Genetic crosses in the apicomplexan parasite Cryptosporidium parvum define recombination parameters

Recombinant progeny lines of Cryptosporidium parvum were generated by coinfecting immunosuppressed mice with two genetically distinct isolates of C. parvum. Progeny lines were obtained from a cross of parental lines MD x TU114 through targeted propagation in mice of progeny oocysts originating from populations lacking one parental allele at one or more loci. For each infection lineage this process was repeated until only a single allele remained for each marker, indicating that the progeny line was clonal. To study genetic recombination, 16 progeny clones were genotyped at 40 loci located on each of the eight chromosomes. The inheritance of parental alleles was significantly skewed towards the more virulent parent isolate MD. A contiguous 476 kb segment of chromosome V displayed MD allele in all progeny recovered, while MD and TU114 alleles were detected at other loci throughout the genome. The absence of alleles from one parental isolate in this chromosomal region may indicate phenotypic selection for the MD allele during the generation of these lines. A range for the meiotic crossover frequency was determined on the basis of 40 markers and the number of meioses estimated to have taken place during the crossing experiment. C. parvum exhibits a high rate of recombination commensurate with other Apicomplexa.     

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changes in parasite virulence induced by the disruption of a single member of the 235 kDa rhoptry protein multigene family of Plasmodium yoelii

Invasion of the erythrocyte by the merozoites of the malaria parasite is a complex process involving a range of receptor-ligand interactions. Two protein families termed Erythrocyte Binding Like (EBL) proteins and Reticulocyte Binding Protein Homologues (RH) play an important role in host cell recognition by the merozoite. In the rodent malaria parasite, Plasmodium yoelii, the 235 kDa rhoptry proteins (Py235) are coded for by a multigene family and are members of the RH. In P. yoelii Py235 as well as a single member of EBL have been shown to be key mediators of virulence enabling the parasite to invade a wider range of host erythrocytes. One member of Py235, PY01365 is most abundantly transcribed in parasite populations and the protein specifically binds to erythrocytes and is recognized by the protective monoclonal antibody 25.77, suggesting a key role of this particular member in virulence. Recent studies have indicated that overall levels of Py235 expression are essential for parasite virulence. Here we show that disruption of PY01365 in the virulent YM line directly impacts parasite virulence. Furthermore the disruption of PY01365 leads to a reduction in the number of schizonts that express members of Py235 that react specifically with the mcAb 25.77. Erythrocyte binding assays show reduced binding of Py235 to red blood cells in the PY01365 knockout parasite as compared to YM. While our results identify PY01365 as a mediator of parasite virulence, they also confirm that other members of Py235 are able to substitute for PY01365.     

Recycling selectable markers in mouse embryonic stem cells.

As a result of gene targeting, selectable markers are usually permanently introduced into the mammalian genome. Multiple gene targeting events in the same cell line can therefore exhaust the pool of markers available and limit subsequent manipulations or genetic analysis. In this study, we describe the combined use of homologous and CRE-loxP-mediated recombination to generate mouse embryonic stem cell lines carrying up to four targeted mutations and devoid of exogenous selectable markers. A cassette that contains both positive and negative selectable markers flanked by loxP sites, rendering it excisable by the CRE protein, was constructed. Homologous recombination and positive selection were used to disrupt the Rep-3 locus, a gene homologous to members of the mutS family of DNA mismatch repair genes. CRE-loxP-mediated recombination and negative selection were then used to recover clones in which the cassette had been excised. The remaining allele of Rep-3 was then subjected to a second round of targeting and excision with the same construct to generate homozygous, marker-free cell lines. Subsequently, both alleles of mMsh2, another mutS homolog, were disrupted in the same fashion to obtain cell lines homozygous for targeted mutations at both the Rep-3 and mMsh2 loci and devoid of selectable markers. Thus, embryonic stem cell lines obtained in this fashion are suitable for further manipulation and analysis involving the use of selectable markers.     

