Further progress towards a catalogue of all Arabidopsis genes: analysis of a set of 5000 non-redundant ESTs

Nearly 7000 Arabidopsis thaliana -expressed sequence tags (ESTs) from 10 cDNA libraries have been sequenced, of which almost 5000 non-redundant tags have been submitted to the EMBL data bank. The quality of the cDNA libraries used is analysed. Similarity searches in international protein data banks have allowed the detection of significant similarities to a wide range of proteins from many organisms. Alignment with ESTs from the rice systematic sequencing project has allowed the detection of amino acid motifs which are conserved between the two organisms, thus identifying tags to genes encoding highly conserved proteins. These genes are candidates for a common framework in genome mapping projects in different plants.     

Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts.

Ran is a small GTPase that is required for protein import, mRNA export, and the maintenance of nuclear structures. To gain a better understanding of Ran's role in the nucleus, we have sought to use Xenopus egg extracts for the purification and characterization of proteins from egg extracts bound with a high affinity to a glutathione-S-transferase-Ran fusion protein (GST-Ran). We found that GST-Ran associates specifically with at least 10 extract proteins. We determined the identifies of six Ran-interacting proteins (Rips), and found that they include RanBP2/Nup358, Nup153, Importin beta, hsc70, RCC1, and RanBP1. On the basis of peptide sequence, a seventh Rip (p88) seems to be similar but not identical to Fug1/RanGAP1, the mammalian Ran-GTPase-activating protein. Gel filtration analysis of endogenous extract proteins suggests that Importin beta acts as a primary GTP-Ran effector. Both Ran and Importin beta are coimmunoprecipitated by anti-p340RanBP2 antibodies in the presence of nonhydrolyzable GTP analogues, suggesting that Ran-Importin beta complexes interact with p340RanBP2. Two other Rips, p18 and p88, are coprecipitated with p340RanBP2 in a nucleotide-independent manner. Analysis of the Ran-GTPase pathway in Xenopus extracts allows the examination of interactions between Ran-associated proteins under conditions that resemble in vivo conditions more closely than in assays with purified components, and it thereby allows additional insights into the molecular mechanism of nuclear transport.     

Inhibition of muscle force by vanadate.

Vanadate (Vi), an analogue of inorganic phosphate (Pi), is known to bind tightly with a long half life to the myosin MgATPase site, producing a complex which inhibits force. Both of these ligands bind to an actin.myosin.ADP state that follows the release of Pi in the enzymatic cycle, and their effects on muscle fibers and proteins in solution provide information on the properties of this state. The inhibition of active force generation began to occur at a [Vi] of 5 microM and was 90% complete at a [Vi] of 1 mM. Hill plots of the inhibition of force by Vi approximated that expected for a simple binding isotherm. Similar plots were obtained at both 25 degrees C and 5 degrees C. A simple binding isotherm is not expected to occur in a muscle fiber where steric constraints imposed by the intact filaments should introduce more complexity into the energetics of ligand binding. The inhibition of MgATPase activity for acto-subfragment-1 to 50% of controls occurred at a [Vi] which was only 20-fold higher than that required to inhibit force generation in fibers to the same level. Some models of actomyosin interactions would predict that the range of [Vi] required for complete force inhibition in fibers and the difference in the [Vi] required for inhibition in fibers and of myosin in solution would both be much larger.     

CENP-B binds a novel centromeric sequence in the Asian mouse Mus caroli.

