Miroslav M. Konstantinovic

The penicillin amidase of Arthrobacter viscosus (ATCC 15294)

The nucleotide (nt) sequence of the gene encoding penicillin G amidase (PA) of Arthrobacter viscosus strain ATCC 15,294 was determined. The sequence contained an open reading frame of 2406 nt with a G+C content of 37%. The deduced amino-acid sequence shows significant homology with other so far identified beta-lactam amidases of Gram- bacteria.

The primary structure of Providencia rettgeri penicillin G amidase gene and its relationship to other gram negative amidases

The nucleotide sequence of Penicillin G amidase (PA,E.C.3.5.1.11) of Providencia rettgeri was determined. We aligned our P. rettgeri PA with other known Gram negative periplasmically located beta-lactam amidases. The analysis revealed a high homology with other Enterobacteric amidases (60%-65%), while with similar Pseudomonas sp. amidases the homology exceeded 25%. These homologies indicate their common ancestry.

The Metallothionein‐Like Gene from Buckwheat: Structural and Functional Analysis of the Promoter Region1

The buckwheat (Fagopyrum esculentum Moench) metallothionein‐like gene was cloned and its 5′ regulatory region was examined. Computer analysis of this region using overlapping data from three different databases predicted the existence of regulatory sequences that could be involved in responses to different hormonal and external stimuli (ERE, heat shock‐HSE, light and stress‐GT‐1, I‐box, GATA, G‐box, metal‐MRE) as well as the presence of putative binding sites for plant‐specific transcription factors (Dof1, NtBBF1, Athb‐1). Further investigation included analysis of the interactions of distal and proximal parts of the defined regulatory region and purified Dof1ΔC domain of maize Dof1 and the HD‐Zip‐1 domain of Arabidopsis thaliana Athb‐1 transcription factors as well as with buckwheat leaf nuclear extract. The identity of putative Dof1‐ and Athb‐1‐binding sites was confirmed in the proximal region. More important, we found that some proteins from buckwheat leaf nuclear extract compete for the Dof1‐binding sites, indicating the presence of a similar protein type in that extract. We also showed that buckwheat leaf nuclear extract itself interacted with both proximal and distal promoter regions. Interaction with the proximal region resulted in the formation of a single HMW complex, while five separate complexes with the distal region were detected, indicating interaction with predicted G‐ and I‐boxes, which are probably involved in light‐ and/or stress‐regulated MT3 gene expression. The promoter ability of the cloned buckwheat MT gene regulatory region was characterized by measuring the activity of the GUS reporter gene in the leaves of transgenic tobacco. Buckwheat MT3 promoter activity was also analyzed for its response to stress produced by either hydrogen peroxide or UV treatment. We found that both treatments increased GUS activity.