Polar mobilization of the Escherichia coli chromosome by the ColE1 transfer origin

Summary Mobilization of the plasmid ColE1 from cells containing a conjugative plasmid (such as F) requires the synthesis of ColE1 mob proteins, and the presence, in cis, of bom (basis of mobility), a region of ColE1 containing the origin of transfer (oriT). The process of ColE1 transfer is thought to resemble that of the conjugative plasmid F, although the plasmids share little sequence homology. In F, conjugation is preceded by a strand-specific nicking event at oriT. The nicked strand is then conducted to the recipient with the 5 end leading. This is believed also to occur with ColE1, but direct biochemical confirmation has been precluded by its small size (6.65 kb). To test this hypothesis genetically, a novel method, using a dv-based vector, has been devised to site-specifically integrate bom (or any other cloned sequence) into the chromosome of Escherichia coli. When provided with suitable mobilizing plasmids, such strains were found to transfer the chromosome in a polar way. From these data, the orientation of transfer of ColE1 was deduced and shown to be analogous to F.     

Transglutaminase and the Neuronal Cytoskeleton in Alzheimer''s Disease

Transglutaminase [EC 2.3.2.13, (R)-glutaminyl-peptide:amine gamma-glutamyltransferase], an enzyme that catalyzes the introduction of glutamine-lysine cross-links into proteins, was purified. Neurofilament and microtubule proteins were substrates for this enzyme but the insoluble neurofibrillary tangles (NFT) isolated from Alzheimer's disease brain were not substrates. In vitro cross-linking of neurofilaments and microtubules by the enzyme did not produce paired helical filaments (PHF), which are the major ultrastructural component of NFT. These results make it unlikely that PHF are formed by the straightforward cross-linking of neurofilaments or microtubules.     

Sperm-oocyte membrane fusion in the human during monospermic fertilization

Sperm-oocyte membrane fusion has been observed during monospermic fertilization of a human oocyte in vitro. Women were stimulated with both clomiphene citrate and human menopausal gonadotropin and were given human chorionic gonadotropin before a LH-surge. Twelve oocytes, collected at laparoscopy from six women who became pregnant by IVF, were allowed to mature for 7–14 hours in vitro and inseminated with preincubated sperm, fixed between 1–3 hours after insemination, and examined by transmission electron microscopy. Membrane fusion had occurred in one ovum 2 hours after insemination, and the oocyte had resumed maturation and was at anaphase II of meiosis. Cortical granules had been exocytosed, and some of their contents were visible at the surface close to the oolemma all around the oocyte. The sperm that fused with this oocyte was acrosome-reacted and had been partly incorporated into the ooplasm, while the anterior two-thirds of its head was phagocytosed by a tongue of cortical ooplasm. Membrane fusion had occurred between the oolemma and the plasma membrane overlying the postacrosomal segment of the sperm head, posterior to the equatorial vestige. Sperm chromatin had not decondensed, and serial sections revealed a midpiece attached to the basal plate and a tail located deeper in the ooplasm, all devoid of plasma membrane. Supplementary sperm penetrating the inner zona, approaching the perivitelline space, had undergone the acrosome reaction but had a persistent vestige of the equatorial segment of the acrosome with intact plasma membrane. Evidence of sperm chromatin decondensation was seen in other oocytes, 3 hours after insemination, which were at telophase II of meiosis. Eight oocytes penetrated by sperm were monospermic, while four were unfertilized. The general pattern of sperm fusion and incorporation appears to conform to that seen in most other mammals. The study also reveals that sperm have to complete the acrosome reaction before fusing with the egg.     

Untargeted mutagenesis induced by UV in the lacI gene of Escherichia coli

Summary Using a nonselective method, we have estimated the proportion of untargeted mutations in the lacI gene of E. coli by transferring either irradiated or unirradiated F pro lac plasmids from an excision deficient donor to an excision deficient pro lac deleted recipient that had been irradiated and allowed to induce recA dependent functions for 30 min. We find that about 10 percent of the mutations induced by either 3.5 Jm^-2 or 7 Jm^-2 UV are untargeted.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

New mutations affecting induced mutagenesis in yeast

Previously isolated mutations in baker's yeast, Saccharomyces cerevisiae, that impair induced mutagenesis were all identified with the aid of tests that either exclusively or predominantly detect base-pair substitutions. To avoid this bias, we have screened 11 366 potentially mutant clones for UV-induced reversion of the frameshift allele, his4–38, and have identified 10 mutants that give much reduced yields of revertants. Complementation and recombination tests show that 6 of these carry mutations at the previously known REV1, REV1 and REV3 loci, while the remaining 4 define 3 new genes, REV4 (2 mutations), REV5 and REV6. The rev4 mutations are readily suppressed in many genetic backgrounds and, like the rev5 mutation, impart only a limited deficiency for induced mutagenesis: it is likely, therefore that the REV4⁺ and REV5⁺ gene functions are only remotely concerned with this process. The rev6 mutants have a more general deficiency, however, as well as marked sensitivity to UV and an increased spontaneous mutation rate, properties that suggest the REV6 gene is directly involved in mutation induction. The REV5 gene is located about 1 cM proximal to CYC1 on chromosome X.     

