Assessment of potential neuropathic changes in cattle after cautery disbudding.

Disbudding of calves is a standard husbandry procedure to reduce the risk of injuries to other cattle and to workers. Whereas acute pain resulting from disbudding has been studied extensively, little is known about chronic pain as a potential long-term consequence. The goal of the present study was to investigate possible morphological changes in the cornual nerve as a function of disbudding. Samples were collected from 17 randomly selected bulls and from 21 calves from a prospective clinical study. Among the calves, 13 were disbudded and 8 were sham-disbudded. Out of the disbudded calves, 4 showed signs of chronic pain. In all the animals, the infraorbital nerve was used as a methodological check. Morphological analysis included measuring minimal diameters of the axons present in both the cornual and infraorbital nerves. Sympathetic fibers were identified as based on the presence of Tyroxine hydroxylase (TH). TH-negative fibers were considered afferents. Trigeminal ganglia from the calves were immunostained for glial fibrillary acidic protein (GFAP) and Activating transcription factor 3 (ATF3). R. cornualis and N. infraorbitalis differed in terms of axon diameters and proportion of TH-positive fibers. Weak evidence (p > .091) of a difference in axon diameters between control and disbudded calves was found in R. cornualis, but the proportion of TH-positive fibers was alike in both groups. Average glial envelope and the percentages of ATF3-positive neurons revealed no difference between calves with and without signs of pain. Thus, available evidence is insufficient to support neuropathic changes as a result of disbudding in calves.

The wis1 signal transduction pathway is required for expression of cAMP-repressed genes in fission yeast.

The wis1 protein kinase of Schizosaccharomyces pombe is a member of the MAP kinase kinase family. Loss of wis1 function has previously been reported to lead to a delay in the G2-mitosis transition, loss of viability in stationary phase, and hypersensitivity to osmotic shock. It acts at least in part by activating the MAP kinase homologue sty1; loss-of-function sty1 mutants share many phenotypes with wis1 deletion mutants. We show here that, in addition, loss of wis1 function leads to defective conjugation, and to suppression of the hyperconjugation phenotype of the pat1-114 mutation. Consistent with this, the induction of the mei2 gene, which is normally induced by nitrogen starvation, is defective in wis1 mutants. In wild-type cells, nitrogen starvation leads to mei2 induction through a fall in intracellular cyclic AMP (cAMP) level and activity of the cAMP-dependent protein kinase. We show here that wis1 function is required for mei2 induction following nitrogen starvation. Expression of the fbp1 gene is negatively regulated by cAMP in response to glucose limitation: induction of fbp1 also requires wis1 and sty1 function. Loss of wis1 is epistatic over increased fbp1 expression brought about by loss of adenylate cyclase (git2/cyr1) or cAMP-dependent protein kinase (pka1) function. These observations can be explained by a model in which the pka1 pathway negatively regulates the wis1 pathway, or the two pathways might act independently on downstream targets. The latter explanation is supported, at least as regards regulation of cell division, by the observation that loss of function of the regulatory subunit of the cAMP-dependent protein kinase (cgs1) brings about a modest increase in cell length at division in both wis1+ and wis1 delta genetic backgrounds.

Stress signal, mediated by a Hog1-like MAP kinase, controls sexual development in fission yeast.

We identified the phh1+ gene that encodes a MAP kinase as the effector of Wis1 MAP kinase kinase in fission yeast, which is highly homologous with HOG1 of S. cerevisiae. Heterothalic phh1 dsiruptant is phenotypically indistinguishable from wis1 deletion mutant, both displaying the same extent of partial sterility and enhanced sensitivity to a variety of stress. In phh1 disruptant, nitrogen starvation-induced expression of ste11+, a key controller of sexual differentiation, is markedly diminished. Ectopic expression of ste11+ effectively restores fertility, but not stress resistance, to the phh1 disruptant. These data show that stress signal, mediated by a MAP kinase, is required for efficient start of sexual differentiation.

A mutation in the C31 subunit of Saccharomyces cerevisiae RNA polymerase III affects transcription initiation.

The C31 subunit belongs to a complex of three subunits (C31, C34 and C82) specific to RNA polymerase (pol) III that have no counterparts in other RNA polymerases. This complex is thought to play a role in transcription initiation since it interacts with the general initiation factor TFIIIB via subunit C34. We have obtained a conditional mutation of pol III by partially deleting the acidic C-terminus of the C31 subunit. A Saccharomyces cerevisiae strain carrying this truncated C31 subunit is impaired in in vivo transcription of tRNAs and failed to grow at 37 degrees C. This conditional growth phenotype was suppressed by overexpression of the gene coding for the largest subunit of pol III (C160), suggesting an interaction between C160 and C31. The mutant pol III enzyme transcribed non-specific templates at wild-type rates in vitro, but was impaired in its capacity to transcribe tRNA genes in the presence of general initiation factors. Transcription initiation, but not termination or recycling of the enzyme, was affected in the mutant, suggesting that it could be altered on interaction with initiation factors or on the formation of the open complex. Interestingly, the C-terminal deletion was also suppressed by a high gene dosage of the DED1 gene encoding a putative helicase.

