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.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Plant lectins specific for N-acetyl-β-D-galactosamine

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).     

Comparison of the localization of nucleic acid synthesis during bud formation on leaf fragments and on intact undetached leaves ofBegonia rex Putz.

The budding capacity ofBegonia rex leaf fragments is well known; that of undetached leaves has been shown by us only recently after treating the leaves with 6γγ DMAAP. Benzyladenine is as effective as 6γγ DMAAP in stimulating budding. Lower temperatures (17°, 22–12°, 12°) are also capable of inducing bud formation but only after a small cut has been made in a main vein of the undetached leaf. Root formation can also be provoked on undetached leaves which have a cut in the main leaf vein by higher temperatures (24–22°) or by an IAA treatment. Differences in the first stages of bud formation on leaf fragments and on undetached leaves are observed using histochemical and histoautoradiographic techniques.     

Role of Peroxidase when Hydroxyproline-rich Protein in Plant Cell Walls is increased by Ethylene

Ethylene may control the growth of plant cells by regulating hydroxylation of specific wall proteins.     

Genetic Control of Variable and Constant Regions of Immunoglobulin Heavy Chains

IMMUNOGLOBULIN polypeptide chains consist of two well defined regions designated the “variable region” and the “constant region”. Whereas great diversity exists in amino-acid sequences of variable regions, the constant regions of a given subclass of heavy chains (C_H)^* are essentially invariant in sequence^{1, 2}. Exceptions are the allelic forms, such as the rabbit allotypes A14 and A15^{3, 4}, where a threonine-alanine interchange occurs in the constant region of γ chains (Appella, Chersi, R. G. M. and Dubiski, in preparation). The markers unique to a chains (for example, A14-A15) are closely linked to allotypic markers at the a locus (a1, a2, a3)^{3, 4} which seem to be present on four different Ig heavy chain classes (α, γ, ε, µ)^5–7. These puzzling observations can be explained if the a locus determinants are variable region markers which reflect genetically controlled differences in some relatively constant residues within the V_H region sequences⁷.     

Mitochondrial Ribosomes in HeLa Cells

HeLa cell mitochondria contain 60S ribosomes which seem to consist of subunits of 45S and 35S particles. The 16S and 12S RNA components are coded by mitochondrial DNA.     

Histocompatibility Gene Organization and Mixed Lymphocyte Reaction

TRANSFORMATION of allogenic lymphocytes in mixed cultures depends chiefly on an incompatibility between the lymphocyte donors at the major histocompatibility locus in man (HL-A), mouse (H-2) and rat (H-l)¹. Although the mouse H-2 locus can be divided into several regions each of which controls one or more antigenic specificities² and two or more subloci control HL-A antigens in man³, it is not known whether all parts of the major histocompatibility locus are equally important in eliciting transformation in mixed lymphocyte cultures. We now show that capacity to elicit lymphocyte transformation is different for different parts of the mouse H-2 locus.