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1.
IUCrJ ; 10(Pt 5): 579-583, 2023 Sep 01.
Article in English | MEDLINE | ID: mdl-37493524

ABSTRACT

The great success of single-particle electron cryo-microscopy (cryoEM) during the last decade has involved the development of powerful new computer programs and packages that guide the user along a recommended processing workflow, in which the wisdom and choices made by the developers help everyone, especially new users, to obtain excellent results. The ability to carry out novel, non-standard or unusual combinations of image-processing steps is sometimes compromised by the convenience of a standard procedure. Some of the older programs were written with great flexibility and are still very valuable. Among these, the original MRC image-processing programs for structure determination by 2D crystal and helical processing alongside general-purpose utility programs such as Ximdisp, label, imedit and twofile are still available. This work describes an updated version of the MRC software package (MRC2020) that is freely available from CCP-EM. It includes new features and improvements such as extensions to the MRC format that retain the versatility of the package and make it particularly useful for testing novel computational procedures in cryoEM.

2.
Biotechnol Bioeng ; 75(3): 322-33, 2001 Nov 05.
Article in English | MEDLINE | ID: mdl-11590605

ABSTRACT

The enzymatic hydrolysis of mannan-based hemicelluloses is technologically important for applications ranging from pulp and paper processing to food processing to gas and oil well stimulation. In many cases, thermostability and activity at elevated temperatures can be advantageous. To this end, the genes encoding beta-mannosidase (man2) and beta-mannanase (man5) from the hyperthermophilic bacteria Thermotoga neapolitana 5068 and Thermotoga maritima were isolated, cloned, and expressed in Escherichia coli. The amino acid sequences for the mannosidases from these organisms were 77% identical and corresponded to proteins with an M(r) of approximately 92 kDa. The translated nucleotide sequences for the beta-mannanase genes (man5) encoded polypeptides with an M(r) of 76 kDa that exhibited 84% amino acid sequence identity. The recombinant versions of Man2 and Man5 had similar respective biochemical and biophysical properties, which were also comparable to those determined for the native versions of these enzymes in T. neapolitana. The optimal temperature and pH for the recombinant Man2 and Man5 from both organisms were approximately 90 degrees C and 7.0, respectively. The presence of Man2 and Man5 in these two Thermotoga species indicates that galactomannan is a potential growth substrate. This was supported by the fact that beta-mannanase and beta-mannosidase activities were significantly stimulated when T. neapolitana was grown on guar or carob galactomannan. Maximum cell densities increased by at least tenfold when either guar or carob galactomannan was added to the growth medium. For T. neapolitana grown on guar at 83 degrees C, Man5 was secreted into the culture media, whereas Man2 was intracellular. These localizations were consistent with the presence and lack of signal peptides for Man5 and Man2, respectively. The identification of the galactomannan-degrading enzymes in these Thermotoga species adds to the list of biotechnologically important hemicellulases produced by members of this hyperthermophilic genera.


Subject(s)
Bacteria/enzymology , Bacterial Proteins , Mannans/metabolism , Mannosidases/chemistry , beta-Mannosidase , Amino Acid Sequence , Bacteria/genetics , Bacteria/growth & development , Galactose/analogs & derivatives , Mannosidases/genetics , Mannosidases/metabolism , Microscopy, Electron , Molecular Sequence Data , Sequence Homology, Amino Acid
10.
Mutat Res ; 398(1-2): 143-9, 1998 Feb 26.
Article in English | MEDLINE | ID: mdl-9626974

ABSTRACT

Previously, we developed a model for high incidence, endogenously generated hepatocellular carcinoma (HCC), the human alpha-1-antitrypsin (alpha1AT) Z gene transgenic mouse (Z#2). We now examine the potential utility of a model for endogenous carcinogenesis utilizing the Z#2 mouse also transgenic for the lacI gene, a convenient target for in vivo mutagenesis studies. We crossed the Z#2 line and mice transgenic for lambda/lacI shuttle vector (Big Blue), for determination of lacI mutant frequency during initiation of endogenous carcinogenesis. Five month old double transgenic mice (Z#2+/lacI+) successfully displayed: (1) the expected post-inflammatory stage of Z#2 carcinogenesis; and (2) hepatic lacI mutants measured at frequencies (10(-5)-10(-4)) useful to mutagenesis studies. In this study, hepatic lacI mutation frequencies in Z#2 transgenic mice appeared to be only slightly increased (< 2x) when compared to age matched negative controls. In the future, it may be important to reconcile possibly limited lacI mutagenesis at the time of initiation and demonstrated high incidence of hepatocarcinogenesis.


