Increasing cleavage specificity and activity of restriction endonuclease KpnI

Restriction enzyme KpnI is a HNH superfamily endonuclease requiring divalent metal ions for DNA cleavage but not for binding. The active site of KpnI can accommodate metal ions of different atomic radii for DNA cleavage. Although Mg2+ ion higher than 500 mu M mediates promiscuous activity, Ca2+ suppresses the promiscuity and induces high cleavage fidelity. Here, we report that a conservative mutation of the metal-coordinating residue D148 to Glu results in the elimination of the Ca2+-mediated cleavage but imparting high cleavage fidelity with Mg2+. High cleavage fidelity of the mutant D148E is achieved through better discrimination of the target site at the binding and cleavage steps. Biochemical experiments and molecular dynamics simulations suggest that the mutation inhibits Ca2+-mediated cleavage activity by altering the geometry of the Ca2+-bound HNH active site. Although the D148E mutant reduces the specific activity of the enzyme, we identified a suppressor mutation that increases the turnover rate to restore the specific activity of the high fidelity mutant to the wild-type level. Our results show that active site plasticity in coordinating different metal ions is related to KpnI promiscuous activity, and tinkering the metal ion coordination is a plausible way to reduce promiscuous activity of metalloenzymes.

Type III restriction-modification enzymes: a historical perspective

Restriction endonucleases interact with DNA at specific sites leading to cleavage of DNA. Bacterial DNA is protected from restriction endonuclease cleavage by modifying the DNA using a DNA methyltransferase. Based on their molecular structure, sequence recognition, cleavage position and cofactor requirements, restriction-modification (R-M) systems are classified into four groups. Type III R-M enzymes need to interact with two separate unmethylated DNA sequences in inversely repeated head-to-head orientations for efficient cleavage to occur at a defined location (25-27 bp downstream of one of the recognition sites). Like the Type I R-M enzymes, Type III R-M enzymes possess a sequence-specific ATPase activity for DNA cleavage. ATP hydrolysis is required for the long-distance communication between the sites before cleavage. Different models, based on 1D diffusion and/or 3D-DNA looping, exist to explain how the long-distance interaction between the two recognition sites takes place. Type III R-M systems are found in most sequenced bacteria. Genome sequencing of many pathogenic bacteria also shows the presence of a number of phase-variable Type III R-M systems, which play a role in virulence. A growing number of these enzymes are being subjected to biochemical and genetic studies, which, when combined with ongoing structural analyses, promise to provide details for mechanisms of DNA recognition and catalysis.

The Object Agreement Restriction

[EN]This paper deals with the so-called Person Case Constraint (Bonet, 1991), a universal constraint blocking accusative clitics and object agreement morphemes other than third person when a dative is inserted in the same clitic/agreement cluster. The aim of this paper is twofold. First, we argue that the scope of the PCC is considerably broader than assumed in previous work, and that neither its formulation in terms of person (1st/2nd vs. 3rd)-case (accusative vs. dative) restrictions nor its morphological nature are part of the right descriptive generalization.We present evidence (i) that the PCC is triggered by the presence of an animacy feature in the object’s agreement set; (ii) that it is not case dependent, also showing up in languages that lack dative case; and (iii) that it is not morphologically bound. Second, we argue that the PCC, even if it is modified accordingly, still puts together two different properties of the agreement system that should be set apart: (i) a cross linguistic sensitivity of object agreement to animacy and (ii) a similarly widespread restriction on multiple object agreement observed crosslinguistically. These properties lead us to propose a new generalization, the Object Agreement Constraint (OAC): if the verbal complex encodes object agreement, no other argument can be licensed through verbal agreement.

What mechanism of niche segregation allows the coexistence of sympatric sibling rhinolophid bats?

