Comparison of promoter-specific events during transcription initiation in mycobacteria

DNA protein interactions that occur during transcription initiation play an important role in regulating gene expression. To initiate transcription, RNA polymerase (RNAP) binds to promoters in a sequence-specific fashion. This is followed by a series of steps governed by the equilibrium binding and kinetic rate constants, which in turn determine the overall efficiency of the transcription process. We present here the first detailed kinetic analysis of promoter RNAP interactions during transcription initiation in the sigma(A)-dependent promoters P-rrnAPCL1, P-rrnB and P-gyr of Mycobacterium smegmatis. The promoters show comparable equilibrium binding affinity but differ significantly in open complex formation, kinetics of isomerization and promoter clearance. Furthermore, the two rrn promoters exhibit varied kinetic properties during transcription initiation and appear to be subjected to different modes of regulation. In addition to distinct kinetic patterns, each one of the housekeeping promoters studied has its own rate-limiting step in the initiation pathway, indicating the differences in their regulation.

Crystal structure of L-lysyl-L-glutamic acid dihydrate

L-Lysyl-L-glutamic acid dihydrate, C11N3O5H21·2H2O, crystallizes in the monoclinic space group P21 with a = 12.474(2), b = 5.020(1), c = 13.157(2) Å, β= 114.69(1)° and Z = 2. The crystal structure was solved by direct methods and refined to an R value of 0.037 using full matrix least-squares method. The molecule exists as a double zwitterion with both the amino and carboxyl groups ionised. The peptide has a folded conformation with its Lys residue trans and Glu residue gauche−gauche+. The side chains of the Lys and Glu residues correspond to all trans and folded (g−g−g−) conformations respectively. The terminal carboxyl group forms hydrogen bonds with the ξ-amino group of the lysine side chain. The head-to-tail interaction often seen in peptide crystals is absent in the present structure. In the extended crystal structure water molecules form channels along the b direction and are enclosed within helically arranged hydrogen bonds formed by the lysine side chain and the peptide backbone.

Quantitation of serological cross-reactivity between two geographical isolates of Oryctes baculovirus by a modified ELISA

An assay was developed for quantitation of the antigenic relationship between viruses, by modification of the indirect ELISA. The principle of this method is to estimate the epitopes not shared between the related viruses, after titration of the antibodies specific to the common epitopes as in a blocking ELISA. In practice, varying concentrations of purified virus are preincubated with a fixed dilution of heterologous or homologous antiserum and the unbound antibodies present in the mixture are back titrated with virus particles bound to microtitre plates. The antigenic relationship is described in terms of differentiation index (DI) and total antigenic reactivity (TAR). This method has been used to quantitate cross-reactivity between two geographically different isolates of Oryctes baculovirus.

Direct somatic embryogenesis in zygotic embryos of nutmeg (Myristica fragrans Houtt.)

Direct somatic embryogenesis from isolated intact as well as broken zygotic embryos and in vitro plantlets of nutmeg (Myristica fragrans Houtt.) was obtained. Enhanced embryogenic response was associated with broken zygotic embryos. Activated charcoal and light were the critical factors for induction of somatic embryogenesis in nutmeg. Histological evaluation revealed the presence of globular and cotyledonary stages. The somatic embryos underwent partial germination after a six-month lag period. A wide range of abnormal embryos were observed. The somatic embryos synthesised chlorophyll, exhibited phenylalanine ammonia lyase activity, synthesised phenolics, and could serve as a stable source of secondary metabolites of nutmeg which are commercially important.

Studies on hypomethylation of liver DNA during early stages of chemical carcinogenesis in rat liver

