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).     

A wheat COP9 subunit 5‐like gene is negatively involved in host response to leaf rust

The COP9 (constitutive photomorphogenesis 9) signalosome (CSN) is a protein complex involved in the ubiquitin proteasome system and a common host target of diverse pathogens in Arabidopsis. The known derubylation function of the COP9 complex is carried out by subunit 5 encoded by AtCSN5A or AtCSN5B in Arabidopsis. A single CSN5‐like gene (designated as TaCSN5) with three homeologues was identified on the long arms of wheat (Triticum aestivum L.) group 2 chromosomes. In this study, we identified and characterized the function of TaCSN5 in response to infection by the leaf rust pathogen. Down‐regulation of all three TaCSN5 homeologues or mutations in the homeologues on chromosomes 2A or 2D resulted in significantly enhanced resistance to leaf rust. Enhanced leaf rust resistance corresponded to a seven‐fold increase in PR1 (pathogenesis‐related gene 1) expression. Collectively, the data indicate that the wheat COP9 subunit 5‐like gene acts as a negative regulator of wheat leaf rust resistance.     

A mutation at the Ala122 position of acetohydroxyacid synthase (AHAS) located on chromosome 6D of wheat: improved resistance to imidazolinone and a faster assay for marker assisted selection

A new mutation at the acetohydroxyacid synthase (AHAS) locus on chromosome 6D of wheat was analyzed in detail because it conferred an improved resistance to the imidazolinone group of herbicides. Sequence analysis showed that the mutation was at the Ala122 position (A122T), a position in AHAS which has not to date been identified in imidazolinone resistant wheat lines even though the position has been identified in other plants and is associated with resistance. An allele-specific assay for the mutation (in the wheat line Brookton-8) was developed and used in a genetic analysis. Two mapping populations were analysed and the doubled haploid progeny from the cross Brookton-8 × Clearfield STL proved to be most informative. The AHAS^Ala122 mutation (A122T) was allelic to the AHAS^Ser653 mutation (S653N) in Clearfield STL (Imi1, on chromosome 6D) and hence was assigned to the chromosome 6D locus. The analysis of the doubled haploid lines in the mapping population demonstrated the greater resistance conferred by the A122T mutation because lines from the same cross and carrying either the A122T or S653N mutations could be directly compared. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

KLF4在内毒素血症小鼠中的表达模式及其意义

目的探讨Kruppel样因子4（Kruppel-like factor 4，KLF4）在内毒素血症小鼠中的表达模式及意义。方法运用实时荧光PCR技术和Western blot技术，分别从mRNA水平和蛋白水平探讨内毒素血症小鼠肝脏和肺脏中KLF4的表达；运用生物信息学技术，对启动子区含有KLF4的结合位点的炎症介质基因进行了预测；运用RT-PCR技术，从mRNA水平探讨内毒素血症小鼠肝脏和肺脏中IL1β的表达模式。结果内毒素血症小鼠肝脏和肺脏中KLF4 mRNA的表达下凋，KLF4蛋白的表达先下凋后升高；IL-18、IL-15、IL-12、IL-18、IL-10等炎症介质基因的启动子区均含有KLF4的结合元件，这些炎症基因的表达可能直接受到KLF4的调控；内毒素血症小鼠肝脏和肺脏中IL-IB的表达模式与KLF4的表达模式呈相反趋势。结论内毒素血症小鼠肝脏和肺脏中KLF4表达下调，KLF4在炎症介质基因表达调控中可能具有重要作用。     

Focus: A Multifaceted Battle Against Cancer: Extracting Knowledge from Chemical Imaging Data Using Computational Algorithms for Digital Cancer Diagnosis

Fourier transform infrared (FTIR) spectroscopic imaging is an emerging microscopy modality for clinical histopathologic diagnoses as well as for biomedical research. Spectral data recorded in this modality are indicative of the underlying, spatially resolved biochemical composition but need computerized algorithms to digitally recognize and transform this information to a diagnostic tool to identify cancer or other physiologic conditions. Statistical pattern recognition forms the backbone of these recognition protocols and can be used for highly accurate results. Aided by biochemical correlations with normal and diseased states and the power of modern computer-aided pattern recognition, this approach is capable of combating many standing questions of traditional histology-based diagnosis models. For example, a simple diagnostic test can be developed to determine cell types in tissue. As a more advanced application, IR spectral data can be integrated with patient information to predict risk of cancer, providing a potential road to precision medicine and personalized care in cancer treatment. The IR imaging approach can be implemented to complement conventional diagnoses, as the samples remain unperturbed and are not destroyed. Despite high potential and utility of this approach, clinical implementation has not yet been achieved due to practical hurdles like speed of data acquisition and lack of optimized computational procedures for extracting clinically actionable information rapidly. The latter problem has been addressed by developing highly efficient ways to process IR imaging data but remains one that has considerable scope for progress. Here, we summarize the major issues and provide practical considerations in implementing a modified Bayesian classification protocol for digital molecular pathology. We hope to familiarize readers with analysis methods in IR imaging data and enable researchers to develop methods that can lead to the use of this promising technique for digital diagnosis of cancer.     

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.