Introduction: Our purpose was to assess how pairs of sibling horseshoe bats coexists when their morphology and echolocation are almost identical. We collected data on echolocation, wing morphology, diet, and habitat use of sympatric Rhinolophus mehelyi and R. euryale. We compared our results with literature data collected in allopatry with similar protocols and at the same time of the year (breeding season). Results:Echolocation frequencies recorded in sympatry for R. mehelyi (mean = 106.8 kHz) and R. euryale (105.1 kHz) were similar to those reported in allopatry (R. mehelyi 105–111 kHz; R. euryale 101–109 kHz). Wing parameters were larger in R. mehelyi than R. euryale for both sympatric and allopatric conditions. Moths constitute the bulk of the diet of both species in sympatry and allopatry, with minor variation in the amounts of other prey. There were no inter-specific differences in the use of foraging habitats in allopatry in terms of structural complexity, however we found inter-specific differences between sympatric populations: R. mehelyi foraged in less complex habitats. The subtle inter-specific differences in echolocation frequency seems to be unlikely to facilitate dietary niche partitioning; overall divergences observed in diet may be explained as a consequence of differential prey availability among foraging habitats. Inter-specific differences in the use of foraging habitats in sympatry seems to be the main dimension for niche partitioning between R. mehelyi and R. euryale, probably due to letter differences in wing morphology. Conclusions: Coexistence between sympatric sibling horseshoe bats is likely allowed by a displacement in spatial niche dimension, presumably due to the wing morphology of each species, and shifts the niche domains that minimise competition. Effective measures for conservation of sibling/similar horseshoe bats should guarantee structural diversity of foraging habitats.

Flood circulation in oligomictic lakes: an undervalued reason for restriction of zooplankton production [Translation from: Memorie dell'Istituto Italiano di Idrobiologia Dott.Marco de Marchi 22 9-52, 1967]

Dormancy has been observed in several species of Cyclops. Animals in dormancy are characterised by almost complete inactivity of the main appendages and sluggish intestinal peristalsis. In Cyclops vicinus dormancy can take place in copepod stages III (19%), IV (78%) and V (3%) but one and the same animal only in one developmental stage. The author gives his own results between frequency of dormancy and photoperiod (20 C, 1000 lux). He concludes that dormancy in C. vicinus can be influenced by day length and can be ended prematurely by short day length.

Unveiling the factors shaping the distribution of widely distributed alpine vertebrates, using multi-scale ecological niche modelling of the bat Plecotus macrobullaris

Several alpine vertebrates share a distribution pattern that extends across the South-western Palearctic but is limited to the main mountain massifs. Although they are usually regarded as cold-adapted species, the range of many alpine vertebrates also includes relatively warm areas, suggesting that factors beyond climatic conditions may be driving their distribution. In this work we first recognize the species belonging to the mentioned biogeographic group and, based on the environmental niche analysis of Plecotus macrobullaris, we identify and characterize the environmental factors constraining their ranges. Distribution overlap analysis of 504 European vertebrates was done using the Sorensen Similarity Index, and we identified four birds and one mammal that share the distribution with P. macrobullaris. We generated 135 environmental niche models including different variable combinations and regularization values for P. macrobullaris at two different scales and resolutions. After selecting the best models, we observed that topographic variables outperformed climatic predictors, and the abruptness of the landscape showed better predictive ability than elevation. The best explanatory climatic variable was mean summer temperature, which showed that P. macrobullaris is able to cope with mean temperature ranges spanning up to 16 degrees C. The models showed that the distribution of P. macrobullaris is mainly shaped by topographic factors that provide rock-abundant and open-space habitats rather than climatic determinants, and that the species is not a cold-adapted, but rather a cold-tolerant eurithermic organism. P. macrobullaris shares its distribution pattern as well as several ecological features with five other alpine vertebrates, suggesting that the conclusions obtained from this study might be extensible to them. We concluded that rock-dwelling and open-space foraging vertebrates with broad temperature tolerance are the best candidates to show wide alpine distribution in the Western Palearctic.

Comparing the identification of southern California juvenile rockfishes (genus Sebastes spp.) by restriction site analysis of the mitochondrial ND3/ND4 region and by morphological characteristics

Rockfish (Sebastes spp.) juveniles are often difficult to identify by using morphological characters. This study independently applies morphological characters and a key based on mitochondrial restriction site variation to identify juvenile rockf ishes collected in southern California during juvenile rockfish surveys. Twenty-four specimens of Sebastes were examined genetically without knowledge of the morphological assignment. Seventeen fish were identified genetically as S. semicinctus, S. goodei, S. auriculatus, S. jordani, S. levis, S. rastrelliger, and S. saxicola. Identities for the remaining fish were narrowed to two or three species: 1) three fish were either S. carnatus or S. chrysomelas; 2) one fish was either S. chlorosticus, S. eos, or S. rosenblatti; and 3) three fish could have been either S. hopkinsi or S. ovalis, the latter for which we now have distinguishing mitochondrial markers. The genetic and morphological assignments concurred except for the identity of one fish that could only be narrowed down to S. hopkinsi or S. semicinctus by using morphological characters. Genetics excluded more species from multispecies groupings than did the morphological approach, especially species within the subgenus Sebastomus. Species in the genetically unresolvable groups may be similar because of recent divergence or because of interspecies introgression.

