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We are building a framework physical infrastructure across the soybean genome by using SSR (simple sequence repeat) and RFLP (restriction fragment length polymorphism) markers to identify BACs (bacterial artificial chromosomes) from two soybean BAC libraries. The libraries were prepared from two genotypes, each digested with a different restriction enzyme. The BACs identified by each marker were grouped into contigs. We have obtained BAC- end sequence from BACs within each contig. The sequences were analyzed by the University of Minnesota Center for Computational Genomics and Bioinformatics using BLAST algorithms to search nucleotide and protein databases. The SSR-identified BACs had a higher percentage of significant BLAST hits than did the RFLP-identified BACs. This difference was due to a higher percentage of hits to repetitive-type sequences for the SSR-identified BACs that was offset in part, however, by a somewhat larger proportion of RFLP-identified significant hits with similarity to experimentally defined genes and soybean ESTs (expressed sequence tags). These genes represented a wide range of metabolic functions. In these analyses, only repetitive sequences from SSR-identified contigs appeared to be clustered. The BAC-end sequences also allowed us to identify microsynteny between soybean and the model plants Arabidopsis thaliana and Medicago truncatula. This map-based approach to genome sampling provides a means of assaying soybean genome structure and organization.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.

We constructed a soybean bacterial artificial chromosome (BAC) library suitable for map-based cloning and physical mapping in soybean. This library consists of approximately 40 000 clones (4-5 genome equivalents) stored individually in 384-well microtiter dishes. A random sampling of 224 clones yielded an average insert size of 150 kb, giving a 98% probability of recovering any specific sequence. We screened the library for seven single or very low copy genie or genomic sequences using the polymerase chain reaction (PCR) and found between one and seven BACs for each of the seven sequences. When testing the library with a portion of the soybean psbA chloroplast gene, we found less than 1% chloroplast DNA representation. We also screened the library for eight different classes of disease resistance gene analogs (RGAs) and identified BACs containing all RGAs except class 8. We arranged nine of the class 1 RGA BACs and six of the class 3 RGA BACs into individual contigs based on fingerprint patterns observed after Southern probing of restriction digests of the member BACs with a class-specific sequence. This resulted in the partial localization of the different multigene family sequences without precise definition of their exact positions. Using PCR-based end rescue techniques and RFLP mapping of BAC ends, we mapped individual BACs of each contig onto linkage group J of the soybean public map. The class 1 contig mapped to the region on linkage group J that contains several disease resistance genes. The class 1 contig extended approximately 400 kb. The arrangement of the BACs within this contig has been confirmed using PCR. One end of the class 1 contig core BAC mapped to two positions on linkage group J and cosegregated with two class 1 RGA loci, suggesting that this segment is within an area of regional duplication.

Various growth and physiological parameters were measured in germinating, presenescent, and senescing soybean (Glycine max [L.] Merr.) cotyledons and in cotyledons rejuvenated by epicotyl removal 18 days after planting. The maximal measured carbon dioxide exchange rates (CER) in the cotyledons were in the range of those reported for field-grown soybean leaves. Rejuvenated cotyledons accumulated total chlorophyll in excess of the maximum observed in presenescent cotyledons. When photosynthetic rates were expressed per cotyledon, the CER in rejuvenated tissue recovered to the maximal rates observed in presenescent cotyledons. Ribulose-1,5-bisphosphate carboxylase/oxygenase in rejuvenated cotyledons also recovered to the maximal amount seen in presenescent cotyledons so that CER appeared to be a function of ribulose-1,5-bisphosphate carboxylase/oxygenase content during most of the period studied. Observations of the postillumination outburst of CO(2) and (14)C label in glycine indicated that photorespiration was occurring in the cotyledons and that photorespiration relative to photosynthesis was different in rejuvenated compared with presenescent cotyledons.

