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At least 919 records · Page 51Linked to original sources
PubMed1989

Transformation of Mycobacterium smegmatis with E. coli plasmids.

One limiting factor in the studies of tuberculosis and leprosy is the lack of a versatile system for genetic analysis and manipulation of mycobacteria. One strategy used in constructing a plasmid vector for transforming Mycobacterium smegmatis was to insert fragments of a mycobacterial plasmid into an Escherichia coli plasmid. We found that the parental E. coli plasmid is capable of self-replication in M. smegmatis yielding chloramphenicol-resistant colonies. Plasmids from different passages of one M. smegmatis transformant were recovered and characterised by restriction digest analysis. The plasmid from the earlier passage was found to be indistinguishable from the original plasmid by restriction analysis. Plasmids from later preparations, however, were found to have undergone modifications in the M. smegmatis host resulting in an apparent increase in transformation efficiency for M. smegmatis. These plasmids can be used as a shuttle vector for the genetic manipulation of mycobacterial species.

DNA, Bacterial↗
PubMed1997

Effect of Bcr sequences on the cellular function of the Bcr-Abl oncoprotein.

In Philadelphia chromosome (Ph1)-positive human leukemia, the c-Abl tyrosine kinase is activated by fusion to sequences encoded by the breakpoint cluster region (bcr) gene. Two major types of Bcr-Abl fusion proteins have been found in human leukemia. Fusion of the N-terminal 426 amino acids of Bcr generates p190(Bcr-Abl) which is mostly found in acute lymphocytic leukemia (ALL), whereas fusion of the N-terminal 902 or 927 amino acids of Bcr generates p210(Bcr-Abl) mostly found with chronic myelogenous leukemia (CML). Previous studies have demonstrated that both the Bcr and the Abl functional domains contribute to the oncogenic activity of Bcr-Abl proteins. Present in both p190 and p210 is the N-terminal coiled-coil of Bcr (aa 1-63), which is shown here to be functionally replaceable with the leucine zipper of the yeast transcription factor GCN4. The ZIP-Bcr-Abl protein transforms Rat-1/myc cells, is autophosphorylated on tyrosine and localized predominantly to actin filaments. Thus, formation of homo-oligomers through either Bcr or GCN4 coiled-coil can activate the tyrosine kinase and F-actin binding functions of Abl. We also found that a Bcr-Abl fusion containing only Bcr amino acids (1-191) can efficiently transform Rat-1/myc cells. Fusion of additional Bcr sequences (aa 192-923) did not affect the transformation of Rat-1/myc cells but progressively reduced the disruptive effect on the actin cytoskeleton. In particular, the Dbl homology domain present in p210(Bcr-Abl) but not in p190(Bcr-Abl) contributes to the stabilization of actin fibers. The modulatory effect of Bcr sequences on actin structure may underlie the apparent pathogenic variations between the different Bcr-Abl fusion proteins.

Actins↗
PubMed1997

Distinct roles for DH and PH domains in the Lbc oncogene.

Members of the Dbl-homology (DH) family of proteins promote guanine nucleotide exchange on Rho GTPases. Lbc, which specifically acts on Rho but not Rac or Cdc42, was isolated as a transforming oncogene and is composed of a DH and a Pleckstrin-homology (PH) domain. We show here that the Lbc DH domain alone is capable of stimulating new DNA synthesis in quiescent fibroblasts and Rho-dependent actin stress fiber assembly. However, the PH domain is required for subcellular localization of Lbc along actin stress fibers and for efficient transformation of NIH3T3 cells. The results show that, in contrast to other Dbl-homology proteins, the PH domain of Lbc is dispensable for activation of Rho in vivo. The PH domain-dependent subcellular localization of Lbc may, however, be important for growth factor activation of endogenous Lbc and for oncogenic transformation.

3T3 Cells↗
PubMed2003

Interaction of the HPV E7 proteins with the pCAF acetyltransferase.