A key to selected rockfishes (Sebastes spp.) based on mitochondrial DNA restriction fragment analysis

Larval and juvenile rockfishes (Sebastes spp.) are difficult to identify using morphological characters. We developed a key based on sizes of restriction endonuclease fragments of the NADH dehydrogenase-3 and -4 (ND3/ND4) and 12S and 16S ribosomal RNA (12S/16S) mitochondrial regions. The key makes use of variation in the ND3/ND4 region. Restriction endonuclease Dde I variation can corroborate identifications, as can 12S/16S variation. The key, based on 71 species, includes most North American taxa, several Asian species, and Sebastolobus alascanus and Helicolenus hilgendorfi that are closely related to rockfishes. Fifty-eight of 71 rockfish species in our database can be distinguished unequivocally, using one to five restriction enzymes; identities of the remaining species are narrowed to small groups: 1) S. polyspinis, S. crameri, and S. ciliatus or variabilis (the two species could not be distinguished and were considered as a single species) ; 2) S. chlorostictus, S. eos, and S. rosenblatti; 3) S. entomelas and S. mystinus; 4)S. emphaeus, S. variegatus, and S. wilsoni; and 5) S. carnatus and S. chrysomelas.

Niche measures and feeding strategies of Barbodes gonionotus Bleeker and Oreochromis spp. from a ricefield in Bangladesh

Intra-and interspecific virtual niche measures and feeding strategies of Barbodes gonionotus and Oreochromis spp. were studied from a rice field in Bangladesh. Appropriateness and ease of interpretation of different indices were evaluated. Small sizes of both species had a relatively wider dietary breadth and used many of the resource categories available to them than the large size groups, though none were generalist feeder. The dietary overlap of large B. gonionotus on the small was greater than the reverse, but biologically insignificant. While the dietary overlap of large Oreochromis spp. on the small was significantly greater. Interspecific dietary width was relatively broader for B. gonionotus than Oreochromis spp. and overlap of B. gonionotus on Oreochromis spp. was significantly greater than the reverse. Evidence of significant intraspecific dietary overlap between the two sizes of tilapia reflects strong competition and cautions for mixed-size stocking in rice-fish system. Besides, there seem fewer opportunities for habitat segregation between B. gonionotus and Oreochromis spp., due to the significant interspecific dietary overlap of the former on the latter in rice-fish system. Tilapia specialized on periphytic detrital aggregate while silver barb tended to consume aquatic macrophytes and molluscs. Small sizes of B. gonionotus should be preferred for rice-fish integration over the Oreochromis spp. due to their broader niche width and pronounced ontogenetic dietary shifts with the aging of the stock.

Effect of feed quality restriction followed by realimentation on growth, nutrient utilization, biochemical changes and haematological profiles of Indian major carp, rohu (Labeo rohita H.)

