Reciprocal Combination Research Articles

New reporter gene-based replication assay reveals exchangeability of replication factors of porcine circovirus types 1 and 2.

Two types of porcine circovirus (PCV), which differ in their pathogenicity, are known. PCV type 2 (PCV2) is the etiological agent of postweaning multisystemic wasting syndrome in swine, while PCV1 has not yet been linked to a disease. Corroborating earlier observations in PCV1, transcript mapping revealed that the rep gene of PCV2 encodes two products, the full-length protein Rep and the spliced version Rep' and that the simultaneous expression of Rep and Rep' proteins is essential for initiation of replication of PCV2. The interchangeability of the replication factors of PCV1 and PCV2 was examined. The rep gene products of PCV2 were not only able to bind the PCV2 origin but also the origin of PCV1 and vice versa. To investigate the competence of the Rep/Rep' proteins to initiate replication at the heterologous origin, a new replication assay was developed. It measures the expression of a luc reporter gene present on a plasmid carrying the origin of the investigated replicon. Replication is initiated by expression of the appendant replicase from a second plasmid and results in replication of the origin plasmid coupled with an increase in the Luc activity. Using this method to compare replication of PCV1 and PCV2 in cell culture, it was shown that the Rep/Rep' protein of PCV2 initiated replication at the origin of PCV1, as did the reciprocal combination. Our results indicate that the cis- and trans-acting replication factors of the two viruses are functionally exchangeable.

Evaluation of Soybean Resistance to Sclerotinia Stem Rot Using Reciprocal Grafting.

Sclerotinia stem rot of soybean is one of the major soybean diseases in the north central region of the United States. One disease management option is to plant cultivars that have resistance. Some sources of partial resistance have been identified, but information pertaining to the nature of resistance is limited. The objective of this study was to determine if the expression of resistance is dictated by shoots of resistant plants and if this can be altered by using resistant and susceptible soybean genotypes grafted in different shoot and rootstock combinations of self-, single-, or double-shoot grafts. After successful grafts were made, several experiments were conducted using different inoculation techniques and soybean genotypes. In one experiment, cotyledons were inoculated with a plug of fungal mycelium, plants were incubated in a mist chamber for 23 h, and plant survival was recorded over time. Based on seven grafting combinations of cross- and self-grafted plants using two soybean cultivars, grafts with NKS19-90 (partially resistant) as shoots had greater (P ≤ 0.05) plant survival at 3, 4, and 5 days after inoculation than the other graft combinations. In another experiment, a total of 17 graft combinations were generated using resistant plant introductions and two susceptible cultivars. Resistant self-grafts of the plant introductions had greater (P ≤ 0.05) plant survival (mean = 75%) than self-grafts of the susceptible cultivars (mean = 15%) at 5 days after inoculation. Inter-genotypic grafts with resistant shoots had greater (P ≤ 0.05) plant survival (mean = 65%) than those in reciprocal combinations (mean = 8%) 5 days after inoculation. A cut stem inoculation method was used to test graft combinations of one resistant and two susceptible cultivars. Grafts with susceptible shoots of cvs. Williams 82 and Asgrow 2242 had greater (P < 0.05) lesion lengths (mean = 13.2 cm) than shoots of NKS19-90 (mean = 9.2 cm) regardless of the rootstock 15 days after inoculation. In a double-graft experiment, shoots of both NKS19-90 and Williams 82 were grafted to either NKS19-90 or Williams 82 rootstocks. Regardless of the rootstock, the shoots of Williams 82 died while shoots of NKS19-90 survived. For all the experiments, resistance was greater when the grafted shoot came from a resistant source on a susceptible rootstock compared with the reciprocal combination regardless of the type of grafting technique or inoculation method.