Subject(s)
Escherichia coli Proteins , Liver Neoplasms, Experimental/genetics , Animals , Bacterial Proteins/genetics , Genetic Vectors , Humans , Lac Repressors , Mice , Mice, Transgenic , Models, Biological , Repressor Proteins/genetics , alpha 1-Antitrypsin/genetics
11.
Nature ; 392(6674): 353-8, 1998 Mar 26.
Article in English | MEDLINE | ID: mdl-9537320

ABSTRACT

Aquifex aeolicus was one of the earliest diverging, and is one of the most thermophilic, bacteria known. It can grow on hydrogen, oxygen, carbon dioxide, and mineral salts. The complex metabolic machinery needed for A. aeolicus to function as a chemolithoautotroph (an organism which uses an inorganic carbon source for biosynthesis and an inorganic chemical energy source) is encoded within a genome that is only one-third the size of the E. coli genome. Metabolic flexibility seems to be reduced as a result of the limited genome size. The use of oxygen (albeit at very low concentrations) as an electron acceptor is allowed by the presence of a complex respiratory apparatus. Although this organism grows at 95 degrees C, the extreme thermal limit of the Bacteria, only a few specific indications of thermophily are apparent from the genome. Here we describe the complete genome sequence of 1,551,335 base pairs of this evolutionarily and physiologically interesting organism.


Subject(s)
Genome, Bacterial , Gram-Negative Aerobic Rods and Cocci/genetics , Chromosome Mapping , Chromosomes, Bacterial , Citric Acid Cycle , DNA Repair , DNA, Bacterial/biosynthesis , DNA, Bacterial/genetics , Gram-Negative Aerobic Rods and Cocci/metabolism , Molecular Sequence Data , Oxidative Stress , Phylogeny , Protein Biosynthesis , Temperature , Transcription, Genetic
15.
Mutat Res ; 352(1-2): 9-22, 1996 Jun 10.
Article in English | MEDLINE | ID: mdl-8676922

ABSTRACT

Transgenic animals offer a powerful tool to study the mechanisms of spontaneous and induced mutagenesis in vivo. Herein we used a test version of a growth selectable assay to obtain spontaneous mutants in a lacI target transgene recovered from lacI transgenic B6C3F1 mice (Big Blue). This selection system may have certain advantages relative to the more established plaque screening system for mutation detection because: (1) the plating density of the phage is up to 60 times higher in the selectable assay, reducing the number of plates needed to be screened for a comparable amount of mutants; and (2) the mutant frequency obtained from the selectable assay is higher compared to the plaque assay, possibly due to a higher sensitivity for weaker mutants. However, the longer incubation time of the growth selectable assay might allow E. coli host derived mutants to appear. To address this issue, we investigated the sequence changes in the amino-terminal domain of the lacI gene of 405 mutants derived from the liver, spleen, brain, germ cells and skin of five untreated 6-week-old mice. The mutant colonies were isolated after 60, 84, 108 and 150 h of incubation under growth selectable conditions. Tissue-specific differences in the mutational pattern obtained after 60 and 84 h disappear after a longer time of incubation, possibly due to an increasing contribution of E. coli derived mutants. The evolving selectable systems offer the potential to increase screening efficiency, but the results suggest caution in interpreting data from this system because repair by E. coli of DNA lesions or mismatched heteroduplexes either originating in mouse in vivo or produced by ex vivo manipulation as well as de novo mutations in E. coli might contribute significantly to the observed mutational spectra at each timepoint.


Subject(s)
Bacterial Proteins/genetics , Escherichia coli Proteins , Mice, Transgenic/genetics , Mutation , Repressor Proteins/genetics , Animals , Bacteriophage lambda/genetics , Base Sequence , Brain , DNA Mutational Analysis , Escherichia coli/genetics , Genes, Bacterial/genetics , Genetic Vectors/genetics , Germ Cells , Lac Repressors , Liver , Mice , Molecular Sequence Data , Organ Specificity , Skin , Spleen
16.
Environ Mol Mutagen ; 28(4): 342-7, 1996.
Article in English | MEDLINE | ID: mdl-8991062