The activity of two photorespiratory enzymes, phosphoglycolate phosphatase (PGPase) and glycolate dehydrogenase (glycolate DH), changes when CO(2)-enriched wild-type (WT) Chlamydomonas reinhardtii cells are transferred to air levels of CO(2). Adaptation to air levels of CO(2) by Chlamydomonas involves induction of a CO(2)-concentrating mechanism (CCM) which increases the internal inorganic carbon concentration and suppresses oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase. PGPase in cell extracts shows a transient increase in activity that reaches a maximum 3 to 5 hours after transfer and then declines to the original level within 48 hours. The decline in PGPase activity begins at about the time that physiological evidence indicates the CCM is approaching maximal activity. Glycolate DH activity in 24 hour air-adapted WT cells is double that seen in CO(2)-enriched cells. Unlike WT, the high-CO(2)-requiring mutant, cia-5, does not respond to limiting CO(2) conditions: it does not induce any known aspects of the CCM and it does not show changes in PGPase or glycolate DH activities. Other known mutants of the CCM show patterns of PGPase and glycolate DH activity after transfer to limiting CO(2) which are different from WT and cia-5 but which are consistent with changes in activity being initiated by the same factor that induces the CCM, although secondary regulation must also be involved.

A mutant strain of Chlamydomonas reinhardtii, designated 18-7F, has been isolated and characterized. 18-7F requires a high CO(2) concentration for photoautrophic growth in spite of the apparent induction of a functional CO(2) concentrating mechanism in air-adapted cells. In 2% O(2) the photosynthetic characteristics of 18-7F and wild type are similar. In 21% O(2), photosynthetic O(2) evolution is severely inhibited in the mutant by preillumination in limiting CO(2), although the apparent photosynthetic affinity for inorganic carbon is similar in preilluminated cells and in cells incubated in the dark prior to O(2) evolution measurements. Net CO(2) uptake is also inhibited when the cells are exposed to air (21% O(2), 0.035% CO(2), balance N(2)) for longer than a few minutes. [(14)C]Phosphoglycolate accumulates within 5 minutes of photosynthetic (14)CO(2) fixation in cells of 18-7F. Phosphoglycolate does not accumulate in wild type. Phosphoglycolate phosphatase activity in extracts from air-adapted cells of 18-7F is 10 to 20% of that in wild-type Chlamydomonas. The activity of phosphoglycolate phosphatase in heterozygous diploids is intermediate between that of homozygous mutant and wild-type diploids. It was concluded that the high-CO(2) requiring phenotype in 18-7F results from a phosphoglycolate phosphatase deficiency. Genetic analyses indicated that this deficiency results from a single-gene, nuclear mutation. We have named the locus pgp-1.

The rate of respiration in sectioned leaves of Bryophyllum tubiflorum Harv. increases with decreasing section thickness. The rates of uninhibited respiration in 2- and 8-millimeter-thick sections are 74 and 46 microliters of O(2) per gram fresh weight of unruptured tissue per hour at 20 C, whereas the rate in the presence of cyanide is 31 microliters of O(2) in each case. The rates are unaffected by salicylhydroxamic acid, but cyanide and salicylhydroxamic acid together completely eliminate O(2) uptake. The capacity of the alternative respiratory pathway is thus initially high (estimated at 84% of the uninhibited respiratory rate in whole leaves) and remains constant but probably unexpressed subsequent to the rapid induction of wound respiration.

Sertoli cells, cultured from testes of 2-3-week-old Balb/c mice, contain tripartite nucleoli, exhibit phagocytic function, and have the typical morphologic appearance of Sertoli cells by light microscopy, transmission and scanning electron microscopy. Fluorescence-activated cell sorter analysis indicated the presence on mouse. Sertoli cells of H-2 but not Ia antigens. Alpha-D-mannose and N-acetyl-D-glucosamine determinants are detected on Sertoli cell surface by inhibition of lectin binding using appropriate sugars. Interaction of Sertoli cells with concanavalin A (Con A) or wheatgerm agglutinin (WGA) results in rapid patching of the labeled lectin, which become internalized as perinuclear vesicles. These changes are accompanied by rounding of the Sertoli cell, mimicking cellular changes known to occur when fat Sertoli cells are stimulated in vitro by FSH or cyclic AMP. Thus, Sertoli cells have surface alloantigens that permit them to serve as target to cytotoxic T lymphocytes, but not as antigen presenting cells.

