The frequency of two-base tracts in eukaryotic genomes

The frequency of two-base tracts is surveyed in a wide range of eukaryotic genomes using the special program TRACTS. All three two-base families are surveyed: R.Y (A,G.C,T), K.M (A,C.G,T), and S;W (A.T and G.C). Data for the human β-globin complex, for the tobacco chloroplast, and for 247 nt mammalian promoter regions are presented. All two-base tracts longer than three or four bases are overrepresented to an extent surpassing by far their occurrence in a randomized DNA population in the majority of the genomic regions analyzed; 20–30 long tracts are quite frequent, against the statistical odds. R.Y tracts are found at the largest excess, K.M tract to a slightly lesser extent, while S.W tracts are found at a moderate yet significant excess. The majority of the tracts manifest only a limited extent of tandem repeat structures. The idea that the two base tracts serve as unwinding elements is considered. Preseented at the NATO Advanced Research Workshop onGenome Organization and Evolution, Spetsai, Greece, 16–22 September 1992     

Occurrence of oligopurine.oligopyrimidine tracts in eukaryotic and prokaryotic genes.

A program to analyse the length and frequency distribution of specific base tracts in genomic sequences is described. The frequency of oligopurine.oligopyrimidine tracts (R.Y. tracts) in a data base of 163 transcribed genes is analysed and compared. The complete genomes of SV40 virus, N. tobacum chloroplast, yeast 2 micron plasmid, bacteriophage lambda, plasmid pBR322 and the E. coli lac operon are also analyzed. A highly significant overrepresentation of oligopurine and oligopyrimidine tracts is observed in all eukaryotic genes examined, as well as in the chloroplast genome. The overrepresentation is evident in all gene subregions of the chloroplast, in the following order: intergenic regions, 3' downstream and 5' upstream (promoter), 5' and 3' untranslated, introns and coding regions. In genes coding for basic proteins, oligopurine rather than oligopyrimidine tracts are found on the coding stand. In prokaryotic genes only the longest R.Y. tracts (greater than or equal to 12) are found in excess, and are concentrated near regulatory regions. While a structural role for R.Y. tracts is most likely in intergenic regions, a functional role, as initiation sites for strand separation, is proposed for regulatory gene regions.     

TRACTS: A program to map oligopurine.oligopyrimidine and other binary DNA tracts

A program to map the locations and frequencies of DNA tracts composed of only two bases ('Binary DNA') is described. The program, TRACTS (URL http://bioportal.weizmann.ac.il/tracts/tracts.html and/or http://bip.weizmann.ac.il/miwbin/servers/tracts) is of interest because long tracts composed of only two bases are highly over-represented in most genomes. In eukaryotes, oligopurine.oligopyrimidine tracts ('R.Y tracts') are found in the highest excess. In prokaryotes, W tracts predominate (A,T 'rich'). A pre-program, ANEX, parses database annotation files of GenBank and EMBL, to produce a convenient one-line list of every gene (exon, intron) in a genome. The main unit lists and analyzes tracts of the three possible binary pairs (R.Y, K.M and S;W). As an example, the results of R.Y tract mapping of mammalian gene p53 is described.     

In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region

Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M). In vivo activities for all mutants were characterized with UV-sensitive E. coli. The results showed that substitution of Thr-127 with Met or Lys-130 with Leu did not have an effect on the survival of the bacteria but substitution of aromatic amino acids (128-132) had various effects on survival.     

