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PubMed2006

Proteomics in clinical interventions: achievements and limitations in biomarker development.

Development of toxicological and clinical biomarkers for disease diagnosis, quantification of toxicant/drug responses and rapid patient care are major concerns in modern biology. Even after human genome sequencing, identification of specific molecular signatures for unambiguous correlation with toxicity and clinical interventions is a challenging task. Differential protein expression patterns and protein-protein interaction studies have started unraveling rigorous molecular explanation of multi-factorial and toxicant borne diseases. Proteome profiling is extensively used to investigate etiology of diseases, develop predictive biomarkers for toxicity and therapeutic interventions and potential strategies for treatment of complex and toxicant mediated diseases. In this review, achievements and limitations of proteomics in developing predictive biomarkers for toxicological and clinical interventions have been discussed.

Animals↗
PubMed2001

New LightCycler PCR for rapid and sensitive quantification of parvovirus B19 DNA guides therapeutic decision-making in relapsing infections.

Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically >or=5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus.

Child↗
PubMed1994

Parental origin of transcription from the human GNAS1 gene.

Variation in the phenotypic expression of Albright's hereditary osteodystrophy (AHO) determined by the parent of transmission, suggests that the human Gs alpha gene (GNAS1), in which mutations occur in AHO, may be under imprinted control. GNAS1 is also known to map to a chromosomal region (20q13.11) showing syntenic homology with the imprinted mouse region 2E1-2H3. To establish if GNAS1 is indeed imprinted, we have examined the parental origin of GNAS1 transcription in human fetal tissues. Of 75 fetuses genotyped, at gestational ages ranging from 6 to 13 weeks, 13 heterozygous for a FokI polymorphism in exon 5 of GNAS1 were identified whose mothers were homozygous for one or other allele. RNA from up to 10 different tissues from each fetus was analysed by RT-PCR. In all cases expression from both parental alleles was shown by FokI digestion of RT-PCR products and quantification of the resulting fragments. No tissue specific pattern of expression was discerned in these experiments. If genomic imprinting regulates the expression of the human GNAS1 gene, our data suggest that the effect must either be subtle and quantitative, or be confined to a small subset of specialised hormone responsive cells within the target tissues.

Alleles↗
PubMed2006

A novel approach to simultaneously scan genes at fragile sites.

BACKGROUND: Fragile sites are regions of the genome sensitive to replication stress and to exposure to environmental carcinogens. The two most commonly expressed fragile sites FRA3B and FRA16D host the histidine triad (FHIT) and WW domain containing oxidoreductase (WWOX) genes respectively. There is growing evidence that both genes contribute to cancer development and they are frequently altered by allelic and homozygous deletions in a variety of tumors. Their status is linked to prognosis in several malignancies and they are thought to be involved in early tumorigenesis. The loci for FHIT and WWOX both span over a megabase but the genes encode for small transcripts. Thus the screening of intragenic deletion can be difficult and has relied on loss of heterozygosity LOH assays, or genomic arrays. METHODS: Multiplex ligation dependent probe amplification MLPA, allows for the detection of deletions/duplications and relative quantification of up to 40 specific probes in a single assay. A FHIT/WWOX MLPA assay was designed, applied and validated in five esophageal squamous cell carcinoma ESCC, cell lines established in South Africa where this cancer is of high prevalence. Sixteen probes covered all FHIT exons and 7 probes covered WWOX. RESULTS: Both homozygous and hemizygous deletions were detected in FHIT, in four of the cell lines with a preferential deletion of exons 5 and 4. Chromosome 3 short arm was present in normal copy number indicating that deletions were site specific. In contrast WWOX was not altered in any cell lines. RT-PCR expression pattern paralleled the pattern of deletions. Ten primary ESCC tumor specimens were subsequently screened with this assay. FHIT exon deletions were found in four of them. CONCLUSION: This method offers an alternative to loss of heterozygosity studies. Simultaneous scanning of FHIT and WWOX exons in the context of early tumorigenesis and tumor progression, may help clarify the mechanistic events related to cancer development which are not revealed by immuno histochemistry assays. The presence of site specific deletions of FHIT in these cell lines and primary tumors support its possible role in South African ESCC and justifies a wider screening.

