Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5* end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P 5 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Genetic resources in wild and cultured stocks of the Asian mud crab, Scylla paramamosain

The mud crab (Scylla spp.) aquaculture industry has expanded rapidly in recent years in many countries in the Indo - West Pacific (IWP) region as an alternative to marine shrimp culture because of significant disease outbreaks and associated failures of many shrimp culture industries in the region. Currently, practices used to produce and manage breeding crabs in hatcheries may compromise levels of genetic diversity, ultimately compromising growth rates, disease resistance and stock productivity. Therefore, to avoid “genetic pollution” and its harmful effects and to promote further development of mud crab aquaculture and fisheries in a sustainable way, a greater understanding of the genetic attributes of wild and cultured mud crab stocks is required. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations for multiple purposes including for commercial production, recreation and conservation and to increase profitability and sustainability of newly emerging crab culture industries. Phylogeographic patterns and the genetic structure of Asian mud crab populations across the IWP were assessed to determine if they were concordant with those of other widespread taxa possessing pelagic larvae of relatively long duration. A 597 bp fragment of the mitochondrial DNA COI gene was amplified and screened for variation in a total of 297 individuals of S. paramamosain from six sampling sites across the species’ natural geographical distribution in the IWP and 36 unique haplotypes were identified. Haplotype diversities per site ranged from 0.516 to 0.879. Nucleotide diversity estimates among haplotypes were 0.11% – 0.48%. Maximum divergence observed among S. paramamosain samples was 1.533% and samples formed essentially a single monophyletic group as no obvious clades were related to geographical location of sites. A weak positive relationship was observed however, between genetic distance and geographical distance among sites. Microsatellite markers were then used to assess contemporary gene flow and population structure in Asian mud crab populations sampled across their natural distribution in the IWP. Eight microsatellite loci were screened in sampled S. paramamosain populations and all showed high allelic diversity at all loci in sampled populations. In total, 344 individuals were analysed, and 304 microsatellite alleles were found across the 8 loci. The mean number of alleles per locus at each site ranged from 20.75 to 28.25. Mean allelic richness per site varied from 17.2 to 18.9. All sites showed high levels of heterozygosity as average expected heterozygosities for all loci ranged from 0.917 – 0.953 while mean observed heterozygosity ranged from 0.916 – 0.959. Allele diversities were similar at all sites and across all loci. The results did not show any evidence for major differences in allele frequencies among sites and patterns of allele frequencies were very similar in all populations across all loci. Estimates of population differentiation (FST) were relatively low and most probably largely reflect intra – individual variation for very highly variable loci. Results from nDNA analysis showed evidence for only very limited population genetic structure among sampled S. paramamosain, and a positive and significant association for genetic and geographical distance among sample sites. Microsatellite markers were then employed to determine if adequate levels of genetic diversity has been captured in crab hatcheries for the breeding cycle. The results showed that all microsatellite loci were polymorphic in hatchery samples. Culture populations were in general, highly genetically depauperate, compared with comparable wild populations, with only 3 to 8 alleles recorded for the same loci set per population. In contrast, very high numbers of alleles per locus were found in reference wild S. paramamosain populations, which ranged from 18 to 46 alleles per locus per population. In general, this translates into a 3 to 10 fold decline in mean allelic richness per locus in all culture stocks compared with wild reference counterparts. Furthermore, most loci in all cultured S. paramamosain samples showed departures from HWE equilibrium. Allele frequencies were very different in culture samples from that present in comparable wild reference samples and this in particular, was reflected in a large decline in allele diversity per locus. The pattern observed was best explained by significant impacts of breeding practices employed in hatcheries rather than natural differentiation among wild populations used as the source of brood stock. Recognition of current problems and management strategies for the species both for the medium and long-term development of the new culture industry are discussed. The priority research to be undertaken over the medium term for S. paramamosain should be to close the life cycle fully to allow individuals to be bred on demand and their offspring equalised to control broodstock reproductive contributions. Establishing a broodstock register and pedigree mating system will be required before any selection program is implemented. This will ensure that sufficient genetic variation will be available to allow genetic gains to be sustainably achieved in a future stock improvement program. A fundamental starting point to improve hatchery practices will be to encourage farmers and hatchery managers to spawn more females in their hatcheries as it will increase background genetic diversity in culture stocks. Combining crablet cohorts from multiple hatcheries into a single cohort for supply to farmers or rotation of breeding females regularly in hatcheries will help to address immediate genetic diversity problems in culture stocks. Application of these results can provide benefits for managing wild and cultured Asian mud crab populations more efficiently. Over the long-term, application of data on genetic diversity in wild and cultured stocks of Asian mud crab will contribute to development of sustainable and productive culture industries in Vietnam and other countries in the IWP and can contribute towards conservation of wild genetic resources.