Our finding that the inhibitors of DNA methylation, 5-azacytidine, 5-azadeoxycytidine or adenosine dialdehyde, given after a carcinogen all potentiated initiation suggested that hypomethylation of DNA during repair synthesis of DNA might play a role in the initiation of the carcinogenic process. To examine this aspect further, we have asked the question, do the nodules which develop from initiated cells after promotion with 1% orotic acid exhibit an altered methylation pattern in their DNA? The methylation status of the DNA from nodules has been examined using the restriction endonucleases HpaII/MspI and HhaI which distinguish between methylated and unmethylated cytosines in their nucleotide recognition DNA 5'-CCGG and 5'-GCGC respectively. The proto-oncogenes, c-myc, c-fos and c-Ha-ras, in the DNA were primarily studied in this investigation because of their possible involvement in cell proliferation and/or in cell transformation and tumorigenesis. The results indicate that in the nodule DNA, c-myc and c-fos are hypomethylated in the sequence of CCGG while the c-Ha-ras shows hypomethylation in the alternating GCGC sequence. This methylation pattern seen in the nodule DNA is not found in the DNA of the non-nodular surrounding liver or liver tissue after exposure to promoter or carcinogen alone. It is also not found in the DNA of regenerating liver. It is particularly significant that the methylation patterns in the c-myc and c-Ha-ras regions are similar to those found in several cancer tissues. The results suggest that this methylation pattern is acquired early in the carcinogenic process and raises the question whether it has any bearing on the process.

Growth of and trehalase activity in the thermophilic fungusThermomyces lanuginosus

The thermophilic fungus,Thermomyces lanuginosus, was grown in a glucose-asparagine liquid medium. Optimal mycelial growth occurred at 50°C. The conditions for sporulation were different from those required for vegetative growth. the former being favoured by lower nitrogen level and temperature. Trehalase (α, α-glu coside-l-glucohydrolase, EC 3.2.1.28) was one of the most active glycosidases at 50°C. Non-sporulating mycelium had higher levels of this enzyme than the sporulating mycelium. Trehalase was synthesized constitutively and its activity appears to be controlled by catabolite repression.

Preparation and characterization of monoclonal antibodies to nucleocapsid protein N and H glycoprotein of rinderpest virus

Fifteen stable mouse spleen cell myeloma hybrids (hybridomas) producing monoclonal antibodies to rinderpest virus proteins were produced. The specificity of these monoclonal antibodies was established by radioimmunoprecipitation followed by polyacrylamide gel analysis and immunofluorescence. Nine antibodies were specific for the surface glycoprotein H. All the nine clones showed inhibition of haemagglutination by measles virus. The antibodies from two clones (A7D2 and B2F6) neutralise infectious virus. Six clones produce antibodies reacting with the nucleocapsid protein N. Three antigenic sites designated I–III, with sites I and II partially overlapping, were topographically mapped on the H molecule by competitive binding assay. Similarly, two antigenic sites I and II were delineated on the N protein. The monoclonal antibodies were used to study the antigenic relationships of H and N proteins of rinderpest virus, measles virus and canine distemper virus.

Differentiation of rinderpest and peste des petits ruminants viruses using specific cDNA clones

The morbilliviruses which infect ruminants, rinderpest (RPV) and peste des petits ruminants (PPRV), are difficult to distinguish serologically. They can be distinguished by differential neutralisation tests and by the migration of the major virus structural protein, the nucleocapsid protein, on polyacrylamide gels. Both these methods are time consuming and require the isolation of live virus for identification; they are not suitable for analysis of material directly from post-mortem specimens. We describe a rapid method for differential diagnosis of infections caused by RPV or PPRV, which uses specific cDNA probes, derived from the mRNAs for the nucleocapsid protein of each virus, which can be used to distinguish unequivocally the two virus types rapidly.

Antibodies raised against denatured DNA bind to double-stranded DNA

Antibodies raised against denatured DNA complexed with methylated bovine serum albumin have been reported to react with ssDNA but not with dsDNA. Using a highly sensitive avidin-biotin microELISA, we report that such antibodies also bind to dsDNA. Antibodies which reacted with ssDNA and dsDNA were found to be IgG type. The antibodies did not react with tRNA and rRNA. The binding of antibodies to dsDNA was partially inhibited dy individual deoxyribonucleotides. ssDNA as well as dsDNA inhibited the binding of antibodies to dsDNA. The binding of these antibodies to supercoiled and relaxed forms of pBR322 DNA was demonstrated by gel retardation assay. The cross-reaction with ssDNA was observed even after affinity purification on native DNA-cellulose. The antibodies were also shown to bind to poly(dA-dT)·poly(dA-dT)

Structure of uracil-DNA glycosylase from Mycobacterium tuberculosis: insights into interactions with ligands

