Series Of Polymerase Chain Reactions Research Articles

Occurrence and characterization of ESKAPE organisms on the hands of veterinary students before patient contact at a veterinary academic hospital, South Africa

ObjectiveThis study aimed to investigate the presence of ESKAPE organisms on the hands of students working in the intensive care unit (ICU) at a veterinary academic hospital.MethodsA cross-sectional study was conducted among students working in an ICU at a veterinary academic hospital in South Africa. Students were sampled before the start of the ICU shift using a modified glove-juice method. Standard microbiological techniques and a series of polymerase chain reaction (PCR) assays were used to identify and characterize the bacteria. All the isolates were tested for resistance against a specific panel of antibiotics using the disk diffusion method. Proportions of bacterial species and their antimicrobial-susceptibility profiles were calculated.ResultsAt screening, all the veterinary students (n = 62) carried at least one of the ESKAPE organisms on their hands. Escherichia coli was the most isolated organism (76%, 47/62), followed by P. aeruginosa (48%, 30/62), A. baumannii (47%, 29/62), E. faecium (35%, 22/62), K. pneumoniae (27%, 17/62), and S. aureus (24%, 15/62). A reduced proportion of isolates were recovered from the samples, E. coli (26%, 12/47), E. faecium (23%, 5/22), P. aeruginosa (43%, 13/30), A. baumannii (24%,7/29), K. pneumoniae (41%, 7/17), and S. aureus (20%, 3/15). Most of the organisms showed a high proportion of resistance to at least one antibiotic. Multidrug resistance was reported among just over half (56%, 5/9) of E. coli, 40% (2/5) of E. faecium, 100% (13/13) of P. aeruginosa, and 33% (1/3) of S. aureus isolates.ConclusionStudents working in the ICU carry several organisms belonging to the ESKAPE group of organisms before contact with patients. Moreover, MDR resistance was common among this group of organisms. The findings of the present study underscore the importance of infection prevention and control (IPC) strategies to help reduce the likelihood of the spread of these organisms to personnel, owners, family members, and patients.

Effect of DNA Template Concentration on Standard Polymerase Chain Reaction

The polymerase chain reaction (PCR) was an essential fundamental procedure widely used in molecular biology to amplify specific deoxyribonucleic acid (DNA) sequences. To investigate the efficiency and specificity of PCR using DNA extracted from human blood cells as sample, several concentrations of DNA templates were used to obtain accurate and reproducible results during this optimisation process. The principal objective was to identify which concentration was the best for amplifying the DNA target sequence and study the effects of the scale of concentrations to standard PCR. For this study, a series of PCR were conducted using various concentrations of DNA template ranging from 1 ng/μL to 100 ng/μL per reaction under optimized conditions of primer concentrations, annealing temperature and extension time. The results demonstrated that DNA template concentration significantly influenced the efficiency and specificity of PCR as the intensity of the target gene amplification band was increased on the agarose gel electrophoresis under ultraviolet light indicating the PCR product yield. Formation of primer-dimers became more prominent as the DNA template concentration reduced resulting in non-specific amplification. The concentrations ranging from 10 ng/μL to 70 ng/μL were the most optimal reactions profiting a remarkable feat of target gene amplification while concentrations less than 1 ng/μL produced none and non-specific outcomes retaining little amount of target gene amplification. In conclusion, our study highlights the pivotal role of DNA template concentration optimises PCR to increase reliability and reproducibility, extending our understanding of genetic analysis, diagnostic, and forensic sciences.

Mobile genetic elements associated with utilization of dichloromethane and methanol as energy sources in Cupriavidus metallidurans.

Cupriavidus metallidurans strain PD11 isolated from laboratory waste drainage can use C1 compounds, such as dichloromethane (DCM) and methanol, as a sole carbon and energy source. However, strain CH34 (a type-strain) cannot grow in the medium supplemented with DCM. In the present study, we aimed to unravel the genetic elements underlying the utilization of C1 compounds by strain PD11. The genome subtraction approach indicated that only strain PD11 had several genes highly homologous to those of Herminiimonas arsenicoxydans strain ULPAs1. Moreover, a series of polymerase chain reaction (PCR) to detect the orthologs of H. arsenicoxydans genes and the comparative study of the genomes of three strains revealed that the 87.9 kb DNA fragment corresponding to HEAR1959 to HEAR2054 might be horizontally transferred to strain PD11. The 87.9 kb DNA fragment identified was found to contain three genes whose products were putatively involved in the metabolism of formaldehyde, a common intermediate of DCM and methanol. In addition, reverse transcription PCR analysis showed that all three genes were significantly expressed when strain PD11 was cultivated in the presence of DCM or methanol. These findings suggest that strain PD11 can effectively utilize the C1 compounds because of transfer of the mobile genetic elements from other bacterial species, for instance, from H. arsenicoxydans.