Analysis of tryptophan fluorescence lifetimes in a series of human serum albumin mutants with substitutions in subdomain 2 A

Tryptophan 214, the only tryptophan residue in human serum albumin, is located in the physiologically important subdomain 2A ligand binding site. In the present study the fluorescence lifetime of tryptophan 214 in the following human serum albumin (HSA) mutants with substitutions in subdomain 2A were determined: K195M, K199M, F211V, R218M, R218H, R218A, R222M, H242V, and R257M. An HSA mutant in which tryptophan was moved from subdomain 2A to subdomain 3A (W214L/Y411W) was also examined. Additionally, the fluorescence lifetime of tryptophan 214 in an HSA fragment consisting of subdomains 1A, 1B, and 2A (1A-1B-2A HSA) was determined. For those species expected to have the most dramatic changes in tryptophan microenvironment, W214L/Y411W and 1A-1B-2A HSA, clear changes in tryptophan lifetimes were observed. Significant changes were also seen for those species with mutations at position 218, which is next to tryptophan in the X-ray structure of HSA. However, significant changes were also observed for H242V and R257M, which contain substitutions at positions not immediately adjacent to tryptophan 214, highlighting the conformational flexibility of subdomain 2A.     

Role of alpha-helix seven of Bacillus thuringiensis Cry1Ab delta-endotoxin in membrane insertion, structural stability, and ion channel activity

Domain I of the Cry1Ab insecticidal toxic protein has seven alpha-helices and is considered to be involved in the ion channel activity. While other alpha-helices, particularly alpha-4 and alpha-5, have been extensively explored, the remaining alpha-helices have been slightly studied. Site-directed mutagenesis was used to generate mutations throughout sequences encoding the alpha-helix 7 to test its role in ion channel function. Every amino acid residue in alpha-helix 7 was mutated to alanine. Most resultant proteins, e.g., D225A, W226A, Y229A, N230A, R233A, R234A, D242A, and F247A yielded no protoxin or were sensitive to degradation by trypsin or Manduca sexta midgut juice. Other mutant proteins, R224A, R228A, and E235A, were resistant to degradation to the above proteases but were 8, 30, and 12 times less toxic to M. sexta, respectively, than the wild-type Cry1Ab. Circular dichroism spectroscopy indicated a very small change in the R228A spectrum, while R224A and E235A display the same spectrum as the wild-type protein. These three mutant proteins showed little differences from Cry1Ab when analyzed by saturation binding and competition binding kinetics with (125)I-labeled toxin or by surface plasmon resonance to M. sexta brush border membrane vesicles. More conservative amino acid substitutions were introduced into alpha-helix 7 residues: R228K, F232Y, E235Q, and F247Y. In comparison with wild-type Cry1Ab, mutant proteins R228K, F232Y, E235A, and E235Q selectively discriminate between K+ and Rb+, while R224A and R228A had reduced inhibition of short-circuit current for both ions, when analyzed by voltage clamping of M. sexta midguts.     

Drug resistance-conferring mutations in Mycobacterium tuberculosis from Madang, Papua New Guinea

ABSTRACT: BACKGROUND: Monitoring drug resistance in Mycobacterium tuberculosis is essential to curb the spread of tuberculosis (TB). Unfortunately, drug susceptibility testing is currently not available in Papua New Guinea (PNG) and that impairs TB control in this country. We report for the first time M. tuberculosis mutations associated with resistance to first and second-line anti-TB drugs in Madang, PNG. A molecular cluster analysis was performed to identify M. tuberculosis transmission in that region. RESULTS: Phenotypic drug susceptibility tests showed 15.7% resistance to at least one drug and 5.2% multidrug resistant (MDR) TB. Rifampicin resistant strains had the rpoB mutations D516F, D516Y or S531L; isoniazid resistant strains had the mutations katG S315T or inhA promoter C15T; streptomycin resistant strains had the mutations rpsL K43R, K88Q, K88R), rrs A514C or gidB V77G. The molecular cluster analysis indicated evidence for transmission of resistant strain. CONCLUSIONS: We observed a substantial rate of MDR-TB in the Madang area of PNG associated with mutations in specific genes. A close monitoring of drug resistance is therefore urgently required, particularly in the presence of drug-resistant M. tuberculosis transmission. In the absence of phenotypic drug susceptibility testing in PNG, molecular assays for drug resistance monitoring would be of advantage.     

Roles of tyrosine 158 and lysine 165 in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis.

