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SUMMARY The microbial community of the gastrointestinal system has an enormous impact on the vertebrate host. The relationship begins at birth or hatch and evolves to a stable ecosystem in which diverse, and unique, niches are created and inhabited by microorganisms. These microbial populations tend to be similar within a host species (and even across host species), but each system is a unique construct resulting from its individual history of mutual influence. The development of the system, both microbial and host, begins in the perinatal animal. The timing of this developmental process is suggestive of imprinting, the process of epigenetic evolution of somatic stem cells. (Imprinted changes are thought not to involve the germ line, i.e., are not inherited by the next generation of the host animal, but are genetic changes that can be passed on to the daughter cells of the imprinted proliferative stem cell.) This review briefly discusses the development of the gastrointestinal system, including both the microbiota and its perinatal host. The effects of the microflora on enteric and immune cells are described. Effects of attempts to restrict contact between the mucosal immune system and the microbiota are addressed, along with further data that would be required to demonstrate that the effects of the gut microbiota on mucosal immune development are restricted to an ontogenetic window. Finally, the consequences of a failure to achieve a relationship of mutual tolerance between the microbiota and the host and some mechanisms to facilitate this process are discussed.

The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

The current status of transgenic studies in mosses is reviewed with particular attention being given to the mosses Physcomitrella patens and Ceratodon purpureus. This paper reviews the advantages of using mosses as models for higher plants in the study of plant development, and includes developmental processes, already partially characterized at the genetic level by mutant analysis, for which transgenic studies may be applicable. The P. patens transformation process is being studied in this laboratory and details are given for a class of transformants which contain extrachromosomal plasmid DNA. Publications which present the nucleic acid and/or protein sequence for nuclear, chloroplast and mitochondrial genes are reviewed. Areas of research in which transgenic studies promise to complement existing cell biological and physiological approaches are discussed. These include the measurement of calcium levels in mutant and wild-type transformants expressing the apoaequorin gene and a role for phytochrome gene expression in the establishment of polarity.

We report two situations in which the polarity of gravitropism of single protonemal cells of the moss Physcomitrella patens is reversed. Dark-grown protonemata of wild-type P. patens grow negatively gravitropically. Time-lapse video-microscopy reveals that a temporary reversal of growth polarity occurs during mitotic division which is independent of the cells’ growth rate. A transitory reversal of growth direction is also observed when the unidirectional gravitropic stimulus is interrupted by a period of growth on a clinostat. A third situation, in which a mutant class responds by growing positively gravitropically, has been described previously (Jenkins, Courtice & Cove, 1986). These observations are discussed in terms of possible mechanisms for cell morphogenesis and tropic growth.

Seven hundred and twenty hatchling broilers were allotted to 12 treatment groups. Groups 1 and 2 were fasted for 48 h posthatch; groups 3 and 4 were fasted for 48 h followed by ad libitum access to a 1% glutamine (Gln) diet; groups 5 and 6 had ad libitum access to a common diet; groups 7 and 8 had access to a 1% Gln diet posthatch; groups 9 and 10 were fed regular Oasis hatchling supplement; and groups 11 and 12 were fed Oasis sprayed with 1% Gln for the first 48 h posthatch. The birds in treatment groups 2, 4, 6, 8, 10, 11, and 12 were vaccinated with Eimeria maxima posthatch, and all birds were orally challenged with high dose E. maxima on d 22. During the first 2 wk, birds in group 7 had the highest gain and feed efficiency among treatments (P0.01). Compared with birds in the nonGln groups, birds in the Gln group had higher gain, feed efficiency, and livability (P0.05). Among the Fast (groups 1 to 4), Feed (groups 5 to 8), and Oasis (groups 9 to 12) groups, birds in the Feed groups had the highest gain during d 0 to 21 (P0.01). During d 22 to 28, birds in the Fast groups had the lowest BW and livability (P0.01), and the nonvaccinated birds had lower gain and feed efficiency relative to vaccinated birds (P0.01). Birds in the Feed and Oasis groups had higher villus height (VH) of mid small intestine than Fast groups at d 2 and 7 (P0.05), and nonvaccinated birds had higher VH than vaccinated birds (P0.01) at d 7 after hatch. On d 14, there were differences in serum interferon-gamma (P0.05) levels among treatments. During d 22 to 28, vaccinated birds had lower lesion scores in the mid small intestine than nonvaccinated birds (P0.01), and birds in the Feed or Oasis groups had lower lesion scores compared with the Fast groups (P0.02). These results indicated the importance of immediate access to feed posthatch, the beneficial effects of feeding Oasis hatching supplement and Gln after hatch, as well as the necessity of the vaccination program against coccidiosis challenge.

