Nucleic Acid Hybridization Analysis Research Articles

Advances in Studies on Horizontal Gene Transfer Promoting the Evolution of Resistance Genes of Periodontal Pathogens

Horizontal gene transfer (HGT) plays an important role in bacterial evolution. HGT is an important way for bacteria to rapidly obtain genetic improvement. HGT promotes the transmission of biological characteristics such as virulence, antibiotic resistance and allometabolism in microorganisms. The bacteria in the mouth are abundant and abundant. Bacterial virulence gene (Virulence genes, VGs) and antibiotic resistance genes (Antibiotic resistance genes, ARGs) besides through vertical transmission by parental, also can through HGT in oral symbiotic species or genus within the transmission. HGT promotes the spread of virulence genes and resistance genes, becomes an important driving force for the adaptive evolution of oral microorganisms, increases the difficulty of the treatment of oral bacterial infectious diseases (periodontal disease, periradicular lesions, Periimplantitis, etc.), and brings serious harm to human oral health and even general health. With modern microbiology, genetics, molecular biology, the macro development of disciplines, such as genomics, electron microscope and laser confocal microscope, nucleic acid hybridization (especially the 16 sRNA gene sequencing) and other modern analysis technology has been widely applied in periodontal pathogens resistance genes, through the study of HGT way spread and made some progress. This paper will analyze and summarize the literature on the spread of resistance genes of common periodontal pathogens through HGT method in recent years, in order to provide reference for the follow-up study of resistance of periodontal pathogens, as well as for the solution of resistance of periodontal pathogens and the treatment of periodontal diseases.

Fine Tuning of Pyrene Excimer Fluorescence in Molecular Beacons by Alteration of the Monomer Structure.

Oligonucleotide probes labeled with pyrene pairs that form excimers have a number of applications in hybridization analysis of nucleic acids. A long excited state lifetime, large Stokes shift, and chemical stability make pyrene excimer an attractive fluorescent label. Here we report synthesis of chiral phosphoramidite building blocks based on (R)-4-amino-2,2-dimethylbutane-1,3-diol, easily available from an inexpensive d-(-)-pantolactone. 1-Pyreneacetamide, 1-pyrenecarboxamide, and DABCYL derivatives have been used in preparation of molecular beacon (MB) probes labeled with one or two pyrenes/quenchers. We observed significant difference in the excimer emission maxima (475-510 nm; Stokes shifts 125-160 nm or 7520-8960 cm-1) and excimer/monomer ratio (from 0.5 to 5.9) in fluorescence spectra depending on the structure and position of monomers in the pyrene pair. The pyrene excimer formed by two rigid 1-pyrenecarboxamide residues showed the brightest emission. This is consistent with molecular dynamics data on excimer stability. Increase of the excimer fluorescence for MBs after hybridization with DNA was up to 24-fold.

CONTEMPORARY MOLECULAR-GENETIC METHODS USED FOR ETIOLOGIC DIAGNOSTICS OF SEPSIS

Etiologic diagnostics of sepsis is one of the most difficult problems of contemporary medicine due to a wide variety of sepsis causative agents, many of which are components of normal human microflora. Disadvantages of contemporary "golden standard" of microbiologic diagnostics of sepsis etiology by seeding of blood for sterility are duration of cultivation, limitation in detection of non-cultivable forms of microorganisms, significant effect of preliminary empiric antibiotics therapy on results of the analysis. Methods of molecular diagnostics that are being actively developed and integrated during the last decade are deprived of these disadvantages. Main contemporary methods of molecular-biological diagnostics are examined in the review, actualdata on their diagnostic characteristic are provided. Special attention is given to methods of PCR-diagnostics, including novel Russian developments. Methods of nucleic acid hybridization and proteomic analysis are examined in comparative aspect. Evaluation of application and perspectives of development of methods of molecular diagnostics of sepsis is given.

