four of these probes were polymorphic, with 46 showing codominance. These probes were hybridized with DNA from the two contrasting bulks. Three RFLPs on chromosome 8 distinguished the bulks on the basis of band intensity. DNA of individuals from the bulks was hybridized with these probes and showed the presence of heterozygous individuals in each bulk. These results suggest that in maize there is a region related to aluminum tolerance on chromosome 8

The X-X-/E+E+ genotype of the XbaI/EcoRI polymorphisms of the apolipoprotein B gene as a marker of coronary artery disease in a Brazilian sample

Studies that consider polymorphisms within the apolipoprotein B (apo B) gene as risk factors for coronary artery disease (CAD) have reported conflicting results. The aim of the present study was to search for associations between two DNA RFLPs (XbaI and EcoRI) of the apo B gene and CAD diagnosed by angiography. In the present study we compared 116 Brazilian patients (92 men) with CAD (CAD+) to 78 control patients (26 men) without ischemia or arterial damage (CAD-). The allele frequencies at the XbaI (X) and EcoRI (E) sites did not differ between groups. The genotype distributions of CAD+ and CAD- patients were different (chi²(1) = 6.27, P = 0.012) when assigned to two classes (X-X-/E+E+ and the remaining XbaI/EcoRI genotypes). Multivariate logistic regression analysis showed that individuals with the X-X-/E+E+ genotype presented a 6.1 higher chance of developing CAD than individuals with the other XbaI/EcoRI genotypes, independently of the other risk factors considered (sex, tobacco consumption, total cholesterol, hypertension, and triglycerides). We conclude that the X-X-/E+E genotype may be in linkage disequilibrium with an unknown variation in the apo B gene or with a variation in another gene that affects the risk of CAD.