Diplonemid glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and prokaryote-to-eukaryote lateral gene transfer.

Lateral gene transfer refers to the movement of genetic information from one genome to another, and the integration of that foreign DNA into its new genetic environment. There are currently only a few well-supported cases of prokaryote-to-eukaryote transfer known that do not involve mitochondria or plastids, but it is not clear whether this reflects a lack of such transfer events, or poor sampling of diverse eukaryotes. One gene where this process is apparently active is glyceraldehyde-3-phosphate dehydrogenase (GAPDH), where lateral transfer has been implicated in the origin of euglenoid and kinetoplastid genes. We have characterised GAPDH genes from diplonemids, heterotrophic flagellates that are closely related to kinetoplastids and euglenoids. Two distinct classes of diplonemid GAPDH genes were found in diplonemids, however, neither class is closely related to any other euglenozoan GAPDH. One diplonemid GAPDH is related to the cytosolic gapC of eukaryotes, although not to either euglenoids or kinetoplastids, and the second is related to cyanobacterial and proteobacterial gap3. The bacterial gap3 gene in diplonemids provides one of the most well-supported examples of lateral gene transfer from a bacterium to a eukaryote characterised to date, and may indicate that diplonemids have acquired a novel biochemical capacity through lateral transfer.     

The flexibility during the juxtaposition of reacting groups and the upper limits of enzyme reactions

The combinations between enzymes and substrates occur only after their reacting groups are in juxtaposition with each other. This will greatly reduce the probability of their effective encounters. However, the results calculated with the finite element method show that the reaction limits will not decrease substantially if van der Waal's forces and a reasonable flexibility during such a juxtaposition are taken into account.     

Transfer RNA genes associated with the 16S and 23S rRNA genes of Euglena chloroplast DNA

Hybridization studies of Euglena chloroplast ¹²⁵I-labeled tRNAs to restriction fragments of Euglena chloroplast DNA have shown that the spacer between the 16S and 23S rRNA genes, in two and possibly all three of the ribosomal DNA units, contains genes for tRNA^Ile and tRNA^Ala, whereas a tRNA gene (for either tRNA^Trp or tRNA^Glu) is located before probably all four 16S rRNA genes present on the chloroplast DNA molecule.     

Worldwide distribution of the conjugative Clostridium perfringens tetracycline resistance plasmid, pCW3

The aim of this study was to test the hypothesis that all conjugative R-plasmids of Clostridium perfringens are closely related to the previously characterized tetracycline resistance plasmid, pCW3. Fourteen conjugative R-plasmids derived from 11 C. perfringens strains isolated in Australia, the United States, France, Belgium, and Japan were analyzed. Eleven of the plasmids encoded tetracycline resistance while three carried both tetracycline and chloramphenicol resistance. Each of these plasmids was compared, by restriction analysis, to the reference plasmid, pCW3. Seven of the tetracycline resistance plasmids had EcoRI, XbaI, and ClaI restriction profiles that were identical to those of the corresponding pCW3 digests. The seven remaining R-plasmids were different from pCW3. Comparison of partial restriction maps of these plasmids with a complete map of pCW3 indicated that they contained at least 17 kb of DNA that also was present in pCW3. Hybridization analysis confirmed that these plasmids shared substantial homology with pCW3. The three tetracycline and chloramphenicol resistance plasmids frequently lost a 6-kb chloramphenicol resistance segment during conjugation. Cloning experiments showed that the chloramphenicol resistance determinant was expressed in Escherichia coli and that the chloramphenicol resistance gene of one of these plasmids, pIP401, was contained within a 1.5-kb region of the 6-kb deletion segment. Hybridization analysis indicated that the deletion segment of pIP401 was related to those of the other two chloramphenicol resistance plasmids. During the course of this study, conjugative R-plasmids which appear to be identical to pCW3 or closely related to pCW3 were identified from C. perfringens strains from human, animal and environmental sources in five countries. It is concluded that C. perfringens strains in humans and animals throughout the world have overlapping gene pools and that all the conjugative C. perfringens R-plasmids examined probably evolved from a pCW3-like element.     

Early steps in specific tumor cell lysis by sensitized mouse T lymphocytes. V. Evidence that manganese inhibits a calcium-dependent step in programming for lysis

The mechanism of T-lymphocyte-mediated cytolysis consists of three successive steps: adhesion formation, programming for lysis, and killer-cell-independent lysis. Mg²⁺, but not Ca²⁺, is required for adhesion formation, whereas programming for lysis is strongly Ca²⁺ dependent. We have previously reported that the transition metal manganese can substitute for Mg²⁺ in supporting adhesion formation. In the present paper, we demonstrate that manganese inhibits programming for lysis. The inhibitory effect of Mn²⁺ on cytolysis can be reduced by increasing the concentration of Ca²⁺. Furthermore, inhibitor sequencing experiments were unable to distinguish the step blocked by Mn²⁺ from the Ca²⁺-dependent step. These results suggest that Mn²⁺ blocks a Ca²⁺-dependent step(s) in programming for lysis. Present evidence does not distinguish whether the action of Ca²⁺ in programming for lysis is via a Ca²⁺ influx (as a “second messenger?”) or whether Ca²⁺ simply serves as a cofactor at the cell exterior.     

