Dictyostelium discoideum plasmid containing an AP-endonuclease upstream sequence: bleomycin induction of a luciferase reporter.

A sequence of 1624 bp 5' to the apurinic/apyrimidinic (AP) endonuclease structural gene of Dictyostelium discoideum (APEA) has been inserted upstream of the luciferase reporter gene in pVTL2, an autonomously replicating nuclear plasmid in this organism. Cells transformed with this plasmid, designated pVTL-AL, displayed strong luciferase induction during treatment with the DNA-damaging agent bleomycin. For example, a luciferase activity of 45-fold above the constitutive level was observed for 20 hours of growth in axenic medium with 0.002 U/mL of bleomycin. The response was bleomycin concentration-dependent. Cell survival was greater than 90% for all treatments. The level of luciferase expression was highly dependent on the cell growth conditions, with the greatest induction observed for stationary phase axenically-grown cells. This effect may be related to a variation of plasmid copy number with growth conditions.

Validation of a 5-log10 reduction of Listeria monocytogenes following simulated commercial processing of Lebanon bologna in a model system.

Recently, numerous product recalls and one devastating outbreak that claimed 21 lives were attributed to Listeria monocytogenes in ready-to-eat meat products. Consequently, the Food Safety and Inspection Service published a federal register notice requiring manufacturers of ready-to-eat meat and poultry products to reassess their hazard analysis and critical control point plans for these products as specified in 9 CFR 417.4(a). Lebanon bologna is a moist, fermented ready-to-eat sausage. Because of undesirable quality changes. Lebanon bologna is often not processed above 48.9 degrees C (120 degrees F). Therefore, the present research was conducted to validate the destruction of L. monocytogenes in Lebanon bologna batter in a model system. During production, fermentation of Lebanon bologna to pH 4.7 alone significantly reduced L. monocytogenes by 2.3 log10 CFU/g of the sausage mix (P < 0.01). Heating the fermented mix to 48.9 degrees C in 10.5 h destroyed at least 7.0 log10 CFU of L. monocytogenes per g of sausage mix. A combination of low pH (5.0 or lower) and high heating temperatures (> or =43.3 degrees C, 115 degrees F) destroyed more than 5 log10 CFU of L. monocytogenes per g of sausage mix during the processing of Lebanon bologna. In conclusion, an existing commercial process, which was validated for destruction of Escherichia coli O157:H7, was also effective for the destruction of more than 5 log10 CFU of L. monocytogenes.

Apurinic/apyrimidinic (AP) endonuclease from Dictyostelium discoideum: cloning, nucleotide sequence and induction by sublethal levels of DNA damaging agents.

We have cloned an AP endonuclease gene (APEA) from Dictyostelium discoideum, along with 1.8 kb of the 5' flanking region. There are no introns. The sequence predicts a protein of 361 amino acids, showing high homology to the major human/Escherichia coli exonuclease III family of AP endonucleases. There is 47% identity and 64% similarity to the Ape endonuclease of human cells using the C-terminal 257 amino acids of the Dictyostelium protein. The 104 amino acids on the N-terminus show only low homology with other AP endonucleases. Instead, this region shows high homology with the acid-rich regions of proteins associated with chromatin, such as nucleolins and HMG proteins. The gene is transcriptionally activated up to 7-fold after treatment of cells with sublethal levels of DNA damaging agents, including ultraviolet light, MNNG and bleomycin. Induction does not occur following blocking of replication fork polymerases with aphidicolin. It is not eliminated by treatment with kinase or phosphatase inhibitors. Four DNA damage-sensitive mutants all retained the DNA damage-induced up-regulation.

Some repair-deficient mutants of Dictyostelium discoideum display enhanced susceptibilities to bleomycin.

Dictyostelium discoideum, a soil eukaryote, is highly resistant to DNA-damaging agents; repair mutants are more susceptible. Susceptibility to bleomycin, produced by Streptomyces verticillus, is greater for mutants which are susceptible to other agents than for resistant strains. The high potential for DNA repair may result from the need to cope with chemicals produced by other soil microorganisms.

Uracil-DNA glycosylase activity from Dictyostelium discoideum.

