Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.     

The Tc5 Family of Transposable Elements in Caenorhabditis Elegans

We have identified Tc5, a new family of transposable genetic elements in the nematode Caenorhabditis elegans. All wild-type varieties of C. elegans that we examined contain 4-6 copies of Tc5 per haploid genome, but we did not observe transposition or excision of Tc5 in these strains. Tc5 is active, however, in the mut-2 mutant strain TR679. Of 60 spontaneous unc-22 mutations isolated from strain TR679, three were caused by insertion of Tc5. All three Tc5-induced mutations are unstable; revertants result from precise or nearly precise excision of Tc5. Individual Tc5 elements are similar to each other in size and structure. The 3.2-kb element is bounded by inverted terminal repeats of nearly 500 bp. Eight of the ten terminal nucleotides of Tc5 are identical to the corresponding nucleotides of Tc4. Further, both elements recognize the same target site for insertion (CTNAG) and both cause duplication of the central TNA trinucleotide upon insertion. Other than these similarities to Tc4, Tc5 is unrelated to the three other transposon families (Tc1, Tc3 and Tc4) that transpose and excise at high frequency in mut-2 mutant strains. Mechanisms are discussed by which four apparently unrelated transposon families are all affected by the same mut-2 mutation.     

The biogeochemistry of a north-temperate grassland with native ungulates: Nitrogen dynamics in Yellowstone National Park

Nutrient dynamics of large grassland ecosystems possessing abundant migratory grazers are poorly understood. We examined N cycling on the northern winter range of Yellowstone National Park, home for large herds of free-roaming elk (Cervus elaphus) and bison (Bison bison). Plant and soil N, net N mineralization, and the deposition of ungulate fecal-N were measured at five sites, a ridgetop, mid-slope bench, steep slope, valley-bottom bench, and riparian area, within a watershed from May, 1991 to April, 1992.Results indicated similarities between biogeochemical properties of Yellowstone grassland and other grassland ecosystems: (1) landscape position and soil water affected nutrient dynamics, (2) annual mineralization was positively related to soil N content, and (3) the proportion of soil N mineralized during the year was negatively related to soil C/N.Grazers were a particularly important component of the N budget of this grassland. Estimated rates of N flow from ungulates to the soil ranged from 8.1 to 45.6 kg/ha/yr at the sites (average = 27.0 kg/ha/yr), approximately 4.5 times the amount of N in senescent plants. Rates of nitrogen mineralization for Yellowstone northern range grassland were higher than those measured in other temperate grassland ecosystems, possibly due to grazers promoting N cycling in Yellowstone.     

Dispersion and site fidelity of breeding female grey seals (Halichoerus grypus) on North Rona, Scotland

Female grey seals ( Halichoerus grypus ) formed breeding aggregations on the island of North Rona, Scotland. Aggregations of females were associated particularly with gullies leading from the sea, leaving large areas of available space unoccupied. Changes in the degree of aggregation of females during the breeding season were similar in 1987, 1988 and 1989. Pronounced aggregations occurred in the early and late parts of each breeding season.
Of 67 breeding females marked in 1985, 62 (93%)) returned to N. Rona to breed in at least one season up to 1989, but 18 (27%) were present in all five years. Females came ashore up to 14 days before giving birth and 82% were observed first in the vicinity of their subsequent pupping site. Between 1985 and 1989, marked females which returned were faithful to their previous pupping sites, even when the previous pup had died. There was no evidence of a gradual change in the location of individual pupping sites over time. This pupping site fidelity may generate aggregations whose location, timing and composition is predictable.     

Using patient and general practice characteristics to explain variations in cervical smear uptake rates.

OBJECTIVES--To produce practice and patient variables for general practices from census and family health services authority data, and to determine the importance of these variables in explaining variation in cervical smear uptake rates between practices. DESIGN--Population based study examining variations in cervical smear uptake rates among 126 general practices using routine data. SETTING--Merton, Sutton, and Wandsworth Family Health Services Authority, which covers parts of inner and outer London. MAIN OUTCOME MEASURE--Percentage of women aged 25-64 years registered with a general practitioner who had undergone a cervical smear test during the five and a half years preceding 31 March 1992. RESULTS--Cervical smear uptake rates varied from 16.5% to 94.1%. The estimated percentage of practice population from ethnic minority groups correlated negatively with uptake rates (r = -0.42), as did variables associated with social deprivation such as overcrowding (r = -0.42), not owning a car (r = -0.41), and unemployment (r = -0.40). Percentage of practice population under 5 years of age correlated positively with uptake rate (r = 0.42). Rates were higher in practices with a female partner than in those without (66.6% v 49.1%; difference 17.5% (95% confidence interval 10.5% to 24.5%)), and in computerised than in non-computerised practices (64.5% v 50.5%; 14.0% (6.4% to 21.6%)). Rates were higher in larger practices. In a stepwise multiple regression model that explained 52% of variation, five factors were significant predictors of uptake rates: presence of a female partner; children under 5; overcrowding; number of women aged 35-44 as percentage of all women aged 25-64; change of address in past year. CONCLUSIONS--Over half of variation in cervical smear uptake rates can be explained by patient and practice variables derived from census and family health services authority data; these variables may have a role in explaining variations in performance of general practices and in producing adjusted measures of practice performance. Practices with a female partner had substantially higher uptake rates.     

