Neutron fibre diffraction study of DNA hydration

Interactions with water are crucial to the conformation assumed by the DNA double helix. The location of water around the D conformation has been investigated in a neutron fibre diffraction study which shows that water is ordered in the minor groove of the DNA. The D conformation is important since its occurrence is limited to specific DNA base pair sequences which have been identified as functionally significant. This study is of particular interest because the D conformation has not been reported in single crystal studies of oligonucleotides.     

Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.

Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell.     

Increased band sharing in DNA fingerprints of an inbred human population

Summary We have compared band sharing between the DNA fingerprints of members of an inbred human population with band sharing between members of an outbred population. It had not previously been determined whether the high rate of mutation at minisatellite loci is sufficient to prevent an increase in band sharing in moderately inbred populations. We have found that there is an increase in band sharing in the 2-kb to 9-kb size range, but not in the >9-kb size range, in the inbred population. The difference was consistently observed using four different multi-locus probes, viz. 33.6, 33.15, (CAC)₅ and M13. Thus, we have demonstrated that moderate but prolonged inbreeding can lead to increased similarity in human DNA fingerprints. This should be considered when analysing DNA fingerprints in forensic or paternity cases involving members of an inbred community.     

Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been used to determine the nucleotide sequences of the gene that determines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length and possess 5'- and 3'-terminal untranslated regions of 23 and 142 nucleotides, respectively. The inferred amino acid sequence reveals VP6 to be a polypeptide of 397 amino acids in which more than 90% of the amino acid sequence is conserved between the two viruses. There are 34 amino acid changes between the subgroup 1 and 2 polypeptides, most clustered in three regions of the molecule at residues 39 through 62, 80 through 122, and 281 through 315.     

Coding assignment and nucleotide sequence of simian rotavirus SA11 gene segment 10: location of glycosylation sites suggests that the signal peptide is not cleaved.

A cloned DNA copy of simian rotavirus SA11 genomic segment 10 was used to confirm the assignment of the nonstructural glycoprotein NCVP5 to this gene. Determination of the nucleotide sequence for gene 10 indicated that NCVP5 is 175 amino acids in length and has an N-terminal hydrophobic region with the characteristics of a signal sequence for membrane translocation. Unexpectedly, this region was also the location for the only two potential glycosylation sites within the molecule, asparagine residues 8 and 18. The carbohydrates carried by NCVP5 were of the high-mannose type, Man9GlcNAc and Man8GlcNAc, with the mannose 9 species predominating; no complex oligosaccharides were present. If these asparagine residues are the sites for carbohydrate attachment, this implies that cleavage of the putative signal peptide does not occur during the maturation of this nonstructural glycoprotein.     

The immunogenicity of VP7, a rotavirus antigen resident in the endoplasmic reticulum, is enhanced by cell surface expression.

The glycoprotein VP7, the major serotype antigen of rotaviruses, is localized to the endoplasmic reticulum (ER) of the cell, where it is retained as a membrane-associated protein before assembly into mature virus particles. Wild-type VP7 expressed by a recombinant vaccinia virus was also located internally and was poorly antigenic. Using recombinant techniques, a correctly processed, secreted form of VP7 (S.C. Stirzaker and G.W. Both, Cell 56:741-747, 1989) was modified by addition to its C terminus of the membrane anchor and cytoplasmic domains from the influenza virus hemagglutinin. The hybrid protein was directed to the surface of cells, where it was anchored in the plasma membrane. When expressed in mice and rabbits by a recombinant vaccinia virus, the surface-anchored antigen stimulated a level of rotavirus-specific antibodies that was greater than 100-fold above the level induced by wild-type VP7. T-cell responses to the novel antigen were also elevated in comparison with the wild-type, intracellular protein. Cell surface anchoring may provide a strategy to increase the immunogenicity of intracellular antigens from other parasites and viruses.