Detecting cellulase and esterase enzyme activities encoded by novel genes present in environmental DNA libraries

A genomic DNA library was made from the alkaliphilic cellulase-producing Bacillus agaradhaerans in order to prove our technologies for gene isolation prior to using them with samples of DNA isolated directly from environmental samples. Clones expressing a cellulase activity were identified and sequenced. A new cellulase gene was identified. Genomic DNA libraries were then made from DNA isolated directly from the Kenyan soda lakes, Lake Elmenteita and Crater Lake. Crater Lake clones expressing a cellulase activity and Lake Elmenteita clones expressing a lipase/esterase activity were identified and sequenced. These were encoded by novel genes as judged by DNA sequence comparisons. Genomic DNA libraries were also made from laboratory enrichment cultures of Lake Nakuru and Lake Elmenteita samples. Selective enrichment cultures were grown in the presence of carboxymethylcellulose (CMC) and olive oil. A number of new cellulase and lipase/esterase genes were discovered in these libraries. Cellulase-positive clones from Lake Nakuru were isolated at a frequency of 1 in 15,000 from a library made from a CMC enrichment as compared to 1 in 60,000 from a minimal medium enrichment. Esterase/lipase-positive clones from Lake Elmenteita were isolated with a frequency of 1 in 30,000 from a library made from an olive-oil enrichment as compared to 1 in 100,000 from an environmental library.Communicated by K. Horikoshi     

A preputial odor imprinted on female mice

Female mice (Mus musculus) which were reared by a preputialectomized female and exposed to an intact male from 14 to 18 days of age preferred the odor of an intact male over that of a preputialectomized male when tested at 15 weeks of age. However, those females which were exposed to an intact male from 28 to 32 days of age preferred the odor of a preputialectomized male over that of an intact male, and those females which were exposed to an intact male from 0 to 4 days of age showed no reliable preferences. Females which were reared by an intact female and exposed a preputialectomized male for 4 days tended to reverse preferences. Females which were exposed to a preputialectomized male from 14 to 18 days of age preferred the odor of a preputialectomized male. However, females which were exposed to a preputialectomized male from 28 to 32 days of age preferred the odor of an intact male, as did females which were exposed to a preputialectomized male from 0 to 4 days.     

Nematodes from river drift and surface drinking water supplies in southern Ontario

Sixty-six genera of nematodes representing 37 families were collected over a 13-month period from untreated and treated water from three water treatment plants in southern Ontario. Two plants receive water from the Grand River which drains agricultural, residential and industrial regions, the third from a small stream in an agricultural district. Specimens were isolated by filtration from weekly samples of untreated and treated water, most were identified to genus but a few to species.Estimated mean density in the organic drift of the river ranged from 0.58 individuals per litre in winter, when it was under ice, to 10.57 per litre during the spring thaw. Most of the 66 genera found passed through the treatment plants and over 50% of these were still motile.Eleven genera were abundant at some time of the year, six known only from soil-dwelling forms and five from aquatic or soil/aquatic species. There was no indication that sewage purification works were a major source of any genus. There were a few plant parasites.Aquatic nematodes occurred during most of the year although they were more abundant during the warmer months, high densities of soil nematodes were a feature of the spring thaw and rains and during heavy autumn rains. More than 50% of the specimens were juveniles and some common genera lacked males.     

Isolation and characterization of 12 dinucletiotide microsatellite loci in Paratrechalea galianoae (Araneae, Trechaleidae), a nuptial gift-spider

Twelve microsatellite loci from the Neotropical spider Paratrechalea galianoae were isolated from a microsatellite-enriched genomic library, and were tested in a sample of 27 individuals from a population that occurs in Brazil. All loci were polymorphic and three to 14 (mean 7.58) alleles per locus were identified. The observed and expected heterozygosities per locus ranged from 0.417 to 0.885 and from 0.595 to 0.878, respectively. Two loci deviated significantly from Hardy-Weinberg equilibrium and one pair showed evidence of linkage disequilibrium. These loci are being used to study the population structure and genetic diversity in P. galianoae.     

Pure acceleration is the primary determinant of speed to first-base in major-league baseball game situations

