An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model. 相似文献
Summary The transformation of streptomycin resistance in Rhizobium japonicum was studied. The susceptible strain 211 was selected from sixty strains and one step mutant resistant to streptomycin in concentration 1 mg per 1 ml was used as the donor. The peak of the competence curve appeared at the ninth hour of growth; the frequency, when the homologous strain had been used was 0.01 p.c. The transformed resistance was of the same level as in the donor strain.This investigation forms part of a contribution prepared by the Czechoslovak National Committee for the International Biological Programme (Section PP: Production Processes). 相似文献
A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles. 相似文献
The R1 allele confers on potato a race-specific resistance to Phytophthora infestans. The corresponding genetic locus maps on chromosome V in a region in which several other resistance genes are also located. As part of a strategy for cloning R1, a high-resolution genetic map was constructed for the segment of chromosome V that is bordered by the RFLP loci GP21 and GP179 and includes the R1 locus. Bulked segregant analysis and markers based on amplified fragment length polymorphisms (AFLP markers) were used to select molecular markers closely linked to R1. Twenty-nine of approximately 3200 informative AFLP loci displayed linkage to the R1 locus. Based on the genotypic analysis of 461 gametes, eight loci mapped within the GP21–GP179 interval. Two of those could not be seperated from R1 by recombination. For genotyping large numbers of plants with respect to the flanking markers GP21 and GP179 PCR based assays were also developed which allowed marker-assisted selection of plants with genotypes Rr and rr and of recombinant plants. 相似文献
Summary Monocalcium phosphate (MCP), the salt of concentrated superphosphate, applied to a Buganda soil increased the amount of manganese taken up by ryegrass. To investigate the cause of this effect the derivatives of MCP hydrolysis, dicalcium phosphate (DCP) and triple-point solution (TPS) were separated and applied independently. Both derivatives, and a synthetic dicalcium phosphate dihydrate, increased the concentration of manganese in ryegrass showing that dissolution of soil manganese by TPS (pH 1.48) was not a unique cause.DCP derived by hydrolysis of MCP supplied little phosphorus to early crops of ryegrass but a larger proportion of its P was taken up by later crops; TPS supplied more of its P to earlier than to later crops. During 42 weeks the proportions of P taken up from DCP and TPS were similar to the proportions of phosphorus in these forms when MCP hydrolyzes.Phosphorus in DCP derived by hydrolysis of MCP was more available to ryegrass than phosphorus in a synthetic dicalcium phosphate dihydrate, showing that results may be misleading when synthetic materials are used to simulate the compounds that form from fertilizers in soil. re]19760401 相似文献
Summary A cytophotometric study of DNA content in Purkinje cells of the cerebellum of rats, cats, chicken and humans (Feulgen staining) revealed that in a certain number of cells the amount of DNA ranged between the diploid and tetraploid level (H2C cells). The incidence of H2C Purkinje cells varied among the species studied. In rats, which were studied most thoroughly, these cells amounted on average to 3%. In some rats, as well as in some cats and chickens H2C Purkinje cells were entirely absent. In the group of animals possesing H2C Purkinje cells, great interindividual differences were observed. In rats for instance, the incidence of these cells varied from 1 to 23 per cent. Topographic analyses carried out in rat and human cerebellum revealed that H2C Purkinje cells occurred more frequently in the hemispheres than in the vermis. No significant differences were found in the number of H2C Purkinje cells in healthy and Kilham-DNA-virus infected rats.Densitometric analysis of the distribution of nuclear chromatin showed that H2C Purkinje cells were richer in condensed chromatin, especially in the region of the nucleolus, which apparently contains the hyperploid surplus of DNA. It is proposed that the phenomenon of DNA hyperdiploidy arises as a result of either incomplete S-phase in some immature Purkinje cell precursors or the amplification of some DNA sequences particularly those localized in the nucleolar region. 相似文献
Summary Intact and denervated extensor digitorum longus (EDL) muscles of 20-day-old inbred Lewis-Wistar rats were labelled with 3H-thymidine. Ninety minutes after the injection of the isotope 4.0% of the nuclei were labelled in the intact (i.e. innervated) and 9.6% in the muscles, denervated 3 days before administration of the isotope. The labelled EDL muscles were grafted into the bed of the previously removed EDL muscles of inbred animals and these isografts were studied 30 days later.In the EDL muscles, regenerated from innervated isografts only occasionally labelled endothelial cells were found whereas in the muscles regenerated from denervated isografts also parenchymal muscle nuclei were regularly labelled. The incidence of labelled nuclei in the regenerated EDL muscles was, however, about 20 times lower than in the donor EDL muscles. The present experiments provide a direct proof of utilization of donor satellite cell nuclei for regeneration in grafted muscle tissue. With respect to the low incidence of labelled nuclei in regenerated EDL muscles, other sources of cells apparently also contribute to the regeneration process. 相似文献
Protecting habitats for charismatic vertebrates can provide an ‘umbrella’ for less conspicuous organisms, especially when these are threatened by the same processes. However, such a conservation scheme is vulnerable to the extirpation of the focal species. We studied wider biodiversity values in long protected black stork (Ciconia nigra) nest sites, which were abandoned by the bird and thus legally subject to de-listing. In 20 abandoned nest sites in Estonia, we (i) mapped breeding birds within 600 m from the stork nest, and (ii) carried out time-limited surveys of lichens, polypore fungi, vascular plants and bryophytes in 2-ha plots. The breeding bird assemblages (64 species recorded) included 19 red-listed species, and showed no clear aggregation to the immediate surroundings of the stork nest. We recorded 740 plant and fungal species, of which 134 (18%) were of conservation concern (nationally protected, red-listed or extremely rare). Across the 2-ha plots, the numbers of the species of conservation concern varied more than three-fold (maximum 42 species), being affected notably by dead wood accumulation over time and presence of nemoral broad-leaved trees. The results demonstrate that many abandoned nest sites of the black stork have broader biodiversity significance, both due to the bird’s habitat requirements and the natural development during the protection. Expanding the umbrella function to sites abandoned by a focal species, but intact from anthropogenic degradation, can thus be a cost-effective conservation approach due to its low additional administrative burden. In most jurisdictions, the assessment procedure for such situations should be formalized, however.