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Challenge studies were carried out to evaluate the safety of a reformulated sous-vide processed spaghetti and meat-sauce product (a w 0.992-0.972, pH 4.5-6) inoculated with Clostridium botulinum types A and B spores. Following processing at 75°C for 36 min (equivalent to a 13 D process for Streptococcus faecium ), samples were stored at 15°C and analyzed at selected time intervals for toxin production and visible signs of spoilage. Toxin was detected in samples of pH > 5.5 after 14-21 days and in products of pH 5.25 after 35 days. Toxin was not detected in any samples of pH < 5.25 within 42 days storage at 15°C. All products of pH 5.75 and 6 were visibly spoiled, i.e., swollen due to CO 2 production, prior to toxigenesis. However, for products of pH 5.5 and 5.25, toxigenesis preceded spoilage. Subsequent studies were done to test the effect of additional salt (1-3% w/w) on the safety of a product of pH 5.5 at mild temperature-abuse storage conditions (i.e., 15°C). Toxin production was not delayed in samples containing < 1.5% added salt, while > 1.5% salt (w/w) prevented toxin production throughout the 42-day storage period at 15°C. Microwave heating of products for 5-10 min at full or half power in a domestic microwave (800 watts) inactivated the preformed toxin in all samples.

Restriction endonuclease analysis has revealed extensive mtDNA polymorphism in two species of rats, Rattus rattus and Rattus norvegicus. Sequence divergence values for the eight detected R. norvegicus variants range from 0.2% to 1.8% and for the eight R. rattus variants, from 0.2% to 9.6%. Three of the most closely related R. norvegicus mtDNA's appear to differ by deletions/insertions of about 4 base pairs apiece. Restriction sites for seven enzymes have been mapped for 11 of these variants. The 31 intraspecific and 41 interspecific variant sites appear to be evenly distributed on the mtDNA molecule outside of the rRNA cistrons. The location of sites present in all the DNAs suggests that the rRNA genes and possibly the light strand origin of replication may be more highly evolutionarily conserved than other parts of the molecule. The sequence divergences among the mtDNAs of animals whose geographic origins are separated by major barriers, such as oceans, were significantly greater than those among animals found within large land masses, such as the continental United States. Dendrograms (phenograms), which have been constructed to depict the relationships among the various DNAs, indicate that East Asian members of the R. rattus species are more closely related to American rats of this species than to other Asian R. rattus animals from Sri Lanka. Moreover, it appears that R. norvegicus comprises a group taxonomically distinct from any of the R. rattus subspecies.

A topoisomerase (nicking-closing enzyme) has been isolated from rat liver mitochondria. It has purified by double-stranded DNA-cellulose chromatography approximately 50,000-fold, based on the crude mitochondrial extract. It possesses a minimum specific activity of 1.9 x 10(5) units/mg. The enzyme has been shown to be distinctly mitochondrial, differentiated from the nuclear topoisomerase by its sensitivity to the intercalating drug, ethidium bromide, and to the non-intercalating trypanocidal drug, Berenil.

Incorporation of labeled deoxynucleoside triphosphates into mtDNA by isolated rat liver mitochondria has been shown previously to reflect DNA replication. We have used this system to seek evidence for a mtDNA gyrase. Coumermycin, novobiocin, nalidixic acid, and oxolinic acid are known to be inhibitors of Escherichia coli gyrase, to inhibit E. coli DNA replication, to abolish colicin E1 replication, and to depress the supercoiling of phage lambda DNA, the last two via inhibition of the DNA gyrase. Our results show that these agents inhibit [3H]dATP incorporation into bulk mtDNA at concentrations similar to those used for E. coli. Analysis by sucrose gradient sedimentation confirms the inhibition and shows further that the synthesis of the highly supercoiled form of mtDNA (i.e. 39 S DNA) is depressed relative to other mtDNA forms (i.e. 27 S DNA), suggesting an inhibition of the supercoiling process. Analysis of the DNA by CsCl/propidium diiodide centrifugation shows, in addition, that incubation with coumermycin results in the appearance of a mtDNA form shown to be relaxed mtDNA. The results are consistent with the occurrence of a mtDNA gyrase and its operation in mtDNA replication.

Restriction enzyme analysis of mtDNAs for the purpose of determining sequence divergence rests on the assumption that variant recognition sites differ with respect to sequence and not methylation. This assumption was tested on two mtDNAs, A and B, which are distributed throughout the laboratory rat population and which can be distinguished by a number of restriction enzymes. The mtDNAs were cloned and the nucleotide sequences of corresponding small HindIII fragments, in which a variant EcoRI site occurs, were determined. Evidence that the fragments differ in sequence and not methylation is as follows: (i) The cloned mtDNA yielded the same fragment pattern as did native mtDNA when treated with EcoRI, Hha I, HinfI, and Hae III; (ii) three nucleotide replacements were found in the 169-base pair fragment, A.T in equilibrium G.C, A.T in equilibrium G.C, T.A in equilibrium G.C; (iii) one of these replacements, A.T in equilibrium G.C at position 80, accounts for the presence of the EcoRI site in the type A and its absence in the type B mtDNA. Examination of the sequence leads to the suggestion that these three nucleotide replacements are silent; i.e., they would not lead to amino acid substitutions in a possible encoded protein.