Immunoregulation in murine malaria. Susceptibility of inbred mice to infection with Plasmodium yoelii depends on the dynamic interplay of host and parasite genes

Inbred and H-2 congenic mouse strains were tested for their ability to resist infections with the non-lethal 17X or with the lethal YM isolates of Plasmodium yoelii. DBA/2 and B10.D2 mice, which best resisted infections with non-lethal P. yoelii, were exquisitely susceptible to infection with lethal isolates of this malaria species. In contrast, B6 and B10 mice, which were susceptible to infection with non-lethal P. yoelii, were resistant to infection with the lethal isolates. This reversal of host response phenotype was influenced by H-2 genes, as evidenced by the divergent responses of the H-2 congenic strains B10 and B10.D2. However, a survey of mouse strains sharing common H-2 genes, but expressing different genetic backgrounds, demonstrated that genes outside the H-2 complex also influence the outcome of P. yoelii infections. By enumerating the numbers of P. yoelii-specific antibody-secreting cells in the spleens of infected mice, it was demonstrated that B6 mice, although susceptible to infection with non-lethal P. yoelii, nonetheless made a far stronger anti-parasite response after infection than did resistant DBA/2 mice. Using FACS analysis it was shown that infected B6 mice also produced large amounts of antibodies which bound to the surface of uninfected RBC. Thus, in B6 mice infected with non-lethal P. yoelii, a strong parasite-induced immune response was associated with susceptibility rather than resistance to infection. When T cell-deficient nude mice and their normal littermates were infected with the different isolates of P. yoelii, the nude mice had lower levels of parasitemia and higher RBC counts during the early stages of these infections, and lived longer than did normal littermates after infection with the lethal isolate. These data and the data from studies of B6 and DBA/2 mice support the idea that a strong immune response may be associated with susceptibility rather than resistance to P. yoelii, at least during the early stages of the infection. The finding that a single strain of mouse may present as resistant to infection with one P. yoelii isolate yet be exquisitely susceptible to infection with another suggests that the outcome of these murine malaria infections is dependent on a dynamic interplay between host and parasite genes. Thus, when genetic variability exists in both the host and the parasite populations, as would occur in nature, there may be little directed evolutionary change toward one phenotype or another.(ABSTRACT TRUNCATED AT 400 WORDS)     

RAPDs as an aid to evaluate the genetic integrity of somatic embryogenesis-derived populations of Picea mariana (Mill.) B.S.P.

Summary The usefulness of random amplified polymorphic DNA (RAPD) in assessing the genetic stability of somatic embryogenesis-derived populations of black spruce [Picea mariana (Mill.) B.S.P.] was evaluated. Three arbitrary 11-mer primers were successfully used to amplify DNA from both in-vivo and in-vitro material. Twenty-five embryogenic cell lines, additional zygotic embryos and megagametophytes from three controlled crosses involving four selected genotypes of black spruce were used for the segregation analysis of RAPD variants. Ten markers were genetically characterized and used to evaluate the genetic stability of somatic embryos derived from three embryogenic cell lines (one cell line per cross, 30 somatic embryos per cell line). No variation was detected within clones. The utilization of RAPD markers both for the assessment of genetic stability of clonal materials and to certify genetic stability throughout the process of somatic embryogenesis is discussed.     

Plasmodium yoelii: Genetic analysis of crosses between two rodent malaria subspecies

New electrophoretic variants of the enzyme adenosine deaminase (ADA) are described from two malaria subspecies. Differences were found in the mobility of ADA enzymes from Plasmodium yoelii yoelii (isolate 17X) and P. y. nigeriensis (isolate N67). Five attempts to cross these two subspecies were made, each of which demonstrated successful hybridization. The four genetic markers differentiating the parent lines appeared to segregate independently of each other. There was no marked preference for the gametes of one subspecies to self-fertilize rather than cross-fertilize with the other subspecies.     

Evidence for asexual genetic recombination in sunflower downy mildew,Plasmopara halstedii

Field isolates and single sporangium lines of the biotrophic Oomycete Plasmopara halstedii, differing in host preference and fungicide sensitivity, were used simultaneously for infection of sunflower. Dual infections led to asexually formed zoosporangia which gave rise to a new phenotype combining the characteristics of the parental strains. The new phenotype showed the metalaxyl-tolerance of one parent and virulence behaviour characteristic of the other, thus being able to infect a specific and fungicide treated sunflower line that neither of the parental strains could infect alone. These characteristics were inherited over many generations and did not occur spontaneously when parental strains were propagated separately. DNA fingerprints with minisatellite and simple sequence repeat primers showed characteristic differences between the patterns of the parental strains and the new phenotype. PCR experiments with mixed parental DNA resulted in additive patterns, but did not show the amplification product specific for the new phenotype. Since sexual reproduction was excluded under the experimental conditions used, the results provide evidence for genetic recombination through parasexual events in dual infections of sunflower downy mildew.     