Neonatal androgen levels and avoidance learning in prepubescent and adult male rats

Neonatal male rats were injected with 1.25 mg of testosterone propionate (TP) and compared with oil-injected controls on the acquisition of an active and passive avoidance response at 25 days of age. The TP treated animals acquired the active avoidance response significantly faster than controls, but no differences were found between groups tested on the step-down passive avoidance task. The active-avoidance paradigm was repeated at 70 days of age, with experimental and control animals receiving the same neonatal treatment as the prepubescent subjects. Again the TP group showed facilitated acquisition of the active avoidance response. The TP treatment also produced an increase in activity levels and aversion threshold to footshock in the prepubescent animals. Therefore the active avoidance effect may be interpreted more parsimoniously as a reflection of these latter effects, rather than learning per se.     

tRNA genes are found between 16S and 23S rRNA genes in Bacillus subtilis.

There are at least nine, and probably ten, ribosomal RNA gene sets in the genome of Bacillus subtilis. Each gene set contains sequences complementary to 16S, 23S and 5S rRNAs. We have determined the nucleotide sequences of two DNA fragments which each contain 165 base pairs of the 16S rRNA gene, 191 base pairs of the 23S rRNA gene, and the spacer region between them. The smaller space region is 164 base pairs in length and the larger one includes an additional 180 base pairs. The extra nucleotides could be transcribed in tRNAIIe and tRNA Ala sequences. Evidence is also presented for the existence of a second spacer region which also contains tRNAIIe and tRNA Ala sequences. No other tRNAs appear to be encoded in the spacer regions between the 16S and 23S rRNA genes. Whereas the nucleotide sequences corresponding to the 16S rRNA, 23S rRNA and the spacer tRNAs are very similar to those of E. coli, the sequences between these structural genes are very different.     

Cascade control of E. coli glutamine synthetase. I. Studies on the uridylyl transferase and uridylyl removing enzyme(s) from E. coli.

The state of adenylylation of glutamine synthetase in Escherichia coli is regulated by the adenylyl transferase, the PII regulatory protein, uridylyl transferase (UTase), and the uridylyl removing enzyme (UR). The regulatory protein exists in an unmodified state (PII) which promotes adenylylation and in a uridylylated form (PII·UMP) which promotes deadenylylation of glutamine synthetase. The UR and UTase enzymes catalyze the interconversion of PII and PII·UMP. The UR and UTase have been partially purified by chromatography over DEAE-cellulose, AH-Sepharose 4B, Sephadex G-200, and gel electrophoresis. The two activities co-purify at all steps in the isolation although preparations containing different ratios of UTase:UR activities have been isolated. These UR·UTase activities have apparent molecular weight of 140,000. Both activities are inactivated by sulfhydryl reagents, both activities are heat inactivated, and both are stabilized by high salt concentrations. Both activities are inhibited in the crude extract by dialyzable inhibitors, but the UR is also inhibited by a nondialyzable inhibitor. This endogenous inhibitor is of molecular weight greater than 100,000 daltons, and binds CMP and UMP which are the apparent inhibitory agents. CMP and UMP are antagonistic in their effects on the UR activity. No effect of the CMP, UMP, or the large inhibitor on the other steps in the cascade could be demonstrated. The Mn²⁺-supported UR activity was also shown to be inhibited by a number of divalent cations, particularly Zn²⁺.     

Adjuvant effect of poly(A:U) upon T cell-mediated in vitro cytotoxic allograft responses

The effect of complexes of polyadenylic acid and polyuridylic acid [poly(A:U)] on thymus-processed lymphocytes was studied using a tissue culture system in which T cells responded to cell bound alloantigens. The in vitro activation of T cells into cytotoxic lymphocytes was assessed with the aid of the ⁵¹Cr cytotoxic assay. Introduction of poly(A:U) into cultures or pretreatment of thymus cells prior to culture resulted in a reduction in the time required for the development of maximal cytotoxic activity as well as a reduction in the dose of allogeneic cells required for maximum stimulus. Poly(A:U) had no influence on the ability of differentiated cytotoxic T cells to lyse ⁵¹Cr-labeled target cells. The amplification of cytotoxic activity caused by poly(A:U) was specific to the antigens used to activate the thymus lymphocytes.     

The digital pads of the tree frog, Hyla cinerea. I. The epidermis

As the digital pad cells grow from the germinal epithelium, the desmosomes on the distal surface oF tile cells become aligned and form pegs filled with tonofilaments on the surface of the outer row of cells. The outer pad cells are separated from each other distally, thus the cell and the pegs form two sizes of protrusions in series on the pad surface which can fit into substratum/irregularities. A dense material, apparently derived from transforming bodies in the cells, coats the plasma membrane of the surface cells, presumably to strengthen them.     

