ELISA detection of restriction site polymorphisms in the pig ryanodine receptor locus

We compare two strategies for ELISA detection of restriction site polymorphisms (EDRSP) that are suitable for high-throughput genotyping of the pig ryanodine receptor point mutation (RYR1 ^hal ). In both procedures, target DNA is amplified by PCR with one primer that is 5′ biotinylated and a second primer that is 5′ fluoresceinylated. PCR products are captured in duplicate wells on a streptavidin-coated, 96-well plate. The duplicates may be treated in two ways. In a single restriction enzyme assay, one duplicate is exposed to a restriction enzyme that cuts one allele specifically, and the second duplicate is exposed to no restriction enzyme. In a dual restriction enzyme assay, the second replicate is exposed to a second restriction enzyme that cuts the alternate allele specifically. Thereafter, the two procedures are similar; anti-fluorescein antibodies conjugated to peroxidase are allowed to bind to the fluoresceinylated ends, the plate is washed, and a substrate is converted to a colored end product. The ratio of the absorbances in the two wells is used to classify subjects by genotype. When the dual restriction enzyme assay is run, three genotype groups are easily distinguishable. When the single restriction enzyme assay is run, heterozygotes generate values that may overlap with those of the homozygotes that are not cut by the restriction enzyme. Dual restriction enzyme assays are more accurate than single restriction enzyme assays; however, single restriction enzyme assays are sufficient for identifying pigs that carry RYR1 ^hal . Received: 30 December 1997 / Accepted: 20 April 1998     

Restriction of streptomyces phage SH5 by endonuclease ShyI from Streptomyces hygroscopicus 0477

Summary DNA of the temperate Streptomyces phage SH5 (DNA molecular weight 27x10⁶) is subject to restriction-modification mediated by S. hygroscopicus 0477, S. levoris 1331 and 2340. The restriction endonuclease ShyI (isoschizomeric with SacII) isolated from S. hygroscopicus 0477 is involved in restriction of SH5·1331 and SH5·2340 DNAs in S. hygroscopicus 0477.     

Disentangling effects of growth and nutritional status on seabird stable isotope ratios

A growing number of studies suggest that an individual’s physiology affects its carbon and nitrogen stable isotope signatures, obscuring a signal often assumed to be only a reflection of diet and foraging location. We examined effects of growth and moderate food restriction on red blood cell (RBC) and feather δ¹⁵N and δ¹³C in rhinoceros auklet chicks (Cerorhinca monocerata), a piscivorous seabird. Chicks were reared in captivity and fed either control (75 g/day; n = 7) or ~40% restricted (40 g/day; n = 6) amounts of high quality forage fish. We quantified effects of growth on isotopic fractionation by comparing δ¹⁵N and δ¹³C in control chicks to those of captive, non-growing subadult auklets (n = 11) fed the same diet. To estimate natural levels of isotopic variation, we also collected blood from a random sample of free-living rhinoceros auklet adults and chicks in the Gulf of Alaska (n = 15 for each), as well as adult feather samples (n = 13). In the captive experiment, moderate food restriction caused significant depletion in δ¹⁵N of both RBCs and feathers in treatment chicks compared to control chicks. Growth also induced depletion in RBC δ¹⁵N, with chicks exhibiting lower δ¹⁵N when they were growing the fastest. As growth slowed, δ¹⁵N increased, resulting in an overall pattern of enrichment over the course of the nestling period. Combined effects of growth and restriction depleted δ¹⁵N in chick RBCs by 0.92‰. We propose that increased nitrogen-use efficiency is responsible for ¹⁵N depletion in both growing and food-restricted chicks. δ¹⁵N values in RBCs of free-ranging auklets fell within a range of only 1.03‰, while feather δ¹⁵N varied widely. Together, our captive and field results suggest that both growth and moderate food restriction can affect stable isotope ratios in an ecologically meaningful way in RBCs although not feathers due to greater natural variability in this tissue.     

