Fatty acid remodeling: a novel reaction sequence in the biosynthesis of trypanosome glycosyl phosphatidylinositol membrane anchors.

The trypanosome variant surface glycoprotein (VSG) is anchored to the plasma membrane via a glycosyl phosphatidylinositol (GPI). The GPI is synthesized as a precursor, glycolipid A, that is subsequently linked to the VSG polypeptide. The VSG anchor is unusual, compared with anchors in other cell types, in that its fatty acid moieties are exclusively myristic acid. To investigate the mechanism for myristate specificity we used a cell-free system for GPI biosynthesis. One product of this system, glycolipid A', is indistinguishable from glycolipid A except that its fatty acids are more hydrophobic than myristate. Glycolipid A' is converted to glycolipid A through highly specific fatty acid remodeling reactions involving deacylation and subsequent reacylation with myristate. Therefore, myristoylation occurs in the final phase of trypanosome GPI biosynthesis.     

In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication.

Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.     

Kinetoplast maxicircle DNA replication in Crithidia fasciculata and Trypanosoma brucei.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5',8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle theta structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from theta structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.     

Resistance of Escherichia coli to penicillins. IX. Genetics and physiology of class II ampicillin-resistant mutants that are galactose negative or sensitive to bacteriophage C21, or both

Ampicillin-resistant mutants of class II are determined by a doubling of chromosomally and episomally mediated ampicillin resistance on agar plates. Several mutants were isolated from a female as well as from an Hfr strain. The mutants differed from each other in various properties such as response to colicin E2 and sodium cholate, response to the phages T4 and C21, and fermentation of galactose. By conjugation and transduction experiments, it was shown that mutations in at least four loci gave the class II phenotype. The mutations were found to be in the galU gene, the ctr gene, and two new genes close to mtl denoted lpsA and lpsB. The carbohydrate compositions of the lipopolysaccharides of the mutants were investigated and found to be changed compared to the parent strains. GalU mutants lacked rhamnose and galactose and had 11% glucose compared to the parent strain. The lpsA mutant also lacked rhamnose and had only traces of galactose and 58% glucose, whereas the lpsB mutant contained 14% rhamnose, traces of galactose, and 81% glucose compared to the parent strain.     

Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine

Non-acetylated glucosamine is an unusual structural feature shared by all glycosyl phosphatidylinositol (GPI) lipids, including a variety of membrane anchors, the leishmanial lipophosphoglycan, and a mediator of insulin action. We proposed previously a pathway for biosynthesis of glycolipid A, the precursor of the GPI membrane anchor of the trypanosome variant surface glycoprotein (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800). In this paper we characterize in more detail the initial steps of GPI assembly. The first and committed step in the pathway is the transfer of GlcNAc, from UDP-GlcNAc, to endogenous phosphatidylinositol to form N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). The GlcNAc-PI is then efficiently deacetylated to form glucosaminyl phosphatidylinositol (GlcN-PI), the substrate for subsequent reactions en route to glycolipid A.     

Replication of kinetoplast DNA in Trypanosoma equiperdum. Minicircle H strand fragments which map at specific locations

The mitochondrial DNA of trypanosomes, kinetoplast DNA, is a network containing thousands of topologically interlocked minicircles. Minicircles are replicated as free molecules after being detached from the network. The minicircle L strand appears to be synthesized continuously and the H strand discontinuously. This paper describes properties of Trypanosoma equiperdum minicircle H strand fragments which could be Okazaki fragments. These fragments constitute a family of molecules of discrete sizes (ranging from about 70 to 1000 nucleotides) which map to specific locations. Three of the most prominent fragments, a 73-mer, 83-mer, and 138-mer, map at contiguous or overlapping sites. Based on their position relative to the initiation site for L strand synthesis, the 73-mer may be the first Okazaki fragment to be synthesized and either the 83-mer or the 138-mer may be the second. The 5' end of the 73-mer lies within a sequence, GGGCGT, found at a similar location in minicircles of all trypanosomatid species. During the maturation of free minicircles and after their reattachment to the networks there appears to be continued extension and ligation of the H strand fragments. However, the ligation of the 73-mer, 83-mer, and 138-mer to the rest of the H strand is delayed; their eventual ligation results in covalent closure of the minicircles.     

Positive identification of a measles virus cDNA clone encoding a region of the phosphoprotein

A measles virus-specific cDNA clone, C1-G, that was previously assigned as a hemagglutinin-specific clone has been reassigned as a phosphoprotein-specific clone. The nucleotide sequence of C1-G was used to deduce the amino acid sequence. A synthetic peptide was constructed from a portion of the deduced sequence, and antisera were prepared. The antibodies directed against this synthetic peptide specifically precipitated the phosphoprotein of measles virus and not the hemagglutinin.