The purposes of this research were to (a) quantify interval sprint times between Home-Plate and the Foul-Line and the Foul-Line and First-Base, (b) determine if differences exist in interval velocities and acceleration between left- and right-handed batters or between-position groupings, and (c) to quantify determinants of time to First-Base in Major-League Baseball players during actual games. A total of 1,896 sprint times to the Foul-Line (13.7 m) and First-Base (27.4 m) were recorded in 302 baseball players by a single coach, positioned in the dugout with a hand-held stopwatch. Interval velocities and accelerations were computed between Home-Plate and the Foul-Line and the Foul-Line and First-Base; average velocity and acceleration were also determined over the entire 27.4 m. Velocity and acceleration for left-handed batters were greater than for right-handed batters from Home-Plate to the Foul-Line and from Home-Plate to First-Base; however, there were no differences in velocity or acceleration from the Foul-Line to First-Base. Interval velocity was significantly greater for outfielders and infielders compared with that for catchers from Home-Plate to the Foul-Line and from the Home-Plate to First-Base. Outfielders were faster than catchers from the Foul-Line to First-Base; no other between-group differences were evident. Accelerations from Home-Plate to the Foul-Line and from Home-Plate to First-Base were greater for outfielders compared with infielders and catchers. Infielders accelerated at greater rates than did catchers between these intervals. There were no between-position differences in acceleration from the Foul-Line to First-Base. These data indicate that time to First-Base is most affected by acceleration from Home-Plate to the Foul-Line. Coaches should implement strategies that encourage players to sprint maximally over the first 13.7 m to maximize chances of successfully reaching First-Base.     

Isolation and characterization of microsatellite markers for <Emphasis Type="Italic">Cedrela odorata</Emphasis> L. (Meliaceae), a high value neotropical tree

We describe 9 primers for amplification of microsatellite loci for the Neotropical tree Cedrela odorata L. (Meliaceae). Loci were isolated from an enriched library derived from a single DNA sample from a tree in Costa Rica. Levels of polymorphism were determined using samples from a large progeny trial. Across loci, the number of alleles ranged from 14 to 30. Observed heterozygosity levels ranged from 0.61 to 0.88. No linkage disequilibria were detected although some departures from Hardy-Weinberg equilibrium (HWE) were found, probably due to a Wahlund effect.     

Molecular analysis of transferable tetracycline resistance plasmids from Clostridium perfringens.

Conjugative tetracycline resistance plasmids from 15 Clostridium perfringens isolates from piggeries were analyzed by restriction endonuclease digestion and agarose gel electrophoresis. Seven isolates from one farm were found to carry a 47-kilobase pair (kb) plasmid, pJIR5, which had EcoRI, XbaI, and ClaI profiles that were identical to those of a previously characterized plasmid, pCW3. An isolate from a second farm was found to carry a plasmid, pJIR6, which also was indistinguishable from pCW3. Five additional isolates from a third farm carried a 67-kb plasmid, pJIR2, which had at least 29 kb of DNA in common with pCW3. Finally, two isolates from a fourth farm were found to carry a 50-kb plasmid pJIR4, which appeared to consist of an entire pCW3 molecule with a 3-kb insertion. Comparative restriction maps of pCW3, pJIR2, and pJIR4 that identified the regions of homology among these plasmids were constructed. We suggest that many conjugative tetracycline resistance plasmids in C. perfringens may contain a pCW3-like core.     

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants.

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.     

Plasmid profiles as indicators of the source of contamination of Staphylococcus aureus endemic within poultry processing plants

A total of 530 strains of Staphylococcus aureus were isolated from the defeathering machinery of a chicken processing plant and from neck skin samples of carcasses at different stages of processing in two visits 4 weeks apart. Eleven different plasmid profiles were detected in the isolates, eight being common to both visits. The plasmid profiles of the strains forming the majority of the population on the freshly slaughtered birds were rarely present in the strains isolated from the pluckers (except at the entry to the first plucker) and were present in only a small proportion of the strains isolated from carcasses after plucking. However, the profiles from the strains isolated from the pluckers on both visits were different from those forming the majority of the population on the incoming birds but formed the major part of the carcass flora after plucking, suggesting that such strains were endemic. These strains were found as a small proportion of the isolates made from the incoming birds, suggesting that this was the route by which the endemic strains were introduced into the plant. Such endemic strains exhibited a clumping growth, even in liquid shake culture, which may have made it easier for them to become established on the pluckers and to resist cleaning and disinfection. This clumping phenotype was correlated with the presence of a 7.5-megadalton plasmid.     

Forces in the knee joint whilst rising from a seated position

Knee joint forces were determined by kinesiological techniques, using a high speed cine camera, a force platform, a specially constructed dynamometerized chair, and EMG recorders; so that a comparison could be made for rising from a normal chair with and without the aid of arms, and for rising from high and low chairs. For rising from the seated position, the knee joint forces parallel to the long axis of the tibia at the point of contact between the tibia and femur were found to be up to seven times body weight at about the time when the body left contact with the chair. When rising from a chair with the aid of arms, the knee joint forces were reduced to less than three times body weight. Knee joint and muscle forces were also reduced when rising from a high seat compared with rising from a low seat.     

