Isolation of cDNA clones for human erythrocyte membrane sialoglycoproteins alpha and delta.

We have isolated almost full-length cDNA clones corresponding to human erythrocyte membrane sialoglycoproteins alpha (glycophorin A) and delta (glycophorin B). The predicted amino acid sequence of delta differs at two amino acid residues from the sequence determined by peptide sequencing. The sialoglycoprotein delta clone we have isolated contains an interrupting sequence within the region that gives rise to the cleaved N-terminal leader sequence for the protein and represents a product that is unlikely to be inserted into the erythrocyte membrane. Comparison of the cDNA sequences of alpha and delta shows very strong homology at the DNA level within the coding regions. The two mRNA sequences are closely related and differ by a number of clearly defined insertions and deletions.     

Monoclonal antibodies that recognize different membrane proteins that are deficient in Rhnull human erythrocytes. One group of antibodies reacts with a variety of cells and tissues whereas the other group is erythroid-specific.

1. Rhnull human erythrocytes lack all of the antigens of the Rh and LW blood group systems and have abnormal shape and an increased osmotic fragility. In this paper two murine monoclonal antibodies raised against intact human erythrocytes were used to investigate further the abnormalities in these cells. BRIC 125 reacts weakly with Rhnull erythrocytes and BRIC 69 does not react at all. The results showed that BRIC 125 reacts with a component of Mr 47,000-52,000 which has a substantial content of N-glycans. In contrast, BRIC 69 reacted with a band of Mr 31,000 together with a very diffuse band of Mr 35,000-52,000. Treatment of BRIC 69 immunoprecipitates with endoglycosidase F/peptidyl-N-glycosidase F resulted in the loss of both BRIC 69 reactive components and the appearance of a new band of Mr similar to that of the Rh(D) polypeptide. 2. BRIC 125 had a broad reactivity with cells in peripheral blood, whereas the reactivity of BRIC 69 was confined to erythrocytes. BRIC 125, but not BRIC 69, reacted with human kidney tissue and bound to endothelium in peritubular capillaries, arteries and veins as well as the epithelial tissue of distal tubules. BRIC 125 stained haemopoietic cells, foetal hepatocytes and megakaryocytes in foetal liver and sinusoidal cells, hepatocytes and portal tracts in adult liver. In contrast, BRIC 69 reactivity was confined to haemopoietic cells in foetal liver. The BRIC 125 epitope has a wide tissue distribution, suggesting the occurrence of a related group of polypeptides which have a general functional role on cell surfaces. 3. Rhnull erythrocytes are deficient in at least four different membrane polypeptides.     

Additions to the silicified upper devonian/lower carboniferous flora from ballyheigue,Ireland

Continuing study of the uppermost Devonian flora from Ballyheigue, County Kerry, Ireland (Tn1a-Tn1b) has resulted in the addition of four taxa. Two plants, Tristichia sp. and cf. Tetrastichia bupatides, are described for the first time from Ireland. Tetrastichia also occurs in Scotland and Tristichia has been described from Scotland and France. New genera include Kerryoxylon hexalobatum gen. et sp. nov. (called “Plant A” in a previous paper), a probable lyginopterid pteridosperm, and Rhizoxylon ambiguum gen. et sp. nov., a new organ genus for seed fern roots similar to Kaloxylon.     

Immunologic differentiation between E. coli and CHO cell-derived recombinant and natural human beta-interferons

The products of the human IFN-beta gene expressed in E. coli, Chinese hamster ovary (CHO) cells, and human fibroblasts appear similar when purified on a monoclonal antibody column and analyzed by reverse-phase HPLC, indicating little difference in their hydrophobic nature. SDS-PAGE differentiates E. coli-rHuIFN-beta ser (Mr = 17,000) from CHO-rHuIFN-beta and HuIFN-beta (Mr = 23,000), with glycosylation accounting for 26% of the apparent m.w. of the latter two proteins. CHO-rHuIFN-beta is preferentially neutralized by mouse monoclonal and monospecific rabbit polyclonal anti-HuIFN-beta antibodies, whereas E. coli-rHuIFN-beta ser is preferentially neutralized by goat polyclonal anti-E. coli-rHuIFN-beta antibodies. Adsorption measurements by a sensitive radioimmunoassay indicate that the binding of the three proteins to anti-HuIFN-beta antibodies is similar. The results show that all three molecules can be differentiated by the heteroclitic cross-reactivities of anti-HuIFN-beta and anti-E. coli-rHuIFN-beta antibodies to the antigens.     

The Distribution and Spreading of Rare Variants in the Histone Multigene Family of Drosophila Melanogaster

We surveyed the distribution of rare variant restriction sites within and among histone gene arrays of Drosophila melanogaster using restriction fragment length polymorphism (RFLP) analysis. Seventy-three naturally occurring arrays were digested with restriction enzymes that had no recognition sites in the published histone sequence. Of the arrays surveyed, 68.5% had at least two nonconsensus restriction sites present as indicated by the presence of a small band or bands on the autoradiographs. These bands were almost always the length of a single repeat in the histone multigene family or a multiple of this length. In arrays with more than one band, intensity of the bands almost always decreased with increasing size. This shows that within these arrays variant restriction sites were predominantly located on adjacent repeats. If these bands are caused by spreading of variant sites, as is most likely, then variants spread along the array as an inverse function of distance. Overall, if a sequence spread it had a 92% probability of ending up in its nearest neighbor. This pattern may result from the noncontiguous nature of the histone family.     