The fluorescent reagent N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-AEDANS) was employed to detect and study the previously reported conformational change in the Escherichia coli ribosome induced by streptomycin. Labeling of ribosomes with this probe, which results in the derivatization of proteins S18 and L31', described earlier, inhibits neither their ribosomal protein synthesizing nor misreading ability. To calculate the amount of streptomycin bound to the ribosome, we determined the K'D for streptomycin, which is 0.24 micron, indicating that under our conditions, bound streptomycin/ribosome molar ratios are low, not in excess of 1. Under these conditions, streptomycin addition induces fluorescence quenching by 15% but does not affect streptomycin-resistant ribosomes. Maximal misreading occurs at these same ratios. Removal of AEDANS-L31' from the ribosomes drastically reduces streptomycin-induced quenching indicating the involvement of the environment of this protein in streptomycin action. The finding that streptomycin decreases AEDANS-L31' affinity for the ribosome supports this view. Streptomycin has been shown to bind to the 30 S subunit protein S4 while the 50 S protein L31' has been shown to be localized at the subunit interface. Thus, the observation that streptomycin influences this 50 S subunit protein L31', combined with the tight correlation between the effects of streptomycin on quenching and on misreading, strongly suggests that this antibiotic induces a conformational change at the subunit interface of the ribosome, and that this results in misreading. Polyuridylic acid also induces a conformational change in the ribosome but the polynucleotide and streptomycin seem to act independently. Streptomycin-resistant ribosomes, which undergo neither streptomycin-induced fluorescence nor streptomycin-induced misreading, are resistant to misreading induced by high Mg2+ as well.

When incubated with isolated intact rat liver mitochondria, novobiocin and nalidixic acid act as uncouplers of oxidative phosphorylation; they stimulate oxygen uptake and inhibit ATP synthesis. Novobiocin is about as powerful an uncoupler as is 2,4-dinitrophenol, nalidixic acid is somewhat less powerful, and oxolinic acid exerts no inhibition whatsoever at the concentrations used. The three inhibitors are without effect on oxidative phosphorylation in Escherichia coli nor does novobiocin affect this process in a novobiocin-permeable mutant of yeast. While it would appear that oxolinic acid may be a relatively specific tool for the manipulation of the superhelicity of DNA in complex systems such as mammalian mitochondria and intact mammalian cells, the specificity of each of these inhibitors may depend upon the particular conditions and species used and such experiments require adequate controls on oxidative phosphorylation.

The structure of Na,K-ATPase has been studied by electron microscopy and image reconstruction. A three-dimensional structure of this enzyme has been obtained to an overall resolution of 2.5 nm using data from specimens of negatively stained dimer sheets tilted through a range of angles +/- 60 degrees. The reconstruction shows a complex mass distribution consisting of ribbons of paired molecules extending approximately 6.0 nm from the cytoplasmic side of the membrane. The molecular envelope consists of a massive "body" with "lobe" and "arm" structures projecting from it. The body has a columnar shape and is tilted with respect to the plane of the membrane. The region of interaction responsible for dimer formation is located between two bodies and is clearly visible in the reconstruction. It has been identified as a segment in the amino-terminal portion of the alpha subunit. The arms that interconnect the ribbons are located close to the membrane and are most probably formed by the beta subunits.

The sequence of the region of the mitochondrial genome that encodes cytochrome oxidase subunit II (COII) has been determined for each of two closely related rat species, Rattus norvegicus and R. rattus. Comparison of the two sequences shows that 94.4% of the nucleotide substitutions are silent. The occurrence of this high proportion of silent substitutions leads us to propose that the rapid evolution of mtDNA relative to nuclear DNA is due only to silent changes and that amino acid-altering substitutions accumulate in nuclear and mtDNA at comparable rates. Other novel features of the nucleotide substitution pattern in the rat COII gene are a high transition/transversion ratio (8.0:1) and a strong bias toward C in equilibrium T transitions in the light strand. Comparison of the R. norvegicus COII sequence with the bovine and human sequences show that there may be selective constraints on some silent positions within the gene and that its rate of evolution may be different in different mammalian lineages.

The use of rapid steam distillation followed by redox iodine titration provides a rapid and accurate determination of total sufite residual in shrimp. Values obtained for sulfite-treated shrimp using the rapid distillation method gave comparable results to those of the officially recognized Monier-Williams method. Values for the rapid distillation method ranged from 6 to 212 ppm while those of the Monier-Williams procedure ranged from 6 to 241 ppm for untreated and treated shrimps, respectively. Statistical analysis using two-sample Student's t-test indicated that there were no significant differences (p>0.05) for residual levels below 100 ppm but the values obtained by the rapid distillation method and the Monier-Williams procedure were significantly different (p<0.05) at concentrations near and above 100 ppm.

