Restriction Fragmentation Research Articles

Home ranges of box turtles in a rural woodland and an urban park in Saint Louis, MO; implications for turtle conservation

The negative consequences of fragmentation, infrastructure development, and urbanization on biodiversity are well known. However, careful urban planning can provide viable habitat for some species and communities. Generally, r-selected species are more likely to persist in urbanizing landscapes, while long lived, mobile species, such as turtles, are likely to decline toward extinction. Understanding species responses across urban gradients is important for developing mitigation planning. We used VHF telemetry to quantify ranging behavior of three-toed box turtles in fragmented urban forest patches and a semi-contiguous large rural forest in Saint Louis, Missouri. We then simulated movement trajectories based on empirical data from the two turtles with the largest and smallest home ranges, overlaid on forest cover and road network maps of Missouri, to quantify the state-wide probability of turtles encountering roads. Home range (HR) size varied from 1–250Ha. The mean home range estimate of rural turtles was > 9 times larger than that of urban turtles. Simulations indicated that the least mobile turtle (HR 1Ha) would have a 22% likelihood of encountering a road if placed randomly in suitable habitat anywhere in the state. The likelihood increased to 90% for the most mobile turtle (HR 250Ha), with 10 road crossings per year. High turtle mortality, range restriction and population fragmentation are likely even in rural areas. In urban cityscapes, large, roadless forest patches offer the highest conservation potential for this species.

Association of HTLV-1 infection prevalence with TLR7 single nucleotide polymorphisms (rs179008 & rs179009) in asymptomatic blood donors in Khorasan Province of Iran

BackgroundHuman T-lymphotropic virus type 1 (HTLV-1) cause two diseases in humans such as adult T-cell leukemia/lymphoma (ATL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). About, 15–20 million persons are infected with HTLV-1 in worldwide. Single nucleotide polymorphisms (SNPs) are a genetic variation that occurs in >1% of the general population; they can alter promoter activities, gene expression, mRNA stability, and protein structure and function.Our aim in this study was to investigate the association between SNPs within Toll like receptor 7 (TLR7) (rs179008 and rs179009) and susceptibility to HTLV-1 infection among asymptomatic blood donors in Iran. MethodsA total of 100 asymptomatic HTLV-1 carriers and 118 healthy blood donors were studied. After extracting genomic DNA from all samples, amplification was performed using specific primers. Then, the restriction fragmentation length polymorphism (RFLP) method was used to evaluate the SNPs within TLR7. Data analysis was administered using SPSS version 22. ResultsThe frequency of the AA, AT, TT genotypes of TLR7 (rs178008) was 73%, 22%, and 5% in the patient group; while for those in the control group the values were 50%, 40.7%, and 9.3%, respectively. Moreover, the frequency of the TLR7 rs179008 A allele was 84% in the HTLV-1 patients group and 69.6 % in the control group. Also, the two groups were significantly different concerning the frequency distribution of the TLR7 (rs178008) genotypes and alleles (p = 0.001). ConclusionThere was a significant association between TLR7 (rs178008) SNP and HTLV-1 infection and the T allele of TLR7 (rs178008) SNP might be considered as a protective factor against HTLV-1 virus infection although additional evidence is needed.

Role of MT1A Polymorphism and Environmental Mercury Exposure on the Montreal Cognitive Assessment (MoCA).

