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G Temple

Publications and source records attributed to G Temple.

6 recordsLinked to original sources

Directional cDNA library construction assisted by the in vitro recombination reaction.

We report here a new directional cDNA library construction method using an in vitro site-specific recombination reaction, based on the integrase-excisionase system of bacteriophage lambda. Preliminary experiments revealed that in vitro recombinational cloning (RC) provided important advantages over conventional ligation-assisted cloning: it eliminated restriction digestion for directional cloning, generated low levels of chimeric clones, reduced size bias and, in our hands, gave a higher cloning efficiency than conventional ligation reactions. In a cDNA cloning experiment using an in vitro synthesized long poly(A)(+) RNA (7.8 kb), the RC gave a higher full-length cDNA clone content and about 10 times more transformants than conventional ligation-assisted cloning. Furthermore, characterization of rat brain cDNA clones yielded by the RC method showed that the frequency of cDNA clones >2 kb having internal NotI sites was approximately 6%, whereas these cDNAs could not be cloned at all or could be isolated only in a truncated form by conventional methods. Taken together, these results indicate that the RC method makes it possible to prepare cDNA libraries better representing the entire population of cDNAs, without sacrificing the simplicity of current conventional ligation-assisted methods.

Animals↗

Use of phage antibodies to distinguish closely related species of protozoan parasites.

Acanthamoeba are typically identified in the laboratory using culture and microscopic observation. In this paper we describe the isolation and specificity of antibody fragments that can be used for the identification of Acanthamoeba. A phage library expressing a large repertoire (approx. 5 x 10(9)) of antibody fragments was used to generate two libraries one enriched for bacteriophage that exhibit genus specific binding and the other containing bacteriophage that bind specifically to pathogenic Acanthamoeba. Individual clones were isolated on the basis of binding by ELISA, and then flow cytometry and immunofluorescence were used for further characterisation. Four monoclonal antibodies were isolated, specific for Acanthamoeba at the generic level with clone HPPG6 exhibiting the highest level of binding. Furthermore clone HPPG55 was specific for pathogenic species of Acanthamoeba.

Animals↗

Testing hypotheses concerning the phenotypic plasticity of escape performance in fish of the family Cottidae.

A knowledge of the natural history of two species of marine Cottidae was used to construct a set of complex a priori hypotheses to test the idea that seasonal temperature acclimation conferred a fitness advantage and to examine whether acclimation responses were constant through development. The species studied were the short-horn sculpin (Myoxocephalus scorpius L.) and the long-spined sea scorpion (Taurulus bubalis Euphr.). Fast-start escape performance was measured as a proxy of fitness, and maximum body-length-specific speed (Umax) and acceleration (Amax), and angular velocity (max) and cumulative turning angle (CTA) were quantified. Fish were acclimated to 5, 15 and 20 degreesC and filmed using high-speed cinematography at 0.8, 5.0, 15.0 and 20.0 degreesC. The following hypotheses were tested: (1) improvements in escape performance following acclimation to high temperature in adult short-horn sculpin that live offshore would involve a trade-off in performance at low temperature, (2) the escape response of the sea scorpion, which experiences large daily temperature variation, would exhibit low thermal sensitivity and be unchanged by acclimation between 5 and 15 degreesC, and (3) in short-horn sculpin, the ability to acclimate maximum speed thermally is acquired during ontogeny in parallel with the offshore migration of the late juvenile stage. At 20.0 degreesC, in adult short-horn sculpin, Umax and Amax were 110 % and 55 % higher, respectively, in 15 degreesC- than in 5 degreesC-acclimated fish. No evidence was obtained for improved fast-start performance at 0.8 degreesC or 5.0 degreesC following cold acclimation. In the long-spined sea scorpion, acclimation to 5 and 15 degreesC did not improve Umax or Amax compared with fish acutely exposed to these temperatures, although acclimation to 5 degreesC increased max (P=0.005). When tested over the most extreme thermal range found in the field, all variables were improved at a test temperature of 0.8 degreesC in 5 degreesC- compared with 15 degreesC-acclimated sea scorpion. Acclimation therefore appeared to be beneficial in some instances in both species. How this affects relative fitness is uncertain. The scaling of Umax with acclimation to 5 and 15 degreesC was examined in both species over the test range 5.0-15.0 degreesC. Temperature acclimation did not affect scaling relationships of Umax in long-spined sea scorpion ranging in total body length (L) from 45 to 160 mm. At a test temperature of 15.0 degreesC, the scaling of Umax for short-horn sculpin ranging in total body length from 43 to 270 mm changed from aL-0.98 in 5 degreesC-acclimated fish to aL-0.50 (where a is the proportionality coefficient in the regression equation) in 15 degreesC-acclimated fish (P<0.01). In short-horn sculpin, therefore, the ability to modify escape performance with temperature acclimation was found to vary during ontogeny, potentially paralleling a migration from a more variable to a more stable thermal environment.

