Molecular cloning of cDNA for rat L-type pyruvate kinase and aldolase B

Two double-stranded cDNA recombinant pBR322 plasmid libraries were constructed starting from high carbohydrate diet rat liver poly(A)+ mRNA, either fractionated by denaturing sucrose gradient centrifugation for the cloning of L-type pyruvate kinase cDNA, or nonfractionated for aldolase B. Both libraries were screened with single-stranded cDNA probes reverse transcribed from fasted or high carbohydrate diet rat liver mRNAs. mRNAs from fasted animals were also fractionated by sucrose gradient centrifugation and mRNAs from the fed animals were, in addition, further purified by high performance liquid gel filtration chromatography. Those clones hybridizing with the "positive" probe (from animals fed the high carbohydrate diet) and not with the "negative" one (from fasted animals) were preselected and their plasmid DNA was purified and analyzed by positive hybridization-selection. Thirty of 4500 bacteria colonies transformed by recombinant plasmids were preselected by differential screening for pyruvate kinase, and 8 of 864 colonies for aldolase B. Twenty-two recombinant plasmids for pyruvate kinase and two for aldolase B were shown to contain specific cDNA inserts by positive hybridization-selection. Plasmids DNAs of some pyruvate kinase and aldolase B clones (whose inserts ranged from 700 to 1050 bases in length) were labeled by nick translation and used as probes for Northern blot hybridization. The pyruvate kinase cDNA probes recognized mainly a 3400-base RNA species which was detected in high carbohydrate diet rat liver, but not in fasted rat liver and in tissues which do not synthesize L-type pyruvate kinase. In addition, some pyruvate kinase probes hybridized with minor RNA species of about 2000 bases in length, only observed after carbohydrate diet. For aldolase B, the recombinant plasmid DNA hybridized with a single RNA species of 1750 bases. This RNA, detected in kidney, small intestine and liver, was induced by a high carbohydrate diet and increased with liver development. The rat probe cross-hybridized with human aldolase B messenger RNA.     

A multicomponent analysis of amino acid transport systems in human lymphocytes. 1. Kinetic parameters of the A and L systems and pathways of uptake of naturally occurring amino acids in blood lymphocytes

We have determined the kinetic parameters of natural and system-specific synthetic amino acid transport by human blood lymphocytes, using a multi-component computer analysis that separates carrier-mediated uptake from diffusion. These studies were initiated in order to provide the basis for studies of human blood T and B lymphocytes and malignant lymphocytes. Methylaminoisobutyric acid (methyl-AIB) and 2-amino-2-carboxy-bicyclo (2,2,1) heptane (BCH) uptakes into lymphocytes were measured as prototypes of A- and L-system amino acid transport. The Michaelis constant for methyl-AIB uptake was 540 microM; the maximal velocity of uptake was 28 mumol/L cell water/min, and the diffusion coefficient was .004 min-1. In contrast, the Michaelis constant for BCH uptake was 63 microM; the maximal velocity was 969 mumol/L cell water/min, and the diffusion coefficient was .141 min-1. The transport of the naturally occurring amino acids, alanine, proline, and leucine was defined by studies of: (1) competitive inhibition with the system-specific synthetic amino acids, methyl-AIB and BCH, (2) the effect of the transcellular sodium gradient on transport, and (3) evaluation of the time-dependent increase of transport in amino acid-deficient medium (adaptation). Alanine was transported principally (approximately 70%) by the ASC-system, and leucine was transported principally (70%) by the L-system in lymphocytes. The analysis of proline transport was more complex because of a large component of uptake by diffusion even at low amino acid concentrations. Taken together, the kinetics of sodium-sensitive uptake and the results of competitive inhibition studies indicated that proline was transported by the A-system (30%), the ASC system (30%), and also by the L-system (15%).     

Interstitial fluid pressure changes in pregnant rats

In pregnant rats significant interstitial fluid pressure changes could be detected by means of capsules chronically implanted into the subcutaneous tissue. The capsular pressure increased significantly from a control value of -4.3 +/- 0.5 mmHg to -0.7 +/- 0.5 mmHg during the first period of pregnancy. Immediately before parturition the capsular pressure returned to the control level. During lactation the pressure rose as high as + 0.5 +/- 0.9 mmHg. After lactation the pressure returned again to the control value. By determining the extracellular fluid and plasma volume, as well as protein concentration in plasma and capsular fluid, the hydrostatic and colloid osmotic forces operating in the extracellular space could be analysed. It has been concluded that the observed capsular pressure changes during pregnancy are not solely of volumetric or colloid osmotic origin.     

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.     

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.