Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide

A simple assay for O-glycosylated glycoproteins involving the liberation of oligosaccharides by beta-elimination with dilute alkali and the subsequent derivatization of the reducing end with 2-cyanoacetamide is reported. The method can be used to quantitate microgram amounts of mucin within 30 min. The assay is 30 times less sensitive to protein or N-linked glycoproteins and 100 times less sensitive to DNA than to the corresponding weight of canine tracheal mucin. Of the substances tested, only cesium chloride and potassium thiocyanate caused substantial interference, but in neither case was this sufficiently serious to prevent the method being used for monitoring mucin purification schemes utilizing these reagents. The coefficients of variation for replicate analyses of canine tracheal mucin (14.0, 5.0, and 2.0 micrograms) were 3.6, 6.5, and 12.3%, respectively.     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Delayed DNA methylation is an integral feature of DNA replication in mammalian cells

In the majority of sites of methylation in the DNA of mammalian cells, the symmetry of methylation is restored within a few minutes of the passage of a replication fork. However, it has been shown that daughter strand methylation in immortalised cell lines is delayed in a substantial minority of sites for up to several hours after replication. We report here the results of two new approaches to the determination of the functional significance of delayed DNA methylation in mammalian cells. Firstly, we demonstrate that normal, nontransformed cells (human peripheral lymphocytes in short-term primary culture) have comparable proportions of delayed DNA methylation to many immortalised cell lines, showing that delayed DNA methylation is not just a secondary consequence of abnormally high methionine requirements commonly observed in transformed cells and that delayed DNA methylation would be unlikely not to occur in vivo. Secondly, we have used 5-aza-2'-deoxycytidine (5azadCyd) to derive subclones of cells from the Chinese hamster ovary cell line which have stably hypomethylated DNA. In three of these subclones which had lost on average one fourth of the methylation sites from their genomes, the proportion of daughter strand methylation which was delayed after replication was reduced by less than 10%. If delayed DNA methylation were site-specific, this implies that of the order of twice the number of "immediate" methylation sites than delayed methylation sites had been lost from the genomes of these hypomethylated subclones. Thus, delayed DNA methylation is an integral part of the process whereby replicating mammalian cells maintain the pattern of methylation in their genomes. These observations are discussed in relation to the significance of delayed DNA methylation for the accurate maintenance of methylation patterns in the genome and the consequent implications for the possible role of methylated deoxycytidines in mammalian gene control.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

Three-dimensional structure of clathrin cages in ice.

We have collected tilt series of electron micrographs from unstained clathrin cages embedded in vitreous ice. From these micrographs we have generated three-dimensional reconstructions of individual hexagonal barrels, which show details of the internal structure. Four types of preparation have been examined: (i) coated vesicles; (ii) cages reassembled from clathrin heavy and light chains; (iii) reassembled cages treated with elastase to remove the light chains; and (iv) reassembled cages treated with trypsin to remove the light chains and the terminal domains of the clathrin heavy chains. In the intact and elastase-treated cages, the clathrin extends from the vertices into the interior of the polyhedron and forms an inner shell of material. Limited digestion with trypsin removes the inner shell, which indicates that this material corresponds to the terminal domains of the clathrin heavy chains.     

Location of the 100 kd-50 kd accessory proteins in clathrin coats.

We present a three-dimensional map of the clathrin coat of coated vesicles, generated from tilt series of electron micrographs of unstained specimens embedded in vitreous ice. We have examined native placental coated vesicles and coats reassembled from their purified constituents, namely clathrin triskelions and accessory proteins of approximate mol. wts 100 kd and 50 kd. Our results show that the accessory proteins contribute a further shell of density within the double shell of the clathrin cage, extending from the terminal domains of the clathrin to the membrane of the vesicle. The thickness of the complete coat is approximately 22 nm.     

Combination Chemotherapy using L-Asparaginase,Daunorubicin, and Cytosine Arabinoside in Adults with Acute Myelogenous Leukaemia

Cytosine arabinoside and daunorubicin used in an intensive intermittent regimen have been shown to be an effective combination for the induction of complete remissions in 14 out of 23 adult patients with acute myelogenous leukaemia. This gives an overall complete remission rate of 60%. A further patient had a good partial remission. The addition of L-asparaginase to the regimen has not increased the incidence of remission and there were more side effects in the L-asparaginasetreated group. Of the 10 patients treated with L-asparaginase in addition to cytosine arabinoside and daunorubicin, five achieved a complete remission. Of the 13 patients treated with cytosine arabinoside and daunorubicin without L-asparaginase, nine achieved a complete remission and one a good partial remission.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.