Acid Anhydride Hydrolases↗
PubMed2026

Near-Whole-Genome Sequencing of Peste Des Petits Ruminants Virus Lineage IV From the Savannah District, Northern Côte d'Ivoire in 2023.

Peste des petits ruminants (PPR) is a highly contagious viral disease affecting sheep and goats, causing substantial economic losses in endemic countries. In the Savannah district of Côte d'Ivoire, knowledge of the genetic diversity and molecular epidemiology of the PPR virus (PPRV) remains limited. This study investigated the genetic diversity and phylogenetic relationships of PPRV circulating in this region using whole-genome sequencing (WGS). A cross-sectional survey was conducted between September and December 2023. Nasal swabs collected from sheep and goats were screened for PPRV ribonucleic acid (RNA) using real-time reverse transcription polymerase chain reaction (RT-qPCR). Samples with low quantification cycle (Cq) values of less than 35 and successful multiplex PCR amplification profiles were selected for sequencing using the Oxford Nanopore MinION platform. Near-complete consensus genomes were generated through reference-based assembly and analysed alongside representative strains from all recognised PPRV lineages. Of the 355 samples analysed, 25 (7.0%) tested positive for PPRV RNA, with positive detections in all three surveyed regions (Poro, Tchologo and Bagoué). The four samples with the lowest Cq values, originating from all three administrative regions, were successfully sequenced, generating genomes that covered 82.0%-86.2% of the reference genome at a depth of ≥ 10 ×. The missing regions were mainly located at the 5' and 3' genomic termini, as well as in limited internal regions associated with amplicon dropout. Phylogenetic analysis revealed that all four sequences belonged to lineage IV and exhibited high nucleotide similarity (98.1%-99.9%). The Ivorian strains clustered with recent lineage IV viruses from West, North and Central Africa, whereas historical Ivorian lineages I and II formed distinct clades. These findings confirm the predominance of lineage IV in northern Côte d'Ivoire and provide baseline genomic data to support molecular epidemiological surveillance in the region.

PPRV↗
PubMed2006

Internally controlled real-time PCR method for quantitative species-specific detection and vapA genotyping of Rhodococcus equi.

We developed a novel quantitative real-time PCR (Q-PCR) method for the soil actinomycete Rhodococcus equi, an important horse pathogen and emerging human pathogen. Species-specific quantification was achieved by targeting the chromosomal monocopy gene choE, universally conserved in R. equi. The choE Q-PCR included an internal amplification control (IAC) for identification of false negatives. A second Q-PCR targeted the virulence plasmid gene vapA, carried by most horse isolates but infrequently found in isolates from other sources. The choE-IAC and vapA assays were 100% sensitive and specific as determined using 178 R. equi isolates, 77 nontarget bacteria, and a panel of 60 R. equi isolates with known vapA+ and vapA-negative (including vapB+) plasmid genotypes. The vapA+ frequency among isolate types was as follows: horse, 85%; human, 20%; bovine and pig, 0%; others, 27%. The choE-IAC Q-PCR could detect up to one genome equivalent using R. equi DNA or 100 bacteria/ml using DNA extracted from artificially contaminated horse bronchoalveolar lavage (BAL) fluid. Quantification was linear over a 6-log dynamic range down to approximately 10 target molecules (or 1,000 CFU/ml BAL fluid) with PCR efficiency E of >0.94. The vapA assay had similar performance but appeared unsuitable for accurate (vapA+) R. equi quantification due to variability in target gene or plasmid copy number (1 to 9). The dual-reaction Q-PCR system here reported offers a useful tool to both medical and veterinary diagnostic laboratories for the quantitative detection of R. equi and (optional) vapA+ "horse-pathogenic" genotype determination.

Animals↗
PubMedDate not supplied

Genetic determinants of addiction to opioids and cocaine.

