DNA polymerase III holoenzyme of Escherichia coli. I. Purification and distinctive functions of subunits tau and gamma, the dnaZX gene products

Escherichia coli dnaZX, the gene which when mutant blocks DNA chain elongation, was cloned into a lambda PL promoter-mediated expression vector. In cells carrying this plasmid, the activity that complements a mutant dnaZ extract in replicating a primed single-stranded DNA circle was increased about 20-fold. Two polypeptides of 71 and 52 kDa were overproduced. Upon fractionation, two complementing activities were purified to homogeneity and proved to be the 71- and 52-kDa polypeptides. Immunoassays revealed their respective identities with the tau and gamma subunits of DNA polymerase III holoenzyme. The N-terminal amino acid sequences of the first 12 residues were identical in both subunits, as were their molar specific activities in dnaZ complementation. Thus, the tau subunit complements the defect in the mutant holoenzyme from the dnaZts strain as efficiently as does the gamma subunit. Inasmuch as the 71-kDa subunit (tau) can also overcome the enzymatic defect in a dnaX mutant strain, this polypeptide has dual replication functions, only one of which can be performed by the gamma subunit. Availability of pure tau and gamma subunits for study has provided the basis for proposing an asymmetry in the structure and function of a dimeric DNA polymerase III holoenzyme.     

Platelet-activating factor induces glycogen degradation in fetal rabbit lung in utero

The role of platelet-activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in initiating glycogen breakdown in the fetal rabbit lung was assessed by intraperitoneal administration of this potent ether-linked glycerophospholipid. Forty-five min after in utero injection of PAF (2.5 X 10(-7) mol), fetal pulmonary and hepatic glycogen concentrations were reduced from 326 to 256 and from 9.8 to 6.6 micrograms of glycogen/mg protein, respectively. Glycolytic activity was similarly increased as judged by an elevation of lactate (2-fold) in lung, liver, and plasma upon PAF injection. These actions of PAF were dose- and time-dependent. The glycogenolytic response did not occur when an equimolar dose of the inactive enantiomer, D-PAF was injected. Pretreatment of the fetus with a specific PAF receptor antagonist, SRI-63-441, prevented the PAF response. We have previously demonstrated (Hoffman, D. R., Truong, C. T., and Johnston, J. M. (1986) Biochim. Biophys. Acta 879, 88-96) that PAF biosynthesis and PAF concentrations increase significantly on day 24 of fetal rabbit lung development. A concurrent decrease in pulmonary glycogen concentration at this point of gestation is potentially reflective of the PAF-induced action. Thus, these observations would suggest a role for PAF in the normal physiology of fetal lung maturation.     

Recovery and diversity of heterotrophic bacteria from chlorinated drinking waters.

Heterotrophic bacteria were enumerated from the Seattle drinking water catchment basins and distribution system. The highest bacterial recoveries were obtained by using a very dilute medium containing 0.01% peptone as the primary carbon source. Other factors favoring high recovery were the use of incubation temperatures close to that of the habitat and an extended incubation (28 days or longer provided the highest counts). Total bacterial counts were determined by using acridine orange staining. With one exception, all acridine orange counts in chlorinated samples were lower than those in prechlorinated reservoir water, indicating that chlorination often reduces the number of acridine orange-detectable bacteria. Source waters had higher diversity index values than did samples examined following chlorination and storage in reservoirs. Shannon index values based upon colony morphology were in excess of 4.0 for prechlorinated source waters, whereas the values for final chlorinated tap waters were lower than 2.9. It is not known whether the reduction in diversity was due solely to chlorination or in part to other factors in the water treatment and distribution system. Based upon the results of this investigation, we provide a list of recommendations for changes in the procedures used for the enumeration of heterotrophic bacteria from drinking waters.     

Contra-IL 2; a suppressor lymphokine that inhibits IL 2 activity

Suppressive activity of culture supernatant of AS-9 (AS-9 CS), a T cell hybridoma line that was derived from fusion of BW5147 thymoma and splenic T cells of anti-lymphocyte serum-treated C3H mice, was analyzed. AS-9 CS inhibited allogeneic cytotoxic T lymphocyte (CTL) generation as well as T cell proliferation to alloantigens and mitogens, but failed to inhibit B cell response to lipopolysaccharide or growth of tumor and fibroblast cells. Although addition of AS-9 CS to the allogeneic sensitization culture as late as on day 2 of incubation resulted in maximal inhibiton of CTL generation, removal of AS-9 CS on day 3 of incubation abolished its inhibitory effect. Addition of purified IL 2 together with AS-9 CS to the allogeneic sensitization cultures only partially abrogated the suppression. Experiments with IL 2-dependent cytotoxic T cell line (CTLL) showed that AS-9 CS suppressed the IL 2-induced proliferation of CTLL. Preincubation of AS-9 CS with CTLL removed its inhibitory effect on CTL generation. These results indicate that AS-9 CS interferes with the mechanism of T cell activation by IL 2. On this basis, AS-9 CS was named contra-IL 2.     

