Spatial behaviour of sheep during the neonatal period: Preliminary study on the influence of shelter.

Effective shelter has been demonstrated to reduce neonatal lamb mortality rates during periods of inclement weather. Periods of high wind speed and rainfall have been shown to influence shelter usage; however, it is not yet known how ewe factors such as breed, age and body condition score influence shelter-seeking behaviour. This study, conducted on a working upland farm in the UK, examined impact of artificial shelter on the biological and climatic factors that influence peri-parturient ewe behaviour. Pregnant ewes (n = 147) were randomly allocated between two adjacent fields which were selected for their similarity in size, topography, pasture management, orientation to the prevailing wind and available natural shelter. In one field, three additional artificial shelters were installed to increase the available shelter for ewes, this field was designated the Test field; no additional artificial shelter was provided in the second field which was used as the Control field. Individual ewes were observed every 2 h between 0800 and 1600 for 14 continuous days to monitor their location relative to shelter. Ewe breed (Aberfield and Highlander), age (2-8 years) and body condition score were considered as explanatory variables to explain flock and individual variance in shelter-seeking behaviour and the prevalence of issues which required the intervention of the shepherd, termed 'shepherding problems'. Any ewe observed with dystocia, a dead or poor vigour lamb or who exhibited mismothering behaviour was recorded as a shepherding problem. The prevalence of these shepherding problems which necessitate human intervention represents arguably the most critical limiting factor for the successful management of commercial sheep flocks in outdoor lambing systems. Overall, ewes in the Test field with access to additional artificial shelter experienced fewer shepherding problems than those in the Control field (P < 0.05). A significant breed effect was also observed, with Highlander ewes more likely to seek shelter than Aberfield ewes (P < 0.001), and experiencing significantly fewer shepherding interventions (P < 0.05). These findings demonstrate the substantial and significant benefits to animal welfare and productivity that can be achieved through the provision of shelter in commercial, upland, outdoor lambing systems in the UK.

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains.

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

CREB binding protein interacts with nucleoporin-specific FG repeats that activate transcription and mediate NUP98-HOXA9 oncogenicity.

Genes encoding the Phe-Gly (FG) repeat-containing nucleoporins NUP98 and CAN/NUP214 are at the breakpoints of several chromosomal translocations associated with human acute myeloid leukemia (AML), but their role in oncogenesis is unclear. Here we demonstrate that the NUP98-HOXA9 fusion gene encodes two nuclear oncoproteins with either 19 or 37 NUP98 FG repeats fused to the DNA binding and PBX heterodimerization domains of the transcription factor HOXA9. Both NUP98-HOXA9 chimeras transformed NIH 3T3 fibroblasts, and this transformation required the HOXA9 domains for DNA binding and PBX interaction. Surprisingly, the FG repeats acted as very potent transactivators of gene transcription. This NUP98-derived activity is essential for transformation and can be replaced by the bona fide transactivation domain of VP16. Interestingly, FG repeat-containing segments derived from the nucleoporins NUP153 and CAN/NUP214 functioned similarly to those from NUP98. We further demonstrate that transactivation by FG repeat-rich segments of NUP98 correlates with their ability to interact functionally and physically with the transcriptional coactivators CREB binding protein (CBP) and p300. This finding shows, for the first time, that a translocation-generated fusion protein appears to recruit CBP/p300 as an important step of its oncogenic mechanism. Together, our results suggest that NUP98-HOXA9 chimeras are aberrant transcription factors that deregulate HOX-responsive genes through the transcriptional activation properties of nucleoporin-specific FG repeats that recruit CBP/p300. Indeed, FG repeat-mediated transactivation may be a shared pathogenic function of nucleoporins implicated human AML.

Analysing genetic information with DNA arrays.

The large amount of DNA sequence information produced in recent years has created a need for high-throughput methods in biology and genetics. These include sequencing, comparing gene sequences and genotyping. DNA arrays promise a highly parallel means for analysis of DNA that is fast and cost-effective, and offers scope for application to complex systems and processes. Recent years have seen continued transfer of technology from the microelectronics industry. Rapid application of the technology to genotyping, antisense oligonucleotide selection and gene expression analysis has illustrated the general power of this approach.