OBJECTIVE: The completion of the human genome sequence has spurred investigation of the genetic contribution to substance dependence. In this article some of the recent scientific evidence for genetic determinants of opioid and cocaine dependence is reviewed. METHOD: An electronic search of the medical literature was conducted to locate published studies relevant to the genetics of opioid and cocaine dependence. The collected information judged to be most pertinent is described and discussed. RESULTS: Genetic epidemiologic studies support a high degree of heritable vulnerability for both opioid and cocaine dependence. Polymorphisms in the genes coding for dopamine receptors and transporter, opioid receptors, endogenous opioid peptides, cannabinoid receptors, and serotonin receptors and transporter all appear to be associated with the phenotypic expression of this vulnerability once opioids or cocaine are consumed. CONCLUSIONS: Despite this initial progress, identification of specific genes and quantification of associated risk for the expression of each gene remain to be elucidated. While alteration of an individual's genome to change the phenotype seems remote, future interventions for treatment of opioid and cocaine dependence may include precise medications targeted to block the effects of proteins that have been identified through genetic research.

Cocaine-Related Disorders↗
PubMed2026

A modular class-aware workflow for small RNA sequencing analysis using mouse sperm as a case study.

BACKGROUND: Small RNA sequencing analysis is challenging because RNA classes differ in biogenesis, sequence redundancy, genomic organization, and annotation reliability. Integrated workflows accommodating these constraints remain limited, particularly for fragment-level and cluster-level analysis. METHODS: We present a reproducible, containerized, class-aware workflow for small RNA sequencing analysis, using mouse sperm as a case study. The workflow combines standardized preprocessing with complementary annotation and quantification strategies for microRNAs (miRNAs), transfer RNA-derived small RNAs (tsRNAs), ribosomal RNA-derived small RNAs (rsRNAs), and PIWI-interacting RNA (piRNA)-enriched genomic clusters. Using sperm small RNA data from offspring of lipopolysaccharide (LPS)-exposed male mice, we compared integrated-reference mapping, multi-class annotation, fragment-level tsRNA profiling, and genome-based piRNA cluster analysis, with custom modules for locus-aware harmonization and condition-specific cluster analysis. RESULTS: Integrated-reference mapping aligned 88.17% of reads and retained 690 features after filtering. It identified 11 differentially expressed miRNAs between LPS and controls, while other classes showed limited signal. Fragment-level profiling improved tsRNA resolution. piRNA cluster analysis identified 958 control and 940 LPS clusters, with 18 control-specific and no LPS-specific clusters. CONCLUSION: This workflow supports transparent, reproducible, class-aware interpretation of small RNA sequencing data while emphasizing cautious interpretation of piRNA-enriched signals from total small RNA sequencing.

Small non-coding RNA analysis↗
PubMed2002

Use of gDNA as internal standard for gene expression in staphylococci in vitro and in vivo.

An internal RNA standard proved less suitable in bacterial gene expression experiments. We therefore developed a method for simultaneous RNA and gDNA (genomic DNA) isolation from in vitro and in vivo samples containing staphylococci and combined it with quantitative PCR. The reliability of gDNA for bacterial quantification and for standardisation in gene expression experiments was evaluated. Quantitative PCR proves equivalent to quantitative culture for in vitro samples, and superior for in vivo samples. In gene expression experiments, gDNA permits a good standardisation for the initial amount of bacteria. The average interassay variability of the in vitro expression is 20.1%. The in vivo intersample variability was 73.3%. This higher variability can be attributed to the biological variation of gene expression in vivo. This method permits exact quantification of the number of bacteria and the expression of genes in staphylococci in vivo (e.g., in biofilms, evolution in time) and in vitro.

Colony Count, Microbial↗
PubMed1985

Identification and quantification of a messenger ribonucleic acid induced by polynuclear aromatic hydrocarbons--using a cloned human cytochrome P-450 gene.

