Molecular cloning of complementary DNA encoding maize nitrite reductase: molecular analysis and nitrate induction

Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Transient Accumulation of Nitrite Reductase mRNA in Maize following the Addition of Nitrate

Expression of the gene coding for nitrite reductase (NiR) is induced upon the addition of nitrate. We have analyzed this induction process in hydroponically grown maize (Zea mays L.) seedlings where the level of nitrate in the medium can be easily manipulated. There is a rapid induction of NiR mRNA upon addition of nitrate, increasing first in the roots and then in the leaves. The rapidity of the response depends on the nitrate concentration and the growth medium. However, the general pattern of expression is the same: the mRNA level increases, reaches a maximum, and then decreases, despite the fact that the nitrate concentration in the medium remains constant. This decline in mRNA level can be quite rapid, particularly in root tissue. If the nitrate is given as a pulse, the mRNA levels decrease even more rapidly. It is clear that the NiR mRNA is short-lived, with a half-life in the roots of less than 30 minutes. The NiR protein level, on the other hand, increases gradually somewhat after the increase in mRNA and remains at high levels at least for 24 hours after the addition of nitrate.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background  

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.     

Synthesis and secretion of wheat alpha-amylase in Saccharomyces cerevisiae

A wheat alpha-amylase cDNA clone has been fused to the phosphoglycerate kinase initiator methionine to enable synthesis in the yeast Saccharomyces cerevisiae of an alpha-amylase enzyme that is identical in size to the wild-type alpha-amylase. The alpha-amylase is synthesized with an N-terminal plant signal peptide which is recognized in the yeast host, leading to efficient processing and secretion into the medium. The secretion of alpha-amylase into the medium is quite efficient in rich medium, but barely detectable in a minimal medium.     

Genomic analysis of the relationship between gene expression variation and DNA polymorphism in Drosophila simulans

Background

Understanding how DNA sequence polymorphism relates to variation in gene expression is essential to connecting genotypic differences with phenotypic differences among individuals. Addressing this question requires linking population genomic data with gene expression variation.

Results

Using whole genome expression data and recent light shotgun genome sequencing of six Drosophila simulans genotypes, we assessed the relationship between expression variation in males and females and nucleotide polymorphism across thousands of loci. By examining sequence polymorphism in gene features, such as untranslated regions and introns, we find that genes showing greater variation in gene expression between genotypes also have higher levels of sequence polymorphism in many gene features. Accordingly, X-linked genes, which have lower sequence polymorphism levels than autosomal genes, also show less expression variation than autosomal genes. We also find that sex-specifically expressed genes show higher local levels of polymorphism and divergence than both sex-biased and unbiased genes, and that they appear to have simpler regulatory regions.

Conclusion

The gene-feature-based analyses and the X-to-autosome comparisons suggest that sequence polymorphism in cis-acting elements is an important determinant of expression variation. However, this relationship varies among the different categories of sex-biased expression, and trans factors might contribute more to male-specific gene expression than cis effects. Our analysis of sex-specific gene expression also shows that female-specific genes have been overlooked in analyses that only point to male-biased genes as having unusual patterns of evolution and that studies of sexually dimorphic traits need to recognize that the relationship between genetic and expression variation at these traits is different from the genome as a whole.     

Promoter cassettes, antibiotic-resistance genes, and vectors for plant transformation

We have constructed a set of plant transformation vectors, promoter cassettes, and chimeric antibiotic-resistance genes for the transformation and expression of foreign genes in plants sensitive to Agrobacterium infection. The different vectors allow for either concurrent or consecutive selection for kanamycin and hygromycin resistance and have a number of unique restriction sites for the insertion of additional DNA. The promoter cassettes utilize the CaMV 19S and CaMV 35S promoters and are constructed to allow for the easy insertion of foreign genes. The cloned gene can then easily be inserted into the transformation vectors. We have utilized the promoter cassettes to express the hygromycin-resistance gene either from the CaMV 35S or the CaMV 19S promoters, with both chimeric resistance genes allowing for the selection of hygromycin-resistant tobacco plants.     

Nitrate effects on nitrate reductase activity and nitrite reductase mRNA levels in maize suspension cultures

Nitrate reductase (NR) activity and nitrite reductase (NiR) mRNA levels were monitored in Black Mexican Sweet maize (Zea mays L.) suspension cultures after the addition of nitrate. Maximal induction occurred with 20 millimolar nitrate and within 2 hours. Both NR and NiR mRNA were transiently induced with levels decreasing after the 2 hours despite the continued presence of nitrate in the medium. Neither ammonia nor chlorate prevented the induction of NR. Furthermore, removal of nitrate, followed by its readdition 22 to 48 hours later, did not result in reinduction of activity or message. NR was synthesized de novo, since cycloheximide completely blocked its induction. Cycloheximide had no effect on the induction of NiR mRNA or on the transient nature of its induction. These results are similar to those reported previously for maize seedlings.