By screening with the fluorescent stain Calcofluor, we have isolated 26 independent transposon TnS insertion mutants ofRhizobium meliloti that are deficient in the production of a known extracellular polysaccharide (Exo-). The mutants belonged to six distinct genetic groups based on the ability of their Exo-phenotype to be complemented by different recombinant plasmids from a R. melilot clone bank. With few exceptions, all of the mutants formed ineffective (non-nitrogen-fixing) nodules on alfalfa. For all but one group, the complementing plasmids restored effective nodulation. These results establish a firm and extensive correlation between the ability ofRhizobium to produce a particular polysaccharide and symbiotic proficiency. The ineffective nodules appeared to contain no bacteroids and to form without shepherds' crooks or infection threads; this symbiotic phenotype matches that described for a set of independently isolated mutants that belong phenotypically and genetically to the group B exopolysaccharide mutants described previously [Finan et al. (1985) Cell 40, 869-877]. Apparently the exopolysaccharide, although not required for nodule formation, is involved in wild-type nodule invasion.The interactions between rhizobia and legumes that result in the development of nitrogen-fixing root nodules entail a complex series of events (1-3). Bacteria attach to a root hair, which curls to form a shepherd's crook. Bacterial penetration of the root hair occurs and a tubular infection thread forms, which carries the invading bacteria toward the base of the root hair. Cells deeper in the root cortex divide, and the bacteria are eventually released from the infection thread into the newly formed nodule cells. Nitrogen fixation occurs in the mature intracellular bacteria, called "bacteroids. " Recently it has become clear that the events of nodule formation (the development of root cortical tissue into a differentiated nodule structure) can be uncoupled from the events of nodule invasion (shepherd's crook formation, infection thread formation, and entry of bacteria into host cell cytoplasm). Strains ofAgrobacterium tumefaciens and Escherichia coli containing Rhizobium meliloti nodulation genes (4-7) and a set of R. meliloti mutants at a locus that is also involved in extracellular polysaccharide synthesis (this paper; ref. 8) all form empty nodules (containing no intracellular bacteroids) without (or before) the development of shepherd's crooks or infection threads. It was recently reported that root cortical cell division in soybean can occur independent of infection thread formation, root hair curling, or even bacterial attachment (9, t).A possible role for Rhizobium polysaccharides in the nodulation process has been a subject of great interest for a number of years (11. 12) but has not been demonstrated conclusively (13), although a single mutant that produced no water-soluble polysaccharide and formed ineffective (nonnitrogen-fixing) nodules was reported recently (14). In this paper we describe the isolat...

Using physical and genetic data, we have demonstrated that Rhizobium meliloti SU47 has a symbiotic megaplasmid, pRmeSU47b, in addition to the previously described nod-nif megaplasmid pRmeSU47a. This plasm'id includes four loci involved in exopolysaccharide (exo) synthesis as well as two loci involved in thiamine 1'iosynthesis. Mutations at the exo loci have previously been shown to result in the formation of nodules which lack infection threads (Inf)' and fail to fix nitrogen (Fix-). Thus, both megaplasmids contain genes involved in the formation of nitrogen-fixing root nodules. Mutations at two other exo loci were not located on either megaplasmid. To mobilize the megaplasmids, the oriT of plasmid RK2 was inserted into them. On alfalfa, Agrobacterium tumefaciens strains containing pRmeSU47a induced marked root hair curling with no infection threads and Fix-nodules, as reported by others. This plant phenotype was not observed to change with A. tumefaciens strains containing both pRmeSU47a and pRmeSU47b megaplasmids, and strains containing pRmeSU47b alone failed to curl root hairs or form nodules.A great many natural isolates of rhizobia, agrobacteria, and pseudomonads have been shown to carry a variety of large plasmids (for examples, see references 8, 21, and 38). Some of these are megaplasmids, with molecular masses over 450 megadaltons (38). Genes for a few functions have been localized to these large plasmids, notably including pathogenicity gepes for the Ti and Ri plasmids of agrobacteria (6, 21) and symbiotic nodulation (nod) and nitrogen fixation (nif) genes for the Sym megaplasmids of the fast-growing rhizobia (3,8,28,33,37,38). Nevertheless, the significance of this genomic organization remains obscure, although the fact that these are all plant-associated soil bacteria does suggest an underlying evolutionary cause.Rhizobial Sym megaplasmids are being characterized extensively with regard to symbiosis. In addition, Rhizobium meliloti 41 has recently been shown to carry a second megaplasmid, with a molecular weight very nearly that of pSym, on which a region for surface exclusion (although none for symbiotic functions) has tentatively been identified (2). A second megaplasmid has also been identified in R.

Huntington's disease (HD) is one of the few neurodegenerative diseases with a known genetic cause, knowledge that has enabled the creation of animal models using genetic manipulations that aim to recapitulate HD pathology. The study of behavioral and neuropathological phenotypes of these HD models, however, has been plagued by inconsistent results across laboratories stemming from the lack of standardized husbandry and testing conditions, in addition to the intrinsic differences between the models. We have compared different HD models using standardized conditions to identify the most robust phenotypic differences, best suited for preclinical therapeutic efficacy studies. With a battery of tests of sensory-motor function, such as the open field and prepulse inhibition tests, we replicate previous results showing a strong and progressive behavioral deficit in the R6/2 line with an average of 129 CAG repeats in a mixed CBA/J and C57BL/6J background. We present the first behavioral characterization of a new model, an R6/2 line with an average of 248 CAG repeats in a pure C57BL/6J background, which also showed a progressive and robust phenotype. The BACHD in a FVB/N background showed robust and progressive behavioral phenotype, while the YAC128 full-length model on either an FVB/N or a C57BL/6J background generally showed milder deficits. Finally, the Hdh Q111 knock-in mouse on a CD1 background showed very mild deficits. This first extensive standardized cross-characterization of several HD animal models under standardized conditions highlights several behavioral outcomes, such as hypoactivity, amenable to standardized preclinical therapeutic drug screening.

No abstract

In several plant systems expression of structurally intact genes may be silenced epigenetically when a transgenic construct increases the copy number of DNA sequences. Here we report epigenetic silencing in Arabidopsis lines containing transgenic inserts of defined genetic structure, all at the same genomic locus. These comprise an allelic series that includes a single copy of the primary insert, which carries repeated drug resistance transgenes, and a set of its derivatives, which as a result of recombination within the insert carry different numbers and alleles of resistance genes. Although the drug resistance genes remained intact, both the primary and some recombinant lines nevertheless segregated many progeny that were partly or fully drug-sensitive because of silencing. As in other systems silencing was reversible, and correlated with decreased steady-state mRNA and increased DNA methylation. Each different number and combination of genes, on the same or different (i.e., homologous) chromosomes, conditioned its own idiosyncratic segregation pattern. Strikingly, lines with a single gene segregated only a few slightly drug-sensitive progeny whereas multi-gene lines segregated many highly sensitive progeny, indicating dependence of silencing at this locus on repeated sequences. This argues strongly against explanations based on antisense RNA, but is consistent with explanations based on ectopic DNA pairing. One possibility is that silencing reflects the interaction of paired homologous DNA with flanking heterologous DNA, which induces condensation of chromatin into a non-transcribable state.