Biochemical Investigation into Cadmium‐Induced Diminished Function of a Thermal Stable DNA Polymerase

The environmental metal cadmium is a carcinogen that has been shown to increase the rate of mutations by inhibiting DNA mismatch repair, and to replace zinc ions within the active site of certain endonucleases. Here, the mutagenic property of cadmium was further explored with a model DNA polymerase, Thermus aquaticus DNA Polymerase I (Taq Pol I). Diminished efficiency of Taq Pol I was observed as a function of cadmium concentration. Using a series of polymerase chain reactions (PCR) and circular dichroism spectroscopic assays with full-length Taq Pol I, structural changes in the presence of low cadmium concentration was a significant factor in the observed change in enzyme efficiency. These structural changes provide new insights into the mutagenic property of cadmium.

A Phylogenetic and Genotyping Study of Bovine Herpesvirus Type 4 (BHV-4) in Turkey

Infertility and reproductive infections are the huge problems for the diary management throughout the world. Bovine herpesviruses act an enormous role in these complicated problems. Bovine Herpesvirus Type 1 (BHV-1) is the most outstanding herpesvirus causing genital and uterine tracts infections among other reproductive viral agents, however, Bovine Herpesvirus Type 4 (BHV-4) is also responsible in terms of similar symptoms and diseases. The main aims of the study are both to investigate the underlying potential presence of BHV-4 in subclinical uterine tract infection, and both to perform molecular and recombination analyses. A herd including 25 repeat breeder cows were investigated by BHV-4. Two out of them were determined BHV-4 infected after a series of Polymerase Chain Reactions (PCRs) tests which able to amplify partial Glycoprotein B (gB) and Thymidine Kinase (TK) gene regions. Obtained sequences were analyzed by using phylogenetic and recombinational software, and two Maximum Likelihood (ML) tree have been constructed. To results, novel Turkish BHV-4 sequences fell into Genotype I in both constructed Maximum Likelihood (ML) phylogenetic trees, however, no recombination evidence has been observed in relevant software. This report is the first genotyping study of BHV-4 from Turkey. This study showed that Turkish BHV-4 substantially originated from European strains and might be observed in different clinical presentations. This suggests that BHV-4 should be deeply investigated by further molecular techniques and included in diagnostic panels for reproductive diseases viruses.

Seasonality of Acute Retinal Necrosis.

PurposeTo study the seasonal variability in the occurrence of acute retinal necrosis (ARN) in a series of polymerase chain reaction (PCR)-positive patients.Methods Consecutive patients clinically diagnosed with ARN and a positive PCR result of aqueous humor during a seven-year period were studied retrospectively. Patients' demographics, causative viral agent(s), and the date of disease onset were extracted from medical records.ResultsTwenty eyes of 20 patients were enrolled; the mean age at presentation was 39.6 14.4 (range, 6–62) years. Nine patients were female. The most common causative agent was varicella-zoster virus in 16 patients (80%), followed by herpes simplex virus in two patients (10%). The disease onset was in winter in 10 patients (50%), and the highest incidence was in February (five patients, 25%). The cumulative occurrence of ARN was significantly higher in the first half of the year (winter and spring) compared to the second half of the year (summer and fall) (P = 0.030). In general, seasons with a high incidence of ARN were preceded by cold seasons.Conclusion In our series, we observed seasonal variability in the incidence of ARN, with the highest incidence during winter and spring. However, further epidemiologic studies in different geographical areas are required to elucidate the true seasonal nature of ARN.

Mistaking infection for inflammation

SummaryAmong the first steps in diagnosing any patient with uveitis is to identify infection. Common infections that may masquerade as autoimmune disease include herpes simplex virus, varicella zoster virus or cytomegalovirus (CMV) anterior uveitis, chronic post‐surgical endophthalmitis, endogenous endophthalmitis, acute retinal necrosis, CMV retinitis in immunocopme patients, syphilis, atypical toxoplasmosis and tuberculosis. Patient history and clinical exam can often make the diagnosis. The utility of diagnostic tests such as treponemal tests, quantiferon testing, Mantoux skin test, and toxoplasmosis serology will be covered. Case series of polymerase chain reaction (PCR) testing of anterior chamber and/or vitreous aspirate for anterior segment and posterior segment uveitis will be reviewed, with an emphasis on demonstrated clinical utility. More invasive biopsy such as vitreous biopsy via pars plana vitrectomy or chorioretinal biopsy may be necessary in some settings. Appropriate treatment and criteria for treating specific infections will be covered. Cases of polymicrobial infection will be reviewed as well. Emphasis will be placed on distinguishing infection from autoimmune disease.