PurposeMany age-related structural and functional changes in the brain have important consequences. Long-term exposure to mercury and the impact of functional polymorphisms of metal-regulating proteins such as metallothioneins (MTs) can result in neurological-neurobehavioral effects in elderly individuals. Therefore, the aims of this study are to examine the associations between biomarkers of mercury exposure and cognitive impairment and to investigate the effect of the rs8052394 single nucleotide polymorphism (SNP) of the potential modifier gene MT1A on different domains of the Montreal Cognitive Assessment (MoCA).Materials and MethodsWe studied 436 participants aged ≥55 years from the Electricity Generating Authority of Thailand study. They underwent a physical examination, an extensive cognitive assessment with the MoCA (cutoff <26 points), and a biochemical analysis related to diabetes and dyslipidemia. The blood mercury level was determined by inductively coupled plasma mass spectrometry. Genotyping of the MT1A rs8052394 SNP was performed by the restriction fragmentation length polymorphism method.ResultsThe mean age of the study population was 58.8±3.01 years, and most had ≥12 years of education (75.7%). The primary study finding was that the prevalence of mild cognitive impairment (MCI) in older Thai adults was 39.7%. The frequency distributions of the G allele of the rs8052394 SNP of the MT1A gene were significantly associated with the total and sub-domain MoCA scores. The prevalence of MCI was significantly associated with increased age, hypertriglyceridemia, hyperhomocysteinemia, the third tertile of blood mercury concentration, and the rs8052394 variant genotype of MT1A (P values for all odds ratios <0.05).ConclusionThese findings suggested that neurocognitive effects associate with mercury exposure and genetic susceptibility in toxicokinetics. Public health strategies can be used to implement as a comprehensive action plan to educate vulnerable populations on how to reduce mercury exposure. Concurrently, impact of such genetic predisposition requires replication for identifying and protecting susceptible individuals from mercury toxicity.

0282 Correlation Between Sleep Depth in the Right and Left Cerebral Hemispheres Following Sleep Deprivation, Restriction or Noise Exposure

Abstract Introduction The Odds-Ratio-Product (ORP) is a highly-validated continuous index of sleep depth (range 0=deep sleep; 2.5=full wakefulness). ORP values fluctuate within this range as sleep state changes between wake and different sleep stages. In healthy non-sleep deprived adults, intra-class correlation coefficient of concurrent right vs. left ORP values (R / L coefficient) is typically &amp;gt;0.80. In a recent study R / L coefficient was markedly reduced in many critically-ill patients and these patients failed to be weaned from mechanical ventilation. Given the high prevalence of sleep loss in such patients we hypothesized that reduction in R/L coefficient might result from sleep loss. This retrospective EEG analysis of data from 3 independent research studies investigated if R / L coefficient decreases in pure models of sleep deprivation, restriction or noise exposure during sleep in healthy subjects. Methods Polysomnograms were obtained from three studies: A) 200 subjects who underwent 36 hours of total sleep deprivation; B) 21 subjects who underwent 4 consecutive nights of sleep restriction (5 hrs. / night); C) 72 subjects who were exposed to intermittent traffic noise events with maximum sound pressure levels ranging from 45–65 dB(A) for 10 consecutive nights. For study A, R / L coefficient was calculated from pre- and post-deprivation sleep studies and the two values were compared. For study B, coefficient was calculated at baseline and in each restriction night. For study C, the coefficient was calculated in each of the 10 exposure nights and the slope of the change was calculated. Results In study A, the coefficient decreased from 0.82±0.12 at baseline to 0.74±0.16 after sleep deprivation (p &amp;lt 0.0001). In study B, the coefficient decreased from 0.83±0.11 at baseline to 0.75±0.15 on the 4th restriction night (p &amp;lt 0.01). In study C, coefficient decreased at a rate of 0.003±0.001 per exposure night (p &amp;lt 0.001). Conclusion The correlation between sleep depth in the right and left hemispheres deteriorates following sleep deprivation, restriction or noise-induced sleep fragmentation. Support NIH P50 HL060287

Association of TLR3 single nucleotide polymorphisms with susceptibility to HTLV-1 infection in Iranian asymptomatic blood donors

INTRODUCTION: The human T-lymphotropic virus type 1 (HTLV-1) has a single-stranded RNA genome and expresses specific proteins that have oncogenic potential. Approximately 15 to 20 million people worldwide have been infected by this virus. Changes in protein or gene expression are the effects of single nucleotide polymorphisms (SNPs) within the Toll-like receptor 3 (TLR3) gene. The function and efficacy of signal transduction also lead to modified immune responses. The present study aimed to investigate the association of SNPs within TLR3 (rs3775291 and rs3775296) with susceptibility to HTLV-1 infection in Iranian asymptomatic blood donors. METHODS: This study was performed on 100 HTLV-1-infected asymptomatic blood donors and 118 healthy blood donors. Genomic DNA from all participants was purified and then amplified using specific PCR primers. SNPs within TLR3 were evaluated using the restriction fragmentation length polymorphism technique, and the results were analyzed using SPSS software (version 22). RESULTS: The frequencies of the TLR3 (rs3775296) CC, CA, AA genotypes were 70%, 24%, and 6% in the patient group, and 50.8%, 44.9%, and 4.2% in the control group, respectively. There was a significant difference in the frequency distribution of TLR3 (rs3775296) genotypes and alleles, but not in the frequency distribution of TLR3 (rs3775291) genotypes between the patient and control groups. CONCLUSIONS: The TLR3 SNP rs3775296 was significantly associated with HTLV-1 infection and may be a protective factor against this viral infection.

