Search PubMed⌕ Search

Biomedical subjects

P Humphries

Publications and source records attributed to P Humphries.

99 records · Page 6Linked to original sources
PubMed1978

Recombinant plasmids containing Xenopus laevis globin structural genes derived from complementary DNA.

Details are presented of the in vitro synthesis of double-stranded DNA complementary to purified Xenopus globin messenger RNA, using a combination of reverse transcriptase, fragment 'A' of E. coli DNA polymerase 1 and S1 endonuclease. After selection of duplex DNA molecules approaching the length of Xenopus globin messenger RNA by sedimentation of the DNA through neutral sucrose gradients, the 3'-OH termini of the synthetic globin gene sequences were extended with short tracts of oligo dGMP using terminal transferase. This material was integrated into oligo dCMP-extended linear pCR1 plasmid DNA and amplified by transfection of E. coli. Plasmids carrying globin sequences were identified by hybridization of 32P-labelled globin mRNA to total cellular DNA in situ, by hybridization of purified plasmids to globin cDNA in solution, by analysis of recombinant DNA on polyacrylamide and agarose gels, and by heteroduplex mapping. The results show that extensive DNA copies of Xenopus globin mRNA have been integrated into recombinant plasmids.

Animals↗
PubMed1977

Molecular cloning of extensive sequences of the in vitro synthesized chicken ovalbumin structural gene.

Double-stranded DNA molecules complementary to ovalbumin chicken messenger RNA were synthesized in vitro and integrated into the E. coli plasmid pCR1 using an oligodG-dc tailing procedure. The resultant hybrid plasmids, amplified by transfection of E. coli, were shown by hybridization and gel electrophoresis to contain extensive DNA sequences of the ovalbumin structural gene.

Animals↗
PubMedDate not supplied

Comparative structural and functional analysis of photoreceptor neurons of Rho-/- mice reveal increased survival on C57BL/6J in comparison to 129Sv genetic background.

To explore the possible influence of defined genetic backgrounds on photoreceptor viability and function in mice carrying a targeted disruption of the rhodopsin gene, the severities of retinopathies in Rho-/- mice on C57BL/6J and 129Sv congenic backgrounds were compared by light microscopy and electroretinography and qualitatively by in situ end labeling of DNA in apoptotic photoreceptor nuclei of retinal sections. Cone photoreceptor viability and function were shown to deteriorate more slowly on the C57BL/6J background in comparison to that of the 129Sv, with significantly greater numbers of outer nuclear layer nuclei in the retinas of C57BL/6J mice at 3 and 4 months of age. Both amplitude and waveform features of the ERG were shown to be remarkably different in the two strains, indicating an approximately 6-fold difference in C57BL/6J Rho-/- mice compared to 129Sv Rho-/- mice at 80 days. Thus, in comparison with the 129Sv strain, genetic modifiers appear to constitute a component of the C57BL/6J background, the expression of which significantly protects cone photoreceptors from apoptotic death in a mutation-induced murine retinopathy. The differences in phenotype revealed in this study are sufficient in principle to provide a basis for comparisons to be made between QTLs in light-induced and mutation-induced systems.

Aging↗
PubMedDate not supplied

The electroretinogram of the rhodopsin knockout mouse.

The electroretinogram (ERG) of the rhodopsin knockout (rho-/-) mouse of Humphries et al. (1997) (Humphries et al., 1997) was studied for evidence of light-evoked rod activity and to describe the cone function. The rho-/- retina develops normal numbers of rod and cone nuclei, but the rods have no outer segments, and no rhodopsin is found by immunohistochemistry. The dark-adapted ERG threshold was elevated 4.7 log units above wild-type (WT) control mice, indicating that any residual rod responses were reduced >50,000-fold, consistent with a complete functional knockout. The dark-adapted rho-/- ERG had a cone waveform, and the spectral sensitivity peaked near 510 nm for both dark-adapted and light-adapted conditions, without evidence of a Purkinje shift. The light-adapted ERG b-wave amplitude of young rho-/- mice was the same as WT. The amplitude remained steady up to postnatal day P47, but thereafter it declined to only 1-2% by P80 when no cone outer segments remained. Cone b-wave threshold of dark-adapted rho-/- mice was -1.07 +/- 0.39 log cd-s/m2 (n = 17), which is 1.27 log units more sensitive than light-adapted thresholds against a rod-suppressing Ganzfeld background of 1.61 log scotopic cd/m2. This indicates that dark-adapted WT responses to still dimmer stimuli are exclusively rod driven with minimal cone intrusion. Above this cone threshold intensity, the dark-adapted b-wave of WT will be a summation of rod and cone responses. Threshold versus intensity (TVI) studies gave no evidence of a rod influence on the mouse cone b-wave.

Animals↗