Sequencing and quantifying plastid DNA fragments stored in sapwood and heartwood of Torreya nucifera

The selection of wood species and the styles of sculpture play key roles in the characterization of Buddhist statues. After Jianzhen, a Chinese Buddhist monk, visited Japan in the mid-eighth century, wood of the genus Torreya had been frequently used to produce single-bole statues. Establishing measures for the accurate identification of wood in the genus Torreya is effective for investigating the drastic change in the production of statues during this period. Analyzing the plastid deoxyribonucleic acid (DNA) fragments extracted from wood is considered helpful in the identification of species in the same genus. This study analyzed the sequences and residual amounts of plastid DNA fragments in the wood of Torreya nucifera. Nucleotide substitutions in the plastid DNA were clearly identified between T. nucifera and the species distributed in China, indicating that the wood of Torreya sp. can be discriminated based on the plastid DNA sequences. DNA polymorphism analyses revealed sequence diversity for the intergenic spacers on the T. nucifera plastid DNA. A series of polymerase chain reaction (PCR) analyses demonstrated that the plastid DNA fragments with a length of approximately 100 bp could be amplified from the residual DNA extracted from the T. nucifera sapwood with longer elapsed years after cutting. Therefore, an identification of wood species in the genus Torreya based on their plastid DNA is considered to be one of the most effective measures taken in the study regarding the historical changes of Buddhist statues.

Detection of respiratory pathogens in pediatric acute otitis media by PCR and comparison of findings in the middle ear and nasopharynx

We conducted a series of polymerase chain reactions (PCRs) in order to detect bacteria (7 species) and viruses (17 species) in middle ear fluid (MEF) and nasopharynx (Nph) of children with acute otitis media (AOM; n=179). Bacterial and viral nucleic acids were detected in MEF of 78.8% and 14.5% patients, respectively. The prevalence was as follows: Streptococcus pneumoniae, 70.4%; Haemophilus influenzae, 17.9%; Staphylococcus aureus, 16.8%; Streptococcus pyogenes, 12.3%; Moraxella catarrhalis, 9.5%; rhinovirus, 9.5%; and adenovirus, 3.4%. The overall rate of PCR-positive specimens for bacterial pathogens was 2.6 times higher, compared to culture results. The rate of PCR-positive results and the distribution of pathogens in the Nph were similar to those in the MEF. Nph PCR results had variable positive predictive values and high negative predictive values in predicting MEF findings. Our results indicate that Nph PCR could be a practical tool for examining respiratory pathogens in children with acute infections.

Analysis of ER Resident Proteins in Saccharomyces cerevisiae: Implementation of H/KDEL Retrieval Sequences

An elaborate quality control system regulates endoplasmic reticulum (ER) homeostasis by ensuring the fidelity of protein synthesis and maturation. In budding yeast, genomic analyses and high‐throughput proteomic studies have identified ER resident proteins that restore homeostasis following local perturbations. Yet, how these folding factors modulate stress has been largely unexplored. In this study, we designed a series of polymerase chain reaction (PCR)‐based modules including codon‐optimized epitopes and fluorescent protein (FP) variants complete with C‐terminal H/KDEL retrieval motifs. These conserved sequences are inherent to most soluble ER resident proteins. To monitor multiple proteins simultaneously, H/KDEL cassettes are available with six different selection markers, providing optimal flexibility for live‐cell imaging and multicolor labeling in vivo. A single pair of PCR primers can be used for the amplification of these 26 modules, enabling numerous combinations of tags and selection markers. The versatility of pCY H/KDEL cassettes was demonstrated by labeling BiP/Kar2p, Pdi1p and Scj1p with all novel tags, thus providing a direct comparison among FP variants. Furthermore, to advance in vitro studies of yeast ER proteins, Strep‐tag II was engineered with a C‐terminal retrieval sequence. Here, an efficient purification strategy was established for BiP under physiological conditions.