PCR-Mediated Recombination between Cryptosporidium spp. of Lizards and Snakes

ABSTRACT: The presence or absence of genetic recombination has often been used as one of the criteria for Cryptosporidium species designation and population structure delineation. During a recent study of cryptosporidiosis in reptiles that were housed in the same room, 4 lizards were found to have concurrent infections of C. serpentis (a gastric parasite) and C. saurophilum (an intestinal parasite), and 6 snakes were concurrently infected with C. serpentis, C. sattrophitmm and a new Cryptosporidium as indicated by PCR-RFLP analysis of the SSU rRNA gene. DNA sequence analysis of cloned PCR products confirmed the diagnosis of mixed infections. Surprisingly, it appeared that 11 of the 22 clones (8 and 14 clones from a lizard and a snake, respectively) had chimeric sequences of two Cryptosporidium spp. BootScan analysis indicated the existence of recombinants among the cloned sequences and detection of the informative sites confirmed the BootScan results. Because the probability for genetic recombination between gastric and intestinal parasites is small, these hybrid sequences were likely results of PCR artifacts due to the presence of multiple templates. This was confirmed by PCR-sequencing analysis of single-copy templates using diluted DNA samples. Direct sequencing of 69 PCR products from 100-to 1.000-fold diluted DNAs from the same snake and lizard produced only sequences of C. serpentis, C. saurophilum and the unnamed Cryntosnoridium. , sp. Thus, care should be taken to eliminate PCR artifacts when determining the presence of genetic recombination or interpreting results of population genetic studies.     

Unequal Exchange and Meiotic Instability of Disease-Resistance Genes in the Rp1 Region of Maize

The Rp1 region of maize was originally characterized as a complex locus which conditions resistance to the fungus Puccinia sorghi, the causal organism in the common rust disease. Some alleles of Rp1 are meiotically unstable, but the mechanism of instability is not known. We have studied the role of recombination in meiotic instability in maize lines homozygous for either Rp1-J or Rp1-G. Test cross progenies derived from a line that was homozygous for Rp1-J, but heterozygous at flanking markers, were screened for susceptible individuals. Five susceptible individuals were derived from 9772 progeny. All five had nonparental combinations of flanking markers; three had one combination of recombinant flanking markers while the other two had the opposite pair. In an identical study with Rp1-G, 20 susceptible seedlings were detected out of 5874 test cross progeny. Nineteen of these were associated with flanking marker exchange, 11 and 8 of each recombinant marker combination. Our results indicate that unequal exchange is the primary mechanism of meiotic instability of Rp1-J and Rp1-G.     

Comparing single- vs. mixed-genotype infections of Mycosphaerella graminicola on wheat: effects on pathogen virulence and host tolerance

Mixed-genotype infections (infections of a host by more than one pathogen genotype) are common in plant-pathogen systems. However their impact on the course of the infection and especially on pathogen virulence and host response to infection is poorly understood. We investigated the effects of mixed-genotype infections on several parameters: host resistance and tolerance, as well as pathogen aggressiveness and virulence. For these purposes, we inoculated three wheat lines with three Mycosphaerella graminicola genotypes, alone or in mixtures, in a greenhouse experiment. For some of the mixtures, disease severity and virulence were lower than expected from infection by the same genotypes alone, suggesting that competition between genotypes was reducing their aggressiveness and virulence. One host line was fully resistant, but there were differences in resistance in the other lines. The two host lines that became infected differed slightly in tolerance, but mixed-genotype infections had no effect on host tolerance.     

