Pasteurella pestis Bacteriophage H and Escherichia coli Bacteriophage φII Are Nearly Identical

Electron microscopy examination of II-H deoxyribonucleic acid heteroduplexes, together with polyacrylamide gel electrophoretic analyses of phage ribonucleic acid species and proteins labeled in II or H-infected cells, demonstrates that Pasteurella pestis phage H is nearly identical to coliphage II.     

Specificity of the STAT4 genetic association for severe disease manifestations of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a genetically complex disease with heterogeneous clinical manifestations. A polymorphism in the STAT4 gene has recently been established as a risk factor for SLE, but the relationship with specific SLE subphenotypes has not been studied. We studied 137 SNPs in the STAT4 region genotyped in 4 independent SLE case series (total n=1398) and 2560 healthy controls, along with clinical data for the cases. Using conditional testing, we confirmed the most significant STAT4 haplotype for SLE risk. We then studied a SNP marking this haplotype for association with specific SLE subphenotypes, including autoantibody production, nephritis, arthritis, mucocutaneous manifestations, and age at diagnosis. To prevent possible type-I errors from population stratification, we reanalyzed the data using a subset of subjects determined to be most homogeneous based on principal components analysis of genome-wide data. We confirmed that four SNPs in very high LD (r²=0.94 to 0.99) were most strongly associated with SLE, and there was no compelling evidence for additional SLE risk loci in the STAT4 region. SNP rs7574865 marking this haplotype had a minor allele frequency (MAF)=31.1% in SLE cases compared with 22.5% in controls (OR=1.56, p=10⁻¹⁶). This SNP was more strongly associated with SLE characterized by double-stranded DNA autoantibodies (MAF=35.1%, OR=1.86, p<10⁻¹⁹), nephritis (MAF=34.3%, OR=1.80, p<10⁻¹¹), and age at diagnosis<30 years (MAF=33.8%, OR=1.77, p<10⁻¹³). An association with severe nephritis was even more striking (MAF=39.2%, OR=2.35, p<10⁻⁴ in the homogeneous subset of subjects). In contrast, STAT4 was less strongly associated with oral ulcers, a manifestation associated with milder disease. We conclude that this common polymorphism of STAT4 contributes to the phenotypic heterogeneity of SLE, predisposing specifically to more severe disease.     

Neuropeptide Y Gene Polymorphisms Confer Risk of Early-Onset Atherosclerosis

Neuropeptide Y (NPY) is a strong candidate gene for coronary artery disease (CAD). We have previously identified genetic linkage to familial CAD in the genomic region of NPY. We performed follow-up genetic, biostatistical, and functional analysis of NPY in early-onset CAD. In familial CAD (GENECARD, N=420 families), we found increased microsatellite linkage to chromosome 7p14 (OSA LOD=4.2, p=0.004) in 97 earliest age-of-onset families. Tagged NPY SNPs demonstrated linkage to CAD of a 6-SNP block (LOD=1.58–2.72), family-based association of this block with CAD (p=0.02), and stronger linkage to CAD in the earliest age-of-onset families. Association of this 6-SNP block with CAD was validated in: (a) 556 non-familial early-onset CAD cases and 256 controls (OR 1.46–1.65, p=0.01–0.05), showing stronger association in youngest cases (OR 1.84–2.20, p=0.0004–0.09); and (b) GENECARD probands versus non-familial controls (OR 1.79–2.06, p=0.003–0.02). A promoter SNP (rs16147) within this 6-SNP block was associated with higher plasma NPY levels (p=0.04). To assess a causal role of NPY in atherosclerosis, we applied the NPY1-receptor–antagonist BIBP-3226 adventitially to endothelium-denuded carotid arteries of apolipoprotein E-deficient mice; treatment reduced atherosclerotic neointimal area by 50% (p=0.03). Thus, NPY variants associate with atherosclerosis in two independent datasets (with strong age-of-onset effects) and show allele-specific expression with NPY levels, while NPY receptor antagonism reduces atherosclerosis in mice. We conclude that NPY contributes to atherosclerosis pathogenesis.     

Isolation and Characterization of Five Erwinia amylovora Bacteriophages and Assessment of Phage Resistance in Strains of Erwinia amylovora

Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms. Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages Ea1 and Ea7 and 3 novel phages named Ea100, Ea125, and Ea116C, were identified based on differences in genome size and restriction fragment pattern. Ea1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates. Phages Ea100, Ea7, and Ea125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns. Ea116C contained an approximately 75-kb genome. Ea1, Ea7, Ea100, Ea125, and Ea116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E. amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E. amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan. Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage. Ea116C was more effective than the other phages at lysing E. amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10⁵ CFU.     

