Isolation and characterization of microsatellite markers from three species of swallows in the genus Tachycineta: T. albilinea, T. bicolor and T. leucorrhoa.

We describe 30 microsatellite loci developed from three species of swallows in the genus Tachycineta: T. bicolor (tree swallow), T. albilinea (mangrove swallow), and T. leucorrhoa (white-rumped swallow). These commonly studied birds nest in secondary cavities and are distributed from Alaska to Argentina. Primer pairs were designed for each species individually and tested for cross-amplification in 40-48 individuals of all three species. Polymorphism ranged from 5 to 65 alleles per locus (mean = 19.1). These markers will allow comparative studies of extra-pair paternity rates among members of the genus as well as the assessment of population structure.

Isolation and characterization of microsatellite markers from the acacia-ant Crematogaster mimosae.

We describe 10 microsatellite loci developed from Crematogaster mimosae, an ant species that nests mutualistically in Acacia drepanolobium trees in east Africa. Polymorphism ranged from 4 to 16 alleles per locus (mean = 7.3). Observed and expected heterozygosities ranged from 0.485 to 0.813 (mean 0.626), and from 0.502 to 0.894 (mean 0.674), respectively. These markers will foster studies of the population structure, colony structure, and reproductive strategies of these ants.

Isolation and characterization of SNP variation at 90 anonymous loci in the banded wren (Thryothorus pleurostictus).

Single nucleotide polymorphisms (SNPs) are becoming more commonly used as molecular markers in conservation studies. However, relatively few studies have employed SNPs for species with little or no existing sequence data, partly due to the practical challenge of locating appropriate SNP loci in these species. Here we describe an application of SNP discovery via shotgun cloning that requires no pre-existing sequence data and is readily applied to all taxa. Using this method, we isolated, cloned and screened for SNP variation at 90 anonymous sequence loci (51kb total) from the banded wren (Thryothorus pleurostictus), a Central American species with minimal pre-existing sequence data. We identified 168 SNPs (a mean of one SNP/305 bp, with SNPs unevenly distributed across loci). Further characterization of variation at 41 of these SNP loci among 256 individuals including 37 parent-offspring families suggests that they provide substantial information for defining the genetic mating system of this species, and that SNPs may be generally useful for this purpose when other markers are problematic.

Congruent population structure inferred from dispersal behaviour and intensive genetic surveys of the threatened Florida scrub-jay (Aphelocoma coerulescens).

The delimitation of populations, defined as groups of individuals linked by gene flow, is possible by the analysis of genetic markers and also by spatial models based on dispersal probabilities across a landscape. We combined these two complimentary methods to define the spatial pattern of genetic structure among remaining populations of the threatened Florida scrub-jay, a species for which dispersal ability is unusually well-characterized. The range-wide population was intensively censused in the 1990s, and a metapopulation model defined population boundaries based on predicted dispersal-mediated demographic connectivity. We subjected genotypes from more than 1000 individual jays screened at 20 microsatellite loci to two Bayesian clustering methods. We describe a consensus method for identifying common features across many replicated clustering runs. Ten genetically differentiated groups exist across the present-day range of the Florida scrub-jay. These groups are largely consistent with the dispersal-defined metapopulations, which assume very limited dispersal ability. Some genetic groups comprise more than one metapopulation, likely because these genetically similar metapopulations were sundered only recently by habitat alteration. The combined reconstructions of population structure based on genetics and dispersal-mediated demographic connectivity provide a robust depiction of the current genetic and demographic organization of this species, reflecting past and present levels of dispersal among occupied habitat patches. The differentiation of populations into 10 genetic groups adds urgency to management efforts aimed at preserving what remains of genetic variation in this dwindling species, by maintaining viable populations of all genetically differentiated and geographically isolated populations.

Eleven microsatellite loci isolated from the banded wren (Thryothorus pleurostictus).

We describe 11 microsatellite loci isolated from the Banded Wren (Thryothorus pleurostictus), a Neotropical species for which understanding the genetic mating system is important for testing questions about the species' unusual vocal behavior. Screening of these loci revealed extremely low allelic variation in a Costa Rican population. Allelic variation at these and other previously developed loci is substantially higher in two other wren species, the southern house wren (Troglodytes aedon bonariae) and rufous-and-white wren (Thryothorus rufalbus), suggesting that the low allelic diversity in the banded wren results from demographic bottlenecks rather than locus-sampling artifacts.

Genome analysis of Marek's disease virus strain CVI-988: effect of cell culture passage on the inverted repeat regions.

The genomes of different derivatives of Marek's disease virus (MDV) strain CVI-988, a low oncogenic isolate of a serotype 1 MDV, were analyzed by restriction enzyme analyses to detect whether alterations occurred after passages in cell culture. DNA molecules of strain 988 isolated directly from blood cells contained mainly two copies of the 132-bp repeat sequence previously reported within BamH1-H and -D fragment as previously reported for more virulent MDV strains. Although a minority of virus particles showed repeat amplification was already at the fifth passage level, amplification mainly occurred between passages 17 and 34 in cell culture. In addition, a 400-bp deletion was detected within the BamH1-A fragment of two derivatives of CVI-988, 988C and 988C/R6.

