A466
895
Biochemical Society Transactions (2000) Volume 28, Part 5
Molecular studies of drug resistant Mycobacterium tuberculosis in Latvia
V.Baumanis*, T.PanEuka*, I.Jansone*, L.Broka***, V.Leimane***, O.Marga** *- Biomed. Res. & Study Centre, Univers. of Latvia, Ratsupites I , Riga, Latvia, LV-1067, **- Med. Academy of Latvia, ***- Tub.Center of Latvia (TC)
The problem of tuberculosis (TB) became actual in the Baltic Republics due to changes in the economical and political life. TB incidence has been increased in Latvia from 27,4 per 100.000 population in 1990 till 74,O in 1998. Antibiotic resistance is another problem. The primary and total drug resistance in 1998 reached 32,3% and 49,4% respectively. Considerably, that in 1998 primary multidrug resistance was increased to 8%. Smear microscopy and BACTEC cultivation are the mainly used methods in Latvia for TB laboratory diagnostics. Modem molecular approaches are lygase chain reaction of 38kDa protein gene (Abbott) and amplification of IS6110 fragment by in-house polymerase chain reaction (PCR), where 245bp labelled fragment has been applied to restriction fragment length polymorphism (RFLP) analysis to sensitive and drug resistant Mycobacteria strains as well. RFLP patterns of these strains were analyzed in comparison with data obtained by studying rifampin resistance using INNO-LiPA test. From 10-15 better known today resistance determining genes, more detailed are studies of RNA polymerase subunit encoding rpoB gene responsible for rifampin resistance. INNO-LiPA test is used for detection of most frequently common mutations in rpoB gene 260bp fragment. From resistant strains studied mutations AspS16Va1, Ser53 1Leu and Leu533Pro were detected more often. Another approach for mutation detection in rpoB gene is single strand conformation polymophism (SSCP) which was applied as well. Mutations in katG gene leading to izoniasid resistance have been studied using PCR and enzyme digestion techniques. Results were compared with rifampin resistance.
1ag6 Exgresston, purification. charactensatlon. and reCOnSUtuUon of the large an small subunits of Yeast acctohydroxyacid synthase . Pane S.S.,Duggleby R.G. Centre for Protem Structure, Function and Engineering, Department of Biochemistry, The University of Queensland, Brisbane Qld 4072 ~
Acetohydroxyacid synthase (AHAS) catalyzes the first reaction that is common to the branched-chain amino acid biosynthesis. The enzyme is able to catalyze two slightly different reactions yielding 2-acetolactate or 2-aceto-2-hydroxybutyate. The former product is the precursor for valine and leucine biosynthesis, while the latter leads to isoleucine. This enzyme is also the target site for several families of herbicides including sulfonylureas and imidazolinones. AHAS activity is found in bacteria, fungi and plants. Biochemical and genetic analysis have shown that the prokaryotic enzymes have a a& structure. consisting of two catalytic large subunits and two small subunits that have a stimulator)' and regulatory role conferring sensitivity to branched-chain amino acid inhibition. Several AHAS genes from yeasts and lants, homologous to the bacterial large subunit, have been clonef and expressed in Escherichia coli. The expressed enzymes function as homodimers and show no end-product feedback regulation. The evidence for a regulatory subunit in eukaryotes is largely indirect and circumstantial. We investigated the possibility that the yeast open reading frame (ORF) YCLOO9c is an AHAS small subunit. In vivo studies show that ORF YCuM9c is expressed in yeast, and the gene product targeted to and processed in the mitochondria, where the biosynthetic pathway occurs. This putative small subunit protein (ILV6) and !he catal 'c subunit of yeast AHAS (ILV2) were each over-expressed in E. c o p a n d purified to near homogeneity. The pure catalytic subunit alone is active but when combined with the regulatory subunit at high concentrations of potassium phosphate ( E 1 M)at neutral pH results in a 10-fold increase in the specific activity. The reconstltuted enzyme also acquires sensitivity to inhibition by valine (K,,pp c lo4 M). Various valine analogs, including leucine and isoleucine, have little or no effect. We also discovered that the valine-inhibited enzyme is activated by ATF'. Neither the large subunit alone, nor the reconstituted enzyme in the absence of valine is affected by ATF'. This effect is quite specific and is not mimicked by AMP, ADP,, E-ATP, GTP,, CW or UTP. CD spectral analysis shows that ATP IS capable of blnding directly to the small subunit and the association is MgZ+dependent.
0 2000 Biochemical Society
Identification of some parameters, responsible for halophily in black
897 yeast Hortaea werneckii Y.Petrovicl, N.Gunde-Cimerman*,
A. Plemenitasl 'Institute of Biochemistry, Medical Faculty, University of Ljubljana, Vrazov trg 2,1000 Ljubljana, Slovenia; 2Department of Biology, Biotechnical Faculty, University of Ljubljana, Vema pot I l l , 1000 Ljubljana, Slovenia. (
[email protected])
Halophilic organisms of Archaea and Bacteria are well known and characterised. However, halophilic representatives of the third domain, Eukaryota, are much less studied. Recently halophilic fungi have been isolated from their natural habitat and characterised, according to their ecophysiology, as halophiles rather than halotolerants. Taxonomically, they belong to a group of black yeasts. To obtain somewhat general picture about the molecular basis of halophily in eukaryotes, differential display method was used to detect differences in mRNA expression in the cells of halophilic black yeast Hortaea werneckii, grown under different conditions. Messenger RNA expression from cells grown in optimal salinity, in hyposaline, and in hypersaline environment, respectively, was compared, and some differences were observed. Using this approach, hydroxymethyl-glutaryl coenzyme A reductase, a key regulatory enzyme of sterol biosynthesis, was identified as a protein which responded to both hypo- and hyperosmotic stresses. Changes in environmental salinity affected its transcription, degradation, and activity. Thus we were able to identify one part of adaptations to high salt content on a biochemical level and we will employ this same approach to study also other proteins involved. The observed differences in mRNA expression are the first step towards identification of the pattern of mechanisms that represent adaptation to different salinities in Hortaea werneckii - in other words, enable its halophily. This may also be the first step towards the possibility to transfer tolerance to high salt content of the environment to other, non-adaptable, eukaryotic organisms, which could be of a great biotechnological importance.
1898 The exceptional properties of extremophilic NDP kinase. Polosina Ya.Yu., Zamyatkin D.F., Kostyukova A.S., Filimonov V.V.,Fedorov O.V. Institute of Protein Research, Russian Academy of Sciences, Pushchino, 142292 Russia.
The proteins of halophilic archaea are highly adapted to function at high ionic strength. Because of this extreme saline environment, halophilic proteins represent a valuable resource for understanding processes of natural selection and adaptive evolution. We isolated and characterised the NDP kinase from haloalkaliphilic archaeon Narronobacterium magadii. The main function of NDP kinase is to exchange y-phosphates between nucleoside triphosphates and diphosphates. It is a cytosolic protein forming a hexamer similar to eucariotic NDP kinases. MW of monomer is 23000. At natural salt conditions the NDP kinase is more thermostable than eucaryotic and bacterial homologs and, unlike other kinases, the natronobacterial protein unfolds reversibly. We have studied kinase structure and functional stability dependence on following parameters: temperature, pH, ionic strength, urea concentration and Iigand presence. It was shown that kinase keeps its functional activity and structure at NaCl concentration from 90 mM to 3.5 M; KCI concentration from 3M to at least 0.5 M; pH range from 5.0 to 12.0; urea concentration up to at least 6M,temperature up to 95OC.
