Alcaligenes eutrophus hydrogenase genes (Hox)

Mutants of Alcaligenes eutrophus H16 lacking catalytically active soluble hydrogenase (Hos-) grew very slowly lithoautotrophically with hydrogen. Mutants devoid of particulate hydrogenase activity (Hop-) were not affected in growth with hydrogen. The use of Hos- and Hop- mutants as donors of hydrogen-oxidizing ability in crosses with plasmid-free recipients impaired in both hydrogenases (Hox-) resulted in transconjugants which had inherited the plasmid and the phenotype of the donor. This indicates that the structural genes which code for the hydrogenases reside on plasmid pHG1. The Hox function of one class of Hox- mutants could not be restored by conjugation. These mutants exhibited a pleiotropic phenotype since they were unable to grow with hydrogen and also failed to grow heterotrophically with nitrate (Hox- Nit-). Nitrate was scarcely utilized as electron acceptor or as nitrogen source. Hox- Nit- mutants did not act as recipients but could act as donors of the Hox character. Transconjugants derived from those crosses were Hox+ Nit+, indicating that the mutation which leads to the Hox- Nit- phenotype maps on the chromosome. Apparently, the product of a chromosomal gene is involved in the expression of plasmid-encoded Hox genes. We observed that the elimination of plasmid pHG1 coincided with the occurrence of multiple resistances to various antibiotics. Since Hox+ transconjugate retained the antibiotic-resistant phenotype, we conclude that this property is not directly plasmid associated.     

A general method for visualizing enzymes releasing adenosine or adenosine-5′-monophosphate

Biochemical Genetics -     

Genetic analysis of clear-plaque mutations induced in bacteriophage lambda by 9-aminoacridine

Clear-plaque mutations were induced in the cI and cII genes of λ by treating lysogenic cells with 9-aminoacridine (9AA). Mapping of the mutations revealed that there were two hot spots for 9AA mutagenesis in cI, and one strong hot spot in cII. The hot spots in cI mapped close to 1 of the 3 runs of 4 G/C base-pairs and near the only run of 5 G/Cs, respectively, in this gene. Of 36 cI mutations tested, at most one mapped near a run of 6 A/T base-pairs. By analogy, the sequence responsible for the strong hot spot in cII may be the run of 6 G/Cs in this gene.     

Molecular analysis of the envelope gene and long terminal repeat of Friend mink cell focus-inducing virus: implications for the functions of these sequences.

We sequenced the envelope (env) gene and 3' long terminal repeat of a Friend mink cell focus-inducing virus (F-MCFV). We also sequenced the gp70 coding regions for two cDNA clones of another F-MCFV. The deduced amino acid sequence of the env gene products of both F-MCFVs were compared to the corresponding sequences of other MCFVs and of ecotropic viruses. The env polypeptides of the different viruses showed long stretches of homology in the carboxy-terminal half of gp70 and in p15env ("constant region"). The amino-terminal half of gp70 was very similar in all MCFVs, but showed extensive variations relative to the ecotropic viruses ("differential region"). This differential region in all MCFVs is of endogeneous origin. We show evidence that this region carries determinants for ecotropic or polytropic host range. No indication could be found that the env gene products determine the histological type of disease caused by particular MCFVs. When the long terminal repeats of F-MCFV and Friend murine leukemia virus were compared with those of other viruses causing either lymphatic leukemia or erythroleukemia, several nucleotides were localized which might determine the histological type of disease caused by these viruses.     

Hypoxia-induced alterations of norepinephrine vascular reactivity in isolated perfused cat lung

Past work in the isolated perfused cat lung has shown that acute hypoxia (H) changes the response to norepinephrine (NE) from vasoconstriction to vasodilation but has no effect on the response to serotonin (S). These results could be related to the increase in pulmonary arterial pressure or vascular resistance during the hypoxic pressor response or a direct effect of H. We addressed this question, in the same preparation, by comparing responses to NE under four conditions in each experimental animal (n = 12): 1) NE infused during normoxia; 2) NE infused after vascular resistance (Rpv) was increased with serotonin; 3) NE infused after Rpv was increased by H; 4) NE infused after lobar pressure was raised by an increase in flow (P/F). PO2 values during H were varied (27-56 Torr). S and H produced a 137 +/- 35 and 43 +/- 8% delta Rpv increase in lobar vascular resistance, respectively. P/F increased lobar pressure 91 +/- 10%. Only NE infusion during H demonstrated significant differences in lobar pressure and Rpv compared with control normoxic periods. There was no correlation between responses to NE during S, H, and P/F and degree to which each stimulus increased Rpv or lobar pressure (r = 0.003, 0.28, 0.24). A significant relationship between response to NE during H vs. PO2 during H was observed (r = 0.78; P less than 0.001). In a subset of animals, we repeated the infusion of NE during H and P/F post-beta-blockade. The decrease in vascular response to NE during H and the correlation of PO2 with NE response were abolished (n = 7).(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate-binding proteins of tumor lines with different growth properties

Summary A tumor model system of clones of myeloproliferative sarcoma virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and metastatic potential was studied. The relationship between metastatic behavior and composition of carbohydrate-binding proteins (lectins) was analyzed by affinity chromatography. The metastatic variant differs qualitatively from its parental clone in the presence of galactoside-binding proteins at apparent molecular weights of 80 kDa, 70 kDa, 22 kDa, 18 kDa and 16 kDa and of a fucose-binding protein at apparent molecular weight of 42 kDa. The -glucosyl-binding proteins at apparent molecular weights of 67 kDa and 53 kDa and a galactoside-binding protein of apparent molecular weight of 34 kDa, however, are not detectable in the metastatic variant in comparison to its parental clone. In this respect the parental clone shows closer resemblance to the clone 5–8#1 with different growth properties and low metastatic potential than to its own metastatic variant. Furthermore, only the parental clone has a melibiose- and a mannan-binding protein of an apparent molecular weight of 64 kDa and 14 kDa, respectively. Rosette formation as model system for intercellular interaction reveals differences in the inhibition pattern with sugar between the two clones 5–8#1 and 5–20#20, whereas the metastatic variant 5–20#20 (s) exhibits drastically reduced capability to form rosettes. Initial experiments demonstrate the feasibility of drug targeting to transformed fibroblasts via carbohydrate-binding proteins.     

Cyclic nucleotide phosphodiesterases in larval brain of wild type and dunce mutant strains of Drosophila melanogaster: isoenzyme pattern and activation by Ca2+/calmodulin

Like adult heads and whole flies, larval brains of wild type Drosophila melanogaster contain two major soluble cyclic nucleotide phosphodiesterases, forms I and II. Larval brains of the learning-defective mutant strain, dunceM11, contain only the form I enzyme. In both wild type and dunce strains the form I enzyme is activated by Ca2+/calmodulin. A time-dependent loss of this Ca2+ activation was observed.     

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.