Abstract. Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals, yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif. We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian, plant, and gramnegative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian clade.
cDNA clones for mRNA sequences regulated by isoprenaline in mouse parotid glands were identified by differential colony hybridisation and all hybridised to a diagnostic proline-rich protein (PRP) oligonucleotide. They were divided into two cross-hybridisation groups, A and B, which were shown by hybrid-selected translations to encode acidic PRP and basic PRP, respectively.The A-type subgroup consisted of sequences homologous to the previously identified mouse PRP genes MP2 and MP3. The B-type subgroup comprised clones for the previously identified cDNA pUMP125 (MP4) as well as other PRP sequences. Six of the B-type clones contained a novel PRP cDNA (MPS) and these were sequenced. The composite MP5 cDNA was 897 nucleotides long and contained an open reading frame capable of encoding a 260-residue-long salivary PRP precursor (30% Pro, 19% Gln and 18% Gly), containing nine variant repeat units of consensus PGNQQGP-PPQGGPQQ(GPP)R(PPQ). MP5 was 80% identical to the sequence of MP4 and had a high degree of similarity (60%) at its 3'-untranslated region to rat salivary glutamate/glutamine-rich protein (GRP) cDNA. Two MP5 clones contained a 273-bp intron-like insertion in the 3' untranslated region, being derived, therefore, from incompletely spliced MP5 transcripts.Northern blotting showed that, although PRP mRNA species were induced by isoprenaline, a B-type PRP mRNA was present in normal parotid glands. RNA dot-blots probed with PRP-genespecific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.Multiple salivary proline-rich proteins (PRP) [l] are induced by dietary plant polyphenols in rats and mice [2, 31. PRP bind polyphenols and protect rats against the toxic effects of polyphenols [4]. The induction of salivary PRP can be mimicked by treatment of rodents with the P-adrenergic agonist isoprenaline [2, 31.The multiple PRP species are encoded by a multigene family and in humans both differential RNA splicing and proteolytic cleavages generate more than 20 PRP from only six genes [5].In mice, induction of PRP by isoprenaline is due, at least in part, to increased transcription of PRP genes in parotid acinar cells [6]. Isoprenaline activates P-adrenoceptors and indirect evidence suggests that CAMP is the intracellular messenger [7], although the precise details of how induction occurs are still undetermined.Evidence from in vitro translations shows that in Balb/C mice there are six PRP mRNA species which are induced by isoprenaline, two of which encode acidic or A-type PRP and four of which encode basic or B-type PRP [8]. There are also at least two distinct constitutive PRP [9].
A mouse genomic B-type proline-rich protein (PRP) cosmid clone was isolated by cDNA hybridisation and mapped, the gene region was subcloned and 3770 bp were sequenced. This gene (MP4) contained three introns and encoded a 1020-nt (nt, nucleotide) mRNA for a PRP precursor 300 amino acids long arranged with 11 imperfect 18-residue proline-rich repeats. The transcriptional start point was determined by S1 nuclease mapping and primer extension to be 26 bp downstream of a TATAA sequence. Sequence comparisons revealed that only two regions from positions -650 bp --30 bp were highly conserved in all other PRP genes, PRP boxes 1 and 2. Box 1 at positions -112 to -135 contained ets-like and rel/NFkB-like elements and was 74% conserved over 23 bp. Box 2 at positions -33 --51 was 53% conserved over 19 bp. A search of the EMBL and GenBank sequence libraries indicated that PRP box 1 was only present upstream of the known mammalian PRP gene sequences and was absent from other genes. These conserved sequences may thus be relevant to the tissue-specific and P-adrenergic regulation of PRP gene transcription.Mammals contain multiple salivary proline-rich protein (PRP) genes arranged as a single locus which is on chromosome 8 in mice and is on chromosome 12 in humans [l]. The PRP genes occur as two sub-families termed H and B in humans [2] and A and B in rodents [3]. The genes are expressed in salivary glands and the submucosal glands of the trachea and nasopharyngial tract [4]. In mouse and rat the amounts of PRPs are normally low but are increased by feeding the animals polyphenols and this is mimicked by 8-adrenergic stimulation [5]. PRPs serve important dental functions, protect animals against toxic polyphenols in the diet and also influence the visco-elastic properties of the mucus [4]. P-Adrenergic induction is due to an increase in PRP gene transcription [6] but the mechanism for this is unknown although PRP gene expression was regulated in a hamster parotid cell culture by treatment with dibutyryl cyclic AMP, suggesting that the genes are under CAMP control in vivo [7]. However, induction of PRP mRNA only occurs after a 4-h lag [6, 71 and was cycloheximide sensitive suggesting that the synthesis of a protein was required for induction [7]. Thus the PRP genes fall into the category of group 3 CAMP-responsive genes which show slow induction, cycloheximide sensitivity and do not contain known CAMP-responsive elements in their 5' flanking regions [8].The promoter and upstream regions of five PRP genes have been sequenced so Far; two extremely closely related variants from human (Hl and H2,[9]) and mouse (A-type MP2, [lo] and A-type M14, [11]) and one hamster gene (Btype H29,[12]). Elements important to the common regulation of PRP genes would have been conserved through evolution of the PRP family, thus we cloned and sequenced the first example of a mouse PRP B-type PRP gene, determined the transcriptional start site and compared its upstream regions with the mouse A-type genes, the hamster B-type gene and the human H-type g...
The gene encoding the human transketolase enzyme (TKT) was localized by fluorescence in situ hybridization to normal and FRA3B human chromosomes. Southern blot analysis of a series of human × mouse and human × hamster hybrid cell lines confirmed this localisation. TKT maps to 3p 14 and distal to FRA3B, localizing TKT to 3p14.3.
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