Kinetic Complexity, Homogeneity, and Copy Number of Chloroplast DNA from the Marine Alga Olisthodiscus luteus

The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar⁻¹ and second⁻¹ (Cot_½ 0.24 molar second) under conditions equivalent to 180 millimolar Na⁺ and 60°C. Given the 92 × 10⁵ dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.     

DNA analysis of insect iridescent virus 6: evidence for circular permutation and terminal redundancy

DNA analysis of small insect iridovirus 6 was performed. Combined exonuclease-restriction endonuclease digestions revealed that all resulting fragments were degraded without preference for any one DNA fragment. Upon denaturation and reannealing of native linear Chilo iridescent virus DNA (158 × 10⁶ daltons), duplex DNA circles of a smaller size (140 × 10⁶ daltons) with protruding tails were formed.     

Synthesis and Stability of Chloroplast Ribosomal-RNA's

The chloroplast ribosomal-RNAs (1.1 × 10⁶ and 0.56 × 10⁶ mol wt) are synthesized in the normal ratio of 2:1. The non-ribosomal distribution observed after extraction and fractionation results from the lability of the 1.1 × 10⁶ component, and a correction for this breakdown can be applied in certain cases. Newly synthesized 1.1 × 10⁶ RNA is more stable than the older accumulated 1.1 × 10⁶ RNA. Accumulation of the chloroplast RNA during growth of radish cotyledons occurs at a later time than the accumulation of cytoplasmic RNA, and its turnover is much less than that of the cytoplasmic ribosomal-RNA.     

Marker Rescue Studies of the Transfer of Recombinant DNA to Streptococcus gordonii In Vitro, in Foods and Gnotobiotic Rats

A plasmid marker rescue system based on restoration of the nptII gene was established in Streptococcus gordonii to study the transfer of bacterial and transgenic plant DNA by transformation. In vitro studies revealed that the marker rescue efficiency depends on the type of donor DNA. Plasmid and chromosomal DNA of bacteria as well as DNA of transgenic potatoes were transferred with efficiencies ranging from 8.1 × 10⁻⁶ to 5.8 × 10⁻⁷ transformants per nptII gene. Using a 792-bp amplification product of nptII the efficiency was strongly decreased (9.8 × 10⁻⁹). In blood sausage, marker rescue using plasmid DNA was detectable (7.9 × 10⁻¹⁰), whereas in milk heat-inactivated horse serum (HHS) had to be added to obtain an efficiency of 2.7 × 10⁻¹¹. No marker rescue was detected in extracts of transgenic potatoes despite addition of HHS. In vivo transformation of S. gordonii LTH 5597 was studied in monoassociated rats by using plasmid DNA. No marker rescue could be detected in vivo, although transformation was detected in the presence of saliva and fecal samples supplemented with HHS. It was also shown that plasmid DNA persists in rat saliva permitting transformation for up to 6 h of incubation. It is suggested that the lack of marker rescue is due to the absence of competence-stimulating factors such as serum proteins in rat saliva.     

Herpes Simplex Virus: Genome Size and Redundancy Studied by Renaturation Kinetics

Herpes simplex virus subtype 1 deoxyribonucleic acid (DNA) was sheared in a French press to uniform fragments, denatured by heating, then allowed to reassociate. The renaturation reaction followed second-order kinetics with a single rate constant indicating that at least 95% of the genome was unique and that repetitive sequences, if present, were not detectable by this technique. The kinetic complexity of the herpes simplex genome was determined by DNA renaturation kinetics to be (95 ± 1) × 10⁶ daltons. Since this value is in excellent agreement with the molecular weight of viral DNA [(99 ± 5) × 10⁶ daltons] obtained from velocity sedimentation studies, it is concluded that virions contain only one species of double-stranded DNA molecules 95 × 10⁶ to 99 × 10⁶ daltons in molecular weight.     