Oligonucleotide derivatives in nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insertion

Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

Distance-Dependent Metal-Enhanced Quantum Dots Fluorescence Analysis in Solution by Capillary Electrophoresis and Its Application to DNA Detection

Here the distance dependence of metal-enhanced quantum dots (QDs) fluorescence in solution is studied systematically by capillary electrophoresis (CE). Complementary DNA oligonucleotides-modified CdSe/ZnS QDs and gold nanoparticles (Au NPs) were connected together in solution by the hybridization of complementary oligonucleotides, and a model system (QD-Au) for the study of metal-enhanced QDs fluorescence was constructed, in which the distance between the QDs and Au NPs was controlled by adjusting the base number of the oligonucleotide. In our CE experiments, the metal-enhanced fluorescence of the QDs solution was only observed when the distance between the QDs and Au NPs ranged from 6.8 to 18.7 nm, and the maximum enhancement by a factor of 2.3 was achieved at 11.9 nm. Furthermore, a minimum of 19.6 pg of target DNA was identified in CE based on its specific competition with the QD-DNA in the QD-Au system. This work provides an important reference for future study of metal-enhanced QDs fluorescence in solution and exhibits potential capability in nucleic acid hybridization analysis and high-sensitivity DNA detection.

Oligonucleotide derivatives in the hybridization analysis of nucleic acids: II. Isothermal signal amplification in DNA analysis by minisequencing

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.

Oligonucleotide derivatives in the hybridization analysis of nucleic acids. I. Covalent immobilization of oligonucleotide probes on nylon

The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

Microcoding and flow cytometry as a high-throughput fungal identification system for Malassezia species

Yeasts of the genus Malassezia have been associated with a variety of dermatological disorders in humans and domestic animals. With the recent recognition of new members of the genus, new questions are emerging with regard to the pathogenesis and epidemiology of the new species. As new species are recognized, a precise and comprehensive identification system is needed. Herein is described a bead suspension culture-based array that combines the specificity and reliability of nucleic acid hybridization analysis with the speed and sensitivity of the Luminex analyser. The developed 16-plex array consisted of species- and group-specific capture probes that acted as 'microcodes' for species identification. The probes, which were designed from sequence analysis in the D1/D2 region of rRNA and internal transcribed spacer (ITS) regions, were covalently bound to unique sets of fluorescent beads. Upon hybridization, the biotinylated amplicon was detected by the addition of a fluorochrome coupled to a reporter molecule. The hybridized beads were subsequently analysed by flow cytometric techniques. The developed array, which allowed the detection of species in a multiplex and high-throughput format, was accurate and fast, since it allowed precise identification of species and required less than 1 h following PCR amplification. The described protocol, which can integrate uniplex or multiplex PCR reactions, permitted the simultaneous detection of target sequences in a single reaction, and allowed single mismatch discrimination between probe and non-target sequences. The assay has the capability to be expanded to include other medically important pathogenic species in a single or multiplex array format.

Temporal and spatial response of bacterioplankton lineages to annual convective overturn at the Bermuda Atlantic Time-series Study site

We used terminal restriction fragment length polymorphism (T-RFLP), clone library, phylogenetic, and bulk nucleic acid hybridization analyses to identify and characterize spatial and temporal patterns in marine bacterioplankton communities at the Bermuda Atlantic Time-series Study (BATS) site. Nonmetric multidimensional scaling of monthly surface and 200-m bacterial 16S rDNA T-RFLP fragments from 1992 to 2002 revealed temporal trends in bacterial community structure in different depth horizons. A 200-m 16S rRNA gene clone library was used to identify fragments increasing in relative abundance following mixing events and to link observed terminal restriction fragments with those predicted from sequence data. T-RFLP fragments matching those of cloned OCS116, SAR11, and marine Actinobacteria rRNA genes exhibited the strongest increases at 200 m following convective overturn, and fragments attributable to SAR11, SAR86, and SAR116 rRNA genes exhibited the strongest increases at the ocean surface during summer time periods. Variability in the distribution and relative abundance of fragments assigned to different SAR11 and SAR86 subclusters was also evident. Quantitative hybridization of extracted 16S rRNA with radiolabeled, taxonspecific oligonucleotide probes provided additional data supporting spatial and temporal patterns of lineage distributions and abundances suggested by ordination. Overall increases in the relative abundance of T-RFLP fragments attributable to the OCS116, SAR11, and marine Actinobacteria clusters following convective overturn suggest that members of these groups may play important roles in dissolved organic carbon dynamics at BATS.

Soybean Seedcoat Mottling: Association with Soybean mosaic virus and Phomopsis spp. Seed Infection.