Successful Identification of Pathogens by Polymerase Chain Reaction (PCR)-Based Electron Spray Ionization Time-of-Flight Mass Spectrometry (ESI-TOF-MS) in Culture-Negative Periprosthetic Joint Infection

The diagnosis of periprosthetic joint infection poses many challenges, one of which is the difficulty of isolating the infecting organism. Recently, a sophisticated modality (the Ibis Biosciences T5000 biosensor system) has been introduced that uses pan-domain primers in a series of polymerase chain reactions (PCRs) to identify and speciate essentially all bacteria and fungi as well as to identify key antibiotic resistance genes. We investigated the role of the Ibis in identifying infecting organisms in cases of known and suspected periprosthetic joint infection. Synovial fluid specimens were collected prospectively from eighty-two patients undergoing eighty-seven arthroplasty procedures (sixty-five knee revisions, fifteen hip revisions, and seven primary knee arthroplasties) and were sent for both conventional culture and Ibis analysis. The surgeon's clinical determination of the cause for revision arthroplasty was failure due to infection in twenty-three cases and noninfectious failure in fifty-seven cases. In the twenty-three cases that were considered on clinical grounds to involve a periprosthetic joint infection, the Ibis detected the same pathogen isolated by conventional culture in seventeen of eighteen cases and also detected one or more organisms in four of the five culture-negative cases. In addition, the Ibis detected organisms in fifty (88%) of the fifty-seven cases in which revision arthroplasty was performed for a presumed noninfectious failure. The Ibis technology was not only effective at detecting organisms in cases of suspected periprosthetic joint infection in which cultures were negative, but it also suggested that many of the revision arthroplasty cases that have previously been considered to be purely aseptic may have a component of unrecognized, subclinical infection.

Improvement of Amorpha-4,11-diene Production by a Yeast-Conform Variant of Vitreoscilla Hemoglobin

Amorpha-4,11-diene is the precursor of the antimalarial compound artemisinin. The effect of Vitreoscilla hemoglobin (VHb) and its yeast-conform variant (VHbm) on amorpha-4,11- diene production in engineered Saccharomyces cerevisiae was investigated. First, the VHb gene was mutated to the yeast-conform variant VHbm based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial VHbm gene contained codons preferred by the yeast translation machinery. Two yeast expression vectors containing VHb or VHbm gene were constructed and introduced into the amorpha-4,11-diene-producing strain S. cerevisiae WK1 to form WK1[VHb] and WK1[VHbm], respectively. Western blot and CO-difference spectrum absorbance assay showed that VHb and VHbm were successfully expressed. In shake fl asks, VHbm expression conferred higher cell growth than VHb expression. GC-MS results indicated the amorpha- 4,11-diene production in WK1[VHbm] and WK1[VHb] was 3- and 2-fold higher than that in WK1, respectively. This suggests that VHb might improve the amorpha-4,11-diene production in engineered S. cerevisiae

Improvement of Amorpha-4,11-diene Production by a Yeast-Conform Variant of Vitreoscilla Hemoglobin

Amorpha-4,11-diene is the precursor of the antimalarial compound artemisinin. The effect of Vitreoscilla hemoglobin (VHb) and its yeast-conform variant (VHbm) on amorpha-4,11-diene production in engineered Saccharomyces cerevisiae was investigated. First, the VHb gene was mutated to the yeast-conform variant VHbm based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial VHbm gene contained codons preferred by the yeast translation machinery. Two yeast expression vectors containing VHb or VHbm gene were constructed and introduced into the amorpha-4,11-diene-producing strain S. cerevisiae WK1 to form WK1[VHb] and WK1[VHbm], respectively. Western blot and CO-difference spectrum absorbance assay showed that VHb and VHbm were successfully expressed. In shake flasks, VHbm expression conferred higher cell growth than VHb expression. GC-MS results indicated the amorpha-4,11-diene production in WK1[VHbm] and WK1[VHb] was 3- and 2-fold higher than that in WK1, respectively. This suggests that VHb might improve the amorpha-4,11-diene production in engineered S. cerevisiae.

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

BackgroundIn sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.MethodsWe have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.Principal FindingsWe have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.ConclusionWe have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.

Mechanisms of CRLF2 Rearrangement with IGH and P2RY8 in B-Cell Acute Lymphoblastic Leukemia.

