Mapping initiation sites for simian virus 40 DNA synthesis events in vitro.

Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro. The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex. Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin. To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined. Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis. At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer. In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal. Initiation signals are present, on average, once every 19 nucleotides. These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events.     

Use of Genetic and Physical Mapping to Locate the Spinal Muscular Atrophy Locus between Two New Highly Polymorphic DNA Markers

The gene for autosomal recessive forms of spinal muscular atrophy (SMA) has recently been mapped to chromosome 5ql3, within a 4-cM region between the blocks D5S465/D5S125 and MAP-1B/D5S112. We identified two new highly polymorphic microsatellite DNA markers—namely, AFM265wf5 (D5S629) and AFM281yh9 (D5S637)—which are the closest markers to the SMA locus. Multilocus analysis by the location-score method was used to establish the best estimate of the SMA gene location. Our data suggest that the most likely location for SMA is between locus D5S629 and the block D5S637/D5S351/MAP-1B/D5S112/D5S357. Genetic analysis of inbred SMA families, based on homozygosity by descent and physical mapping using mega-YACs, gave additional information for the loci order as follows: cen–D5S6–D5S125/D5S465–D5S435–D5S629–SMA–D5S637–D5S351–MAP–1B/D5S112–D5S357–D5S39–tel. These data give the direction for bidirectional walking in order to clone this interval and isolate the SMA gene.     

Changes in in vivo fluorescence quenching in rye and barley as a function of reduced PSII light harvesting antenna size

The relationship between the size of the light harvesting antenna to photosystem II (LHCII) and quenching of non-photochemical and dark level fluorescence was studied in wild-type rye (Secale cereale L. cv. Musketeer) and barley (Hordeum vulgare L. cv. Gunilla) as well as in the barley chlorophyll b-less chlorina F2 mutant (H. vulgare L. cv. Dornaria, chlorina-F2). Exposure for 10 min to an irradiance of 500 μmol m^?2 s^?1 resulted in a strong (0.71–0.73) non-photochemical (q_s) quenching of the fluorescence yield in wild-type (WT) material, while the barley chlorina F2-mutant was quenched to 75% of this level. Relaxation of q_s in darkness revealed a fast initial decay, related to relaxation of the high-energy-state dependent (q_E) part of q_s. Etiolated seedlings of rye and barley exposed to intermittent light (IML) for 36 cycles of 2 min light and 118 min darkness had suppressed Chl b and LHCII-production in both WT rye and barley, while the barley chlorina F2-mutant became totally devoid of all LHCII-polypeptides. It was found that the levels of q_s and q_s were similar in control grown barley chlorina F2 and IML-grown WT rye and barley, but q_s was reduced by 30 to 35% and q_s by 50 to 65%, respectively, as compared to control-grown. WT plants. No significant q_s could be detected in IML-grown barley chlorina F2. It is clear, from these changes in in vivo fluorescence quenching in rye and barley that a significant level of q_s is detectable even in the absence of LHCII. Only when the proximal antennae are totally absent, does q_E completely disappear. We conclude that the presence of LHCII is not an absolute requirement for q_E-quenching and suggest that distal as well as proximal antenna may contribute to q_E in vivo.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes

Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg²⁺-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase -dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergenta availability and cation sensitivity on the apparent distribution of PAPH in neutrophil sub-cellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzyme was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.     

Submaximal,aerobic exercise training exacerbates the cardiomyopathy of postweanling Cu-depleted rats

To determine the dual effect of exercise training and copper depletion on myocardial function and ultrastructure, postweanling rats were either trained or sedentary while fed copper-adequate or copper-deficient diets for 8 wk. Rats developed characteristic myocardial subcellular degeneration and increased cardiac mitochondrial volume density when copper depleted, despite lack of overt cardiac hypertrophy, hypertension, or anemia. Training combined with copper depletion induced mild left ventricular hypertrophy. Basal laminae appeared fractionated in areas at capillary-myocyte interface, with focal pericapillary and interstitial collagen accumulation, where-as overt fibrosis was absent or minimal. Electrocardiograms revealed increased QRS wave and QT duration and notching of QRS complex with copper depletion, consistent with intraventricular conductance disturbances. The oxidative capacity of soleus muscle increased with training in copper-adequate rats, but was reduced with progressive copper depletion. These data suggest that copper depletion and training are synergistic in effecting focal accumulation of collagen, with deleterious effect on exercise capacity.     

The Response to Exercise with Constant Energy Intake in Identical Twins

Seven pairs of young adult male identical twins completed a negative energy balance protocol during which they exercised on cycle ergometers twice a day, 9 out of 10 days, over a period of 93 days while being kept on a constant daily energy and nutrient intake. The total energy deficit caused by exercise above the estimated energy cost of body weight maintenance reached 244 ± 9.8 MJ (Mean ± SEM). Baseline energy intake was estimated over a period of 17 days preceding the negative energy balance protocol. Mean body weight loss was 5.0 kg (SEM = 0.6) (p <0.001) and it was entirely accounted for by the loss of fat mass (p <0.001). Fat-free mass was unchanged. Body energy losses reached 191 MJ (SEM = 24) (p <0.001) which represented about 78% of the estimated energy deficit. Subcutaneous fat loss was slightly more pronounced on the trunk than on the limbs as estimated from skinfolds, circumferences, and computed tomography (CT). The reduction in CT-assessed abdominal visceral fat was quite striking, from 81 cm² (SEM = 5) to 52 cm² (SEM = 6) (p <0.001). At the same submaximal power output level, subjects oxidized more lipids than carbohydrates after the program as indicated by the changes in the respiratory exchange ratio (p <0.05). Intrapair resemblance was observed for the changes in body weight (p <0.05), fat mass (P <0.01), percent fat (p <0.01), body energy content (p <0.01), sum of 10 skinfolds (p <0.01), abdominal visceral fat (p <0.01), fasting plasma triglycerides (p <0.05) and cholesterol (p <0.05), maximal oxygen uptake (p <0.05), and respiratory exchange ratio during submaximal work (p <0.01). We conclude that even though there were large individual differences in response to the negative energy balance and exercise protocol, subjects with the same genotype were more alike in responses than subjects with different genotypes particularly for body fat, body energy, and abdominal visceral fat changes. High lipid oxidizers and low lipid oxidizers during sub-maximal exercise were also seen despite the fact that all subjects had experienced the same exercise and nutritional conditions for about three months.