David O. F. Skibinski

Protein heterozygosity, protein structure, and taxonomic differentiation

Soon after the introduction of electrophoretic techniques to study the genetic structure of natural populations of animals and plants came the realization that enzymes differed in their levels of variability. Some enzymes, such as esterases and phosphoglucomutases, were frequently polymorphic, whereas others, such as superoxide dismutase and glutamate dehydrogenase, were so only rarely. Initial explanations focused on physiological considerations, such as the degree of substrate specificity (Gillespie and Kojima, 1968; Gillespie and Langley, 1974) and the regulatory nature of the enzyme (Johnson, 1974). Correlations were observed in some groups of organisms but not others, and there were some conflicts of opinion concerning the proper allocation of enzymes to particular categories (Selander, 1976).

The consequences of sample pooling in proteomics: An empirical study

Pooling of samples in proteomics experiments might help overcome resource constraints when many individuals are analysed. The measured biological variation should be reduced giving increased power to detect treatment differences. Pooling has been advocated in microarray work but there are few tests of its potential in proteomics. In this study, we examine three issues on which the success of the pooling approach might hinge and provide evidence that: (i) the protein expression in a pool matches the mean expression of the individuals making up the pool for the majority of proteins, although for some proteins the pool expression is different; (ii) the biological variance between pools is reduced compared with that between individuals, as predicted in theory, but this reduction is not as large as expected. A practical consequence of this is that power could be reduced; (iii) proteins detectable in individual samples are usually but not always visible when samples are pooled. We conclude that pooling of samples in proteomics work is a valid and potentially valuable procedure but consideration should be given to these issues in experimental design.

Environmental sex determination: the effect of temperature and salinity on sex ratio in Oreochromis niloticus L

Abstract This paper reports the effects of environmental conditions during the period of sex differentiation on the sex ratio of the Nile tilapia (Oreochromis niloticus). Different sex genotypes were exposed to varying temperatures (putative all-female, all-male and all-YY males) and salinities (putative all-female progeny only) for a minimum period of 21 days after first feeding and were on grown prior to sexing by gonad squash. The majority of the putative all-female progeny exposed to high temperature (36.54±0.39°C) produced significantly higher percentages of males compared to controls reared at ambient temperature (27.87±1.40°C). Similarly, at high temperature, some of the all-male and YY male progenies had significantly lower percentage of males compared to controls. Sex differentiation in YY males appears to be more labile than in normal XY males although this could possibly be attributable to different levels of inbreeding. Low temperature (25.78±0.24°C) and varying levels of salinity (11.30 to 26.65 ppt) did not significantly affect sex ratios. The apparent sensitivity of sex differentiation to some environmental factors is considered in the context of a predominantly monofactorial genetic sex determining mechanism. Implications for sex control technologies are discussed.

Detection of damage to the mitochondrial genome in the oncocytic cells of Warthin's tumour.

Warthins tumour of the salivary glands is composed of oncocytic cells containing excessive numbers of mitochondria which show frequent structural abnormalities and reduced metabolic function. Recent evidence of a strong association between cigarette smoking and the occurrence of Warthins tumour prompted this study, to look for evidence of damage to mitochondrial DNA (mtDNA) that could be the result of an increase in oxidative stress; two‐colour fluorescence in situ hybridization (FISH) was developed to show the distribution of mitochondria with deleted mtDNA in paraffin wax‐embedded material. Approximately 10% of mtDNA bears the ‘common’ 4977 bp deletion. Using the polymerase chain reaction (PCR), the 4977 bp deletion was further quantified, in Warthins tumour and age‐matched normal parotid control tissue. Whilst the deletion was present in all parotid tissue, its presence was significantly higher in oncocytic tumour cells. In a small number of controls, there was a trend towards higher concentrations of the deletion in smokers. Copyright

Multiple Hypothesis Testing in Proteomics: A Strategy for Experimental Work

In quantitative proteomics work, the differences in expression of many separate proteins are routinely examined to test for significant differences between treatments. This leads to the multiple hypothesis testing problem: when many separate tests are performed many will be significant by chance and be false positive results. Statistical methods such as the false discovery rate method that deal with this problem have been disseminated for more than one decade. However a survey of proteomics journals shows that such tests are not widely implemented in one commonly used technique, quantitative proteomics using two-dimensional electrophoresis. We outline a selection of multiple hypothesis testing methods, including some that are well known and some lesser known, and present a simple strategy for their use by the experimental scientist in quantitative proteomics work generally. The strategy focuses on the desirability of simultaneous use of several different methods, the choice and emphasis dependent on research priorities and the results in hand. This approach is demonstrated using case scenarios with experimental and simulated model data.