The gene for apoaequorin has been used previously to indicate cytosolic calcium changes in higher plants. Here we report the transformation of the moss Physcomitrella patens with the cDNA for apoaequorin. Stable transformants were obtained in the wild type which reconstitute the calcium-sensitive luminescent protein aequorin in vivo after incubation in coelenterazine, and continue to grow normally. The wild type responds to cold-shock (0-10 degrees C) with increases in cytosolic calcium. Mechanical perturbation, in the form of touch, also induces transient increases in cytosolic calcium. A smaller response to pH, distinct from the touch response and exhibiting different kinetics, can also be detected.

Animal feeding studies were conducted with rats, broiler chickens, catfish and dairy cows as part of a safety assessment program for a soybean variety genetically modified to tolerate in-season application of glyphosate. These studies were designed to compare the feeding value (wholesomeness) of two lines of glyphosate-tolerant soybeans (GTS) to the feeding value of the parental cultivar from which they were derived. Processed GTS meal was incorporated into the diets at the same concentrations as used commercially; diary cows were fed 10 g/100 g cracked soybeans in the diet, a level that is on the high end of what is normally fed commercially. In a separate study, laboratory rats were fed 5 and 10 g unprocessed soybean meal 100 g diet. The study durations were 4 wk (rats and dairy cows), 6 wk (broilers) and 10 wk (catfish). Growth, feed conversion (rats, catfish, broilers), fillet composition (catfish), and breast muscle and fat pad weights (broilers) were compared for animals fed the parental and GTS lines. Milk production, milk composition, rumen fermentation and nitrogen digestibility were also compared for dairy cows. In all studies, measured variables were similar for animals fed both GTS lines and the parental line, indicating that the feeding value of the two GTS lines is comparable to that of the parental line. These studies support detailed compositional analysis of the GTS seeds, which showed no meaningful differences between the parental and GTS lines in the concentrations of important nutrients and antinutrients. They also confirmed the results of other studies that demonstrated the safety of the introduced protein, a bacterial 5-enolpyruvyl-shikimate-3-phosphate synthase from Agrobacterium sp. strain CP4.

The Galileo Probe Atmosphere Structure Instrument will make in-situ measurements of the temperature and pressure profiles of the atmosphere of Jupiter, starting at about 10−10 bar level, when the Probe enters the upper atmosphere at a velocity of 48 km s−1, and continuing through its parachute descent to the 16 bar level. The data should make possible a number of inferences relative to atmospheric and cloud physical processes, cloud location and internal state, and dynamics of the atmosphere. For example, atmospheric stability should be defined, from which the convective or stratified nature of the atmosphere at levels surveyed should be determined and characterized, as well as the presence of turbulence and/or gravity waves. Because this is a rare opportunity, sensors have been selected and evaluated with great care, making use of prior experience at Mars and Venus, but with an eye to special problems which could arise in the Jupiter environment. The temperature sensors are similar to those used on Pioneer Venus; pressure sensors are similar to those used in the Atmosphere Structure Experiment during descent of the Viking Landers (and by the Meteorology Experiment after landing on the surface); the accelerometers are a miniaturized version of the Viking accelerometers. The microprocessor controlled experiment electronics serve multiple functions, including the sequencing of experiment operation in three modes and performing some on-board data processing and data compression.

In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.

We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

Three nodulation-deficient (nod) mutants of Rhizobium leguminosarum were isolated following insertion of the transposon Tn5 into pRL1JI, the R. leguminosarum plasmid known to carry the nodulation genes. DNA adjacent to the nod: Tn5 alleles was subcloned and used to probe a cosmid clone bank containing DNA from a Rhizobium strain carrying pRL1JI. Two cosmid clones which showed homology with the probe contained about 10 kb of DNA in common. The R. leguminosarum host-range determinants were found to be present within this 10 kb common region since either of the cosmid clones could enable a cured R. phaseoli strain to nodulate peas instead of Phaseolus beans, its normal host. Electron microscopy of nodules induced by Rhizobium strains cured of their normal symbiotic plasmid but containing either of the two cosmid clones showed bacteroid-forms surrounded by a peri-bacteroid membrane, indicating that normal infection had occurred. Thus it is clear that this 10 kb region of nodDNA carries the genes that determine host range and that relatively few bacterial genes may be involved in nodule and bacteroid development.