Uracil N-glycosylase (Ung) is the most thoroughly studied of the group of uracil DNA-glycosylase (UDG) enzymes that catalyse the first step in the uracil excision-repair pathway. The overall structure of the enzyme from Mycobacterium tuberculosis is essentially the same as that of the enzyme from other sources. However, differences exist in the N- and C-terminal stretches and some catalytic loops. Comparison with appropriate structures indicate that the two-domain enzyme closes slightly when binding to DNA, while it opens slightly when binding to the proteinaceous inhibitor Ugi. The structural changes in the catalytic loops on complexation reflect the special features of their structure in the mycobacterial protein. A comparative analysis of available sequences of the enzyme from different sources indicates high conservation of amino-acid residues in the catalytic loops. The uracil-binding pocket in the structure is occupied by a citrate ion. The interactions of the citrate ion with the protein mimic those of uracil, in addition to providing insights into other possible interactions that inhibitors could be involved in.

Assessment of the mutagenic potential of a fungicide Bavistin using multiple assays

The fungicide Bavistin was assessed for mutagenic potential by various assays. Bavistin was found to be unable to induce gene mutation in Salmonella typhimurium, but it was able to induce transfection inhibition in Mycobacterium smegmatis. Bavistin was able to induce immediate genotoxic effects in plants but these were not carried through in development as in the long term no genotoxic effects were observed by the progeny test. Bavistin did induce micronuclei formation and did cause an increase in the ratio of normochromatic to polychromatic erythrocytes in mice. It was able to induce a very low frequency of sister-chromatid exchange in human lymphocytes and in addition, it was observed that the chemical affected the mitotic index but did not affect the cell cycle duration. Present studies indicate that the pesticide shows a positive response in 4 out of 5 different test systems (Table 8) and most of the observations support that Bavistin is genotoxic.

Binding of deoxyadenylate and deoxycytidylate antibodies to double-stranded DNA

Antibodies raised against deoxyadenylate and deoxycytidylate were found to react with double stranded DNA as assessed by highly sensitive avidin-biotin microELISA. The binding was specific as it was completely inhibited by the homologous hapten. The antibodies did not react with tRNA and rRNA. These antibodies were also shown to react with supercoiled and relaxed forms of pBR322 DNA as demonstrated by gel retardation assay. ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; CT DNA, calf thymus DNA; AB microELISA, avidin-biotin microELISA; dpA, deoxyadenylate; dpC, deoxycytidylate; avidin-HRP, avidin-horseradish peroxidase

Characterization of foot-and-mouth disease virus types Ο and Asia 1 RNA

Foot-and-mouth disease is an acute and highly contagious febrile disease affecting cloven-footed animals. Identification of the foot-and-mouth disease virus (FMDV), the causative agent of the disease, posed problems because of the occurrence of many types and subtypes of the virus. A molecular approach based on oligonucleotide mapping of FMDV RNA has been used for the identification and characterization of virus isolates obtained in a disease outbreak (King et al., 1981). One-dimensional oligonucleotide mapping was used for rapid analysis of FMDV RNA (LaTorre et al., 1982). FMDV types Ο and Asia 1 of Indian origin are being routinely used for vaccine production in India. This report presents the differences between FMDV types Ο and Asia 1 at molecular level based on one-dimensional oligonucleotide mapping of virus-induced poly (A) RNA.

Cytotoxic activity of daunomycin and adriamycin encapsulated in immunoliposomes against avian myeloblastosis virus-infected cells

Immunoliposomes were prepared using the antibody raised against the avian myeloblastosis virus envelope glycoprotein, gp80. Adriamycin was encapsulated into immunoliposomes. More drug was delivered into target cells when the drug encapsulated in immunoliposomes was incubated with the cells. The drug encapsulated in immunoliposomes was able to inhibit the RNA synthesis twice more than free drug in the virus-transformed myeloblasts. Pre-treatment of cells with ammonium chloride, reversed the effect of drug encapsulated in immunoliposomes. The drugs encapsulated in immunoliposomes had marginal effect on the RNA synthesis of non-target cells, the yolk sac cells. Colony formation by virus-transformed cells and focus formation by virus-infected yolk sac cells was inhibited significantly by the drug encapsulated in immunoliposomes.