Development of High-Frequency Delivery System for Transposon Tn919 in Lactic Streptococci: Random Insertion in Streptococcus lactis subsp. diacetylactis 18-16

The conjugative transposon Tn919, originally isolated in Streptococcus sanguis FC1, is capable of low-frequency transfer (10⁻⁷ and 10⁻⁸ per recipient) on membrane filters to a wide number of streptococcal recipients including the industrially important lactic streptococci. The introduction of pMG600 (Lac⁺ Lax⁻; a lactose plasmid capable of conjugative transfer at high frequencies and which, in certain hosts, confers an unusual clumping phenotype) into a Streptococcus lactis CH919 donor, generating S. lactis CH001, resulted in a significant improvement in the transfer frequency of Tn919 to S. lactis CK50 (1.25 × 10⁻⁴ per recipient). In addition, these matings could be performed on agar surfaces, allowing the recovery of a greater number of recipients than with filter matings. Tn919 also transferred at high frequency to S. lactis subsp. diacetylactis 18-16S but not to Streptococcus cremoris strains. Insertion in 18-16S transconjugants generated from filter matings with an S. lactis CH919 donor was random, occurring at different sites on the chromosome and also in plasmid DNA. Thus, the conditions necessary for the practical exploitation of Tn919 in the targeting and cloning of genes from a member of the lactic streptococci, namely, high-frequency delivery and random insertion in host DNA, were achieved.     

Microelectrode studies of potential difference responses to changes in stromal K+ in bullfrog cornea

The effects of changing stromal K+ were studied using microelectrodes in an in vitro preparation of frog cornea. The intracellular potential (V0) responded in two opposite ways under short-circuit conditions: (1) depolarization (normal response) when stromal K+ was increased from 4 to 20 or to 79 mM, about 30 mV per 10-fold K+ concn. change; (2) a hyperpolarization (anomalous response) of 10 mV maximum when stromal K+ was increased from 0 to 4 mM. The increase from 4 to 20 or 79 mM decreased or even reversed the short-circuit current (Isc). The transepithelial conductance (gt) increased when K+ was increased to 79 mM but no change occurred in the apical membrane fractional resistance (fRo). Increase of stromal K+ from 0 to 4 mM increased Isc and minimally changed gt and fRo. Ouabain (10(-3) M) abolished the anomalous responses, that is, the increases in V0 and Isc when stromal K+ was increased from 0 to 4 mM. These results are interpreted in terms of two K+ conductive pathways in the basolateral membrane of the corneal epithelium, a Nernstian conductance and an electrogenic (Na+ + K+)-ATPase pump transporting more Na+ than K+ ions per cycle. The normal or anomalous potential difference responses to changes in stromal K+ appear to depend on the relative resistance of the two pathways at the time stromal K+ is changed.     

A study of cytoplasmic and nuclear estrogen and progestin receptors in gynecologic neoplasms

A rapid method for simultaneous preparation of cytosol and nuclear estrogen (E) and progestin (P) receptors and their in vitro determination is described. The method was applied to several uterine or ovarian surgical specimens to evaluate their steroid hormone "dependence". The results suggest that low cytoplasmic E receptor levels (ERc) are associated with higher nuclear E receptor (ERn) levels but no apparent correlation was observed between PRc and ERn levels. The method appeared to be suitable for screening steroid hormone receptor content in tumor tissues and may provide better estimation of steroid dependence since both cytoplasmic and nuclear compartments can be studied simultaneously.     

Immunogold cytochemistry of cytochromes P-450 in porcine adrenal cortex. Two enzymes (side-chain cleavage and 11 beta-hydroxylase) are co-localized in the same mitochondria

In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 side-chain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunoelectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.     

A functional analysis of the repeated methionine initiator tRNA genes (IMT) in yeast

Summary Standard laboratory yeast strains have from four to five genes encoding the methionine initiator tRNA (IMT). Strain S288C has four IMT genes with identical coding sequences that are colinear with the RNA sequence of tRNA _I ^Met . Each of the four IMT genes from strain S288C is located on a different chromosome. A fifth IMT gene with the same coding sequence is present in strain A364A but not in S288C. By making combinations of null alleles in strain S288C, we show that each of the four IMT genes is functional and that tRNA _I ^Met is not limiting in yeast strains with three or more intact genes. Strains containing a single IMT2, 3 or 4 gene grow only after amplification of the remaining IMT gene. Strains with only the IMT1 gene intact are viable but grow extremely slowly; normal growth is restored by the addition of another IMT gene by transformation, providing a direct test for IMT function.Abbreviations IMT and imt (imt=initiator methionine tRNA), designate the genotype of the wild-type and the mutant alleles respectively, of the initiator methionine transfer RNA gene - met-tRNA _I ^Met methionylated initiator methionine transfer RNA - eIF-2 eukaryotic initiation factor two - GTP guanosine 5-triphosphate The calculation of Td values (the temperature at which half of the duplex is dissociated) for oligonucleotides used as probes in hybridizations was based on the assumption that the increase in Td value was 4° C for each G:C base pair and 2° C for each A:T base pair (Wallace et al. 1981)     