Restriction map of the antibiotic resistance plasmid R1drd-19 and its derivatives pKN102 (R1drd-19B2) and R1drd-16 for the enzymes BamHI, HindIII, EcoRI and SalI.

Summary The conjugative R plasmid R1drd-19, mediating antibiotic resistance to ampicillin (Ap), chloramphenicol (Cm), kanamycin (Km), streptomycin (Sm) and sulfonamides (Su) was mapped using the restriction endonucleases BamHI, HindIII, EcoRI and SalI. BamHI generates 5 fragments (A-E) with molecular weights between 46×10⁶ dalton (representing mainly the RTF) and 0.25×10⁶ dalton, and HindIII 8 (A-H) between 42×10⁶ dalton (representing the main part of the RTF) and 0.1×10⁶ dalton. EcoRI recognises 17 sites and produces fragments (A-Q) with molecular weights between 11.7 and 0.1×10⁶ dalton. SalI yields 7 fragments (A-G) of 16.5 to 2.0×10⁶ dalton.A physical map was constructed from fragments obtained by partial digestion of R1drd-19 with one restriction enzyme, by double and triple digestion of the DNA with two or three enzymes with and without isolation of individual bands from preparative gels. In addition the restriction patterns of several mutants of R1drd-19 were compared with it.Evidence is presented which indicates that the derivatives of R1 investigated are generated by extende deletions, namely the copy mutant pKN102 which has lost the Km resistance, R1 drd-16, which has lost all resistances other than Km and the Km^s derivative of R1drd-16, which represents the pure RTF. The map of R1drd-19 is remarkably different from those of R100 and R6-5. Its molecular weight was estimated to be 62.5 Md. The circular fragment order for BamHI is: A-C-B-D-E, for HindIII: A-D-C-B-F-H-E-G, for EcoRI: A-C-K-B-F-J-O-D-H-L-G-P-Q-N-I-E-M-and for SalI A-B-C-D-G-F-E.     

Restriction in vivo

Summary Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degradation of restricted nonglucosylated T4 DNA by exonuclease V delayed cell division and induced filament formation and mutagenesis in lexA ⁺ but not in lexA ^- cells. Delay of cell division was also dependent upon recA and recBC funtions. Such degradation of DNA also dramatically increased mutagenesis in tif ^- Sfi^- cells at 42°C. The synthesis of recA protein continued in the restricting host after infection by the nonglucosylated T4 phage, but enhanced synthesis is not induced to the extent seen in SOS induced tif ^- cells grown at 42°. We also found that restriction of nonglucosylated T4 was alleviated in UV irradiated cells. The UV induced alleviation of rgl and r _K restriction depended upon post irradiation protein synthesis and was not observed in recA, lexA or recBC mutants.     

Effect of water restriction on energy and water balance and osmoregulation of the fruit bat Rousettus aegyptiacus

The energy budget, water balance and osmoregulation of the fruit bat, Rousettus aegyptiacus, were studied during normal hydration and during water restriction (oven-dried apple diet). The water input and output were balanced during both normal hydration and water restriction. The kidney of the fruit bat is well adapted to handle the water load from its fruit diet by excreting large volumes (14% of the body mass per day) of dilute urine (113±25 mosmol·kg H₂P^-1) as well as reducing urine volume (-95%) and increasing urine osmotic concentration (555±280 mosmol·kg H₂O^-1) during water restriction. The haematocrit, plasma haemoglobin and total protein concentrations did not increase during water restriction and heat exposure, suggesting the conservation of plasma volume. Gross energy intake was not alfected by water restriction. However, digested energy intake and digestibility were significantly reduced. The effective regulation of energy and water budgets during water restriction suggests that the fruit bat can cope with seasonal climatic changes and with variable fruit supply during various seasons.Abbreviations BM body mass - DEI digested energy intake - EWL evaporative water loss - GEL gross energy intake - NH normal hydration - T _a ambient temperature - WR water restriction     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.     