Interactions in Syntrophic Associations of Endospore-Forming, Butyrate-Degrading Bacteria and H2-Consuming Bacteria

Butyrate is an important intermediate in the anaerobic degradation of organic matter. In sulfate-depleted environments butyrate is oxidized to acetate and hydrogen by obligate proton reducers, in syntrophic association with hydrogen-consuming methanogens. This paper describes two enrichments of endospore-forming bacteria degrading butyrate in consortia with methanogens. The isolates are readily established in coculture with H₂-consuming, sulfate-reducing bacteria by pasteurizing the culture. The two original enrichments differed in that one grew to an optically dense culture while the second grew in clumps. Examination by scanning electron microscopy showed that clumping resulted from the production of large amounts of extracellular polymer. Several H₂-consuming methanogens were identified in the enrichments. Some of them grew closely associated to the butyrate degraders. This attachment to the hydrogen producer may permit some methanogens to compete for the growth substrate against other bacteria having higher substrate affinity.     

Estrogen-mediated induction of a vitellogenin-specific nonhistone chromatin protein in the male chicken liver

Summary Non-histone chromatin proteins prepared from the livers of estrogen-treated and nontreated male chickens were compared by reverse-phase high performance liquid chromatography (RP-HPLC), followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results revealed that the hormone-treated male liver chromatin contained a specific protein corresponding to the vitellogenin-specific protein previously purified from the liver of egg-laying hens (Nakayama 1985). The chicken protein, purified further by gel-filtration high performance liquid chromatography (GF-HPLC), showed specific binding activity to DNA fragments carrying a part of the vitellogenin gene. On the basis of similarities in the elution patterns from GF-HPLC and RP-HPLC as well as in the mobility on SDS-PAGE, we concluded that this hormone-induced protein in the male chicken liver was identical to the vitellogenin-specific protein identified in the hen liver, and assumed it to be a specific regulatory protein for the vitellogenin gene expression. The amino acid composition of this chicken protein has been determined.     

Correlation between limitation of growth ofPachysolen tannophilus on D-xylose with the formation of ethanol and other products

The formation of ethanol, xylitol, ribitol, arabitol and acetic acid from D-xylose byPachysolen tannophilus correlated with the limitation of growth. The correlation was consistent with these products being secondary metabolites.Issued as NRCC Publication Number 24259.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.     

Climate and vegetation in China II. Distribution of main vegetation types and thermal climate

The authors examined relationships between Kira's warmth index (WI) and four other important thermal indices: the sums of daily mean temperatures above 5°C and 10°C, Thornthwaite's potential evapotranspiration (PE) and Holdridge's annual biotemperature. The thermal records of 671 meteorological stations evenly located all over China were used to make these comparisons. Close correlations were found within the four relationships, and accordingly WI was used to analyse the thermal distributions of the main vegetation types. Vegetation types around the 671 stations were read from a vegetation map with a scale of 1/4000000. Vegetation types at 269 stations corresponded to the natural or seminatural vegetation, and 29 vegetation types were distinguished by arranging the 269 data into the same or similar types. The geographical distribution of these 29 types and the corresponding main climatic features were described. The relations between WI and distribution of these vegetation types were discussed in detail. As a result, WI values (°C month) corresponding to the vegetation zones could be summarized as follows: (1) arctic or alpine vegetation zone: 0–15; (2) boreal or subalpine vegetation zone: 15-(50–55); (3) cool-temperate vegetation zone: (50–55)–(80–90); (4) warm-temperate vegetation zone: (80–90)–(170–180). These values almost coincided with Kira's values. Chinese postgraduate student in Japan sent by the Chinese Government.     

Carbon-cycling changes during regeneration of a decinduous broadleaf forest after clear-cutting II. Aboveground net production

Growth and death rates of aboveground plant parts were measured in a mature forest and four different-aged deciduous broadleaf forests regeneratede after clear-cutting, with special reference to rates for woody parts (stems and branches) of different diameters (ø) in rerms of the pipe model theory (Shinozaki et al., 1964). The total biomass increment of woody parts of trees higher than 1.3 m varied within a range of 2.1-4.6 ton ha^?1 yr^?1, the increase beingdue largely to the growth of canopy trees exposed to direct sunlight. Biomass increments of small (ø<1 cm) and medium (1≤ø<5 cm) woody parts were negligibly small except in the youngest forest, and changes in aboveground woody biomass with forest age after clear-cutting mainly resulted from accumulation of large (5 cm<ø) woody parts of canopy trees. Biomass loss of trees due to death and grazing increased with forest age from 4.0 to 8.3 ton ha^?1 yr^?1. Recovery of leaf and small wood falls was observed at the early stage of regeneration, while large wood falls increased during regeneration. Flower and fruit fall was markedly higher in the mature forest than in the other four forest types. Mortality of woody parts became higher with forest age and was 20, 5.0 and 0.46% yr^?1 for small, medium and large parts, respectively, at the mature stage. Aboveground net production of the forest was in therange 7.6-13.3 ton ha^?1 yr^?1 with the undergrowth vegetation lower than 1.3 m being 0.4-1.4 ton ha^?1 yr^?1. Production recovered rapidly at an early stage of regeneration and was highest in mature forest.