Aluminum-resistant Arabidopsis mutants that exhibit altered patterns of aluminum accumulation and organic acid release from roots.

Al-resistant (alr) mutants of Arabidopsis thaliana were isolated and characterized to gain a better understanding of the genetic and physiological mechanisms of Al resistance. alr mutants were identified on the basis of enhanced root growth in the presence of levels of Al that strongly inhibited root growth in wild-type seedlings. Genetic analysis of the alr mutants showed that Al resistance was semidominant, and chromosome mapping of the mutants with microsatellite and random amplified polymorphic DNA markers indicated that the mutants mapped to two sites in the Arabidopsis genome: one locus on chromosome 1 (alr-108, alr-128, alr-131, and alr-139) and another on chromosome 4 (alr-104). Al accumulation in roots of mutant seedlings was studied by staining with the fluorescent Al-indicator dye morin and quantified via inductively coupled argon plasma mass spectrometry. It was found that the alr mutants accumulated lower levels of Al in the root tips compared with wild type. The possibility that the mutants released Al-chelating organic acids was examined. The mutants that mapped together on chromosome 1 released greater amounts of citrate or malate (as well as pyruvate) compared with wild type, suggesting that Al exclusion from roots of these alr mutants results from enhanced organic acid exudation. Roots of alr-104, on the other hand, did not exhibit increased release of malate or citrate, but did alkalinize the rhizosphere to a greater extent than wild-type roots. A detailed examination of Al resistance in this mutant is described in an accompanying paper (J. Degenhardt, P.B. Larsen, S.H. Howell, L. V. Kochian [1998] Plant Physiol 117: 19-27).

Characterization of cauliflower mosaic virus (CaMV) resistance in virus-resistant ecotypes of Arabidopsis.

Two Arabidopsis ecotypes are resistant to systemic infection by cauliflower mosaic virus (CaMV), a plant para-retrovirus. Arabidopsis ecotype Enkheim-2 (En-2) is highly resistant to CaMV infection while Bla-14 is more weakly resistant. CaMV resistance in En-2 can be largely attributed to the action of a single semidominant gene called cauliflower mosaic virus resistance1 (CAR1), located at a locus on chromosome 1. Resistance in Bla-14 is tightly linked to CAR1 and may be due to a weak allele at the same locus or another gene in a gene cluster. A quantitative polymerase chain reaction assay in conjunction with replication- and movement-incompetent viral mutants was used to determine whether virus replication or movement is affected in the resistant ecotypes. The pattern of accumulation of the wild-type virus in the resistant ecotype, En-2, was similar to that of a movement-incompetent CaMV mutant, suggesting that CAR1 interferes with or fails to support CaMV movement. CaMV-inoculated En-2 plants do not show visible signs of a hypersensitive response. However, indicators of an induced defense response do appear in CaMV-infected En-2 plants, such as the activation of pathogenesis-related protein gene expression and the production of camalexin, an Arabidopsis phytoalexin. Defense responses induced chemically or by mutation in the susceptible ecotypes delayed and reduced the severity of a CaMV infection. These findings suggest that CAR1 acts either in the susceptible ecotype to support virus movement or in the resistant ecotype to signal a defense response.

Cytokinins and auxins control the expression of a gene in Nicotiana plumbaginifolia cells by feedback regulation.

Both cytokinin (N6-benzyladenine [BA]) and auxin (2,4-dichlorophenoxyacetic acid [2,4-D]) stimulate the accumulation of an mRNA, represented by the cDNA pLS216, in Nicotiana plumbaginifolia suspension culture cells. The kinetics of RNA accumulation were different for the two hormones; however, the response to both was transient, and the magnitude of the response was dose dependent. Runoff transcription experiments demonstrated that the transient appearance of the RNA could be accounted for by feedback regulation of transcription and not by the induction of an RNA degradation system. The feedback mechanism appeared to desensitize the cells to further exposure of the hormone. In particular, cells became refractory to the subsequent addition of 2,4-D after the initial RNA accumulation response subsided. A very different response was observed when the second hormone was added to cells that had been desensitized to the first hormone. Under such conditions, BA produced a heightened response in cells desensitized to 2,4-D and vice versa. These findings support a model in which cytokinin further enhances the auxin response or prevents its feedback inhibition. The hormone-induced RNA accumulation was blocked by the protein kinase inhibitor staurosporin. On the other hand, the protein phosphatase inhibitor okadaic acid stimulated expression, and, in particular, okadaic acid was able to stimulate RNA accumulation in cells desensitized to auxin. This suggests that hormone activation involves phosphorylation of critical proteins on the hormone signaling pathway, whereas feedback inhibition may involve dephosphorylation of these proteins. The sequence of pLS216 is similar to genes in other plants that are stimulated by multiple agonists such as auxins, elicitors, and heavy metals, and to the gene encoding the stringent starvation protein in Escherichia coli. It is proposed that this gene family in various plants be called multiple stimulus response (msr) genes.

