Two early nodulin 40 (enod40) genes, ENOD40-1, the shortest legume ENOD40 gene, and ENOD40-2, were isolated from Lupinus luteus, a legume with indeterminate nodules. Both genes were expressed at similar levels during symbiosis with nitrogen-fixing bacteria. ENOD40 phylogeny clustered the L. luteus genes with legumes forming determinate nodules and revealed peptide similarities. The ENOD40-1 small ORF A fused to a reporter gene was efficiently expressed in plant cells, indicating that the start codon is recognized for translation. The ENOD40-1 RNA structure predicted based on Pb(II)-induced cleavage and modeling revealed four structurally conserved domains, an absence of domain 4 characteristic for legumes of indeterminate nodules, and interactions between the conserved region I and a region located upstream of domain 6. Domain 2 contains Mg(II) ion binding sites essential for organizing RNA secondary structure. The differences between L. luteus and Glycine max ENOD40 RNA models suggest the possibility of a switch between two structural states of ENOD40 transcript.
Several different isolates of Tomato black ring virus (TBRV) have been collected in Poland from cucumber, tomato, potato and black locust plants. Biological tests showed some differences in the range of infected plants and the type of symptoms, which was the basis for selection of seven the most biologically different TBRV isolates. According to the sequence of TBRV-MJ, several primer pairs were designed and almost the entire sequence of both genomic RNAs was amplified. The RT-PCR products derived from all tested TBRV isolates were digested by restriction enzymes. On the basis of the restriction patterns, the variable and the conserved regions of the TBRV genome were defined and the relationships between the Polish TBRV isolates established.
MADS-box genes encode transcription factors in all eukaryotic organisms thus far studied. Plant MADS-box proteins contain a DNA-binding (M), an intervening (I), a Keratin-like (K) and a C-terminal C-domain, thus plant MADS-box proteins are of the MIKC type. In higher plants most of the well-characterized genes are involved in floral development. They control the transition from vegetative to generative growth and determine inflorescence meristem identity. They specify floral organ identity as outlined in the ABC model of floral development. Moreover, in Antirrhinum majus the MADS-box gene products DEF/GLO and PLE control cell proliferation in the developing flower bud. In this species the DEF/GLO and the SQUA proteins form a ternary complex which determines the overall "Bauplan" of the flower. Phylogenetic reconstructions of MADS-box sequences obtained from ferns, gymnosperms and higher eudicots reveal that, although ferns possess already MIKC type genes, these are not orthologous to the well characterized MADS-box genes from gymnosperms or angiosperms. Putative orthologs of floral homeotic B- and C-function genes have been identified in different gymnosperms suggesting that these genes evolved some 300-400 million years ago. Both gymnosperms and angiosperms also contain a hitherto unknown sister clade of the B-genes, which we termed Bsister. A novel hypothesis will be described suggesting that B and Bsister might be involved in sex determination of male and female reproductive organs, respectively.
The coding sequences of two S-adenosyl-L-homocysteine hydrolases (SAHases) were identified in yellow lupine by screenig of a cDNA library. One of them, corresponding to the complete protein, was sequenced and compared with 52 other SAHase sequences. Phylogenetic analysis of these proteins identified three groups of the enzymes. Group A comprises only bacterial sequences. Group B is subdivided into two subgroups, one of which (B1) is formed by animal sequences. Subgroup B2 consist of two distinct clusters, B2a and B2b. Cluster B2b comprises all known plant sequences, including the yellow lupine enzyme, which are distinguished by a 50-residue insert. Group C is heterogeneous and contains SAHases from Archaea as well as a new class of animal enzymes, distinctly different from those in group B1.
What is the microbe that we are dealing with? Whether it is cholera or anthrax, we want to know the disease-causing microorganism as quickly as possible since prompt identification of the causative organism would help control disease spread - and potentially save lives through provision of appropriate care and medication. Yet, despite the advent of rapid microbial identification tools – particularly those based on mass spectrometry – most undergraduate curricula continue to focus on culture- and nucleic acid-based identification techniques since they are widely used for detecting and identifying microbes in clinical and environmental samples. Mass spectrometry-based methods, however, have increasingly complemented traditional approaches in clinical and research laboratories - but are rarely featured in undergraduate curricula. Motivated by the desire to address the curriculum gap, the author of this study developed an inquiry-based laboratory exercise for introducing students to the operating principles and methodology of mass spectrometry-based microbial identification. By requiring students to identify microbes in environmental water samples – a real-life problem with unknown answers – the exercise piqued the students’ interest in learning, while helping to stir their curiosity through an interesting field activity in which they could put on a scientist’s hat in solving a mystery. This synopsis article summarizes a piece of published educational research and expands on the discussion of concepts underlying matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based microbial identification. Herein, the article discusses the relative advantages and disadvantages of the pattern recognition and proteome database search approaches for analyzing mass spectra data. Additionally, the effect of general and tailored sample preparation protocols on identification accuracy is also elaborated. Finally, the pedagogical utility of field- and inquiry-based educational tools is also discussed in greater detail from a post-publication perspective. A full-length synopsis of the work and a structured abstract can be found in the accompanying PDF file, the original article being entitled: “Teaching Microbial Identification with Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Bioinformatics Tools”.
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