We have isolated four overlapping human genomic clones associated with the polynuclear aromatic hydrocarbon-induced form of cytochrome P-450. The form of P-450 most closely associated with polynuclear aromatic hydrocarbons induction has been defined as P1-450. These four overlapping genomic clones span a total of 31.0 X 10(3) base pairs in length with the coding sequence lying in the center of these clones. Translation in vitro of 3-methylcholanthrene-induced mRNA, selected with the human P1-450 genomic clone, detect a protein with Mr 52000, which is immunoprecipitable by the anti-(mouse P1-450) antibody. The isolated human P1-450 genomic clone hybridizes to 3-methylcholanthrene-induced mRNA from monkey liver, benzanthracene and 3-methylcholanthrene-treated human mammary tumor cells (MCF-7), but not to isosafrole-treated human cells. Upon treatment with polynuclear aromatic hydrocarbons there is a positive correlation between induced arylhydrocarbon hydroxylase (flavoprotein-linked monoxygenase) activity and the amount of mRNA that hybridizes to the isolated human genomic clone for P1-450. The size of mRNA, induced from human cells and monkey liver by polynuclear aromatic hydrocarbons, is around 3.3 X 10(3) base pairs, which is the same as the larger of two mRNA induced by polynuclear aromatic hydrocarbons in the inbred strain of mouse (C57BL/6N). Our data also showed that the isolated DNA clone can detect a mRNA size of 3.3 X 10(3) base pairs from phytohemagglutinin-activated, benzanthracene-treated human lymphocytes. Densitometer scanning indicated the presence of a 3.6-fold variation (highest-lowest) in the levels of lymphocyte P1-450 mRNA contents among six individuals studied.

Animals↗
PubMed2000

Influence of DNA target melting behavior on real-time PCR quantification.

BACKGROUND: Quantitative real-time PCR is increasingly used to quantify copy numbers of nucleic acids for clinical applications. We observed that the measurements of allele imbalances of the tumor suppressor gene p16 and the oncogene ErbB-2 yielded results with variable precision under certain experimental conditions. METHODS: We used the LightCycler(TM) real-time PCR system to quantify different genomic target sequences using hybridization probes or SYBR Green for detection. RESULTS: With two primer/template systems (p16 and ErbB-2), we observed sinusoidal scattering of the threshold cycle values depending on the capillary position in the thermostated reaction chamber. This scattering depended on the denaturation temperature only when complete genomic DNA was used as template and did not occur when PCR product or restricted or boiled genomic DNA was used or the denaturation temperature in the first cycles was increased (and other targets, such as p53, HBB, IGF-1, GAPDH, and PBGD, did not show this behavior). CONCLUSIONS: Before a primer system is used for precise quantitative real-time PCR, the dependence of the quantification results on the positions of reaction tubes in the thermocycler should be tested. Our data indicate that amplification efficiencies, especially in the first cycles, depend not only on the priming efficiencies of the primers and the melting temperature of the amplicon, but also on the melting behavior of the amplicon's genomic vicinity. Complete denaturation of genomic DNA is necessary to maximize precision of quantitative PCR. Higher denaturation temperatures in the initial cycles or boiling of DNA before the PCR can improve the accuracy of quantification in some cases.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2006

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard.

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.

Food, Genetically Modified↗
PubMed2005

Quantification of mitochondrial DNA using real-time polymerase chain reaction in patients with premature ovarian failure.

OBJECTIVE: To quantify mitochondrial DNA using real-time PCR in women with premature ovarian failure (POF) and a control group. DESIGN: Prospective study. SETTING: Genome Research Center for Reproductive Medicine and Infertility, Korea Ministry of Health & Welfare. PATIENT(S): Thrity patients with POF and 30 control individuals. INTERVENTION(S): The mitochondrial DNA content was quantified using real-time PCR. The effectiveness of the assay was determined by relative quantification using the comparative threshold cycle (CT) method. MAIN OUTCOME MEASURE(S): Relative quantification of mitochondrial DNA content. RESULT(S): The mitochondrial DNA content was significantly lower in the POF group than in the control group (0.58 +/- 0.38 vs. 1.15 +/- 0.67; P < .01). In both groups, there was a significant positive correlation between the mitochondrial DNA/28S rRNA ratio and mitochondrial DNA CT (control group: r = 0.774; P < .001; POF group: r = 0.556; P = .001) and a significant negative correlation between the mitochondrial DNA/28S rRNA ratio and 28S rRNA CT (control group: r = -0.677; P < .001; POF group: r = -0.627; P = .001). CONCLUSION(S): This study has established the clinical feasibility of quantifying amounts of mitochondrial DNA, relative to an internal standard, using real-time PCR. Further studies are warranted to elucidate the roles of apoptosis and mitochondrial function in the pathogenesis of POF.