Most cervical carcinomas express the E6 and E7 proteins of a high-risk human papillomavirus (HPV). These proteins affect growth control by interfering with the functions of cell regulatory proteins, promoting oncogenic transformation. A key target of E7 is the tumor suppressor protein pRb, which directly interacts with E7. However, binding to additional cellular regulatory proteins is clearly required for oncogenesis, as mutants of E7 have been identified that bind to pRb, yet fail to transform efficiently. Here we demonstrate the interaction of the HPV 6, 16 and 18 E7 proteins with the pCAF acetyltransferase, which has been reported to function as a coactivator for a variety of transcription factors including p53. Mutation of a highly conserved leucine residue within the zinc finger region of HPV 16 E7 disrupts binding to pCAF and also impairs transformation and transcriptional activation. HPV 16 E7 interacts with the acetyltransferase domain of pCAF, and reduces its acetyltransferase activity in vitro. Our analysis of the interaction between the pCAF acetyltransferase and E7 provides new insight into the mechanisms by which the E7 oncoproteins can alter cellular gene expression and growth.

Acetylation↗
PubMed1981

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.

Base Sequence↗
PubMed1981

Competence for DNA transfer of ouabain resistance and thymidine kinase: clonal variation in mouse L-cell recipients.

We have used the calcium phosphate precipitation technique to study the competence of mammalian cell recipients for transformation with genomic mammalian cell DNA. The transformation efficiency for thymidine kinase (tk) varies 10- to 20-fold (up to 10(-4) transformants/recipient) among different subclones of the LM tk- CL 1D mouse fibroblast cell line. Analysis of this phenotype among second-generation subclones indicates that subclones exhibiting high competence tend to breed true, whereas those with low competence do not. Isolation of Tk- revertants from TK+ transformants results in the selection of cells with a high-competence phenotype as measured by their subsequent transformation for tk. This phenotype appears to be a general characteristic of such cells because recipients more competent for transfer of a second marker, ouabain resistance (ouaR). This codominant marker coding for the Na K+-ATPase can be transferred at frequencies of 10(-5) in the high-competence recipients. These results indicate that competence for DNA-mediated gene transfer can be determined in part by genetic factors.

Animals↗
PubMed1996

Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation.

Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [32P]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [Dt] and [Cat] of DNA and of calcium, respectively. At [Cat] = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at [Dt] = 2.7 nM and 10(8) cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3. The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size ø approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive translocation of the inserted DNA into the cell interior. For practical purposes of optimal transformation efficiency, 1 mM calcium is necessary for sufficient DNA binding and the relatively long pulse duration of 20-40 ms is required to achieve efficient electrodiffusive transport across the cell wall and into the outer surface of electroporated cell membrane patches.

Adsorption↗
PubMed2003

Mycobacteriophage Bxb1 integrates into the Mycobacterium smegmatis groEL1 gene.

Mycobacteriophage Bxb1 is a temperate phage of Mycobacterium smegmatis and forms stable lysogens in which the Bxb1 genome is integrated into the host chromosome. Bxb1 encodes an integrase of the large serine recombinase family that catalyses integration and excision of the Bxb1 genome. We show here that Bxb1 integrates into a chromosomal attB site located within the 3' end of the groEL1 gene such that integration results in alteration of the C-terminal 21 amino acid residues. An integration-proficient plasmid vector containing the Bxb1 integrase gene and flanking DNA sequences efficiently transforms M. smegmatis via integration at attB. Bxb1-integrated recombinants are stable and fully compatible with L5 integration vectors. Strand exchange occurs within an 8 bp common core sequence present in attB and within an attP site situated immediately upstream of the phage integrase gene. Establishment of a defined in vitro system for Bxb1 integration shows that recombination occurs efficiently without requirement for high-energy cofactors, divalent metals, DNA supercoiling or additional proteins.

Amino Acid Sequence↗
PubMed1996

Interaction between T antigen and TEA domain of the factor TEF-1 derepresses simian virus 40 late promoter in vitro: identification of T-antigen domains important for transcription control.

