Twintrons are not unique to the Euglena chloroplast genome: structure and evolution of a plastome cpn60 gene from a cryptomonad

Introns within introns (twintrons) are known only from the Euglena chloroplast genome. Twintrons are group II or III introns, into which another group II or III intron has been transposed. In this paper we describe a non-Euglena twintron structure within a plastid-encoded chaperone gene (cpn60) of the cryptomonad alga Pyrenomonas salina. In addition, the evolutionary relationships between members of the Cpn60 protein family are determined. Our findings permit the inclusion of cryptomonad plastomes in phylogenetic studies of intron evolution and present further evidence for the origin of modern plastids from a cyanobacterial ancestor.This paper is dedicated to Prof. Dr. Peter Sitte on the occasion of his 65th birthday     

Sensitivity of the ELISA Test to Detect Phytophthora fragariae var. rubi in Raspberry Roots

A commercial serological, multiwell assay kit was used to assess the detection limits of Phytophthora fragariae var. rubi in raspberry roots. Detection limits in time lapse after inoculation, were assessed after inoculation of root systems of raspberry plants by zoospores of P. fragariae var. rubi. In extracts taken 3-9 days after inoculation, the pathogen was detected from the fourth day after inoculation. In a test series of simulated P. fragariae var. rubi infection where 0.25, 0.5, 1.0 and 1.5% of infected root tissue respectively, were mixed with healthy tissue (w/w), it was possible to detect the pathogen at 0.25% of simulated infection level. The results obtained show the possibility of an early detection of small amounts of antigen by the ELISA test procedure used. This enhance possibilities for early diagnosis and thereby more effective prevention of Phytophthora diseases in raspberry.     

Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant

The dioecious white campion (Silene latifolia) has been chosen as a working model for sexual development. In this species, sexual dimorphism is achieved through two distinct developmental blocks: inhibition of carpel development in male flowers, and early arrest of anther differentiation in female flowers. The combined advantages of the dioecious system and the availability of a sexual mutant lacking both male and female reproductive organs have been exploited in a molecular subtraction approach using male and asexual flower buds. This resulted in the cloning of 22 cDNA clones expressed in stamens at distinct stages of development. Fourteen of these clones corresponded to genes whose expression was detected in pre-meiotic stamens, a stage of development for which very little information is presently available. Furthermore, the absence of similarities with database sequences for ten clones suggests that they represent novel genes. Functional analysis of each clone will enable their positioning within the reproductive organ developmental pathway(s). In parallel, these clones are being exploited as developmental markers of early differentiation within the flower.     

Male mating success, reproductive success and multiple paternity in a natural population of sand lizards: behavioural and molecular genetics data

Sand lizard Lacerta agilis females characteristically mate with several males which, in staged mating experiments, results in multiple paternity of the offspring. In order to investigate multiple paternity in a natural population and interpret male reproductive behaviours in terms of sired young, we sampled the blood of females, potential fathers and hatchlings, and determined paternity using multilocus DNA fingerprinting as well as the variation at a single locus detected by the probe (TC) _n . The paternity analyses were preceded by a laboratory experiment in which we established that the parental alleles identified by the single locus probe were inherited in a Mendelian way. Our molecular data demonstrated that 12 out of 13 males (92%) that sired offspring were correctly identified from the 56 sexually mature males in the population. Also smaller males were accepted as sexual partners by the females, but sired fewer young in competition with larger males and were less able to maintain prolonged post-copulatory mate guarding. This may result in that some sexually successful males are only observed inside a female's home range, but never in pair-association with the female.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997     

The 85-kilodalton phosphoprotein (pp85) of human herpesvirus 7 is encoded by open reading frame U14 and localizes to a tegument substructure in virion particles.

A family of antigenically related proteins present in cells infected with human herpesvirus 7 (HHV-7), designated phosphoprotein 85 (pp85), comprises a complex of proteins, of which the 85-kDa species is phosphorylated. pp85 is a major determinant of human response to HHV-7 infection (L. Foà-Tomasi, E. Avitabile, L. Ke, and G. Campadelli-Fiume, J. Gen. Virol. 75:2719-2727, 1994; L. Foà-Tomasi, M. P. Fiorilli, E. Avitabile, and G. Campadelli-Fiume, J. Gen. Virol. 77:511-518, 1996; J. B. Black et al., Clin. Diagn. Lab. Immunol. 3:79-83, 1996). By immunoscreening of a cDNA library from HHV-7-infected cells with monoclonal antibody (MAb) 5E1, directed to the proteins of the pp85 complex, we mapped the gene encoding pp85 to the U14 open reading frame of the HHV-7 genome. A prokaryotically expressed fusion protein containing the U14 open reading frame reacted with MAb 5E1 in an immunoblot assay. A functional role for pp85 was defined by immunoelectron microscopy studies. Immunogold labeling of cryosections of HHV-7-infected cord blood mononuclear cells at high resolution localized the reactivity of MAb 5E1 to the outer surface of the virion tegument. This finding demonstrates that pp85, the product of the U14 gene, is a component of the HHV-7 tegument and suggests that the HHV-7 tegument is not a homogeneous structure but rather is composed of substructures, including an outermost layer containing pp85. The present findings, together with previously reported properties of MAb 5E1, including its ability to react with formalin-fixed paraffin-embedded samples, make this antibody a specific tool useful for etiopathogenetic studies of HHV-7 infection in humans and provide the basis for further development of pp85 into a specific recombinant diagnostic reagent.     

