Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements.TRANSPOSABLE elements have a rich history as mutagenesis tools in Drosophila melanogaster (reviewed in Ryder and Russell 2003). Initially, researchers focused their efforts on the use of nonautonomous P-element transposons for gene disruption (Cooleyet al. 1988). However, P elements have insertion biases, preferring to transpose into euchromatic regions, the 5′ regions of genes (Tsubotaet al. 1985; Kelleyet al. 1987), and to target sequence motifs similar to the octamer GGCCAGAC (O''Hare and Rubin 1983). These biases make it unlikely that full genome saturation will be reached using P-element mutagenesis. Therefore, mutational systems that utilize transposable elements with different insertion biases have been developed. These include Hobo (Smithet al. 1993); the lepidopteran-derived piggyBac element, which inserts at TTAA sites (Hackeret al. 2003; Hornet al. 2003); and Minos, a Tc-1/mariner-like element originally isolated from Drosophila hydei that inserts at TA dinucleotides (Franz and Savakis 1991; Loukeriset al. 1995). Using a combination of these transposons, the Drosophila Gene Disruption Project has generated inserts in ∼60% of the 14,850 annotated genes (Spradlinget al. 1999; Bellenet al. 2004).In spite of the growing number of transposon insertions in the Drosophila genome, many are inserted in regions that do not completely abolish gene function, such as 5′-UTRs and introns. This can make it difficult to discern the true null phenotypes of genes. Furthermore, there still exist a sizable number of genes for which no transposon insertions are available. To address these issues, many transposons have been constructed with additional characteristics, such as FRT sites, that make generation of molecularly defined deletions by site-specific recombination relatively straightforward (Parkset al. 2004; Thibaultet al. 2004; Ryderet al. 2007). However, until saturation of the genome with these designer transposons is achieved, their utility in creating single-gene deletions remains limited.A more general approach for generating single-gene deletions that has proven successful is the use of P elements in imprecise excision screens. Excision of a P element creates a DNA double-strand break with 17 nucleotide noncomplementary ends (Beall and Rio 1997). If the ends of the break are degraded prior to repair, a deletion of DNA flanking the original insertion site is created (reviewed in Hummel and Klambt 2008). On average, the frequency of flanking deletions recovered from imprecise excision screens is ∼1%. However, this frequency varies tremendously by locus and depends on a multitude of factors that are not well understood, including chromatin structure and local sequence context. Therefore, generation of suitable deletion mutants frequently involves screening many hundreds of independent lines.An alternative method that uses P elements to generate deletions involves screening for events associated with male recombination. These events, which probably arise through a hybrid element insertion mechanism, generate one-sided deletions of sizes ranging from several base pairs to several kilobases (Preston and Engels 1996). This method, although powerful, involves screening a large number of flies and requires two sequential screens to generate bidirectional deletions.P-element-induced double-strand breaks are preferentially repaired through homologous recombination using a sister chromatid or a homologous chromosome as a template (Engelset al. 1990). Previously, we and others have demonstrated that the Drosophila Bloom protein ortholog (DmBlm), a RecQ DNA helicase encoded by mus309, is involved in homology-directed repair of these breaks (Beall and Rio 1996; McVeyet al. 2004a). In the absence of DmBlm, repair of a P-element-induced break on a plasmid or at a chromosomal locus frequently results in a large, flanking deletion. Several groups have applied this observation to imprecise excision screens using P elements and have successfully recovered multiple deletions (Astromet al. 2003; Johanssonet al. 2007; Y. Rong, unpublished data). However, a direct comparison between imprecise excision screens carried out in wild-type vs. mus309 mutant backgrounds has not been published, and little is known regarding the use of this technique with other types of transposable elements. In this study, we used three different transposons to test the hypothesis that the use of a mus309 mutant background in imprecise excision screens would result in a greater yield of deletions and that these deletions would be larger than those recovered from a wild-type background.