Cytochrome-<Emphasis Type="Italic">b</Emphasis> variation in <Emphasis Type="Italic">Apis mellifera</Emphasis> samples and its association with COI–COII patterns

Five Mbo I (Mbo-A, Mbo-M, Mbo-C¹, Mbo-C² and Mbo-C³) and Hinf I (Hinf-1 to Hinf-5) patterns were observed in Apis mellifera samples after restriction of a 485 bp fragment of the mitochondrial cytochrome-b (cyt-b) gene. Associating the cyt-b Restriction fragment length polymorphism (RFLP) pattern of each sample to its respective previously established COI–COII (Dra I sites) pattern, five restriction patterns (Mbo-C¹, Mbo-C², Mbo-C³, Hinf-1 and Hinf-4) were observed in samples of maternal origin associated to the evolutionary branch C. No deletions or insertions were observed and the nucleotide substitution rate was estimated at 5.4%. Higher nucleotide diversity was observed among the branch C-haplotypes when compared with A and M lineages. Further studies are needed to confirm if the cyt-b + COI–COII haplotypes help to assign certain phylogeographic patterns to the branch C and to clarify phylogenetic relationships among A. mellifera subspecies.     

Restriction endonuclease activity in Clostridium thermocellum and Clostridium thermosaccharolyticum

 Clostridium thermocellum cell extracts exhibit specific endonuclease activity with very little non-specific exonuclease activity at 55°C. The Dam methylation system of Escherichia coli offers complete protection from digestion by C. thermocellum ATCC 27405 cell extracts for all DNA tested (totaling >100 kb, insuring that most potential restriction sequences have been exposed). Based on both the Dam recognition sequence and the similarity of cell extract and MboI DNA digests, the C. thermocellum restriction enzyme recognition sequence appears to be 5′ GATC 3′. Cell extracts made from a second thermophile, C. thermosaccharolyticum ATCC 31960 do not exhibit specific endonuclease activity under the conditions tested. Genomic DNA from C. thermocellum exhibits a Dam⁺ phenotype while genomic DNA from C. thermosaccharolyticum exhibits a Dam^- phenotype. Received: 10 March 1995/Received revision: 4 September 1995/Accepted: 13 September 1995     

Comparative macromolecular analysis of the cytoplasms of normal and cytoplasmic male sterile Brassica napus

Summary Chloroplast (cp) and mitochondrial (mt) compartments of normal (N) and cytoplasmic male sterile (cms) lines of Brassica napus have been characterized and compared on the basis of cp and mt DNA restriction enzyme analysis and in vitro protein synthesis by isolated mitochondria. Cytoplasmic male sterility of B. napus (rape) comes from cms Raphanus sativus (radish) through intergeneric crosses.Cp DNAs isolated from N and cms lines had distinct restriction patterns with Sal I, Kpn I and Sma I enzymes. The size of the two cp DNAs measured from the restriction patterns was found to be identical and of about 95 × 10⁶ d. N and cms lines of B. napus were characterized by specific mt DNAs, as shown from Sal I, Kpn I, Pst I and Xho I cleavage patterns. The small number of well-separated restriction fragments obtained with Sal I enabled us to determine precisely mt DNA sizes. The values of 136.5 and 140.3 × 10⁶ d, obtained from restriction patterns with N and cms DNAs respectively, are smaller than any of those previously obtained from studies on other genera. With molecular hybridization experiments, it was possible to distinguish N and cms lines by the different locations of rRNA genes on the cp and mt DNAs.Two lines of B. napus are characterized by specific mt translation products formed in isolated mitochondria.     

