The stability of oligodeoxyribonucleotide duplexes containing degenerate bases.

Oligodeoxyribonucleotides containing N4-methoxycytosine (mo4C), N4-methoxy-5-methylcytosine (mo4m5C) and other base-analogues were synthesised and used to compare the stabilities of duplexes containing mo4C.A and mo4C.G base pairs with those containing normal and mismatch pairs. The Tm values and other thermodynamic parameters are recorded. The otherwise identical duplexes containing a mo4C.A and a mo4C.G base pair have closely similar stabilities to each other and to the corresponding duplexes containing normal base pairs, considerably greater than the stabilities of those containing mismatch pairs. Corresponding observations are recorded in dot-blot experiments using M13 cloned DNA carrying an insert complementary to the oligonucleotides; approximate Td values are given.     

Water and polypeptide conformations in the gramicidin channel. A molecular dynamics study.

Theoretical studies of ion channels address several important questions. The mechanism of ion transport, the role of water structure, the fluctuations of the protein channel itself, and the influence of structural changes are accessible from these studies. In this paper, we have carried out a 70-ps molecular dynamics simulation on a model structure of gramicidin A with channel waters. The backbone of the protein has been analyzed with respect to the orientation of the carbonyl and the amide groups. The results are in conformity with the experimental NMR data. The structure of water and the hydrogen bonding network are also investigated. It is found that the water molecules inside the channel act as a collective chain; whereas the conformation in which all the waters are oriented with the dipoles pointing along the axis of the channel is a preferred one, others are also accessed during the dynamics simulation. A collective coordinate involving the channel waters and some of the hydrogen bonding peptide partners is required to describe the transition of waters from one configuration to the other.     

A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.     

A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.     

The WHI1+ gene of Saccharomyces cerevisiae tethers cell division to cell size and is a cyclin homolog.

WHI1-1 is a dominant mutation that reduces cell volume by allowing cells to commit to division at abnormally small sizes, shortening the G1 phase of the cell cycle. The gene was cloned, and dosage studies indicated that the normal gene activated commitment to division in a dose-dependent manner, and that the mutant gene had a hyperactive but qualitatively similar function. Mild over-expression of the mutant gene eliminated G1 phase, apparently entirely relaxing the normal G1 size control, but revealing hitherto cryptic controls. Sequence analysis showed that the hyperactivity of the mutant was caused by the loss of the C-terminal third of the wild-type protein. This portion of the protein contained PEST regions, which may be signals for protein degradation. The WHI1 protein had sequence similarity to clam cyclin A, to sea urchin cyclin and to Schizosaccharomyces pombe cdc13, a cyclin homolog. Since cyclins are inducers of mitosis, WHI1 may be a direct regulator of commitment to division. A probable accessory function of the WHI1 activator is to assist recovery from alpha factor arrest; WHI1-1 mutant cells could not be permanently arrested by pheromone, consistent with a hyperactivation of division.     

Evidence that the functional beta-actin gene is single copy in most mice and is associated with 5'' sequences capable of conferring serum- and cycloheximide-dependent regulation.

Hybridization to synthetic oligonucleotides representing conserved regions in the promoter and first intron of several vertebrate beta-actin genes was used to discriminate between what appears to be a single functional beta-actin gene and numerous pseudogenes in the mouse genome. Sequences derived from the 5' end of this gene were shown to confer serum-inducible expression upon a heterologous reporter gene when transfected into mouse fibroblasts. Moreover, these sequences rendered reporter gene expression superinducible by a combination of serum and cycloheximide. These experiments indicate that the 5' end of the mouse beta-actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and which are responsive to both positive and negative regulators of gene expression.     

Effect of Within-field Variation in Soil Texture on Heterodera glycines and Soybean Yield

The influence of soil texture on Soybean yield in the presence of Heterodera glycines was investigated by comparing yields of susceptible cultivars with a resistant cultivar for 2 years. Soybean yield was negatively correlated with increasing sand content (P = 0.05). Yields of susceptible cultivars were suppressed with increasing sand content. Final nematode population densities were lowest in plots with greatest sand content. Soybean infection by SCN, as determined by the number of cysts 30 days after planting, was not consistently related to soil texture over 2 years. Initial nematode population density was positively related to soybean yield the first year and negatively related to soybean yield the second, probably a result of greater yield suppression by H. glycines in plots with greater sand content.     

Putrescine uptake regulation in response to alpha-difluoromethylornithine treatment in Leishmania infantum promastigotes

Summary The putrescine uptake/efflux regulation and their regulatory role on intracellular polyamine pools have been studied in the parasitic protozoa Leishmania infantum. Putrescine uptake was age-dependent with maximal values in logarithmic phase promastigotes and minimal in stationary phase. Moreover, putrescine uptake was activated in response to depletion of intracellular polyamines by alpha-difluoromethylornithine (DFMO) — a well known irreversible enzyme-activated inhibitor of ornithine decarboxylase. Kinetic studies of putrescine uptake induction showed a notable rise in Vmax without Km changes, suggesting a de novo synthesis of putrescine carriers. Putrescine uptake was able to replenish polyamine content and also to recover the proliferative rate in cells treated during 24 hours with DFMO.     

Effect of cycloheximide and growth factors on gene expression in quiescent mouse embryo fibroblasts

Gene expression in quiescent mouse embryo fibroblasts was studied by labelling the cells with [14C] amino acids and analysing the proteins by electrophoresis in polyacrylamide gradient gels containing sodium dodecyl sulfate. Cycloheximide (CH) pretreatment of the cells was found to induce the synthesis of four proteins of molecular weights 72,000, 68,000, 42,000, and 29,000. These proteins were induced by CH both in serum-arrested and serum-stimulated cells. Addition of platelet-derived growth factor to serum-arrested quiescent cells also induced the synthesis of these proteins. Addition of CH and fetal calf serum (20%) to quiescent cells resulted in a dramatic increase in the synthesis of actin and another protein of molecular weight 29,000. The 29,000-dalton protein was present in higher quantities in the nuclei of induced cells. This protein appeared to be an early protein whose synthesis was transiently induced in quiescent cells within 3 hours of addition of 20% fetal calf serum (FCS). The synthesis of this protein was virtually turned off at 5-6 hours after the addition of serum. However, if CH or a combination of CH and FCS was present, a continuous synthesis of the 29 K protein was observed.     

Studies of potential filaricides: Part 14--Activity of 1-iso-butoxycarbonyl-4-methylpiperazine against experimental filariasis

The synthesis and filaricidal activity of 1-iso-butoxycarbonyl-4-methylpiperazine against Litomosoides carinii in Sigmodon hispidus and Dipetalonema viteae in Mastomys natalensis is reported. At an intraperitoneal or oral dose of 3 mg/kg given for 6 days, the compound removed 91% of the circulating microfilariae but had no effect on adult L. carinii. However, it killed all microfilariae and adults of D. viteae at a subcutaneous dose of 50 mg/kg given for 6 days. The compound also possessed chemoprophylactic activity against the larvae of L. carinii and D. viteae at a dose of 30 and 50 mg/kg respectively.