To investigate the effect of protein restriction with subsequent re-alimentation on nutrient utilization, hematological and biochemical changes of Indian major carp, Rohu (Labeo rohita H.), 150 acclimatized Rohu fingerlings (average 20.74 ± 0.13 g) divided into five experimental groups (30 fingerlings in each groups with three replications with 10 fingerlings in each) for experimental trial of 90 days using completely randomized design. Control group (T sub(CPR)) was fed with feed having 30% crude protein at 3% of body weight for 90 days trial period. Other experimental groups T sub(1PR) was alternatively 3 days fed with feed having 20% CP and 30% CP at 3% of body weight, T sub(2PR) was alternatively 7 days fed with feed having 20% CP and 30% CP at 3% of body weight, T sub(3PR) was alternatively 15 days fed with feed having 20% CP and 30% CP at 3% of body weight and T sub(4PR) was alternatively 25 days fed with feed having 20% CP and 30% CP at 3% of body weight during 90 days trial period with daily ration in two equal halves at morning and afternoon. It was noticed that retention of different nutrients was almost similar among all treatment groups indicated improvement of digestibility of nutrients might not be the mechanisms for recovery growth in carps. Increased percent feed intake of body weight (hyperphagia) (4.14 ± 0.30 or 4.94 ± 0.46 and 3.33 ± 0.29), improved specific growth rate (1.86 ± 0.09 or 2.26 ± 0.05 and 1.43 ± 0.01), absolute growth rate (1.57 ± 0.08 or 1.84 ± 0.18 and 1.36 ± 0.12), protein efficiency ratio (1.19 ± 0.11 or1.16 ± 0.12 and 1.05 ± 0.09) were the important mechanism showing better performance index (21.60 ± 1.09 or 23.80 ± 0.21 and 19.45 ± 0.37) through which the experimental groups which were protein restricted and re-alimented at 3 or 7 days alternatively during 90 days trial period could able to compensate the growth retardation and to catch up the final body weight of control (128.68 ± 11.53 g/f) but other experimental groups failed to compensate during 90 days trial period. Result of the present study indicated that deprived fish i.e., fish received alternate 3 or 7 days protein restriction and re-alimentation showed recovery growth had still lower values of Hb (10.21 ± 0.02, and 9.88 ± 0.04 g/dl), hematocrit value (30.62 ± 0.05 and 26.64 ± 0.11%), total erythrocytic count (3.40 ± 0.01 and 3.29 ± 0.01 X10super(6) mm³), plasma glucose (126.93 ± 0.20 and 126.67 ± 0.05 mg/dl), total plasma lipid (1.04 ± 0.01 and 1.02 ± 0.01 g/dl) and liver glycogen (290.10 ± 0.80 and 288.99 ± 0.95 mg/kg) in comparison to control (10.56 ± 0.08 g/dl, 31.68 ± 0.24%, 3.52 ± 0.03 X10super(6) mm³, 128.23 ± 0.25 mg/dl, 1.07 ± 0.01g/dl and 292.00 ± 0.23 mg/kg) at the end of 90 days trial but total plasma protein in deprived group was compensated with advancement of trial period. All hematological and biochemical parameters studied were proportionately lowered in the experimental group got higher degree of deprivation. These findings suggested that with the increase of trial length complete compensation of hematological and biochemical profiles of rohu might be achieved. The results indicated that the implementation of alternative 7 days low and high protein diet feeding during aquaculture of carps could make economize the operation through minimizing the feed input cost.

GENETIC DIVERSITY IN THE CHINESE PANGOLIN (MANIS-PENTADACTYLA) - INFERRED FROM RESTRICTION ENZYME ANALYSIS OF MITOCHONDRIAL DNAS

Two different forms of Chinese pangolins can be recognized according to the color of their scales, i.e., brown and dusky. We analyzed mitochondrial DNA (mtDNA) purified from the livers of seven dusky and six brown Chinese pangolins from the same locality, using cleavage patterns from 19 restriction enzymes. From the 19 6-bp recognition enzymes used, 51-56 sites were observed. By combining the cleavage patterns for each enzyme, the 13 samples were classified into four restriction types: two in dusky and two in brown Chinese pangolins. The estimated number of nucleotide substitutions per site in dusky and brown types is 0.002, and that between dusky and brown types is 0.012. Divergence between brown and dusky forms began 0.6 Myr ago, provided the mean rate of sequence divergence is 0.02 per Myr in mtDNA. Our results suggest that there is considerable divergence in Chinese pangolins, and brown and dusky Chinese pangolins may be quite different forms or, at least, belong to different maternal groups.

PHYLOGENY OF RHESUS-MONKEYS (MACACA-MULATTA) AS REVEALED BY MITOCHONDRIAL-DNA RESTRICTION ENZYME ANALYSIS

Mitochondrial DNA, purified from 36 samples of 23 local populations which are widely distributed in Vietnam, Burma, and 10 provinces of China, has been analyzed to model the phylogeny of rhesus monkeys. The 20 local populations of China may represent nearly all major populations in China. Using 20 restriction endonucleases of 6-bp recognition, we observed a total of 50-61 sites in the various samples. By combining the cleavage patterns for each enzyme, the 36 samples were classified into 23 restriction types, each of which was found exclusively in the respective population from which samples were obtained By combining the earlier study of Indian rhesus monkeys, phylogenetic trees, which have been constructed on the basis of genetic distance, indicate that rhesus monkeys in China, Vietnam, India, and Burma can be divided into seven groups. Integrating morphological and geographical data, we suggest that rhesus monkeys in China, Vietnam, and Burma may be classified into six subspecies-M. m. mulatta, M. m. brevicaudus, M. m. lasiotis, M. m. littoralis, M. m. vestita, and M. m. tcheliensis-and rhesus monkeys in India may be another valid subspecies. M. m. tcheliensis is the most endangered subspecies in China. Divergence among subspecies may have begun 0.9-1.6 Ma. The radiation of rhesus monkeys in China may have spread from the southwest toward the east. The taxonomic status of the Hainan monkey and the Taiwan monkey require further investigation.