Vaccination and the population structure of antigenically diverse pathogens that exchange genetic material.

Populations of antigenically diverse pathogens undergoing genetic exchange may be categorized into strains on the basis of a set of principal protective antigens. The extent to which polyvalent vaccines based on these protective antigens can alter the population structure of the pathogen is determined by the degree of cross-protection between strains. In the case where there is no cross-protection, vaccinating against a particular strain will have no effect on the others. As cross-protection increases, the strains containing the antigenic variants included in the vaccine will be diminished in prevalence, and those that do not will increase in prevalence. The rise in prevalence of the latter will become more and more exaggerated as cross-protection increases. However, beyond a critical level of cross-protection, in the absence of vaccination, the steady state of the system is asymmetric in that a certain subset of strains (with non-overlapping repertoires of antigenic variants) will dominate over the others in terms of prevalence. Under these circumstances, a vaccine consisting of the most immunogenic combinations of antigenic variants can cause a dramatic increase in frequency of a subset of rare strains.     

Expression of a cloned gene segment of poliovirus in E. coli: evidence for autocatalytic production of the viral proteinase

The poliovirus polyprotein is proteolytically processed predominantly by a virus-encoded proteinase (P3-7c) that cleaves glutamine-glycine amino acid pairs. The biosynthesis of the viral proteinase, itself a product of glutamine-glycine cleavages, was studied by constructing a bacterial expression plasmid that contained a cloned segment of the poliovirus genome slightly larger than the coding region for P3-7c. The induction of expression of this plasmid in E. coli produced several poliovirus-specific polypeptides. One polypeptide, an unstable protein called 3i, was the product of fortuitous in-phase initiation of translation within the coding region of P3-7c. Three other induced polypeptides were products of proteolytic cleavages, the smallest (polypeptide 3) having the properties (amino-terminal amino acids, carboxy-terminal amino acids, size, antigenicity) of P3-7c. Insertion of a DNA linker into the P3-7c coding region results in the loss of P3-7c-specific glutamine-glycine cleavage activity. We conclude that P3-7c was produced by autocatalytic cleavage.     

Differences Between Lipopolysaccharide Compositions of Plant Pathogenic and Saprophytic Pseudomonas Species

Lipopolysaccharides (LPS) were obtained by washing cells of plant pathogenic and saprophytic Pseudomonas species with saline (fraction 1) and then with saline-EDTA (fraction 2). The cells subsequently were extracted with phenol to yield a third aqueous preparation (fraction 3). Each fraction type contained the LPS components, lipid A, heptose, 2-keto-3-deoxy sugar, and neutral and amino sugars. The neutral sugar compositions of fractions 1, 2, and 3, although similar within a species, differed between the Pseudomonas species. The LPS of two pathovars (pv.) of Pseudomonas syringae had glucose and rhamnose as major components: 13 (±3)% glucose and 87 (±3)% rhamnose for P. syringae pv. pisi and 18 (±5)% glucose and 76 (±2)% rhamnose for P. syringae pv. syringae. Fucose was present in addition to glucose and rhamnose for P. syringae pv. phaseolicola (68 [±8]% rhamnose, 14 [±1]% fucose, and 14 [±5]% glucose) and P. syringae pv. tabaci (24 [±2]% rhamnose, 54 [±3]% fucose, and 17 [±1]% glucose). The LPS from different races of P. syringae pv. pisi and P. syringae pv. phaseolicola could not be distinguished by neutral sugar composition. Three saprophytic species, P. aeruginosa, P. fluorescens, and P. putida, also produced LPS which had different proportions of rhamnose, fucose, and glucose. The LPS from three isolates of P. putida were distinct in possessing a high proportion of amino sugar and containing glucose as the major neutral sugar component (86 to 100%). The LPS fractions from plant pathogenic and saprophytic Pseudomonas species did not elicit browning or phytoalexin production in treated dark red kidney bean cotyledons or red Mexican bean leaves. Rather, chlorosis of the LPS-treated leaf tissue was observed.     

Photosynthetic electron transport in guard cells of diverse species

Guard cells of plants representing 18 species were assayed qualitatively for potential to conduct photosynthetic linear electron transport. These plants included C₃ pteridophytes, C₃ and C₄ monocots, and C₃, C₄, and Crassulacean acid metabolism dicots. By use of a microfluorospectrophotometer, guard cell samples in epidermal peels were isolated optically. Chlorophyll fluorescence was monitored from the onset of excitation light. For guard cells of all these species, fluorescence intensity increased during illumination. When samples were preincubated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, however, there was a more rapid increase in fluorescence. These results indicate that all tested guard cells conduct photosynthetic electron transport through the reaction center of photosystem II.