Genetic variation in zinc-tolerant populations of Glyceria fluitans

Amplified fragment length polymorphism (AFLP) was used to conduct a study of the genetic diversity of zinc-tolerant populations of Glyceria fluitans from 10 sites from across Europe. Six different primer combinations were used on five to nine plants from each of the 10 sites to generate a total of 796 bands, of which 670 were polymorphic. These data were then used to calculate a dendrogram by agglomerative clustering using the unweighed pair group method with average linkage (UPGMA). The dendrogram contained two distinct clusters, with little overlap between populations. Genetic diversity between populations of G. fluitans did not always correlate with geographical distances, for example, plants from the Navan population from Ireland were more genetically similar to populations from Poland than other populations from Ireland. In other instances, geographical origin was significant, for example, all Polish populations were genetically similar to each other. Populations from two English sites only 160 km apart, showed such a high degree of genetic diversity that they were placed in different clusters in the dendrogram. They were more closely related to Irish and Polish populations than to each other. Plants from different zinc-contaminated sites were found not to cluster together. The conclusion was that the cluster groupings were not related to exposure to zinc at the sites of origin, and that the drive to generate distinct metal-tolerant populations may not occur in this species due to the existence of a constitutive tolerance to metals.     

Metabolic diversity in myxobacteria: identification of the myxalamid and the stigmatellin biosynthetic gene cluster of Stigmatella aurantiaca Sg a15 and a combined polyketide-(poly)peptide gene cluster from the epothilone producing strain Sorangium cellulosum So ce90.

Myxobacterial strains producing polyketides (PKs) assumed to be biosynthesized by a type I polyketide synthase (PKS) were analysed. Myxobacteria also produce a variety of polypeptides (PP) and PKs with incorporated amino acids ('mixed PK-PP'). In order to be able to identify the biosynthetic gene clusters for these metabolites a PCR based approach has been developed to clone ketosynthase (KS) domains of PKS genes from these organisms. Conserved regions of peptide synthetases of the non-ribosomal type (NRPS) were also amplified via PCR. KS fragments from Stigmatella aurantiaca Sg a15 were used for chromosomal gene inactivation experiments resulting in a series of mutants including such that were unable to produce stigmatellins and myxalamids. A NRPS fragment and PKS fragments from Sorangium cellulosum So ce90 were used to identify cosmids hybridizing with both types of probes from a genomic library. Both a NRPS and a PKS fragment were cloned and sequenced from a relatively short restriction fragment of one of these cosmids. The method described here should be very useful to clone and identify PKS, NRPS and mixed PKS-NRPS from myxobacteria in general and thereby open opportunities to use the biochemical diversity of these bacteria for genetic engineering and combinatorial biosynthesis.     

Y Chromosome aneuploidy,micronuclei, kinetochores and aging in men

This investigation was conducted to determine the relationship between Y chromosome loss and increased micronucleus formation with age. We also investigated the status of kinetochore proteins in the micronuclei. Umbilical cord blood samples were obtained from 18 newborn males, and peripheral blood was obtained from 35 adult males ranging in age from 22 to 79 years. Isolated lymphocytes from all 53 donors were cultured and blocked with cytochalasin B. Two thousand binucleate cells per donor were scored using a modified micronucleus assay to determine the kinetochore status of each micronucleus. This assay showed 23.8% of the micronuclei to be kinetochore-positive, while 76.2% of the micronuclei were kinetochore-negative. Cells were then hybridized with a 3.56-kb biotinylated Y chromosome-specific probe. All micronucleate cells were relocated and their Y probe status was determined. A significant mcrease in Y-bearing micronuclei with age was observed. Metaphase cells from the same samples were analyzed for the presence or absence of Y chromosome. The relationship between Y chromosome-positive micronuclei and Y chromosome-negative metaphase cells was highly significant, suggesting that Y chromosome-deficient metaphase cells result from cells which had previously lost a Y chromosome due to micronucleation. The cause of micronucleus formation from a lagging Y chromosome appears probably to be either a faulty or a diminished amount of kinetochore protein.     

Predisposing factors in systemic and central nervous system candidiasis: Histopathological and cultural observations in the rat

Four atypical isolates of Microsporum canis, three from humans and one from a cat, were obtained from North-West London. These and a further human isolate were compared with each other and with a typical isolate of the fungus. Immediately after isolation the atypical isolates were very labile, but were stabilised after a few subcultures from selected sectors. The stable forms differed from each other, but all had a tendency to brown rather than yellow pigmentation, to feathery submerged mycelium and to abnormal macroconidia. The macroscopic appearance and texture of the colonies depended on the density, orientation and branching pattern of the submerged mycelium.In recent years similar brown, feathery forms of M. canis have been reported from monkeys but not from cats. It is suggested that all such isolates may be culturally stable forms of a very unstable strain, probably feline in origin, which has yet to be described.     

