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. In 2001, this proved very disruptive to the wider rural economy, such as the recreational and tourism sectors. The policy also caused severe animal welfare problems and psychological problems among farmers and their families. This raised questions about the wider, and not only veterinary or agricultural, implications of control strategies of foot and mouth disease virus (FMDV). The technology seems to be in place for a return to the use of protective vaccination against FMDV during an outbreak, provided the Office International des Epizooties (OIE: World organisation for animal health) and European Commission (EC) receive data that substantiate the reliability of differentiating tests such as the 3ABC enzyme-linked immunosorbent assay (ELISA) for use in individual animals. Research is in progress but may not be able to produce these data until 2003 or 2004. High potency vaccines should be used to elicit sufficient immunity within three to four days. During an FMD crisis, farmers should be assisted to find markets for products from areas affected by FMDV. The human dimension of any FMD outbreak must be dealt with sufficiently in any contingency plan.

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. Modelling of the three-dimensional structures of TIM, PGK and GAPDH using the known structural coordinates of homologous enzymes from other organisms indicates that all three may have in common two 'hot spots' about 40 A apart, which themselves include a pair of basic amino acid residues separated by a distance of about 7 A. The sequence of glycosomal ALDO, for which no three-dimensional information is available, is compatible with the presence of the same configuration on the surface of this enzyme. We propose that this feature plays an essential role in the import of enzymes into glycosomes.

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. By a novel method, suitable for low abundance transcripts, we demonstrate that TIM mRNA contains the 35-nucleotide leader sequence (mini-exon) also found on several other trypanosome mRNAs. The TIM gene and a DNA segment of at least 6 kbp upstream of the gene are transcribed at an equal rate in isolated nuclei, suggesting that the gene is part of a much larger transcription unit. The predicted protein is of the same size as TIMs from other organisms and shares approximately 50% amino acid homology with other eukaryote TIMs, somewhat less with prokaryote TIMs. Trypanosome TIM is the most basic of all TIMs sequenced thus far. This is, in part, due to the presence of two clusters of positively charged residues in the molecule which may act as a signal for entry into glycosomes.

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. It virtually coincides with a highly conserved stretch of nine nucleotides that is found immediately upstream of all transcriptional start sites described thus far. Two different point mutations within this nonanucleotide sequence drastically reduce promoter function. Conversely a single point mutation that results in the formation of a nonanucleotide sequence 99 nucleotides upstream of the large rRNA gene leads to a new, efficient transcription initiation site. MtRNA polymerase can be resolved into two different components by chromatography on Blue Sepharose: one retaining the capacity to synthesize RNA, the other conferring the correct specificity of initiation to the catalytic component.

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. wt of 38.9 kd. This is considerably larger than all other GAPDH proteins studied so far, including the enzyme that is located in the cytosol of the trypanosome. The glycosomal enzyme shows 52-57% homology with known sequences of GAPDH proteins from 10 other organisms, both prokaryotes and eukaryotes. The residues that are involved in NAD+ binding, catalysis and subunit contacts are well conserved between all these GAPDH molecules, including the trypanosomal one. However, the glycosomal protein of T. brucei has some distinct features. Firstly, it contains a number of insertions, 1-8 amino acids long, which are responsible for the high mol. wt of the protein. Secondly, an unusually high number of positively charged amino acids confer a high isoelectric point (pI 9.3) to the protein. Part of the additional basic residues are present in the insertions. We discuss the genomic organization of the genes for the glycosomal GAPDH and the possibility that the particular features of the protein are involved in its transfer from the cytoplasm, where it is synthesized, into the glycosome.

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. Gene B codes for the cytosolic and gene C for the glycosomal isoenzyme. Genes B and C are 95% homologous, and the predicted protein sequences share approximately 45% amino acid homology with other eukaryote PGKs. The microbody isoenzyme differs from the cytosolic form and other PGKs in two respects: a high positive charge and a carboxy-terminal extension of 20 amino acids. Our results show that few alterations are required to redirect a protein from cytosol to microbody. From a comparison of our results with the unpublished data for three other glycosomal glycolytic enzymes we infer that the high positive charge represents the major topogenic signal for uptake of proteins into glycosomes.

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. This result is still compatible with the single copy nature of variant surface glycoprotein (VSG) genes in Trypanozoon, if different VSG genes are present in corresponding positions on paired chromosomes. Using pulse field gradient gel electrophoresis, we show that the genes for the three glycolytic enzymes are all located in very large DNA molecules, but the gene for triosephosphate isomerase is in another fraction from the genes for the other two enzymes. Since all three enzymes are located in glycosomes, which are trypanosome microbodies, the genes for glycosomal enzymes are not all clustered in one chromosomal segment of the trypanosome genome.