Adult↗
PubMed2007

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule.

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.

Base Sequence↗
PubMed2024

Detecting inbreeding depression in structured populations.

Measuring inbreeding and its consequences on fitness is central for many areas in biology including human genetics and the conservation of endangered species. However, there is no consensus on the best method, neither for quantification of inbreeding itself nor for the model to estimate its effect on specific traits. We simulated traits based on simulated genomes from a large pedigree and empirical whole-genome sequences of human data from populations with various sizes and structures (from the 1,000 Genomes project). We compare the ability of various inbreeding coefficients ([Formula: see text]) to quantify the strength of inbreeding depression: allele-sharing, two versions of the correlation of uniting gametes which differ in the weight they attribute to each locus and two identical-by-descent segments-based estimators. We also compare two models: the standard linear model and a linear mixed model (LMM) including a genetic relatedness matrix (GRM) as random effect to account for the nonindependence of observations. We find LMMs give better results in scenarios with population or family structure. Within the LMM, we compare three different GRMs and show that in homogeneous populations, there is little difference among the different [Formula: see text] and GRM for inbreeding depression quantification. However, as soon as a strong population or family structure is present, the strength of inbreeding depression can be most efficiently estimated only if i) the phenotypes are regressed on [Formula: see text] based on a weighted version of the correlation of uniting gametes, giving more weight to common alleles and ii) with the GRM obtained from an allele-sharing relatedness estimator.

Humans↗
PubMed2006

Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women.

PURPOSE: Methods to isolate cell-free fetal DNA from maternal plasma are critical in developing noninvasive fetal DNA testing strategies. Given that plasma consists of heterogeneous DNA-size fragments in a complex mix of proteins, recovery and analysis of this DNA are understandably inefficient. To facilitate recovery, we performed qualitative and quantitative analysis of DNA isolated from maternal plasma. METHODS: DNA isolated from maternal blood (n = 15) was compared using five different DNA isolation protocols: two conventional, two column-based, and one magnetic-bead based. Purity and concentration of DNA recovered were determined with a NanoDrop spectrophotometer. Real-time polymerase chain reaction quantification of the beta-globin and DYS1 loci was performed to determine total and fetal-specific genome equivalents, respectively. RESULTS: DNA quality and quantity were different among the five methods tested. Although purity and concentration of total DNA were greatest with the conventional boiling-lysis approach, correct detection of a male fetus was achieved in only 62.5% of cases. DNA isolation using the magnetic beads yielded the highest quantity of total DNA (2018.83 +/- 4.09 GEq/mL), with 100% fetal DNA detection. CONCLUSIONS: Optimal plasma DNA recovery protocols must take into account DNA purity and concentration. We confirm that the magnetic-beads method provides a fast, simple, sensitive, and specific approach to purify plasma DNA. The resulting high-quality DNA facilitates efficient examination of fetal DNA sequences.

DNA↗
PubMed1991

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.

Acyltransferases↗
PubMed2005

Recombination every day: abundant recombination in a virus during a single multi-cellular host infection.

Viral recombination can dramatically impact evolution and epidemiology. In viruses, the recombination rate depends on the frequency of genetic exchange between different viral genomes within an infected host cell and on the frequency at which such co-infections occur. While the recombination rate has been recently evaluated in experimentally co-infected cell cultures for several viruses, direct quantification at the most biologically significant level, that of a host infection, is still lacking. This study fills this gap using the cauliflower mosaic virus as a model. We distributed four neutral markers along the viral genome, and co-inoculated host plants with marker-containing and wild-type viruses. The frequency of recombinant genomes was evaluated 21 d post-inoculation. On average, over 50% of viral genomes recovered after a single host infection were recombinants, clearly indicating that recombination is very frequent in this virus. Estimates of the recombination rate show that all regions of the genome are equally affected by this process. Assuming that ten viral replication cycles occurred during our experiment-based on data on the timing of coat protein detection-the per base and replication cycle recombination rate was on the order of 2 x 10(-5) to 4 x 10(-5). This first determination of a virus recombination rate during a single multi-cellular host infection indicates that recombination is very frequent in the everyday life of this virus.

Caulimovirus↗