ABSTRACT

The genotoxic response of benzene and tris(2,3-dibromopropyl)-phosphate (TDBP) have been evaluated in several tissues using the standardized lambda/lacI (Big Blue) transgenic mouse mutation assay. Separate groups of four to five male B6C3F1 transgenic lambda/lacI mice were given oral administrations of benzene or TDBP at varying concentrations. Tissues evaluated include lung, bone marrow, and spleen in benzene-treated animals, and liver, kidney, and stomach in TDBP-treated animals. Significant increases in lacI mutations were observed in the spleen and bone marrow of benzene treated mice, and the kidneys of TDBP-treated mice. Where applicable, mutagenesis patterns of tissue sensitivity were consistent with what has been observed previously in other assays. In addition, mutagenicity in tissues not traditionally evaluated for mutations correlated to sites of carcinogenicity for the chemicals tested.


Subject(s)
Bacterial Proteins/genetics , Benzene/toxicity , Escherichia coli Proteins , Mice, Transgenic/genetics , Mutation , Organophosphates/toxicity , Repressor Proteins/genetics , Animals , Bacterial Proteins/drug effects , Carcinogens/toxicity , Dose-Response Relationship, Drug , Female , Lac Repressors , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mutagenicity Tests/methods , Mutagens/toxicity , Repressor Proteins/drug effects , Tissue Distribution
17.
Environ Mol Mutagen ; 28(4): 447-58, 1996.
Article in English | MEDLINE | ID: mdl-8991077

ABSTRACT

Elements of the lactose operon were used to study parameters affecting gene expression in cultured cells and transgenic animals. A Lac repressor protein containing a nuclear transport signal was shown to inhibit expression of a reporter gene by interacting with lac operator sequences. In cultured cells, operator sequence, operator placement and induction parameters were all shown to be important for obtaining tight repression of a reporter gene followed by high level expression upon induction. Induction levels were also dependent on the reporter gene, with the luciferase gene yielding higher induction levels than the chloramphenicol acetyltransferase gene. In transgenic animals, the lacI mRNA was not detected in the C57BL/6 mouse strain until the animal was exposed to a demethylating agent. After 5-azacytidine treatment, expression of lacI mRNA was detected in the brain, heart, kidney, lung and ovary. In the FVB transgenic mouse strain, expression of lacI mRNA was detected without 5-azacytidine treatment in the kidney, liver, lung, and testes. Preliminary experiments with double transgenic animals containing both lacI and operator/luciferase transgenes showed a decrease in luciferase expression compared to the luciferase-only animals in both tissue extracts and transgenic fetal primary cultures, although IPTG induction was not achieved in these animals or primary cultures. The applicability and challenges of the system for regulation of gene expression are discussed.


Subject(s)
Bacterial Proteins/biosynthesis , Bacterial Proteins/genetics , Escherichia coli Proteins , Genetic Vectors/genetics , Mice, Transgenic/genetics , Repressor Proteins/biosynthesis , Repressor Proteins/genetics , Animals , Bacterial Proteins/drug effects , Base Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cells, Cultured , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , Cloning, Molecular/methods , Dose-Response Relationship, Drug , Eukaryotic Cells , Female , Fetus/cytology , Fetus/metabolism , Gene Expression Regulation , Genes, Reporter , Genetic Vectors/metabolism , Isopropyl Thiogalactoside/pharmacology , Lac Repressors , Male , Methylation , Methylgalactosides/pharmacology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/drug effects , Thiogalactosides/pharmacology , Time Factors , Tissue Distribution
18.
Mutat Res ; 334(2): 161-5, 1995 Apr.
Article in English | MEDLINE | ID: mdl-7885368

ABSTRACT

A rat embryo cultured cell line was generated that carries stably integrated copies of a lambda/lacI shuttle vector, containing the lacI gene as a mutational target. After the desired treatment of the cells, this vector can be rapidly and efficiently recovered from the cell DNA by in vitro packaging and then screened for mutations in the lacI gene, using bacterial detection systems. The vector is identical to that integrated into the Big Blue transgenic mouse, which was developed for in vivo mutation analysis. Characterization of the cell line by fluorescence in situ hybridization showed that the phage DNA is integrated at two distinct sites on separate chromosomes at approximately 50-70 copies per cell and the cell line is polyploid. The rescue efficiency is approximately 100,000 pfu/micrograms of genomic DNA. To examine the ability of the cell line to detect mutations in the lacI gene, the cells were treated with 100 micrograms/ml of the direct-acting alkylating agent N-methyl-N-nitrosourea (MNU) for 30 min at 37 degrees C and grown to confluence. The shuttle vector was rescued from untreated and mutagen treated cells, and spontaneous and induced mutant frequencies were determined to be 4.0 x 10(-5) and 92.7 x 10(-5), respectively. The cell line can be used to detect mutations in the lacI gene, followed by recovery of mutants for sequence analysis. The cell line may be valuable for short-term in vitro mutagenesis studies, oncogene and tumor suppressor studies, and DNA repair studies.


