Search

Previous analyses of tso1 mutants revealed a loss of control of directional cellular expansion and coordination of growth of adjacent cells, and defects in karyokinesis and cytokinesis. We isolated TSO1 using a map-based approach, and show that it is a member of a family of at least three genes in Arabidopsis. Consistent with the mutant phenotype, TSO1 transcript was most abundant in flowers, where it accumulated to the highest levels in developing ovules and microspores. The putative TSO1 protein has two cysteine-rich regions that are similar to the CXC domains of a variety of proteins from plants and animals, including a class of kinesins involved in chromosome segregation, and enhancer of zeste-type proteins. Visualization of TSO1-fusion proteins indicated that TSO1 is a nuclear protein. The tso1 mutant phenotypes and the novelty of the TSO1 sequence suggest the existence of previously unknown participants in regulation of directional processes in eukaryotic cells.

The salt-sensitive phenotype of yeast cells deficient in the phosphoprotein phosphatase, calcineurin, was used to identify genes from the higher plant Arabidopsis thaliana that complement this phenotype. cDNA clones corresponding to two different sequences, designated STO (salt tolerance) and STZ (salt tolerance zinc finger), were found to increased tolerance of calcineurin mutants and of wild-type yeast to both Li+ and Na+ ions. STZ is related to Cys2/His2-type zinc-finger proteins found in higher plants, and STO is similar to the Arabidopsis CONSTANS protein in regions that may also be zinc fingers. Although neither protein has sequence similarity to any protein phosphatase, STO was able to at least partially compensate for all tested additional phenotypic effects of calcineurin deficiency, and STZ compensated for a subset of these effects. Salt tolerance produced by STZ appeared to be partially dependent on ENA1/PMR2, a P-type ATPase required for Li+ and Na+ efflux in yeast, whereas the effect of STO on salt tolerance was independent of ENA1/PMR2. STZ and STO were found to be expressed in Arabidopsis roots and leaves, whereas only STO message was detectable in flowers. An apparent increase in the level of STZ mRNA was observed in response NaCl exposure in Arabidopsis seedlings, but the level of STO mRNA was not altered by this treatment.

We have used the technique of DNA-mediated gene transfer to introduce dihydrofolate reductase genes into dihydrofolate reductase-deficient Chinese hamster ovary cells. The transferred sequences include: dihydrofolate reductase minigenes, dihydrofolate reductase cDNA clones, and genomic DNA from mouse cells with highly amplified dihydrofolate reductase genes. The hamster cells were capable of utilizing the murine transcription initiation sites, splice junctions, and polyadenylation sites in complete murine dihydrofolate reductase genes. Only a short region of 5'-flanking sequence (160 base pairs (bp] was sufficient for proper initiation of dihydrofolate reductase transcription. In contrast only those clones with all of the dihydrofolate reductase introns and extensive (greater than 6.5 kilobase pairs) 3'-flanking regions utilized the dihydrofolate reductase polyadenylylation sites. Four of nine clones tested regulate the transfected genes normally, synthesizing dihydrofolate reductase preferentially at the onset of S phase. Regulated genes include one which entirely lacks intervening sequences, two with fewer than 420 bp of 5'-flanking sequence, and two in which polyadenylylation occurs predominantly in the adjacent hamster sequences. Two of the five cell lines which do not exhibit normal regulation contain genes which appear to be transcribed primarily from promoters present in the flanking hamster DNA. The other genes which fail to regulate do not appear to differ significantly in the structure of their transcripts or protein products from genes which regulate normally. We conclude that only the coding sequences, a region of less than 340 bp of sequence 5' of the translation initiation codon, and a short region of 3'-flanking sequence are required for regulated production of dihydrofolate reductase. The regulation can be abolished, however, by unknown properties of the site of insertion.

Our understanding of the molecular mechanisms that regulate and integrate the temporal and spatial control of cell proliferation during organ ontogenesis, particularly of floral organs, continues to be primitive. The ovule, the progenitor of the seed, of Arabidopsis thaliana has been used to develop an effective model system for the analysis of plant organogenesis. A typical feature of a generalized ovule is the linear arrangement of at least three distinct elements, the funiculus, chalaza and nucellus, along a proximal-distal axis. This pattern is supposed to be established during the early proliferative phase of ovule development. We provide genetic evidence that the young ovule primordium indeed is a composite structure. Two genes, HUELLENLOS and AINTEGUMENTA have overlapping functions in the ovule and differentially control the formation of the central and proximal elements of the primordium. The results indicate that proximal-distal pattern formation in the Arabidopsis ovule takes place in a sequential fashion, starting from the distal end. Furthermore, we show that HUELLENLOS also regulates the initiation and/or maintenance of integument and embryo sac ontogenesis and interestingly prevents inappropriate cell death in the young ovule.