Journal Article↗

A comparison study of human papillomavirus prevalence by the polymerase chain reaction in low risk women and in a gynaecology referral group at elevated risk for cervical cancer.

The reported prevalence of human papillomavirus (HPV) type 16 in the genital tracts of women with various gynaecological conditions is highly variable. In particular, some results with the polymerase chain reaction (PCR) technique have suggested that HPV-16 is a ubiquitous or very common virus. We undertook this study to help clarify the current confusion. PCR with HPV consensus L1 primers and specific E6 primers was used to study 89 women attending two gynaecology referral clinics, as well as 99 women attending a health maintenance organization (HMO) clinic; 70 of these latter women had no current or prior history of genital HPV disease. HPV-16 was detected in less than 5% of cytologically normal women from either group and in 17% (6/36) and 31% (9/29) of women with cervical intraepithelial neoplasia (CIN) from the referral clinic and the HMO, respectively. The other high-risk or intermediate-risk HPVs (types 18, 31, 33 or 35) were less prevalent than HPV 16 in all groups of women. A majority of the HPV types detected by the L1 primers in normal women were uncharacterized HPVs. Overall these uncharacterized HPVs were detected in 37% (46/123) of the normal women and in 48% (31/65) of the women with CIN. Using the most sensitive PCR product detection method employed in the study, HPV DNA was detected in 36% (4/11) of swab specimens obtained from the external abdomen.

Base Sequence↗

Sexually transmitted papillomaviral infections. I. The anatomic distribution and pathologic grade of neoplastic lesions associated with different viral types.

Multiple colposcopic biopsy specimens were collected from 160 women, with sampling of principal cervical and vulvar lesions as well as secondary areas of either minor acetowhitening or normal epithelium. Papillomaviral deoxyribonucleic acid was detected by Southern blot hybridization in 197 (90%) of the 218 principal biopsy specimens and 93 (46%) of 198 secondary biopsy specimens. Although different papillomaviruses were found at different sites in 31 women, only six of 416 specimens contained multiple types within the same sample. Specific viral types were associated with specific disease patterns. Only one of 80 type 6 or 11 infections had a diagnosis greater than cervical intraepithelial neoplasia, grade 2. In contrast, 42 of 48 (90%) biopsy specimens of cervical intraepithelial neoplasia, grade 3, or invasive cancer contained type 16, 18, or 31. Nonetheless, 12 of 124 (10%) cases of condyloma and cervical intraepithelial neoplasia, grade 1, were associated with types 16, 18, and 31 infections. Of 58 women with multicentric disease, 46 had positive hybridizations for both cervical and vulvar lesions (32 showing the same type in both samples and 14 showing different viruses). Differing patterns of papillomavirus-induced disease arise partly from the predilection of specific viral types for certain anatomic sites and partly through variations in host response. Detection of viral deoxyribonucleic acid in 46% of the secondary biopsy specimens suggests that disease expression may represent focal breakdown of host surveillance within a field of latent papillomaviral infection.

Biopsy↗