The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant phenotype

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 (hp-1) and the high pigment-1^w (hp-1^w) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 (hp-2), high pigment-2^j (hp-2^j) and dark green (dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1^w mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A⁹³¹-to-T⁹³¹ base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1^w mutant, on the other hand, a single G²³⁹²-to-A²³⁹² transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F₂ population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25<LOD score<26, R²=62.8%). These results strongly support the hypothesis that DDB1 is the gene encoding the hp-1 and hp-1^w mutant phenotypes.Communicated by R. Hagemann     

Are selective serotonin reuptake inhibitors cardiac teratogens? Echocardiographic screening of newborns with persistent heart murmur

BACKGROUND : Selective serotonin reuptake inhibitors (SSRIs) have been suspected of cardiac teratogenicity, but reports have been inconsistent. Our aim was to investigate the rate of nonsyndromic congenital heart defects in newborns exposed in utero to SSRIs compared with unexposed controls. METHODS : This prospective study of women who gave birth at our tertiary center from 2000 to 2007 yielded 235 women who reported first‐trimester SSRI use during pregnancy. All newborns born during the study period and found to have a persistent cardiac murmur on day 2 or 3 of life were referred for examination by a pediatric cardiologist and by echocardiography. The findings were compared between the newborns who were exposed to SSRIs and those who were not. RESULTS : Nonsyndromic congenital heart defects were identified by echocardiography in 8 of 235 (3.40%) newborns exposed in utero to SSRIs and in 1083 of 67,636 (1.60%) non‐exposed newborns. The difference in prevalence between the two groups was significant (relative risk, 2.17; 95% confidence interval, 1.07–4.39). The prevalence rates for paroxetine and fluoxetine exposure were 4.3% and 3.0%, respectively. All cardiac defects in the study group were mild: ventricular septal defect (6), bicuspid aortic valve (1) and right superior vena cava to coronary sinus (1). CONCLUSIONS : Newborns exposed in utero to SSRIs, have a twofold higher risk of mild nonsyndromic heart defects than unexposed infants. The data suggest that women who require SSRI treatment during pregnancy can be reassured that the fetal risk is low and possible cardiac malformations will probably be mild. Late‐targeted ultrasound and fetal echocardiography at 22 to 23 weeks' gestation are recommended in this patient group. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Cognitive function in adolescence and the risk for premature diabetes and cardiovascular mortality in adulthood

Background

Epidemiological studies have demonstrated a relationship between cognitive function in youth and the future risk of death. Less is known regarding the relationship with diabetes related death. This study assessed the relationship between cognitive function in late adolescence and the risk for diabetes, cardiovascular- (CVD) and all-cause mortality in adulthood.

Methods

This retrospective study linked data from 2,277,188 16–19 year olds who had general intelligence tests (GIT) conducted during pre-military recruitment assessment with cause of death as coded by the Israel Central Bureau of Statistics. The associations between cognitive function and cause-specific mortality were assessed using Cox models.

Results

There were 31,268 deaths that were recorded during 41,916,603 person-years of follow-up, with a median follow-up of 19.2 (IQR 10.7, 29.5) years. 3068, 1443, 514 and 457 deaths were attributed to CVD, CHD, stroke, and diabetes, respectively. Individuals in the lowest GIT vs. highest GIT quintiles in unadjusted models had the highest risk for all-cause mortality (HR 1.84, 95% CI 1.78, 1.91), total CVD (HR 3.32, 95% CI 2.93, 3.75), CHD (HR 3.49 95% CI 2.92, 4.18), stroke (HR 3.96 95% CI 2.85, 5.5) and diabetes-related (HR 6.96 95% CI 4.68, 10.36) mortality. These HRs were attenuated following adjustment for age, sex, birth year, body-mass index, residential socioeconomic status, education and country of origin for all-cause (HR 1.23, 95% CI 1.17, 1.28), CVD (HR 1.76, 95% CI 1.52, 2.04), CHD (HR 1.7 95% CI 1.37, 2.11), stroke (HR 2.03, 95% CI 1.39, 2.98) and diabetes-related (HR 3.14 95% CI 2.00, 4.94) mortality. Results persisted in a sensitivity analyses limited to participants with unimpaired health at baseline and that accounted competing risk.

Conclusions

This analysis of over 2 million demonstrates a strong relationship between cognitive function at youth and the risk for diabetes, all-cause and CVD-related mortality independent of adolescent obesity.

