Assembly of long chain phosphatidylcholines at a liquid-liquid interface.

The molecular-level organization of mixed and pure saturated symmetric chain 1,2-diacyl-sn-glycero-3-phosphocholines (PCs) adsorbed at a carbon tetrachloride-aqueous interface is explored by probing the hydrocarbon chain conformation within the adsorbed layer. PCs of the chain lengths found most frequently in biological systems, which in pure form are seen to form either very well-ordered or disordered layers, are observed in these studies to assemble into interfacial layers ranging from disordered to ordered states when mixed in various proportions. Independently, while C(16) and shorter chain PCs tend to form disordered layers, a strong increase in ordering is observed for C(18) and longer chain PCs in which the hydrocarbon chains are found to be primarily in an all trans conformation. Pure C(17)-PCs adsorbed at the interface produce layers with an intermediate degree of chain ordering. The ability to tune interfacial layer properties in mixed systems as a function of molecular composition, including PC chain length as demonstrated here, is an important mechanism by which surface characteristics of oil-water emulsion systems can be controlled both in vivo and in numerous commercial applications.

Expression of a retroposon-like sequence upstream of the putative Trypanosoma brucei variant surface glycoprotein gene expression site promoter.

We have cloned the region spanning the putative promoter from two variant surface glycoprotein gene expression sites that are at each end of chromosome M4 of Trypanosoma brucei IsTat 7. Both expression sites contain a retroposon-like sequence (ESR) pseudogene whose 3' end is approximately 30 bp upstream of the putative expression site promoter. The ESRs from both expression sites share considerable sequence homology and are related to LINE-like elements, especially the T. brucei ingi retroposon. Other ESRs are located on large, but not intermediate or mini-, chromosomes in the IsTaR 1 serodeme, and the total copy number is 10 to 20, similar to that estimated for variant surface glycoprotein expression sites. No DNA rearrangements in the vicinity of the ESR and putative expression site promoter were detected following antigenic switches in the IsTaR 1 serodeme. ESR transcripts are present in bloodstream, but not procyclic, forms. Variation in transcript size and sequence between bloodstream variant antigenic types implies that only the ESR from the active expression site is transcribed. This pattern of expression reflects that of sequences downstream of the putative expression site promoter, suggesting that the region of coordinately controlled expression extends upstream of this promoter.

Hepatic protein tyrosine phosphatases in the rat.

Regulation of cell growth and metabolism by protein tyrosine phosphorylation involves dephosphorylation via the action of protein tyrosine phosphatases (PTPases). We have characterized the membrane PTPases in rat liver, monitoring their activity by measuring the dephosphorylation of P-Tyr-reduced, carboxyamidomethylated and maleylated lysozyme (P-Tyr-RCML) and P-Tyr-myelin basic protein (P-Tyr-MBP). Separation of membrane PTPases by poly (L-lysine) chromatography yielded three peaks of PTPase, termed I, II and III. PTPases I and II were most active with P-Tyr-RCML, whereas PTPase III showed greater activity with P-Tyr-MBP than with P-Tyr-RCML (ratio of activities 4:1). Separation of membrane proteins by gel-filtration chromatography yielded two peaks of activity. Based on substrate specificity, sensitivity to inhibitors and requirement for thiol-containing compounds, the activity peak with an Mr of approximately 400,000 corresponded to PTPase III, whereas that with an Mr of approx. 40,000 contained PTPases I and II. All three PTPases dephosphorylated epidermal growth factor receptors and insulin receptors, but only PTPases I and II were active with P-Tyr-asialoglycoprotein receptors. Although none of the above characteristics distinguished between PTPases I and II, only PTPase I reacted in a Western immunoblotting procedure with anti-peptide antibodies directed towards human placental PTPase. We conclude that the membrane fraction from rat liver contains at least three distinct PTPases.

The trypanosome leucine repeat gene in the variant surface glycoprotein expression site encodes a putative metal-binding domain and a region resembling protein-binding domains of yeast, Drosophila, and mammalian proteins.

