Long distance animal transport: the way forward.

Too often, the issue of animal welfare during transport is the subject of emotional debates. For farmers within the International Federation of Agricultural Producers, it is important that the economic, scientific and practical aspects be taken into account when setting international rules for animal welfare. Farmers also stress the need to combine scientific data with their practical experience.

The position of the Dutch Farmers' Union on lessons learned and future prevention and control of foot and mouth disease.

Foot and mouth disease (FMD) has devastated animal husbandry in The Netherlands frequently in the past and still constitutes a threat. The use of vaccination reduced the number of outbreaks in The Netherlands in the 20th Century. However, the desire of some member states of the European Community not to use vaccination led to a new strategy based on stamping-out of infected and contagious farms and to strict transportation regulations.

Common elements on the surface of glycolytic enzymes from Trypanosoma brucei may serve as topogenic signals for import into glycosomes.

In Trypanosoma brucei, a major pathogenic protozoan parasite of Central Africa, a number of glycolytic enzymes present in the cytosol of other organisms are uniquely segregated in a microbody-like organelle, the glycosome, which they are believed to reach post-translationally after being synthesized by free ribosomes in the cytosol. In a search for possible topogenic signals responsible for import into glycosomes we have compared the amino acid sequences of four glycosomal enzymes: triosephosphate isomerase (TIM), glyceraldehyde-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK) and aldolase (ALDO), with each other and with their cytosolic counterparts. Each of these enzymes contains a marked excess of positive charges, distributed in two or more clusters along the polypeptide chain.

Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei. Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome. We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides.

Characterization of the promoter of the large ribosomal RNA gene in yeast mitochondria and separation of mitochondrial RNA polymerase into two different functional components.

We have characterized a DNA sequence that functions in recognition of the promoter of the mitochondrial large rRNA gene by the yeast mtRNA polymerase. Promoter-containing DNA fragments were mutagenized and used as templates to study initiation of transcription in vitro with a partially purified mtRNA polymerase preparation. Deletion mutants, in which increasing stretches of DNA were removed from regions flanking the promoter, define a short area essential for correct initiation of transcription.

Two tandemly linked identical genes code for the glycosomal glyceraldehyde-phosphate dehydrogenase in Trypanosoma brucei.

Trypanosoma brucei contains two isoenzymes for glyceraldehyde-phosphate dehydrogenase (GAPDH); one enzyme resides in a microbody-like organelle, the glycosome, the other one is found in the cytosol. We show here that the glycosomal enzyme is encoded by two tandemly linked genes of identical sequence. These genes code for a protein of 358 amino acids, with a mol.

Topogenesis of microbody enzymes: a sequence comparison of the genes for the glycosomal (microbody) and cytosolic phosphoglycerate kinases of Trypanosoma brucei.

To determine how microbody enzymes enter microbodies, we are studying the genes for cytosolic and glycosomal (microbody) isoenzymes in Trypanosoma brucei. We have found three genes (A, B and C) coding for phosphoglycerate kinase (PGK) in a tandem array in T. brucei.

Trypanosomes of subgenus Trypanozoon are diploid for housekeeping genes.

The ploidy of trypanosomes has until now remained undetermined, although isoenzyme studies and direct measurements of DNA content and complexity suggest diploidy. Direct cytogenetic analysis is not possible, because the chromosomes do not condense at any stage of the cell cycle. We now present evidence from analysis of restriction site polymorphisms in and around three glycolytic enzyme genes (phosphoglycerate kinase, triosephosphate isomerase, glyceraldehyde phosphate dehydrogenase) and the tubulin gene cluster, that trypanosomes of subgenus Trypanozoon are diploid for these housekeeping genes.

Splicing of large ribosomal precursor RNA and processing of intron RNA in yeast mitochondria.

We have studied splicing of precursors to the large ribosomal RNA and processing of the excised intron in yeast mitochondria using primer extension with reverse transcriptase and electron microscopy. Structural features of the following intermediates are described: first, a linear RNA carrying a 5'-terminal G that is not encoded in mitochondrial DNA; second, a circular RNA in which the 3' and 5' intron borders are covalently linked. Three nucleotides of the 5' intron border are absent from the site of circle closure.

Processing of yeast mitochondrial messenger RNAs at a conserved dodecamer sequence.

The yeast mitochondrial genes coding for cytochrome c oxidase subunit I ( COX1 ) and the ATPase subunits 8 and 6 are organized in one transcription unit. Precise mapping of RNA termini with S1 nuclease and primer extension analysis shows that the 3' end of the COX1 mRNA and the 5' end of the ATPase precursor RNA are juxtaposed within a conserved dodecamer sequence (5'- AAUAAUAUUCUU -3'). Sequence comparison reveals that this motif is present downstream of nearly all protein-encoding genes, including extragenic unassigned reading frames ( URFs ) and two URFs located within introns.