Subject(s)
Bacteriophage lambda/genetics , Cell Line/virology , Genetic Vectors , Lac Operon/genetics , Virus Integration , Animals , DNA, Viral , Escherichia coli/genetics , Genes, Bacterial , In Situ Hybridization, Fluorescence , Mutagenesis , Rats , Repressor Proteins , Transfection/methods , beta-Galactosidase/genetics
19.
Mutat Res ; 327(1-2): 201-8, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7870088

ABSTRACT

Statistical analysis of the lambda/lacI transgenic mutagenicity assay was used to determine optimal sample size and resource allocation in terms of number of animals and number of recovered target genes (recovered phage) required to demonstrate a statistically significant induction in mutant frequency. Statistical assumptions as applied to mutagenicity data are discussed for a number of frequently used statistical analyses. Log transformations are suggested as a means of meeting statistical assumptions and examples are given on interpreting results of analyses of log transformed data. The data analyzed in this study indicate that 300,000 lambda plaques from each of five animals should be analyzed per treatment group in order to detect a doubling of mutant frequencies. Additional sensitivity is gained primarily through increase of animal number and not the number of phage rescued, due to inherent animal-to-animal variability.


Subject(s)
Bacterial Proteins/genetics , Escherichia coli Proteins , Genes, Reporter , Genes, Synthetic , Mutagenicity Tests/statistics & numerical data , Repressor Proteins/genetics , Animals , Bacteriophage lambda/drug effects , Bacteriophage lambda/genetics , Escherichia coli/genetics , Female , Genetic Vectors/drug effects , Genetic Vectors/genetics , Lac Repressors , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Sampling Studies , Sensitivity and Specificity , Specific Pathogen-Free Organisms
20.
Mutat Res ; 327(1-2): 67-73, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7870100

ABSTRACT

Spontaneous mutant frequency in livers of two transgenic mouse strains, each carrying identical lambda shuttle vectors with a lacI target gene, was evaluated by two laboratories. These studies investigated variability in spontaneous mutant frequency between animals and as a function of the number of phage screened. Liver DNA was independently isolated from 7-11 week old C57BL/6 and B6C3F1 Big Blue transgenic mice. At least 500,000 phage were screened for mutation at lacI for each animal using standardized assay procedures. In the two labs, the C57BL/6 liver spontaneous mutant frequency was 45 +/- 9 x 10(-6) and 41 +/- 7 x 10(-6). The B6C3F1 liver spontaneous mutant frequency was 42 +/- 10 x 10(-6) at one lab and 43 +/- 12 x 10(-6) and 41 +/- 8 x 10(-6) in two trials at the second lab. Mean mutant frequency data from both labs, calculated in increments of 100,000 plaque forming units (pfu) scored for each mouse strain, show stabilized mean mutant frequency and standard deviation after approximately 200,000-300,000 pfu screened. The frequency of spontaneous lacI mutants was reproducible both within and between labs and was comparable between the two transgenic mouse strains.


Subject(s)
Bacterial Proteins/genetics , DNA, Recombinant/genetics , Escherichia coli Proteins , Genes, Reporter , Genes, Synthetic , Liver/metabolism , Mice, Transgenic/genetics , Mutagenicity Tests/standards , Mutation , Repressor Proteins/genetics , Animals , Bacterial Proteins/biosynthesis , Chromogenic Compounds , DNA, Recombinant/isolation & purification , Enzyme Induction , Escherichia coli/genetics , Female , Galactosides , Gene Expression Regulation, Bacterial , Indoles , Laboratories , Lac Repressors , Liver/chemistry , Male , Mice , Mice, Inbred C57BL , Recombinant Fusion Proteins/genetics , Reproducibility of Results , beta-Galactosidase/biosynthesis
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