Mutator Gene Studies in Escherichia coli

An Escherichia coli mutator gene, mutT, has been shown by P1-mediated crosses to map between the leucine and azide loci. The mutT1 and azi-r alleles cotransduce with a frequency of >92%. In mutT1/mutT⁺ merodiploids, the mutT1 phenotype is recessive; in mutT1/F′trp or mutT1/F′lac merodiploids, the mutT1 allele has a trans effect. The gene can mutate λ and T7 phage but not T1, T3, T4, T5, and S13.     

The effect of pancreatic ribonuclease on rabbit reticulocyte ribosomes and its interpretation in terms of ribosome structure

1. Parts of the 16s and 30s RNA species of reticulocytes are readily hydrolysed by pancreatic ribonuclease. The biological activity of the ribosomes is diminished after treatment with low concentrations of the enzyme (e.g. 1ng. of ribonuclease/2.5mg. of polyribosome fraction/ml.). A high proportion of the chain scissions are ;hidden' owing to the secondary structure of the RNA moiety. 2. As the concentration of ribonuclease is increased RNA is lost from the ribosome. About 20-30% of the RNA may be removed from the ribosome without altering appreciably its sedimentation coefficient or its appearance in the electron microscope. 3. The amount of RNA removed from the ribosome is not increased by raising the concentration of enzyme from about 1mug. to 2.5mg. of ribonuclease/2.5mg. of polyribosome fraction/ml., or by increasing the temperature from 0 degrees to 30 degrees , or by first converting the RNA moiety into a single-stranded form before exposure to ribonuclease. 4. Untreated polyribosomes aggregate at about 75 degrees , whereas ribosomes treated with ribonuclease aggregate at about 45 degrees . The aggregates that are found on heating ribosomes after enzymic hydrolysis contain about 40-50% of the complement of RNA of intact ribosomes. 5. From the size of the fragments of RNA isolated from RNA-depleted ribosomes it is inferred that there is one site/60-100 nucleotides that is sensitive to ribonuclease. 6. The RNA moiety of RNA-depleted ribosomes has some double-helical character as shown by the optical properties and X-ray-diffraction pattern of ribonuclease-treated ribosomes and by the ;melting' properties of the isolated RNA. 7. Subparticles prepared by titration with an excess of EDTA are readily hydrolysed by ribonuclease to fragments of S(20,w) less than 4s, in contrast with the intact particle.     

Cell population density and phenotypic expression of tissue culture fibroblasts from heterozygotes of Lesch-Nyhan's disease (inosinate pyrophosphorylase deficiency)

Radioautographic examination of skin fibroblasts grown in tissue culture from normal donors revealed heavy labeling of almost all cells following incubation with tritiated hypoxanthine. Cells from patients with Lesch-Nyhan's disease, lacking inosinate pyrophosphorylase, had only 10 grains or less per cell. When normal and abnormal cells were mixed prior to culture, there was a progressive increase, with culture time, in the percentage of heavily labeled cells so that by 96 hr, when the cells were confluent, over 95% of the cells were heavily labeled. Reduction of cell density by subculture produced a reversion to original values. Cultures from three obligatory heterozygotes revealed the expected mixed population of cells. This appears to be a practical approach to the identification of the heterozygote.Aided by USPHS CA08748 and GM15508, and the Health Research Council of the City of New York.     

Indentification and Localization of the Fatty Acids in Haemophilus parainfluenzae

Haemophilus parainfluenzae was capable of synthesizing 22 fatty acids. These fatty acids were equivalent to 4% of the bacterial dry weight. These fatty acids were localized in the membrane-wall complex, which contained the respiratory pigments, the quinone, and the phospholipids. The fatty acids which could be extracted with organic solvents comprised 86% of the total fatty acids of the cell. These fatty acids were distributed as 98% in the phospholipids and 1.9% in the neutral lipids, of which 0.5% were free fatty acids. Palmitic, palmitoleic, oleic, and vaccenic acids comprised 72% of the total fatty acids and were found almost exclusively in the phospholipids. The phospholipids also contained the cyclopropane fatty acids. The neutral lipids contained significant proportions of the odd-numbered branched and straight-chain fatty acids. The principal free fatty acids were n-dodecanoic and pentadecenoic acids. The nonextractable wall complex contained 14% of the total fatty acids. These wall fatty acids were rendered soluble only after saponification. The wall fraction contained all of the beta-hydroxymyristic acid and most of the myristoleic and pentadecenoic acids. The significance of the distribution of fatty acids between nonesterified, neutral lipid, phospholipid, and nonextractible wall remains to be determined.     