Soybean mosaic virus (SMV) infection predisposes soybean (Glycine max) seed to Phomopsis spp. seed infection and may induce seedcoat mottling. The extent of seedcoat mottling associated with the accumulation of SMV or Phomopsis spp. infection of the seedcoat was investigated in 1996 and 1997. Plants of two SMV-susceptible cultivars, 'Clark' and 'Williams', were sap inoculated with the G2 strain of SMV at growth stage R2. Control plants (SMV-resistant isolines L78-434 and L78-379, respectively) were not inoculated. Harvested seed were evaluated visually for seedcoat mottling and sorted into four categories (severe, banded, blemish, and none) according to the degree of seedcoat pigmentation. Seed infection with Phomopsis spp. was determined and SMV accumulation was assessed by enzyme-linked immunosorbent assay and nucleic acid hybridization analysis. Phomopsis spp. were isolated from seed of all mottling categories, with the incidence of infection increasing as the degree of seedcoat mottling increased. SMV was detected in both mottled and nonmottled seedcoats, with generally higher levels of SMV in mottled seedcoats. However, the relationship of SMV titer to seedcoat mottling was inconsistent across cultivars and years. Thus, the extent of seedcoat mottling was not directly related to the accumulation of SMV in the seedcoat.

Diversity Among Isolates ofBean pod mottle virus

ABSTRACT Isolates of Bean pod mottle virus (BPMV), a member of the genus Comovirus, collected from soybean fields in Kentucky, Virginia, Arkansas, and Iowa were classified into two distinct subgroups, I and II, based on nucleic acid hybridization analysis using cloned cDNA probes to RNA-1 from BPMV strains K-G7 and K-Ha1. Slot blot hybridization analysis using cloned cDNA probes to RNA-2 from the same two strains (K-G7 and K-Ha1), however, revealed that some of the isolates, initially classified as belonging to subgroup I after analysis with RNA-1 probes, are in fact natural reassortants between the two strain subgroups. This was corroborated by nucleotide sequence analysis of full-length cDNA clones of both RNA-1 and RNA-2 from a putative reassortant strain (K-Ho1). These results indicate that BPMV strain diversity is more complex than initially anticipated, and that the use of cloned probes to both genomic RNAs during nucleic acid hybridization analysis is required to unravel the extent of such diversity. In a field plot experiment, BPMV isolates that belong to distinct strain subgroups induced symptoms that varied in severity and in the level of yield losses. In this regard, the reassortant strain K-Ho1 caused the most serious damage compared with four other BPMV isolates tested. Furthermore, the soybean alleles Rsv(1) and Rsv(4), known to confer resistance against Soybean mosaic virus, a member of the genus Potyvirus, did not provide any protection against BPMV. Additionally, we developed a reverse transcription-polymerase chain reaction procedure based on the sequence of a highly conserved region in the capsid polyprotein coding sequence that provides efficient and highly sensitive detection of all BPMV isolates tested, regardless of their strain classification.

Some properties of site-specific nickase BspD6I and the possibility of its use in hybridization analysis of DNA.

A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55 degrees C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA-DNA duplexes and therefore cannot be used for detection of RNA targets.

Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes.

Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2-4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68 degrees C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.

Hybridization analysis of chesapeake bay virioplankton

It has been hypothesized that, by specifically lysing numerically dominant host strains, the virioplankton may play a role in maintaining clonal diversity of heterotrophic bacteria and phytoplankton populations. If viruses selectively lyse only those host species that are numerically dominant, then the number of a specific virus within the virioplankton would be expected to change dramatically over time and space, in coordination with changes in abundance of the host. In this study, the abundances of specific viruses in Chesapeake Bay water samples were monitored, using nucleic acid probes and hybridization analysis. Total virioplankton in a water sample was separated by pulsed-field gel electrophoresis and hybridized with nucleic acid probes specific to either single viral strains or a group of viruses with similar genome sizes. The abundances of specific viruses were inferred from the intensity of the hybridization signal. By using this technique, a virus comprising 1/1,000 of the total virioplankton abundance (ca. 10(4) PFU/ml) could be detected. Titers of either a single virus species or a group of viruses changed over time, increasing to peak abundance and then declining to low or undetectable levels, and were geographically localized in the bay. Peak signal intensities, i.e., peak abundances of virus strains, were 10-fold greater than the low background level. Furthermore, virus species were found to be restricted to a particular depth, since probes specific to viruses from bottom water did not hybridize with virus genomes from surface water at the same geographical location. Overall, changes in abundances of specific viruses within the virioplankton were episodic, supporting the hypothesis that viral infection influences, if not controls, clonal diversity within heterotrophic bacteria and phytoplankton communities.