The digital pads of the tree frog, Hyla cinerea. II. The mucous glands

The mucous glands consist of a single row of cells surrounded by smooth muscle. The cells are attached at their apical and basal regions and only cytoplasmic projections loosely link the lateral aspects of adjacent cells. Material accumulates in the cisternae of the rough endoplasmic reticulum, appears to form into dense granules in the Golgi apparatus, and then before being secreted, undergoes chemical and morphological alterations. Since some glands secrete onto the dorsal epidermis of the digits, the mucous is believed to function as a wetting agent for the skin as well as an aid to climbing.     

Effects of a mixture of a saturated with an unsaturated fatty acid on secretion of the very low density lipoprotein by the liver.

Palmitic acid (16:0) and palmitoleic acid (16:1), as the complex with bovine serum albumin, were infused at rates of 62 and 124 μmoles/hr into an albumin-buffer medium perfusing livers isolated from normal fed male rats. In other experiments, equimolar mixtures (124 μmoles/hr, total) of 16:0 + 16:1, or myristate (14:0) + 16:1 were infused. The output of triglyceride when 16:1 was infused was greater than when equivalent amounts of 14:0 or 16:0 were infused; output with equimolar mixtures of 14:0 and 16:1, or 16:0 and 16:1 was intermediate between that of saturated and unsaturated fatty acids alone. Rate-zonal mobility of the VLDL in the ultracentrifuge was more rapid as the quantity of 16:1 available to the liver increased, but did not change with increasing amounts of 16:0. The rate-zonal mobility of the mixtures of 14:0 and 16:1, or of 16:0 and 16:1, was not different than that of 16:1 alone. The ratios of phospholipid and cholesterol relative to triglyceride in the VLDL decreased with increasing output of triglyceride and with unsaturation of the fatty acid. Ratios resulting from mixtures of the fatty acids appeared to be in an intermediate position. The composition and properties of the secreted VLDL clearly are dependent on the structure and quantity of FFA available to the liver; with mixtures of saturated and unsaturated fatty acids, the unsaturated fatty acid seems to exert a dominant effect.     

Biogenesis of mitochondria. The effects of catabolite repression on the in vitro phospholipid synthetic capacities of subcellular fractions of respiratory-competent and respiratory-deficient strains of Saccharomyces cerevisiae.

A respiratory-competent wild-type strain and a nuclear isogenic, mitochondrial DNA-less, petite mutant strain of Saccharomyces cerevisiae were grown under conditions of catabolite repression in batch cultures and under conditions of catabolite derepression in chemostat cultures. Subcellular fractions were isolated and the capacity of these fractions to incorporate sn-[2-³H]glycerol 3-phosphate into phospholipids was studied. Neither catabolite repression nor loss of mitochondrial DNA appreciably altered the total in vitro lipid synthesized by mitochondrial fractions during the incubation. Mitochondria isolated from catabolite-derepressed wild-type and petite cells had approximately the same specific activity in vitro for the synthesis of phosphatidylinositol. phosphatidic acid, phosphatidylethanolamine, phosphatidylserine, and neutral lipids. Mitochondria isolated from the petite cells retained the capacity to synthesize phosphatidylglycerol and diphosphatidylglycerol, although the synthesis of these phospholipids was far less extensive than that by the mitochondria isolated from the wild-type cells. In both cases, mitochondria prepared from catabolite-repressed cells synthesized a greater proportion of phosphatidylserine than did mitochondria from catabolite-derepressed cells. The proportions of phospholipid species synthesized in vitro by the microsomal fractions studied were not grossly affected by catabolite repression or loss of mitochondrial DNA.     

Linkage of 5S RNA and 16S+23S RNA genes on the E. coli chromosome.

Summary Episomes carrying limited regions of the chromosome where 5S RNA genes have previously been located are described. The DNA purified from each of these episomes contains one gene per molecule for each of the three ribosomal RNA species as shown by hybridization experiments.This work was supported in part by a grant from the C.E.A.     

Identification and restriction endonuclease mapping of the threonine operon regulatory region.

A DNA fragment carrying the thrP region of Escherichia coli has been identified and characterized. Starting from a secondary site lysogen of bacteriophage λ, where the position of the prophage is either within thrP or between thrP and thrP structural genes (Gardner et al., 1974; Gardner &; Smith, 1975) it has been possible to isolate transducing phages which carry bacterial DNA from either side of the prophage. By Int-Xis mediated site-specific recombination we have generated recombinant transducing phages which carry an intact thrP region. A phage carrying a regulatory mutation, which maps in the thrP region, has also been constructed.Mapping with several restriction endonucleases has allowed us to construct a map of the cleavage sites of the thrP region. A 1700 base-pair HaeIII fragment carrying the secondary attachment site (attΔOΔ′) was isolated and its position was localized within a 180 base-pair TaqI-HhaI restriction fragment. The location of attΔOΔ′ in the HaeIII fragment suggests that the entire thrP region is carried by this fragment.