Analysis of DNA from various species and strains of malaria parasites by restriction endonuclease fingerprinting

1. DNA from various rodent Plasmodium species and strains and from P. falciparum, the human parasite, were analysed by agarose gel electrophoresis following digestion with restriction endonucleases EcoRI, Hind III and Bam Hl. Complex patterns of ethidium-stained bands were obtained, which showed similarity but reproducible differences among the various parasite species (P. chabaudi, P. yoelii, P. berghei and P. falciparum). 2. No differences could be discerned among two cloned strains of P. yoelii (33X, and YM) and among pyrimethamine-resistant (pyrimethamine + chloroquine)-resistant and the drug-sensitive P. chabaudi clone from which the resistant clones were derived. 3. From the known complexity of Plasmodium DNA it could be concluded that the visible bands were derived from repetitive DNA fractions.     

A high-density rice genetic linkage map with 2275 markers using a single F2 population.

A 2275-marker genetic map of rice (Oryza sativa L.) covering 1521.6 cM in the Kosambi function has been constructed using 186 F2 plants from a single cross between the japonica variety Nipponbare and the indica variety Kasalath. The map provides the most detailed and informative genetic map of any plant. Centromere locations on 12 linkage groups were determined by dosage analysis of secondary and telotrisomics using > 130 DNA markers located on respective chromosome arms. A limited influence on meiotic recombination inhibition by the centromere in the genetic map was discussed. The main sources of the markers in this map were expressed sequence tag (EST) clones from Nipponbare callus, root, and shoot libraries. We mapped 1455 loci using ESTs; 615 of these loci showed significant similarities to known genes, including single-copy genes, family genes, and isozyme genes. The high-resolution genetic map permitted us to characterize meiotic recombinations in the whole genome. Positive interference of meiotic recombination was detected both by the distribution of recombination number per each chromosome and by the distribution of double crossover interval lengths.     

Protective antigens of rodent and human bloodstage malaria

Bloodstage malaria parasites are antigenically complex, but individual antigens can be identified and analysed using monoclonal antibodies. Two monoclonal antibodies that recognize a 235 000 molecular mass Plasmodium yoelii rhoptry protein provide some protection when injected into mice against a challenge infection. The purified rhoptry protein also provides protective immunity against P. yoelii YM when used to vaccinate mice and fulminating infections are converted into self-limiting, reticulocyte-restricted infections. Another monoclonal antibody immunoprecipitates a 230 000 molecular mass protein and a series of proteolytic processing fragments. At least one of these processing fragments, probably a 90 000 molecular mass species, is located on the merozoite surface. Mice immunized with the purified protein were protected against challenge infection with P. yoelii YM. This antigen may provide protection by inducing a cell-mediated immune response. A monoclonal antibody raised against P. falciparum schizonts reacts with a 195 000 molecular mass protein which is synthesized in schizonts and subsequently cleaved. Fragments of the 195 000 molecular mass protein are expressed as major antigens on the merozoite surface. The 195 000 molecular mass P. falciparum protein and the 230 000 molecular mass P. yoelii protein belong to a class of malaria parasite antigens which probably is important in the induction of a protective immune response in the host.     

Relative clonal density of malaria parasites in mixed-genotype infections: validation of a technique using microsatellite markers for Plasmodium falciparum and P. mexicanum

Quantifying the relative proportion of coexisting genotypes (clones) of a malaria parasite within its vertebrate host's blood would provide insights into critical features of the biology of the parasite, including competition among clones, gametocyte sex ratio, and virulence. However, no technique has been available to extract such data for natural parasite-host systems when the number of clones cycling in the overall parasite population is likely to be large. Recent studies find that data from genetic analyzer instruments for microsatellite markers allow measuring clonal proportions. We conducted a validation study for Plasmodium mexicanum and Plasmodium falciparum by mixing DNA from single-clone infections to simulate mixed infections of each species with known proportions of clones. Results for any mixture of DNA gave highly reproducible results. The relationship between known and measured relative proportions of clones was linear, with high regression r2 values. Known and measured clone proportions for simulated infections followed over time (mixtures) were compared with 3 methods: using uncorrected data, with uncorrected data and confidence intervals constructed from observed experimental error, and using a baseline mixture of equal proportions to calibrate all other results. All 3 methods demonstrated value in studies of mixed-genotype infections sampled a single time or followed over time. Thus, the method should open new windows into the biology of malaria parasites.     

