A single-nucleotide polymorphism that accounts for allelic variation in the Lr34 gene and leaf rust reaction in hard winter wheat

Leaf rust, caused by Puccinia triticina Eriks, is one of the most common and persistent wheat diseases in the US Great Plains. We report that the Lr34 gene was mapped in the center of a QTL for leaf rust reaction and explained 18–35% of the total phenotypic variation in disease severity of adult plants in a Jagger × 2174 population of recombinant inbred lines (RILs) field-tested for 3 years. The sequence of the complete Lr34 gene was determined for the susceptible Jagger allele and for the resistant 2174 allele. The two alleles had exactly the same sequence as the resistant allele reported previously in Chinese Spring at three polymorphic sites in intron 4, exon 11, and exon 12. A G/T polymorphism was found in exon 22, where a premature stop codon was found in the susceptible Jagger allele (Lr34E22s), confirming a previous report, due to a point mutation compared with the resistant 2174 allele (Lr34E22r). We have experimentally demonstrated a tight association between the point mutation at exon 22 of Lr34 and leaf rust susceptibility in a segregating biparental population. A PCR marker was developed to distinguish between the Lr34E22r and Lr34E22s alleles. A survey of 33 local hard winter wheat cultivars indicated that 7 cultivars carry the Lr34E22s allele and 26 cultivars carry the Lr34E22r allele. This study significantly improves our genetic understanding of allelic variation in the Lr34 gene and provides a functional molecular tool to improve leaf rust resistance in a major US wheat gene pool.     

CCA paper: a new two-dimensional cyanuric chloride-activated matrix for universal application in molecular biology

A novel two-dimensional cyanuric chloride-activated (CCA) paper has been developed. It is composed of a cellulosic base, covalently bound cyanuric chloride, and microprecipitated complex cyanuric chloride-sodium chloride crystals on its surface. CCA paper covalently binds nucleic acids and proteins. Its binding capacity for nucleic acids is about 400 micrograms/cm2. Sealed into nitrogen-filled bags and stored at -20 degrees C, it retains its binding activity for at least a year and is always ready for use. CCA paper has been successfully used for capillary and electroblotting of DNA, RNA, and proteins (Southern, Northern, and Western blotting) as well as for dot tests. Furthermore, it was applied to colony and plaque hybridization. A unique property of it is that it permits the staining of proteins after blotting and subsequent performance of radioimmunological detection of specific protein components. This has proven advantageous in two-dimensional Western blotting experiments. Of further importance is its ability to bind DNA fragments from one up to several hundred bp from polyacrylamide sequencing gels.     

Pyruvate Formate-Lyase Interacts Directly with the Formate Channel FocA to Regulate Formate Translocation

The FNT (formate-nitrite transporters) form a superfamily of pentameric membrane channels that translocate monovalent anions across biological membranes. FocA (formate channel A) translocates formate bidirectionally but the mechanism underlying how translocation of formate is controlled and what governs substrate specificity remains unclear. Here we demonstrate that the normally soluble dimeric enzyme pyruvate formate-lyase (PflB), which is responsible for intracellular formate generation in enterobacteria and other microbes, interacts specifically with FocA. Association of PflB with the cytoplasmic membrane was shown to be FocA dependent and purified, Strep-tagged FocA specifically retrieved PflB from Escherichia coli crude extracts. Using a bacterial two-hybrid system, it could be shown that the N-terminus of FocA and the central domain of PflB were involved in the interaction. This finding was confirmed by chemical cross-linking experiments. Using constraints imposed by the amino acid residues identified in the cross-linking study, we provide for the first time a model for the FocA–PflB complex. The model suggests that the N-terminus of FocA is important for interaction with PflB. An in vivo assay developed to monitor changes in formate levels in the cytoplasm revealed the importance of the interaction with PflB for optimal translocation of formate by FocA. This system represents a paradigm for the control of activity of FNT channel proteins.     

Copy number alterations and allelic ratio in relation to recurrence of rectal cancer

Background

In rectal cancer, total mesorectal excision surgery combined with preoperative (chemo)radiotherapy reduces local recurrence rates but does not improve overall patient survival, a result that may be due to the harmful side effects and/or co-morbidity of preoperative treatment. New biomarkers are needed to facilitate identification of rectal cancer patients at high risk for local recurrent disease. This would allow for preoperative (chemo)radiotherapy to be restricted to high-risk patients, thereby reducing overtreatment and allowing personalized treatment protocols. We analyzed genome-wide DNA copy number (CN) and allelic alterations in 112 tumors from preoperatively untreated rectal cancer patients. Sixty-six patients with local and/or distant recurrent disease were compared to matched controls without recurrence. Results were validated in a second cohort of tumors from 95 matched rectal cancer patients. Additionally, we performed a meta-analysis that included 42 studies reporting on CN alterations in colorectal cancer and compared results to our own data.