The role of tyrosine 158 (Y158) and lysine 165 (K165) in the catalytic mechanism of InhA, the enoyl-ACP reductase from Mycobacterium tuberculosis, has been investigated. These residues have been identified as putative catalytic residues on the basis of structural and sequence homology with the short chain alcohol dehydrogenase family of enzymes. Replacement of Y158 with phenylalanine (Y158F) and with alanine (Y158A) results in 24- and 1500-fold decreases in k(cat), respectively, while leaving K(m) for the substrate, trans-2-dodecenoyl-CoA, unaffected. Remarkably, however, replacement of Y158 with serine (Y158S) results in an enzyme with wild-type activity. Kinetic isotope effect studies indicate that the transfer of a solvent-exchangeable proton is partially rate-limiting for the wild-type and Y158S enzymes, but not for the Y158A enzyme. These data indicate that Y158 does not function formally as a proton donor in the reaction but likely functions as an electrophilic catalyst, stabilizing the transition state for hydride transfer by hydrogen bonding to the substrate carbonyl. A conformational change involving rotation of the Y158 side chain upon binding of the enoyl substrate to the enzyme is proposed as an explanation for the inverse solvent isotope effect observed on V/K(DD-CoA) when either NADH or NADD is used as the reductant. These data are consistent with the recently published structure of a C16 fatty acid substrate bound to InhA that shows Y158 hydrogen bonded to the substrate carbonyl group and rotated from the position it occupies in the InhA-NADH binary complex [Rozwarski, D. A., Vilcheze, C., Sugantino, M., Bittman, R., and Sacchettini, J. C. (1999) J. Biol. Chem. 274, 15582-15589]. Finally, the role of K165 has been analyzed using site-directed mutagenesis. Replacement of K165 with glutamine (K165Q) and arginine (K165R) has no effect on the enzyme's catalytic ability or on its ability to bind NADH. However, the K165A and K165M enzymes are unable to bind NADH, indicating that K165 has a primary role in cofactor binding.     

The complete amino acid sequence of the low molecular weight cytosolic acid phosphatase

This paper presents the complete amino acid sequence of the low molecular weight acid phosphatase from bovine liver. This isoenzyme of the acid phosphatase family is located in the cytosol, is not inhibited by L-(+)-tartrate and fluoride ions, but is inhibited by sulfhydryl reagents. The enzyme consists of 157 amino acid residues, has an acetylated NH2 terminus, and has arginine as the COOH-terminal residue. All 8 half-cystine residues are in the free thiol form. The molecular weight calculated from the sequence is 17,953. The sequence was determined by characterizing the peptides purified by reverse-phase high performance liquid chromatography from tryptic, thermolytic, peptic, Staphylococcus aureus protease, and chymotryptic digests of the carboxymethylated protein. No sequence homologies were found with the two known acylphosphatase isoenzymes or the metalloproteins porcine uteroferrin and purple acid phosphatase from bovine spleen (both of which have acid phosphatase activity). Two half-cystines at or near the active site were identified through the reaction of the enzyme with [14C] iodoacetate in the presence or in the absence of a competitive inhibitor (i.e. inorganic phosphate). Ac-A E Q V T K S V L F V C L G N I C R S P I A E A V F R K L V T D Q N I S D N W V I D S G A V S D W N V G R S P N P R A V S C L R N H G I N T A H K A R Q V T K E D F V T F D Y I L C M D E S N L R D L N R K S N Q V K N C R A K I E L L G S Y D P Q K Q L I I E D P Y Y G N D A D F E T V Y Q Q C V R C C R A F L E K V R-OH.     