A RFLP linkage map of Sorghum bicolor (L.) Moench

A RFLP linkage map of sorghum composed principally of markers detected with sorghum low-copy-number nuclear DNA clones has been constructed. The map spans 1789 cMs and consists of 190 loci grouped into 14 linkage groups. The 10 largest linkage groups consist of from 10 to 24 markers and from 103 to 237 cMs, and the other 4 linkage groups consist of from 2 to 5 markers and from 7 to 62 cMs. The map was derived in Sorghum bicolor ssp. bicolor by analysis of a F₂ population composed of 50 plants derived from a cross of IS 3620C, a guinea line, and BTx 623, an agronomically important inbred line derived from a cross between a zera zera (a caudatum-like sorghum) and an established kafir line. The restriction fragment length polymorphism (RFLP) frequency detected in this population using polymerase chain reaction (PCR)-amplifiable low-copy-number sorghum clones and five restriction enzymes was 51%. A minimal estimate of the number of clones that detect duplicate sequences is 11 %. Null alleles occurred at 13% of the mapped RFLP loci.     

Rapid isolation of CA microsatellites from the tilapia genome

We have developed (CA)n microsatellite markers for the cichlid fish, Oreochromis niloticus using a variation of the hybrid capture method. The resulting genomic library was highly enriched in repetitive DNA with 96% of clones containing CA repeats. The number of repeats ranged from four to 45 with an average of 19. Two-thirds of the sequenced clones had 12 or more repeats and sufficient flanking sequence to design primers. The resulting markers were tested in an F2 cross of O. niloticus x O. aureus. Nearly 90% of the markers amplified in this cross and 74% of these were informative. This work demonstrates the importance of minimizing the number of polymerase chain reaction (PCR) amplification cycles before and after the enrichment steps to reduce PCR recombination and the generation of chimaeric clones.     

Diversity array technology markers: genetic diversity analyses and linkage map construction in rapeseed (Brassica napus L.)

We developed Diversity Array Technology (DArT) markers for application in genetic studies of Brassica napus and other Brassica species with A or C genomes. Genomic representation from 107 diverse genotypes of B. napus L. var. oleifera (rapeseed, AACC genomes) and B. rapa (AA genome) was used to develop a DArT array comprising 11 520 clones generated using PstI/BanII and PstI/BstN1 complexity reduction methods. In total, 1547 polymorphic DArT markers of high technical quality were identified and used to assess molecular diversity among 89 accessions of B. napus, B. rapa, B. juncea, and B. carinata collected from different parts of the world. Hierarchical cluster and principal component analyses based on genetic distance matrices identified distinct populations clustering mainly according to their origin/pedigrees. DArT markers were also mapped in a new doubled haploid population comprising 131 lines from a cross between spring rapeseed lines 'Lynx-037DH' and 'Monty-028DH'. Linkage groups were assigned on the basis of previously mapped simple sequence repeat (SSRs), intron polymorphism (IP), and gene-based markers. The map consisted of 437 DArT, 135 SSR, 6 IP, and 6 gene-based markers and spanned 2288 cM. Our results demonstrate that DArT markers are suitable for genetic diversity analysis and linkage map construction in rapeseed.     

Genetic Variation in Clones of Pseudomonas pseudoalcaligenes After Ten Months of Selection in Different Thermal Environments in the Laboratory

The random amplification of polymorphic DNA (RAPD) method was used to examine genetic variation in experimental clones of Pseudomonas pseudoalcaligenes in two experimental groups, as well as their common ancestor. Six clones derived from a single colony of P. pseudoalcaligenes were cultured in two different thermal regimes for 10 months. Three clones in the Control group were cultured at constant temperature of 35°C and another three clones in the High Temperature (HT) group were propagated at incremental temperature ranging from 41 to 47°C for 10 months. A total of 45 RAPD primers generated 146 polymorphic markers. Analysis of molecular variance (AMOVA) revealed mild (11%) but significant (P < 0.001) genetic difference between the Control and the HT clones. Phylogenetic analysis based on pairwise genetic distances showed that the HT clones were more divergent from the ancestor and from each other than the Control clones, implying that the HT clones of P. pseudoalcaligenes may have evolved faster than the Control clones.