Eucalyptus camaldulensis × globulus hybrids

Controlled pollination techniques were used to produce hybrids between Eucalyptus camaldulensis and E. globulus, with E. camaldulensis as the female parent. There were substantial barriers to hybridisation, with a 28% reduction in the percentage capsule set, a 78% reduction in the number of seeds produced per capsule, a 99% increase in the number of inviable abnormal seedlings and overall a 92% reduction in the number of normal seedlings produced per pollinated flower compared with outcrossed E. camaldulensis. The number of normal seedlings per pollinated flower varied from 0.1 to 4.7 and was most strongly affected by the source of the pollen. Selfing E. camaldulensis showed it to be partially self-incompatible, with a 29% reduction in the percentage capsule set and an 86% reduction in the number of seeds produced per capsule compared with outcrosses. One reciprocal combination of E. camaldulensis clones originating from Erudina and Broken Hill gave a very low seed�set.Cotyledon shape of the E. camaldulensis × globulus hybrids was intermediate between the two parents and an excellent way of checking that seedlings were hybrids. Shape and size of juvenile leaves did not distinguish the hybrids, but hybrid leaves started to change from being opposite and sessile to alternate and petiolate at a lower node than in the E. globulus parent, although not as quickly as for E. camaldulensis. In adult trees, although the hybrid leaves were longer than leaves of the parent species, it was difficult to identify the hybrids in the field on this basis. Flowers of the hybrid were more similar to E. camaldulensis, being in pedicellate clusters of 3–6. Flower size was intermediate between the parents (operculum height and width, stamen and style length). Fruits were also intermediate in size but valves were exserted as in E. camaldulensis. Hybrids were fertile and overlapped in flowering time with E. camaldulensis, so that if planted adjacent to natural stands of E. camaldulensis, some introgression may occur.

Reassortment between genetically distinct Japanese and US strains of Soil-borne wheat mosaic virus: RNA1 from a Japanese strain and RNA2 from a US strain make a pseudorecombinant virus.

Soil-borne wheat mosaic virus (SBWMV), the type species of the genus Furovirus, has a plus-sense bipartite RNA genome. Japanese and US strains of SBWMV are genetically distantly related, despite their biologically identical properties. Here we report formation of a pseudorecombinant virus consisting of RNA1 from a Japanese strain and RNA2 from a US strain, using infectious in vitro transcripts for both strains. Full-length infectious cDNA clones for a Japanese strain were previously constructed (Yamamiya and Shirako [38]). For RNA1 of a US strain, due to instability of full-length cDNA clones in Escherichia coli cells, it was necessary to prepare a full-length template DNA for in vitro transcription by combining overlapping 5'-terminal and 3'-terminal cDNAs individually cloned in two plasmids, whereas for RNA2 a full-length cDNA clone was the template. For infectivity assays, Chenopodium quinoa, a local lesion host, and wheat, a systemic host, were used. A mixture of Japanese RNA1 transcripts and US RNA2 transcripts caused formation of local lesions on C. quinoa leaves and systemic infection to wheat plants. The nucleotide sequence of the progeny viral RNA2 was identical to that of the US RNA2. The reciprocal combination was not infectious to either host. These results confirm that the Japanese and US SBWMV are genetically distantly related strains belonging to a single species.

Hybridization in larch-alternating Melampsora epitea (M. larici-epitea)

