The gene of a cytoplasmic 18 S ribosomal RNA (18 S rDNA) of the dicotyledonous plant tomato (Lycopersicon esculentum) cv. Rentita has been cloned, and its complete primary structure has been determined. The tomato 18 S rDNA is 1805 bp long with a G + C content of 49.6%. Its sequence exhibits 94%-96% positional identity when it is colinearly aligned with the previously reported sequences of the 17-18 S rDNAs of the dicot soybean and the monocots maize and rice. A model of the secondary structure of the 18 S rRNA of angiosperms is presented and its genera-specific structural features are compared with a current eukaryotic 18 S rRNA consensus model.
The isolation of the rearranged immunoglobulin genes from a hybridoma cell line, which is a prerequisite for the construction of a recombinant antibody, can easily be achieved by polymerase chain reaction. Here we demonstrate that this method, which was originally described for cloning murine immunoglobulin genes from cDNA, is also applicable for rat genes. We show that the procedure also works with crude cell lysates as starting material, thereby greatly reducing the time required for sample preparation. In addition we have sequenced the nonfunctional heavy chain variable gene of the fusion partner X63Ag8.653, which was readily amplified from our hybridoma cells, and whose sequence has been so far unknown.
A chromatographic procedure for the size fractionation of DNA fragments ranging from 20 to 30000 base pairs within a single chromatographic run is described. The procedure was developed from a method communicated about two years ago [Muller, W. et al. (1979) Nucleic Acids Res. 7, 2483 -24991 which applies the aqueous two-phase system formed by poly(ethy1ene glycol) and dextran using cellulose as support for the lower, dextranrich stationary phase. The parameters found to exert a marked influence on the resolution and the capacity of a column include the ratio of mobile phase to bound phase in the column (phase ratio), the temperature, and the ionic strength in the phases. Concerning the support, no better material than microgranular cellulose has yet been found. A slight preference of dA+dT-rich fragments for the mobile phase manifests itself by elution anomalies comparable in extent to the migration anomalies of the same fragments in polyacrylamide gels. The resolution observed approaches that of polyacrylamide and agarose gels, but the capacity is 200-500 times higher.The DNA fragments passed through the fractionation procedure seem to be fully susceptible to the enzymatic restriction and modification reactions.The separation of DNA fragments of the size range covered by the common restriction fragments still presents considerable problems if larger amounts (5 -100 mg) of material are to be fractionated. No satisfactory chromatographic procedure of sufficient resolving power for fragments larger than 350 base pairs exists, a fact which seems to hold for all neutral or charged linear polymers of comparable molecular mass. The electrophoretic procedures for DNA suffer from the fact that there is no gel matrix which allows one to separate fragments ranging from 20 base pairs to the size of the I genome, i.e. 50000 base pairs, in a single gel. The practical need for such fractionation methods might thus evoke more than mere academic interest in their development.The procedure to be presented results from the further development of a chromatographic procedure reported by us about two years ago [l]. It is based on the aqueous two-phase system formed by poly(ethy1ene glycol) (PEG) and dextran [2], using the dextran-rich lower phase as the stationary phase while the PEG-rich upper phase forms the mobile one. Cellulose or Celite were used as supports for the stationary phase which was produced as a thin film on the support particles during the coating process. The resolution of such columns was limited with respect to the size range of DNA. Fragments smaller than 160 base pairs migrated with the front while fragments above 3000 base pairs were barely resolved.In our first communication we pointed out [l] that the system had not been studied sufficiently with respect to all parameters which may influence the resolution. The present 1.23.17), Hindlll (EC 3.1.23.21), BumHI (EC 3.1.23.6).paper will show that much better resolution can be achieved in the size range for small as well as for large fragments if the phase rat...
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