Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

var1 Gene on the mitochondrial genome of Torulopsis glabrata

We have cloned and sequenced a region of the Torulopsis glabrata mitochondrial genome homologous to the Saccharomyces cerevisiae var1 gene (var1Sc). An open reading frame that could encode a protein of 339 amino acids was found with 72.7% amino acid and 85.3% nucleotide sequence homology to the S. cerevisiae var1 gene. The T. glabrata gene (var1Tg) is transcribed yielding two stable RNAs, a more abundant 13.5 S RNA and a less abundant 18 S species. We have also identified a candidate for a T. glabrata var1 protein among mitochondrial translation products labeled in isolated mitochondria. The var1Tg gene is even more A + T-rich (93%) than var1Sc (89.6%) and has conserved the strong codon bias of var1Sc. Major differences between the two sequences were found. Significant among these are that no GC clusters are found in var1Tg and the sequences surrounding each of the sites where known polymorphisms exist in var1Sc have deletions at the corresponding sites in var1Tg. These data are discussed with respect to possible origins of these var1 genes and translocation of GC clusters in S. cerevisiae mitochondrial DNA.     

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.     

Genetic reconstruction and functional analysis of the repeating lipoyl domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli

The dihydrolipoamide acetyltransferase component (E2p) of the pyruvate dehydrogenase complex of Escherichia coli contains three highly homologous sequences of about 100 residues that are tandemly repeated to form the N-terminal half of the polypeptide chain. All three sequences include a lysine residue that is a site for lipoylation and they appear to form independently folded functional domains. These lipoyl domains are in turn linked to a much larger (about 300 residues) subunit-binding domain of the E2p chain that aggregates to form the octahedral inner core of the complex and also contains the acetyltransferase active site. In order to investigate whether individual lipoyl domains play different parts in the enzymic mechanism, selective deletions were made in vitro in the dihydrolipoamide acetyltransferase gene (aceF) so as to excise one or two of the repeating sequences. This was facilitated by the high degree of homology in these sequences, which allowed the creation of hybrid lipoyl domains that closely resemble the originals. Pyruvate dehydrogenase complexes incorporating these genetically reconstructed E2p components were purified and their structures were confirmed. It was found that the overall catalytic activity, the system of active site coupling, and the ability to complement pyruvate dehydrogenase complex mutants, were not significantly affected by the loss of one or even two lipoyl domains per E2p chain. No special role can be attached thus far to individual lipoyl domains. On the other hand, certain genetic deletions affecting the acetyltransferase domain caused inactivation of the complex, highlighting particularly sensitive areas of that part of the E2p chain.     

Structure and polymorphism of bipolar isopranyl ether lipids from archaebacteria

We describe in this work the structure and polymorphism of a variety of lipids extracted from Sulfolobus solfataricus, an extreme thermoacidophilic archaebacterium growing at about 85 °C and pH 2. These lipids are quite different from the usual fatty acid lipids of eukaryotes and prokaryotes: each molecule consists of two C₄₀ ω-ω′ biphytanyl residues (with 0 to 4 cyclopentane groups per residue), ether linked at both ends to two (variably substituted) glycerol or nonitol groups. Four lipid preparations were studied; the total and the polar lipid extracts, and two hydrolytic fractions, the symmetric glycerol dialkyl glycerol tetraether and the asymmetric glycerol dialkyl nonitol tetraether, as a function of water content and temperature, using X-ray scattering techniques. The main conclusions from the study of the four lipid preparations can be summarized as follows. (1) As with other lipids, a remarkable number and variety of phases are observed over a temperature-concentration range close to “physiological” conditions. The possibility is discussed that this polymorphism reflects a fundamental property of lipids, closely related to their physiological rôle. (2) As in other lipids, two types of chain conformations are observed: a disordered one (type α) at high temperature; at lower temperature, a more ordered packing of stiff chains, all parallel to each other (type β′). At temperatures and degrees of hydration approaching the conditions prevailing in the living cell, the conformation is of type α. (3) In all the phases with chains in the α conformation, the unsubstituted glycerol headgroups, whose concentration is high in these lipids, segregate in the hydrocarbon matrix, away from the other polar groups. This property may have interesting biological consequences: for example, the chains of a fraction of the bipolar lipid molecules can span hydrocarbon gaps as wide as 75 Å. (4) Two cubic phases are observed in the total and the polar lipid extracts, which display a remarkable degree of metastability, most unusual in lipid phase transitions involving structures with chains in the α conformation. This phenomenon can be explained by the interplay of the physical structure of the cubic phases (the two contain two intertwined and unconnected three-dimensional networks of rods) and the chemical structure of the lipid molecules: the two headgroups of most molecules being anchored on each of the two networks of rods, the migration of the lipid molecules is hindered by the two independent diffusion processes and by the entanglement of the chains. The possibility is discussed that this phenomenon may reflect an evolutionary response to a challenge of the natural habitat of these archaebacteria.     

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.     

Mutation induction in Escherichia coli incubated in the reaction mixture of NADPH-dependent lipid peroxidation of rat-liver microsomes

Experiments were carried out to examine mutation induction in E. coli cells incubated in the reaction mixture of NADPH-dependent lipid peroxidation of microsomes isolated from rat liver. The results obtained were as follows: (1) Lipid peroxidation of microsomes occurred extensively on incubation with NADPH and Fe2+. In the E. coli WP2uvrA(pKM101) system, the mutation frequency to streptomycin resistance increased markedly when the cells were incubated in the reaction mixture of microsomal lipid peroxidation. The induced mutation frequencies were dependent on the extent of the lipid peroxidation. (2) It was also found that the mutations were induced at the same rate as in the case of (1) when the cells were added to the microsomal suspensions after the reactions due to the short-lived free radicals had terminated. (3) The cytotoxicity of the lipid peroxidation products was larger in the DNA repair-defective mutant, E. coli SR18 (uvrArecA) than the wild-type strain, SR749. From these results it is concluded that some DNA-damaging and mutagenic substances are indeed produced in the degradation process of peroxidized polyunsaturated fatty acids in liver microsomal lipids.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.