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).     

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.     

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).     

DNA sequence analysis of serologically detected ELA class II haplotypes at the equine DQP locus

The genetic diversity at the ELA DQβ locus was investigated using polymerase chain reaction and DNA sequencing. Based upon serological methods 16 class II homozygous animals were selected and their genomic DNA was used. A DQβ gene from an equine cDNA library was also sequenced. Our methology and the similarity between the genomic and the cDNA sequences suggest that the studied locus is expressed on equine lymphocytes. In the predicted amino acid sequence the most extensive variation is located at residues 56–60. The pattern of these five amino acids is strongly correlated to the serological ELA class II specificities (W13, W22, W23, Be200). The alleles corresponding to the W23 specificity are the most divergent among the equine DQβ alleles and also from other mammalian DQβ sequences.     

Characterization of tetranucleotide microsatellite loci and development and validation of multiplex reactions for the study of right whale species (genus Eubalaena)

A North Atlantic right whale (Eubalaena glacialis) genomic library was developed and screened with a (GATA)₈ probe to identify tetranucleotide microsatellite loci. Sixteen characterized loci were polymorphic in North Atlantic and/or South Atlantic (Eubalaena australis) right whales, 12 being polymorphic in E. glacialis, and 15 in E. australis. Fourteen of these were combined with 21 other previously identified loci for a suite of 35 loci which can be used to increase resolution of genetic analyses of these species. Multiplex reactions were developed for genotyping samples at these loci, providing a method that is rapid, reliable and cost‐effective.     

Genetic variation, real-time PCR, metabolites and mycotoxins ofFusarium avenaceum and related species

In this paper the latest studies dealing with genetic variation and mycotoxins ofF. avenaceum and related species are reviewed and compared to the data from chromatographic image analyses. Forty-three European strains ofFusarium avenaceum and related species were classified by chromatographic image analysis on full chromatographic matrices. The results were in most cases in agreement with those from morphological and molecular analyses and supported the separation betweenF. avenaceum, F. arthrosporioides andF. tricinctum and betweenF. avenaceum groups I and II. The mycotoxin profiles of the FinnishF. avenaceum, F. arthrosporioides andF tricinctum strains were very similar to each other. Moniliformin and enniatins were the main mycotoxins produced. A fluorogenic TaqMan PCR assay (qPCR) was used for the detection ofF. avenaceum/ F. arthrosporioides DNA in Finnish barley and wheat. The qPCR results obtained from grain samples were compared to mycotoxin levels. A correlation was found betweenF. avenaceum/F. arthrosporioides DNA and moniliformin (MON) and enniatin (ENNs) levels in barley. A correlation was also found between the combinedF. avenaceum/F. arthrosporioides/F. tricinctum contamination and MON and ENNs levels in barley in 2002, but not in 2003. This was probably due to the higher MON and ENNs levels in 2002 than in 2003. It was possible to use the DNA levels ofF. avenaceum/F. arthrosporioides to distinguish between most barley samples containing high amounts of MON and ENNs from those containing low levels of the mycotoxins. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: Grants from the National Technology Agency of Finland (No. 40168/03) and the Academy of Finland (No. 52104); travel grants from NorFA and the European Commission to the Laboratory of Dr. Ulf Thrane