Identifying ribozyme-accessible sites using NUH triplet-targeting gapmers.

Accurately identifying accessible sites in RNA is a critical prerequisite for optimising the cleavage efficiency of hammerhead ribozymes and other small nucleozymes. Here we describe a simple RNase H-based procedure to rapidly identify hammerhead ribozyme-accessible sites in gene length RNAS: Twelve semi-randomised RNA-DNA-RNA chimeric oligonucleotide probes, known as 'gapmers', were used to direct RNase H cleavage of transcripts with the specificity expected for hammerhead ribozymes, i.e. after NUH sites (where H is A, C or U). Cleavage sites were identified simply by the mobility of RNase H cleavage products relative to RNA markers in denaturing polyacrylamide gels. Sites were identified in transcripts encoding human interleukin-2 and platelet-derived growth factor. Thirteen minimised hammerhead ribozymes, miniribozymes (Mrz), were synthesised and in vitro cleavage efficiency (37 degrees C, pH 7.6 and 1 mM MgCl2) at each site was analysed. Of the 13 Mrz, five were highly effective, demonstrating good initial rate constants and extents of cleavage. The speed and accuracy of this method commends its use in screening for hammerhead-accessible sites.

A transfection compound series based on a versatile Tris linkage.

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.

Rough genes with Deformed homeobox substitutions exhibit rough regulatory specificity during Drosophila eye development.

In certain cases, homeobox genes with different in vivo roles encode proteins with similar in vitro DNA binding specificities. To test the role of the homeobox in the regulatory specificity of such genes, rough homeobox sequences were changed in part or entirely to those of the Deformed gene, and the modified rough genes tested for their ability to rescue the rough mutant phenotype. Surprisingly, the chimaeric genes retained levels of rough regulatory specificity but acquired no novel functions. These results suggest that factors other than the DNA binding specificity of the homeodomain play crucial roles in determining the target, and thus the regulatory specificity, of such proteins.

The rough (ro+) gene as a dominant P-element marker in germ line transformation of Drosophila melanogaster.

We describe a new vector for the P-element-mediated introduction of gene constructs into the germ line of Drosophila melanogaster. The P-element vector carries 6.8 kb of genomic DNA containing the rough gene (ro) from D. melanogaster and a polylinker (MCS) containing ten unique cloning sites. To demonstrate its utility, we have cloned into the MCS of this vector, the firefly luciferase (Luc)-encoding gene (luc) under the control of the D. melanogaster hsp70 promoter and have transformed flies with the resultant P-element. Single insertions of this element, whether in the hemizygous or homozygous condition, completely rescued the ro- mutation and directed heat-inducible synthesis of Luc mRNA and enzyme.

Small, efficient hammerhead ribozymes.

The hammerhead ribozyme is able to cleave RNA in a sequence-specific manner. These ribozymes are usually designed with four basepairs in helix II, and with equal numbers of nucleotides in the 5' and 3' hybridizing arms that bind the RNA substrate on either side of the cleavage site. Here guidelines are given for redesigning the ribozyme so that it is small, but retains efficient cleavage activity. First, the ribozyme may be reduced in size by shortening the 5' arm of the ribozyme to five or six nucleotides; for these ribozymes, cleavage of short substrates is maximal. Second, the internal double-helix of the ribozyme (helix II) may be shortened to one or no basepairs, forming a miniribozyme or minizyme, respectively. The sequence of the shortened helix + loop II greatly affects cleavage rates. With eight or more nucleotides in both the 5' and the 3' arms of a miniribozyme containing an optimized sequence for helix + loop II, cleavage rates of short substrates are greater than for analogous ribozymes possessing a longer helix II. Cleavage of gene-length RNA substrates may be best achieved by miniribozymes.

Serum concentrations of brain-derived neurotrophic factor (BDNF) are decreased in colorectal cancer patients.

OBJECTIVE: To determine the usefulness of brain-derived neurotrophic factor (BDNF) as a diagnostic biomarker for colorectal cancer (CRC). MATERIALS AND METHODS: ELISA immunoassay was used to examine BDNF concentrations in the sera of two different retrospective cohorts consisting of CRC patients and age/gender matched controls. Cohort 1 consisted of 99 controls and 97 CRC patients, whereas cohort 2 consisted of 47 controls and 91 CRC patients. RESULTS: In cohort 1, the median concentration of BDNF was significantly (p< 0.0001) lower in CRC patient samples (18.8 ng/mL, range 4.0-56.5 ng/mL) than control samples (23.4 ng/mL, range 3.0-43.1 ng/mL). This finding was validated in an independent patient cohort (CRC patients: 23.0 ng/mL, range 6.0-45.9 ng/mL; control patients: 32.3 ng/mL, range 14.2-62.4 ng/mL). BDNF concentrations did not differ significantly between Dukes' staging in the patient cohort, however patients with Stages A, B, C and D (p< 0.01 for each stage) tumours had significantly reduced BDNF levels compared to healthy controls. Receiver operating characteristic analysis was performed to determine the ability of BDNF to discriminate between healthy controls and those with CRC. At 95% specificity, BDNF concentrations distinguished CRC patients with 25% and 18% sensitivity, respectively, in cohorts 1 and 2 (cohort 1: AUC=0.79, 95% CI 0.70-0.87; cohort 2: AUC =0.69, 95% CI 0.61-0.76). CONCLUSION: The serum levels of BDNF were significantly lower in colorectal cancer patients when compared to a control population, and this did not differ between different Dukes' stages.