Crossing and selfing experiments were carried out with six field collections and five isolates belonging to three formae speciales, larici-epitea typica (LET), larici-daphnoides (LD), and larici-retusae (LR), of willow rust, Melampsora epitea . European larch ( Larix decidua ) was inoculated with basidiospores produced on overwintered telial leaves and the resulting spermagonial lesions on larch needles subsequently paired in vitro . In two crosses between LR and LD involving a total of 439 lesion pairs, only two cultures obtained were identified as hybrids. These two cultures were non-pathogenic to the maternal host Salix burjatica cv. Korso and weakly pathogenic to the paternal host S. daphnoides cv. Meikle. No identifiable hybrids were obtained from 56 lesion pairs between LET and LD. In a cross between LET as receptor and LD as donor, one-third of the lesions formed aecia. In the reciprocal combination, however, less than 1% developed aecia. All F 1 cultures between LET and LR were weakly pathogenic to the parental hosts S. viminalis cv. Mullatin and S. burjatica cv. Korso. When three of the F 1 cultures were used to produce telia, only one developed mature teliospores. Subsequent selfing experiments showed that this culture was predominantly self-sterile, with only 1 % of lesions producing aecia. In selfing and crossing two pathotypes within the same f. spp. , 20–40 % of needles produced aecia and the rate of aecial formation was similar in both directions. The results obtained suggest that M. epitea is heterothallic and the sexual compatibility is controlled by a pair of alleles at a locus. It is concluded that the three f. spp. are genetically different populations and ecologically fit new pathotypes cannot arise easily as a result of hybridization between them. Within a f. sp. , however, many pathotypes exist or will occur due to exchange of genes for virulence during the sexual life-cycle.

Cooperativity between free and N-(2-hydroxypropyl) methacrylamide copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine induced photodynamic therapy in human epithelial ovarian carcinoma in vitro.

The purpose of this study was to determine the interaction between free (unbound) and N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer bound adriamycin and meso-chlorin e6 monoethylene diamine (Mce6) induced photodynamic therapy in combination in their cytotoxic activities against human ovarian epithelial carcinoma (OVCAR-3) in vitro. The effects of each agent (free drugs and HPMA copolymer bound) alone and in combination were measured simultaneously utilizing two measures of cell viability: a) mitochondrial respiration via the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide reduction (MTT) assay; and b) thymidine incorporation via the tritiated thymidine incorporation (TI) assay. These were performed at 72 and 144 h after drug exposure. Forty-eight hours from time zero (24 h after drug addition), the cells treated with Mce6 (free and HPMA copolymer bound) and controls were exposed to 650 nm light (13 min at 15 mW/cm2, 11.7 J/cm2). The calculated ED50 values by the MTT 72 h assay for adriamycin (A) and Mce6/light (C) were 1.5 microg/ml and 209 ng/ml, respectively. Adriamycin demonstrated progressive cellular toxicity over time in both assays. Mce6/light demonstrated initial damage at 72 h by MTT and TI which recovered by 144 h. Adriamycin and Mce6/light acted cooperatively to increase the percentage of cells inhibited. In combination, 21.3+/-1.5% MTT reduction activity was observed by free adriamycin and Mce6/light compared to the expected 27+/-5% (p<0. 0001) based on additivity. Twice the ED50 of adriamycin (2A=3 microg/ml) or Mce6/light (2C=418 ng/ml) resulted in only 42+/-3.6% and 39.2+/-2.0% activity, respectively (both p<0.0001 vs. combination). When Mce6/light at 10x ED50 (10C) was combined with 1x ED50 of adriamycin (1A), or the reciprocal combination, additional cooperativity was demonstrated. Compared to free drugs, both HPMA copolymer bound adriamycin (P-A) and HPMA copolymer bound Mce6/light (P-C) required a 10-fold increase in drug concentration to show equivalency with free drugs (A or C). Dose response curves demonstrated a reduced slope compared to free drugs in the same dose ranges. When P-A was added (1-10x free adriamycin ED50) to an effective concentration of P-C (10P-C: equivalent to 10x free Mce6 ED50) an improved long-term inhibition of OVCAR-3 cell multiplication was noted in both the MTT and TI 144 h assays. P-C (1-10x free Mce6 ED50) added to an effective concentration of P-A (10P-A: equivalent to 10x free adriamycin ED50) did not appear to significantly improve the efficacy profile of P-A. A and C in vitro appear to act independently and are cooperative in their combined toxicity against the human ovarian epithelial carcinoma cell line OVCAR-3. HPMA copolymer-adriamycin and Mce6 conjugates (P-A and P-C, respectively) inhibited growth of OVCAR-3 in vitro. HPMA copolymer-adriamycin added to HPMA copolymer-Mce6 improved the efficacy of HPMA copolymer-Mce6.