The mus309 mutant background increases the frequency and size of flanking deletions following P-element excision:
Previously, we have shown that repair of a double-strand break created by excision of the P{wa} transposon, located at 13F1–13F4 on the X chromosome, is deletion prone in the absence of DmBlm (McVeyet al. 2004a). This is likely due to a requirement for DmBlm in D-loop unwinding during homologous recombination (Bachratiet al. 2006; Weinert and Rio 2007). We have speculated that an unknown endonuclease may cleave D-loops in the absence of DmBlm, resulting in deletions flanking the P-element insertion site. To determine whether these observations can be generalized to imprecise excision screens, we tested two additional P-element insertions. One of these, P{EPgy2}Trf4-1EY14679, is inserted within a 1-kb intron of the Trf4-1 gene on the X chromosome (Figure 1A). The other, P{EPgy2}mus205EY20083, is inserted in a small intron in the mus205 gene on chromosome 2 (Figure 1B). Both of these EY elements contain wild-type copies of the yellow and white genes (Bellenet al. 2004). Thus, flies possessing EPgy2 elements have a wild-type body color and pigmented eyes. For our excision screens, we generated males containing the P element and a constitutively expressed transposase source, Δ2-3 (Robertsonet al. 1988). To test the effect of DmBlm absence, we also conducted the screens in heteroallelic mus309D2/mus309N1 mutants (Kusanoet al. 2001; McVeyet al. 2007).Open in a separate windowFigure 1.—Frequency and size of deletions accompanying imprecise P-element excision is increased in the mus309 mutant background. Crosses to generate males possessing both the P transposase and the desired P element were carried out in bottles containing standard cornmeal-based food at 25°. Excision events occurring in the male premeiotic germlines of flies carrying (A) P{EPgy2}Trf4-1EY14679 and the P{ry+, Δ2-3}99B transposase or (B) P{EPgy2}mus205EY20083 and the CyO, H{w+, Δ2-3} transposase were recovered in male progeny (for Trf4-1EY14679) or over a deficiency spanning the region (for mus205EY20083). Only one excision per male germline was analyzed to ensure that all events were independent. Genomic DNA was isolated and subjected to PCR analysis using primers specific to the P inverted repeats or to sequences flanking each P element. A and B show a genomic region with genes represented as boxes, intergenic regions as lines, and P elements as inverted triangles. Deletions were recovered from both wild-type (top panels) and mus309D2/mus309N1 mutant males (bottom panels) for Trf4-1EY14679, while deletions were recovered only from mus309 mutant males for mus205EY20083. Solid lines represent confirmed deletions, broken lines represent potential deletions, and arrows represent deletions that extend farther than was tested by PCR. Numbers in parentheses indicate the number of excisions recovered.First, we determined whether loss of DmBlm affected the fertility of males in which P-element excision was occurring. We found no significant difference in the percentage of wild-type vs. mus309 males that were sterile, as defined by an inability of an individual male to produce more than five adult offspring (
Open in a separate windowSterility and excision rates were determined for males possessing one copy of the transposon and the corresponding transposase (described in Figures 1 and and2).2). A male was classified as sterile if it produced fewer than five adult progeny when mated with three or more females.aNumbers in parentheses indicate the number of males tested.Next, we recovered independent excision events from individual male germlines for further analysis. Most independent excision events that resulted in loss of eye pigmentation also resulted in loss of wild-type body color. However, we did recover some events, mostly from wild-type males, which lost the white gene but retained the yellow gene, suggesting that an internal P-element deletion had occurred. We utilized a PCR strategy to determine the percentage of independently derived excision events that resulted in flanking genomic deletions. Chromosomes with an excision event were recovered in hemizygous males (for Trf4-1EY14679) or in trans to a deficiency spanning the relevant locus (for mus205EY20083), and genomic DNA was isolated. Primers flanking the P insertion site were used in initial reactions to determine whether a precise excision had occurred, as indicated by a PCR product equal in size to that obtained from wild-type flies with no insertion. In cases where no product was observed, we paired a primer complementary to the P-element terminal inverted repeats with primers flanking the insertion site in secondary