Quantity and Kinetic Properties of Ribulose 1,5-Bisphosphate Carboxylase in C3, C4, and C3-C4 Intermediate Species of Flaveria (Asteraceae)

The kinetic properties of ribulose 1,5-bisphosphate carboxylase(RuBPC) appear to have been modified during evolution of photosynthesisto adjust to changes in substrate availability. C₄ plants areconsidered to have a higher concentration of CO₂ available toRuBPC than C₃plants. In this study, the K_m(CO₂ and catalyticcapacity (k_cat) of RuBPC and the ratio of RuBPC protein to totalsoluble protein from several Flaveria species, including C₃,C₃-C₄ intermediate, and C₄ species, were determined. The C₃and intermediate species had similar K_m(CO₂) values while theC₄ species on average had higher K_m(CO₂) values. The mean ratioof K_cat/K_m for species of each group was similar, supportingthe hypothesis that changes in K_m and K_cat, are linked. Theallocation of total soluble protein to RuBPC was lowest in theC₄ Flaveria species, intermediate in the C₃-C₄ species, andhighest in the C₃ species. The results suggest that during evolutionof C₄ photosynthesis adjustments may occur in the quantity ofRuBPC prior to changes in its kinetic properties. (Received January 4, 1989; Accepted April 11, 1989)     

Book Review

International Journal of Primatology -     

Mn2+ reduces Yz + in manganese-depleted Photosystem II preparations

Manganese in the oxygen-evolving complex is a physiological electron donor to Photosystem II. PS II depleted of manganese may oxidize exogenous reductants including benzidine and Mn²⁺. Using flash photolysis with electron spin resonance detection, we examined the room-temperature reaction kinetics of these reductants with Y_z ⁺, the tyrosine radical formed in PS II membranes under illumination. Kinetics were measured with membranes that did or did not contain the 33 kDa extrinsic polypeptide of PS II, whose presence had no effect on the reaction kinetics with either reductant. The rate of Y_z ⁺ reduction by benzidine was a linear function of benzidine concentration. The rate of Y_z ⁺ reduction by Mn²⁺ at pH 6 increased linearly at low Mn²⁺ concentrations and reached a maximum at the Mn²⁺ concentrations equal to several times the reaction center concentration. The rate was inhibited by K⁺, Ca²⁺ and Mg²⁺. These data are described by a model in which negative charge on the membrane causes a local increase in the cation concentration. The rate of Y_z ⁺ reduction at pH 7.5 was biphasic with a fast 400 s phase that suggests binding of Mn²⁺ near Y_z ⁺ at a site that may be one of the native manganese binding sites.Abbreviations PS II Photosystem II - Y_D tyrosine residue in Photosystem II that gives rise to the stable Signal II EPR spectrum - Y_z tyrosine residue in Photosystem II that mediates electron transfer between the reaction center chlorophyll and the site of water oxidation - ESR electron spin resonance - DPC diphenylcarbazide - DCIP dichlorophenolindophenol     

Release of PYY from pig intestinal mucosa; luminal and neural regulation

The localization, molecular nature and secretion of Peptide YY (PYY), a putative gut hormone belonging to the Pancreatic Polypeptide family of peptides, was studied in pigs. Immunoreactive PYY was identified in a population of endocrine cells in the mucosal epithelium of the pig ileum. Release of PYY was observed in isolated perfused pig ileum in response to luminal stimulation with glucose and vascular administration of the neuropeptide gastrin-releasing peptide (GRP). Electrical stimulation of the vagus nerve supply to the distal small intestine in intact anaesthetized pigs resulted in release of PYY into the circulation. Stimulation of the splanchnic nerves did not affect the basal release of PYY. PYY-immunoreactivity extracted from ileal tissue or released to plasma or perfusate from the ileum was indistinguishable from synthetic porcine PYY by gel filtration and reverse phase HPLC. It is concluded that the secretion of PYY in the pig ileum may be regulated not only by nutritional luminal factors, but also by postsynaptic parasympathetic nerves.