During analysis of early events in the infection and nodulation of Vicia hirsuta roots inoculated with normal and mutant strains of Rhizobium leguminosarum and strains containing cloned nodulation (nod) genes, a number of novel observations were made. (i) Alternating zones of curled and straight root hairs were seen on roots of V. hirsuta inoculated with the wild-type strain of R. leguminosarum. This phasing of root hair curling was not seen if plants were grown under continuous light or continuous dark conditions. (ii) Reduced nodulation and delayed nodule initiation was observed with a strain carrying a Tn5 mutation in the nodE gene. In addition the phased root hair curling was absent, and root hair curling was observed along the length of the root. (iii) The nodABC genes cloned on a multicopy plasmid in a wild-type strain inhibited nodulation but induced a continuous root hair curling response. Those few nodules that eventually formed were found to contain bacteria which had lost the plasmid carrying the nodABC genes. (iv) With a strain of Rhizobium cured of its indigenous symbiotic plasmid, but containing the cloned nodABCDEF genes, continuous root hair curling on V. hirsuta was observed. However, no infection threads were observed, and surprisingly, it did appear that initial stages of nodule development occurred. Observations of thin sections of these early developing nodules indicated that early nodule meristematic divisions may have occurred but that no bacteria were found within the nodules and no infection threads were observed either within the nodule bumps or within any of the root hairs. It was concluded that for normal infections to occur, precise regulation of the nod genes is required and that overexpression of the root hair curling genes inhibits the normal infection process.

SUMMARY This review will examine the early stages of infection of legume roots by strains of Rhizobium that induce nitrogen-fixing nodules. The object is to show that, at least in terms of ultrastructure, the interactions between the plant and the rhizobia occur at the cell surface interface between these organisms. This situation exists at all stages, from the time the bacteria attach to the surface of the root hairs to the time they occur as the nitrogen-fixing form in the cytoplasm of the nodule cells, enclosed by peribacteroid membranes.

Mutant strains of Rhizobium leguminosarum have been isolated in which the transposon Tn5 was inserted into the cloned nodulation genes. The mutant strains were examined for their ability to induce nodules and to induce normal root hair curling on peas. Five distinct classes of nod mutations were identified on the basis of the phenotypes of the various mutant strains, and of the mapped locations of the nod:: Tn5 alleles. Three classes of mutants (I, II and III) were defective in nodulation and two classes (IV and V) were delayed in nodule appearance. Mutations of classes I, II and III types affected root hair curling and a subcloned 6.6 kb fragment of DNA containing this region conferred the ability to induce root hair curling upon a Rhizobium strain deleted of its nod genes. Analysis of protein products formed after in vitro transcription and translation of the plasmid DNA corresponding to the class I, II, III and IV regions indicated that proteins of molecular weights 48,000, 45,000, 36,000 and 34,000 were formed. A comparison of protein products formed by equivalent plasmids carrying nod:: Tn5 alleles indicated that the class I mutations blocked the synthesis of the 45,000 molecular weight peptide and that the two products of molecular weights 36,000 and 34,000 were absent from the class II mutants. It is concluded that at least five nod genes are involved in nodulation and the polypeptide products of some of these genes have been identified.

This paper describes our current understanding of the organization of the nodulation genes in a strain of R. leguminosarum, the species that nodulates peas, Vicia, Lens and Lanthyrus. In many of the fast-growing species of Rhizobium it is well established that the genes for nodulation and nitrogen fixation are clustered on large “symbiotic” plasmids one of which is the R. leguminosarum plasmid pRL 1JI whose transfer to other species of Rhizobium allows the transconjugants to nodulate peas (Johnston et al., 1978). Further, a region, no more than 10kb in size, was sufficient to confer the ability to nodulate peas when transferred to a strain of R. phaseoli cured of its resident symbiotic plasmid (Downie et al., 1983). The fact that the nodulation and host-range genes of pRL1JI are clustered within such a small region means that they can be analysed in considerable detail using both genetical and physical techniques.

The region of the Rhizobium leguminosarum plasmid pRL1JI involved in nodulation and nitrogen fixation has been cloned on a series of four overlapping cosmid clones. These clones represent ˜60 kb of pRL1JI DNA on which a series of Tn5-induced fix and nod alleles have been identified, with the two most distant alleles being separated by ˜45 kb of DNA. The mutant alleles fell into three groups, two clusters of fix alleles separated by one cluster of nod alleles. Within one group of fix alleles, DNA homologous to the nifA gene of Klebsiella pneumoniae has been identified, whereas the pRL1JI DNA homologous to the K. pneumoniae nitrogenase genes is present within the other group of fix alleles.