Cellulosomes prepared by the cellulose affinity digestion method from Clostridium thermocellum culture supernatant hydrolysed carob galactomannan during incubation at 60 SC and pH 65. A recombinant phage expressing mannanase activity was isolated from a library of C. thermocellum genomic DNA constructed in λZAPII. The cloned fragment of DNA containing a putative mannanase gene (manA) was sequenced, revealing an ORF of 1767 nt, encoding a protein (mannanase A ; Man26A) of 589 aa with a molecular mass of 66 816 Da. The putative catalytic domain (CD) of Man26A, identified by gene sectioning and sequence comparisons, displayed up to 32 % identity with other mannanases belonging to family 26. Immediately downstream of the CD and separated from it by a short proline/threonine linker was a duplicated 24-residue dockerin motif, which is conserved in all C. thermocellum cellulosomal enzymes described thus far and mediates their attachment to the cellulosomeintegrating protein (CipA). Man26A consisting of the CD alone (Man26A') was hyperexpressed in Escherichia coli BL21(DE3) and purified. The truncated enzyme hydrolysed soluble and insoluble mannan, displaying a temperature optimum of 65 SC and a pH optimum of 65, but exhibited no activity against other plant cell wall polysaccharides. Antiserum raised against Man26A' crossreacted with a polypeptide with a molecular mass of 70 000 Da that is part of the C. thermocellum cellulosome. A second variant of Man26A containing the N-terminal segment of 130 residues and the CD (Man26A'') bound to ivory-nut mannan and weakly to soluble Carob galactomannan and insoluble cellulose. Man26A' consisting of the CD alone did not bind to these polysaccharides. These results indicate that the N-terminal 130 residues of mature Man26A may constitute a weak mannan-binding domain. Sequence comparisons revealed a lack of identity between this region of Man26A and other polysaccharidebinding domains, but significant identity with a region conserved in the three family 26 mannanases from the anaerobic fungus Piromyces equi.

Changes in food intake affect the reproductive axis in both sexes, and the nutritional signals involved and the sites that receive those signals are now beginning to be unravelled. Our studies have focussed on the mature male sheep, a model in which high food intake stimulates GnRH-LH pulse frequency for only 10-20 days but continues to promote testicular growth over several months. Different signals and different target organs seem to be responsible for these short- and long-term responses. Short-term dietary treatments lead to changes in blood concentrations of glucose, fatty acids, insulin and leptin, and concentrations of glucose, insulin, leptin and some amino acids in cerebrospinal fluid. It seems unlikely that amino acids affect GnRH-LH secretion directly in sheep. Intracerebroventricular infusions of insulin specifically increase LH pulse frequency, but intravenous, intra-abomasal or intracerebroventricular infusions of glucose have no effect, despite their effects on cerebrospinal fluid insulin concentrations. The addition of fatty acids to the diet also increases LH pulse frequency, but does not affect the concentrations of insulin or leptin in the cerebrospinal fluid. It appears that acute responses to changes in nutrition involve a range of alternative pathways, possibly including interactions among insulin, leptin and energy substrates. Effects of long-term dietary treatments on testicular size are only partly dependent on the GnRH-LH system (that is, on brain control) and so must also depend on other, as yet unknown, pathways. Concepts of 'metabolic sensing and integration' are being developed from the basis of existing knowledge of the central control of appetite and reproduction.

The recombinant clone pBAW101 (in pBluescript SK-) contains the celB endoglucanase gene from Ruminococcus flavefaciens FD-1. Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104). The nucleotide sequence of the celB gene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69,414. A putative Shine-Dalgarno sequence was identified 6 bp upstream from the translation start site. The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences. Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published beta-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44. Primer extension analyses were performed using RNA isolated from R. flavefaciens grown on cellulose and cellobiose, and from Escherichia coli containing the plasmid clone pBAW104. Transcription is initiated at different sites in E. coli and R. flavefaciens. In the case of R. flavefaciens transcription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site. There were no regions resembling E. coli sigma 70-like promoter sequences present upstream from this putative transcription initiation site. In contrast, numerous transcription initiation sites were identified when RNA from E. coli was used in the primer extension analyses.

The endoglucanase gene was sequenced from Prevotella ruminicola AR20, isolated as clone pJW4. The endoglucanase (BrEND) is encoded by an open reading frame (ORF1) of 501 codons, corresponding to a protein of calculated molecular weight 55.7 kDa. Analysis of proteins on SDS-PAGE revealed a protein corresponding to the calculated molecular weight of the processed BrEND. The protein showed substantial homology to members of the A4 sub-family cellulases. Primer extension studies revealed that transcription of celA is initiated at different sites in Escherichia coli and Prevotella ruminicola. E. coli sigma 70 recognition sequences were identified, which were located upstream from the transcription initiation site (TIS) functional in E. coli. A longer extension product was identified using RNA from P. ruminicola, indicating that the gene may normally be transcribed as part of a polycistronic message. The end of the primer extension product corresponded to a site beyond the 5' boundary of the cloned fragment, thus preventing identification of native promoter sequences. A second ORF of 110 codons (ORF2) was identified on the antisense strand, and primer extension indicated that transcription through ORF2 was initiated at an identical site in both E. coli and P. ruminicola. E. coli-like consensus sequences were located at positions -10 and -35 upstream from this site, suggesting that some promoter sequences in P. ruminicola are similar to E. coli consensus sequences, although others recognized by E. coli are non-functional in P. ruminicola.(ABSTRACT TRUNCATED AT 250 WORDS)

A total of 2,600 methane (CH 4 ) and 1,847 CO 2 measurements of sheep housed for 1 h in portable accumulation chambers (PAC) were recorded at 5 sites from the Australian Sheep CRC Information Nucleus, which was set up to test leading young industry sires for an extensive range of current and novel production traits. The final validated dataset had 2,455 methane records from 2,279 animals, which were the progeny of 187 sires and 1,653 dams with 7,690 animals in the pedigree file. The protocol involved rounding up animals from pasture into a holding paddock before the first measurement on each day and then measuring in groups of up to 16 sheep over the course of the day. Methane emissions declined linearly (with different slopes for each site) with time since the sheep were drafted into the holding area. After log transformation, estimated repeatability (rpt) and heritability (h 2 ) of liveweight-adjusted CH 4 emissions averaged 25% and 11.7%, respectively, for a single 1-h measurement. Sire × site interactions were small and nonsignificant. Correlations between EBV for methane emissions and Sheep Genetics Australia EBV for production traits were used as approximations to genetic correlations. Apart from small positive correlations with weaning and yearling weights (r = 0.21-0.25, P < 0.05), there were no significant relationships between production trait and methane EBV (calculated from a model adjusting for liveweight by fitting separate slopes for each site). To improve accuracy, future protocols should use the mean of 2 (rpt = 39%, h 2 = 18.6%) or 3 (rpt = 48%, h 2 = 23.2%) PAC measurements. Repeat tests under different pasture conditions and time of year should also be considered, as well as protocols measuring animals directly off pasture instead of rounding them up in the morning. Reducing the time in the PAC from 1 h to 40 min would have a relatively small effect on overall accuracy and partly offset the additional time needed for more tests per animal. Field testing in PAC has the potential to provide accurate comparisons of animal and site methane emissions, with potentially lower cost/increased accuracy compared to alternatives such as SF 6 tracers or open path lasers. If similar results are obtained from tests with different protocols/seasonal conditions, use of PAC measurements in a multitrait selection index with production traits could potentially reduce methane emissions from Australian sheep for the same production level.