Abstract 3243Poster Board III-180 IntroductionPrecursor B-cell acute lymphoblastic leukemia (B-ALL) is the most common malignancy in children. While the great majority of these children achieve long-term remission, the disease exacts a high mortality in adults, and children with high-risk features. Fusion oncogenes such as BCR-ABL, MLL-AF4, E2A-PBX and TEL-AML1 are present in 60% of B-ALL cases. Cloning of these rearrangements has provided significant insight into the mechanisms involved in their formation. The gene alterations that affect the remaining 40% are poorly understood. We and others have recently identified CRLF2, a subunit of the thymic stromal lymphopoietin receptor, as a novel oncoprotein in B-ALL. Approximately 15% of adult and high-risk pediatric B-ALL that lack other characteristic gene rearrangements overexpress CRLF2, while leukemias with these rearrangements do not. In cases with CRLF2 overexpression, CRLF2 appears to be the driver of STAT activation, either alone or in combination with gain-of-function mutations in Janus Kinases (JAKs). CRLF2 overexpression results from rearrangements involving the CRLF2 locus, which is located in the pseudoautosomal regions on chromosomes X and Y. These rearrangements can either involve translocation with the immunoglobulin heavy chain (IGH) locus (t(X;14)(p22;q32) or t(Y;14)(p11;q32)) or interstitial deletion (del(X)(p22.33p22.33) or del(Y)(p11.32p11.32)). We sought to define the mechanisms of cleavage and repair that mediate these rearrangements. MethodsFor the translocations, we performed a series of polymerase chain reaction (PCR) assays to amplify junctions between IGHJ segments on chr.14 and the region upstream (i.e., centromeric) of CRLF2 on chr.X/Y. For the deletions, we used single nucleotide polymorphism (SNP) arrays and quantitative PCR to define the extent of deletions and then amplified junctions by PCR. ResultsWe successfully amplified IGHJ/CRLF2 translocation junctions from six B-ALL with CRLF2 overexpression. Junctions involved chr.X/Y sequence between 8-16kb upstream of the CRLF2 translation start site. Five of 6 clustered near putative V(D)J recombinase recognition signal sequences (RSS). Additional evidence for involvement of the V(D)J recombinase was identified in all cases, including the presence of nontemplated nucleotides. In contrast, deletions resulted in juxtaposition of the full length CRLF2 coding sequence to P2RY8. A similar event involving t(X;12) that resulted in SOX5 translocation to P2RY8 has been described in a single case of splenic follicular lymphoma. P2RY8 is a member of the purine nucleotide G-protein coupled receptor gene family and is highly expressed in lymphocytes. ConclusionCRLF2 rearrangements result in overexpression through juxtaposition to alternate transcriptional control elements. While translocations appear to be mediated by aberrant V(D)J recombination, deletions likely involve an alternate sequence-dependent mechanism that targets downstream of the P2RY8 promoter. DisclosuresNo relevant conflicts of interest to declare.

The improvement of amorpha-4,11-diene production by a yeast-conform variant

To investigate the effect of the yeast-conform variant of the Artemisia annua gene encoding for amorpha-4,11-diene synthase (ADS) on the production of amorpha-4,11-diene in a transformed yeast. The ADS gene was mutated to the yeast-conform variant ADSm. The ADSm synthesis was performed based on step-by-step extension of a short region of the gene through a series of polymerase chain reactions (PCR). The artificial ADSm gene contained codons preferred by the yeast translation machinery. The sequence was then integrated into a yeast expression vector pYeDP60. The fusion construct was active and the transformed yeast cells produced higher level of amorpha-4,11-diene compared with the plant gene-transformed yeast cells. Strains transformed with the yeast-conform allele (ADSm) were more efficient in terms of production of amorpha-4,11-diene than those transformed with the plant gene. We demonstrated that yeast-conform allele of foreign genes by serial PCR reactions can be a solution to low efficiency of heterologous gene expression in Saccharomyces cerevisiae cells.

Orthopoxvirus pan-genomic DNA assay

A genome-spanning assay is described that enables laboratory confirmation of infections with orthopoxviruses (OPVs), particularly Vaccinia, Monkeypox, and Variola viruses, which can cause vesiculo-pustular rash illnesses in humans. The assay uses a series of polymerase chain reaction (PCR) amplicons that overlap to span the approximately 200 kilobase pair linear DNA genome of OPVs. Corresponding amplicons of different viral isolates can then be compared by matching their restriction fragment length polymorphism (RFLP) gel electrophoresis patterns. The PCR step does not necessarily require viral growth to produce sufficient DNA for the RFLP comparisons. The assay would be useful as a prelude to sequencing entire or partial DNA genome regions of various OPVs, including natural or recombinant OPVs and potentially dangerous bioengineered OPVs designed to express foreign DNA or other viruses.

