Genetical investigations into β-glucan content in barley

Summary Random inbred lines produced by doubled haploidy (DH) and single seed descent (SSD) have been used to investigate the genetics of -glucan (gum) content in barley (Hordeum vulgare). Genetical analyses indicated that gum content is controlled by a simple additive genetic system. Significant negative genetic correlations were observed between -glucan content, thousand grain weight and height in the DH samples. These correlations were much reduced in the SSD samples and would suggest linkage of the genes controlling these characters. The presence of repulsion linkages could be exploited in a barley breeding programme by producing F1 derived DH to generate recombinants with high thousand grain weight and low -glucan content. Genetical parameters estimated from DH and F3 samples have successfully been used to predict the number of inbred lines transgressing the parental range for -glucan content and bivariate combinations involving -glucan.     

The consequences of using inadequate testers in the simplified triple test-cross.

The genetical consequences of common alleles in the L1 and L2 testers of a simplified version of the triple test-cross which is applicable to populations of inbred lines are examined. The test for epistasis under these circumstances becomes ambiguous and can spuriously detect non-allelic interactions when they may not exist although it still provides a test for epistasis and the adequacy of the testers simultaneously. The tests of significance and the estimates of additive variation are biased to an extent related to the dominance and dominance x additive effects of the common loci while the significance and estimates of dominance variation are deflated because they reflect the dominance effects at the non-common loci only. The covariance of sums and differences is also underestimated for the same reasons. These expectations are illustrated by analysing the 190 simplified triple test-crosses that could be extracted from a 20 x 20 diallel set of crosses between pure-breeding lines of Nicotiana rustica.     

The reduced nicotinamide adenine dinucleotide "oxidase" of Acholeplasma laidlawii membranes.

An NADH dehydrogenase possessing a specific activity 3-5 times that of membrane-bound enzyme was obtained by extraction of Acholeplasma laidlawii membranes with 9.0% ethanol at 43 degrees C. This dehydrogenase contained only trace amounts of iron (suggesting an uncoupled respiration), a flavin ratio of 1:2 FAD to FMN and 30-40% lipid. Its resistance to sedimentation is probably due to the high flotation density of the lipids. It efficiently utilized ferricyanide, menadione and dichlorophenol indophenol as electron acceptors, but not O2, ubiquinone Q10 or cytochrome c. Lineweaver-Burk plots of the dehydrogenase were altered to linear functions upon extraction with 9.0% ethanol. A secondary site of ferricyanide reduction could not be explained by the presence of cytochromes, which these membranes lack. In comparison to other respiratory chain-linked NADH dehydrogenases in cytochrome-containing respiratory chains, this dehydrogenase was characterized by similar Km's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, but considerably smaller V's with ferricyanide, dichlorophenol indophenol, menadione as electron acceptors, and smaller specific activities. It was not stimulated or reactivated by the addition of FAD, FMN, Mg2+, cysteine or membrane lipids, and was less sensitive to respiratory inhibitors than unextracted enzyme. The ineffectiveness of ADP stimulation on O2 uptake, the insensitivity to oligomycin and the very low iron content of A. laidlawii membranes were considered in relation to conservation of energy by these cells. Some kinetic properties of the dehydrogenation, the uniquely high glycolipid content and apparently uncoupled respiration at Site I were noteworthy characteristics of this NADH dehydrogenase from the truncated respiratory chain of A. laidlawii.     

Method for exchange of the lipid environment of the membrane-bound (Mg2+ + Na+)-ATPase of Acholeplasma laidlawii B

A method that exchanges the endogenous lipids in the environment of the membrane-bound (Mg2+ + Na+)-ATPase of Acholeplasma laidlawii B with defined exogenous lipids has been devised. Results demonstrate that 99.9% of the original membrane lipids were replaced with phosphatidylcholine and phosphatidic acid. ATPase enzyme activity was maintained throughout the substitution procedure.     

Methylated DNA/RNA in Body Fluids as Biomarkers for Lung Cancer

DNA/RNA methylation plays an important role in lung cancer initiation and progression. Liquid biopsy makes use of cells, nucleotides and proteins released from tumor cells into body fluids to help with cancer diagnosis and prognosis. Methylation of circulating tumor DNA (ctDNA) has gained increasing attention as biomarkers for lung cancer. Here we briefly introduce the biological basis and detection method of ctDNA methylation, and review various applications of methylated DNA in body fluids in lung cancer screening, diagnosis, prognosis, monitoring and treatment prediction. We also discuss the emerging role of RNA methylation as biomarkers for cancer.     

Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development

The products of Hox genes function in assigning positional identity along the anterior–posterior body axis during animal development. In mouse embryos, Hox genes located at the 3′ end of HoxA and HoxB complexes are expressed in nested patterns in the progenitors of the secondary heart field during early cardiogenesis and the combined activities of both of these clusters are required for proper looping of the heart. Using Hox bacterial artificial chromosomes (BACs), transposon reporters, and transgenic analyses in mice, we present the identification of several novel enhancers flanking the HoxB complex which can work over a long range to mediate dynamic reporter expression in the endoderm and embryonic heart during development. These enhancers respond to exogenously added retinoic acid and we have identified two retinoic acid response elements (RAREs) within these control modules that play a role in potentiating their regulatory activity. Deletion analysis in HoxB BAC reporters reveals that these control modules, spread throughout the flanking intergenic region, have regulatory activities that overlap with other local enhancers. This suggests that they function as shadow enhancers to modulate the expression of genes from the HoxB complex during cardiac development. Regulatory analysis of the HoxA complex reveals that it also has enhancers in the 3′ flanking region which contain RAREs and have the potential to modulate expression in endoderm and heart tissues. Together, the similarities in their location, enhancer output, and dependence on retinoid signaling suggest that a conserved cis-regulatory cassette located in the 3′ proximal regions adjacent to the HoxA and HoxB complexes evolved to modulate Hox gene expression during mammalian cardiac and endoderm development. This suggests a common regulatory mechanism, whereby the conserved control modules act over a long range on multiple Hox genes to generate nested patterns of HoxA and HoxB expression during cardiogenesis.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.