Correcting nucleotide-specific biases in high-throughput sequencing data

BackgroundHigh-throughput sequence (HTS) data exhibit position-specific nucleotide biases that obscure the intended signal and reduce the effectiveness of these data for downstream analyses. These biases are particularly evident in HTS assays for identifying regulatory regions in DNA (DNase-seq, ChIP-seq, FAIRE-seq, ATAC-seq). Biases may result from many experiment-specific factors, including selectivity of DNA restriction enzymes and fragmentation method, as well as sequencing technology-specific factors, such as choice of adapters/primers and sample amplification methods.ResultsWe present a novel method to detect and correct position-specific nucleotide biases in HTS short read data. Our method calculates read-specific weights based on aligned reads to correct the over- or underrepresentation of position-specific nucleotide subsequences, both within and adjacent to the aligned read, relative to a baseline calculated in assay-specific enriched regions. Using HTS data from a variety of ChIP-seq, DNase-seq, FAIRE-seq, and ATAC-seq experiments, we show that our weight-adjusted reads reduce the position-specific nucleotide imbalance across reads and improve the utility of these data for downstream analyses, including identification and characterization of open chromatin peaks and transcription-factor binding sites.ConclusionsA general-purpose method to characterize and correct position-specific nucleotide sequence biases fills the need to recognize and deal with, in a systematic manner, binding-site preference for the growing number of HTS-based epigenetic assays. As the breadth and impact of these biases are better understood, the availability of a standard toolkit to correct them will be important.

Association Between Telomere Length, Chronic Kidney Disease, and Renal Traits: A Systematic Review.

Telomere length (TL) is an important biological variable that can influence a variety of disease-related complex traits as well as host-environment interactions such as drug and nutritional responses. Chronic kidney disease (CKD) is a common global health challenge especially with the currently aging world population. We conducted a PubMed database search according to the preferred reporting items for systematic reviews and meta-analysis (PRISMA) guidelines for systematic reviews. Studies in adults (18 years and above) in which TL was determined and correlated with CKD, renal traits, and function were included, while animal model studies were excluded. Nine studies comprising 7829 participants, published between 2005 and 2016, met the inclusion criteria. These included eight observational studies (six being prospective), and one clinical trial. Participants in two studies were diabetic patients with varying stages of CKD, and nondialysis chronic glomerulonephritis CKD patients in two other studies. TL measurements used polymerase chain reaction in five studies, terminal restriction fragmentation in three studies, and quantitative fluorescence in situ hybridization in one study. Short TL was independently associated with increased risk of prevalent microalbuminuria in diabetic men with CKD (p = 0.007). Among CKD patients with heterogeneous etiologies, however, there was an unadjusted lower risk (p < 0.001). Short TL was significantly associated with CKD progression among smokers (p = 0.001) and diabetic patients (p = 0.03). On the other hand, long TL was paradoxically associated with longer diagnosed duration of moderate CKD. We postulate that shortening TL might be associated with CKD prevalence/occurrence or declining kidney function, but this association is likely offset by the cellular telomere reparative process in those surviving longer with CKD. This systematic review underscores the need for future omics and human genetics research to delineate the contribution of TL to CKD, renal dysfunction, and related health outcomes. Telomeres and telomerase activity hold great promise for CKD risk stratification and personalized medicine.

Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci.

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer–promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.

Polymorphism T81C in H-RAS Oncogene Is Associated With Disease Progression in Imatinib (TKI) Treated Chronic Myeloid Leukemia Patients.

