Isolation and characterization of the tubular organelles induced by fumarate reductase overproduction in Escherichia coli

Strains of Escherichia coli amplifying the intrinsic membrane enzyme fumarate reductase accommodate the overproduced enzyme by increasing the amount of membrane material, in the form of intracellular tubular structures. These tubules have been observed in strains harbouring multicopy frd plasmids and in ampicillin hyper-resistant strains. A procedure has been developed for isolation of tubules nearly free of cytoplasmic membrane. Using protein A-gold labelling and optical diffraction of electron micrographs, a model for tubule structure is proposed. The tubules have a lower lipid/protein ratio than the cytoplasmic membrane, with the enzyme accounting for greater than 90% of the protein in the tubules. Both cytoplasmic membranes and tubules from amplified strains are enriched in cardiolipin and have a more fluid fatty acid composition than wild-type strains. Mutants defective in cardiolipin synthesis produce tubules in response to excess fumarate reductase, but these tubules have an altered appearance, indicating that lipid-protein interactions may be important for tubule assembly.     

Living Vessel Elements in the Late Metaxylem of Sheathed Maize Roots

The two types of nodal roots of field-grown maize, sheathedand bare, were found to have such different water conductivitiesthat an investigation of the anatomy of their large metaxylemvessels was made. While the vessels of the bare roots were openfor scores of centimetres, those of the sheathed roots werefound to be not vessels but developing vessel elements, withcross walls at 1 mm intervals, and protoplasts. The cross wallsbetween the elements had several unique histochemical properties.Previous investigators have often failed to find the cross wallsbecause they are very easily dislodged during the usual methodsof tissue preparation. They are best identified by microdissectionof fresh xylem. The living elements persist in the late metaxylemup to 20 – 30 cm from the tip. As the roots become longerthan this both the cross walls and the soil sheaths disappearand there is a transition to a bare root with open vessels inthe proximal region. The soil sheath persists a little longerthan the cross walls. The two types are thus stages in a developmentalsequence through which all nodal roots pass. A fundamental differencebetween the two types is in their water status, since the estimatedconductive capacity of a bare root is about 100 times greaterthan that of a sheathed root. These observations point to theneed for a reassessment of the published work on transport ofions into the xylem of grass roots through a reinvestigationof the ‘maturity’ of their xylem vessels. Grass roots, dimorphic roots, ion secretion to xylem, soil sheaths, xylem vessels, xylem differentiation, water conduction, Zea mays L     

Formation and possible functions of alpha-putrescinylthymine in bacteriophage phi W-14 DNA: analysis of bacteriophage mutants with decreased levels of alpha-putrescinylthymine in their DNAs.

The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.     

Pregnancy rates and sexual behavior under pasture breeding conditions in mares

Pony mares (n=480) and 16 stallions were assigned to four herds of 60 mares and one stallion (large herds) and to 12 herds of 20 mares and one stallion (small herds). The stallions remained with the herds continuously for all of the large herds and seven of the small herds. In the five remaining small herds the stallion was put into a herd for three hours every two days for 12 observation periods. Pregnancy rates and day of ovulation were estimated by size of embryonal enlargements. Mean pregnancy rates of 51% and 54% were obtained in the small herds and 42% in the large herds during a 48-day period (equivalent to two estrous cycles). Pregnancy rates for herds with the stallion present continuously were higher (P<0.01) for the small herds than for the large herds for days 1-24 (42% versus 19%). There was no effect of herd size on number of mares becoming pregnant per herd on days 1-24, but more mares (P<0.01) became pregnant during days 25-48 in the large herds (13.2 mares per herd versus 1.8). In the herds in which the stallion was present intermittently, the number of times that the stallion rebred the same mare when more than one mare was in estrus was greater (P<0.01) than what would be expected to occur by chance (observed, 21%; expected, 11%). Repeated breeding of the same mare seemed related to the availability or activity of the mare, since such mares more frequently followed and positioned themselves in the vicinity of the stallion. Most of the interferences by a mare which involved keeping the stallion and another mare apart were directed at the mare, whereas most of the interferences during mounting were directed at the stallion (P<0.01). Mares were more likely (P<0.01) to interfere when in estrus than when in nonestrus. When interfering mares were in nonestrus, their hostility was usually directed at the stallion (92%), whereas when in estrus their interference was more frequently directed at a mare (73%, P<0.01).     

Studies of the interaction of RecA protein with DNA

Ethidium fluorescence assays were adapted for the rapid and sensitive detection of precA; in addition, fluorescence measurements on binding precA to linear, OC and CCC PM2 DNAs have enabled the stoichiometry of precA binding as well as the precA-induced unwinding angle of DNA to be determined. The stoichiometry of binding was independently confirmed by sedimentation analysis to be one precA molecule per 3 bp. The unwinding angle was also independently confirmed by measurements of fluorescence changes induced by the binding of precA to CCC DNA which was relaxed by topoisomerase to give a precA-induced unwinding angle of 51 degrees. Electron microscopy of OC DNA molecules which bound nonsaturating amounts of precA revealed that the length increase in DNA due to precA was approximately 55%. Finally, examination of negatively stained precA complexes with a variety of linear DNAs showed that the minor groove is the primary site of interaction for this protein.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Studies on the binding of integration host factor (IHF) and TraM to the origin of transfer of the IncFV plasmid pED208

The origin of transfer (oriT) of the IncFV plasmid pED208 contains a region with three binding sites for both the plasmid-encoded TraM protein and the integration host factor (IHF) of Escherichia coli, a sequence-specific DNA-binding protein. One region, containing overlapping TraM and IHF binding sites, could be interpreted as containing two binding sites for each protein. Using gel retardation assays, an affinity constant for IHF binding to the three main sites was estimated in the presence and absence of 0.1 M potassium glutamate, which increased the avidity of IHF binding to the weaker sites by two orders of magnitude. DNase I protection analyses and electron microscopy were used to determine the affinity of IHF for oriT-containing DNA in the presence and absence of TraM. The binding of IHF and TraM was found to be non-cooperative by the two techniques employed. Electron microscopy also demonstrated that IHF bent the oriT region in a manner consistent with its previously determined mode of action, while TraM had no discernible effect on the appearance of the DNA. This suggested that IHF and TraM interact with a 295 by sequence in the oriT region and organize it into a higher order structure that may have a role in the initiation of DNA transfer and control of traM expression.     

Incubation in Mice Provides a Signal for the Differentiation of Trypanosoma cruzi Epimastigotes to Trypomastigotes1

The differentiation of Trypanosoma cruzi epimastigotes into trypomastigotes was studied in diffusion chambers sub-cutaneously implanted in mice. Using epimastigotes of the Tulahuén strain, transformation was first evident at 16 h after implantation and reached its maximum (92% trypomastigotes) by 24 h. Shortly before their differentiation into trypomastigotes, epimastigotes were found to develop resistance to lysis by the alternative pathway of complement. Furthermore, implantation of stationary-phase (as opposed to log-phase) parasites resulted in the accumulation of large numbers of complement-resistant epimastigotes in the chambers. These observations suggest that epimastigotes pass through a complement-resistant transitional stage before differentiating into trypomastigotes and that transformation may require cell division. In a further series of experiments, epimastigotes recovered 7 h after implantation in mice were found to differentiate into trypomastigotes when cultured in vitro for an additional 17 h at 37°C. This observation indicates that the events which trigger the morphologic transformation of epimastigotes into trypomastigotes can be dissociated operationally from the differentiation process itself.