Single amino acid change in the helicase domain of the putative RNA replicase of turnip crinkle virus alters symptom intensification by virulent satellites.

The virulent satellite [satellite C (sat C)] of turnip crinkle virus (TCV) is a small pathogenic RNA that intensifies symptoms in TCV-infected turnip plants (Brassica campestris). The virulence of sat C is determined by properties of the satellite itself and is influenced by the helper virus. Symptoms produced in infections with sat C differ in severity depending on the helper virus. The TCV-JI helper virus produces more severe symptoms than the TCV-B helper virus when inoculated with sat C. To find determinants in the TCV helper virus genome that affect satellite virulence, the TCV-JI genome was cloned and the sequence compared to the TCV-B genome. The genomes were found to differ by only five base changes, and only one of the base changes, at nucleotide position 1025, produced an amino acid change, an aspartic acid----glycine in the putative viral replicase. A chimeric TCV genome (TCV-B/JI) containing four of the five base changes (including the base change at position 1025) and a mutant TCV-B genome (TCV-B1025G) containing a single base substitution at position 1025 converted the TCV-B genome into a form that produces severe symptoms with sat C. The base change a position 1025 is located in the helicase of the putative viral replicase, and symptom intensification appears to result from differences in the rate of replication of the satellite supported by the two helper viruses.

Nonmutant forms of the avirulent satellite D of turnip crinkle virus are produced following inoculation of plants with mutant forms synthesized in vitro.

The avirulent satellite RNA D (sat D) of turnip crinkle virus (TCV) has been cloned as a copy DNA. RNA transcripts synthesized in vitro from the cloned cDNA are infectious when coinoculated with RNA transcripts of the cloned TCV genome. Sat D is the smallest and most likely the progenitor of the other TCV satellites and, in contrast to the virulent satellite RNA C (sat C), does not intensify viral symptoms. Mutant forms of sat D including internal deletions up to 50 bases yielded sat D in infected plants. However, sat D mutants were not recovered in mutant form, but reverted to normal size and sequence in infected turnip plants. Mutations at a site with homology to the catalytic strand of self-cleaving sequences in certain viroids and satellites appeared to confer virulence on sat D in that test plants showed severe crinkling and stunting normally associated with sat C. However, sat C appeared along with a restored form of sat D in the progeny RNAs of these severely infected plants. Sat C was presumably generated by recombination between sat D and the TCV genome. In contrast, when plants were inoculated with transcripts containing the equivalent mutations in sat C, sat C was recovered from infected plants in mutant form. These findings demonstrate the tendency for mutant forms of the avirulent satellite, sat D, to revert, but raise questions about the source of information used in the reversion process.

Identification by a random sequencing strategy of the fowlpoxvirus DNA polymerase gene, its nucleotide sequence and comparison with other viral DNA polymerases.

The nucleotide sequence of the DNA polymerase gene of the avipoxvirus fowlpox is presented and the predicted amino acid sequence compared with that of the orthopoxvirus vaccinia. The results have brought to light an error in the vaccinia sequence which has resulted in the ommission of 44 amino acids from the carboxy-terminus of the vaccinia DNA polymerase. There has been extensive conservation of amino acids throughout the enzymes, and regions identified as being present in DNA polymerases from a wide range of viruses are again present here. The method used to identify the fowlpoxvirus gene could have applications towards defining genomic organisations in other viral systems.

Differentiation molecules of the equine trophoblast.

Monoclonal antibodies raised against horse placenta were tested using an indirect immunoperoxidase-labelling technique for reactivity with a panel of tissues from adult horses and conceptuses of various gestational ages. The pattern of reactivity of 4 of the antibodies (F67.1, F71.3, F71.7, F71.14) on trophoblastic tissues described unique antigenic phenotypes for the non-invasive trophoblast of the allantochorion, the invasive trophoblast of the chorionic girdle, and the mature endometrial cup cells, which are derived from the chorionic girdle. Two of the monoclonal antibodies (F67.1 and F71.3) reacted only with chorionic girdle and the endometrial cups. Antibody F71.7 labelled strongly the non-invasive allantochorion from Day 29 of gestation to term. However, F71.7 failed to label mature endometrial cups and stained chorionic girdle only weakly, suggesting that the ability of the girdle cells to synthesize the molecule identified by F71.7 was gradually lost after development of the girdle. Antibody F71.14 reacted with trophoblastic tissues from all stages of gestation tested, with the exception of chorionic girdle. The other 3 anti-trophoblast monoclonal antibodies (F71.1, F71.2 and F71.8) labelled trophoblast-derived tissues from all stages tested. When the monoclonal antibodies were tested on cultured fetal and placental cells from Day 33 conceptuses recovered non-surgically from pregnant mares, the reactivities of the monoclonal antibodies on cultured cells were mostly identical to their reactivities in situ on tissue samples of similar gestational age; F67.1 and F71.3 were strong, specific markers for chorionic girdle cells, and F71.7 labelled allantochorion weakly in vitro, but failed to label chorionic girdle cells.(ABSTRACT TRUNCATED AT 250 WORDS)