PCR amplification of murine immunoglobulin germline V genes: strategies for minimization of recombination artefacts

Murine immunoglobulin germline V genes exist as multiple sequences arranged in tandem in germline DNA. Because members of V gene families are very similar, they can be amplified simultaneously using the polymerase chain reaction (PCR) with a single set of primers designed over regions of sequence similarity. In the present paper, the variables relevant to production of artefacts by recombination between different germline sequences during amplification are investigated. Pfu or Taq DNA polymerases were used to amplify from various DNA template mixtures with varying numbers of amplification cycles. Pfu generated a higher percentage of recombination artefacts than Taq. The number of artefacts and their complexity increased with the number of amplification cycles, becoming a high proportion of the total number of PCR products once the 'plateau phase' of the reaction was reached. Recombination events were located throughout the approximately 1-kb product, with no preferred sites of cross-over. By using the minimally detectable PCR bands (produced by the minimum number of amplification cycles), recombination artefacts can be virtually eliminated from PCR amplifications involving mixtures of very similar sequences. This information is relevant to all studies involving PCR amplification of members of highly homologous multigene families of cellular or viral origin.     

The comparative anatomical study of the parietal region of the skull of the Korean native goat (Capra hircus)

In the skull of the Korean native goat, the parietal region was classified into four types by the degree of the fusion of the bones, the os interparietale, the os parietale and the squama occipitalis of the os occipitale, and the structural variations of these fusions. The fusion appeared first in the sutura interparietoparietalis and that of the sutura sagittalis of both ossa parietalia was followed. There was no fusion between the os parietale and the squama occipitalis of the os occipitale. These results suggest that the os interparietale developed independently but fused to the os parietale after birth, and the os parietale were developed as paired bones in prenatal life and then fused together according to age.     

Met-enkephalin-Arg6-Phe7 in spinal cord and brain following traumatic injury to the spinal cord: influence of p-chlorophenylalanine. An experimental study in the rat using radioimmunoassay technique

The possibility that trauma to the dorsal horn may affect the release and distribution of enkephalin was examined using the opioid peptide Met-Enk-Arg6-Phe7 (MEAP) as a marker in a rat model. The peptide content of samples of spinal cord and whole brain was measured using a radioimmunoassay (RIA) technique. In addition, the possible functional relation between this peptide and serotonin was evaluated using a pharmacological approach that included depletion of endogenous serotonin. A focal trauma to the right dorsal horn in the T10-11 segments (2 mm deep and 5 mm long) markedly modified the content of MEAP of the adjacent rostral and caudal segments of the cord, as well as the content of MEAP of the brain. Depletion of serotonin with p-CPA (an inhibitor of the synthesis of serotonin) significantly elevated the content of MEAP in the whole brain without affecting the regions of the spinal cord (except T9 level which showed a 25% decrease from an intact control group). Trauma to the spinal cord in the serotonin-depleted animals did not alter the content of MEAP further, as compared to a p-CPA-treated but untraumatized group. These results indicate that enkephalin (i) participates in the pathophysiology of spinal cord trauma and (ii) suggest that the peptide is somehow functionally related with serotonin.