Aniko Pay

An alfalfa cDNA encodes a protein with homology to translationally controlled human tumor protein.

In mouse tumor cell lines, an abundant mRNA species was found to occur largely as untranslated mRNP particle. The corresponding cDNA sequence was isolated from a cDNA library derived from mouse cells [ 1 ] and subsequently from human mammary carcinomas [2]. The two putative proteins are highly identical (96 ~o) and encode proteins of 21 kDa. Here, we report the isolation and characterization of an alfalfa cDNA clone which has considerable homology to these proteins. The alfalfa clone was isolated from an alfalfa cDNA library prepared from suspension culture cells which were induced to form somatic embryos [3]. The probe used for screening was a PCR fragment with homology to phosphoprotein phosphatases (unpublished results). The complete nucleotide sequence with the predicted amino acid sequence is shown in Fig. 1. Comparison of the predicted protein sequence with the SWISS PROT data bank revealed homology over the entire length of 157 amino acids to mouse and human translationally controlled tumor proteins (42.7~o and 40.8~o identities, respectively). When conserved amino acid exchanges were taken into consideration a similarity score of 78.3 ~o was obtained for both mammalian proteins. Figure 2 shows the sequence alignment of the putative alfalfa and mouse proteins. Although the plant sequence appears to lack the first ten amino acids, the two proteins are highly similar over the whole sequence, including the very carboxyl end. Taken together, the data suggest that the alfalfa and the mammalian proteins could be either homologues of each other or belong to a family of closely related proteins. Unfortunately, nothing is known about the function of the translationally controlled human tumor proteins. Since this protein appears to be highly conserved during evolution, it can be expected to be also found in yeast cells. Here, a genetic approach might reveal some unexpected scientific treasures.

Isolation and characterization of a phosphoprotein phosphatase type 2A gene from alfalfa

Phosphoprotein phosphatases are central regulatory components of the cell cycle in eukaryotes. We report the cloning and sequencing of an alfalfa phosphoprotein phosphatase type 2A (pp2aMs) cDNA. The predicted protein sequence shows high similarity to PP2A from Brassica napus, rabbit and Drosophila. No changes in pp2aMs mRNA abundance during the cell cycle were found. During growth of a batch cell culture, mRNA levels decreased gradually. In planta, all organs contained pp2a transcripts but maximal mRNA levels were detected in stems. Since Southern analysis indicated the presence of a small pp2a gene family in alfalfa, it appears that different subtypes may have specialized roles in various tissues and developmental situations which await characterization.

Isolation and sequence determination of the plant homologue of the eukaryotic initiation factor 4D cDNA from alfalfa, Medicago sativa

Eukaryotic translation initiation factor 4D (elF4D) is a protein of 16-18 kDa. The precise function of elF4D in protein synthesis is not known. It appears to be involved either in ribosomal subunit joining or in the formation of the 80S initiation complex [ 1 ]. In mammals, e lF4D is present in considerable amounts in all tissues [2, 3] and is the only protein known to be posttranslationally modified to contain a hypusine (N%(4-amino-2-Hydroxybutyl)lysine) which is required for biological activity [4-6]. Recently, the cDNAs of the human, rabbit, yeast and slime mould elF4D proteins have been isolated [7-9]. Here, we report on the isolation of the first e lF4D cDNA clone from the plant kingdom. The elF4D-Ms cDNA clone was fortuitously isolated from an alfalfa cDNA library prepared from suspension culture cells that had been challenged for 48 h with 100/~M 2,4-dichlorophenoxy-acetic acid to induce somatic embryogenesis [10] (kindly provided by J. GyOrgyey). The probe used for screening was a PCR fragment with homology to phosphoprotein phosphatases (unpublished results). The length of the elF4D-Ms clone is 742 nucleotides. Since preparation of the cDNA library was done by GC-tailing into pGem2, the cDNA inserts are flanked by two GC tails that can often not be sequenced directly. Subcloning leading to only one GC tail per clone can overcome this problem. Therefore, two 350 and 320 nt long Hind III fragments were subcloned into pTzl8 and were sequenced from both sides. The remaining 94nt long insert was religated in pGem2 and was also sequenced. The complete nucleotide sequence with the predicted amino acid sequence is shown in Fig. 1. A computer-assisted search in the Swiss Prott databank for amino acid homologies revealed significant similarity to eIF4D proteins from human, rabbit, yeast and Dictyostelium. Interestingly, the yeast eIF4D showed a considerably higher identity score, i.e. of 58.7 ~o than both the human and rabbit proteins (both 50.6~o identity). Figure 2 shows a sequence alignment of the plant and yeast protein sequences. Among all proteins, the most conserved region consists of a sequence of 12 amino acids at position 46 to 57. This region embeds the post-translational modification site of the lysine residue to hypusine (position 51) that is crucial to eIF4D activity. Another interesting fea-

The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41

SummaryRestriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temparate phage 16-3 have been established. Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, ‘evenly’ distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome. All the restriction maps have been fitted to each other by computer optimalization. Beyond the conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.

Isolation and characterization of phosphoprotein phosphatase 1 from alfalfa

Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

Heterozygosis of phage 2-3 of Rhizobium meliloti: Moderate level of mismatch repair or gene conversion

SummaryAnalysis of clear/turbid mottled (heterozygotic plaques) of Rhizobium meliloti temperate phage 16-3 indicates that the efficiency of repair at three sites (ti3, ti4, and ti5) in the C cistron is 2 to 20-fold less than that observed in E. coli phage λ. In agreement with this conclusion, heterozygotic plaques were observed at similar frequency in crosses where point and small delection mutants were combined, suggesting that in Rhizobium, DNA molecules with short single-stranded loops can escape from repair as efficiently as the simple mismatches.

Recombination deficient mutants of Rhizobium meliloti 41

SummaryTwo mutants deficient in homologous genetic recombination have been isolated from Rhizobium meliloti 41 after Tn5 mutagenesis. Both mutants are defective in the induction of temperate phage 16-3 by UV-light, Mytomycin-C or Bleomycin, their UV sensitivity is more pronounced than that of the wild-type strain, and they lack the ‘SOS activity’ responsible for induced mutations.