Organization of the centromeric region of chromosome XIV in Saccharomyces cerevisiae.

A 15.1 kb fragment of the yeast genome was allocated to the centromeric region of chromosome XIV by genetic mapping. It contained six bona fide genes, RPC34, FUN34, CIT1 (Suissa et al., 1984), RLP7, PET8 and MRP7 (Fearon and Mason, 1988) and two large open reading frames, DOM34 and TOM34. RPC34 and RLP7 define strictly essential functions, whereas CIT1, PET8 and MRP7 encode mitochondrial proteins. The PET8 product belongs to a family of mitochondrial carrier proteins. FUN34 encodes a putative transmembraneous protein that is non-essential as judged from the normal growth of the fun34-::LUK18(URA3) allele even on respirable substrates. TOM34 codes for a putative RNA binding protein, and DOM34 defines a hypothetical polypeptide of 35 kDa, with no significant homology to known proteins. The region under study also contains two divergently transcribed tDNAs, separated only by a chimeric transposable element. This tight tDNA linkage pattern is commonly encountered in yeast, and a general hypothesis is proposed for its emergence on the Saccharomyces cerevisiae genome. RPC34, RLP7, PET8 and MRP7 are unique on the yeast genome, but the remaining genes belong to an extant centromeric duplication between chromosome III and XIV.

A general suppressor of RNA polymerase I, II and III mutations in Saccharomyces cerevisiae.

A multicopy genomic library of Saccharomyces cerevisiae (strain FL100) was screened for its ability to suppress conditionally defective mutations altering the 31 kDa subunit (rpc31-236) or the 53 kDa subunit (rpc53-254/424) of RNA polymerase III. In addition to allele-specific suppressors, we identified seven suppressor clones that acted on both mutations and also suppressed several other conditional mutations defective in RNA polymerases I or II. All these clones harbored a complete copy of the SSD1 gene. The same pleiotropic suppression pattern was found with the dominant SSD1-v allele present in some laboratory strains of S. cerevisiae. SSD1-v was previously shown to suppress mutations defective in the SIT4 gene product (a predicted protein phosphatase subunit) or in the regulatory subunit of the cyclic AMP-dependent protein kinase. We propose that the SSD1 gene product modulates the activity (or the level) of the three nuclear RNA polymerases, possibly by altering their degree of phosphorylation.

Two yeast chromosomes are related by a fossil duplication of their centromeric regions.

A 15 kbp fragment of the Saccharomyces cerevisiae genome was cloned and localised to the centromeric region of chromosome XIV by genetic linkage and DNA sequencing. It had a strong sequence similarity and a conserved gene linkage and transcriptional orientation relatively to the centromeric region of chromosome III, indicating a fossil interchromosomal duplication of several linked genes. On chromosome XIV, the duplicated fragment included the centromere, four genes (FUN34, CIT1 and two tDNAs), one open reading frame (DOM34) and a truncated delta element. Additional inserts bearing unique genes were present on the centromeric region of chromosome III. The level of silent substitutions indicated a relatively ancient genetic separation, pre-dating the emergence of S. cerevisiae and S. douglasii as distinct species. The ensuing evolution of the duplicated regions retained strict sequence identity for the two tDNAs pairs, but was partially divergent for CIT1 and FUN34, and generated a probable pseudogenic equivalent of DOM34 on chromosome III. Extant multigenic duplications of this type might play an important role in the evolution of eukaryotic genomes.

An essential and specific subunit of RNA polymerase III (C) is encoded by gene RPC34 in Saccharomyces cerevisiae.

The RPC34 gene of Saccharomyces cerevisiae was cloned by immunological screening, using antibodies raised against the C34 polypeptide of the RNA polymerase III (C). This single copy gene was located near the centromere of chromosome XIV. It included a coding sequence of 317 amino acids that strictly matched two internal oligopeptides of C34. This polypeptide is a specific component of RNA polymerase III, with no significant homology to any other RNA polymerase subunit known so far. It is an essential subunit, since inactivation by deletion or nonsense mutations led to a recessive lethal phenotype. Moreover, a partially blocked mutant, rpc34-F297, had a reduced tRNA synthesis in vivo but no detectable effect on 5 S RNA synthesis. The latter phenotype was observed for all conditionally defective RNA polymerase III mutants isolated so far.