COMPARATIVE MORPHOLOGY, CROSSABILITY, AND TAXONOMY WITHIN THE CALOGLOSSA CONTINUA (DELESSERIACEAE, RHODOPHYTA) COMPLEX FROM THE WESTERN PACIFIC

ABSTRACT Systematic studies were undertaken on the Caloglossa continua (Okamura) King et Puttock complex from Japan, Singapore, and Australia, based on morphology and reproductive compatibility. Specimens from Japan had two to six cell rows derived from a nodal axial cell, at the margin opposite the branch, whereas those from Australia possessed only a single cell row. Specimens from Singapore formed one to four cell rows per nodal axial cell and always contained at least one single cell row on any one thallus. These differences were maintained in cultured materials over a range of temperatures or salinities. Type material of C. continua had the same morphology as the Japanese specimens in this study. Carpospores discharged from the Japanese isolate germinated at 10°C, whereas those from Singapore and Australia died at this temperature. In hybridization experiments, the Japanese entity was completely nonfertile with both the Singaporean and Australian isolates. Many pseudocystocarps were produced in the crossing between the male from Australia and the female from Singapore, although the reciprocal combination did not produce any such structures. On the basis of the discontinuous morphology coupled with the complete reproductive isolation, the entities from Singapore and Australia are described here as C. monosticha sp. nov. The entities with multiple cell rows likely expanded their geographic range from tropical regions, where the majority of Caloglossa species are now distributed, to high‐latitude regions, and such an expansion would be associated with acquiring low‐temperature tolerance.

CROSSING EXPERIMENTS, LIPID COMPOSITION, AND THE SPECIES CONCEPT IN ECTOCARPUS SILICULOSUS AND E. FASCICULATUS (PHAEOPHYCEAE, ECTOCARPALES)1

ABSTRACT Sporophytes of Ectocarpus siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey were collected in the vicinity of Roscoff Brittany, France. Gametophytes derived from meiospores were used for intra‐ and interspecific crosses. Intraspecific gamete combinations gave viable zygotes, which developed into fertile sporophytes. Interspecific crosses were unsuccessful. Gamete fusions did not occur between female gametes of E. fasciculatus and male gametes of E. siliculosus. Hybrid zygotes were formed in the reciprocal combination but died soon after germination. We conclude that the two species of Ectocarpus at Roscoff represent distinct taxonomic entities, which are separated by pre‐ and postzygotic compatibility barriers. These biological findings are confirmed by the differential occurrence of the chemotaxonomic marker betaine‐lipid diacetylglycerylhydroxymethyltrimethyl‐β‐alanine, which is present in our cultures of E. fasciculatus but absent in E. siliculosus.

Role of intrathymic clonal deletion and peripheral anergy in transplantation tolerance induced by bone marrow transplantation in mice conditioned with a nonmyeloablative regimen.

We have investigated the mechanism of tolerance induced by allogeneic bone marrow transplantation (BMT) in mice conditioned with a nonmyeloablative regimen. Permanent mixed chimerism and skin allograft tolerance were achieved when recipient B10 (H-2b) mice were pre-treated with anti-CD4 and anti-CD8 mAbs, thymic irradiation, and 3 Gy whole body irradiation, followed by injection of B10.A (H-2a) bone marrow cells. V beta 11+ T cells are normally deleted in I-E+ B10.A mice, but not in I-E- B10 mice. In spleens of mixed B10.A-->B10 chimeras, V beta 11+ B10 T cells were reduced but detectable in the first 6 wk after BMT, and later became undetectable. Splenic V beta 11+ T cells were unresponsive to receptor cross-linking with V beta 11-specific mAb, demonstrating anergy. B10 V beta 11+ T cells were also detected in spleens of mice that were thymectomized before BMT. In PBL of chimeras, V beta 11+ T cells were undetectable for at least 1.5 yr. Thymocyte chimerism and extensive clonal deletion of mature V beta 11+ B10 thymocytes were observed as early as 10 days post-BMT and at all subsequent time points in thymi of mixed chimeras. Rare I-E+ donor cells were detected in day 10 thymi, and these increased progressively in frequency at later times. In chimeras prepared in the reciprocal strain combination, B10-->B10.A, B10 donor V beta 11+ T cells were undetectable in PBL. Together, our results implicate both central clonal deletion and peripheral clonal anergy in tolerance induced with our regimen. A very low number of intrathymic donor cells or low density of Ag expression is sufficient to mediate extensive clonal deletion of thymocytes that recognize donor Ags.