Minor satellite DNA, found at Mus musculus centromeres, is not present in the genome of the Asian mouse Mus caroli. This repetitive sequence family is speculated to have a role in centromere function by providing an array of binding sites for the centromere-associated protein CENP-B. The apparent absence of CENP-B binding sites in the M. caroli genome poses a major challenge to this hypothesis. Here we describe two abundant satellite DNA sequences present at M. caroli centromeres. These satellites are organized as tandem repeat arrays, over 1 Mb in size, of either 60- or 79-bp monomers. All autosomes carry both satellites and small amounts of a sequence related to the M. musculus major satellite. The Y chromosome contains small amounts of both major satellite and the 60-bp satellite, whereas the X chromosome carries only major satellite sequences. M. caroli chromosomes segregate in M. caroli x M. musculus interspecific hybrid cell lines, indicating that the two sets of chromosomes can interact with the same mitotic spindle. Using a polyclonal CENP-B antiserum, we demonstrate that M. caroli centromeres can bind murine CENP-B in such an interspecific cell line, despite the absence of canonical 17-bp CENP-B binding sites in the M. caroli genome. Sequence analysis of the 79-bp M. caroli satellite reveals a 17-bp motif that contains all nine bases previously shown to be necessary for in vitro binding of CENP-B. This M. caroli motif binds CENP-B from HeLa cell nuclear extract in vitro, as indicated by gel mobility shift analysis. We therefore suggest that this motif also causes CENP-B to associate with M. caroli centromeres in vivo. Despite the sequence differences, M. caroli presents a third, novel mammalian centromeric sequence producing an array of binding sites for CENP-B.     

Identification of members of gene families in Arabidopsis thaliana by contig construction from partial cDNA sequences: 106 genes encoding 50 cytoplasmic ribosomal proteins

Partial cDNA sequencing to obtain expressed sequence tags (ESTs) has led to the identification of tags to about 8000 of the estimated 20 000 genes in Arabidopsis thaliana . This figure represents four to five times the number of complete coding sequences from this organism available in international databases. In contrast to mammals, many proteins are encoded by multigene families in A. thaliana . Using ribosomal protein gene families as an example, it is possible to construct relatively long sequences from overlapping ESTs which are of sufficiently high quality to be able to unambiguously identify tags to individual members of multigene families, even when the sequences are highly conserved. A total of 106 genes encoding 50 different cytoplasmic ribosomal protein types have been identified, most proteins being encoded by at least two and up to four genes. Coding sequences of members of individual gene families are almost always very highly conserved and derived amino acid sequences are almost, if not completely, identical in the vast majority of cases. Sequence divergence is observed in untranslated regions which allows the definition of gene-specific probes. The method can be used to construct high-quality tags to any protein.     

Breeding success and chronology of Wood Storks Mycteria americana in northern and central Florida, U.S.A.

The breeding success and chronology of Wood Storks Mycteria americana were studied at eight colonies in northern and central Florida during 1981–1985. Mean ± s.d. clutch size for all colony-years was 3.07 ± 0.56 (n = 2694 nests), with three-egg clutches (72%) most frequent. Mean clutch size among all colonies and years ranged from 2.73 ± 0.55 to 3.41 ± 0.61. Many colonies exhibited significant negative trends in clutch size with, hatching date because of a proportional decrease in four-egg clutches later in the season. Mean colony clutch size was not correlated with nest numbers, nesting density or mean hatching date within most years. Mean ± s.d. number of fledglings for all colonies and years was 1.29 ± 1.16 fledglings per nest (n = 2812 nests). Mean annual fledging rates in colonies ranged from 0 (colony failed) to 2.66 fledglings per nest. Most breeding failure occurred prior to egg hatching, and the second highest mortality occurred between hatching and 2 weeks of age. Four-egg clutches fledged more storks than three-egg clutches, which in turn were more successful than two-egg clutches. However, all clutch sizes showed similar fledgling per egg rates. The seasonal decline in productivity was associated proportionally with smaller clutch sizes later in the breeding season. An increase in mean hatching date was correlated with an increase in latitude. There was greater within-year breeding synchrony among colonies than interyear breeding synchrony within each colony. Breeding synchrony was not correlated with mean hatching date, latitude, longitude, nest numbers or nesting density.     

