Two poplar cellulose synthase-like D genes, PdCSLD5 and PdCSLD6, are functionally conserved with Arabidopsis CSLD3

Root hairs are tip-growing long tubular outgrowths of specialized epidermal cells, and are important for nutrient and water uptake and interaction with the soil microflora. Here we characterized two poplar cellulose synthase-like D (CSLD) genes, PdCSLD5 and PdCSLD6, the most probable orthologs to the Arabidopsis AtCSLD3/KOJAK gene. Both PdCSLD5 and PdCSLD6 are strongly expressed in roots, including in the root hairs. Subcellular localization experiments showed that these two proteins are located not only in the polarized plasma membrane of root hair tips, but also in Golgi apparatus of the root hair and non-hair-forming cells. Overexpression of these two poplar genes in the atcsld3 mutant was able to rescue most of the defects caused by disruption of AtCSLD3, including root hair morphological changes, altered cell wall monosaccharide composition, increased non-crystalline β-1,4-glucan and decreased crystalline cellulose contents. Taken together, our results provide evidence indicating that PdCSLD5 and PdCSLD6 are functionally conserved with AtCSLD3 and support a role for PdCSLD5 and PdCSL6 specifically in crystalline cellulose production in poplar root hair tips. The results presented here also suggest that at least part of the mechanism of root hair formation is conserved between herbaceous and woody plants.     

Repressive BMP2 gene regulatory elements near the BMP2 promoter

The level of bone morphogenetic protein 2 (BMP2) profoundly influences essential cell behaviors such as proliferation, differentiation, apoptosis, and migration. The spatial and temporal pattern of BMP2 synthesis, particular in diverse embryonic cells, is highly varied and dynamic. We have identified GC-rich sequences within the BMP2 promoter region that strongly repress gene expression. These elements block the activity of a highly conserved, osteoblast enhancer in response to FGF2 treatment. Both positive and negative gene regulatory elements control BMP2 synthesis. Detecting and mapping the repressive motifs is essential because they impede the identification of developmentally regulated enhancers necessary for normal BMP2 patterns and concentration.     

A cathepsin L-like protease from Strongylusvulgaris: An orthologue of Caenorhabditiselegans CPL-1

Cathespin L-like proteases (CPLs), characterized from a wide range of helminths, are significant in helminth biology. For example, in Caenorhabditiselegans CPL is essential for embryogenesis. Here, we report a cathepsin L-like gene from three species of strongyles that parasitize the horse, and describe the isolation of a cpl gene (Sv-cpl-1) from Strongylusvulgaris, the first such from equine strongyles. It encodes a protein of 354 amino acids with high similarity to other parasitic Strongylida (90-91%), and C.elegans CPL-1 (87%), a member of the same Clade. As S.vulgariscpl-1 rescued the embryonic lethal phenotype of the C.eleganscpl-1 mutant, these genes may be orthologues, sharing the same function in each species. Targeting Sv-CPL-1 might enable novel control strategies by decreasing parasite development and transmission.     

Cellular localization of mitotic RAD21 with repetitive amino acid motifs in Allium cepa

Onion can be used in experimental observation of mitotic cell division in plant science because its chromosome is large and easy to observe. However, molecular genetic studies are difficult in onion because of its large genome size, and only limited information of onion genes has been available to date. Here we cloned and characterized an onion homologue of mitotic RAD21 gene, AcRAD21-1, to develop a molecular marker of mitosis. The N-terminal, middle, and C-terminal regions of deduced AcRAD21-1 protein sequence were conserved with Arabidopsis SYN4/AtRAD21.3 and rice OsRAD21-1, whereas three characteristic types of repetitive motifs (Repeat-1, Repeat-2/2′, and Repeat-3) were observed between the conserved regions. Such inserted repetitive amino acid sequences enlarge the AcRAD21-1 protein into almost 200 kDa, which belongs to the largest class of plant proteins. Genomic organization of the AcRAD21-1 locus was also determined, and the possibility of tandem exon duplication in Repeat-2 was revealed. Subsequently, the polyclonal antiserum was raised against the N-terminal region of AcRAD21-1, and purified by affinity chromatography. Immunohistochemical analysis with the purified antibody successfully showed localization of AcRAD21-1 in onion mitosis, suggesting that it can be used as a molecular marker visualizing dynamic movement of cohesin.     