We have isolated and partially characterized a uracil-DNA glycosylase activity from the cellular slime mold, Dictyostelium discoideum. This glycosylase has a broad pH optimum (6.5-8.5) and is fully active in 10 mM EDTA or in 5 mM Mg2+. Its molecular weight by gel filtration is about 55 000. This enzyme activity may work in concert with previously described apurinic/apyrimidinic (AP) endonuclease activities in the excision repair of uracil from the DNA of this lower eukaryote.

Acid DNAase activity from Dictyostelium discoideum.

We have isolated and partially characterized an acid endonuclease activity from the cellular slime mold, Dictyostelium discoideum. This activity comprises more than 90% of the nonspecific DNA-endonuclease activity of the vegetative cells. Its molecular weight is about 44 000, and its activity is enhanced 7-fold by Mg2+. The pH optimum for the nicking activity depends upon NaCl concentrations, being at pH 5.0 in 207 mM NaCl, and at pH 5.8 in 7 mM NaCl. Large quantities of this enzyme activity are released into the growth medium or buffer, with detectable amounts appearing within 15 min of incubation.

Apurinic/apyrimidinic-specific endonuclease activities from Dictyostelium discoideum.

Two apurinic/apyrimidinic- (AP-) specific endonuclease activities have been isolated from the cells of Dictyostelium discoideum by fractionation on DEAE-cellulose, CM-cellulose and Sephadex G-75. These activities, designated A and B, have apparent molecular weights of 49000 and 40000, respectively. Although their precise reaction optima differ somewhat, both A and B quantitatively nick AP DNA best at pH 8.0-8.5 in low salt (less than 100 mM NaCl); both require Mg2+. These activities are apparently specific only for AP sites in DNA. The low activities observed on heavily ultraviolet-irradiated DNA, gamma-irradiated DNA and osmium tetroxide-treated DNA are consistent with the small numbers of secondary AP sites expected in these DNAs. Both A and B produce single-strand nicks in AP DNA that result in termini that serve as good primers for Escherichia coli polymerase I. Hence, A and B appear to be Class II AP endonucleases which yield 3'-OH termini at nicks on the 5' side of baseless sugars. It is unclear whether A and B are independently coded proteins, different post-translational modifications of the same gene product, or whether one is an artifact arising from the isolation. Many of the properties of these D. discoideum AP endonuclease activities are similar to those of the predominant AP endonucleases observed in bacterial, plant and animal cells. They will be of use in the characterization of excision repair in this organism.

Bone parameters of thick and thin eggshell lines of chickens.

Comparative measurements of bone parameters were made on chickens from thick and thin eggshell lines that were maintained on a normal diet containing 3.5% calcium, injected i.v. with 45CaCl2 and sacrificed 30 min later. There were no significant differences between shell lines for the following measurements on the left femur: specific gravity, ash wt, total calcium, percent calcium and 45Ca content. There was deposition of 45Ca in the femur of all hens--even those in the process of eggshell formation. Skeletal metabolism was not a limiting factor in determining whether a hen produced a thick or thin eggshell.

Avian riboflavinuria--XI. Immunological quantitation of cross-reacting liver proteins from normal, heterozygous, and mutant hens.

A genetic defect, avian riboflavinuria, was discovered in a strain of Single Comb White Leghorn chickens and has been attributed to the absence of functional riboflavin-binding protein (RBP). The ratio of functional RBP in blood, egg yolk, and egg white was 2 (RdRd) : 1 (Rdrd) : 0 (rdrd). The present study on non-riboflavin-binding, cross-reacting proteins (CRPs) from RdRd, Rdrd, and rdrd hens involved partial purification and immunochemical quantitation using antiserum to RBP. Immunoreactivities (microgram/g liver) of CRPs were found to be 2.6 (RdRd) : 1.3 (Rdrd) : 0.02 (rdrd). Reciprocal cross-reactions were observed with rabbit sera directed toward both RdRd CRP and RBP. Reaction of the CRPs with antiglycopeptide serum (specific for beta-linked galactose) showed that they were glycosylated. CRPs from RdRd and Rdrd hens had relative antigenicities similar to that of RBP (Krel approximately or equal to 1), while rdrd CRP had a significantly lower antigenicity (Krel = 0.003). The molecular weights of the CRPs as determined by SDS-polyacrylamide gel electrophoresis were as follows: RdRd = 31,6000, Rdrd = 30,900, and rdrd = 27,500. The molecular weight of egg yolk RBP was 34,7000 by the same method. The conclusion is drawn that the rd gene codes for a nonfunctional mutant protein, possibly an "altered precursor", that is different from RBP.