Effects of base mismatches on joining of short oligodeoxynucleotides by DNA ligases.

The requirement for Watson-Crick base pairing surrounding a nick in duplex DNA to be sealed by DNA ligase is the basis for oligonucleotide ligation assays that distinguish single base mutations in DNA targets. Experiments in a model system demonstrate that the minimum length of oligonucleotide that can be joined differs for different ligases. Thermus thermophilus (Tth) DNA ligase is unable to join any oligonucleotide of length six or less, while T4 DNA ligase and T7 DNA ligase are both able to join hexamers. The rate of oligonucleotide ligation by Tth DNA ligase increases between heptamer and nonamer. Mismatches which cause the duplex to be shortened by fraying, at the end distal to the join, slow the ligation reaction. In the case of Tth DNA ligase, mismatches at the seventh and eighth position 5'to the nick completely inhibit the ligation of octamers. The results are relevant to mechanisms of ligation.

Update in diabetes: applications for clinical practice from the Diabetes Control and Complications Trial.

Diabetes mellitus is a complex disease that affects 15 million Americans. Although the pathogenesis of type I and type II varies, a person may have the disease for years prior to onset of the clinical manifestations. Because there is presently no reliable screening tool for this disease, aggressive management beginning at disease onset is crucial in preventing and reducing long-term microvascular complications. The Diabetes Control and Complications Trial unequivocally demonstrates the benefit of intensive management in the reduction of these complications in patients with type I diabetes. It is therefore imperative that health care professionals are aware of the significance and the implications of this landmark study.

Methylphosphonate mapping of phosphate contacts critical for RNA recognition by the human immunodeficiency virus tat and rev proteins.

The HIV-1 regulatory proteins tat and rev are both RNA binding proteins which recognize sequences in duplex RNA which are close to structural distortions. Here we identify phosphate contacts which are critical for each binding reaction by use of a new method. Model RNA binding sites are constructed carrying substitutions of individual phosphodiesters by uncharged methylphosphonate derivatives isolated separately as Rp and Sp diastereoisomers and tested for protein binding by competition assays. In the binding of tat to the trans-activation response region (TAR), three phosphates, P21 and P22 which are adjacent to the U-rich bulge and P40 on the opposite strand, are essential and in each case both isomers inhibit binding. Similarly, in the interaction between the HIV-1 rev protein and the rev-responsive element (RRE) both methylphosphonate isomers at P103, P104, P124 and P125 interfere with rev binding. At P106, only the Rp methylphosphonate isomer is impaired in rev binding ability and it is proposed that the Rp oxygen is hydrogen-bonded to an uncharged amino acid or to a main chain hydrogen atom. Synthetic chemistry techniques also provide evidence for the conformations of non-Watson-Crick G106:G129 and G105:A131 base-pairs in the RRE 'bubble' structure upon rev binding. Almost all functional groups on the 5 bulged residues in the bubble have been ruled out as sites of contact with rev but, by contrast, the N7-positions of each G residue in the flanking base-pairs are identified as sites of likely hydrogen-bonding to rev. The results show that both tat and rev recognize the major groove of distorted RNA helixes and that both proteins make specific contacts with phosphates which are displaced from the sites of base-pair contact.

The synthesis of oligonucleotides that contain 2,4-dinitrophenyl reporter groups.

The synthesis of non-nucleoside-based phosphoramidites that bear the 2,4-dinitrophenyl group is reported. These labelled phosphoramidites, which have been used in solid-phase oligonucleotide synthesis to attach single and multiple dinitrophenyl groups to the 5'-end of oligonucleotides, are entirely compatible with the normal oligonucleotide synthesis cycle. Multiple labelling can be performed readily in high yield and the resulting oligonucleotides can be purified readily by reversed-phase h.p.l.c. The labelled oligonucleotides have been detected using monoclonal and polyclonal anti-dinitrophenyl antibodies.