Regulation of parental alloreactivity by reciprocal F1 hybrids. The role of lactation

Adult reciprocal F1 hybrids differ in their susceptibility to parental graft versus host (GvH) reactions. These reactions were lower when the donor strain was syngeneic with the maternal one. Splenocytes from the member of the reciprocal pair in which the GvH reactions were lower also induced a decreased response of parental cells in cytotoxicity assays and in mixed lymphocyte reactions (MLR). The treatment with anti-CD8 plus complement was able to abrogate the different stimulatory ability of the reciprocal F1 spleen populations. Foster-nursing of F1 hybrids on mothers from the paternal strain was able to induce permanent alterations in the ability of their splenocytes to induce both parental anti-F1 MLR and CTL. The stimulatory ability was indistinguishable from that observed in the reciprocal F1 combination nursed on its own mother. Moreover, lactation was able to alter the ability of CD8+ spleen cells to regulate CTL and parental anti-F1 MLR. The results reported herein show the existence of a maternal effect acting though milk capable of altering the regulation of parental alloreactive T reactions towards self histocompatibility antigens.

Advantages of donor-specific blood transfusion in the rat via the portal vein for renal transplantation.

It is well known that blood transfusion can promote immune unresponsiveness to a renal allograft in both animals and humans. The present study examined the differences between either portal venous (PV) or intravenous (IV) donor-specific transfusion (DST) alone on the survival of rat renal allografts without other immunosuppression. Seven days before LEW (RT1(1)) renal transplantation, recipient WKA rats (RT1u) were inoculated with graded doses (1.0 to 0.0001 mL) of LEW whole blood through either the portal vein or the tail vein. A dose of 0.001 mL whole blood PV resulted in significantly greater prolongation of renal allograft survival than occurred with IV preimmunization (38.5 +/- 10.6 days v 12.5 +/- 2.7 days). Although recipient survival was not significantly different for the two routes for a lower dose (0.1 mL) of whole blood preimmunization 7 days before transplantation, azotemia and rejection were prevented when the PV route, but not the IV route, was used. However, when 0.1 mL of whole blood was administered 5 days before transplantation, survival was prolonged only in animals preimmunized by the PV route (54.7 +/- 14.0 days v 14.3 +/- 4.0 days). In the stronger reciprocal combination of WKA to LEW, there was an advantage of PV with 0.1 mL whole blood, but not with 1 mL. These results indicate that the beneficial effects of DST on renal allograft survival can be obtained when a smaller volume of blood is given via the PV rather than the IV route and a shorter interval elapses between preimmunization and transplantation.

Histological Observations on Interspecific Crosses in the Genus Lupinus

AbstractThe hybridization barriers of interspecific lupin crosses were determined by histological investigations. The cross Lupinus hartwegii×L. mutabilis (both of American origin) revealed embryos in the cotyledonary stage as well as cellular endosperm. In the reciprocal combination, L. mutabilis×L. harwegii, embryo development stopped at the end of the globular stage, and simultaneously the coenocytic endosperm did no differentiate into the cellular state. In the combination L. luteus (European origin) ×L. hartwegii, embryo as well as endosperm development were initiated. Embryo development ended at any early globular stage. Twelve days after pollination the coenocytic endosperm had almost degenerated. With L. hartwegii as the female, no pollen germination could be observed.