Initiation of transcription in yeast mitochondria: analysis of origins of replication and of genes coding for a messenger RNA and a transfer RNA.

The initiation of transcription of the yeast mitochondrial genes coding for subunit I of cytochrome c oxidase (COX1) and for tRNA1Thr has been examined. COX1 messenger RNA synthesis is initiated in a conserved nonanucleotide sequence (ATATAAGTA) which we have previously found immediately upstream of ribosomal RNA genes at positions at which RNA synthesis starts. The 5'-end of the precursor of tRNA1Thr is located in a variant nonanucleotide motif (TTATAAGTA), which may be characteristic for tRNA genes.

In vitro site-directed mutagenesis with synthetic DNA oligonucleotides yields unexpected deletions and insertions at high frequency.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events.

Initiation of transcription of the yeast mitochondrial gene coding for ATPase subunit 9.

We have determined transcriptional initiation sites for the ATPase subunit 9 gene on the yeast mitochondrial genome. Using S1 nuclease mapping, in vitro capping of primary transcripts with GTP and guanylyl transferase, and in vitro transcription analysis with purified mitochondrial RNA polymerase, we find the major site of transcriptional initiation to be at a point 630 nucleotides upstream of the coding region for the gene. In addition, we find much lower levels of initiation at a second site 78 nucleotides downstream of the first.

A nonanucleotide sequence involved in promotion of ribosomal RNA synthesis and RNA priming of DNA replication in yeast mitochondria.

We have examined the initiation of transcription of the mitochondrial genes for ribosomal RNA (rRNA) in the yeast Kluyveromyces lactis and show that these are transcribed independently from individual promoters. The mature large rRNA contains a 5' di- or triphosphate end which can be labelled in vitro with [alpha-32P]GTP using guanylyltransferase and this enabled us to determine the nucleotide sequence of its 5' terminus. For the small rRNA, a minor in vitro capped RNA species hybridizes in the region where--as judged from S1 nuclease protection experiments--the precursor of this RNA starts.

Initiation of transcription of genes for mitochondrial ribosomal RNA in yeast: comparison of the nucleotide sequence around the 5'-ends of both genes reveals a homologous stretch of 17 nucleotides.

The DNA sequence around the beginning of the genes coding for the large and small ribosomal RNAs in yeast mitochondria has been established. In order to determine the 5'-end points of the ribosomal RNAs, DNA fragments were labelled in vitro at a restriction site within each gene and hybridized with ribosomal RNA. The hybrids were then treated with S1 nuclease and the products analysed for size by gel electrophoresis.

Use of a synthetic DNA oligonucleotide to probe the precision of RNA splicing in a yeast mitochondrial petite mutant.

In some strains of Saccharomyces cerevisiae the mitochondrial gene coding for 21S rRNA is interrupted by an intron of 1143 bp. This intron contains a reading frame for 235 amino acids: Unassigned Reading Frame (URF). In order to check whether expression of this URF is required for proper splicing of precursors to 21S rRNA, the precision of RNA splicing was analysed in a petite mutant, where no mitochondrial protein synthesis is possible anymore.

A putative precursor for the small ribosomal RNA from mitochondria of Saccharomyces cerevisiae.

We have characterized a putative precursor RNA (15.5S) for the 15S ribosomal RNA in mitochondria of Saccharomyces cerevisiae. Hybrids were formed with mitochondrial RNA and mtDNA fragments terminally labelled at restriction sites located within the gene coding for 15S ribosomal RNA and treated with S1 nuclease (Berk, A.

Splice point sequence and transcripts of the intervening sequence in the mitochondrial 21S ribosomal RNA gene of yeast.

By S1 nuclease mapping we have located the intervening sequence in the large ribosomal RNA gene of Saccharomyces cerevisiae omega+ strains 570 bp from the 3' end of the rRNA gene. No intervening sequence was detected at this position in S. carlsbergensis, but the sequences of the mature 21S rRNAs of these two strains appear to be identical in this region.

Nucleotide sequence of the mitochondrial structural genes for cysteine-tRNA and histidine-tRNA of yeast.

We have determined the nucleotide sequence of a segment of Saccharomyces cerevisiae mtDNA that contains the structural genes for a cysteine-tRNA and a histidine-tRNA. The genes are approximately 85 bp apart, they do not contain intervening sequences or sequences coding for the 3'-CCA terminus and they are surrounded by nearly pure AT segments. The tRNAs deduced are very AT-rich, 74 and 75 nucleotides long, respectively, and contain one or more unusual features not found in tRNAs from other sources.