BackgroundMammalian cells contain three functional RAS proto-oncogenes, known as H-RAS, K-RAS, and N-RAS, which encode small GTP-binding proteins in terms of p21rass. RAS genes have been elucidated as major participants in the development and progression of cancer. A single nucleotide polymorphism (SNP) at H-RAS cDNA position 81 T→C (rs12628) has been found to be associated with the risk of many human cancers like gastrointestinal, oral, colon, bladder and thyroid carcinomas. Therefore, we hypothesized that this polymorphisms in H-RAS could influence susceptibility to chronic myeloid leukemia as well, and we conducted this study to test the hypothesis in Indian population.MethodH-RAS polymorphism was studied in 100 chronic myeloid leukemia (CML) patients and 100 healthy controls by restriction fragmentation length polymorphism (RFLP-PCR). Associations between polymorphism and clinicopathological features of CML patients were investigated.ResultsIn CML patients, the TT, TC and CC genotype frequency was 38%, 61% and 1% respectively, compared to 92%, 8% and 0% in healthy controls respectively. Compared to TT genotype, CT was significantly associated with increased risk of CML (odds ratio (OR): 8.4, P < 0.00001). There was a statistically significant correlation of H-RAS polymorphism with phases (P < 0.0003), molecular response (P < 0.0001), hematological response (P < 0.04) and thrombocytopenia (P < 0.003). However, there was no correlation of this polymorphism found with other clinical parameters.ConclusionH-RAS T81C polymorphism was found to be associated with CML risk and prognosis of CML. These results suggest that C heterozygosis may be considered a potential risk factor for CML development in the North Indian population.

CpG Motif in Phage Genome DNA Enhanced the Efficacy of Phage Therapy by Immunostimulation

Lytic phages for multiple-drug resistant E. coli and Pseudomonas aeruginosa were isolated. They belong to Siphoviridea and Myoviridea families, respectively. Lytic life cycle of the phage has therapeutic implications. Concurrent immune-stimulation of adaptive immunity against the etiologic agent, the MDR bacteria in the victim would revolutionize the field of phage therapy. In this pretext, methylated and unmethylated CpG motifs were identified in genome DNA of these phages by MspI and HpaII restriction fragmentation. To test the immune-stimulatory effects, two groups of BALB/c mice and two groups of rabbits were vaccinated with soluble antigen of Salmonella typhi with or without fragmented genome DNA of these phages. In addition, adjuvant efficacy of CpG motifs was compared with mineral oil. In antisera, types of antibodies were determined by immunoelectrophoresis and Western blotting. ELISA was done for quantitative analysis and has shown IgG immunoglobulin was produced by BALB/c and rabbit when fragmented DNA was used as adjuvant. Strong and specific immunogenicity has been attributed to the presence of CpG motifs. However, IgG antibody titer in antiserum raised by Pseudomonas' Phage DNA was high as compared to E. coli . The plausible reason for the variation in immune response is that this DNA has additional AACGAT motifs to enhance immunopotential of its CpG motifs. Strong and discrete bands were highlighted due to IgG Fab on the Western blots. We predict that efficacy of phage therapy will be enhanced by concurrent immune stimulation with CpG motif DNA injection with the respective MDR pathogen’s antigen for the control of disease.

Clinical Benefit of High-Sensitivity KRAS Mutation Testing in Metastatic Colorectal Cancer Treated with Anti-EGFR Antibody Therapy

Objective: We compared high-sensitivity KRAS mutation testing with direct sequencing for predicting the efficacy of antiepidermal growth factor receptor antibodies in patients with metastatic colorectal cancer (mCRC). Methods: We analyzed the KRAS status in 61 tumors from cetuximab-treated mCRC patients by both direct sequencing and a high-sensitivity method: 2-step PCR restriction fragmentation length polymorphism (RFLP). Therapeutic effects in each mutational status were evaluated. Results: The incidences of KRAS mutations determined by direct sequencing and 2-step PCR RFLP were 34.4 and 52.5%, respectively (p = 0.02). Patients were categorized into 3 groups [W/W, wild-type by both methods (n = 29); W/M, wild-type by direct sequencing, detected mutation by 2-step PCR RFLP (n = 11); M/M, mutant-type by both methods (n = 21)]. The response rate for cetuximab in the W/M group (0%) was the same as that in the M/M group, and was significantly lower than in the W/W group (41.4%) (p < 0.001). Progression-free survival in the W/M group (11.0 weeks) was similar to that in the M/M group (8.0 weeks), and was significantly shorter than in the W/W group (18.0 weeks) (p < 0.002). Conclusion: High-sensitivity KRAS mutation testing is useful for selecting true responders to cetuximab.