Functional and phenotypic analyses of thymic low density adherent cells from murine bone marrow chimeras--influence on thymocyte differentiation.

Previous studies from this laboratory demonstrated a malfunction of the thymus of C3H mice to induce normal level of thymocyte differentiation. Thymocytes developed in the C3H thymus showed depressed proliferative responses to stimulation with anti-CD3 antibody compared with those developed in the other strains. This study was conducted to analyze immunological functions of the thymic stromal cell population in the C3H mice. Using allogeneic bone marrow (BM) chimeras established by reciprocal combination of AKR and C3H mice as donor or recipient, antigen presenting cell (APC) function of low density adherent cells (LDAC) in the thymus was analyzed. The thymic LDAC from C3H mice or [AKR----C3H] BM chimeras where AKR were BM donors and C3H were recipients contained high proportion of Mac-1+ cells as compared to AKR mice or [C3H----AKR] chimeras. The proportion of Mac-1+ cells paralleled the IL-1 secreting ability of the LDAC. Thus, the higher proportion of Mac-1+ cell in the thymus may be responsible for low accessory function observed in C3H thymuses. However, when APC function was analyzed using various T cell hybridomas or a T cell line, the APC functions did not necessarily correlate to the proportions of Mac-1+ cells and amounts of IL-1 produced by the LDAC. When proliferative responses of thymocytes to anti-CD3 stimulation were analyzed in the presence of prostaglandins, PGE-2 inhibited more profoundly the responses of [AKR----C3H] and normal C3H mice than those of [C3H----AKR] and normal AKR mice. Furthermore, a prostaglandin inhibitor, indomethacin, reversed the depressed responses of the former thymocytes which had developed in the C3H thymus. These findings suggest that the hyporesponsiveness of thymocytes from [AKR----C3H] chimeras to anti-CD3 stimulation may be attributable to their increased sensitivity to prostaglandin produced by LDAC.

Influence of Rootstock, Scion, and Water Deficits on Growth of `Colt' and `Meteor' Cherry Trees

Growth and physiological characteristics were evaluated in autografted and reciprocally grafted plants of Prunus avium L. ×pseudocerasus Lindl. `Colt' and Prunus cerasus L. `Meteor'. Containerized plants were grown for 150 days in a greenhouse under either well-watered or water-stressed conditions. Both the scion and rootstock influenced growth (relative growth rate, R̄), morphological [leaf area : root surface area (LARSA) and specific leaf area (SLA)], and physiological (mean net assimilation rate, Ē) characteristics of grafted plants. Regardless of the watering regime, plants with `Meteor' scions and `Colt' rootstocks maintained higher R̄ than plants with `Colt' scions and `Meteor' rootstocks. This enhanced growth occurred as a result of higher Ē. Measurements on water-stressed plants also showed that the graft combination of `Meteor' on `Colt' had the lowest LARSA, while the reciprocal combination of `Colt' on `Meteor' had the highest. Differences in LARSA among water-stressed plants primarily reflected changes in SLA, as influenced by both rootstock and scion, and not in partitioning of dry weight between these organs.