PCR reactions to determine whether any P sequence remained. For events in which one or both of the P-element ends was missing, additional reactions were performed to determine if unidirectional or bidirectional deletions had occurred. In cases in which we were able to obtain a PCR product spanning the deletion junction, DNA sequencing was performed to determine the exact size of the deletion.The vast majority of excisions (>95%) obtained in a wild-type background were precise excisions or internally deleted P elements. We recovered two deletions (4% of total excisions) of <170 bp from the Trf4-1EY14679 excision in wild-type males, but none following mus205EY20083 excision (Figure 2A). In contrast, Trf4-1EY14679 excision in mus309 mutants resulted in 20 deletions (28% of total excisions), and mus205EY20083 excision created 8 deletions (20% of total excisions). The minimum size of the deletions obtained in mus309 mutants varied from tens of base pairs to >10 kb (Figure 2B), and many were bidirectional, extending multiple kilobases in both directions. Of 6 deletions whose exact breakpoints were identified, 3 retained a portion of P-element sequence, suggesting that homologous recombination repair initiated but then failed, resulting in a one-sided deletion. The other 3 deletions appeared to involve end-joining repair; 1 deletion had an insertion of 18 nucleotides, suggesting an alternative end-joining process. From these comparisons, we conclude that excision of P elements in a mus309 mutant background increases both the number and the size of flanking genomic deletions relative to excision that occurs in wild-type flies.Open in a separate windowFigure 2.—Number and size of deletions following transposon excision is increased in mus309 mutants. (A) Histogram showing the percentage of excisions accompanied by flanking deletions in wild-type and mus309 mutants. Solid bars indicate unidirectional deletions; hatched bars indicate bidirectional deletions. (B) Histogram showing the minimum size of deletions, as determined by the absence of a PCR product, in wild-type and mus309 mutants.
Absence of DmBlm also increases the yield of large deletions following imprecise excision of Minos elements:
Recently, Metaxakiset al. (2005) demonstrated that remobilization of Minos transposons can also be used to produce deletions adjacent to the original insertion site. However, the proportion of deletions recovered relative to total excisions was small, and the largest confirmed deletion was only 800 bp. Approximately 25% of Minos-induced double-strand breaks in females heterozygous for the insertion are repaired by nonhomologous end joining and mismatch repair, frequently resulting in a 6-bp insertion, or “footprint,” relative to the original target sequence (Arcaet al. 1997). The other 75% are likely repaired by homology-directed repair.Because DmBlm is required to prevent deletions during homologous recombination, we tested whether imprecise excision of Minos in flies lacking DmBlm would also result in an increased probability of recovering large deletions in nearby sequence. Males containing the Minos transposase driven by a heat-shock promoter and Mi{ET1} insertions on chromosomes X, 2, and 3 (located in the Pvf1, dp, and Tequila genes, respectively) were generated (Figure 3). To test the effects of DmBlm loss, the mus309D2 and mus309N1 alleles were used in combination with the Pvf1MB01242 and dpMB00453 insertions, and the mus309D2 and mus309D3 alleles (Kusanoet al. 2001) were used with the TequilaMB00537 insertion (the mus309D3 allele was crossed onto the TequilaMB00537-bearing chromosome by standard genetic methods). For these three screens, we compared mus309 heteroallelic males to mus309 heterozygous males. Because mus309 heterozygotes behave as wild types in double-strand break repair assays (McVeyet al. 2007), we will hereafter refer to them as wild type.Open in a separate windowFigure 3.—Frequency and size of deletions accompanying imprecise Minos excision is increased in the mus309 mutant background. Crosses to generate males possessing both the Minos transposase and each Minos element were done in bottles containing standard cornmeal-based food at 25°. Parental flies were moved to new bottles every 2 days for three consecutive broods. To induce transposase expression, cleared bottles were heat-shocked for one hour in a 37° incubator every day until adults eclosed. Excision events occurring in male premeiotic germlines of flies carrying the (A) Mi{ET1}Pvf1MB01242, (B) Mi{ET1}dpMB00453, or (C) Mi{ET1}TequilaMB00537 transposons, together with the SM6a,P{hsMi\T}2.4transposase, were recovered in male progeny (for Pvf1MB01242), over a deficiency (for TequilaMB00537), or