Meta-Analysis of Genome-Wide Scans for Human Adult Stature Identifies Novel Loci and Associations with Measures of Skeletal Frame Size

Recent genome-wide (GW) scans have identified several independent loci affecting human stature, but their contribution through the different skeletal components of height is still poorly understood. We carried out a genome-wide scan in 12,611 participants, followed by replication in an additional 7,187 individuals, and identified 17 genomic regions with GW-significant association with height. Of these, two are entirely novel (rs11809207 in CATSPER4, combined P-value=6.1×10⁻⁸ and rs910316 in TMED10, P-value=1.4×10⁻⁷) and two had previously been described with weak statistical support (rs10472828 in NPR3, P-value=3×10⁻⁷ and rs849141 in JAZF1, P-value=3.2×10⁻¹¹). One locus (rs1182188 at GNA12) identifies the first height eQTL. We also assessed the contribution of height loci to the upper- (trunk) and lower-body (hip axis and femur) skeletal components of height. We find evidence for several loci associated with trunk length (including rs6570507 in GPR126, P-value=4×10⁻⁵ and rs6817306 in LCORL, P-value=4×10⁻⁴), hip axis length (including rs6830062 at LCORL, P-value=4.8×10⁻⁴ and rs4911494 at UQCC, P-value=1.9×10⁻⁴), and femur length (including rs710841 at PRKG2, P-value=2.4×10⁻⁵ and rs10946808 at HIST1H1D, P-value=6.4×10⁻⁶). Finally, we used conditional analyses to explore a possible differential contribution of the height loci to these different skeletal size measurements. In addition to validating four novel loci controlling adult stature, our study represents the first effort to assess the contribution of genetic loci to three skeletal components of height. Further statistical tests in larger numbers of individuals will be required to verify if the height loci affect height preferentially through these subcomponents of height.     

Admixture Mapping of 15,280 African Americans Identifies Obesity Susceptibility Loci on Chromosomes 5 and X

The prevalence of obesity (body mass index (BMI) ≥30 kg/m²) is higher in African Americans than in European Americans, even after adjustment for socioeconomic factors, suggesting that genetic factors may explain some of the difference. To identify genetic loci influencing BMI, we carried out a pooled analysis of genome-wide admixture mapping scans in 15,280 African Americans from 14 epidemiologic studies. Samples were genotyped at a median of 1,411 ancestry-informative markers. After adjusting for age, sex, and study, BMI was analyzed both as a dichotomized (top 20% versus bottom 20%) and a continuous trait. We found that a higher percentage of European ancestry was significantly correlated with lower BMI (ρ=−0.042, P=1.6×10⁻⁷). In the dichotomized analysis, we detected two loci on chromosome X as associated with increased African ancestry: the first at Xq25 (locus-specific LOD=5.94; genome-wide score=3.22; case-control Z=−3.94); and the second at Xq13.1 (locus-specific LOD=2.22; case-control Z=−4.62). Quantitative analysis identified a third locus at 5q13.3 where higher BMI was highly significantly associated with greater European ancestry (locus-specific LOD=6.27; genome-wide score=3.46). Further mapping studies with dense sets of markers will be necessary to identify the alleles in these regions of chromosomes X and 5 that may be associated with variation in BMI.     

Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000

RSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the RSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the RSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A RSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these RSA1 genes as 5′ TGTTGT-(X)₁₃-ACAACA. The genomic sequence similarity between RSA1 and related phages 52237 and CTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. RSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the RSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a RSA1-related prophage (designated RSX). RSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. RSX ORFs shared very high amino acid identity with their RSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.     

Prophage Origin of a Virulent Phage Appearing on Fermentations of Lactobacillus casei S-1

For protection from the abnormal fermentation of Lactobacillus casei S-1 caused by contamination of a virulent phage, FSV, the origin of this phage was studied. Morphologies, viral structural proteins, and DNA structures of three independent isolates of FSV were compared with those of FSW, which is lysogenized in strain S-1. The results showed (i) that the morphology of FSV phages is indistinguishable from that of FSW and (ii) that all viral structural components found in FSW are present in the particles of FSV's. In addition, restriction endonuclease analyses of viral DNA showed that the HindIII-digested fragments of FSW DNA, the sum of which covered at least 94.7% of this phage genome, were conserved in the FSV DNA digests. Results of Southern filter hybridization of the S-1 and prophage-cured cell (C239) DNAs with FSV DNA as a probe revealed that C239 had lost most of the FSV DNA sequence, whereas S-1 had about one copy of the FSV DNA sequence. These results indicate that virulent phage FSV is derived from the lysogenized phage FSW. Therefore, the appearance of FSV can be eliminated by using the prophage-cured derivative of S-1.     

