A 3347-locus genetic recombination map of sequence-tagged sites reveals features of genome organization, transmission and evolution of cotton (Gossypium)

We report genetic maps for diploid (D) and tetraploid (AtDt) Gossypium genomes composed of sequence-tagged sites (STS) that foster structural, functional, and evolutionary genomic studies. The maps include, respectively, 2584 loci at 1.72-cM ( approximately 600 kb) intervals based on 2007 probes (AtDt) and 763 loci at 1.96-cM ( approximately 500 kb) intervals detected by 662 probes (D). Both diploid and tetraploid cottons exhibit negative crossover interference; i.e., double recombinants are unexpectedly abundant. We found no major structural changes between Dt and D chromosomes, but confirmed two reciprocal translocations between At chromosomes and several inversions. Concentrations of probes in corresponding regions of the various genomes may represent centromeres, while genome-specific concentrations may represent heterochromatin. Locus duplication patterns reveal all 13 expected homeologous chromosome sets and lend new support to the possibility that a more ancient polyploidization event may have predated the A-D divergence of 6-11 million years ago. Identification of SSRs within 312 RFLP sequences plus direct mapping of 124 SSRs and exploration for CAPS and SNPs illustrate the "portability" of these STS loci across populations and detection systems useful for marker-assisted improvement of the world's leading fiber crop. These data provide new insights into polyploid evolution and represent a foundation for assembly of a finished sequence of the cotton genome.     

Reduction of menin expression enhances cell proliferation and is tumorigenic in intestinal epithelial cells

Menin, the product of the tumor suppressor gene MEN1, is widely expressed in mammalian endocrine and non-endocrine tissues, including intestine. Its known abundant expression in several types of cells with high proliferative capacity led us to investigate the physiological function of the protein menin in intestinal epithelium, one of the most rapidly growing epithelia. Here we showed that the Men1 gene is mainly expressed in the crypt compartment of the proximal small intestine and that its expression was increased during fasting in vivo, both suggesting a role of menin in the control of cell growth. Indeed, specific reduction of menin expression by transfected antisense cDNA in the rat duodenal crypt-like cell line, IEC-17, increased cell proliferation. The latter is correlated to a loss of cell-cycle arrest in G(1) phase by resting cells and an overexpression of cyclin D1 and cyclin-dependent kinase (Cdk)-4. Furthermore, these cells lost the inhibition of proliferation induced by transforming growth factor-beta1, associated with a decrease of transforming growth factor-beta type II receptor expression. As a result of deregulated proliferation, antisense menin transfected IEC-17 cells became tumorigenic as shown in vitro as well as in vivo in immunosuppressed animals. These results indicate that menin contributes to proliferation control in intestinal epithelial cells. The present study reveals an unknown physiological function for menin in intestine that may be important in the regulation of epithelial homeostasis.     

Effects of dietary alpha linolenic acid on cholesterol metabolism in male and female hamsters of the LPN strain

N-3 polyunsaturated fatty acids and estrogens are recognized as protective factors of atherosclerosis, however their interactions on cholesterol metabolism remain unclear. Male and female hamsters were fed for 9 weeks diets containing 12.5% lipids and rich in either alpha-linolenic acid ("linseed" diet) or saturated fatty acids ("butter" diet). Hamsters fed the "linseed" diet exhibited lower plasma concentrations of cholesterol (-29%), total LDL (-35%) and HDL (-17%), glucose (-20%), insulin (-40%) and of the LDL-cholesterol/HDL-cholesterol ratio (-27%) than those fed the "butter" diet. In the liver, cholesterol content was 2.7-fold lower in response to the "linseed" diet, whereas the concentration of HDL receptor (SR-BI) and the activities of HMGCoA reductase and cholesterol 7alpha-hydroxylase were 30 to 50% higher than with the "butter" diet. By contrast, the LDL receptor concentration did not vary with the diet. Females exhibited higher concentration of LDL (+24%), lower concentration of plasma triglycerides (-34%), total VLDL (-46%) and VLDL-cholesterol (-37%) and of biliary phospholipids (-19%). Besides, there was also an interaction between gender and diet: in males fed the "butter" diet, plasma triglycerides and VLDL concentration, were 2 to 4 fold higher than in the other groups. These data suggest that gene and/or metabolic regulations by fatty acids could interact with that of sex hormones and explain why males are more sensitive to dietary fatty acids.     