A Simple and Rapid Method for Genetic Transformation of Lactic Streptococci by Electroporation

An electroporation procedure for the plasmid-mediated genetic transformation of intact cells of Streptococcus cremoris and Streptococcus lactis was performed. Ten different strains were transformed. The method was simple and rapid and yielded transformant colonies in 14 to 24 h. The method was optimized for S. lactis LM0230, and transformation frequencies of between 1 × 10⁴ and 5 × 10⁵ transformants per μg of purified plasmid (pMU1328) were achieved routinely. The optimized procedure involved lysozyme treatment of cells. Transformation of LM0230 occurred at comparable frequencies with pLS1 (4.4 kilobase pair [kbp]), pMU1328 (7.4 kbp), and pAMβ1 (26.5 kbp). Plasmid DNA isolated from transformants had not undergone detectable deletions or rearrangements. Transformation was possible with plasmid DNA which was religated after restriction endonuclease digestion. Phage DNA-dependent transfection of S. lactis LM0230 and S. lactis C6 was also achieved.     

Laser activation of rapid absorption changes in spinach chloroplasts and chlorella

The kinetics of the 520 mμ absorption change in spinach chloroplasts and Chlorella vulgaris following a flash from the ruby laser have been determined as follows: rise halftime ≤ 0.3 × 10⁻⁶ second; rapid recovery halftime = 5 to 6 × 10⁻⁶ second; intermediate recovery halftime = 4 × 10⁻⁴ second (spinach chloroplasts only); slow recovery halftime = 12 to 170 × 10⁻³ second, dependent on the measuring light intensity and aerobicity of the suspension.

The rapid phase of the 520 mμ reaction is approximately independent of temperature, from 295° to 77° Absolute.

With increasing oxygenation of the sample, the extent of the rapid phase decreases, the extent of the slow phase increases, while the extent of the intermediate phase in spinach chloroplasts remains constant.

In spinach chloroplasts, no recovery halftime of the 3 recovery phases for the 520 mμ absorption change was observed to correspond to the halftime for oxidation of cytochrome f (t_½ = 1.3 × 10⁻³ second).

     

Gene Expression During the Development of Bacillus subtilis Bacteriophage φ29 II. Resolution of Viral-Specific Ribonucleic Acid Molecules

The ribonucleic acid (RNA) specified by bacteriophage 29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 × 10⁶, 0.44 × 10⁶, 0.37 × 10⁶, 0.25 × 10⁶, 0.09 × 10⁶, and 0.04 × 10⁶, accounting for 35% of the coding capacity of 29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 × 10⁶ were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 × 10⁶) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 × 10⁶, 0.93 × 10⁶, and 0.07 × 10⁶, appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins.     

Microbial Flora of Pond-Reared Brown Shrimp (Penaeus aztecus)

Agar plate counts and microbial types are reported for brown shrimp reared in 2-acre natural marshland and in 0.5-acre artificial ponds during June to October 1970. Bacterial counts of pond-reared shrimp ranged from 5 × 10⁴ to 5.5 × 10⁶ per g. At final harvest in October, bacterial counts ranged from 2 × 10⁵ to 5.5 × 10⁶ per g. In marsh ponds, bacterial counts of shrimp and pond water were lowest in August when both water temperature and salinity were high. Coryneform bacteria and to a lesser extent Vibrio were the predominant isolates from fresh pond shrimp. Shrimp stored at 3 to 5 C for 7 days were acceptable as judged by appearance and odor. Between 7 and 14 days of refrigerated storage, bacterial counts increased sharply and about 50% of the samples became unacceptable. Refrigerated storage of pond shrimp caused increases in coryneform bacteria and micrococci and decreases in Vibrio, Flavobacterium, Moraxella, and Bacillus species. Pseudomonas species were not significant in fresh or stored pond shrimp. The microbial flora of pond water usually was dominated by coryneform bacteria, Flavobacterium, Moraxella, and Bacillus species.     

Properties of Light Particles Produced During Growth of Type 4 Adeno-Associated Satellite Virus

Adeno-associated satellite virus type 4, obtained by repeated undiluted passage, failed to produce distinct bands at the expected density of 1.43 g/cm³ after density gradient centrifugation in CsCl. This phenomenon occurred regardless of the hemagglutinating activity of the starting material. Sharp bands were found at a density of 1.34 to 1.35 g/cm³. These bands contained adenovirions and numerous satellite particles. These latter particles could be distinguished by electron microscopy from standard dense satellite particles by their flattened profiles and deep penetration of negative stains. Dense bands of satellite virus at 1.43 g/cm³ were constantly observed when the inoculum was comprised of highly diluted seed virus. Light satellite particles had a particle to HA ratio comparable with dense particles, but possessed low infectivity. Measurements of contour lengths of extracted deoxyribonucleic acid (DNA) indicate that light particles contain only a small amount of DNA, possibly less than 0.5 × 10⁶ daltons, compared to 1.4 × 10⁶ for the complete satellite DNA molecule.     