Response of compressed skinned skeletal muscle fibers to conditions that simulate fatigue

Myburgh, Kathryn H., and Roger Cooke. Response ofcompressed skinned skeletal muscle fibers to conditions that simulate fatigue. J. Appl. Physiol. 82(4):1297-1304, 1997.During fatigue, muscles become weaker, slower,and more economical at producing tension. Studies of skinned musclefibers can explain some but not all of these effects, and, inparticular, they are less economical in conditions that simulatefatigue. We investigated three factors that may contribute to thedifferent behavior of skinned fibers. 1) Skinned fibers have increasedmyofilament lattice spacing, which is reversible by osmoticcompression. 2) A myosin subunit becomes phosphorylated during fatigue.3) Inosine 5-monophosphate (IMP) accumulates during fatigue. We tested the response ofphosphorylated and unphosphorylated single skinned fibers (isometrictension, contraction velocity, and adenosinetriphosphatase activity) to changes in lattice spacing (0-5% dextran) and IMP (0-5 mM)in the presence of altered concentrations ofP_i (3-25 mM),H⁺ (pH 7-6.2), and ADP(0-5 mM). The response of maximally activated skinned fibers tothe direct metabolites of ATP hydrolysis is not altered by osmoticcompression, phosphorylating myosin subunits, or increasing IMPconcentration. These factors, therefore, do not explain the discrepancybetween intact and skinned fibers during fatigue.

     

The role of calcium in luteinizing hormone-releasing hormone agonist (ICI 118630)-stimulated steroidogenesis in rat Leydig cells.

The luteinizing hormone-releasing hormone (LHRH) agonist ICI 118630 was found to increase testosterone production in purified rat testis Leydig cells in a concentration- and time-dependent manner, but no consistent changes in cyclic AMP levels were detectable. The stimulation of steroidogenesis by LHRH agonist was found to be dependent on the concentration of Ca2+ in the incubation medium; at least 1 mM was required. The calcium ionophore A23187 mimicked the effects of the LHRH agonist on steroidogenesis, and addition of both compounds together did not further increase testosterone production. The calcium ionophore caused a small increase in cyclic AMP which was independent of the concentration of the ionophore and of the calcium concentrations. The evidence obtained in this study indicates that LHRH agonist-stimulated steroidogenesis in rat testis Leydig cells is primarily mediated by calcium and not cyclic AMP.     

A New Malaria Parasite Plasmodium (Sauramoeba) heischi in Skinks (Mabuya striata) from Nairobi, with a Brief Discussion of the Distribution of Malaria Parasites in the Family Scincidae

ABSTRACT. A new species of malaria parasite, Plasmodium (Sauramoeba) heischi , is described from African skinks (Mabuya striata). Eleven individuals of 90 specimens collected in Nairobi were found to be infected. The new parasite is a large species, characterized by spindle-shaped gametocytes, the female often with a subterminal nucleus. The schizonts produce up to 65 nuclei and cause great hypertrophy and distortion of the host cell. Although similar to P. (Sauramoeba) giganteum in dimensions and merozoite numbers, P. heischi is easily distinguished by gametocyte and schizont shapes.     

Toward a complete linkage map of the human X chromosome: regional assignment of 16 cloned single-copy DNA sequences employing a panel of somatic cell hybrids.

Closely linked restriction fragment length polymorphisms (RFLPs) are potentially useful as diagnostic markers of genetic defects, and, in principle, RFLPs can be employed to construct a complete linkage map of the human genome. On the X chromosome, linkage studies are particularly rewarding because in man more than 120 X-linked genes are known. Thus, it is probable that each X-specific RFLP will be of use as a genetic marker of one or several X-linked disorders. To facilitate the search for closely linked RFLPs, we have regionally assigned 16 cloned DNA sequences to various portions of the human X chromosome, employing a large panel of somatic cell hybrids. These probes have been used to correlate genetic and physical distances on Xp, and it can be extrapolated from these data that the number and distribution of available Xq sequences will also suffice to span the long arm of the X chromosome.