Molecular characterization of two y-type high molecular weight glutenin subunit alleles 1Ay12 and 1Ay8 from cultivated einkorn wheat (Triticum monococcum ssp. monococcum)

Two y-type high molecular weight glutenin subunits (HMW-GSs) 1Ay12^? and 1Ay8^? from the two accessions PI560720 and PI345186 of cultivated einkorn wheat (Triticum monococcum ssp. monococcum, AA, 2n = 2x = 14), were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The mobility of 1Ay12^? and 1Ay8^? was similar to that of 1Dy12 and 1By8 from common wheat Chinese Spring, respectively. Their ORFs respectively consisted of 1812 bp and 1935 bp, encoding 602 and 643 amino acid residues with the four typical structural domains of HMW-GS including signal peptide, conserved N-, and C-terminal and central repetitive domains. Compared with the most similar active 1Ay alleles previous published, there were a total of 15 SNPs and 2 InDels in them. Their encoding functions were confirmed by successful heterogeneous expression. The two novel 1Ay alleles were named as 1Ay12^? and 1Ay8^? with the accession No. JQ318694 and JQ318695 in GenBank, respectively. The two alleles were classed into the two distinct groups, Phe-type and Cys-type, which might be relevant to the differentiation of Glu-A1-2 alleles. Of which, 1Ay8^? belonged to Cys-type group, and its protein possessed an additional conserved cysteine residue in central repetitive region besides the six common ones in N- and C-terminal regions of Phe-type group, and was the second longest in all the known active 1Ay alleles. These results suggested that the subunit 1Ay8^? of cultivated einkorn wheat accession PI345186 might have a potential ability to strengthen the gluten polymer interactions and be a valuable genetic resource for wheat quality improvement.     

The sterol-based transcriptional control of human 7-dehydrocholesterol reductase (DHCR7): Evidence of a cooperative regulatory program in cholesterol synthesis

The enzyme 7-dehydrocholesterol reductase (DHCR7) catalyzes the final step of cholesterol synthesis via the Kandutsch–Russell pathway, and is crucial in maintaining cellular cholesterol levels. Its absence leads to the devastating fetal developmental disorder Smith–Lemli–Opitz Syndrome (SLOS). How this enzyme is regulated has implications in controlling not only cholesterol synthesis, but also the synthesis of Vitamin D — another product of 7-dehydrocholesterol.     

The functional VNTR of IGH enhancer HS1.2 associates with human longevity and interacts with TNFA promoter diplotype in a population of Central Italy

The dysregulation of both immune and inflammatory responses occurring with aging is believed to substantially contribute to morbidity and mortality in humans. We have already reported the association of the functional Variable Number of Tandem Repeat (VNTR) at the Immunoglobulin heavy chain (IGH) enhancer HS1.2 with Immunoglobulin levels and with several autoimmune diseases. Herein we tested the association of the VNTR at the HS1.2 enhancer with human longevity, also evaluating the possible modulatory effect of TNFA promoter diplotype (rs361525/rs1800629). HS1.2 enhancer genotypes have been determined for 193 unrelated healthy individuals from Central Italy divided into two groups: Group 1 (18–84 yrs, mean age 56.8 ± 19.4) and Group 2 (85–100 yrs, mean age 93.0 ± 3.5). Homozygous subjects for *2 allele were significantly disadvantaged in reaching higher life-expectancy (OR = 0.457, p = 0.021). A significant interaction between TNFA promoter diplotype status, HS1.2 2/2 genotype and the two Groups was found (p = 0.014). Of note, TNFA − 308A allele seems to exert a protective effect in HS1.2 2/2 carriers. These results support the hypothesis of an important role of HS1.2 VNTR in the puzzle of the immune-system regulation, evidenced also by the potential interaction with TNFA. Moreover, the previous results showing the association of HS1.2 *2 allele with inflammatory phenomena are consistent with the hypothesis that this allele is a detrimental factor in reaching advanced age.     

Ssa1 Overexpression and [PIN] Variants Cure [PSI] by Dilution of Aggregates

Several cellular chaperones have been shown to affect the propagation of the yeast prions [PSI⁺], [PIN⁺] and [URE3]. Ssa1 and Ssa2 are Hsp70 family chaperones that generally cause pro-[PSI⁺] effects, since dominant-negative mutants of Ssa1 or Ssa2 cure [PSI⁺], and overexpression of Ssa1 enhances de novo [PSI⁺] appearance and prevents curing by excess Hsp104. In contrast, Ssa1 was shown to have anti-[URE3] effects, since overexpression of Ssa1 cures [URE3]. Here we show that excess Ssa1 or Ssa2 can also cure [PSI⁺]. This curing is enhanced in the presence of [PIN⁺]. During curing, Sup35-GFP fluorescent aggregates get bigger and fewer in number, which leads to their being diluted out during cell division, a phenotype that was also observed during the curing of [PSI⁺] by certain variants of [PIN⁺]. The sizes of the detergent-resistant [PSI⁺] prion oligomers increase during [PSI⁺] curing by excess Ssa1. Excess Ssa1 likewise leads to an increase in oligomer sizes of low, medium and very high [PIN⁺] variants. While these phenotypes are also caused by inhibition of Hsp104 or Sis1, the overexpression of Ssa1 did not cause any change in Hsp104 or Sis1 levels.     