Biological Characterization of a Nucleopolyhedrovirus of Spodoptera litura (Lepidoptera: Noctuidae) Isolated from the Philippines

Abstract A Philippine Spodoptera litura nucleopolyhedrovirus (SltMNPV) was isolated by plaque purification from an uncloned NPV population in a formulated product that exhibits high insecticidal activity against the onion-attacking common cutworm, S. litura. Its estimated genome size was approximately 142 kb. Comparison with Autographa californica MNPV and Spodoptera exigua NPV by restriction fragmentation analysis showed that it was a distinct isolate. Light microscopy examination showed that SltMNPV-P7 infected insect cell lines derived from Spodoptera littoralis (CLS-79), Spodoptera frugiperda (Sf9), and S. litura (TUAT-SpLi221). It produced typical cytopathic effects in cell lines established from Bombyx mori and S. exigua and induced apoptotic-like cytopathology in Lymantria dispar -derived cell line; it exerted no observable cytopathic effect on Spilosoma imparilis cell line. Significant increase in budded virus (BV) production was observed in CLS-79, Sf9, and TUAT-SpLi221 cell lines, in which TUAT-SpLi221 cell line supported the highest BV titer. No significant BV production was observed in the remaining four cell lines. Similarly, viral DNA replication and polyhedrin production proceeded only in these three spodopteran cell lines. Results showed that SltMNPV-P7 is a unique isolate which can be propagated in vitro for further studies and thus has a potential for development into a more effective microbial insecticide.

Propagation of TEM- and PSE-type beta-lactamases among amoxicillin-resistant Salmonella spp. isolated in France.

A survey conducted between 1987 and 1994 at the University Hospital of Besançon, France, demonstrated a dramatic increase (from 0 to 42. 5%) in the prevalence of amoxicillin resistance among Salmonella spp. Of the 96 resistant isolates collected during this period (including 77 Typhimurium), 54 were found to produce TEM-1 beta-lactamase, 40 produced PSE-1 (equivalent to CARB-2), one produced PSE-1 plus TEM-2, and one produced OXA-1 in isoelectric focusing and DNA hybridization experiments. Plasmids coding for these beta-lactamases were further characterized by (i) profile analysis, (ii) restriction fragmentation pattern analysis, (iii) hybridization with an spvCD-orfE virulence probe, and (iv) replicon typing. In addition, isolates of S. typhimurium were genotypically compared by pulsed-field gel electrophoresis of XbaI-macrorestricted chromosomal DNA. Altogether, these methods showed that 40 of the 41 PSE-1 producers were actually the progeny of a single epidemic S. typhimurium strain lysotype DT104. Isolates of that strain were found to harbor RepFIC virulence plasmids with somewhat different restriction profiles, but which all carried the bla(PSE-1) gene. Of these virulence/resistance plasmids, 15 were transmissible to Escherichia coli. TEM-1-producing S. typhimurium displayed much greater genotypic and plasmidic diversities, suggesting the acquisition of the bla(TEM-1) gene from multiple bacterial sources by individual strains. In agreement with this, 32 of the 35 S. typhimurium plasmids encoding TEM-1 were found to be conjugative. These data show that development of amoxicillin resistance among Salmonella, especially in serovar Typhimurium, results from both gene transfers and strain dissemination.

Molecular characterization of penicillin-resistant Streptococcus pneumoniae isolates from Bulgaria.

