Specific expression of a myeloid-associated CMP-NeuAc:Gal beta 1----3GalNAc alpha-R alpha 2----3-sialyltransferase and the sialyl-X determinant in myeloid human-mouse cell hybrids containing human chromosome 11.

Human-murine myeloid somatic cell hybrids were assayed for the expression of the myeloid-associated sialyl-X determinant. This determinant is expressed at the surface of hybrid cells containing human chromosome 11, but its expression could not be correlated with the presence of the sialyltransferase which is involved in the sialyl-X synthesis. The sialyl-X determinant, however, is simultaneously expressed with another alpha 2----3-sialyltransferase activity, which is involved in the sialylation of the O-linked Gal beta 1----3GalNAc alpha-R core structure. Chromosomal analyses and enzymatic data suggest that human chromosome 11 is involved in the expression of both the sialyl-X antigen and this alpha 2----3-sialyltransferase.

A GDP-fucose:[Gal beta 1----4]GlcNAc alpha 1----3-fucosyltransferase activity is correlated with the presence of human chromosome 11 and the expression of the Lex, Ley, and sialyl-Lex antigens in human-mouse cell hybrids.

By fusion of human leukocytes and cells of the murine myeloid cell line WEHI-TG, we produced human-mouse myeloid cell hybrids. Hybrids which contain human chromosome 11 have been demonstrated to express the myeloid-associated carbohydrate antigen Lex (Geurts van Kessel, A. H. M., Tetteroo, P. A. T., Von dem Borne, A. E. G. Kr., Hagemeijer, A., and Bootsma, D. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3748-3752). In this paper, we report that the hybrids that contain chromosome 11 also expressed the Lex-related antigens Ley and sialyl-Lex. Glycosyltransferase activities were measured in a panel of six such hybrid cell lines, and the correlation to antigen expression and to the presence of human chromosomes was investigated. GDP-fucose:[Gal beta 1----4]GlcNAc alpha 1----3-fucosyltransferase activity in the hybrids tested correlated with the expression of Lex, Ley, and sialyl-Lex and with the occurrence of chromosome 11. No such correlation was found for several other glycosyltransferases involved in the synthesis of these antigens. These findings suggest that the gene for alpha 3-fucosyltransferase is located on chromosome 11 and that it is through the activity of this enzyme that the expression of Lex, Ley, and sialyl-Lex in human myeloid cells is regulated.

Biosynthesis of disialylated beta-D-galactopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopy ran osyl oligosaccharide chains. Identification of a beta-D-galactoside alpha-(2----3)- and a 2-acetamido-2-deoxy-beta-D-glucoside alpha-(2----6)-sialyltransferase in regenerating rat liver and other tissues.

Regenerating rat liver microsomes contain a beta-D-galactoside alpha-(2----3)- and a 2-acetamido-2-deoxy-beta-D-glucoside alpha-(2----6)-sialyltransferase that are involved in the synthesis of the terminal alpha-NeuAc-(2----3)-beta-D-Galp-(1----3)-alpha-[NeuAc-(2----6)]-beta- D-GlcpNAc-(1----R) group occurring in human milk oligosaccharides and the glycan chains of several N-glycoproteins. Analysis by liquid chromatography and methylation of the products of sialylation obtained when lacto-N-tetraose [beta-D-Galp-(1----3)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4) -D-Glc] was used as a substrate in the incubations in vitro indicated that the disialylated sequence is formed for greater than 95% through the tetrasaccharide alpha-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----3)-beta-D-G al- (1----4)-D-Glc as one of two possible intermediates. This indicates that in the synthesis of the disialylated sequence the alpha-(2----3)- and the alpha-(2----6)-sialyltransferase act in a highly preferred order in which the alpha-(2----3) enzyme acts first. This order is imposed by the specificity of the alpha-(2----6)-sialyltransferase, which requires an alpha-NeuAc-(2----3)-beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----R) sequence for optimal activity, and shows very low and no activity with beta-D-Gal-(1----3)-beta-D-GlcNAc-(1----R) and beta-D-GlcNAc-(1----R) acceptor structures, respectively. Results obtained with normal rat, fetal calf, rabbit and human liver, and human placenta indicated that very similar or identical sialyltransferases occur in these tissues. It is suggested that these enzymes differ from the sialyltransferases that previously had been identified in fetal calf liver and human placenta.

Repeated sequences on mitochondrial DNA ofSpirodela oligorhiza.

Mitochondrial DNA ofSpirodela oligorhiza (duck weed) was analyzed with restriction enzymes. The genome size appears to be at least 250 kbp. Four different PstI fragments were cloned. These four clones contain a sequence which is reiterated about 100-fold on theSpirodela mitochondrial DNA. Hybridization analysis showed that a similar sequence is present onZea mays mitochondrial DNA, although much less reiterated here. The presence of these reiterated sequences might contribute to the physical heterogeneity of plant mitochondrial DNA.

The nucleotide sequences of the genes coding for tRNAArgUCU, tRNAArgACG and tRNAAsnGUU on Spirodela oligorhiza chloroplast DNA.

The nucleotide sequences of the genes coding for tRNAArgUCU, tRNAArgACG and tRNAAsnGUU on chloroplast DNA of Spirodela oligorhiza have been determined. All three genes are expressed. 5' Proximal to these genes sequences are found homologous to prokaryotic promoter sequences, which might be involved as transcriptional start motifs.

Protein synthesis during chloroplast development in Spirodela oligorhiza. Coordinated synthesis of chloroplast-encoded and nuclear-encoded subunits of ATPase and ribulose-1,5-bisphosphate carboxylase.

We have studied qualitative and quantitative changes of several parameters during chloroplast development in Spirodela oligorhiza (duckweed). On a dry weight basis, the amount of protein increases from 2.5% (w/w) in dark-grown to 7.8% (w/w) in light-grown fronds. At the same time the amount of starch drops from 50% to 27% (w/w). Using an immunochemical quantification method we have found that during greening of etiolated plants the amount of all subunits of the ATPase complex per frond increases 10-fold, whereas the level of the subunits of ribulose-1,5-biphosphate carboxylase increases 50-fold. Cytochrome f was found to be present in dark-grown Spirodela and the amount of this polypeptide per frond increases about 30-fold. The concentration of a polypeptide that possibly represents a cytochrome b6 subunit increases about 10-fold upon greening. The molar ratio of the CF1-beta and CF1-gamma subunits of the ATPase complex varies over 2-3, while in all stages of chloroplast development studied the molar ratio of the carboxylase subunits is about 1. As these values are in agreement with the stoichiometrical amounts in the native protein complexes, we conclude that the synthesis of CF1-beta and CF1-gamma, as well as the synthesis of the large and small carboxylase subunits, are strictly coordinated during chloroplast biogenesis in Spirodela oligorhiza.

Chloroplast DNAs of Spinacia, Petunia and Spirodela have a similar gene organization.

We have located the positions of the genes coding for the α, ß and ε subunits of the ATPase complex on Spirodela oligorhiza chloroplast DNA by means of heterologous hybridization with Spinacia cpDNA fragments.The overall cpDNA sequence organization of Petunia hybrida and Spirodela was compared. We hybridized well-characterized, cloned Spirodela cpDNA fragments with size fractionated Petunia cpDNA digested by Sall. It appears that the monocotyledonous Spirodela and the dicotyledonous Petunia cpDNA share a common sequence organization around their entire circumference. These observations, together with data reported in the literature, indicate a strikingly similar genetic organization of the chloroplast genome in widely divergent plants.