BUCHBESPRECHUNGEN

Book reviewed in this article:
J arman , M artha V.: Impala Social Behavior: Territory, Hierarchy, Mating and the Use of Space. "Fortschritte der Verhaltensforschung - Advances in Ethology", Beihefte Z. Tierpsychologie, H. 21.
Handbook of Sensory Physiology. Ed. Board: H. A utrum , R. J ung , W. R. L oewenstein , D. M. M ackay and H.-L. T eüber . Vol. 7. Part 6: Comparative Physiology and Evolution of Vision in Invertebrates. A. Invertebrate Photoreceptors. By H. A utrum , M. F. B ennet , B. D iehn , K. H amdorf , M. H eisenberg , M. J arvilehto , P. K unze , R. M enzel , W. H. M iller , A. W. S nyder , D. G. S tavenga and M. Y oshida . Ed. by H. A utrum . Berlin, Heidelberg, New York:
Leitfaden für das zoologische Praktikum. Begründet von W. K ükenthal . Überarbeitet von M. R enner . 18., überarb. Auflage.     

Consequences of cAMP-binding site mutations on the structural stability of the type I regulatory subunit of cAMP-dependent protein kinase

The regulatory (R) subunit of cAMP-dependent protein kinase (cAPK) is a multidomain protein with two tandem cAMP-binding domains, A and B. The importance of cAMP binding on the stability of the R subunit was probed by intrinsic fluorescence and circular dichroism (CD) in the presence and absence of urea. Several mutants were characterized. The site-specific mutants R(R209K) and R(R333K) had defects in cAMP-binding sites A and B, respectively. R(M329W) had an additional tryptophan in domain B. Delta(260-379)R lacked Trp260 and domain B. The most destabilizing mutation was R209K. Both CD and fluorescence experiments carried out in the presence of urea showed a decrease in cooperativity of the unfolding, which also occurred at lower urea concentrations. Unlike native R, R(R209K) was not stabilized by excess cAMP. Additionally, CD revealed significant alterations in the secondary structure of the R209K mutant. Therefore, Arg209 is important not only as a contact site for cAMP binding but also for the intrinsic structural stability of the full-length protein. Introducing the comparable mutation into domain B, R333K, had a smaller effect on the integrity and stability of domain A. Unfolding was still cooperative; the protein was stabilized by excess cAMP, but the unfolding curve was biphasic. The R(M329W) mutant behaved functionally like the native protein. The Delta(260-379)R deletion mutant was not significantly different from wild-type RIalpha in its stability. Consequently, domain B and the interaction between Trp260 and cAMP bound to site A are not critical requirements for the structural stability of the cAPK regulatory subunit.     

Conformational changes in the alpha- and beta-subunits of the insulin receptor identified by anti-peptide antibodies

The structure of the insulin receptor was studied with polyclonal antibodies obtained from rabbits which were immunized with synthetic peptides having a sequence identity to three regions of the alpha-subunit and five regions of the beta-subunit. None of the alpha-subunit antibodies including alpha-Pep8 (residues 40-49 (Ullrich, A., Bell, J.R., Chen, E.Y., Herrera, R., Petruzzelli, L.M., Dull, T.J., Gray, A., Coussens, L., Liao, Y.-C., Tsubokawa, M., Mason, A., Seeburg, P.H., Grunfeld, C., Rosen, O.M., and Ramachandran, J. (1985) Nature 313, 756-761), alpha-Pep7 (12 amino acid C-terminal extension (Ebina, Y., Ellis, L., Jarnagin, K., Ederly, M., Graf, L., Clauser, E., Ou, J.-H., Masiar, F., Kan, Y.W., Goldfine, I.D., Roth, R.A., and Rutter, W.J. (1985) Cell 313, 747-758], or alpha-Pep6 (residues 1-7, 9) immunoprecipitated the human insulin receptor solubilized from IM-9 lymphocytes; however, alpha-Pep8 immunoprecipitated the dithiothreitol-reduced receptor. Antibodies prepared against the N terminus of the beta-subunit (alpha-Pep5, residues 780-790) and the ATP binding site (alpha-Pep3, residues 1013-1022) did not react with the intact receptor under any conditions; however, antibodies to the C terminus of the beta-subunit (alpha-Pep1, residues 1314-1324) and to the juxta-membrane region (alpha-Pep3, residues 952-962) immunoprecipitated the solubilized receptor in both its phosphorylated and nonphosphorylated forms. In contrast, the antibody reactive with the regulatory region of the beta-subunit which contains the major autophosphorylation sites (alpha-Pep2, residues 1143-1154) only precipitated the phosphorylated form. Thus the conformation of the extracellular domain of the receptor is rigid and stabilized by disulfide bonds, whereas several regions of the intracellular domain are accessible to antibodies and undergo conformational changes during autophosphorylation.     