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase is an enzyme of the shikimate pathway which is located in the chloroplasts in higher plants. This enzyme is the target of the nonselective herbicide glyphosate. We have isolated and sequenced cDNA clones encoding EPSP synthase from petunia and tomato. The deduced amino acid sequences of the two enzyme precursors show 93% identity in the mature protein regions and 58% identity in the transit peptides. The sequences of the plant enzymes show significantly greater similarity to bacterial EPSP synthases than to fungal EPSP synthases. A genomic clone containing an EPSP synthase gene was isolated from a library of petunia DNA and was shown to contain seven intervening sequences. This gene is expressed approximately 25-fold higher in flower petals than in other organs of petunia. Transcription of this gene is initiated at multiple sites in petunia leaves and in a glyphosate-tolerant petunia cell line overproducing EPSP synthase mRNA. In petals, however, transcription of this gene is initiated almost entirely from only one of these sites. In contrast to petunia, the levels of EPSP synthase mRNA in different organs of tomato do not differ significantly.

We have constructed a number of abbreviated dihydrofolate reductase (DHFR) genes by using cloned mouse genomic and cDNA sequences. These genes contain 1.0 kilobase of 5' flanking genomic sequence and varying portions of the 3' non-coding region. Two of the genes contain the first two introns of the DHFR gene; the other three lack introns. Transfection of DHFR-deficient Chinese hamster ovary cells with any of these constructed genes results in cells with the DHFR+ phenotype. Treatment of the transfectants with methotrexate, a folate antagonist, leads to the emergence of methotrexate-resistant colonies which have amplified the transfected genes.

In a case of primary atypical pneumonia (blood grounp O), incomplete antibodies of the cold agglutinin type with a very wide thermal spectrum (35 to 37 C.) led to a severe immunopancytopenia in the form of severe hemolytic anemia, agranulocytosis, and thrombocytopenia. It is obvious that hemolysis was not induced by cold agglutinins which were active 1:1024 at 4 C. and only 1:32 at 22 C. The indirect cold antibodies which were probably responsible for the hemolysis which occurred were thermolabile (1:16 at 22 C., 1:8 at 37 C.) and could be demonstrated only in the antiglobulin plasma test. During the acute phase, too, a hemolysin of the Dacie type active for normal erythrocytes, but even more active for trypsinized cells, at 37 C. and at a pH of 6.8 and 7.6 could be demonstrated. The severe hemolysis could be stopped by ACTH treatment but the titer of the complete and incomplete cold antibodies showed no distinct change and the direct and indirect Coombs test remained positive for a long time. However, there was a distinct diminution of the thermal amplitude of the incomplete antibodies. A severe agranulocytosis (140 granulocytes) developed which could be improved each time by cortisone. However, permanent improvement resulted only after the disappearance of the incomplete antibodies one year later. A leukopenic factor could be demonstrated by transfusion of 300 cc. blood to a recipient of the same blood group producing a pronounced fall in the granulocytes of from 4200 to 910. Control transfusions from a normal inidividual to the same recipient showed no change and the same is true for the transfusion experiment performed one year later from the now cured patient to the same recipient. Leukocyte agglutination tests were negative, but it is presumed that agglutinins of the incomplete type may have been present. Opsonins could not be demonstrated. The thrombocytopenia never descended below 50,000. The bone marrow showed a distinct shift to the left, i.e. a predominance of unripe forms. On the basis of our previous experimental work (amidopyrine agranulocytosis) it is believed that the changes in the marrow are brought about by a depletion and exhaustion of the bone marrow due to the enormously increased peripheral destruction of the granulocytes by agglutination rather than to an actual inhibition of the marrow. The relation of this immunoleukopenia in our previous findings to the occurrence of leukocyte agglutination in agranulocytosis of allergic origin is discussed. Agranulocytosis and leukopenia may be produced on an immunologic basis in many other cases and thus some form of agranulocytosis may be related to the mechanism of erythrocyte destruction in acquired hemolytic anemia and to the agglutination of thrombocytes ins essential thrombocytopenia on an immunologic basis.