A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates.

A method for the efficient preparation of high quality bacteriophage lambda DNA from cleared lysates is described. Advantages of the method include high DNA yields (typically around 0.8 micrograms of DNA/1 ml of cleared lysate), speed of processing (approximately 2 h from lysate to DNA), economy, and the absence of any requirement for phenol or chloroform extractions. The technique involves the concentration of phage particles by standard polyethylene glycol precipitation followed by enzymatic treatment to remove contaminating RNA and DNA. Phage particles are then lysed with sodium dodecyl sulfate (SDS) at elevated pH and temperature. Contaminating protein/SDS complexes are rendered insoluble by the addition of potassium acetate and removed by centrifugation. The quality of the resultant DNA is comparable to that prepared by cesium chloride banding for all standard molecular biological purposes providing that spermidine is included in all restriction endonucleases digestions.

SV40 enhancer activation during retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

The transient expression vector pSV2CAT, which carries the bacterial chloramphenicol acetyl transferase (CAT) gene under the control of the SV40 early promoter, was used to transfect the murine embryonal carcinoma cell line F9 at various times during the retinoic acid-induced differentiation of these cells. Expression of the CAT gene under SV40 promoter control was found to increase markedly on F9 cell differentiation, measured relative to expression from the thymidine kinase promoter in the same cells. A series of constructs was prepared to identify the features of the SV40 early promoter required for transcription in differentiated and undifferentiated cells, as well as the factors limiting transcription in each case. The increased transcription seen on F9 cell differentiation was not observed when cells were transfected with molecules lacking a functional enhancer. It appears that as embryonal carcinoma cells differentiate, increased SV40 transcription results from enhancer sequence activation. In both differentiated and undifferentiated cell types the level of transcription was found to be limited by the availability and/or activity of cellular factors necessary for enhancer function.

Antisense inhibition of beta-actin mRNA localization and its effect on smooth muscle cell migration.

A crucial step in cell migration involves changes in the actin cytoskeleton in response to extracellular signals. We have previously shown that beta-actin transcripts are associated with mobile regions of mouse 3T3 fibroblasts when grown in the presence of serum. In the current study we used in situ hybridization and laser scanning confocal microscopy to show that cultured rat smooth muscle cells also localize beta-actin mRNA to the cell periphery and that this peripheral pool of beta-actin mRNA is dependent on the presence of growth factors in the culture medium. We also show that antisense phosphorothioated oligonucleotides directed against sequences in the 3' untranslated region of rat beta-actin mRNA block peripheral localization of beta-actin mRNA while the corresponding control oligonucleotides have no effect. Time-lapse video analysis demonstrates that the antisense oligonucleotides inhibit rat smooth muscle cell migration in culture and analysis of beta-actin mRNA confirms this is not due to changes in beta-actin gene expression or instability of the message. Our results suggest that depletion of beta-actin transcripts from the cell periphery is associated with suppression of SMC migration.

Temporal and spatial utilization of the alcohol dehydrogenase gene promoters during the development of Drosophila melanogaster.

The enzyme alcohol dehydrogenase of Drosophila melanogaster is encoded by a single structural gene (Adh) with two promoters, distal and proximal (PD and PP). During development these two promoters are used differently: the major Adh transcript of larvae is from PP, the major transcript of adult flies is from PD. At a few discrete times in development transcription occurs simultaneously from both promoters. In situ hybridization has been used to investigate the spatial and temporal aspects of promoter activity at these stages of development. Maternally inherited Adh transcripts are not localized in the embryo; they decay very rapidly after fertilization. Zygotic expression of Adh RNA begins after germ-band retraction, 10.5 hr after fertilization. Expression is confined to the fat body, but occurs from both distal and proximal promoters. By 15 hr expression is first seen in the gut, from PP. By the same time fat body expression from PD has ceased, and transcription in this tissue is exclusively from PP for the next 4 days. The steady-state level of Adh transcript begins to decline at the end of larval development. There is then the transient accumulation of transcripts from PD, but predominantly in the larval fat body, rather than in the gut. These data illustrate a surprising complexity in the tissue and temporal regulation of Adh expression in D. melanogaster. Moreover, they show that transcripts from two different promoters of the same gene can, at certain well-defined stages of development, accumulate in the same cells.