atRA Regulation of NEDD9, a gene involved in neurite outgrowth and cell adhesion

We previously identified NEDD9 (RAINB2/HEF1/Cas-L) as a new downstream target of all-trans retinoic acid (atRA) and its receptors in the human neuroblastoma cell line, SH-SY5Y [R.A. Merrill, A.W.-M. See, M.L. Wertheim, M. Clagett-Dame, Dev. Dyn. 231 (2004) 564-575; R.A. Merrill, J.M. Ahrens, M.E. Kaiser, K.S. Federhart, V.Y. Poon, M. Clagett-Dame, Biol. Chem. 385 (2004) 605-614]. We now provide functional evidence that NEDD9 is directly regulated by atRA through a complex retinoic acid response element (RARE) located in the NEDD9 proximal promoter and consisting of four conserved half-sites separated by 1, 5, and 1 intervening base pairs. We show that a region of the human NEDD9 promoter from −1670 to +15 is sufficient to confer atRA-responsiveness and that a complex RARE located from −475 to −445 is necessary for this effect. While mutation of any one half-site does not eliminate complex formation in electrophoretic mobility shift assays (EMSA); these same mutations, when tested in transient transfection assays, markedly decrease atRA-responsiveness. Finally, chromatin immunoprecipitation (ChIP) assays demonstrate that RAR and RXR are bound to the RARE in cells.     

Bombyx mori silk fibroin liquid crystallinity and crystallization at aqueous fibroin-organic solvent interfaces.

A banded morphology has been observed for Bombyx mori silk fibroin films obtained from an aqueous hexane interface; the period of the banding is approximately 1 microm. Morphology and diffraction from different regions of the banded structure suggest that it is a free surface formed by a cholesteric liquid crystal. Truncated hexagonal lamellar crystallites of B. mori silk fibroin have been observed in films formed in the surface excess layer of fibroin at the interface between aqueous fibroin and hexane or chloroform. Based on initial crystallographic evidence, a three-fold helical conformation has been ascribed to the fibroin chains within the crystals. The chain conformation and crystalline habit appear to be similar to the silk III structure previously observed at the air-water interface (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Valluzzi R, Gido S, Zhang W, Muller W, Kaplan D. Macromolecules 1996;29:8606-8614) but the crystalline packing is different. Diffraction data obtained for the crystallites are similar to diffraction behavior for a collagen-like model peptide. Diffraction patterns obtained from crystallized regions of the banded morphology can be indexed using the same unit cell as the hexagonal lamellar crystallites. Surfactancy of fibroin and subsequent aggregation and mesophase formation may help to explain the liquid crystallinity reported for silk, which is long suspected to play a role in the biological silk spinning process (Valluzzi R, Gido SP. Biopolymers 1997;42:705-717; Willcox, P. J.; Gido, SP, Muller W, Kaplan DL. Macromolecules 1996:29:5106-5110; Magoshi J, Magoshi Y, Nakamura S. In: Kaplan D, Adams W, Farmer B, Viney C, editors, Mechanism of Fiber Formation of Silkworm. Washington, DC: American Chemical Society 1994:292-310; Magoshi J, Magoshi Y, Nakamura S. J Appl Polym Sci Appl Polym Symp 1985;41:187-204; Magoshi J, Magoshi Y, Nakamura S. Polym Commun 1985;26:309.).