Results

The genomic profiles in our study were comparable to other rectal cancer studies. Results of the meta-analysis supported the hypothesis that colon cancer and rectal cancer may be distinct disease entities. In our discovery patient study cohort, allelic retention of chromosome 7 was significantly associated with local recurrent disease. Data from the validation cohort were supportive, albeit not statistically significant, of this finding.

Conclusions

We showed that retention of heterozygosity on chromosome 7 may be associated with local recurrence in rectal cancer. Further research is warranted to elucidate the mechanisms and effect of retention of chromosome 7 on the development of local recurrent disease in rectal cancer.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1550-0) contains supplementary material, which is available to authorized users.     

Expression and function of the SDF-1 chemokine receptors CXCR4 and CXCR7 during mouse limb muscle development and regeneration

The chemokine, SDF-1/CXCL12, and its receptor, CXCR4, have been implied to play major roles during limb myogenesis. This concept was recently challenged by the identification of CXCR7 as an alternative SDF-1 receptor, which can either act as a scavenger receptor, a modulator of CXCR4, or an active chemokine receptor. We have now re-examined this issue by determining whether SDF-1 would signal to C2C12 myoblasts and subsequently influence their differentiation via CXCR4 and/or CXCR7. In addition, we have analyzed CXCR7, CXCR4, and SDF-1 expression in developing and injured mouse limb muscles. We demonstrate that in undifferentiated C2C12 cells, SDF-1-dependent cell signaling and resulting inhibitory effects on myogenic differentiation are entirely mediated by CXCR4. We further demonstrate that CXCR7 expression increases in differentiating C2C12 cells, which in turn abrogates CXCR4 signaling. Moreover, consistent with the view that CXCR4 and CXCR7 control limb myogenesis in vivo by similar mechanisms, we found that CXCR4 expression is the highest in late embryonic hindlimb muscles and drops shortly after birth when secondary muscle growth terminates. Vice versa, CXCR7 expression increased perinatally and persisted into adult life. Finally, underscoring the role of the SDF-1 system in muscle regeneration, we observed that SDF-1 is continuously expressed by endomysial cells of postnatal and adult muscle fibers. Analysis of dystrophin-deficient mdx mice additionally revealed that muscle regeneration is associated with muscular re-expression of CXCR4. The apparent tight control of limb muscle development and regeneration by CXCR4 and CXCR7 points to these chemokine receptors as promising therapeutic targets for certain muscle disorders.     

Ultrasensitive enzymatic radioimmunoassay using a fusion protein of protein A and neomycin phosphotransferase II in two-chamber-well microtiter plates

A new sensitive method for antigen detection employing a phosphorylation reaction is described using human serum albumin as a model. The antigen is initially bound to the surface of polystyrene microtiter plates and reacted with an antibody (rabbit). A microbiologically produced bifunctional fusion protein of protein A and neomycin phosphotransferase II (NPT II) serves as a second immunological reagent by virtue of its protein A component. The detection is based on the phosphorylation of an aminoglycoside antibiotic by the NPT II moiety of the fusion protein using [gamma-32P]ATP as a cosubstrate. This reaction is performed in solution and the evaluation is accomplished by dotting aliquots of the reaction mixture onto phosphocellulose paper, washing with water, and autoradiography. Microtiter plates with a specially designed 10 microliter-volume reaction chamber are particularly advantageous for this procedure. The sensitivity of detection is currently 10 fg (1 pg/ml) of antigen.     

Reisolation and growth conditions of Bacillus agar-exedens

Several agarolytic Bacillus strains have been isolated. Their properties agree with those described by Wieringa (1941) for Bacillus agar-exedens. These strains are the first reisolates since the original cultures were lost. A second group of isolates is related to the agarolytic B. palustris var. gelaticus of Sickles and Shaw (1934). B. agar-exedens requires carbohydrates for growth. In mineral-glucose media growth is inhibited by peptone at pH values of about 7 or less. Under alkaline conditions no inhibition by peptone is observed. A method for the enrichment of B. agar-exedens is described.