Australian lungfish (Neoceratodus forsteri: Dipnoi) have low genetic variation at allozyme and mitochondrial DNA loci : a conservation alert?

Genetic variation at allozyme and mitochondrial DNA loci was investigated in the Australian lungfish, Neoceratodus forsteri Krefft 1870. Tissue samples for genetic analysis were taken non-lethally from 278 individuals representing two spatially distinct endemic populations (Mary and Burnett rivers), as well as one population thought to be derived from an anthropogenic translocation in the 1890's (Brisbane river). Two of 24 allozyme loci resolved from muscle tissue were polymorphic. Mitochondrial DNA nucleotide sequence diversity estimated across 2,235 base pairs in each of 40 individuals ranged between 0.000423 and 0.001470 per river. Low genetic variation at allozyme and mitochondrial loci could be attributed to population bottlenecks, possibly induced by Pleistocene aridity. Limited genetic differentiation was detected among rivers using nuclear and mitochondrial markers suggesting that admixture may have occurred between the endemic Mary and Burnett populations during periods of low sea level when the drainages may have converged before reaching the ocean. Genetic data was consistent with the explanation that lungfish were introduced to the Brisbane river from the Mary river. Further research using more variable genetic loci is needed before the conservation status of populations can be determined, particularly as anthropogenic demands on lungfish habitat are increasing. In the interim we recommend a management strategy aimed at conserving existing genetic variation within and between rivers.

Association analysis of a highly polymorphic CAG Repeat in the human potassium channel gene KCNN3 and migraine susceptibility

Background Migraine is a polygenic multifactorial disease, possessing environmental and genetic causative factors with multiple involved genes. Mutations in various ion channel genes are responsible for a number of neurological disorders. KCNN3 is a neuronal small conductance calcium-activated potassium channel gene that contains two polyglutamine tracts, encoded by polymorphic CAG repeats in the gene. This gene plays a critical role in determining the firing pattern of neurons and acts to regulate intracellular calcium channels. Methods The present association study tested whether length variations in the second (more 3') polymorphic CAG repeat in exon 1 of the KCNN3 gene, are involved in susceptibility to migraine with and without aura (MA and MO). In total 423 DNA samples from unrelated individuals, of which 202 consisted of migraine patients and 221 non-migraine controls, were genotyped and analysed using a fluorescence labelled primer set on an ABI310 Genetic Analyzer. Allele frequencies were calculated from observed genotype counts for the KCNN3 polymorphism. Analysis was performed using standard contingency table analysis, incorporating the chi-squared test of independence and CLUMP analysis. Results Overall, there was no convincing evidence that KCNN3 CAG lengths differ between Caucasian migraineurs and controls, with no significant difference in the allelic length distribution of CAG repeats between the population groups (P = 0.090). Also the MA and MO subtypes did not differ significantly between control allelic distributions (P > 0.05). The prevalence of the long CAG repeat (>19 repeats) did not reach statistical significance in migraineurs (P = 0.15), nor was there a significant difference between the MA and MO subgroups observed compared to controls (P = 0.46 and P = 0.09, respectively), or between MA vs MO (P = 0.40). Conclusion This association study provides no evidence that length variations of the second polyglutamine array in the N-terminus of the KCNN3 channel exert an effect in the pathogenesis of migraine.