Oncogene expression in differentiating F9 mouse embryonal carcinoma cells.

Differentiation of F9 mouse embryonal carcinoma cells in culture is accompanied by a decrease in growth rate and loss of tumorigenicity. Cells differentiating in monolayer culture (to parietal endoderm-type cells) or in aggregates (to visceral endoderm-type cells) show qualitatively similar changes in transcript levels from several c-oncogenes. In contrast with other studies with F9 cells, we find an early decrease in c-myb RNA but not in c-myc RNA. This and a later increase in c-src RNA may be associated with decreasing cell growth rate. Before differentiation, induction and maintenance of elevated c-abl RNA levels depend on the presence of retinoic acid in the medium. After differentiation c-abl RNA levels decline only partially when retinoic acid is removed. Increased RNA from c-fos is seen late in differentiation in monolayer cultures only, a change also seen with appearance of similar endoderm cell types in the developing mouse embryo.

Selected classes of minimised hammerhead ribozyme have very high cleavage rates at low Mg2+ concentration.

In vitro selection was used to enrich for highly efficient RNA phosphodiesterases within a size-constrained (18 nt) ribonucleotide domain. The starting population (g0) was directed in trans against an RNA oligonucleotide substrate immobilised to an avidin-magnetic phase. Four rounds of selection were conducted using 20 mM Mg2+to fractionate the population on the basis of divalent metal ion-dependent phosphodiesterase activity. The resulting generation 4 (g4) RNA was then directed through a further two rounds of selection using low concentrations of Mg2+. Generation 6 (g6) was composed of sets of active, trans cleaving minimised ribozymes, containing recognised hammerhead motifs in the conserved nucleotides, but with highly variable linker domains (loop II-L.1-L.4). Cleavage rate constants in the g6 population ranged from 0.004 to 1.3 min-1at 1 mM Mg2+(pH 8.0, 37 degrees C). Selection was further used to define conserved positions between G(10.1) and C(11.1) required for high cleavage activity at low Mg2+concentration. At 10 mM MgCl2the kinetic phenotype of these molecules was comparable to a hammerhead ribozyme with 4 bp in helix II. At low Mg2+concentration, the disparity in cleavage rate constants increases in favour of the minimised ribozymes. Favourable kinetic traits appeared to be a general property for specific selected linker sequences, as the high rates of catalysis were transferable to a different substrate system.

The mechanism of the hammerhead ribozyme.

The rate of cleavage of a 13-mer substrate by an all-RNA ribozyme with 2 10-mer hybridising arms (TAT RA), appears to be limited by a conformational change involving the longer than necessary arms. The same limitation does not apply to a similar ribozyme with DNA arms (TAT RB) or to an all-RNA ribozyme with only 6 hybridising bases on each arm (TAT RA 6x2). The limitation does not arise in the hybridisation step nor in the dissociation of products. An extra step involving a conformational change of the pre-formed ribozyme-substrate prior to cleavage is introduced to explain the observed kinetics.

Pattern formation in the developing eye of Drosophila melanogaster is regulated by the homoeo-box gene, rough.

Homoeo-box genes play a central role in the regulation of embryogenesis in Drosophila melanogaster. Their widespread phylogenetic distribution, and the tissue and stage specificity of their expression in other organisms, argue that they play a general and significant role in animal development. In D. melanogaster, all homoeo-box genes characterized to date are involved in major aspects of embryogenesis. We report here the molecular characterization of a Drosophila homoeo-box gene that has no apparent involvement in early embryogenesis. The gene appears to be rough, a gene implicated in pattern formation in the developing eye. It is expressed in cells within, and posterior to, the morphogenetic furrow, the site of the primary pattern forming events in the developing retina, and also in a region of the brain of the third instar larva. We have found no genetic or molecular evidence of a role for this gene in other aspects of fly development.

The Notch locus of Drosophila melanogaster.

The Notch locus appears to correspond to a 37 kb transcription unit. Homologous poly(A)+ RNA is about 11.7 kb. Nine RNA coding regions have been detected and localized within the transcription unit by S1 nuclease analyses. These range in size from 130 to 7250 bp. Sequences within the 3' coding region are variably used. Twenty-four Notch locus "point" mutations have been examined. Seven are associated with DNA insertions. Four insertions are associated with dominant mutations and are located within or very near RNA coding sequences of the 37 kb transcription unit. Three insertions are associated with recessive mutations and fall within intervening sequences. The positions of all insertions agree with the genetic map. It is estimated that, in the Notch region, one map unit corresponds to approximately 275 kb. On this basis, most of the 17 mutations that were not associated with DNA insertions can be placed within coding sequences of the 37 kb transcription unit.