in homozygotes (for dpMB00453). Genomic DNA was isolated for independent excisions and analyzed by PCR using primers specific to Minos or to sequences flanking each insertion. Genomic regions with genes are represented as boxes, intergenic regions as lines, and Minos elements as triangles. The 412 endogenous retrotransposon is located adjacent to TequilaMB00537. For all three insertions, deletions were recovered from both wild-type (top panels) and mus309 mutant males (bottom panels). Solid lines represent confirmed deletions, broken lines represent potential deletions, and arrows represent deletions that extend farther than was tested by PCR. Numbers in parentheses indicate the number of excisions recovered.Similar to what we observed in the P-element screens, loss of DmBlm had no significant effect on the percentage of males that were sterile (Figure 2A). The percentage of genomic deletions that resulted from repair following Pvf1MB01242 excision was similar for mus309 heteroallelic and heterozygous males (6% vs. 7%). These data are consistent with the model that double-strand breaks created by Minos excision can be repaired either by nonhomologous end joining or by homologous recombination and that DmBlm is required for efficient gap repair during homologous recombination.For all three Minos insertions, the size of deletions was also increased when recovered from mus309 mutants (Figure 2B). Of 9 deletions isolated from wild type, only 1 (11%) had a minimum size >1 kb. In contrast, 11 of 18 deletions (61%) isolated in a mus309 mutant background had a deletion >1 kb, and 4 of 18 (22%) involved deletions of at least 8 kb. During the process of PCR mapping of the deletion breakpoints for the dpMB00453 and TequilaMB00537 excisions, we became aware of the existence of a highly repetitive sequence and an endogenous 412 transposon to one side of each of these respective Minos elements. This impaired our fine-scale mapping and may have caused us to underestimate the minimum size of several of the deletions obtained from mus309 mutants. Notably, the percentage of bidirectional deletions relative to total deletions was also increased for all Minos insertions in the mus309 mutants (71%) compared to wild type (44%).
Loss of DmBlm does not promote deletion formation following piggyBac excision:
PiggyBac elements have also been utilized in genomewide transposon saturation screens (Thibaultet al. 2004). However, no reports of imprecise excision of piggyBac elements have been published, preventing their use in traditional deletion screens. To formally test whether imprecise excision of piggyBac elements can occur in either wild-type or mus309 mutants, we conducted screens with three different piggyBac elements—PBac{RB}WRNexoe04496, PBac{RB}CG6719e00315, and PBac{PB}lig3c03514—in males that also inherited a constitutively expressed piggyBac transposase under the control of the αTub84B promoter. Overall, we found that germline excisions with piggyBac were less frequent than with either P or Minos elements. We obtained 25 excisions from a wild-type background using PBac{RB}WRNexoe04496, all of which were precise. When mus309D2/mus309N1 males were used, we recovered 64 excision events from 25 independent male germlines, only 1 of which was imprecise. This single inaccurate repair event deleted 12 bp directly adjacent to the insertion site. Screens using an alternative piggyBac transposase source driven by the Hsp70 promoter or conducted in mus309 heterozygous females were also unsuccessful in generating any imprecise excisions (data not shown). In addition, no imprecise excisions were obtained from wild-type or mus309 mutant males with the PBac{RB}CG6719e00315 or PBac{PB}lig3c03514 elements (data not shown). We conclude that the absence of DmBlm does not appreciably improve the yield of imprecise excisions or deletions for piggyBac elements.PiggyBac is the first example of a DDE superfamily transposon in eukaryotes (Mitraet al. 2008). Similar to bacterial Tn5 and Tn10, piggyBac transposition involves a transposon hairpin intermediate that is subsequently cleaved, producing four-nucleotide TTAA overhangs on the 5′-ends of both the transposon and the donor DNA. These clean breaks can be easily repaired by nonhomologous end joining. In contrast, the complementary-ended breaks created by the I-SceI endonuclease, which creates 3′ TTAT overhangs, are frequently repaired inaccurately in Drosophila (Prestonet al. 2006). Therefore, it seems likely that the piggyBac transposase itself may promote accurate rejoining of the double-strand break created during transposition and may prevent other repair pathways, such as homologous recombination, from acting upon the break.