Synthesis and characterization of 2′-modified-4′-thioRNA: a comprehensive comparison of nuclease stability

We report herein the synthesis and physical and physiological characterization of fully modified 2′-modified-4′-thioRNAs, i.e. 2′-fluoro-4′-thioRNA (F-SRNA) and 2′-O-Me-4′-thioRNA (Me-SRNA), which can be considered as a hybrid chemical modification based on 2′-modified oligonucleotides (ONs) and 4′-thioRNA (SRNA). In its hybridization with a complementary RNA, F-SRNA (15mer) showed the highest T_m value (+16°C relative to the natural RNA duplex). In addition, both F-SRNA and Me-SRNA preferred RNA as a complementary partner rather than DNA in duplex formation. The results of a comprehensive comparison of nuclease stability of single-stranded F-SRNA and Me-SRNA along with 2′-fluoroRNA (FRNA), 2′-O-MeRNA (MeRNA), SRNA, and natural RNA and DNA, revealed that Me-SRNA had the highest stability with t_1/2 values of>24h against S1 nuclease (an endonuclease) and 79.2min against SVPD (a 3′-exonuclease). Moreover, the stability of Me-SRNA was significantly improved in 50% human plasma (t_1/2=1631min) compared with FRNA (t_1/2=53.2min) and MeRNA (t_1/2=187min), whose modifications are currently used as components of therapeutic aptamers. The results presented in this article will, it is hoped, contribute to the development of 2′-modified-4′-thioRNAs, especially Me-SRNA, as a new RNA molecule for therapeutic applications.     

Interaction between Light Quality and Light Quantity in the Photoregulation of Anthocyanin Production

The interaction between phytochrome photoequilibrium () and photon flux in the photoregulation of anthocyanin production under prolonged irradiation was studied in seedlings of Brassica oleracea L. and Lycopersicon esculentum Mill. In cabbage, anthocyanin production increases with decreasing , reaching a maximum at the lowest value ( = 0.13) used in this study; in tomato, the extent of the response is higher at intermediate values, reaching a maximum at = 0.46. In cabbage, the response increases with increasing photon flux at all values; however, the response to changes in photon flux is minimal at = 0.85, and, at = 0.13, minimal at photon fluxes higher than 5 micromolar per square meter per second. In tomato, the response increases with increasing photon flux at = 0.46, 0.65, and 0.85, the response to changes in photon fluxes being minimal at = 0.85; at = 0.13 and 0.29 the response first increases (significantly at = 0.29 and minimally at = 0.13) and then decreases with increasing photon fluxes, the transition occurring at about 1 micromolar per square meter per second at = 0.13, and at 5 micromolar per square meter per second at = 0.29. The patterns of light quality-quantity interaction in the photoregulation of anthocyanin production are significantly different in cabbage and tomato and are also significantly different than those observed for other photomorphogenic responses to prolonged irradiations.     

Meta-Analysis of 28,141 Individuals Identifies Common Variants within Five New Loci That Influence Uric Acid Concentrations

Elevated serum uric acid levels cause gout and are a risk factor for cardiovascular disease and diabetes. To investigate the polygenetic basis of serum uric acid levels, we conducted a meta-analysis of genome-wide association scans from 14 studies totalling 28,141 participants of European descent, resulting in identification of 954 SNPs distributed across nine loci that exceeded the threshold of genome-wide significance, five of which are novel. Overall, the common variants associated with serum uric acid levels fall in the following nine regions: SLC2A9 (p=5.2×10⁻²⁰¹), ABCG2 (p=3.1×10⁻²⁶), SLC17A1 (p=3.0×10⁻¹⁴), SLC22A11 (p=6.7×10⁻¹⁴), SLC22A12 (p=2.0×10⁻⁹), SLC16A9 (p=1.1×10⁻⁸), GCKR (p=1.4×10⁻⁹), LRRC16A (p=8.5×10⁻⁹), and near PDZK1 (p=2.7×10⁻⁹). Identified variants were analyzed for gender differences. We found that the minor allele for rs734553 in SLC2A9 has greater influence in lowering uric acid levels in women and the minor allele of rs2231142 in ABCG2 elevates uric acid levels more strongly in men compared to women. To further characterize the identified variants, we analyzed their association with a panel of metabolites. rs12356193 within SLC16A9 was associated with DL-carnitine (p=4.0×10⁻²⁶) and propionyl-L-carnitine (p=5.0×10⁻⁸) concentrations, which in turn were associated with serum UA levels (p=1.4×10⁻⁵⁷ and p=8.1×10⁻⁵⁴, respectively), forming a triangle between SNP, metabolites, and UA levels. Taken together, these associations highlight additional pathways that are important in the regulation of serum uric acid levels and point toward novel potential targets for pharmacological intervention to prevent or treat hyperuricemia. In addition, these findings strongly support the hypothesis that transport proteins are key in regulating serum uric acid levels.     

