Evidence for plasmid-mediated chemotaxis ofPseudomonas putidatowards naphthalene and salicylate

A naphthalene (Nap) and salicylate (Sal) degrading microorganism, Pseudomonas putida RKJ1, is chemotactic towards these compounds. This strain carries a 83 kb plasmid. A 25 kb EcoRI fragment of the plasmid contains the genes responsible for Nap degradation through Sal. RKJ5, the plasmid-cured derivative of RKJ1, is neither capable of degradation nor is chemotactic towards Nap or Sal. The recombinant plasmid pRKJ3, which contained a 25 kb EcoRI fragment, was transferred back into the plasmid-free wild-type strain RKJ5, and the transconjugant showed both degradation and chemotaxis. The recombinant plasmid pRKJ3 was also transferred into motile, plasmid-free P. putida KT2442. The resulting transconjugant (RKJ15) showed chemotaxis towards both Nap and Sal. Two mutant strains carrying deletions in pRKJ3 (in KT2442) with phenotypes Nap-Sal+and Nap-Sal-, were also tested for chemotaxis. It was found that the Nap-Sal+mutant strain showed chemotaxis towards Sal only, whereas the Nap-Sal-mutant strain is non-chemotactic towards both the compounds. These results suggest that the metabolism of Nap and Sal may be required for the chemotactic activity.Key words: Pseudomonas putida, plasmid-encoded chemotaxis, naphthalene, salicylate.

Meiotic Transmission Rates Correlate With Physical Features of Rearranged Centromeres in Maize

The centromere of the maize B chromosome was used as a model to study the physical features of a functional centromere. Pulsed-field gel electrophoresis was previously used to determine the organization of a repetitive sequence (referred to as the B-specific repeat) localized in the centromeric region of the maize B chromosome. The centromere is composed mostly of this repeat. In this report, a collection of 25 B chromosome derivatives that suffered from misdivision of the centromere was examined for the content and organization of the B repeat. Meiotic transmission of these derivatives was also determined and compared with rearrangements within the centromere. This analysis revealed that there is a strong correlation between the size of the centromere and meiotic transmission. In addition, the loss of a particular PmeI fragment of 370 kb considerably reduced meiotic transmission. This sequence contains a 55-kb EcoRI fragment that is also present in all but four derivatives. Because the centromere of the maize B chromosome can be divided by successive misdivisions to derivatives with centromeres of <300 kb, it should be possible for artificial chromosomes to be produced in maize.

Panagrellus redivivus ornithine decarboxylase: structure of the gene, expression in Escherichia coli and characterization of the recombinant protein

A Southern blot analysis of the Panagrellus redivivus ornithine decarboxylase (ODC) gene suggests that it is a single-copy gene that resides on a genomic 3.2 kb EcoRI fragment. Phage clones possessing ODC gene sequences were isolated from a genomic EMBL-4 library and purified. The phage DNA inserts were analysed and a 3.2 kb EcoRI fragment containing the entire ODC gene was isolated. The nucleotide sequence analysis of this fragment reveals that the gene is interrupted by two introns of 47 and 49 bp. In the 5´ non-translated region of the gene, putative AP1, VPE2 and c-Myc binding sites were identified. The ODC cDNA was expressed in a bacterial system as a His-fusion protein and the enzyme was purified by Ni2+-chelating affinity chromatography. The subunit molecular mass, as deduced from the cDNA and shown by SDS/PAGE, is 47.1 kDa. On the basis of gel filtration analyses it is shown that the active enzyme is a dimer. The specific enzyme activity was determined to be 4.2 μmol CO2/min/mg protein. The enzyme is dependent on pyridoxal 5-phosphate as a cofactor, and the presence of dithioerythritol or other thiol-reducing agents is essential for maximal activity. The Km value for l-ornithine was determined as 44 μM. The Ki values for putrescine, α-difluoromethylornithine, α-hydrazino-ornithine and α-methylornithine were calculated as 51, 34, 0.34 and 42 μM respectively.

Construction of a DNA probe to detect isoquinoline-degrading bacteria

To facilitate the cloning of DNA encoding isoquinoline degradation an assay was developed that allowed the rapid visual scoring of the isoquinoline degradation phenotype of single colonies. Transposon mutagenesis of one of the isolates, Comamonas acidovorans IQ3, was performed using Tn 5, and nine Isq− mutants deficient in the ability to utilise isoquinoline as the sole nitrogen source were isolated. These mutants were also incapable of utilising the first metabolite of the isoquinoline degradation pathway, 1-hydroxyisoquinoline, as the sole carbon source. For each Isq− mutant, the EcoRI fragment containing the Tn5 insertion was cloned into pBR322. Restriction and Southern analyses of the cloned DNA revealed that of the nine Isq− mutants, six contained Tn5 insertions in a common 8.9-kb EcoRI fragment derived from the wild type, C. acidovorans IQ3. The cloned DNA thought to be involved in the degradation of isoquinoline proved to be specific when used as a probe in colony hybridization to some bacteria possessing the ability to degrade isoquinoline.Key words: DNA probe, isoquinoline degradation, transposon mutagenesis, Comamonas acidovorans.

Chromosomal localization and polymorphisms of ribosomal DNA in oat (Avena spp.)

The 17S/5.8S/26S ribosomal DNA (rDNA) sequences were mapped to the three satellited (SAT) chromosomes in the common hexaploid cultivated oat Avena sativa (2n = 6x = 42, AACCDD genomes). In situ hybridization and Southern hybridization of maize and (or) wheat rDNA probes to DNA from nullisomics derived from the cultivar 'Sun II' allowed the placement of rDNA sequences to the physical chromosomes. A restriction map was produced for the rDNA sequences of 'Sun II' using a maize probe from the transcribed region of the 17S/26S rDNA repeat. The set of rDNA repeats on SAT 2 of 'Sun II' possesses a 10.5-kb EcoRI fragment not found in the rDNA repeats of SAT 1 and SAT 8. This 10.5-kb fragment results from the absence of an EcoRI site in the intergenic spacer (IGS) of SAT 2 repeats. Extensive polymorphisms were demonstrated for three hexaploid Avena species, namely, the Mediterranean-type cultivated oat A. byzantina and the wild species A. sterilis and A. fatua. However, geographically diverse A. sativa cultivars displayed little rDNA variation. In contrast with all of the A. sativa cultivars examined, the A. sterilis accessions generally lacked the 10.5-kb EcoRI fragment. The results support the hypothesis that A. sativa accessions descend from a limited ancestral cultivated population. The rDNA polymorphisms are attributed to differences in lengths and restriction sites of the IGS.Key words: oats, rDNA, RFLPs, nullisomics, in situ hybridization.

A gene for a myosin peptide is disrupted by the inv(16)(p13q22) in acute nonlymphocytic leukemia M4Eo

Chromosome 16 aberrations are well known in acute nonlymphocytic leukemia (ANLL). The most frequent chromosome 16 aberration in ANLL subtype M4Eo is the inv(16)(p13q22). Recently, we showed that in 5 inv(16) patients with ANLL M4Eo the short arm breakpoints are clustered within a 14-kb genomic EcoRI fragment. We report here the identification of a gene situated in the 14-kb fragment. The gene, which codes for a myosin peptide, is disrupted by the inversion of chromosome 16 in the 5 patients. To the best of our knowledge, this is the first report of a myosin gene disrupted in leukemia.