Gene amplification in Bacillus subtilis

A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning'. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.     

Drug protein conjugates--III. Inhibition of the irreversible binding of ethinylestradiol to rat liver microsomal protein by mixed-function oxidase inhibitors, ascorbic acid and thiols

The metabolism of [6,7-3H]ethinylestradiol [( 3H]EE2) by rat liver microsomes was studied in vitro. After incubation of [3H]EE2 with rat liver microsomes for 20 min, 90% of the substrate was metabolised and 18% of the 3H-labelled material irreversibly bound to microsomal protein. Ascorbic acid (1 mM) decreased irreversible binding of 3H and produced an accumulation of 2-hydroxyethinylestradiol (2OH-EE2), while mixed-function oxidase inhibitors (0.5 mM) decreased binding of 3H to protein by inhibiting EE2 2-hydroxylation. Addition of thiols gave water-soluble metabolites which were characterised as 1(4)-thioether derivatives of 2OH-EE2 by co-chromatography with synthetic products. The results are consistent with the hypothesis that the chemically reactive metabolite of EE2 formed in vitro is either a quinone or o-semiquinone derived from 2OH-EE2 [1].     

Control of respiratory pattern in conscious dog: effects of heat and CO2

We measured tidal volume (VT) and inspiratory (TI) and expiratory (TE) durations in five conscious tracheostomized dogs breathing air or 5% CO2 in air either at normal (20 degrees C) or elevated (30 degrees C) ambient temperatures. Respiratory frequency ranged between 16 and 333/min due to changes in both TI and TE. During panting TI exceeded TE. During air inhalation instantaneous ventilation (V) spontaneously ranged from 100 to 1,600 ml . kg-1 . min-1. Hypercapnia, heat stress, or both, increased this range of V by increasing maximum V, primarily due to increases in mean inspiratory flow. Under these conditions, changes in TI accounted for more of the spontaneous changes in breath duration. During inhalation of air and 5% CO2, a positive correlation between VT and TI was obtained for TI between 0.13 and 1.05 s; above 1.05 s VT decreased. Heat stress increased VT at a given TI. We suggest that either the decay rate or position of the inspiratory off-switch threshold curve (Clark and von Euler, J. Physiol. London 222: 267, 1972) varies in conscious dogs. Shifts in either the reset (onset) value or decay rate of the curve yield a positive correlation between VT and TI. This modification to the Clark-von Euler model implies that the primary effect of anesthesia on respiratory control is fixation of the inspiratory off-switch threshold curve.     

Life-cycles of some invertebrate taxa in a small pond together with changes in their numbers over a period of three years

1. Monthly quantitative samples of the invertebrate fauna (except Protozoa) in a small pond were taken over a period of three years. During one year, insect emergence traps were in operation. Water temperatures were recorded during the investigation.
2. The most abundant organisms in the pond were Phaenocora typhlops, Limnodrilus hoffmeisteri and Chaoborus crystallinus. Certain species of Micro-Crustacea and Chironomidae were also abundant but these groups have been dealt with elsewhere (see p. 66). Dendrocoelum lacteum, Polycelis nigra, Helobdella stagnalis, Lumbriculus aariegatus, Tubifex tubifex, Planorbis complanatus, and Asellus meridianus also occurred in considerable though lower numbers; other species occurred in low numbers.
3. The life-cycles and changes in numbers of the more numerous species are considered. The life-histories of D. lacteum, P. nigra, H. stagnalis, P. complanatus, A. meridianus are in agreement with published information. P. typhlops is seasonal in occurrence, being active from May to Sept. inclusive. Times of emergence of adults of various insect species agree with information available in the literature.
4. The life-cycle of L. hoffmeisteri in the pond is as follows: young worms hatch in spring/summer and form the bulk of the population from April to July/Aug; they mature from Aug. onwards and breeding starts in earnest from Feb./March. The life-cycle of T. tubifex is as follows: breeding starts in Feb., recruitment of young takes place from April till June, and these start to mature in Nov./Dec. It is not certain if some animals which breed in the spring/early summer survive to breed the following year.
5. The life-cycle of C. crystallinus appears to be as follows: first instars present from May to Oct., second instars from May to Dec., third instars from June till following Jan., fourth instars all the year round, pupae from May till Aug., and eggs from May to Sept. Adult emergence takes place from late April till mid-Sept.
6. A six-week drought in Oct/beginning Nov. in the second year of the investigation caused considerable mortality in most species, but most survived with only a few exceptions.

     

Inhibition of Transformation of Bacillus subtilis by Heavy Metals

Mercuric ions, as well as organomercuric ions and cadmium ions, can inhibit deoxyribonucleic acid-mediated transformation in Bacillus subtilis 168 without decreasing the viability of the total population. Differences in the inhibition of transformation by mercuric ions are identifiable on a temporal and concentration dependence basis. Sensitivity to low concentrations (9.2 x 10(-8) M) appears early in the uptake of deoxyribonucleic acid before the transformed markers have become insensitive to deoxyribonuclease. Resistance to "low concentrations" of Hg(2+) is kinetically indistinguishable from the requirement for magnesium in the transformation process. This inactivation is not reversed by the mercury-binding compound glutathione. Sensitivity to mercuric ions at a higher concentration (5.52 x 10(-7) M) occurs after the donor deoxyribonucleic acid has become insensitive to deoxyribonuclease. These complex interactions between mercuric ions and the process of transformation are discussed.     

Sodium Content of Community Water Supplies in California

The amount of sodium ion in water used for ingestion may be critical in effective use of a low sodium dietary regimen. Waters containing not over 20 mg of sodium per liter are provided for in the sodium restricted diets set forth by the American Heart Association. For diets containing more than 500 mg of sodium a day, waters of greater sodium content may be used if proper dietary adjustments are made.While assessment of the long-term average sodium content of a community water supply is difficult, the determined values for sodium lend to classification within range categories. The larger community water supplies in California are presented within several range categories of sodium content.The more commonly used water softeners add sodium to water. The sodium-restricted patient should be cautioned against their use. Similar consideration should probably be given to water supplies of retirement communities where the potential for disorders requiring sodium restriction is greater than in the general population.