Cloning, Structural Analysis, and Chromosomal Localization of the HumanCSRP2Gene Encoding the LIM Domain Protein CRP2

TheCSRP2gene encoding the LIM domain protein CRP2 was originally identified in quail based on its strong transcriptional suppression in transformed avian fibroblasts. Here we have isolated a humanCSRP2cDNA clone encoding a 193-amino-acid human CRP2 (hCRP2) protein with 96.4% amino acid sequence identity to the avian homolog. TheCSRP2cDNA clone was used to isolateCSRP2-related clones from λEMBL3 and P1 libraries of human genomic DNA. The complete organization of theCSRP2gene was determined by nucleic acid hybridization, transcriptional mapping, and nucleotide sequence analysis. The gene spans a total of approximately 22 kb and contains six exons. The coding region is confined to exons 2–6 and predicts a hCRP2 protein identical in its amino acid sequence to the protein deduced from theCSRP2cDNA clone. By fluorescencein situhybridization using both λEMBL3 and P1 library clones as hybridization probes and a new method for computerized signal localization,CSRP2was mapped to chromosome subband 12q21.1, a region frequently affected by deletion or breakage events in various tumor types. The library screens also led to the isolation of aCSRP2-related pseudogene,CSRP2P,which carried several extensive deletions and nucleotide substitutions but no intervening sequences in comparison to theCSRP2cDNA sequence. By physical linkage and fluorescencein situhybridization,CSRP2Pwas mapped to chromosome subband 3q21.1.

Surreptitious hepatitis C virus (HCV) infection detected in the majority of patients with cryptogenic chronic hepatitis and negative HCV antibody tests.

Reverse transcription-polymerase chain reaction was used to identify hepatitis C virus (HCV) RNA in peripheral whole blood (WB) and plasma samples from 15 patients with chronic, unexplained hepatitis. These patients were serologically negative for hepatitis A, B, and C and were classified as having chronic non-A, non-B, non-C hepatitis (NANBNC). HCV RNA was repeatedly detected in WB samples from 10 (67%). In contrast, plasma samples from only 5 were intermittently positive. Statistically, HCV RNA detection in WB was significantly more sensitive than in plasma. Nucleic acid hybridization and HCV genotypic analysis confirmed the specificity of the HCV RNA assay. Liver biopsies from these patients suggested histopathologic differences between HCV RNA-positive and -negative groups. These data demonstrate that HCV infection is present in patients with unexplained chronic hepatitis more frequently than previously believed. Additionally, WB HCV RNA detection is more sensitive than plasma assays in identifying antibody-negative HCV infection.

Occurrence of Peach Latent Mosaic Viroid (PLMVd) on Plum in Italy.

Peach latent mosaic disease was originally described in France in 1976 (1). The disease is often latent in peach trees but may induce mosaic symptoms on leaves, irregularly shaped colorless fruit with cracked sutures and enlarged pits, bud necrosis, and delay in foliation, flowering, and fruit maturity. Recently, the disease has been shown to be caused by peach latent mosaic viroid (PLMVd) (2). PLMVd is distributed worldwide on peach. A recent survey in Italian plum orchards has showed the occurrence of PLMVd in two plum cultivars (Black Diamond and Angeleno) at different geographical locations in central Italy. Plum samples showed bark necrosis and bark split on branches and trunk; moreover, 1-or 2-year-old shoots and stem appeared dwarfed because of shortening of internodes and reduced growth. Budwood from plum samples were grafted, in greenhouse, onto peach GF305 indicator plants. Total nucleic acids (TNA) were then extracted from peach GF 305, or plum leaves, after 6 months of postinoculation. The presence or absence of PLMVd was ascertained by bidirectional polyacrylamide gel electrophoretic (dPAGE) analysis and by spot hybridization assay with PLMVd cRNA probe provided by A. Hadidi. A dPAGE analysis of TNA extracted from grafted peach GF305 showed a band with the same electrophoretic mobility as that of D-168 strain of PLMVd positive control. No bands were observed in healthy peach GF305 or from healthy or diseased plum extracts. The lack of detection of a viroid band from diseased plum samples by dPAGE analysis may be due to low titer of the viroid in infected plum. Molecular hybridization analysis of total nucleic acids with PLMVd cRNA probe, however, showed positive hybridization signals with nucleic acids from grafted peach GF305 or infected plum. Thus, PLMVd or a very closely related viroid infects plum trees. Moreover, on the basis of biological and serological assays, apple chlorotic leaf spot trichovirus, in a sample, and prunus necrotic ring spot ilarvirus, in the second one, were found in association with PLMVd. The same symptoms observed in the two PLMVd-infected plums occurred in other plants infected only by the two viruses (alone or in association). These data suggest that, very probably, PLMVd does not play a specific role in the etiology of the field symptomatology described above. In any case, this is the first report for the occurrence of PLMVd on plum in Italy.