The effect of phytoplankton and suspended sediment on the growth of Corbicula fluminea (Bivalvia)

Juvenile Corbicula fluminea Müller (1774) were cultured at 15.3 °C in the laboratory on eight combinations of suspended sediment and phytoplankton. Sediment concentrations were 2.6, 25, 50, and 150 mg l^–1. Chlorophyll a levels were 15.6 and 62.5 µg l^–1. Clam tissue growth was found to be independent of silt concentration but increased at the higher chlorophyll level (p < 0.05). The growth experiment was repeated at 24 °C with chlorophyll a concentrations of 18.9 and 112.6 µg l^–1. Growth was again greater at the higher phytoplankton level (p < 0.05). These results demonstrate that Asiatic clam populations are food-limited most of the growing season in the Northern and Western portions of California's eutrophic Sacramento-San Joaquin Delta where chlorophyll a levels average less than the lower of these values. Comparisons of clam growth in the laboratory and estuary support the food limitation hypothesis as at the higher food concentration laboratory tissue growth was 2.3 and 3.8 times greater during the high and low temperature evaluations than in the estuary.     

Neurospora Trehalase and Its Structural Gene

We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.—Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.—We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.     

Temporal fluctuations of silver,copper and zinc in the bivalve Macoma balthica at five stations in South San Francisco Bay

Concentrations of Cu, Ag and Zn were measured in the soft tissues of the estuarine bivalve Macoma balthica in South San Francisco Bay at near-monthly intervals for periods of two to three years at four stations, and eight years at a metal-enriched station. The amplitude and frequency of fluctuations differed among stations and among metals. Fluctuations were greatest at stations with the greatest metal enrichment and with the least dilution and flushing of wastes. A consistent seasonal pattern of fluctuation in Cu and Ag concentrations was evident in M. balthica at the metal-enriched station. These seasonal changes in tissue metal concentrations appeared to be affected by metal inputs, hydrologic processes that may affect both metal concentrations and bioavailability, and seasonal changes in the weight of the bivalve. The contributions of each of these interacting factors could not be determined quantitatively. At the metal-enriched station significant variation in the amplitude of seasonal fluctuations was also evident from year to year. Interpretation of metal concentrations in bivalves from estuaries will require careful consideration of the processes which affect metal dynamics in these complex environments.     

Multiplicity of peptide permeases in Candida albicans: evidence from novel chromophoric peptides.

Evidence is presented for the presence of multiple peptide permeases in the eucaryotic organism Candida albicans. Instrumental in these studies were the peptides L-alanyl-L-2-thiophenylglycine (Ala-alpha-TPG) and L-alanyl-L-2-thiophenylglycyl-L-alanine (Ala-alpha-TPG-Ala), which contain thiophenol attached to the alpha-carbon of glycine. Subsequent to transport into the fungal cell, enzymatic hydrolysis of these peptides resulted in the release of free thiophenol, which was quantified by using Ellman reagent. Thiophenol release was shown to be directly correlated to peptide transport and hydrolysis, with transport being the rate-limiting step in intact cells. These peptides, whose uptake showed Michaelis-Menten kinetics, have been used to determine peptide uptake in C. albicans. In addition, we found that the intracellular peptidases can readily be assayed in permeabilized cells and that bestatin, an aminopeptidase inhibitor, inhibits all detectable peptidase activity. C. albicans 124 was able to transport and hydrolyze both Ala-alpha-TPG and Ala-alpha-TPG-Ala, whereas the mutant (124NIK5) was able to transport only the tripeptide. The intracellular peptidases of this mutant were unaffected. In wild-type C. albicans 124, oligopeptides were able to compete with uptake of Ala-alpha-TPG-Ala to a far greater extent than with that of Ala-alpha-TPG; dipeptides inhibited uptake of both Ala-alpha-TPG and Ala-alpha-TPG-Ala. These results provide complementary evidence for the existence of distinct transport systems.