Spore Load of Ascosphaera Species on Emerging Adults of the Alfalfa Leafcutting Bee, Megachile rotundata

The spore load of Ascosphaera species spores on larval chalkbrood cadavers and newly emergent adults of the alfalfa leafcutting bee, Megachile rotundata, was determined. The spore content of chalkbrood cadavers ranged from 3 × 10⁶ to 5 × 10⁸. Adults emerging through zero to nine cadavers carried spores on all body parts examined by scanning electron microscopy. Estimates of the total number of spores obtained from a series of adult washes ranged from 9 × 10⁴ to 8 × 10⁷. Some adult males which emerged through no cadavers carried 10⁴ to 10⁵ spores, indicating that nesting materials might also have been contaminated. However, the control of chalkbrood in commercial bee populations may not be accomplished simply by providing clean nesting materials as adults may still emerge through diseased larvae.     

Ultrasound-mediated DNA transfer for bacteria

In environmental microbiology, the most commonly used methods of bacterial DNA transfer are conjugation and electroporation. However, conjugation requires physical contact and cell–pilus–cell interactions; electroporation requires low-ionic strength medium and high voltage. These limitations have hampered broad applications of bacterial DNA delivery. We have employed a standard low frequency 40 kHz ultrasound bath to successfully transfer plasmid pBBR1MCS2 into Pseudomonas putida UWC1, Escherichia coli DH5α and Pseudomonas fluorescens SBW25 with high efficiency. Under optimal conditions: ultrasound exposure time of 10 s, 50 mM CaCl₂, temperature of 22°C, plasmid concentration of 0.8 ng/µl, P. putida UWC1 cell concentration of 2.5 × 10⁹ CFU (colony forming unit)/ml and reaction volume of 500 µl, the efficiency of ultrasound DNA delivery (UDD) was 9.8 ± 2.3 × 10⁻⁶ transformants per cell, which was nine times more efficient than conjugation, and even four times greater than electroporation. We have also transferred pBBR1MCS2 into E. coli DH5α and P. fluorescens SBW25 with efficiencies of 1.16 ± 0.13 × 10⁻⁶ and 4.33 ± 0.78 × 10⁻⁶ transformants per cell, respectively. Low frequency UDD can be readily scaled up, allowing for the application of UDD not only in laboratory conditions but also on an industrial scale.     

Diffusion Rates in Disrupted Bacterial Cells

The viscosity of the material resulting from squeezing Escherichia coli cells through an orifice in a French pressure cell has been shown to be very high and variable with temperature. Diffusion constants in this medium have been determined for sucrose, dextran, and beta galactosidase. The values found are: 1.07 × 10^-6cm²/second for sucrose, 0.36 × 10^-6cm²/second for dextran, and 0.025 × 10^-6cm²/second for beta galactosidase. The results agree with the idea that there is much interstitial space available for diffusion of small molecules in the cell medium in spite of the high viscosity, but that large molecules will be transported less readily.     

Simultaneous Measurement of NH(4) Absorption and N(2) Fixation by Glycine max L. : RESPONSE TO TEMPERATURE, pH, AND EXTERNAL NITROGEN CONCENTRATION