Are Elevated Aminotransferases and Decreased Bilirubin Additional Characteristics of the Metabolic Syndrome?

Abnormal liver tests, as well as morphological changes in the liver, are frequent among obese patients. Other frequent disturbances are visceral fat accumulation, insulin resistance, non-insulin-dependent diabetes mellitus (NIDDM), hypertriglyceridemia, and hypertension; these are a set of aberrations known as the metabolic syndrome. In order to investigate a possible relationship between the metabolic syndrome and impaired liver status we examined associations between liver tests, metabolic variables (insulin, glucose, and triglycerids), body composition and nutrition in 1083 men (BMI 28.8–63.8 kg/m²) and 1367 women (BMI 26.7–68.0 kg/m²) in the ongoing intervention study of Swedish Obese Subjects (SOS). Standard biochemical techniques were used to assess liver status and metabolic variables. Lean body mass (LBM) and masses of visceral and subcutaneous adipose tissue (AT) were estimated by means of computed tomography (CT) calibrated anthropometric equations. In both genders aspartate aminotransferase and alanine aminotransferase were, or tended to be, positively correlated to fasting serum insulin, visceral AT (women), and alcohol intake. In women, the aminotransferases were also correlated with fasting blood glucose. In both genders alkaline phosphatase was, or tended to be, positively associated with visceral AT, insulin (women), and glucose. Bilirubin was negatively correlated to insulin and visceral AT in men and women. Additional multivariate analyses indicated that alcohol had less explanatory power than serum insulin for the examined liver tests, especially among women. These results suggest that pathological liver tests in the obese may represent an expression of the metabolic syndrome.     

Demonstration of retinal afferents in the RCS rat,with reference to the retinohypothalamic projection and suprachiasmatic nucleus

In the Royal College of Surgeons (RCS) rat, characterized by inherited retinal dystrophy, retinal projections to the brain were studied using anterograde neuronal transport of cholera toxin B subunit upon injection into one eye. The respective immunoreactivity was found predominantly contralateral to the injection site in the lateral geniculate nucleus, superior colliculus, nucleus of the optic tract, medial terminal nucleus of the accessory optic tract, and bilateral hypothalamic suprachiasmatic nuclei. Although terminal density was somewhat reduced in dystrophic rats, the projection patterns in these animals appeared similar to those seen in their congenic controls and were comparable to the visual pathways described for the rat previously. In dystrophic rats, the number of cell bodies exhibiting immunoreactivity to vasoactive intestinal polypeptide, viz. a population of suprachiasmatic neurons receiving major retinohypothalamic input, was reduced by one-third, and some differences were observed in the termination pattern of the geniculohypothalamic tract, as revealed by immunoreactivity to neuropeptide Y in the suprachiasmatic nucleus.This study was supported by grants from the DFG (Re 644/2-1) and the NMFZ, Mainz (to S.R.).     

Five new microsatellite polymorphisms at the q21 region of human chromosome 21

Five clones, containing polymorphic CA-repeat sequences, have been isolated from a specific human chromosome 21 phage library and have been localised to band q21 of chromosome 21 using a somatic cell hybrid panel. These highly repetitive sequences (D21S1263, D21S1264, D21S1415, D21S1417 and D21S1420) have been characterised in the CEPH reference parents and have heterozygosities ranging from 0.30 to 0.81 and an average polymorphism information content (PIC) of 0.62. The relative order of these markers, based on the somatic cell hybrid panel, is cen-D21S1417, D21S1420-D21S1263, D21S1415-D21S1264-tel. The most polymorphic marker (D21S1264) has been included in the chromosome 21 genetic map. They have also been localised in the CEPH/ Généthon YAC panel, providing a refined localisation of these polymorphic sequences. These five CA-repeat markers should provide a better characterisation of the q21 region of chromosome 21.