Molecular genetic analyses of RFLPs for PCR-amplified growth hormone gene, renal kallikrein gene and atrial natriuretic factor gene in essential hypertension

The present study examined polymorphisms of genes that might be involved in the onset of essential hypertension (HT). These included the (i) growth hormone gene (GH1), whose locus has recently been linked to elevated blood pressure (BP) in the stroke-prone SHR, although recent sib-pair analysis of a polymorphism near the human chorionic somatomammotropin gene (a member of the GH cluster) was unable to show linkage with HT; (ii) renal kallikrein gene (KLK1); and (iii) atrial natriuretic factor gene (ANF), where a primary defect in production or activity of kallikrein or ANF could cause NaCl retention and vasoconstriction. Association analyses were conducted to compare restriction fragment length polymorphisms (RFLPs) of each gene in 85 HT and 95 normotensive (NT) Caucasian subjects whose parents had a similar BP status at age ≥50 years. The frequency of the minor allele of (i) a RsaI RFLP in the promoter of GH1, amplified from leukocyte DNA by the polymerase chain reaction, was 0.15 in the HT group and 0.14 in the NT group (χ1=0.34, P=0.55); (ii) a TaqI RFLP for KLK1 was 0.035 in the HT group and 0.015 in the NT group (χ2=1.5, P=0.21); and (iii) a XhoI RFLP for ANF was 0.50 in HTs and 0.46 in NTs (χ2=0.20, P=0.65). Studies of HT pedigrees found one family in which the ANF locus and HT were not linked, owing to an obligate recombinant. The present data thus provide no evidence for involvement of the growth hormone, renal kallikrein, nor ANF gene in the causation of essential hypertension.

Gene amplified in oesophageal cancer 1 (GAEC1) amplification in colorectal cancers and its impact on patient's survival

GAEC1 (gene amplified in oesophageal cancer 1) is located at 7q22.1, first identified in oesophageal cancer.1 Initial work indicated that GAEC1 can act as an oncogene.2 Our pilot study found ∼80% of colorectal cancers showing amplification of GAEC1.3 In this research, we will study GAEC1 copy number in colon cancer cell lines and colorectal tissues, and its prognostic significance. Two human colon cancer cell lines (SW480 and SW48) and one normal colonic epithelial cell line (FHC) were obtained from American Type Culture Collection. Culturing conditions for these cell lines were as published previously.4 Tissues were collected from 283 patients (213 Australian; 70 Japanese) diagnosed with colorectal cancers. Ninety surgically removed non-cancer colorectal tissues (diverticular diseases, hyperplastic polyps and volvulus) were used as controls. H&E stained sections from each cancer were checked to select a block with sufficient cancer tissue and representative morphological features for each patient for DNA extraction...

Head and neck squamous-cell cancer and its association with polymorphic enzymes of xenobiotic metabolism and repair