A general strategy for the use of double-strand break repair mutants to create genomic deletions:
DNA double-strand breaks in D. melanogaster can be repaired by multiple pathways, including homologous recombination, single-strand annealing, nonhomologous end joining requiring DNA ligase IV, and DNA ligase IV-independent alternative end joining (Prestonet al. 2006). These four pathways are not mutually exclusive and can compensate for each other if one is disabled. Our data obtained with P and Minos elements suggest that, in the absence of DmBlm, homologous recombination is impaired and break repair proceeds through a deletion-prone alternative end-joining pathway. Similarly, several groups have shown that repair of double-strand breaks created by the I-SceI endonuclease in the absence of Drosophila DNA ligase IV also causes an increase in flanking deletions (Prestonet al. 2006; Wei and Rong 2007). We have not observed any difference in deletion frequency during imprecise excision screens of P elements conducted in wild-type vs. lig4 mutant backgrounds (McVeyet al. 2004b). However, we have not systematically tested the use of a lig4 mutant background for piggyBac or Minos excision screens.Zinc-finger nucleases (ZFNs) have recently emerged as an effective way to induce double-strand breaks in a number of eukaryotic organisms, including Drosophila, Arabidopsis thaliana, Caenorhabditis elegans, and Danio rerio (Bibikovaet al. 2002; Lloydet al. 2005; Mortonet al. 2006; Carrollet al. 2008; Doyonet al. 2008; Menget al. 2008). By utilizing ZFNs in mutants lacking one or more critical components of the different repair pathways, it is possible to bias repair of site-specific breaks toward a desired outcome. For example, inducing breaks in the absence of DNA ligase IV increases the proportion that are accurately repaired by homologous recombination in both Drosophila and C. elegans (Mortonet al. 2006; Bozaset al. 2009). In contrast, loss of both Rad51 and DNA ligase IV causes a majority of ZFN-induced breaks to be repaired by deletion-prone alternative end-joining pathways (Bozaset al. 2009). It will be interesting to determine whether mutation of mus309 similarly increases inaccurate repair and causes large deletions when ZFNs are used as a mutagenic agent.Although the use of transposons to induce genomic deletions is a powerful tool for Drosophila geneticists, transposition can occasionally create second-site mutations that may affect subsequent phenotypic analysis. This might be of particular concern in a mus309 mutant background, which causes elevated genomic instability in the form of mitotic crossovers (McVeyet al. 2007). In a separate study, we have used a lacZ reporter system (Garciaet al. 2007) to measure the spontaneous mutation frequency in mus309 mutants. We find that the overall point mutation frequency is unchanged relative to wild-type flies, while the frequency of genomic rearrangements (deletions, inversions, and translocations) is elevated approximately twofold (A. Garcia, M. Lundell, J. Vijg and M. McVey, unpublished results). These genomic rearrangements are likely a result of the inaccurate repair of endogenous double-strand breaks. Although these data suggest that the probability of a second-site mutation following P-element excision may be slightly elevated in mus309 mutants, such events can easily be discerned by comparing the phenotypes of multiple independent excisions or by transgenic rescue.
Conclusions:
The goal of imprecise transposon excision screens is to create deletions that remove genes or regions of genomic sequence. The studies presented here demonstrate that performing screens with P and Minos insertions in male flies lacking DmBlm improves the chances of obtaining multiple large deletions. This approach does not affect male fertility or overall recovery of germline excisions. Furthermore, by utilizing the mus309N2 allele, which is female fertile but associated with deletion-prone repair of breaks (McVeyet al. 2007), the technique can also be applied to imprecise excision screens in females. We anticipate that this approach will benefit researchers working with Drosophila (and perhaps other model organisms) by significantly reducing the amount of labor required to obtain null alleles of genes for which transposons are inserted far from coding sequences. 相似文献