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation

Summary The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.Abbreviations used Ap^r resistance to ampicillin - Cm^r resistance to chloramphenicol - R/M restriction and modification - Tc^r resistance to tetracycline     

Isolation and characterization of restriction endonuclease EclHKI from an Enterobacter cloacae strain

An Enterobacter cloacae strain, producing restriction enzyme EclHKI, was isolated from a decaying potato. The enzyme is an isoschizomer of Eam1105I, which recognizes and cleaves GACNNN!NNGTC. EclHKI was produced at high activity (4×10⁴ U/g wet cells) and was purified from contaminants which interfere with restriction digestion by passing the cell lysate through DEAE-Sephacel and heparin columns. Activity was optimal at 37°C in a medium salt buffer.H.-Y. Chan, Y.-C. Chan and P.-C. Shaw are with the Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong: K.-M. Kam is with the Institute of Pathology, Sai Ying Pun Jockey Club Clinic, Hong Kong.     

Alleviation of EcoK DNA restriction in Escherichia coli and involvement of umuDC activity.

Summary The activity of the EcoK DNA restriction system of Escherichia coli reduces both the plating efficiency of unmodified phage and the transforming ability of unmodified pBR322 plasmid DNA. However, restriction can be alleviated in wild-type cells, by UV irradiation and expression of the SOS response, so that 10³-to 10⁴-fold increases in phage growth and fourfold increases in plasmid transformation occurred with unmodified DNA. Restriction alleviation was found to be a transient effect because induced cells, which initially failed to restrict unmodified plasmid DNA, later restricted unmodified phage . Although the SOS response was needed for restriction alleviation, constitutive SOS induction, elicited genetically with a recA730 mutation, did not alleviate restriction and UV irradiation was still needed. A hitherto unsuspected involvement of the umuDC operon in this alleviation of restriction is characterized and, by differential complementation, was separated from the better known role of umuDC in mutagenic DNA repair. The need for cleavage of UmuD for restriction alleviation was shown with plasmids encoding cleavable, cleaved, and non-cleavable forms of UmuD. However, UV irradiation was still needed even when cleaved UmuD was provided. The possibility that restriction alleviation occurs by a general inhibition of the EcoK restriction/modification complex was tested and discounted because modification of was not reduced by UV irradiation. An alternative idea, that restriction activity was competitively reduced by an increase in EcoK modification, was also discounted by the lack of any increase in the modification of Ral^–, a naturally undermodified phage. Other possible mechanisms for restriction alleviation are discussed.     

Alleviation of type I restriction in adenine methylase (dam) mutants of Escherichia coli

Summary The host-controlled EcoK-restriction of unmodified phage .O is alleviated in dam mutants of Escherichia coli by 100- to 300-fold. In addition, the EcoK modification activity is substantially decreased in dam ^- strains. We show that type I restriction (EcoB, EcoD and EcoK) is detectably alleviated in dam mutants. However, no relief of EcoRI restriction (Type II) occurs in dam ^- strains and only a slight effect of dam mutation on EcoP1 restriction (Type III) is observed. We interpret the alleviation of the type I restriction in dam ^- strains to be a consequence of induction of the function which interferes with type I restriction systems.     

Serial permeability barriers to water transport in human placental vesicles

Summary Microvillous vesicles were prepared from term human placenta by shearing, differential centrifugation and Mg²⁺ precipitation. Vesicles were purified further on a sucrose density gradient producing two bands with densities of 1.16 to 1.18 g/ml (C1) and 1.13 to 1.15 g/ml (C2). The C2 fraction, which had a 24-fold enrichment of alkaline phosphatase and a three-fold reduction in Na⁺, K⁺-ATPase activity compared to homogenates, was used to measure osmotic water (P _f ) permeability.P _f was measured from the time course of scattered light intensity following exposure of vesicles to specified gradients of impermeant solutes.P _f decreased from 3.0×10^–3 to 0.6×10^–3 cm/sec with increasing gradient size (65 to 730mm; 23°C). Four possible causes of this behavior were examined theoretically and experimentally: an unstirred layer, saturation of water transport, large changes in the vesicle surface area with changes in volume and a structural restriction to vesicle volume change. The measured dependence ofP _f on gradient size and the effect of the channel-forming ionophore gramicidin onP _f fit best to the theoretical dependences predicted by a structural restriction mechanism. This finding was supported by experiments involving the effects onP _f of increased solution viscosity, initial vesicle volume, the magnitude of transmembrane volume flow, and the effects of gradient size on activation energy (E _a) forP _f . The decreasedP _f resulting from a structural restriction limiting vesicle volume change was modeled mathematically as a second barrier in series with the vesicle membrane.E _a measured using a 250-mm inwardly directed sucrose gradient was 5.4±0.6 kcal/mol (T>27°C) and 10.0±0.6 kcal/mol (T<27°C).E _a above 27°C is in the range normally associated with transmembrane passage of water via aqueous channels. Water transport was not inhibited byp-chloromercuribenzenesulfonate.     