Early evolution of the human immunodeficiency virus type 1 subtype C epidemic in rural Malawi

We have tracked the early years of the evolution of the human immunodeficiency virus type 1 (HIV-1) epidemic in a rural district of central east Africa from the first documented introductions of subtypes A, D, and C to the present predominance of subtype C. The earliest subtype C sequences ever reported are described. Blood samples were collected on filter papers from 1981 to 1984 and from 1987 to 1989 from more than 44,000 individuals living in two areas of Karonga District, Malawi. These samples included HIV-1-positive samples from 200 people. In 1982 to 1984, HIV-1 subtypes A, C, and D were all present, though in small numbers. By 1987 to 1989, 152 (90%) of a total of 168 sequences were subtype C and AC, AD, and DC recombinants had emerged. Four of the subtype C sequences from 1983 to 1984 were closely related and were found at the base of a large cluster of low diversity that by the late 1980s accounted for 40% of C sequences. The other two early C sequences fell into a separate and more diverse cluster. Three other clusters containing sequences from the late 1980s were identified. Each cluster contained at least one sample from a person who had recently arrived in the district. From 18 HIV-1-positive spouse pairs, 12 very closely related pairs of sequences were identified. We conclude that there were multiple introductions of HIV-1 with limited spread, followed by explosive growth of a subtype C cluster, probably arising from a single introduction in or before 1983.     

A new approach for study of structural and functional properties of proteins with unknown functions

Selected proteins were produced in Escherichia coli bacterial expression system--three proteins from extremophil bacteria: a putative monooxygenase from Deinococcus radiodurans, a putative nucleotidyltransferase from Thermotoga maritima, a putative oxidoreductase from Exiguobacterium sibiricum; and a shaperon from Homo sapiens DJ-1. The protocol of isolation & purification of recombinant proteins were developed that allowed to obtain expression products with the purity of no less than 96%. Conditions for the crystallization have been selected that allowed a stable growth of crystals. Preliminary x-ray experiments were conducted in order to confirm the quality of produced crystals; the resolution of obtained structural data was from 1.2 to 1.8 angstrom.     

Comparative study of temperate bacteriophages isolated from Yersinia

170 Yersinia strains belonging to various species were investigated for the presence of temperate bacteriophages. By induction with mitomycin C seven phages were isolated from Y. enterocolitica strains and one phage from a Y. frederiksenii strain. The phages were characterized on the basis of their morphology, host range, genome size, DNA homology, and protein composition. They belong to different phage families and reveal narrow to moderate wide host ranges. Some of the isolated phages were able to infect pathogenic as well as nonpathogenic strains of Y. enterocolitica. The genomes of all isolated phages were found to be composed of double stranded DNA ranging from about 40 to 60 kb. In addition to the analysed phages, a number of putative phages were induced in strains of Y. frederiksenii, Y. kristensenii, Y. intermedia, and Y. mollaretii. The putative phages were identified by isolation of phage DNA from cell free lysates but could not be propagated on indicator strains. Southern hybridization experiments revealed relationships between phages belonging to different families. Moreover, DNA homologies were observed between phages isolated from nonpathogenic Yersinia strains and a phage which was isolated from a pathogenic Y. enterocolitica serogroup O:3 strain.     

Isolation of 37 single-copy DNA probes from human chromosome 6 and physical mapping of 11 probes by in situ hybridization

Fifty-five single-copy DNA probes were isolated from the library LL06NS01, which was constructed from a complete HindIII digest of a flow-sorted human chromosome 6. Because chromosomes from a human x Chinese hamster somatic cell hybrid were used as the starting material for the flow-sorting, the library could be expected to contain some contaminating Chinese hamster DNA as well as DNA from human chromosomes other than 6. Thirty-seven of the 55 probes, however, were shown to map to human chromosome 6 by Southern blot hybridization with DNA from a panel of somatic cell hybrids. Eleven of the probes were mapped further by in situ hybridization. Four probes were localized to the short arm of chromosome 6, six to the long arm, and one to the centromeric region.     

Size differences in migrant sandpiper flocks: ghosts in ephemeral guilds

Summary Scolopacid sandpipers were studied from 1980 until 1984 during spring migration in North Dakota. Common species foraging together in mixed-species flocks differed in bill length most often by 20 to 30 percent (ratios from 1.2:1 to 1.3:1). Observed flocks were compared to computer generated flocks drawn from three source pools of Arctic-nesting sandpipers. The source pools included 51 migrant species from a global pool, 33 migrant species from a Western Hemisphere pool, and 13 species that migrated though North Dakota. The observed flocks formed randomly from the available species that used the North Dakota migration corridor but the North Dakota species were not a random selection from the Western Hemisphere and global pools of Arctic-nesting scolopacid sandpipers. In short, the ephemeral, mixed-species foraging flocks that we observed in North Dakota were random mixes from a nonrandom pool. The size-ratio distributions were consistent with the interpretation that use of this migration corridor by sandpipers has been influenced by some form of sizerelated selection such as competition.