Two trials on Mexican cypress Cupressus lusitanica Miller in the North Island of New Zealand were assessed for diameter at breast height and at one site, subjective scores for branch size and stem canker (caused by Seiridium spp.) at age 6 from planting. The trials comprised 15 open-pollinated families, represented by both cloned and seedling progeny. Linear mixed model methodology, using a spatial model for the residuals, was applied to estimate genetic parameters. Estimated narrow-sense heritabilities were moderate to high for diameter at breast height (range from 0.46 to 0.62), stem canker (≈0.30) and branch size (range from 0.23 to 0.45) and did not appear to differ significantly between propagule types for all traits. Clonally replicated progeny led to an increase in accuracy of selection for additive genetic merit when compared with seedling testing, with the improvement being greater for traits with lower narrow-sense heritabilities. Estimated additive genetic correlations between cloned and seedling progeny were moderate to high (≥0.65) for diameter and branch size, indicating that selection decisions would not be substantially changed using either propagule type for progeny testing. All estimates of non-additive genetic variation based on the cloned progeny were non-significant. The use of spatial analysis was effective for diameter and branch size, but not for stem canker. No significant genotype by environment interaction was detected for diameter. Implications of the results for breeding and deployment of C. lusitanica are discussed. 相似文献
Contrasting host and parasite population genetic structures can provide information about the population ecology of each species and the potential for local adaptation. Here, we examined the population genetic structure of the nematode Neoheligmonella granjoni at a regional scale in southeastern Senegal, using 11 microsatellite markers. Using the results previously obtained for the two main rodent species of the host community, Mastomys natalensis and Mastomys erythroleucus, we tested the hypothesis that the parasite population structure was mediated by dispersal levels of the most vagile host. The results showed similar genetic diversity levels between host and parasite populations, and consistently lower levels of genetic differentiation in N. granjoni, with the exception of one outlying locus with a high FST. The aberrant pattern at this locus was primarily due to two alleles occurring at markedly different frequencies in one locality, suggesting selection at this locus, or a closely linked one. Genetic differentiation levels and isolation by distance analyses suggested that gene flow was high and random in N. granjoni at the spatial scale examined. The correlation between pair-wise genetic differentiation levels in the parasite and its main host was consistent with the hypothesis tested. Models of local adaptation as a function of the dispersal rates of hosts and parasites suggest that opportunities for local adaptation would be low in this biological system. 相似文献
In Drosophila, P-GAL4 enhancer trap lines can target expression of a cloned gene, under control of a UASGAL element, to any cells of interest. However, additional expression of GAL4 in other cells can produce unwanted lethality or
side-effects, particularly when it drives expression of a toxic gene product. To target the toxic gene product ricin A chain
specifically to adult neurons, we have superimposed a second layer of regulation on the GAL4 control. We have constructed
flies in which an effector gene is separated from UASGAL by a polyadenylation site flanked by two FRT sites in the same orientation. A recombination event between the two FRT sites,
catalysed by yeast FLP recombinase, brings the effector gene under control of UASGAL. Consequently, expression of the effector gene is turned on in that cell and its descendants, if they also express GAL4.
Recombinase is supplied by heat shock induction of a FLP transgene, allowing both timing and frequency of recombination events
to be regulated. Using a lacZ effector (reporter) to test the system, we have generated labelled clones in the embryonic mesoderm and shown that most recombination
events occur soon after FLP recombinase is supplied. By substituting the ricin A chain gene for lacZ, we have performed mosaic cell ablations in one GAL4 line that marks the adult giant descending neurons, and in a second
which marks mushroom body neurons. In a number of cases we observed loss of one or both the adult giant descending neurons,
or of subsets of mushroom body neurons. In association with the mushroom body ablations, we also observed misrouting of surviving
axons.
Received: 17 December 1995 / Accepted: 6 March 1996 Edited by M. Akam 相似文献
Receptors of the Ly-49 multigene family regulate rodent NK cell functions. Ly-49Rs are highly polymorphic and exist in either activating or inhibitory forms. Examples of both Ly-49 receptor types have been shown to recognize class I MHC ligands. Ly-49Rs can distinguish between class I alleles, but the molecular basis of this discrimination is unknown. Two activating receptors, Ly-49P and Ly-49W, differ in class I recognition, recognizing H-2D(d), or H-2D(d) and D(k), respectively. In this report, we demonstrate that specificity for H-2D(k) can be transferred from Ly-49W to Ly-49P by substituting 3 aa predicted to reside in the beta4-beta5 loop of Ly-49W into Ly-49P. Replacement of these same residues of Ly-49W with corresponding residues in Ly-49P eliminates H-2D(k) recognition while still preserving H-2D(d) recognition. Further mutagenesis indicates that all 3 aa facilitate optimal class I specificity exchange. These results provide the first evidence for a specific site on Ly-49Rs, the beta4-beta5 loop, in determining class I MHC allele specificity. 相似文献