Curvature, bending moment, and second moment of stem cross-sectional area were evaluated from photographic data and used to compute flexural rigidity and Young's modulus in the panicle rachis of rice, Oryza sativa L. `M-101.' Flexural rigidity C, and its components E, Young's modulus, and I, the moment of inertia of the area about the neutral axis, were evaluated 1.5 cm (tip), 9.5 cm (mid), and 16.5 cm (base) from the tip of the panicle rachis. In dynes per square centimeter, C increases from 1.1 × 10³ near the tip to 1.09 × 10⁴ in the middle to 5.35 × 10⁴ in the basal region of the rachis. Of the components of C, the I changes have the larger effect, increasing from 2.12 × 10⁻⁷ centimeters⁴ near the tip to 8.21 × 10⁻⁷ centimeters⁴ in mid regions to 6.0 × 10⁻⁶ centimeters⁴ in the basal regions. Young's modulus increases from 4.8 × 10⁹ dynes per square centimeter near the tip to 1.4 × 10¹⁰ dynes per square centimeter in mid regions then falls to 7.4 × 10⁹ dynes per square centimeter near the base of the main stem. Values of Young's modulus from Instron experiments were in satisfactory agreement with values calculated from the beam bending equation. Flexural rigidity in the curved region of the panicle proved independent of panicle load, indicating that the dissected panicle rachis behaves in some respects as a tapered loaded beam.     

Abundance of narG, nirS, nirK, and nosZ Genes of Denitrifying Bacteria during Primary Successions of a Glacier Foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 × 10⁵ to 8.9 × 10⁵ copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 × 10³ to 2.6 × 10⁴, 7.4 × 10² to 1.4 × 10³, 2.5 × 10² to 6.4 × 10³, and 1.2 × 10³ to 5.5 × 10³, respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Epigenome-Wide DNA Methylation in Hearing Ability: New Mechanisms for an Old Problem

Epigenetic regulation of gene expression has been shown to change over time and may be associated with environmental exposures in common complex traits. Age-related hearing impairment is a complex disorder, known to be heritable, with heritability estimates of 57–70%. Epigenetic regulation might explain the observed difference in age of onset and magnitude of hearing impairment with age. Epigenetic epidemiology studies using unrelated samples can be limited in their ability to detect small effects, and recent epigenetic findings in twins underscore the power of this well matched study design. We investigated the association between venous blood DNA methylation epigenome-wide and hearing ability. Pure-tone audiometry (PTA) and Illumina HumanMethylation array data were obtained from female twin volunteers enrolled in the TwinsUK register. Two study groups were explored: first, an epigenome-wide association scan (EWAS) was performed in a discovery sample (n = 115 subjects, age range: 47–83 years, Illumina 27 k array), then replication of the top ten associated probes from the discovery EWAS was attempted in a second unrelated sample (n = 203, age range: 41–86 years, Illumina 450 k array). Finally, a set of monozygotic (MZ) twin pairs (n = 21 pairs) within the discovery sample (Illumina 27 k array) was investigated in more detail in an MZ discordance analysis. Hearing ability was strongly associated with DNA methylation levels in the promoter regions of several genes, including TCF25 (cg01161216, p = 6.6×10⁻⁶), FGFR1 (cg15791248, p = 5.7×10⁻⁵) and POLE (cg18877514, p = 6.3×10⁻⁵). Replication of these results in a second sample confirmed the presence of differential methylation at TCF25 (p(replication) = 6×10⁻⁵) and POLE (p(replication) = 0.016). In the MZ discordance analysis, twins'' intrapair difference in hearing ability correlated with DNA methylation differences at ACP6 (cg01377755, r = −0.75, p = 1.2×10⁻⁴) and MEF2D (cg08156349, r = −0.75, p = 1.4×10⁻⁴). Examination of gene expression in skin, suggests an influence of differential methylation on expression, which may account for the variation in hearing ability with age.     

High-Frequency Phage-Mediated Gene Transfer among Escherichia coli Cells, Determined at the Single-Cell Level