Sucrose density gradient fractionation of isolated rat liver mitochondrial DNA ordinarily yields two peaks, one at 39 S, the other at 27 S. However, when these mitochondria are first incubated with a labeled DNA precursor, a labeled peak at about 8 S is also observed. Is this low molecular weight 8 S DNA merely an artifact of contamination or breakdown, or is it a functioning part of the mitochondrial genome? That it is not a nuclear contaminant is shown by: (a) the absence of nuclei or nuclear fragments in active mitochondrial preparations; (b) the insensitivity of 8 S DNA synthesis to treatment of mitochondria with DNase and RNase; (c) the ability of inner membrane preparations to synthesize this DNA; (d) the ability of atractyloside to inhibit incorporation of [3H]dATP into 8 S and 39 S or 27 S DNA equally; (e) the labeling of 8 S DNA (as well as 39 S and 27 S DNA) but not of nuclear DNA after the administration in vivo of [3H]thymidine. The evidence that 8 S DNA is not an artifact resulting from DNA breakdown during mitochondrial incubation or DNA isolation is as follows: (a) 8 S DNA can be isolated from unincubated mitochondrial; (b) 8 S DNA becomes labeled when labeled DNA precursors are administered in vivo; (c) 8 S DNA biosynthesis continues in the complete absence of labeled 39 S or 27 S DNA (whose synthesis is repressed by ethidium bromide), making it unlikely that 8 S DNA is formed from the breakdown of 39 S or 27 S DNA; (d) substitution of milder methods of DNA extraction does not decrease 8 S DNA labeling; moreover, the usual conditions of extraction, when applied to purified 39 S and 27 S DNA, do not generate 8 S DNA, nor does an additional mitochondrial washing cycle; (e) the specific radioactivity of 8 S DNA is higher than that of 39 S or 27 S DNA, making it improbable that the latter forms are precursors of 8 S DNA. Since 8 S DNA is double-stranded, it is not identical to the 7 S fragment of D loop DNA. The hypothesis that the artifactual nicking of those DNA molecules which contain opposing D loops leads to the release of double-stranded fragments was tested. The DNA which was released was predominantly (and probably completely) single-stranded. We conclude that 8 S DNA is probably not an artifact and studies are in progress on its function.

Thymidine triphosphate-2-C(14) and other deoxynucleoside triphosphates are incorporated into the DNA of isolated mitochondria from rat liver cells. The reaction is partially dependent on the other deoxynucleoside triphosphates and proceeds in the virtual absence of bacteria. The product has the properties of mitochondrial rather than nuclear DNA, and end-group studies indicate labeling of the interior of the molecule.

The previously observed incorporation of deoxynucleoside triphosphate precursors into DNA by isolated rat liver mitochondria could reflect either replication of DNA or a repair process. Density labeling experiments in cesium chloride demonstrate that DNA synthesized in the presence of 5-bromodeoxyuridine triphosphate instead of thymidine triphosphate shows an appreciable increase in density. In some of the molecules undergoing synthesis, the amount of the density increase indicated a replacement of thymine by bromouracil to the extent of 33 percent. This extensive replacement, which would compute to twice this amount if only a single strand of the duplex is labeled, provides evidence for the synthesis of fairly long pieces of DNA. Such synthesis is characteristic of replication rather than repair, and the results thus suggest that mitochondria are able to replicate their own DNA.

Translocation in ribosomes consists of transposition of peptidyl-tRNA from the aminoacyl to the peptidyl site and, probably concomitantly, the movement of ribosomes on mRNA. Does a conformational change in the ribosome provide the motive force for this process? Hydrogen exchange and sedimentation velocity experiments indicate that the Escherichia coli ribosome does undergo a conformational change associated with translocation. When pretranslocational ribosomes carrying acetyldiphenylalanyl-tRNA in the aminoacyl site were incubated with G factor and GTP, translocation occurred, with a concomitant increase in hydrogen exchange rate and a decrease in sedimentation constant. These changes did not occur when GTP was replaced by a nonhydrolyzable analogue, GDP-CH(2)-P, and they were blocked by the antibiotics fusidic acid and thiostrepton. When posttranslocational ribosomes were cycled back to the pretranslocational state by T factor, GTP, and phenylalanyl-tRNA, the sedimentation constant reverted to the original value. Whether or not this conformation change drives translocation requires further study.

The role played by ribosomal conformation in codon-anticodon recognition has been studied using streptomycin as a probe, inasmuch as streptomycin is known to cause misreading of the genetic code. Changes in ribosomal structure have been followed by the method of hydrogen-tritium exchange. The results show that streptomycin induces two types of change in the hydrogen exchange pattern. At low molar ratios of streptomycin to ribosomes, a stimulation of the hydrogen exchange rate ("loosening" of ribosomal structure) is observed, with a small inhibition of polypeptide synthesis. As the streptomycin: ribosome ratio is increased, a maximum exchange rate is reached, after which the rate decreases ("tightening" of structure); in this region, inhibition of peptide synthesis increases sharply, and misreading of the code begins. None of these effects is observed with streptomycin-resistant ribosomes.