Parental effects on growth in Solanum conditioned at multiple levels

Reciprocal crosses were made between plants that had nuclear genes of S.tuberosum L. ssp tuberosum combinated, by recurrent backrossing, with cytoplasmic factors of S. phureja Juz. & Buk. and, separately, of S. tuberosum ssp. andigena (Juz. & Buk.) Hawkes. Significant differences in growth between 19 of 23 intra-cytoplasmic reciprocal progenies occurred in vigor at one or more growth stages during the season and/or in yield as measured by tuber number and weight. If no transmission of cytoplasmic factors occurred through the pollen, the cytoplasmic factors of the parents were the same, and these reciprocal differences were caused by parental chromosomal gene mechanisms such as maternal effect, pollen-tube selection, and/or imprinting. Early seedling vigor was, in some cases, associated with greater seed weight, but this did not account for all of the reciprocal contrasts. The data do not show whether these parental effects are a result of maternal and/or paternal effects. Analysis to determine whether parental effect was a single gene character or alleles expressed at two levels, or multi-genic and so expressed at multiple levels, was based on a study of progeny of ten parents that were used in more than one reciprocal pair combination. The data showed that the parents could be classified at eight successive levels of relative effectiveness as staminate or pistillate parents. Of 23 crosses, 19 showed significant differences between reciprocal progenies that were consistent with this eight-level array. The data support the interpretation that parental effect is a multi-genic character.

Effects of cell type used for fusion on chromosome elimination and chloroplast segregation in Brassica oleracea (+) Brassica napus hybrids

To determine how the developmental and physiological characteristics of parental cells can influence the fate of chromosomes and chloroplasts in somatic hybrids, fusions were made between Brassica napus and B. oleracea using hypocotyl and/or mesophyll protoplasts in four different combinations. Chromosome number varied greatly among the hybrids obtained. Most hybrids (58%) had more chromosomes than the parental sum, 32% had a DNA-content corresponding to the sum, and only 10% had fewer chromosomes than the sum. The frequencies of symmetric hybrids and chromosome eliminating hybrids obtained in fusions performed by combining mesophyll and hypocotyl protoplasts did not differ significantly from those obtained by combining protoplasts from the same cell type. However, the frequency of hybrids with a DNA-content corresponding to one genome from one of the parental species and two from the other and the frequency of hybrids having the reciprocal combination of three genomes differed significantly between fusion categories. Of 128 hybrids examined, 36 (28%) had B. oleracea chloroplasts and 92 (72%) had B. napus chloroplasts. The pattern of chloroplast segregation did not differ significantly between fusion categories.

Central nervous system neurons migrate on astroglial fibers from heterotypic brain regions in vitro.

In different regions of the developing mammalian brain, neurons follow the processes of radial glial cells over very different trajectories to reach their destinations in specific neuronal layers. To investigate whether the movement of neurons along glial fibers is specified by glia in a given region or whether glia provide a permissive substrate for migration in different brain regions, we purified neurons and astroglial cells from developing cerebellum and hippocampus and analyzed neuronal migration on heterotypic glial fibers with time-lapse, video-enhanced differential interference microscopy in vitro. Granule neurons purified from early postnatal rat cerebellum migrated on astroglial processes of glia purified from late embryonic or early postnatal rat hippocampus with a cytology, neuron-glial relationship, and dynamics of movement that were indistinguishable from those of mouse granule cells migrating on cerebellar astroglial processes in vitro [Edmondson, J. C. & Hatten, M. E. (1987) J. Neurosci. 7, 1928-1934]. In the reciprocal combination, hippocampal neurons migrated on cerebellar glial processes in a manner that was also remarkably similar to migration along homotypic, hippocampal glial fibers [Gasser, U. E. & Hatten, M. E. (1990) J. Neurosci. 10, 1276-1285]. In all cases, migrating neurons had a characteristic appearance, apposing their cell soma against the glial fiber and extending in the direction of migration a motile, leading process that enfolded the glial fiber with short filopodia and lamellipodia. As seen by video microscopy, neurons moved along homotypic and heterotypic glial processes by translocation of the soma and were not "pulled" forward by the leading process. As the neuron moved, the nucleus remained in the posterior portion of the cell and cytoplasmic vesicles moved forward from the soma into the leading process. The dynamics of the movement of neurons along heterotypic glial substrates, including the speed and periodicity of motion, was identical to that of neurons migrating along homotypic glial substrates. These experiments suggest that the mechanism of movement of neurons along glial fibers is conserved in these two brain regions during development.