Recent whole-genome analysis suggests that lateral gene transfer by bacteriophages has contributed significantly to the genetic diversity of bacteria. To accurately determine the frequency of phage-mediated gene transfer, we employed cycling primed in situ amplification-fluorescent in situ hybridization (CPRINS-FISH) and investigated the movement of the ampicillin resistance gene among Escherichia coli cells mediated by phage at the single-cell level. Phages P1 and T4 and the newly isolated E. coli phage EC10 were used as vectors. The transduction frequencies determined by conventional plating were 3 × 10⁻⁸ to 2 × 10⁻⁶, 1 × 10⁻⁸ to 4 × 10⁻⁸, and <4 × 10⁻⁹ to 4 × 10⁻⁸ per PFU for phages P1, T4, and EC10, respectively. The frequencies of DNA transfer determined by CPRINS-FISH were 7 × 10⁻⁴ to 1 × 10⁻³, 9 × 10⁻⁴ to 3 × 10⁻³, and 5 × 10⁻⁴ to 4 × 10⁻³ for phages P1, T4, and EC10, respectively. Direct viable counting combined with CPRINS-FISH revealed that more than 20% of the cells carrying the transferred gene retained their viabilities. These results revealed that the difference in the number of viable cells carrying the transferred gene and the number of cells capable of growth on the selective medium was 3 to 4 orders of magnitude, indicating that phage-mediated exchange of DNA sequences among bacteria occurs with unexpectedly high frequency.     

Useful Host-Vector Systems in Bacillus stearothermophilus

We isolated a highly transformable thermophile, Bacillus stearothermophilus SIC1, which exhibited the following features. The growth temperature ranged from 45 to 65°C in L broth. The maximum cell concentration in 2L broth (2% tryptone, 1% yeast extract, 0.5% NaCl, pH 7.2) was determined as an optical density at 660 nm of 7.8, and the generation time was 11 min at 60°C. Strain SIC1 was a prototroph and was transformed by the protoplast procedure not only with repB plasmids (high-copy-number plasmids such as pTB913 and pUB110) but also with repA plasmids (low-copy-number plasmids such as pTB53). Transformation efficiencies with repB and repA plasmids were about 2 × 10⁶ to 5 × 10⁶ and 5 × 10⁴ transformants per μg of DNA, respectively. The transformant carrying plasmid pTB913Y/K could grow at 63°C in the presence of kanamycin. The regeneration frequency of protoplasts was 60%, and only 1 day was needed for regeneration at 55°C.     

Genetic variation at the FADS1-FADS2 gene locus influences delta-5 desaturase activity and LC-PUFA proportions after fish oil supplement

Delta-5 and delta-6 desaturases (D5D and D6D) are key enzymes in endogenous synthesis of long-chain PUFAs. In this sample of healthy subjects (n = 310), genotypes of single nucleotide polymorphisms (SNPs) rs174537, rs174561, and rs3834458 in the FADS1-FADS2 gene cluster were strongly associated with proportions of LC-PUFAs and desaturase activities estimated in plasma and erythrocytes. Minor allele carriage associated with decreased activities of D5D (FADS1) (5.84 × 10⁻¹⁹ ≤ P ≤ 4.5 × 10⁻¹⁸) and D6D (FADS2) (6.05 × 10⁻⁸ ≤ P ≤ 4.20 × 10⁻⁷) was accompanied by increased substrate and decreased product proportions (0.05 ≤ P ≤ 2.49 × 10⁻¹⁶). The significance of haplotype association with D5D activity (P = 2.19 × 10⁻¹⁷) was comparable to that of single SNPs, but haplotype association with D6D activity (P = 3.39 × 10⁻²⁸) was much stronger. In a randomized controlled dietary intervention, increasing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) intake significantly increased D5D (P = 4.0 × 10⁻⁹) and decreased D6D activity (P = 9.16 × 10⁻⁶) after doses of 0.45, 0.9, and 1.8 g/day for six months. Interaction of rs174537 genotype with treatment was a determinant of D5D activity estimated in plasma (P = 0.05). In conclusion, different sites at the FADS1-FADS2 locus appear to influence D5D and D6D activity, and rs174537 genotype interacts with dietary EPA+DHA to modulate D5D.     

Molecular Weight Determination of Sendai RNA by Dimethyl Sulfoxide Gradient Sedimentation

The molecular weight of the large RNA of Sendai virus has been determined by sedimentation analysis in sucrose gradients containing 99% dimethyl sulfoxide (DMSO) to be 2.3 × 10⁶. Sendai RNA recovered from 99% DMSO was found to cosediment with nondenatured Sendai RNA at 46 to 48s in ordinary sucrose gradients. The molecular weight value of 2.3 × 10⁶ is considerably smaller than the estimates of 6 × 10⁶ to 7 × 10⁶ determined under nondenaturing conditions, suggesting a unique structure for Sendai RNA.