Purification and characterization of the Saccharomyces cerevisiae BGL2 gene product, a cell wall endo-beta-1,3-glucanase.

One of the major proteins of the Saccharomyces cerevisiae cell wall, a beta-glucanase (BGL2 gene product), has been isolated and purified to homogeneity under conditions for preserving enzyme activity. The study of enzyme properties of the protein revealed that it is an endo-beta-1,3-glucanase and not an exoglucanase as reported previously (F. Klebl and W. Tanner, J. Bacteriol. 171:6259-6264, 1989). The examination of the glucanase structure showed that the lower apparent molecular mass of the protein (29 kDa) compared with what was calculated from the amino acid sequence of the enzyme (33.5 kDa) is due to anomalous migration in sodium dodecyl sulfate gels and not to posttranslational processing of the polypeptide chain. Of two potential N glycosylation sites at Asn-202 and Asn-284, only the latter site is glycosylated. The overproduction of the beta-glucanase from the high-copy-number plasmid brought about a significant decrease in the growth rate of transformed yeast cells.     

The PMT gene family: protein O-glycosylation in Saccharomyces cerevisiae is vital.

The transfer of mannose to seryl and threonyl residues of secretory proteins is catalyzed by a family of protein mannosyltransferases coded for by seven genes (PMT1-7). Mannose dolichylphosphate is the sugar donor of the reaction, which is localized at the endoplasmic reticulum. By gene disruption and crosses all single, double and triple mutants of genes PMT1-4 were constructed. Two of the double and three of the triple mutants were not able to grow under normal conditions; three of these mutants could grow, however, when osmotically stabilized. The various mutants were extensively characterized concerning growth, morphology and their sensitivity to killer toxin K1, caffeine and calcofluor white. O-Mannosylation of gp115/Gas1p was affected only in pmt4 mutants, whereas glycosylation of chitinase was mainly affected in pmt1 and pmt2 mutants. The results show that protein O-glycosylation is essential for cell wall rigidity and cell integrity and that this protein modification, therefore, is vital for Saccharomyces cerevisiae.     

7-Mercaptoheptanoylthreonine phosphate substitutes for heat-stable factor (mobile factor) for growth of Methanomicrobium mobile.

Methanomicrobium mobile requires a heat-stable factor present in ruminal fluid and in boiled cell extract from Methanobacterium thermoautotrophicum for growth. By comparing the growth of M. mobile with boiled cell extract with that observed with various methanogenic cofactors, we found that 7-mercaptoheptanoylthreonine phosphate (HS-HTP) supported sustained growth of M. mobile, at an optimal concentration of 100 microM. No derivatives or possible biosynthetic precursors of HS-HTP could replace HS-HTP as the sole source of growth factor. Results suggest that the growth requirement might be satisfied by 7-mercaptoheptanoic acid plus a second, unidentified heat-stable factor.     

Desulfomonile tiedjei gen. nov. and sp. nov., a novel anaerobic,dehalogenating, sulfate-reducing bacterium

An anaerobic, dehalogenating, sulfate-reducing bacterium, strain DCB-1, is described and nutritionally characterized. The bacterium is a Gram-negative, nonmotile, non-sporeforming large rod with an unusual morphological feature which resembles a collar. The microorganism reductively dehalogenates meta substituted halobenzoates and also reduces sulfate, sulfite and thiosulfate as electron acceptors. The bacterium requires nicotinamide, 1,4-naphthoquinone and thiamine for optimal growth in a defined medium. The microorganism can grow autotrophically on H₂:CO₂ with sulfate or thiosulfate as terminal electron acceptors. It can also grow heterotrophically with pyruvate, several methoxybenzoates, formate plus sulfate or benzoate plus sulfate. It ferments pyruvate to acetate and lactate in the absence of other electron acceptors. The bacterium is inhibited by MoO _inf4 ^sup2- or SeO _inf4 ^sup2- as well as tetracycline, chloramphenicol, kanamycin or streptomycin. Cytochrome c₃ and desulfoviridin have been purified from cells grown in defined medium. 16S rRNA sequence analysis indicates the organism is a new genus of sulfate-reducing bacteria in the delta subdivision of the class Proteobacteria. We propose that the strain be named Desulfomonile tiedjei.Non-standard abbreviations PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - MES 2-[N-morpholino]ethanesulfonic acid - TES N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid - HQNO 2-N-heptyl-4-hydroxy-quinoline-N-oxide - CCCP carbonyl-cyanide-m-chlorophenylhydrazine - CM carboxymethyl