Isolation and disruption of the melanin pathway polyketide synthase gene of the softwood deep stain fungus Ceratocystis resinifera

Ceratocystis resinifera hyphae produce a black melanin pigment causing a deep stain in softwood logs. We exploited the homology of polyketide synthases to clone PKS1, a gene responsible for dihydroxynaphthalene-melanin biosynthesis in C. resinifera. Sequence analysis indicated that PKS1 has two introns near its 5(') end and encodes a 2188-amino acid polypeptide with five functional domains: beta-ketoacyl synthase, acyl transferase, two acyl carrier proteins and a thioesterase/Claisen cyclase. A gene disruption construct designed to replace a portion of PKS1 with a hygromycin resistance cassette was transformed into C. resinifera through Agrobacterium tumefaciens-mediated transformation. PKS1 null mutants had an albino phenotype, and pigmentation was restored by the addition of scytalone, a melanin pathway intermediate. The disruption of PKS1 and restoration of pigmentation with scytalone confirmed the presence of a dihydroxynaphthalene-melanin pathway in C. resinifera. The transformation method described in this paper is the first reported for a Ceratocystis species.     

A portable,non-focusing optics spectrophotometer (NoFOSpec) for measurements of steady-state absorbance changes in intact plants

Kinetically-resolved absorbance measurements during extended, or steady-state illumination are typically hindered by large, light-induced changes in the light-scattering properties of the material. In this work, a new type of portable spectrophotometer, the Non-Focusing Optical Spectrophotometer (NoFOSpec), is introduced, which reduces interference from light-scattering changes and is in a form suitable for fieldwork. The instrument employs a non-focusing optical component, called a compound parabolic concentrator (CPC), to simultaneously concentrate and homogeneously diffuse measuring and actinic light (from light-emitting diode sources) onto the leaf sample. Light passing through the sample is then collected and processed using a subsequent series of CPCs leading to a photodiode detector. The instrument is designed to be compact, lightweight and rugged for field work. The pulsed measuring beam allows for high sensitivity (typically < 100 ppm noise) and time resolution (∼ 10 μs) measurements in the visible and near infrared spectral regions. These attributes allow high-resolution measurements of signals associated with energization of the thylakoid membrane (the electrochromic shifting of carotenoid pigments), as well as electron transfer, e.g., the 820-nm changes associated with electron transfer through Photosystem I (PS I). In addition, the instrument can be used as a kinetic fluorimeter, e.g., to measure saturation-pulse fluorescence changes indicative of Photosystem II (PS II) quantum efficiency. The instrument is demonstrated by estimating electron and proton fluxes through the photosynthetic apparatus in an intact tobacco leaf, using respectively the saturation-pulse fluorescence changes and dark-interval relaxation kinetics (DIRK) of the electrochromic shift. A linear relationship was found, confirming our earlier results with the laboratory-based diffused-optics flash spectrophotometer, indicating a constant H⁺/e⁻ stoichiometry for linear electron transfer, and suggesting that cyclic electron flow around PS I is either negligible or proportional to linear electron flow. This type of measurement should be useful under field conditions for estimating the extent of PS I cyclic electron transfer, which is proposed to operate under stressed conditions. This revised version was published online in August 2006 with corrections to the Cover Date.     