Effect of estradiol on calcium and calcium binding in serum of thick and then-shell lines of chickens.

Estradiol (10 mg/bird) was injected intramuscularly into hens of a thick-shell line and a thin-shell line as well as into roosters of both lines on day 0. Serum levels of calcium (Ca), phosphorus (P), and Ca binding involving the proteins vitellogenin (a phosphoprotein) and albumin were measured on day 0 and day 6 or 7. Serum levels of all measured Ca and P parameters and vitellogenin binding of both sexes were significantly greater, while levels of albumin binding were significantly less on day 6 or 7 than on day 0. Serum levels of non-diffusible Ca in hens and vitellogenin binding in both sexes were significantly greater in the thick-shell than in the thin-shell line. There were no significant differences between lines for total serum P of either sex. A study of the hens only revealed that the change in nondiffusible Ca and vitellogenin binding from day 0 to day 6 or 7 was significantly different between lines. The correlations between vitellogenin binding and total P, expressed as either change or percent change, were greater than .8.

Calcium-binding proteins in serum of chickens: vitellogenin and albumin.

The Ca-binding proteins of hen serum were resolved by gel filtration in 45Ca buffer and were named CaBP(1) and CaBP(2). The major calcium-binding protein, CaBP(1), had a molecular size of 6.0 X 10(5) daltons and appears to be vitellogenin. Deeley et al. (1975) described vitellogenin as the precursor of lipovitellin and phosvitin, the Ca-binding proteins of egg yolk. The present paper demonstrates that "native" vitellogenin is a calcium-binding protein in the serum of laying chickens. The CaBP(2) co-eluted with and was immunologically identical to chicken serum albumin which also bound 45Ca. Data from elution profiles and phosphorus assays indicated that CaBP(1) and CaBP(2) were different from phosvitin and the Ca-binding proteins of the duodenum and uterus.

Calcium-binding proteins in serum: quantitative differences between thick and thin shell lines of chickens.

Calcium binding by two proteins, vitellogenin and albumin, was measured in serum of a line of hens producing thick (THK) and a line of hens producing thin shells (THN) as well as a line of commercial hens using gel filtration in 45calcium buffer. Vitellogenin was quantitated in serum of THK and THN using a radial immunodiffusion test. Levels of serum calcium were measured and related to the above mentioned parameters. The binding of vitellogenin was significantly greater in 13 THK than 13 THN (672 vs. 508 cpm/ml X 10(-3), but binding by albumin was not significantly different between the two lines (386 vs. 344 cpm/ml X 10(-3). Binding in 11 commercial hens was similar to that for THN. The THK had significantly greater levels of diffusible, non-diffusible, and total serum calcium than THN. Significant positive correlations between total serum calcium and vitellogenin binding were found in THK (.77) and THN (.81) as well as in the commercial hens (.64). Like vitellogenin binding, levels of vitellogenin were significantly greater in THK than THN (4.0 vs. 1.8 mg/ml). These results suggest that in addition to having more diffusible and non-diffusible serum calcium, THK have more calcium binding and more vitellogenin to perform the binding function than THN.

Urinary excretion of calcium in the presence or absence of shell formation by chickens producing thick or thin shells.

The pattern of urinary calcium excretion before and during shell formation was observed for White Leghorn chickens producing thick or thin shelled eggs. Urine samples were collected at 2.5, 7.5, 12.5, and 20 hr after oviposition. Urine was collected in a tube inserted to cover both ureters within the cloaca and calcium levels were determined by atomic absorption. Urinary calcium was found to decrease similarly in both shell lines from 44 mg % to 5 to 6 mg % during shell formation. When no shell was being produced, hens of both shell lines excreted calcium in the urine at 34 to 53 mg % levels throughout the 20-hr period.