Variable nucleotide tandem repeat (VNTR) typing of two palaeopathological cases of lepromatous leprosy from Mediaeval England

Using ancient DNA methods, we have examined in detail two archaeological cases of leprosy from Mediaeval England. The first was a child skeleton with rhino-maxillary changes typical of lepromatous leprosy (LL). The second case was the skeleton of a male adult who showed both typical rhino-maxillary changes and osteitis/periostitis on the leg and foot bones. Bone powder was sampled from both cases and DNA extracts were prepared. These were subjected to a series of polymerase chain reactions (PCRs) specific for regions on the Mycobacterium leprae genome. The repetitive element RLEP was used for confirmation of M. leprae DNA and then three polymorphic regions were successfully amplified and sequenced to determine the number of variable nucleotide tandem repeats (vntr) at these loci. These were the microsatellite regions ML2344 and ML2172 and the minisatellite region ML0058. Genotyping data from the strains preserved within the skeletal remains were compared with those obtained for a reference strain of M. leprae. Variation at these three loci was found between both burials and the reference strain, indicating that vntr typing of LL cases from the archaeological record is a useful way of confirming disease and an additional means of authenticating aDNA data. This demonstrates the feasibility of targeting multiple loci for phylogenetic studies of leprosy strains from archival sources.

Fungal Diversity in Rock Beneath a Crustose Lichen as Revealed by Molecular Markers

Lichen-forming fungi have been assumed to be more or less restricted to the surface of the substrate on which they grow, Conclusive identification of hyphae or an assessment of the fungal diversity inside lichen-covered rock has not been possible using methods based on direct observation. We circumvented this problem by using a DNA sequencing approach. Cores were drilled from a Devonian arcosic sandstone rock harboring the crustose lichen Ophioparma ventosa (L.) Norman on the surface. The cores were cut vertically, and DNA was extracted from the pulverized rock slices. A series of polymerase chain reactions using fungal-specific primers as well as Ophioparma ventosa specific primers were employed to amplify the internal transcribed spacer region of the nuclear ribosomal DNA. The results show that hyphae of O. ventosa penetrate approximately 10-12 mm into the rock. Consequently, the hyphal layer formed by the lichen fungus inside the rock could be 7-12 times as thick as the symbiotic thallus at the surface of the rock. In addition, eight non-lichenized fungal taxa and five that could not be identified to species were encountered. One fungal species in the order Helotiales occurs in six of the eight cores. The significance of these results to the colonization and weathering of rock by lichenized fungi is discussed.

Binding of daunomycin to diaminopurine- and/or inosine-substituted DNA.

The binding of the anticancer drug daunomycin to double-helical DNA has been investigated by DNase I footprinting and fluorescence titration, using a series of polymerase chain reaction (PCR) synthesized DNA fragments that contained systematic base substitutions to alter the disposition of functional groups within the minor groove. The 160 bp tyrT DNA fragment constituted the starting material. Fragments in which (i) inosine was substituted for guanosine, (ii) diaminopurine was substituted for adenine, and (iii) both inosine and diaminopurine were substituted for guanosine and adenine, respectively, were studied. These fragments permit the role of the 2-amino group in the minor groove to be systematically explored. The results of DNase I footprinting experiments confirmed that daunomycin binds preferentially to 5'(A/T)GC and 5'(A/T)CG triplets in the normal fragment. Substitution of inosine for guanosine, with the concomitant loss of the N-2 in the minor groove, weakened binding affinity but did not dramatically alter the sequence preference associated with daunomycin binding. Complete reversal of the location of the N-2 group by the double substitution, however, completely altered the sequence preference of daunomycin and shifted its binding from the canonical triplets to ones with a 5'IDD motif. These results have critically tested and confirmed the proposed key roles of the daunosamine moiety and the 9-OH group of daunomycin in dictating binding to preferred sites. In a parallel study, both macroscopic and microscopic binding to the normal tyrT fragment were investigated, experiments made possible by using PCR to prepare large quantities of the long, defined DNA sequence. The results of these experiments underscored the complexity of the interaction of the drug with the DNA lattice and revealed unequivocal heterogeneity in its affinity for different binding sites. A class of high-affinity sites, most probably corresponding to the 5'(A/T)GC and 5'(A/T)CG triplets, was identified and characterized in macroscopic binding isotherms.