Cloning and restriction fragment length polymorphism analysis of a cDNA for swine PIT-1, a gene controlling growth hormone expression*

A partial swine cDNA which encodes the functional domain of PIT-1 was isolated by the polymerse chain reaction (PCR). The swine PIT-1 cDNA clone is 95% identical at the protein level to the rat Pit-1 gene. Thus, Pit-l's known function in control of rat growth hormone and prolactin expression is likely to be conserved in swine. This swine cDNA clone was used to investigate genetic variability at PIT-1 in several American and Chinese breeds. Polymorphic BamIII fragments were found in pure-bred Meishan animals (n= 13), but only monomorphic fragments in five American breeds (n= 36).     

Denver Papillae Protocol for Objective Analysis of Fungiform Papillae

The goal of the Denver Papillae Protocol is to use a dichotomous key to define and prioritize the characteristics of fungiform papillae (FP) to ensure consistent scoring between scorers. This protocol builds off of a need that has arisen from the last two decades of taste research using FP as a proxy for taste pore density. FP density has historically been analyzed using Miller & Reedy’s 1990 characterizations of their morphology: round, stained lighter, large, and elevated. In this work, the authors forewarned that stricter definitions of FP morphology needed to be outlined. Despite this call to action, follow up literature has been scarce, with most studies continuing to cite Miller & Reedy’s original work. Consequently, FP density reports have been highly variable and, combined with small sample sizes, may contribute to the discrepant conclusions on the role of FP in taste sensitivity. The Genetics of Taste Lab explored this apparent inconsistency in counting and found that scorers were individually prioritizing the importance of these characteristics differently and had no guidance for when a papilla had some, but not all, of the reported qualities of FP. The result of this subjectivity is highly variable FP counts of the same tongue image. The Denver Papillae Protocol has been developed to remedy this consequence through use of a dichotomous key that further defines and prioritizes the importance of the characteristics put forth by Miller & Reedy. The proposed method could help create a standard way to quantify FP for researchers in the field of taste and nutritional studies.     

Detection of cyanobacterial sxt genes and paralytic shellfish toxins in freshwater lakes and brackish waters on Åland Islands,Finland

Harmful cyanobacteria are a globally growing concern. They produce a large variety of toxic compounds, including saxitoxin and its many structural variants, a group of potent neurotoxins collectively called paralytic shellfish toxins or PST. Nucleic acid based detection methods, such as qPCR, have been proposed as potential screening and monitoring tools for toxic cyanobacteria, but it is not clear how well the presence and quantity of saxitoxin biosynthesis (sxt) genes can be used to predict the production of PST in the environment. In this study, the prevalence of three sxt genes and their co-occurrence with paralytic shellfish toxins in the environment was investigated. The sxtA, sxtG and sxtB genes were present on average in 31% of the samples collected from lakes and brackish coastal waters on Åland Islands, Finland, during the three-year monitoring period. PST detection frequency varied from 13% to 59% from year to year, and concentrations were generally low. On average higher sxtB copy numbers were associated with PST detection, and although a positive correlation between gene copy numbers and toxin concentrations was observed (Spearman rank correlation, ρ = 0.53, P = 0.012), sxt gene presence or quantity didn’t reliably predict PST production. Sequencing of sxtA fragments and identification of main cyanobacteria indicated that the likely candidate responsible for PST production in the samples belonged to the genus Anabaena.     

Conventional and Molecular Identification of Bacteria with Magnesite Enrichment Potential from Local Quarries in Erzurum

In this study, the bacteria having ore enrichment potential were isolated from three different magnesite quarries located in Erzurum-Askale borderlines. The obtained isolates were identified and characterized according to the conventional (morphological, physiological and biochemical tests) and molecular techniques (fatty acid methyl ester profiles (FAME), BOX PCR and 16S rDNA). According to sequence analysis, they were determined as Exiguobacterium aurantiacum (4), Exiguobacterium sibiricum (2), Bacillus sp. (2), Staphylococcus epidermidis (2), Staphylococcus haemolyticus (1), Shewanella baltica (1) and Klebsiella oxytoca (1), respectively.     

Denaturing gradient gel electrophoresis detection of polymorphism in a PCR fragment of sheep epidermal growth factor gene

The ovine map is not yet well-developed, which represents a problem when looking for markers of a region of interest in sheep. A means of circumventing this is to use comparative mapping. In this study primers were determined using consensus sequences for the epidermal growth factor gene of humans, rats and mice, and an ovine epidermal growth factor gene fragment was amplified by polymerase chain reaction (PCR). A new set of specific ovine primers was chosen to study the polymorphism of this DNA fragment by denaturing gradient gel electrophoresis. Eighty-four individuals belonging to seven sheep breeds were studied with this technique and four alleles were detected. The heterozygosity rate was 0.57. Family analysis showed mendelian inheritance of the alleles. Usually, genetic analysis of type-I loci used in the comparative mapping is based on the detection of restriction fragment length polymorphisms in sheep DNA using cDNA probes from other species. Our work shows that another method, based on PCR and denaturing gradient gel electrophoresis techniques, can be efficiently used.     

Absolute nannofossil abundance estimates: Quantifying the pros and cons of different techniques

A quick and inexpensive method to determine absolute nannofossil abundance in deep sea sediments – the “drop” technique (modified dilution method) – was compared to two other available methods – the filtration and random settling techniques. All techniques rely on the same basic principle, under which a volume of known concentration (bulk sediment weight/mL) is distributed evenly over a known total area (glass slide or filter) to then count particles within a set of (randomly) selected fields of view. The three preparation techniques were also calibrated by spiking the samples with microbeads to approach the “real values” as closely as possible. Significant offsets in abundance estimates between methods mainly reflect bias due to the uneven distribution and/or loss of particles. We show that the drop technique is most consistent and accurate in estimating “real values” and offers similar or better reproducibility than the other techniques. The drop method also allows detection of the same trends with or without calibration with microbeads. The filtration method holds the risk to drastically underestimate absolute abundances, while the settling technique is demanding in terms of time and may suffer from advection processes. The composition of nannofossil assemblages can be reliably determined by any of the three different techniques.     

Molecular fingerprinting of peppermint (Mentha piperita) and some Mentha hybrids by sequencing and RFLP analysis of the 5S rRNA Non-Transcribed Spacer (NTS) region

Hybridization of species belonging to the genus Mentha is quite common. However, the indicators of hybridity are many and make Mentha hybrids' identification difficult. By using the same molecular strategy that allowed us to unequivocally identify some Mentha species, we amplified the Not-Transcribed-Spacer (NTS) of the 5S-rRNA gene to characterize the industrial crop peppermint, M. × piperita and some important Mentha interspecific hybrids: M. × dalmatica, M. × dumetorum, M. × rotundifolia, M. × maximilianea, M. × smithiana, M. × verticillata, M. × villosa. DNA amplification, sequence and cluster analysis revealed differences in the 5S-rRNA NTS region of Mentha hybrids. Peppermint and all other hybrids were unequivocally discriminated by RFLP analysis by using TaqI restriction enzyme, while a further discrimination between M. × dumetorum and M. × verticillata was obtained by XhoI restriction enzyme. Essential oil composition showed clustering patterns similar to DNA fingerprint, with a clear discrimination between plants producing menthofuran (e.g. M. aquatica and its related hybrids, including peppermint) and those containing piperitenone oxide (M. longifolia and its related hybrids).