Lack of association between genetic polymorphisms in ROBO1, MRPL19/C2ORF3 and THEM2 with Developmental Dyslexia

Developmental Dyslexia (DD) is a heritable, complex genetic disorder characterized by specific impairment in reading and writing ability that is substantially below the expected reading ability given the person's chronological age, measured intelligence and age-appropriate education. More than ten susceptible genes have been identified for DD. A Single Nucleotide Polymorphism (SNP) of these genes was found to be associated with various phenotypes of DD. To identify the role of SNPs of four candidate genes namely, MRPL19/C2ORF3, ROBO1 and THEM2 in an Indian population, we genotyped eight SNPs of these genes in 157 children with DD and 212 normal readers using a MassARRAY technique with a MALDI-TOF MS analyzer. Power analysis of some of these SNPs showed > 80% of power. Chi-square test, Odds Ratios (ORs), 95% Confidence Intervals (CIs) and Bonferroni's correction were applied to identify the significance of the genotyped SNPs and haplotypes. Our study failed to show any association of SNPs and haplotypes of these genes with DD in an Indian population.     

Evolved Lactococcus lactis Strains for Enhanced Expression of Recombinant Membrane Proteins

The production of complex multidomain (membrane) proteins is a major hurdle in structural genomics and a generic approach for optimizing membrane protein expression is still lacking. We have devised a selection method to isolate mutant strains with improved functional expression of recombinant membrane proteins. By fusing green fluorescent protein and an erythromycin resistance marker (ErmC) to the C-terminus of a target protein, one simultaneously selects for variants with enhanced expression (increased erythromycin resistance) and correct folding (green fluorescent protein fluorescence). Three evolved hosts, displaying 2- to 8-fold increased expression of a plethora of proteins, were fully sequenced and shown to carry single-site mutations in the nisK gene. NisK is the sensor protein of a two-component regulatory system that directs nisin-A-mediated expression. The levels of recombinant membrane proteins were increased in the evolved strains, and in some cases their folding states were improved. The generality and simplicity of our approach allow rapid improvements of protein production yields by directed evolution in a high-throughput way.     

Calcium-induced precipitation of αs1-casein: effect of modification of lysine residues

Turbidity measurements have been used to study the calcium-induced precipitation of α₃₁-casein whose amino groups have been modified by reaction with dansyl chloride and fluorescamine. Provided account is taken of the change in charge wrought by these modifications, the precipitation behaviour of these modified caseins is shown to be no different from that of the native α_s1-casein protein. The results provide further support for the previously suggested isoelectric precipitation model for this reaction.     

Physical mapping and cloning of RAD56

Here we report the physical mapping of the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in Saccharomyces cerevisiae. Mutation of RAD56 causes sensitivity to X-rays, methyl methanesulfonate, zeocin, camptothecin and hydroxyurea, but not to UV light, suggesting that N-terminal acetylation of specific DNA repair proteins is important for efficient DNA repair.     

Functional analysis and tissue-differential expression of four FAD2 genes in amphidiploid Brassica napus derived from Brassica rapa and Brassica oleracea

Fatty acid desaturase 2 (FAD2), which resides in the endoplasmic reticulum (ER), plays a crucial role in producing linoleic acid (18:2) through catalyzing the desaturation of oleic acid (18:1) by double bond formation at the delta 12 position. FAD2 catalyzes the first step needed for the production of polyunsaturated fatty acids found in the glycerolipids of cell membranes and the triacylglycerols in seeds. In this study, four FAD2 genes from amphidiploid Brassica napus genome were isolated by PCR amplification, with their enzymatic functions predicted by sequence analysis of the cDNAs. Fatty acid analysis of budding yeast transformed with each of the FAD2 genes showed that whereas BnFAD2-1, BnFAD2-2, and BnFAD2-4 are functional enzymes, and BnFAD2-3 is nonfunctional. The four FAD2 genes of B. napus originated from synthetic hybridization of its diploid progenitors Brassica rapa and Brassica oleracea, each of which has two FAD2 genes identical to those of B. napus. The BnFAD2-3 gene of B. napus, a nonfunctional pseudogene mutated by multiple nucleotide deletions and insertions, was inherited from B. rapa. All BnFAD2 isozymes except BnFAD2-3 localized to the ER. Nonfunctional BnFAD2-3 localized to the nucleus and chloroplasts. Four BnFAD2 genes can be classified on the basis of their expression patterns.     

Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.