Crossing experiments of lettuce cultivars and species (Lactuca sect.Lactuca, Compositae)

The degree of relationships withinLactuca sativa and three wild relativesL. serriola, L. saligna, andL. virosa was studied by observing the performance, vigour and fertility of the F 1 hybrids obtained from crosses made in and between the four species. The crosses ofL. saligna ×L. virosa and the reciprocal crosses produced no hybrids.L. saligna andL. virosa are the least related of the four species.L. sativa ×L. serriola and the reciprocal crosses were successful and produced fertile hybrids These two species are genetically very closely related.L. saligna is known to produce, as a female parent, hybrids withL. sativa andL. serriola. Now the reciprocal cross was successful for the first time, so the unability to obtain hybrids in the past was based on the choice of accessions and not caused by unilateral incompatibility.L. virosa ×L. sativa and the reciprocal combination produced hybrids. The combinationL. serriola ×L. virosa produced hybrids with very limited fertility. In contrast to earlier reports (sterile hybrids) one combination of the reciprocal cross too produced hybrids with very limited fertility.—Some of theL. saligna ×L. sativa (and reciprocal) hybrids were found to look strikingly likeL. serriola. This adds evidence for the descent ofL. serriola andL. sativa:L. saligna also made part of the ancestral complex of the cultivated lettuce.

Variation in nitrogen fixation among populations ofFrankia sp. andCeanothus sp. in actinorhizal association

Wildland shrub improvement is needed for sound range and disturbed land revegetation practice. The possibility of selecting superior N2-fixingFrankia-Ceanothus spp. actinorhizal associations was examined. Greenhouse tests were used to expose various soil-borne microsymbiont andCeanothus sp. population accessions in reciprocal combination. The acetylene reduction rate was used as a measure of N2-fixation capacity. There was no significant interaction between host and microsymbiont regardless of source for all variables measured. The acetylene reduction rate, nodule number and mass, plant biomass, and root: shoot ratio were significantly different among soil sources. The acetylene reduction rate was not significantly different amongCeanothus sp. accessions. Neither was it strongly correlated with other variables. It was concluded that the N2-fixation rate is more a function ofFrankia sp. than the hostCeanothus sp. in actinorhizal associations. It appears possible to select soil sources with superior N2-fixing microsymbiont populations.

Functional evidence for the recognition of endogenous peptides by autoreactive T cell clones

The fine specificity of two human T cell clones responding to autologous HLA-DR1 expressing antigen-presenting cells (APC) in the absence of nominal antigen has been investigated using Epstein-Barr virus-transformed B cells (BCL) of known DR beta 1 domain sequence. It was found that responsiveness was markedly affected by changes in a limited number of residues in this domain. Substitution of the DR1 beta sequence at one residue, position 74, even conservatively, was found to be particularly significant. Located on the beta 1 domain alpha-helix, this residue is predicted to point into the antigen-binding groove and is therefore unlikely to make contact with the T cell receptor. This finding suggests that these T cells are specific for a bound endogenous peptide within the autologous major histocompatibility (MHC) binding groove. The autospecific T cell clones also responded to murine L cell transfectants expressing DR alpha DR1 beta as well as to transfectants expressing the mouse/human hybrid MHC molecule I-E alpha DR1 beta but not to the reciprocal combination DR alpha I-E beta, thus confirming the importance of the beta 1 domain to T cell recognition. In contrast to the autocytotoxicity observed with BCL, cytolysis of the murine L cells expressing the HLA-DR1 molecule was slight and only found at high effector-target ratios. In addition, although fixation enhanced the recognition of BCL, capacity of the murine L cells bearing the HLA-DR1 molecule to stimulate T cell clone proliferation was markedly reduced by aldehyde fixation. When taken together, these results suggest that the endogenous peptides recognized by these autoreactive T cells are of human origin.