Mechanism of mutation at the aprt locus in Chinese hamster ovary cells: analysis of heterozygotes and hemizygotes.

A two-step model to explain the high frequency of mutation at the diploid adenine phosphoribosyltransferase (aprt) locus in CHO cells has been proposed previously (Simon et al., Mol. Cell. Biol. 2:1126-1133, 1982). This model indicates that two distinct classes of aprt heterozygotes can be isolated. Class 1 heterozygotes, the most abundant class, were defined as those which arose spontaneously and were capable of undergoing mutation to the APRT- phenotype only at a low frequency (putative point mutation). Class 2 heterozygotes arose from a mutation and gave rise at a high frequency to APRT- cells. This high-frequency event has been identified as a deletion of the wild-type allele (A. E. Simon and M. W. Taylor, Proc. Natl. Acad. Sci. U.S.A. 80:810-814, 1983). In this paper we report further analysis of class 1 heterozygotes with respect to genetic structure, gene products, and karyotype. Our study indicated that class 1 heterozygotes contain two different types of mutants. About half have only one copy of the aprt gene and an unaltered karyotype, indicating that a deletion (similar to the high-frequency second-step event observed for class 2 heterozygotes) rather than a loss of the chromosome was responsible for the generation of the aprt+/- genotype. The remainder of the previously designated class 1 heterozygotes still contained two copies of the aprt gene (within the limits of the quantitation technique used) and arose presumably by a point mutation. One of this group, D423, was characterized with respect to aprt gene products and found to produce an electrophoretic variant in addition to the wild-type protein. APRT- mutants derived from D423 retained the same number of aprt gene copies as D423 and still synthesized a protein that comigrated with wild type, unlike APRT- mutants derived from class 2 heterozygotes. D423 and the other heterozygotes with two aprt genes therefore did not fit into either class 1 or 2 and are now designated class 3. The model we present suggests that only one of the two aprt alleles present in wild-type cells can undergo the deletion.     

Legumin Synthesis in Developing Cotyledons of Vicia faba L

The synthesis of legumin in developing cotyledons of Vicia faba L. has been examined as a potential system for approaching the problem of differential gene expression. The pattern of legumin synthesis was determined during the growth of the cotyledon by microcomplement fixation which provided a sensitive and specific assay for legumin in the presence of vicilin. Legumin was detected even in young cotyledons. However, when the cotyledons were about 10 millimeters long, and cell division was essentially complete, there was a sharp increase in the rate of legumin accumulation.     

Purification and Thermal Stability of Intact Bacillus subtilis Flagella

Flagella were prepared and purified in a relatively intact form from bacterial lysates. Immunochemical tests showed that over 95% of the protein in the final preparation consisted of flagellar antigen. These flagella are more stable to thermal denaturation than flagella filaments obtained by shearing. Their thermal properties more closely resemble those of flagella in the native state on bacteria. The presence of the hook structure is responsible for this extra stability.     

The in vitro synthesis and characteristics of ribosomal RNA in imaginal discs of Drosophila melanogaster

Summary Late third instar imaginal discs of Drosophila melanogaster cultured in vitro in Robb's tissue culture medium synthesize 38S, 28S and 18S ribosomal RNAs which are qualitatively indistinguishable from their in vivo synthesized counterparts (Fig. 1). As found in other insect systems, the 38S molecule appears to be the precursor for both the 28S and 18S rRNAs (Figs. 2, 3 and 4). The 28S rRNA and a portion of the 38S pre-rRNA shift in sedimentation value upon exposure to heat or dimethylsulfoxide (Figs. 5 and 8). Studies of the thermal denaturations of these molecules (Figs. 6, 7 and 9) indicate the existence of a single class of 28S rRNA, but three classes of 38S pre-rRNAs. The addition of -ecdysone to the in vitro culture medium stimulates the net amount of rRNA synthesized, increases the rate of processing of the 38S precursor and increases the relative amount of 18S material produced (Figs. 10 and 12).This work was supported in part by grants from the National Science Foundation (GB-8176) and from the Atomic Energy Commission (AT-04-3-34).Predoctoral Trainees, PHS Training Grant No. 2-Tl-GM367 from Research Training Grants Branch, National Institute of General Medical Sciences.1 For purposes of simplification we shall refer to the rRNA molecules of D. melanogaster as being 38S, 30S, 28S and 18S; however, it should be noted that these values are approximate (see Hastings and Kirby, 1966; Greenberg, 1969; Tartof and Perry, 1970).     

Conversion of exogenous insulin into high molecular weight forms in vivo

[¹²⁵I]-insulin, injected in rats, was converted into high molecular weight forms as judged by gel filtration of blood serum samples collected at various intervals. These forms represented 26% (10 min. after injection) to 81% (240 min. after injection) of the total immunoprecipitable radioactivity. Their molecular weights were not affected by rechromatography in 0.1 M borate buffer (pH 8) or in 8 M urea-1 M acetic acid (pH 2.4). On incubation of [¹²⁵I]-insulin with blood serum $\text{in}\text{vitro}$, no high molecular weight forms could be observed.     

Synthesis of Bacterial Flagella: Chromosomal Synchrony and Flagella Synthesis

Synchronous cultures of Bacillus subtilis 168 M were obtained from light-density spores germinated at 46 C and grown at 37 C. This procedure synchronizes both cell division and chromosome replication. The chromosome synchrony was demonstrated by using transformation to measure changes in marker frequency during the cell cycle. The synthesis of two enzymes and of bacterial flagellar protein was also followed. All of the proteins were found to be synthesized continuously with an abrupt doubling in the rate of synthesis at a specific time in the cell cycle. The time at which the doubling occurred for each enzyme corresponded to the time at which the structural gene for the enzyme was replicated. The doubling of the rate of flagella synthesis corresponded to the time of replication of the hisA1 gene. We conclude that the genetic locus for the factors involved in the rate-limiting steps in flagella synthesis are located on the genetic map near the hisA1 locus.