As part of an ongoing surveillance program of antibiotic-resistant Streptococcus pneumoniae in Sofia, Bulgaria, 120 penicillin-resistant strains (PRSP) (most of them recovered from children hospitalized with pneumococcal disease) were analyzed by microbiological and molecular methods. Several unique features of this collection are of particular interest. (i) Most isolates (112 of 120) were also resistant to trimethoprim-sulfamethoxazole (SXT) (97 of 120 isolates, or 80%), and over 70% (86 of 120) of the isolates were resistant to at least three antibiotics in addition to penicillin. (ii) Close to 80% of all isolates were represented by large clusters of bacteria, each with a unique serotype, antibiotype, and chromosomal macrorestriction pattern (determined by pulsed-field gel electrophoresis), as well as unique restriction fragmentation length polymorphisms of the penicillin-binding protein genes pbp1a, pbp2x, and pbp2b. (iii) A large proportion (45 of 120, or 38%) of the strains belonged to two internationally spread epidemic clones of S. pneumoniae, the first expressing capsular type 23F and the second expressing serotype 9. (iv) A unique Bulgarian cluster composed of eight serotype 19F isolates was resistant to tetracycline, SXT, cefotaxime, and extremely high levels of penicillin and erythromycin. Nevertheless, this clone did not react with either the erm or the mef DNA probes, and thus the mechanism of macrolide resistance in this group of PRSP remains to be elucidated.

Stability of clonally related DNA fingerprints and cell-wall peptide patterns in geographic isolates of multiresistant epidemic clones of Streptococcus pneumoniae

Objective: To determine the stability of multidrug-resistant epidemic clones of Streptococcus pneumonia. Methods: Polymerase chain reaction (PCR) was used to amplify the penicillin-binding protein (PBP) genes 1A, 2X, and 2B, followed by fingerprinting after restriction digestion of the amplified molecules. Cell walls were isolated, digested by the autolytic amidase of pneumococcus, and the family of stem peptides was separated by high-pressure liquid chromatography. Results: Twenty-eight isolates of multidrug-resistant capsular type 6B S. pneumoniae recovered in Iceland between 1990 and 1992 and ten isolates of the multidrug-resistant capsular type 23F S. pneumoniae recovered in the United States, Portugal, Croatia, and South Korea between 1989 and 1996 were compared for restriction fragmentation length polymorphism (RFLP) of their PBP genes 1A, 2X, and 2B, using the PCR for amplification. Five isolates each of the multidrug-resistant capsular type 6B pneumococci from Iceland and the capsular type 23F isolates from Cleveland, Ohio, were also compared for their muropeptide profiles, using a high-performance liquid chromatography (HPLC) system to separate the family of stem peptides generated by enzymatic hydrolysis. The isolates representing the two major and internationally spread epidemic clones have retained the clone-specific RFLP fingerprints of their PBP genes and also reproduced distinct cell-wall stem peptide patterns characteristic of the two pneumococcal clones. Conclusions: The data further document the considerable stability of clonal types of the multiresistant epidemic clones of S. pneumoniae over time and over a wide range of geographic isolation sites.

Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR.

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides. All B. anthracis profiles were identical for each of 18 restriction enzymes, each of 10 arbitrary PCR primers, and a repetitive extragenic palindrome-specific PCR primer. The PCR profiles generated with a coliphage M13-based primer exhibited slight pattern variation in a 400- to 500-bp band region. The B. anthracis profiles were unique compared with the profiles of the other species examined. In these other species, strain-to-strain variations were observed. Our results showed that isolates of B. anthracis are almost completely homogeneous, indicating a clonal lineage, and are distinct from other members of the B. cereus group and that B. anthracis, as a species in its own right, may have evolved only relatively recently.

Investigation of an outbreak of methicillin-resistant Staphylococcus aureus in patients with skin disease using DNA restriction patterns.