We have identified a new variant surface glycoprotein expression site-associated gene (ESAG) in Trypanosoma brucei, the trypanosome leucine repeat (T-LR) gene. Like most other ESAGs, it is expressed in a life cycle stage-specific manner. The N-terminal 20% of the predicted T-LR protein resembles the metal-binding domains of nucleic acid-binding proteins. The remainder is composed of leucine-rich repeats that are characteristic of protein-binding domains found in a variety of other eucaryote proteins. This is the first report of leucine-rich repeats and potential nucleic acid-binding domains on the same protein. The T-LR gene is adjacent to ESAG 4, which has homology to the catalytic domain of adenylate cyclase. This is intriguing, since yeast adenylate cyclase has a leucine-rich repeat regulatory domain. The leucine-rich repeat and putative metal-binding domains suggest a possible regulatory role that may involve adenylate cyclase activity or nucleic acid binding.

A retroposon in the 5' flank of a Trypanosoma brucei VSG gene lacks insertional terminal repeats.

A retroposon-like repeated sequence, ingi, occurs in high copy number in the genome of Trypanosoma brucei brucei. An ingi is present in the 5' flank of the 5C gene, an intrachromosomal IsTat 1.5 variant surface glycoprotein (VSG) gene family member. The 5' end of the ingi is located 22 bp upstream of the putative VSG start codon and the ingi open reading frame is in the opposite orientation to that of the VSG gene. The termini of the ingi are not flanked by a short repeat sequence and there are no sequences upstream of the ingi insertion which are homologous to the 5' flanking sequence of other 5 VSG gene family members. Thus, it appears that recombination and/or gene conversion between two ingi sequences may have eliminated the original 5C gene flanking sequence. Similar events may also have occurred with all but one previously reported ingi.

An extensively edited mitochondrial transcript in kinetoplastids encodes a protein homologous to ATPase subunit 6.

The mitochondrial MURF4 gene of T. brucei has pronounced G versus C strand bias and heterogeneously sized transcripts, characteristic of genes encoding extensively edited transcripts. We find that MURF4 transcripts of T. brucei are extensively edited throughout by the addition and deletion of numerous uridines, creating potential initiation and termination codons and a continuous open reading frame. A potential guide RNA sequence occurs in a minicircle between inverted repeats. The 5' region of L. tarentolae MURF4 transcripts is also extensively edited, with a created initiation codon. The predicted MURF4 amino acid sequences have homology to those of mitochondrial ATP-ase subunit 6 genes from a variety of organisms. In addition, their hydropathic profiles are quite similar to those of other species. We therefore conclude that MURF4 encodes ATPase subunit 6 genes.

LR1: a candidate RNA virus of Leishmania.

Although viruses are important biological agents and useful molecular tools, little is known about the viruses of parasites. We report here the discovery of a candidate for an RNA virus in a kinetoplastid parasite. This potential virus, which we term LR1, is present in the promastigote form of the human pathogen Leishmania braziliensis guyanensis CUMC1-1A but not in 11 other stocks of Leishmania that were examined nor in Trypanosoma brucei. The candidate viral RNA has a size of approximately 6000 nucleotides, is single-stranded, and is largely, if not exclusively, located in the cytoplasm. No homologous LR1 sequences are detected in genomic DNA. The candidate viral RNA is associated with a spherical particle 32 nm in diameter that has a sedimentation coefficient of approximately 130 S. There is as yet no evident effect of this potential virus on parasite physiology or the disease caused by the parasite.

Regulation of the rpsU-dnaG-rpoD macromolecular synthesis operon and the initiation of DNA replication in Escherichia coli K-12.

We have fused the E. coli dnaG 5' regulatory region to the TcR structural gene in the promoter probe plasmids pPV33 and pKK175-6 to demonstrate that a promoter activity is located on a 250 bp SacII-HindIII restriction fragment and that a transcription terminator, previously reported by nucleotide sequence (Smiley et al. 1982) to immediately precede the dnaG gene, acts as such in vivo. We have determined the complete nucleotide sequence of this controlling region and report: 1) tandem promoters on the same SacII-HindIII restriction fragment which promotes tet expression in the gene fusion experiments, 2) an open reading frame between these promoters and the dnaG gene which is the rpsU (ribosomal protein S21) gene, 3) a sequence homologous to the lambda nut site, 4) a possible LexA protein binding site on one of the dnaG promoters. This places the order on the E. coli genetic map at 66 min in the clockwise direction as rpsU-dnaG-rpoD which are all contained in a single macromolecular synthesis operon. We postulate a model for regulation of the initiation of DNA replication based on antitermination of the rpsU-dnaG-rpoD operon.

