Isolation, purification, and amino acid composition of the tunicate hemocyte Thy-1 homolog

A serologic cross-reacting homolog to rodent Thy-1 glycoproteins has been isolated from hemocyte cell surfaces of the advanced invertebrate group of tunicates. The Thy-1.1 cross-reacting antigenic activity was followed during purification by inhibiting the binding of MRC OX7 monoclonal antibody to pure rat brain Thy-1 in a soluble phase radioimmunoassay. After solubilization in deoxycholate, tunicate hemocyte Thy-1.1 antigenic activity was purified by affinity chromatography using an MRC OX7 monoclonal antibody affinity column, followed by gel filtration. A 602-fold enrichment in the Thy-1.1 antigenic activity, with a yield of 55.6% compared to the starting crude membrane fraction, was obtained. The antigenic activity was associated with a single glycoprotein of molecular size of 3.1 nm and molecular weight estimated at 27,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% gels). Amino acid composition of the purified molecule was compared by the S delta Q index of differences in composition to mammalian and non-mammalian Thy-1 glycoproteins, Ig, major histocompatibility complex-encoded polypeptides, beta 2-microglobulin, and other recognition molecules. With this parameter, the tunicate hemocyte Thy-1 homology revealed significant relatedness to avian and mammalian Thy-1 molecules and was interestingly more related to mu chains of primitive vertebrates and to HLA class I and II encoded polypeptides than to Thy-1 molecules of higher vertebrates. Based upon these observations, the tunicate hemocyte Thy-1 homolog seems to represent an ancestral Thy-1 molecule which, in structural terms, may represent an invertebrate member of the Ig superfamily.     

Replication and resolution of cloned poxvirus telomeres in vivo generates linear minichromosomes with intact viral hairpin termini.

The covalently closed terminal hairpins of the linear duplex-DNA genomes of the orthopoxvirus vaccinia and the leporipoxvirus Shope fibroma virus (SFV) have been cloned as imperfect palindromes within circular plasmids in yeast cells and recombination-deficient Escherichia coli. The viral telomeres inserted within these recombinant plasmids are equivalent to the inverted-repeat structures detected as telomeric replicative intermediates during poxvirus replication in vivo. Although the telomeres of vaccinia and SFV show little sequence homology, the termini from both viral genomes exist as AT-rich terminal hairpins with extrahelical bases and alternate "flip-flop" configurations. Using an in vivo replication assay in which circular plasmid DNA was transfected into poxvirus-infected cells, we demonstrated the efficient replication and resolution of the cloned imperfect palindromes to bona fide hairpin termini. The resulting linear minichromosomes, which were readily purified from transfected cells, were shown by restriction enzyme mapping and by electron microscopy to have intact covalently closed hairpin termini at both ends. In addition, staggered unidirectional deletion derivatives of both the cloned vaccinia and SFV telomeric palindromes localized an approximately 200-base-pair DNA region in which the sequence organization was highly conserved and which was necessary for the resolution event. These data suggest a conserved mechanism of the resolution of poxvirus telomeres.     

Membrane lipids and ethanol tolerance in the mouse. The influence of dietary fatty acid composition

Mice of the TO Swiss strain received diets containing different amounts of saturated or unsaturated fat throughout life. These diets produced characteristic changes in cardiac phospholipid fatty acid composition, but produced no significant differences in fatty acid composition of phospholipids from a crude membrane fraction of brain. When littermates of these animals were exposed to ethanol vapour in an inhalation chamber it was observed that mice which had received a diet high in saturated fat lost the righting reflex at an estimated concentration of ethanol in blood higher than that required for mice receiving a control diet, or a diet rich in polyunsaturated fat. Analysis of the brain membrane fraction from those animals which had received ethanol revealed that mice receiving the highly saturated fat diet now had a significantly greater proportion of saturated fatty acids in brain membrane phospholipids. These results are discussed in relation to the hypothesis that brain membrane lipid composition may influence the behavioural response to ethanol.     

Algorithms and software for support of gene identification experiments

MOTIVATION: Gene annotation is the final goal of gene prediction algorithms. However, these algorithms frequently make mistakes and therefore the use of gene predictions for sequence annotation is hardly possible. As a result, biologists are forced to conduct time-consuming gene identification experiments by designing appropriate PCR primers to test cDNA libraries or applying RT-PCR, exon trapping/amplification, or other techniques. This process frequently amounts to 'guessing' PCR primers on top of unreliable gene predictions and frequently leads to wasting of experimental efforts. RESULTS: The present paper proposes a simple and reliable algorithm for experimental gene identification which bypasses the unreliable gene prediction step. Studies of the performance of the algorithm on a sample of human genes indicate that an experimental protocol based on the algorithm's predictions achieves an accurate gene identification with relatively few PCR primers. Predictions of PCR primers may be used for exon amplification in preliminary mutation analysis during an attempt to identify a gene responsible for a disease. We propose a simple approach to find a short region from a genomic sequence that with high probability overlaps with some exon of the gene. The algorithm is enhanced to find one or more segments that are probably contained in the translated region of the gene and can be used as PCR primers to select appropriate clones in cDNA libraries by selective amplification. The algorithm is further extended to locate a set of PCR primers that uniformly cover all translated regions and can be used for RT-PCR and further sequencing of (unknown) mRNA.      

Recent evolutionary history of American Onchocerca volvulus, based on analysis of a tandemly repeated DNA sequence family

Polymerase chain reaction (PCR) products were characterized for a repeated sequence family (designated "O-150") of the human filarial parasite Onchocerca volvulus. In phylogenetic inferences, the O-150 sequences clustered into closely related groups, suggesting that concerted evolution maintains sequence homology in this family. Using a novel mathematical model based on a nested application of an analysis of variance, we demonstrated that African rainforest and savannah strain parasite populations are significantly different. In contrast, parasites collected in the New World are indistinguishable from African savannah strains of O. volvulus. This finding supports the hypothesis that onchocerciasis was recently introduced into the New World, possibly as a result of the slave trade.      

Bile acids: antioxidants or enhancers of peroxidation depending on lipid concentration

Using three different assay systems, we have discovered a heretofore unrecognized antioxidant property of bile acids at physiological concentrations. Bile acids inhibit peroxidation of the polyunsaturated lipid, linoleic acid, and of the highly fluorescent protein phycoerythrin. In part, the antioxidant activity results from scavenging of peroxyl radicals by direct oxidation of the bile acids. The most abundant products of the reaction of cholate and chenodeoxycholate with peroxyl radicals were studied in detail and shown to be the keto derivatives formed by oxidation of the 7 alpha-hydroxyl groups. Paradoxically, at linoleate concentrations higher than 1-2 mM, glycocholate up to approximately 10-14 mM enhances lipid peroxidation and inhibits only at higher concentrations. These findings may prove important in understanding the etiology of certain disease states of the biliary tract and intestine where lipid peroxidation may be involved and in providing a rationale for the positive epidemiological correlation between high lipid intake and higher fecal bile acid output and colon cancer.     

Tubulin mRNAs of Trypanosoma brucei

The tubulin genes of Trypanosoma brucei are located in a single, tightly packed cluster of ten tandemly arranged alternating alpha and beta-genes. No tubulin genes are detected outside the clustered array. Therefore, the cluster can be assumed to be the locus of tubulin gene expression. Single bands of alpha and beta-tubulin mRNAs are observed in cultured procyclic as well as in bloodstream trypanosomes. Both alpha and beta-tubulin mRNAs have distinct 5' termini, which carry a 35-nucleotide mini-exon sequence. The 3' termini of both mRNA populations are heterogeneous.