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. The properties of these intermediates fit remarkably well into the mechanism of self-splicing described for the ribosomal precursor RNA from Tetrahymena nuclei. A new feature of the yeast mitochondrial system is that the excised intron can have one of two destinies, circularization or cleavage at an internal position.

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. Also the 3' terminus of an RNA species derived from the URF -containing intron of the large rRNA gene maps within such a dodecamer sequence. It is likely, therefore, that this motif serves as a processing point in the generation of mature mRNA. From a comparison of the various transcription units, we infer that RNAs that originate from an endonucleolytic cleavage at this sequence have stable 3' termini, while further processing of the 5' ends occurs. The efficiency of the initial cleavage varies between the different positions at which the motif is present.

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. Using a partially purified fraction of mtRNA polymerase, we demonstrate that RNA synthesis is precisely initiated in vitro in nonanucleotide sequences preceding both ribosomal RNA-, tRNA- and messenger RNA-encoding genes and origins of replication.

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. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.

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. Both initiation sites occur at the same position within a nonanucleotide sequence which we have previously found associated with initiation of rRNA synthesis. This work further supports the notion that this nonanucleotide sequence is an integral component of mitochondrial promoters and indicates that the same RNA polymerase is used for transcription of both mRNA and rRNA in yeast mitochondria.

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. We have determined the DNA sequence around the beginning of both rRNA genes and this reveals the existence of an identical nonanucleotide sequence (5' -ATATAAGTA- 3') just preceding the positions where the rRNAs start. This sequence is identical to the one preceding the rRNA genes in the mtDNA of the distantly related yeast Saccharomyces cerevisiae (Osinga, K.A. and Tabak, H.F. (1982) Nucl.Acids Res. 10, 3617-3626) and supports our proposal that this sequence motif is part of a yeast mitochondrial promoter. We have noticed that the same sequence is located in the putative origin of replication present in hypersuppressive petite mutants of S. cerevisiae and consider the possibility that this sequence is involved in RNA priming of DNA replication.

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. This enabled us to identify where in the determined DNA sequence the 21S ribosomal RNA and the precursor for 15S ribosomal RNA (15.5S rRNA) start, since both transcripts are initiated de novo (Levens et al. (1981) J.Biol.Chem., 256, 5226-5232). Comparison of the DNA sequences around the start points of transcription reveals the existence of a homologous stretch of 17 nucleotides. This conserved sequence may be an essential element of a promoter in mtDNA.

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. We have devised a new assay to monitor the precision of the splicing event. The method is of general application, provided that the sequence of the splice boundaries is known. In the case of the 21S rRNA it involves the synthesis of the DNA oligonucleotide d(CGATCCCTATTGTC( complementary to the 5' d(CGATCCCTAT) and 3' d(TGTC) borders flanking the intron in the 21S rRNA gene. The oligonucleotide is labelled with 32p at the 5'-end, hybridised to RNA and subsequently subjected to digestion with S1 nuclease. Resistance to digestion will only be observed if the correct splice-junction is made. The petite mutant we have studied contains a 21S rRNA with the same migration behaviour as wildtype 21S rRNA. In RNA blotting experiments, using an intron specific hybridisation probe, the same intermediates in splicing are found both in wild type and petite mutant. Finally the synthetic oligonucleotide hybridises to petite 21S rRNA and its thermal dissociation behaviour is indistinguishable from a hybrid formed with wildtype 21S rRNA. We conclude that expression of the URF, present in the intron of the 21S rRNA gene, is not required for processing and correct splicing of 21S ribosomal precursor RNA.

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.J. and Sharp, J.A. (1977) 12, 721-732). Sites of resistant hybrids were measured by agarose gel electrophoresis and end points of RNAs determined. The 15.5S RNA is approximately 80 nucleotides longer than the 15S ribosomal RNA, with the extra sequences being located at the 5'-end. Both 15S ribosomal RNA and 15.5S RNA are fully localised within a 2000 base pair HapII fragment. This putative precursor and the mature 15S ribosomal RNA are also found in petite mutants which retain the 15S ribosomal RNA gene. The petite mutant with the smallest genetic complexity has its end point of deletion (junction) just outside the HapII site located in the 5' flank of the 15S ribosomal RNA genes as determined by S1 nuclease analysis. This leaves a DNA stretch approximately 300 base pairs long where an initiation signal for mitochondrial transcription may be present.

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. By comparing the DNA sequence of the region of the intervening sequence in an omega+ strain with the corresponding sequence in S. carlsbergensis, we have determined the splice points of the 21S rRNA gene. These sequences show no homology with splice points in nuclear and viral genes or with the splice points in the chloroplast 23S rRNA gene of Chlamydomonas. The external borders of the splice points have a complementary sequence in the intervening sequence. The largest transcript hybridizing with the probe of the intervening sequence has a size corresponding to that expected for an rRNA precursor still containing the intervening sequence; the smallest transcript corresponds in size to the intervening sequence itself.

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.