Ulcers in Atlantic menhaden Brevoortia tyrannus (Latrobe) (Clupeidae), observed along the USA east coast, have been attributed to diverse etiologies including bacterial, fungal and, recently, harmful algal blooms. To understand the early pathogenesis of these lesions, we examined juvenile Atlantic menhaden collected during their seasonal presence in Chesapeake Bay tributaries from April to October 1999 and from March to August 2000. We conducted histopathological examinations of young-of-the-year fish from the Pocomoke River tributary, which has a history of fish mortalities and high lesion prevalence. Kudoa clupeidae (Myxozoa: Myxosporea) spores were present in the muscles of fish collected in both years. Of the fish assessed by histology in April, 5 to 14% were infected, while in May 90 to 96% were infected. Infection rates remained high during the summer. Mature spores were primarily located within myomeres and caused little or no observable pathological changes. Ultrastructure showed spores with capsulogenic cells bearing filamentous projections, and a basal crescentic nucleus with mottled nucleoplasm containing cleaved, condensed chromatin. Also, a highly invasive plasmodial stage of a myxozoan was found in the lesions of juvenile Atlantic menhaden. The plasmodia were observed in fish collected between May and July, with the maximum occurrence in late June 1999 and late May 2000. Plasmodia penetrated and surrounded muscle bundles, causing grossly observable raised lesions in 73% of all fish infected with this invasive stage. Plasmodia were also detected in the visceral organs, branchial arches, and interocular muscles of some fish. Some of the invasive extrasporogonic plasmodial lesions were associated with ulcers and chronic inflammatory infiltrates. The plasmodial stage appeared to slough out of the tissue with subsequent evidence of wound healing. Ultrastructure showed plasmodia with an elaborate irregular surface, divided into distinct ectoplasm and endoplasm; the latter contained numerous spherical vegetative nuclei, secondary generative cells, and occasional cell doublets. Our ultrastructural studies indicate that the plasmodial organisms, which are important in the etiology of the skin lesions, are myxozoans, and they may represent early stages of K. clupeidae. 相似文献
To fully understand how pathogens infect their host and hijack key biological processes, systematic mapping of intra-pathogenic and pathogen–host protein–protein interactions (PPIs) is crucial. Due to the relatively small size of viral genomes (usually around 10–100 proteins), generation of comprehensive host–virus PPI maps using different experimental platforms, including affinity tag purification-mass spectrometry (AP-MS) and yeast two-hybrid (Y2H) approaches, can be achieved. Global maps such as these provide unbiased insight into the molecular mechanisms of viral entry, replication and assembly. However, to date, only two-hybrid methodology has been used in a systematic fashion to characterize viral–host protein–protein interactions, although a deluge of data exists in databases that manually curate from the literature individual host–pathogen PPIs. We will summarize this work and also describe an AP-MS platform that can be used to characterize viral-human protein complexes and discuss its application for the HIV genome. 相似文献
The calcitonin-like receptor (CLR) and the calcitonin receptor (CTR) interact with receptor activity-modifying protein 1 (RAMP1) at the cell surface to form heterodimeric receptor complexes. CLR and CTR are members of the class II (family B) G-protein-coupled receptors (GPCR) and bind calcitonin gene-related peptide (CGRP) with similar affinities when coexpressed with RAMP1. The observation that various nonpeptide CGRP receptor antagonists display a higher affinity for the CLR/RAMP1 complex than for CTR/RAMP1 provided an opportunity to investigate the molecular determinants of the differential receptor affinities of these antagonists. A chimeric receptor approach was utilized to identify key domains within CLR responsible for conferring high-affinity antagonist binding. Initial chimera experiments implicated distinct regions within CLR as responsible for the affinities of structurally diverse CGRP receptor antagonists. Dissection of these key regions implicated amino acids 37-63 located in the amino terminus of CLR as responsible for the high-affinity interaction of one structural class, while transmembrane domain (TM) 7 was responsible for the interaction of a second class of antagonist. A unique binding interaction in the amino terminus of CLR is consistent with the observation that these compounds also interact with the extracellular region of RAMP1 and could suggest the formation of a binding pocket between the two proteins. Conversely, a compound which interacted with TM7 did not display a similar RAMP1 dependence, suggesting an allosteric mechanism of antagonism. Collectively, these data provide insight into two alternative mechanisms of antagonism for this unique heterodimeric receptor complex. 相似文献
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