Sequences of the Escherichia coli dnaG primase gene and regulation of its expression.

The nucleotide sequence of a cloned section of the Escherichia coli chromosome containing the dnaG primase gene [Lupski, J., Smiley, B., Blattner, F. & Godson, G. N. (1982) Mol. Gen, Genet. 185, 120--128] has been determined. The region coding for the dnaG primase has been identified by NH2-terminal and tryptic peptide amino acid analysis of the dnaG protein. The coding region is 1,740 base pairs long (580 amino acids) and is preceded by an unusual ribosome-binding site sequence (G-G-G-G). The dnaG gene is read in the same direction as the adjacent rpoD gene, but no obvious promoter sequences can be found for either gene within several hundred nucleotides upstream. Other unusual features of the dnaG gene that may explain the maintenance of its product at low copy number are the presence of a RNA polymerase terminator 31 nucleotides upstream from the ATG initiator codon and greater use (3--10 times) of certain condons that occur infrequently in other E. coli genes. The nucleotide sequence has also been correlated with data from transposon Tn5 insertional inactivation mapping.

Cloning and characterization of the Escherichia coli chromosomal region surrounding the dnaG Gene, with a correlated physical and genetic map of dnaG generated via transposon Tn5 mutagenesis.

A 24 kilobase pair region of the E. coli chromosome surrounding the dnaG gene has been cloned and characterized. A lambda phage library was constructed by ligating a Sau3A( decreases GATC) partial DNA digest of the entire E. coli chromosome into the lambda BamHI(G decreases GATCC) cloning vector charon 28. Partial digestion was performed to generate overlapping chromosomal fragments and to allow one to walk along the chromosome. This library was probed with a nick-translated plasmid (pRRBl) containing the rpoD gene, which maps adjacent to dnaG at 66 min. Four bacteriophages: lambda 3, lambda 4, lambda 5, lambda 6 that hybridized to the probe were isolated from the 2,500 plaques screened. One phage recombinant lambda 4, was shown to contain the dnaG gene. Three recombinant plasmids containing dnaG: pGL444, pGL445, pBS105, were constructed via subcloning of lambda 4 using different restriction of fragments. Plasmids pGL444 and pBS10 5 were subjected to transposon Tn5 mutagenesis and 88 Tn5 inserts into the cloned region were isolated. The location of the Tn5 inserts were mapped by restriction enzyme analysis of the plasmids and the insertion mutations were checked for ability to complement of dnaGts chromosomal marker at nonpermissive 40 degrees C. In this manner a correlated physical and genetic map of dnaG was determined. A large number of Tn5 inserts map to a specific 900 b.p. region which we propose may be involved in the regulation of dnaG gene expression.

Heteroduplexes of phiX174 and G4 DNAs: orientation to genetic map and comparison with predictions from nucleotide sequences.

Heteroduplexes between the viral DNA of phiX174 and DNA from the replicative form (RF) of phage G4 were examined by electron microscopy. The single Eco RI site of G4-RF was utilized as a physical marker by preparing the heteroduplexes from the denatured, linear DNA obtained by restricting G4-RF with Eco RI endonuclease. Restriction fragments of phiX were used in a separate series of heteroduplexes to align the heteroduplex map and the G4 Eco RI site with the similar genetic maps of the two phages. The positions of the branch migrating junctions of recombinant phiX-G4 figure-8s, previously located only with respect to the G4-Eco RI site, have now been located with high proability within the gene A region of the two genomes. The degree of mismatch between the known nucleotide sequences of phi X and G4 accounts for positions of all of the regions of single-strandedness in the observed heteroduplexes, but unexplained discrepancies were also found.