Molecular analysis of the gene for the human vitamin-D-binding protein (group-specific component): allelic differences of the common genetic GC types

Summary DNA sequence analysis of the polymerase chain reaction products, including the coding region for amino acids 416 and 420, of the vitamin-D-binding protein (DBP, group-specific component, GC) shows allelespecific differences. The GC2 and GC1F phenotypes have an aspartic acid residue at amino acid position 416, whereas the GC1S phenotype has a glutamic acid at this position. In the GC2 phenotype, amino acid 420 is a lysine residue, and in the both common GC1 phenotypes, it is a threonine residue. The nucleotide exchanges involve a HaeIII (position 416) and a StyI (position 420) restriction site: the HaeIII restriction site is specific for the GC^*1S allele and the StyI restriction site is specific for the GC^*2 allele. We have tested 140 individual genomic DNA samples for the HaeIII site and 148 samples for the StyI site by restriction fragment length polymorphism (RFLP) analysis with a DBP-specific direct genomic DNA probe, and have compared these findings with the GC phenotype classification, by isoelectric focusing (IEF) of the corresponding plasma. The results of the HaeIII RFLP analysis and the IEF typing were in complete agreement. By using our DNA probe, we could disclose, in addition to the StyI site at amino acid position 420, two further StyI site downstream: one was specific for the GC^*1S allele and another for the GC^*1F allele. In 147 samples, there was agreement between the IEF GC typing and the analysis of the StyI restriction sites. In a single case, the observed result of the StyI-digest differed from the result expected after IEF classification: homozygous GC 1F-1F by IEF and heterozygous by StyI RFLP analysis. We discuss this finding as a recombination event or a possible silent allele in IEF typing. The GC polymorphism revealed by Southern blot analysis of StyI-digests provides an informative DNA marker system for chromosome 4q11–q13.     

φ20, A Temperate Bacteriophage Isolated from Bacillus anthracis Exists as a Plasmidial Prophage

This study describes the isolation of temperate B. anthracis phages, from 4 out of 20 B. anthracis strains screened, by use of the inducing agents mitomycin C and UV light. Phage φ20 isolated from B. anthracis Sterne 34F₂ (pXO1⁺ pXO2⁻) was shown to have double-stranded DNA of size 48756 bp and a restriction site map showing nine sites for enzymes BamHI, BglII, and SstI is included. The φ20 genome was found to exist as a plasmidial prophage and the phage itself to have a polyhedral head of diameter 65 nm and tail 217 nm long and 15 nm wide.     

Effects of 12 weeks of aerobic exercise plus dietary restriction on body composition, resting energy expenditure and aerobic fitness in mildly obese middle-aged women