A spectrophotometric study of the secondary structure of ribonucleic acid based on a method for diminishing single-stranded base-`stacking'' without affecting multihelical structures

1. On the basis of studies with model compounds it was concluded that in 8m-urea-m-potassium chloride (or 4m-guanidinium chloride) in 0.01m-potassium phosphate buffer, pH7.0, multi-helical structures have about the same stability as in 0.1m-potassium phosphate buffer, pH7.0, whereas the tendency of base residues to ;stack' along a single polynucleotide chain is much decreased. 2. Base-pairing was eliminated whereas base-;stacking' persisted after RNA in 1% formaldehyde-0.1m-potassium phosphate buffer, pH7.0, was heated to 95 degrees . 3. From a study of the thermal denaturation of unfractionated transfer RNA from Escherichia coli and of RNA from the fractionated sub-units of rabbit reticulocyte ribosomes in 8m-urea-m-potassium chloride (or 4m-guanidinium chloride) in 0.01m-potassium phosphate buffer, pH7.0, it was inferred that ;stacked' residues may account for up to 25% of the increase in E(260) found on heating RNA in solvents such as 0.1m-potassium phosphate buffer, pH7.0. 4. Changes in the spectrum with temperature were analysed on the basis of the assumptions that (a) the polynucleotide chain is amorphous on denaturation (which is probable in 8m-urea-m-potassium chloride-0.01m-potassium phosphate buffer, pH7.0) and that (b) the polynucleotide chain adopts a single-stranded ;stacked' conformation on denaturation (which is probable when ordinary solvents such as 0.1m-potassium phosphate buffer, pH7.0, are used).     

A possible method for characterizing the secondary structure of ribonucleic acids

The E(280)/E(260) ratio was found to be suitable for following the ionization of cytosine residues of polynucleotides on the basis of studies with model compounds such as oligoguanylic acid, oligocytidylic acid, a complex formed between polyadenylic acid and polyuridylic acid, and a copolymer of guanylic acid and cytidylic acid, provided that changes in secondary structure were taken into account. The pK of cytosine residues of a polynucleotide in the amorphous form was found to be 4.70 at 25 degrees in 0.1m-sodium phosphate on the basis of titration at 75-85 degrees and on the assumption that the heat of ionization was the same as the value (5.2kcal./mole) found for CMP. In contrast, the pK of cytosine residues in the double-helical form of DNA was found to be about 3.25. These observations were utilized in estimating the fraction of cytosine residues in helical segments of ribosomal RNA, a copolymer of guanylic acid and cytidylic acid, and a copolymer of adenylic acid, guanylic acid, uridylic acid and cytidylic acid. The ionization of guanine and uracil residues was estimated from changes in the E(270)/E(260) ratio and E(230)/E(260) ratio respectively. In the amorphous form of RNA both residues had the same pK, whereas in the double-helical form ionization was suppressed. The fraction of guanine and uracil residues in amorphous segments may be estimated from the titration curves. The difference in the denaturation spectrum of adenine--uracil and guanine--cytosine base pairs at 280mmu was enhanced in acidic solutions whereas E(260) was hardly affected. Hence a comparison of the increments in E(280) and E(260) obtained on increasing the temperature at constant pH may be used to distinguish the melting ranges of helical domains differing in nucleotide composition. In alkaline solutions comparison of the increments in E(260) and E(270) yields similar information. In acidic solutions the fraction of cytosine residues involved in helical secondary structure, the degree of ionization of cytosine residues and the fraction of adenine--uracil base pairs denatured may be estimated from DeltaE(265) and DeltaE(280). In alkaline solutions the fractions of guanine and uracil residues involved in secondary structure and the degrees of ionization of these residues may be estimated from DeltaE(230), DeltaE(245), DeltaE(260) and DeltaE(280).