Isolation and Characterization of Temperate Bacteriophages of Clostridium difficile

The lack of information on bacteriophages of Clostridium difficile prompted this study. Three of 56 clinical C. difficile isolates yielded double-stranded DNA phages C2, C5, C6, and C8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of C2 showed nucleotide homology to the sequenced C. difficile strain CD630.     

Photocontrol of Hypocotyl Elongation in Light-Grown Cucumis sativus L. : Responses to Phytochrome Photostationary State and Fluence Rate

The effects of the calculated photostationary state of phytochrome (_c) and the photon fluence rate on the elongation growth of the hypocotyl of light-grown seedlings of Cucumis sativus L. are examined. Two threshold responses to _c are found at values of 0.06 and 0.43. At _c = 0.06, there is no response at any fluence rate. In the _c range 0.1 to 0.43, elongation growth does not respond to changes in _c. Above the second threshold (_c = 0.43), there is a strong response to changes in _c. At all values of _c at and above 0.1, there is a response to fluence rate. A linear relationship can be demonstrated between a factor comprised of the logarithm of phytochrome cycling rate (a fluence-rate-dependent process) and _c, and the growth response.     

High-yield production of short GpppA- and 7MeGpppA-capped RNAs and HPLC-monitoring of methyltransfer reactions at the guanine-N7 and adenosine-2′O positions

Many eukaryotic and viral mRNAs, in which the first transcribed nucleotide is an adenosine, are decorated with a cap-1 structure, ^7MeG^5′-ppp^5′-A_2′OMe. The positive-sense RNA genomes of flaviviruses (Dengue, West Nile virus) for example show strict conservation of the adenosine. We set out to produce GpppA- and ^7MeGpppA-capped RNA oligonucleotides for non-radioactive mRNA cap methyltransferase assays and, in perspective, for studies of enzyme specificity in relation to substrate length as well as for co-crystallization studies. This study reports the use of a bacteriophage T7 DNA primase fragment to synthesize GpppAC_n and ^7MeGpppAC_n (1≤n≤9) in a one-step enzymatic reaction, followed by direct on-line cleaning HPLC purification. Optimization studies show that yields could be modulated by DNA template, enzyme and substrate concentration adjustments and longer reaction times. Large-scale synthesis rendered pure (in average 99%) products (1≤n≤7) in quantities of up to 100nmol starting from 200nmol cap analog. The capped RNA oligonucleotides were efficient substrates of Dengue virus (nucleoside-2′-O-)-methyltransferase, and human (guanine-N7)-methyltransferase. Methyltransfer reactions were monitored by a non-radioactive, quantitative HPLC assay. Additionally, the produced capped RNAs may serve in biochemical, inhibition and structural studies involving a variety of eukaryotic and viral methyltransferases and guanylyltransferases.     

Saturation Mutagenesis of Toluene ortho-Monooxygenase of Burkholderia cepacia G4 for Enhanced 1-Naphthol Synthesis and Chloroform Degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase α-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V_max of 9.3 versus 4.5 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 ± 0.07 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹ [mean ± standard deviation]) at 91 μM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V_max of 2.61 versus 0.95 nmol·min⁻¹·mg of protein⁻¹ and unchanged K_m) and chloride release (0.034 ± 0.002 versus 0.012 ± 0.001 nmol·min⁻¹·mg of protein⁻¹). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 ± 0.3 versus 1.30 ± 0.06 nmol·min⁻¹·mg of protein⁻¹). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Characterization of Temperate Bacillus Bacteriophage phi105

Temperate Bacillus phage 105 is serologically unrelated to previously described virulent Bacillus phages. Phage 105 is incapable of generalized transduction. Prophage 105 is inducible with mitomycin C. Phage 105 contains double-stranded deoxyribonucleic acid (DNA) with a molecular weight of about 25 × 10⁷ as determined by band sedimentation and electron microscopy. The per cent guanine plus cytosine of 105 DNA is 43.5 as determined by buoyant density in CsCl and by thermal denaturation. Phage 105 DNA may contain complementary single-stranded ends.     