This study investigated the effects of 12 weeks of aerobic exercise plus voluntary food restriction on the body composition, resting metabolic rate (RMR) and aerobic fitness of mildly obese middle-aged women. The subjects were randomly assigned to exercise/diet (n = 17) or control (n = 15) groups. The exercise/diet group participated in an aerobic training programme, 45–60 min · day ^–1 at 50%–60% of maximal oxygen uptake (VO_2max), 3–4 days · week^–1, and also adopted a self-regulated energy deficit relative to predicted energy requirements (–1.05 MJ · day ^–1 to –1.14 MJ · day ^–1 ). After the regimen had been followed for 12 weeks, the body mass of the subjects had decreased by an average of 4.5 kg, due mainly to fat loss, with little change of fat free mass (m _ff). The absolute RMR did not change, but the experimental group showed significant increases in the RMR per unit of body mass (10%) and the RMR per unit of m _ff (4%). The increase in RMR/m _ff was not correlated with any increase in VO_2max/m _ff. The resting heat production per unit of essential body mass increased by an average of 21%, but the resting heat production rate per unit of fat tissue mass remained unchanged. We concluded that aerobic exercise enhances the effect of moderate dietary restriction by augmenting the metabolic activity of lean tissue.     

Identification and chromosomal locations of a family of cytochrome P-450 genes for pisatin detoxification in the fungus Nectria haematococca

Summary The ability to detoxify the phytoalexin, pisatin, an antimicrobial compound produced by pea (Pisum sativum L.), is one requirement for pathogenicity of the fungus Nectria haematococca on this plant. Detoxification is mediated by a cytochrome P-450, pisatin demethylase, encoded by any one of six Pda genes, which differ with respect to the inducibility and level of pisatin demethylase activity they confer, and which are associated with different levels of virulence on pea. A previously cloned Pda gene (PdaT9) was used in this study to characterize further the known genes and to identify additional members of the Pda family in this fungus by Southern analysis. DNA from all isolates which demethylate pisatin (Pda⁺ isolates) hybridized to PdaT9, while only one Pda^– isolate possessed DNA homologous to the probe. Hybridization intensity and, in some cases, restriction fragment size, were correlated with enzyme inducibility. XhoI/BamHI restricted DNA from reference strains with a single active Pda allele had only one fragment with homology to PdaT9; no homology attributable to alleles associated with the Pda^– phenotype was found. Homology to this probe was also limited to one or two restriction fragments in most of the 31 field isolates examined. Some unusual progeny from laboratory crosses that failed to inherit demethylase activity also lost the single restriction fragment homologous to PdaT9. At the chromosome level, N. haematococca is highly variable, each isolate having a unique electrophoretic karyotype. In most instances, PdaT9 hybridized to one or two chromosomes containing 1.6–2 million bases of DNA, while many Pda- isolates lacked chromosomes in this size class. The results from this study of the Pda family support the hypothesis that deletion of large amounts of genomic DNA is one mechanism that reduces the frequency of Pda genes in N. haematococca, while simultaneously increasing its karyotypic variation.     

Induction of multiple shoots in a monopodial orchid hybrid (Aerides vandarum Reichb.f × Vanda stangeana Reichb.f) using thidiazuron and analysis of their genetic stability

The effect of thidiazuron (TDZ) on direct multiple shoot induction in axenic seedlings of a monopodial orchid hybrid Aerides vandarum × Vanda stangeana, using a dual phase culture medium was studied. The culture system consisted of a basal agar solidified half-strength Murashige and Skoog medium overlaid by a liquid fraction of the same composition. Highest regeneration of multiple shoots (15.8 shoots per seedling) was obtained in the medium containing 2% sucrose (w/v) supplemented with 2 mgl⁻¹ TDZ. Genetic stability of the regenerated shoots was assessed using random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), and restriction fragment length polymorphism of the PCR amplified (PCR-RFLP) nrITS region, as well as those of the coding (matK) and non-coding (trnL-F) regions of the cpDNA. Across the randomly selected mother plant and nine of its regenerated shoots, 2,680 bands were generated by 19 RAPD and 12 ISSR primers, exhibiting monomorphic banding profiles. Homogenous PCR-RFLP profiles were generated for nrITS using four restriction enzymes (REs), matK using five REs, and trnL-F using six REs. These molecular analyses showed no genomic alterations in all regenerated shoots obtained on medium containing 2 mgl⁻¹ TDZ.