Growth performance trials of genetically male tilapia (GMT) derived from YY-males in Oreochromis niloticus L.: On station comparisons with mixed sex and sex reversed male populations

Genetic manipulation of predominantly monofactorial sex determining mechanisms in the Nile tilapia Oreochromis niloticus is proposed as a technology for the mass production of all-male progeny for culture. Existing alternative methods for producing all-male tilapia, principally direct hormonal masculinization, have produced inconsistent results and are largely the preserve of scientists and developed country aquaculturists. Successful adoption of these techniques in developing countries is seriously limited by the number and complexity of factors involved in their effectiveness. Novel YY-male genotypes have been shown to be viable and to produce all- or nearly all-male (XY) progeny in crosses with normal XX females. This paper presents the results from a series of on-station growth performance trials of these genetically male tilapia (GMT) in three environments. Two comprehensive and replicated trials in extensively managed earthen ponds, compared the performance of GMT with sex reversed male (SRT) and normal mixed sex tilapia (MST). Three further, smaller experiments, compared the growth of GMT with that of MST populations in rice-cum-fish culture and under intensive culture in closed recirculating systems. These comparisons were made within the ‘Egypt-UCS’ strain of O. niloticus. Results indicate that culture of GMT populations can improve marketable yields under all environments. In the first experiment in ponds, GMT had significantly (P = 0.003) higher yields than both MST (+ 58.8%) and SRT (+ 31.03%). The second experiment, following the same design, showed a similar trend, although differences were smaller and not significant. GMT produced yields 11.9% greater than MST in rice-cum-fish culture and 33.6–47.0% greater in recirculating systems. There was no recruitment in GMT pond populations whereas recruits were present in MST and SRT populations. Coefficient of variance for individual weight in ponds was significantly (P < 0.05) lower for GMT than for MST for both pond experiments. GMT produced from novel YY-males in this strain has considerable potential for increasing yields from tilapia culture.

Multiple paternity assessed using microsatellite markers, in green turtles Chelonia mydas (Linnaeus, 1758) of Ascension Island, South Atlantic

Paternity was determined for three clutches and up to 20 offspring per clutch in the green turtle (Chelonia mydas) from Ascension Island, South Atlantic, using microsatellite markers. All three clutches were sired by at least two different males. The results were compared with those of previous studies of multiple paternity in turtles. No significant difference among studies was observed in the mean contribution of the males siring the largest proportion of progeny per clutch. The present study also provides evidence for segregation distortion (meiotic drive) in turtles.

Non-linear dose-response of DNA-reactive genotoxins: Recommendations for data analysis

Until recently, there has only been a limited amount of data available on the kinetics of mutation induction in the low dose region of exposure. In our publication Doak et al. [S.H. Doak, G.J. Jenkins, G.E. Johnson, E. Quick, E.M. Parry, J.M. Parry, Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens, Cancer Res. 67 (2007) 3904-3911] we showed that the two alkylating agents methyl-methanesulfonate (MMS) and ethyl-methanesulfonate (EMS) possess non-linear dose-response curves with no observed effect levels (NOEL) for mutation or chromosomal damage in vitro. These experiments were carried out in the AHH-1 human lymphoblastoid cell line, using the hypoxanthine phosphoribosyl transferase (HPRT) assay and the cytokinesis-block micronucleus (CBMN) assay, respectively. We have now carried out more advanced statistical analyses to define threshold values, which is critical as it has a dramatic impact on hazard and risk assessment. To do this, we re-analysed the data to see if the linear model or a more complex model (hockey stick or quadratic) gave a significant better fit of the data. For both EMS and MMS cytokinesis-block micronucleus data sets, the hockey stick model gave the most significant fit. The same was true for EMS, MMS and surprisingly ethylnitrosourea (ENU) in the HPRT assay in human AHH-1 cells. However, methylnitrosourea (MNU) was linear in both assays. These further analyses have shown that EMS and MMS have clear thresholds for both gene mutation and chromosome damage, as does ENU for gene mutation in AHH-1 cells. MNU was linear for gene and chromosome mutation and so was ENU for chromosome mutations at the concentrations tested. These findings correlate closely with those in vivo findings of Gocke et al. [E. Gocke, L. Müller, In vivo studies in the mouse to define a threshold for the genotoxicity of EMS and ENU, Mutat. Res. (this issue)] and together these data show a true threshold for EMS both in vitro and in vivo. In this report, we will discuss the approaches that were taken to investigate potential threshold dose-response curves for DNA-reactive genotoxic compounds, with recommendations for further studies.

Doubly Uniparental Inheritance Is Associated With High Polymorphism for Rearranged and Recombinant Control Region Haplotypes in Baltic Mytilus trossulus

Many bivalve species, including mussels of the genus Mytilus, are unusual in having two mtDNA genomes, one inherited maternally (the F genome) and the other inherited paternally (the M genome). The sequence differences between the genomes are usually great, indicating ancient divergence predating speciation events. However, in Mytilus trossulus from the Baltic, both genomes are similar to the F genome from the closely related M. edulis. This study analyzed the mtDNA control region structure in male and female Baltic M. trossulus mussels. We show that a great diversity of structural rearrangements is present in both sexes. Sperm samples are dominated by recombinant haplotypes with M. edulis M-like control region segments, some having large duplications. By contrast, the rearranged haplotypes that dominate in eggs lack segments from this M genome. The rearrangements can be explained by a combination of tandem duplication, deletion, and intermolecular recombination. An evolutionary pathway leading to the recombinant haplotypes is suggested. The data are also considered in relation to the hypothesis that the M. edulis M-like control region sequence is necessary to confer the paternal role on genomes that are otherwise F-like.

The application of DNA fingerprinting in the analysis of gynogenesis in tilapia

Abstract The technique of DNA fingerprinting using human minisatellite probes is applied to the study of gynogenetically derived fish in two tilapia species, Oreochromis aureus and O. niloticus . In O. aureus , analysis of the progeny of a cross between two putative mitogynes provides unexpected evidence of transmission of some paternal DNA during gynogenesis. In O. niloticus , DNA fingerprinting provides confirmation of exclusively maternal transmission during gynogenesis but mitogynes and meiogynes cannot be distinguished unambiguously.