Motility processes in Acantharia. II. A Ca2+ dependent system of contractile 2–4 nm filaments isolated from demembranated myonemes

Myonemes of the acantharians are contractile ribbon-like organelles. As previously shown, their motility is based on the coiling mechanism of double-twisted 2–4 nm nonactin filaments [14]. Myonemes have been isolated and manipulated in vitro. After demembranation, the contraction takes place when the Ca²⁺ concentration is above 10^?7 M, whereas relaxation occurs below this threshold concentration. The response to Ca²⁺ ions is an on/off mechanism. Both contraction and relaxation can be induced repeatedly without fatigue phenomena. Other divalent cations such as Sr²⁺, Ba²⁺, Ma²⁺, CO²⁺, and La³⁺ can replace Ca²⁺ in inducing contraction of the demembranated myonemes although with less efficiency. Contraction and relaxation are ATP-independent and calmodulin is not involved in this in vitro motility process. The myoneme is a strongly resistant structure which is capable of contracting and relaxing under various extreme conditions which indicates very stable proteins and resistant functions.     

Variation in Genes Related to Cochlear Biology Is Strongly Associated with Adult-Onset Deafness in Border Collies

Domestic dogs can suffer from hearing losses that can have profound impacts on working ability and quality of life. We have identified a type of adult-onset hearing loss in Border Collies that appears to have a genetic cause, with an earlier age of onset (3–5 years) than typically expected for aging dogs (8–10 years). Studying this complex trait within pure breeds of dog may greatly increase our ability to identify genomic regions associated with risk of hearing impairment in dogs and in humans. We performed a genome-wide association study (GWAS) to detect loci underlying adult-onset deafness in a sample of 20 affected and 28 control Border Collies. We identified a region on canine chromosome 6 that demonstrates extended support for association surrounding SNP Chr6.25819273 (p-value = 1.09×10⁻¹³). To further localize disease-associated variants, targeted next-generation sequencing (NGS) of one affected and two unaffected dogs was performed. Through additional validation based on targeted genotyping of additional cases (n = 23 total) and controls (n = 101 total) and an independent replication cohort of 16 cases and 265 controls, we identified variants in USP31 that were strongly associated with adult-onset deafness in Border Collies, suggesting the involvement of the NF-κB pathway. We found additional support for involvement of RBBP6, which is critical for cochlear development. These findings highlight the utility of GWAS–guided fine-mapping of genetic loci using targeted NGS to study hereditary disorders of the domestic dog that may be analogous to human disorders.     

The Repetitive Domain of ScARP3d Triggers Entry of Spiroplasma citri into Cultured Cells of the Vector Circulifer haematoceps

Spiroplasma citri is a plant pathogenic mollicute transmitted by the leafhopper vector Circulifer haematoceps. Successful transmission requires the spiroplasmas to cross the intestinal epithelium and salivary gland barriers through endocytosis mediated by receptor-ligand interactions. To characterize these interactions we studied the adhesion and invasion capabilities of a S. citri mutant using the Ciha-1 leafhopper cell line. S. citri GII3 wild-type contains 7 plasmids, 5 of which (pSci1 to 5) encode 8 related adhesins (ScARPs). As compared to the wild-type strain GII3, the S. citri mutant G/6 lacking pSci1 to 5 was affected in its ability to adhere and enter into the Ciha-1 cells. Proteolysis analyses, Triton X-114 partitioning and agglutination assays showed that the N-terminal part of ScARP3d, consisting of repeated sequences, was exposed to the spiroplasma surface whereas the C-terminal part was anchored into the membrane. Latex beads cytadherence assays showed the ScARP3d repeat domain (Rep3d) to be involved, and internalization of the Rep3d-coated beads to be actin-dependent. These data suggested that ScARP3d, via its Rep3d domain, was implicated in adhesion of S. citri GII3 to insect cells. Inhibition tests using anti-Rep3d antibodies and competitive assays with recombinant Rep3d both resulted in a decrease of insect cells invasion by the spiroplasmas. Unexpectedly, treatment of Ciha-1 cells with the actin polymerisation inhibitor cytochalasin D increased adhesion and consequently entry of S. citri GII3. For the ScARPs-less mutant G/6, only adhesion was enhanced though to a lesser extent following cytochalasin D treatment. All together these results strongly suggest a role of ScARPs, and particularly ScARP3d, in adhesion and invasion of the leafhopper cells by S. citri.