Tobacco smoking, alcohol drinking, and occupational exposures to polycyclic aromatic hydrocarbons are the major proven risk factors for human head and neck squamous-cell cancer (HNSCC). Major research focus on gene-environment interactions concerning HNSCC has been on genes encoding enzymes of metabolism for tobacco smoke constituents and repair enzymes. To investigate the role of genetically determined individual predispositions in enzymes of xenobiotic metabolism and in repair enzymes under the exogenous risk factor tobacco smoke in the carcinogenesis of HNSCC, we conducted a case-control study on 312 cases and 300 noncancer controls. We focused on the impact of 22 sequence variations in CYP1A1, CYP1B1, CYP2E1, ERCC2/XPD, GSTM1, GSTP1, GSTT1, NAT2, NQO1, and XRCC1. To assess relevant main and interactive effects of polymorphic genes on the susceptibility to HNSCC we used statistical models such as logic regression and a Bayesian version of logic regression. In subgroup analysis of nonsmokers, main effects in ERCC2 (Lys751Gln) C/C genotype and combined ERCC2 (Arg156Arg) C/A and A/A genotypes were predominant. When stratifying for smokers, the data revealed main effects on combined CYP1B1 (Leu432Val) C/G and G/G genotypes, followed by CYP1B1 (Leu432Val) G/G genotype and CYP2E1 (-70G>T) G/T genotype. When fitting logistic regression models including relevant main effects and interactions in smokers, we found relevant associations of CYP1B1 (Leu432Val) C/G genotype and CYP2E1 (-70G>T) G/T genotype (OR, 10.84; 95% CI, 1.64-71.53) as well as CYP1B1 (Leu432Val) G/G genotype and GSTM1 null/null genotype (OR, 11.79; 95% CI, 2.18-63.77) with HNSCC. The findings underline the relevance of genotypes of polymorphic CYP1B1 combined with exposures to tobacco smoke.

Arsenic trioxide promotes mitochondrial DNA mutation and cell apoptosis in primary APL cells and NB4 cell line

This study aimed to investigate the effects of arsenic trioxide (As2O3) on the mitochondrial DNA (mtDNA) of acute promyelocytic leukemia (APL) cells. The NB4 cell line was treated with 2.0 μmol/L As2O3in vitro, and the primary APL cells were treated with 2.0 μmol/L As2O3in vitro and 0.16 mg kg-1 d-1 As2O3in vivo. The mitochondrial DNA of all the cells above was amplified by PCR, directly sequenced and analyzed by Sequence Navigatore and Factura software. The apoptosis rates were assayed by flow cytometry. Mitochondrial DNA mutation in the D-loop region was found in NB4 and APL cells before As2O3 use, but the mutation spots were remarkably increased after As2O3 treatment, which was positively correlated to the rates of cellular apoptosis, the correlation coefficient: rNB4-As2O3=0.973818, and rAPL-As2O3=0.934703. The mutation types include transition, transversion, codon insertion or deletion, and the mutation spots in all samples were not constant and regular. It is revealed that As2O3 aggravates mtDNA mutation in the D-loop region of acute promyelocytic leukemia cells both in vitro and in vivo. Mitochondrial DNA might be one of the targets of As2O3 in APL treatment.

A comparison of DNA pools constructed following whole genome amplification for two-stage SNP genotyping designs

Genotyping in DNA pools reduces the cost and the time required to complete large genotyping projects. The aim of the present study was to evaluate pooling as part of a strategy for fine mapping in regions of significant linkage. Thirty-nine single nucleotide polymorphisms (SNPs) were analyzed in two genomic DNA pools of 384 individuals each and results compared with data after typing all individuals used in the pools. There were no significant differences using data from either 2 or 8 heterozygous individuals to correct frequency estimates for unequal allelic amplification. After correction, the mean difference between estimates from the genomic pool and individual allele frequencies was .033. A major limitation of the use of DNA pools is the time and effort required to carefully adjust the concentration of each individual DNA sample before mixing aliquots. Pools were also constructed by combining DNA after Multiple Displacement Amplification (MDA). The MDA pools gave similar results to pools constructed after careful DNA quantitation (mean difference from individual genotyping .040) and MDA provides a rapid method to generate pools suitable for some applications. Pools provide a rapid and cost-effective screen to eliminate SNPs that are not polymorphic in a test population and can detect minor allele frequencies as low as 1% in the pooled samples. With current levels of accuracy, pooling is best suited to an initial screen in the SNP validation process that can provide high-throughput comparisons between cases and controls to prioritize SNPs for subsequent individual genotyping.

Development of type I genetic markers from expressed sequence tags in highly polymorphic species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: - 1) priming site polymorphism in the template leads to inferior or erratic amplification; - 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions, and; - 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.