To investigate the genetic basis of differing thermotolerance in the closely related species Drosophila virilis and Drosophila lummei, which replace one another along a latitudinal cline, we characterized the hsp70 gene cluster in multiple strains of both species. In both species, all hsp70 copies cluster in a single chromosomal locus, 29C1, and each cluster includes two hsp70 genes arranged as an inverted pair, the ancestral condition. The total number of hsp70 copies is maximally seven in the more thermotolerant D. virilis and five in the less tolerant D. lummei, with some strains of each species exhibiting lower copy numbers. Thus, maximum hsp70 copy number corresponds to hsp70 mRNA and Hsp70 protein levels reported previously and the size of heat-induced puffs at 29C1. The nucleotide sequence and spacing of the hsp70 copies are consistent with tandem duplication of the hsp70 genes in a common ancestor of D. virilis and D. lummei followed by loss of hsp70 genes in D. lummei. These and other data for hsp70 in Drosophila suggest that evolutionary adaptation has repeatedly modified hsp70 copy number by several different genetic mechanisms.

The sequencing of the moss Physcomitrella patens genome has facilitated studies of the plant proteome. To develop a proteome reference map based on the genome sequence, we conducted 2D electrophoreses of proteins extracted from moss protoplasts, protonemata, and gametophores grown under standard conditions on Petri dishes. On silver-stained gels, depending on the developmental stage of the moss, we resolved from 500 to 600 protein spots that were then excised and digested by trypsin, and 212 proteins were identified by PMF-MALDI-TOF. To enhance the proteome coverage, we performed 1D SDS-PAGE with subsequent separation of tryptic peptides derived from digested gel band slices by LC-ESI-MS/MS. The proposed approach allowed us to identify 186 proteins had not been determined by 2D PMF-MALDI-TOF. Proteins identified by both methods were categorized using a system of clusterization of orthologous genes as metabolism (26%), cellular processes and signaling (16%), and information storage and processing (7%). Proteome analysis by differential gel electrophoresis revealed moderate differences between filamentous protonemata and leafy shoots. Surprisingly, protoplasts isolated from protonema filaments displayed significant differences in protein composition compared with both protonemata and gametophores.

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The interaction of acetyl-CoA fragments with rat liver acetyl-CoA carboxylase has been studied. Dephosphorylated acetyl-CoA did not actually differ from acetyl-CoA in its substrate properties. Non-nucleotide analogues of the substrate, S-acetylpantatheine and it's 4'-phosphate, also possess substrate properties (V,,, = 1.5% and 15% of the maximal rate value of acetyl-CoA carboxylation, respectively). The nucleotide fragment in the acetyl-CoA molecule produces a marked effect on the thermodynamics of the substrate-enzyme interaction, and is apparently involved in activation and appropriate orientation of the acetyl group in the active site. The better substrate properties of S-acetylpantetheine 4-phosphate and the inhibitory properties of pantetheine 4'-phosphate, compared to the unphosphorylated analogues, evidence an important role of the 5'-P-phosphate of 3'-phosphorylated ADP residue in acetyl-CoA binding to the enzyme.Acetyl-CoA carboxylase catalyzes the first step in the pathway of long-chain fatty acid biosynthesis and plays a critical role in the regulation of this process [l, 21. CoA modulates the enzyme activity in the cell, though binding to the specific kinase of carboxylase [3] and directly activating carboxylase at low concentration [4]. The long-chain acyl-CoA are effective competitive inhibitors of the enzyme which assist in the dissociation of the active polymeric form of the enzyme 151. A CoA precursor, pantethine, has found a wide application as a hypolipidaemic drug [6]. According to our data, the 4'-phosphate of D-pantothenic acid possesses a similar activity. Data are available featuring peculiarities of the interaction of CoA, its analogues and precursors with CoA-dependent enzymes [7 -91. However, no data have been reported yet on the specificity of acetyl-CoA carboxylase toward any CoA precursors. The aim of this work was to study the interaction of acetyl-CoA carboxylase with a successive series of CoA precursors and their S-acetyl derivatives, which would allow the evaluation of the individual contributions of different fragments of the CoA molecule to interaction with the enzyme. MATERIALS AND METHODSThe following reagents were used in the study: ATP, phenylmethylsulphonyl fluoride, EDTA from Serva; tris(hydroxymethyl) aminomethane, dithiothreitol, bovine serum albumin (fraction V), tosyl-2-phenylalaninechloromethane, tosyl-L-lysinechloromethane, D-biotin, CoASH, acetyl-CoA, cyanogen bromide purchased from Sigma; avidin from Biolar (USSR), CI-Sepharose 4B from Pharmacia, potassium Dpantothenate and potassium D-pantothenate 4'-phosphate Synthesis of acetyl-CoA and its precursorsD-Pantetheine 4-phosphate was obtained as described previously [8]. Acetyl-CoA, S-acetylpantetheine, and S-acetylpantetheine 4-phosphate were synthesized by acetylation with acetic anhydride (in the latter two cases, after reducing the corresponding disulfides by NaBH4) [lo]. S-Acetylcysteamine was prepared from 2-mercaptoethylamine hydrochloride and acetyl chloride by the procedure of Foyl et al. 1111. Enzyme p...

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