Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.     

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.     

Effect of Calcium on the Structure and Template Activity of Pea Chromatin

The absorption spectrum of native pea chromatin solubilizedunder minimal shearing conditions changed with increasing Ca²⁺concentration; the ratio of maximum to minimum absorption decreasedand the maximum absorption peak shifted to a longer wavelength.The concentration of Ca²⁺ to cause half complete sedimentationof chromatin was much lower for the solubilized native chromatin(more condensed and larger in size) than for the sonicated chromatin(less condensed and smaller in size). Solubilized native chromatinshowed a two-step melting profile in the absence of Ca²⁺. In the presence of Ca²⁺ the two T_ms disappeared and a new higherT_m appeared. Template activity of solubilized native chromatinincreased 3-fold upon dispersion and fragmentation by sonication.Addition of a small amount of ethylene glycol-bis (ß-aminoethylether)-N, N'-tetraacetic acid (EGTA) promoted the template activityof solubilized native chromatin, but not that of sonicated ordenatured DNA. The effect of EGTA was reversed by Ca²⁺. Thechromatin reconstituted in the presence of EGTA showed a lowerT_m than the chromatin reconstituted in the presence of Ca²⁺.The relationship between chromatin structure and its templateactivity is discussed in relation to Ca²⁺. (Received August 12, 1985; Accepted December 7, 1985)     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

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.