Nucleic acid hybridization analyses confirm the presence of a hitherto unknown morbillivirus in mediterranean dolphins

In 1990 an epidemic caused by a morbillivirus was noticed among Mediterranean dolphins. RNA was extracted from the tissues of dolphins and from cell cultures infected with a corresponding dolphin morbillivirus isolate. By nucleic acid hybridization this RNA was compared to RNA extracted from animal tissue or cell cultures infected with canine distemper virus (CDV), phocine distemper virus (PDV) or measles virus (MV). The presence of morbillivirus RNA in the dolphin tissue was demonstrated. Morbillivirus N, P, M and F gene mRNAs were detected in the RNA from dolphin morbillivirus infected cells. These mRNA species seemed to be of approximately the same size as the corresponding mRNA species of CDV, PDV and MV. The results of the comparison demonstrated that the dolphin morbillivirus is genetically different from CDV, PDV and MV. No indication of a close relationship between the dolphin isolate and either CDV, PDV or MV was found.

Chromogranin-A expression in gastric and colon cancer tissues.

We studied the expression of chromogranin A (CgA) in human gastric (n = 17) and colorectal (n = 18) adenocarcinomas by nucleic acid hybridization and immunohistochemical analyses using a specific monoclonal antibody (MAb) to human chromogranin A (CgA). Some corresponding adjacent non-malignant mucosal tissues were also examined. (1) Northern blotting: of 3 normal gastric mucosas examined, 2 (67%) had an easily detected signal for expression of CgA. Only one of 14 gastric carcinomas (7%) and one of 18 colorectal carcinomas (6%) had easily detected RNA signals. (2) Immunohistochemical staining: all non-malignant samples of gastric and colonic mucosa contained CgA-positive neuroendocrine (NE) cells. Two of 17 (12%) gastric adenocarcinomas, and 3 of 18 (17%) of colorectal adenocarcinomas contained CgA-positive tumor cells. Interestingly, the positive cases detected by immunohistochemistry included both cases detected by Northern blotting. Of the 5 cases detected by immunohistochemistry, 2 gastric cancers and 1 rectal carcinoma contained many diffusely scattered positive cells, occurring singly or in small clusters, while 2 colorectal carcinomas contained only occasional single CgA-positive tumor cells. In one of the positive gastric cases, a well-differentiated adenocarcinoma arising in a tubular adenoma, both the adenomatous and the carcinomatous elements contained positively staining cells. Our specific assays for CgA indicate that (1) a NE cell component, either diffusely scattered or occasional, occurs in about 15% of gastric and colorectal tumors; (2) there is no correlation between the presence of NE cells and degree of tumor differentiation; and (3) because only a minority of the tumor cells in positive cases stain for CgA, immunohistochemistry is a more sensitive method than Northern blotting.

Genetic Interrelatedness among Clover Proliferation Mycoplasmalike Organisms (MLOs) and Other MLOs Investigated by Nucleic Acid Hybridization and Restriction Fragment Length Polymorphism Analyses

DNA was isolated from clover proliferation (CP) mycoplasmalike organism (MLO)-diseased periwinkle plants (Catharanthus roseus (L.) G. Don.) and cloned into pSP6 plasmid vectors. CP MLO-specific recombinant DNA clones were biotin labeled and used as probes in dot hybridization and restriction fragment length polymorphism analyses to study the genetic interrelatedness among CP MLO and other MLOs, including potato witches'-broom (PWB) MLO. Results from dot hybridization analyses indicated that both a Maryland strain of aster yellows and a California strain of aster yellows are distantly related to CP MLO. Elm yellows, paulownia witches'-broom, peanut witches'-broom, loofah witches'-broom, and sweet potato witches'-broom may be very distantly related, if at all, to CP MLO. A new Jersey strain of aster yellows MLO, tomato big bud MLO, clover phyllody MLO, beet leafhopper-transmitted virescence MLO, and ash yellows MLO are related to CP MLO, but PWB MLO is the most closely related. Similarity coefficients derived from restriction fragment length polymorphism analyses revealed that PWB and CP MLOs are closely related strains and thus provided direct evidence of their relatedness in contrast to reliance solely on biological characterization.