Progressive subcultivation of IMR-90 cells results in non-proliferative, heterogeneous cultures which may reflect aging of the diploid line (Hayflick, Exp. Cell Res., 37 (1965) 614). We have observed that late passage cells exhibit different rates of spreading and morphogenesis when compared to early passage groups, phenomena which we attribute to altered reassembly of the cytoskeleton in senescent cells (Kelley et al. Mech. Ageing Dev., 13 (1980) 127). To determine whether potential differences in cytoskeletal proteins develop with progressive subcultivation, early and late passage cultures were extracted with 0.5% Triton X-100 for 1 min followed by 1.0% sodium dodecyl sulfate (SDS) prior to separation and characterization of extracted proteins by electrophoresis on 7.5-15% gradient SDS gels. Extractions were made of both culture groups 3, 6 and 24 h after reseeding. Cytoskeletal ultrastructure at each stage of spreading was examined either in replicas of extracted cells or directly by scanning electron microscopy. Although considerable variation in cytoskeletal organization was observed, qualitative differences in gel banding patterns of actin, myosin and tubulin were not apparent at selected time points. However, late passage cells at 6 h and 24 h did not exhibit filamin associated with the Triton insoluble fraction as did early passage cells. Since it has been demonstrated that filamin is capable of cross-linking actin microfilaments into bundles or sheets, we suggest that it is a principal element for the variant cell shape and cytoskeletal morphology observed during altered spreading behavior of late passage human diploid fibroblasts.

The total glycine pool in Zea mays L. Mo17xB73 leaf discs was measured after steady state photosynthesis in 50%, 21% and 1% O(2). The glycine pool was a function of O(2) concentration; it was largest in 50% O(2) and smallest in 1% O(2). Incubation of discs with methyl hydroxybutynoic acid in 21% O(2) in the light caused an accumulation of carbon in glycolate. This accumulation was O(2) sensitive, as subsequent photosynthetic periods in 50%, 21%, and 1% O(2) resulted in the largest glycolate pool in 50% O(2) and the smallest in 1% O(2). At the same time, the O(2)-dependent increase in the glycine pool was eliminated. After untreated leaf discs reached steady state photosynthesis in 21% O(2), measurements made subsequently in darkness, or in 1% O(2) in the light, showed that the glycine pool decreased. On the basis of these results, we conclude that a major portion of the total glycine pool in corn is an intermediate in the photorespiratory glycolate pathway. Considering both the rate of decay of the glycine pool in the dark and the rate of decay of the glycine pool after changing from 21% to 1% O(2), we conclude that this glycine pool is turning over slowly.

The cytoskeletons of early and late passage IMR-90 human diploid fibroblasts have been directly imaged in replicas of Triton X-100 extracted cells during spreading following reseeding. All cells from both young and sensescent cultures exhibit a cytoskeletal network of actin microfilaments, intermediate (10 nm) filaments, microtubules, and interconnecting thin filaments (6-8 nm in diameter) which do not interact with heavy meromyosin. Early passage cells assemble linear aggregates of actin filaments within 1 h of spreading. By 4 h of incubation, these bundles establish a structural bond with the cell membrane which results in resistance by the plasmalemma to detergent extraction at these sites. Furthermore, these membrane regions are associated with developing stress fibers of well-spread cells. In contrast, late passage cells exhibit slower spreading which correlates with a retarded assembly of actin bundles. In addition, by 8 h of spreading, cells of older cultures do not exhibit the regions of membrane-actin interaction which impart detergent resistance to the plasmalemma. We conclude that the ability to reassemble actin-actin and actin-membrane association during cell spreading is reduced with increased serial subcultivation of cells.

Examination of whole mount cells in the transmission electron microscope has been useful in studies of cellular architecture. The common technique is to grow cells directly on formvar-coated, gold grids for direct observation through a cell. We report a technique for obtaining whole mount preparations which requires neither fragile formvar films nor expensive, gold grids. Cells are grown on palladium-coated coverslips and processed for electron microscopy. The cells and the palladium substrate are separated from the coverslip. The cell-palladium complex is then picked up on copper grids as in thin section processing. We compare images of the cytoskeleton using our technique with images using previously described techniques and present preliminary observations of contracting cell models. Such contractions would tear formvar films if attempted on cells grown in the conventional manner for whole mount examination. Our technique allows cells to contract without tearing the underlying substrate.