NRXN3 Is a Novel Locus for Waist Circumference: A Genome-Wide Association Study from the CHARGE Consortium

Central abdominal fat is a strong risk factor for diabetes and cardiovascular disease. To identify common variants influencing central abdominal fat, we conducted a two-stage genome-wide association analysis for waist circumference (WC). In total, three loci reached genome-wide significance. In stage 1, 31,373 individuals of Caucasian descent from eight cohort studies confirmed the role of FTO and MC4R and identified one novel locus associated with WC in the neurexin 3 gene [NRXN3 (rs10146997, p=6.4×10⁻⁷)]. The association with NRXN3 was confirmed in stage 2 by combining stage 1 results with those from 38,641 participants in the GIANT consortium (p=0.009 in GIANT only, p=5.3×10⁻⁸ for combined analysis, n=70,014). Mean WC increase per copy of the G allele was 0.0498 z-score units (0.65 cm). This SNP was also associated with body mass index (BMI) [p=7.4×10⁻⁶, 0.024 z-score units (0.10 kg/m²) per copy of the G allele] and the risk of obesity (odds ratio 1.13, 95% CI 1.07–1.19; p=3.2×10⁻⁵ per copy of the G allele). The NRXN3 gene has been previously implicated in addiction and reward behavior, lending further evidence that common forms of obesity may be a central nervous system-mediated disorder. Our findings establish that common variants in NRXN3 are associated with WC, BMI, and obesity.     

Effect of turgor pressure and cell size on the wall elasticity of plant cells

Direct measurements of the volumetric elastic modulus, , of cells of a higher plant were performed on the epidermal bladder cells of Mesembryanthemum crystallinum using a pressure probe technique. Measurements on giant algal cells (Valonia, Nitellopsis) are given for comparison. Giant celled algae and M. crystallinum bladders have elastic moduli, , which depend strongly on turgor pressure, P, and on cell volume, V. The values of Mesembryanthemum bladders range between 5 bar at zero pressure and 100 bar at full turgor pressure (3-4 bar). increased with cell size (volume) at a given turgor pressure, and this volume dependence was pronounced more in the high pressure range. From the (P) characteristics, complete volume-pressure curves were obtained for Mesembryanthemum bladders and giant algal cells. The results suggest that the (P) and (V) characteristics of all plant cells are similar. The significance of the pressure and volume effects for the water relations and growth processes of plant cells is discussed briefly.     

Differential functional behavior of viral phi29, Nf and GA-1 SSB proteins

DNA replication of 29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage 29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of 29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of 29 DNA polymerase to ssDNA and their stimulatory effect on replication by 29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during 29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by 29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the 29 and Nf SSBs do not show any effect. On the other hand, the 29 SSB is the only one of the three SSBs able to increase the replication rate of 29 DNA polymerase in primed M13 ssDNA replication. From the fact that the 29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of 29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on 29-like DNA replication has been proposed.     

High-Frequency Plasmid Transduction by Lactobacillus gasseri Bacteriophage φadh

The temperate bacteriophage adh mediates plasmid DNA transduction in Lactobacillus gasseri ADH at frequencies in the range of 10^-8 to 10^-10 transductants per PFU. BglII-generated DNA fragments from phage adh were cloned into the BclI site of the transducible plasmid vector pGK12 (4.4 kb). Phage adh lysates induced from Lactobacillus lysogens harboring pGK12 or the recombinant plasmids were used to transduce strain ADH to chloramphenicol resistance. The transduction frequencies of recombinant plasmids were 10²- to 10⁵-fold higher than that of native pGK12. The increase in frequency generally correlated with the extent of DNA-DNA homology between plasmid and phage DNAs. The highest transduction frequency was obtained with plasmid pTRK170 (6.6 kb), a pGK12 derivative containing the 1.4- and 0.8-kb BglII DNA fragments of adh. DNA hybridization analysis of pTRK170-transducing phage particles revealed that pTRK170 had integrated into the adh genome, suggesting that recombination between homologous sequences present in phage and plasmid DNAs was responsible for the formation of high-frequency transducing phage particles. Plasmid DNA analysis of 13 transductants containing pTRK170 showed that each had acquired intact plasmids, indicating that in the process of transduction a further recombination step was involved in the resolution of plasmid DNA monomers from the recombinant pTRK170::adh molecule. In addition to strain ADH, pTRK170 could be transduced via adh to eight different L. gasseri strains, including the neotype strain, F. Gasser 63 AM (ATCC 33323).