To investigate an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) among patients using chromosomal typing of the isolates. Comparison of epidemiological and clinical data to endonuclease restriction fragmentation analysis (RFA) of the MRSA isolates associated with an outbreak. Total DNA from the MRSA isolates was restricted with HINDIII and HAEIII for typing. Tertiary care academic medical center. An epidemiological investigation of an outbreak of MRSA among patients in private rooms was evaluated by routine infection control methods. The MRSA isolates from blood cultures of 7 patients and the nares of a nurse were collected during the outbreak. MRSA isolates from 23 patients not associated with the outbreak also were collected. The total DNA of the MRSA isolates were restricted with HINDIII and HAEIII and electrophoresed on 0.6% agarose gels. MRSA from 4 of the 7 bacteremic patients and the nurse on the outbreak unit had the same endonuclease restriction pattern. The patients were linked in that they were compromised by severe psoriasis or skin ulcers, were on the unit during the same period, and had oatmeal baths in a common bathtub. Of 50 staff members screened, the nurse was the only person detected as colonized by the strain. The other 3 patients on the unit as well as the 23 patients in other locations not associated with the outbreak had MRSA isolates with different RFA patterns. The use of the bathtub was discontinued and further transmission of MRSA was stopped. A comparison of the relatedness of MRSA by RFA demonstrated the uniqueness of the epidemiologically linked isolates and the utility of the RFA technique in the performance of routine infection control investigations.

Investigation of an Outbreak of Methicillin-Resistant Staphylococcus aureus in Patients With Skin Disease Using DNA Restriction Patterns

AbstractObjective:To investigate an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) among patients using chromosomal typing of the isolates.Design:Comparison of epidemiological and clinical data to endonuclease restriction fragmentation analysis (RFA) of the MRSA isolates associated with an outbreak. Total DNA from the MRSA isolates was restricted with HINDIII and HAEIII for typing.Setting:Tertiary care academic medical center.Methods:An epidemiological investigation of an outbreak of MRSA among patients in private rooms was evaluated by routine infection control methods. The MRSA isolates from blood cultures of 7 patients and the nares of a nurse were collected during the outbreak. MRSA isolates from 23 patients not associated with the outbreak also were collected. The total DNA of the MRSA isolates were restricted with HINDIII and HAEIII and electrophoresed on 0.6% agarose gels.Results:MRSA from 4 of the 7 bacteremic patients and the nurse on the outbreak unit had the same endonuclease restriction pattern. The patients were linked in that they were compromised by severe psoriasis or skin ulcers, were on the unit during the same period, and had oatmeal baths in a common bathtub. Of 50 staff members screened, the nurse was the only person detected as colonized by the strain. The other 3 patients on the unit as well as the 23 patients in other locations not associated with the outbreak had MRSA isolates with different RFA patterns. The use of the bathtub was discontinued and further transmission of MRSA was stopped.Conclusions:A comparison of the relatedness of MRSA by RFA demonstrated the uniqueness of the epidemiologically linked isolates and the utility of the RFA technique in the performance of routine infection control investigations.

Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobilizing plasmids isolated ranged from 40 kb to over 200 kb, and 19 of 54 were found to encode mercury resistance. Plasmid-encoded resistance to tetracycline and streptomycin was also found but not resistance to UV light or various heavy metals. Eight plasmids of epilithic bacteria, analyzed by comparing restriction fragmentation patterns, showed significant differences between those isolated from different independent matings. Optimal temperatures for mobilization of pD10 were between 15 and 25 degrees C. Four mercury resistance plasmids were found to be broad host range, transferring mercury resistance and mobilizing pD10 readily to representative species of beta- and gamma-purple bacteria. In general, frequencies of pD10 mobilization by plasmids of epilithic bacteria were 2 to 3 orders of magnitude lower than conjugal transfer frequencies. Thus, there is a high potential for exchange of recombinant genes introduced into the epilithon by mobilization between a variety of bacterial species.

A conserved and unique (AT)-rich segment in yeast mitochondrial DNA

The mtDNA of the cytoplasmic petite mutant of yeast RD1A consists mainly of a perfect head-to-tail repetition of a known sequence of 66 consecutive AT and 2 GC base pairs. We have hybridized complementary RNA made on RD1A mtDNA with the mtDNAs of four different wild-type Saccharomyces strains that differ markedly in restriction fragmentation pattern. The tm's of the four heteroduplexes are identical to the tm of the homoduplex of RD1A mtDNA with complementary RNA of one repeat length. With all four wild-type mtDNAs this complementary RNA hybridizes mainly to a single restriction fragment of about 300 base pairs. This shows the conservation and individuality of at least one (AT)-rich segment in yeast mtDNA. The 300 base pair fragment has been mapped in the vicinity of the oxi-2 locus. The possible role of the (AT)-rich segment in the processing of the primary transcript of this region is discussed.