Joannes Sri Widada

Identification and sequence analysis of IS6501, an insertion sequence in Brucella spp.: relationship between genomic structure and the number of IS6501 copies

An insertion sequence (IS) element of Brucella ovis, named IS6501, was isolated and its complete nucleotide sequence determined. IS6501 is 836 bp in length and occurs 20-35 times in the B. ovis genome and 5-15 times in other Brucella species. Analysis of the junctions at the sites of insertion revealed a small target site duplication of four bases and inverted repeats of 17 bp with one mismatch. IS6501 presents significant similarity (53.4%) with IS427 identified in Agrobacterium tumefaciens, suggesting a common ancestral sequence. A long ORF of 708 bp was identified encoding a protein with a predicted molecular mass of 26 kDa and sharing sequence identity with the hypothetical protein 1 of A. tumefaciens and with the transposase of Mycobacterium tuberculosis. IS6501 is present in all Brucella strains we have tested. Restriction fragment length polymorphism of reference and field strains of two species (B. melitensis and B. ovis) was studied using either pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA or hybridization of EcoRI-digested DNA using IS6501 as a probe. The genome of B. melitensis biovar 3 contains about 10 IS copies per genome and field strains of the same species could not be distinguished either by IS hybridization or by XbaI (PFGE) restriction patterns. In contrast, the number of IS copies in the B. ovis genome is around 30 and the different field strains can be differentiated by both methods.(ABSTRACT TRUNCATED AT 250 WORDS)

IMPLICATION OF ANNEXIN 1 IN PHAGOCYTOSIS: EFFECTS OF N-TERMINAL DOMAIN DELETIONS AND POINT MUTATIONS OF THE PHOSPHORYLATION SITE SER-27

Directed mutagenesis, in the form of deletions and point mutations, was used to investigate the regulatory importance of the N‐terminal domain of annexin 1. Wild‐type and mutant forms were fused to green fluorescent protein (GFP) to track their localization and introduced in to J‐774A.1 cells by transfection. The fusion of annexin 1 to GFP at the N‐ or C‐terminal end did not alter the cellular distribution or co‐localization with phagosomes. The effects of mutations were determined according to these characteristics. The prominent effect resulted from S27E mutation which mimics the phosphorylated state of Ser‐27. Although still retaining the granular structures in the cytoplasm, S27E annexin 1 failed to associate with the phagosomal protein complex. This suggests an essential regulatory role of the phosphorylation of residue 27 in annexin 1 function.

Mutational analysis of the putative PLA2-inhibiting sequence of annexin 1

Annexin 1 has been proposed to inhibit phospholipase A2 by direct interaction through a specific amino acid sequence spanning residues 246–254. The possible role of this region was investigated by protein engineering. Three point mutations and a deletion have been performed. The four mutant proteins have been expressed in E. coli, purified and tested for calcium and lipid binding, and for phospholipase inhibition. All mutant proteins conserved the properties of the wild‐type recombinant protein. This result clearly demonstrates that this part of the molecule is not involved in the inhibition of phospholipase A2.

Primary structure of mature SAG1 gene of an Indonesian Toxoplasma gondii and comparison with other strains

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.

Interdependence of phospholipid specificity and calcium binding in annexin I as shown by site-directed mutagenesis

We have mutated the lysine 128 of domain II of annexin I, which flanks a putative calcium-binding loop, into a glutamic acid residue. The properties of the mutated recombinant protein were compared to those of the wild-type recombinant protein. A change in the isotherm of calcium binding in the presence of lipids was observed. A slight decrease in the affinity for lipids was evident. When tested for the vesicle aggregation property, the mutation induced a change in lipid specificity; unlike the wild-type protein, the mutant protein aggregates vesicles containing phosphatidylserine plus phosphatidylethanolamine better than vesicles containing only phosphatidylserine. These experiments are in agreement with a model which suggests that a lipid molecule is inserted into the calcium-binding loop of annexin I and that the conserved lysine residue is involved in the specificity of annexins for anionic phospholipids.

Structural analysis of human skeletal β-tropomyosin produced in E. coli

We have cloned the cDNA coding the beta-tropomyosin of human muscle in an expression vector whose expression depends upon a promotor that can be induced by isopropyl-beta-thiogalactopyranoside. We show that a new protein was synthesized by bacteria containing the engineered plasmid. This protein was heat stable and reacted with antibodies against tropomyosin. We have purified this protein and further identified it by determining its amino acid composition and sequencing the NH2 terminal. Unlike the native muscle tropomyosin, the NH2 terminal is not acetylated and contains a methionine. The circular dichroism spectrum is compatible with 100% alpha-helices. These results show that the protein synthesized in E. coli possesses a native structure.

Quantitative analysis of the selective pressure exerted on homologous proteins

Evolution analysis is used to locate the regions of a protein that are important for its function or structure. The rate of evolution is generally constant for a given family of homologous sequences. From the starting point of this observation, an algorithm is proposed to establish quantitatively the sequence zones where selective pressure is maximal. A program that computes this pressure has been written in PASCAL. Analysis of results on some sequences validate this theoretical approach, and this knowledge can be used as a starting-point for carrying out site-directed mutagenesis.