     

Linear forms of plasmid DNA are superior to supercoiled structures as active templates for gene expression in plant protoplasts

Introduction of the plasmids pUC8CaMVCAT and pNOSCAT into plant protoplasts is known to result in transient expression of the chloramphenicol acetyl transferase (CAT) gene. Also, transfection with the plasmid pDO432 results in transient appearance of the luciferase enzyme. In the present work we have used these systems to study the effect of DNA topology on the expression of the above recombinant genes. Linear forms of the above plasmids exhibited much higher activity in supporting gene expression than their corresponding super-coiled structures. CAT activity in protoplasts transfected with the linear forms of pUC8CaMVCAT and pNOSCAT was up to ten-fold higher than that observed in protoplasts transfected by the supercoiled template of these plasmids. This effect was observed in protoplasts derived from two different lines of Petunia hybrida and from a Nicotiana tabacum cell line. Transfection with the relaxed form of pUC8CaMVCAT resulted in very low expression of the CAT gene.Northern blot analysis revealed that the amount of poly(A)⁺ RNA extracted from protoplasts transformed with the linear forms of the DNA was about 10-fold higher than that found in protoplasts transformed with supercoiled DNA.Southern blot analysis revealed that about the same amounts of supercoiled and linear DNA molecules were present in nuclei of transfected protoplasts. No significant quantitative differences have been observed between the degradation rates of the various DNA templates used.     

The regulatory control of β-receptor dependent adenylate cyclase

The characteristics of the β-receptor in turkey erythrocyte adenylate cyclase were studied using both kinetics of enzyme activation and direct binding measurement of the β-agonists and antagonists to the β-receptor. The regulatory ligands Gpp(NH)p and Ca²⁺ do not have any direct effect on the β-receptor, but modulate the enzyme activity through the interaction with specific regulatory sites. It was found that the role of the catecholamine hormone is to facilitate the activation of the enzyme by the guanyl nucleotide. The regulatory guanyl nucleotide binds to its allosteric site in the absence of hormone, but the activation of the enzyme is slow in the absence of hormone. This role of the hormone can be described by the scheme: Where R is the receptor, E the enzyme, G the guanyl nucleotide, H the hormone, and E′ the activated form of the enzyme. The binding steps are fast and reversible but the conversion of the inactive enzyme E to its active form occurs with a k～1.0 min^?1 In the absence of the β-agonist (l-catecholamine) at the β-receptor and at physiological free Mg²⁺ concentrations, the activation of the enzyme is insignificant. Thus the presence of a guanyl nucleotide at the allosteric site is obligatory but not sufficient to induce the conversion of the inactive enzyme to its active form. At high (nonphysiological) Mg²⁺ concentration the conversion of E to E′ occurs slowly in the absence of hormone probably by another pathway. There are two classes of Gpp(NH)p regulatory sites: tight sites and loose sites, both of which can be identified kinetically. We have also identified the tight sites by direct binding studies using ³H-Gpp(NH)p. It is not clear, however, whether these are two distinct classes of sites or whether their existence reflects the presence of negative cooperativity among the guanyl nucleotide regulatory sites. Calcium was found to be a negative allosteric inhibitor of adenylate cyclase. The inhibitory effect of Ca²⁺ is exerted on the nonactivated enzyme as well as on the Gpp(NH)p preactivated enzyme.     

FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco

Site-specific recombination systems have been shown to excise transgene DNA sequences positioned between their cognate target sites, and thus be used to generate clonal sectors in transgenic plants. Here we characterized clonal sectors derived from genetic reversion of rolC (A. rhizogenes) – induced vegetative and reproductive phenotypes, mediated by FLP recombinase from S. cerevisiae, in tobacco. The constitutive expression of rolC induces pleiotropic effects including reduced apical dominance and plant height, lanceolate and pale green leaves and small, male-sterile flowers. Two transgenic male-sterile tobacco lines (N. tabacum, Samsun NN) expressing a 35sP-rolC gene construct flanked by two FRT (FLP recombinase target) sites, were cross-pollinated with pollen from a constitutive 35sP-FLP expressing line. Three main phenotypes were generated in result of recombinase-mediated excision of the 35sP-rolC locus in the F₁ (FLP×FRT-35sP-rolC-FRT) hybrid progenies: (a) restoration of male fertility, associated with reversion to normal leaf phenotypes prior to flower bud formation, (b) development of normal and fertile lateral shoot sectors on the background of rolC-type plants, (c) restoration of partially fertile flowers, associated with display of peripheral normal leaf sectors surrounding rolC-type inner-leaf tissues, consistent with periclinal chimeras. These results, supported by DNA molecular analysis, indicate that site-specific recombination might be used as a relatively efficient tool for generation of transgenic periclinal chimeric plants.