The large tumor antigen (TAg) of simian virus 40 regulates transcription of the viral genes. The early promoter is repressed when TAg binds to the origin and DNA replication begins, whereas the late promoter is activated by TAg through both replication-dependent and -independent mechanisms. Previously it was shown that activation is diminished when a site in the viral enhancer to which the factor TEF-1 binds is disrupted. We show here that the NH2-terminal region of TAg binds to the TEA domain of TEF-1, a DNA binding domain also found in the Drosophila scalloped and the Saccharomyces cerevisiae TEC1 proteins. The interaction inhibits DNA binding by TEF-1 and activates transcription in vitro from a subset of naturally occurring late start sites. These sites are also activated by mutations in the DNA motifs to which TEF-1 binds. Therefore, TEF-1 appears to function as a repressor of late transcription, and its involvement in the early-to-late shift in viral transcription is discussed. The mutation of Ser-189 in TAg, which reduces transformation efficiency in certain assays, disrupts the interaction with TEF-1. Thus, TEF-1 might also regulate genes involved in growth control.

Animals↗
PubMedDate not supplied

In vitro transformation of chicken bone marrow cells with avian erythroblastosis virus.

An in vitro transformation of bone marrow cells has been demonstrated for two strains of avian erythroblastosis virus (AEV-R and AEV-ES4). The transformed cells were indistinguishable from in vivo transformed erythroblasts in morphology and staining characteristics and could be propagated to large numbers. The transformation efficiency could be greatly increased by the addition of dimethylsulfoxide (DMSO). The number of foci appearing in the presence of DMSO was proportional to the virus concentration.

Alpharetrovirus↗
PubMed1986

Genetic analysis of the 3' early region transformation and replication functions of bovine papillomavirus type 1.

Cell transformation by BPV-1 is dependent on sequences confined to a region comprising only 69% of the viral DNA. Analysis of this subgenomic fragment of the viral genome has revealed a composite of functions. To further study this region of the BPV-1 genome, we constructed mutants affecting the 3' half of the transforming region. These mutations have revealed a plasmid maintenance function for the E2 ORF and a requirement that the 3' half of E5 be intact for in vitro mouse cell transformation. Cotransfection experiments revealed that phenotypic complementation (wild type transformation efficiency and autonomous replication of viral DNA) resulted in recombination between input plasmids in the majority of cases. However, rare cases were encountered where the wild type phenotype was observed with no evidence of recombination between input plasmids. These data suggest trans-acting functions of the E2 and E5 ORFs in viral DNA replication and the E5 ORF in cell transformation.

Animals↗
PubMed1982

Sequence-specific DNA uptake in transformation of Neisseria gonorrhoeae.

Piliated, competent gonococci are known to preferentially take up homologous transforming DNA into the cell. We examined the mechanism for DNA uptake with pFA10, a hybrid 11.5-kilobase (kb) penicillin-resistant (Pcr) plasmid composed of heterologous DNA from a 7.2-kb Pcr plasmid and homologous DNA from a 4.2-kb gonococcal cryptic plasmid. The presence of the gonococcal cryptic plasmid DNA in the hybrid resulted in markedly increased transformation efficiencies in isogenic crosses as compared with the parent 7.2-kb Pcr plasmid. Uptake of 32P-end-labeled MspI or TaqI restriction fragments of the hybrid was limited to fragments entirely derived from the 4.2-kb gonococcal cryptic plasmid, indicating that DNA uptake was probably dependent on the presence of a specific DNA sequence. Since Haemophilus DNA did not inhibit transformation by the hybrid Pcr plasmid, the gonococcal DNA uptake sequence is different from the known sequence involved in homologous DNA uptake by Haemophilus spp.

Base Sequence↗
PubMed2001

Stable transformation of Pleurotus ostreatus to hygromycin B resistance using Lentinus edodes GPD expression signals.

It was reported that Pleurotus ostreatus was transformed unstably using recombinant plasmids containing a hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans expression signals, and that the plasmids were maintained extrachromosomally in the transformants. Here we report a stable and integrative transformation of the fungus to hygromycin B resistance, using a recombinant hph fused with Lentinus edodes glyceraldehyde-3-phosphate dehydrogenase expression signals. Restriction-enzyme-mediated integration (REMI) was also tried and increased the transformation efficiency about ten-fold.

Anti-Bacterial Agents↗
PubMed1998

Insertion-duplication mutagenesis in Streptococcus pneumoniae: targeting fragment length is a critical parameter in use as a random insertion tool.

To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.

Chimera↗
PubMed1989

dam methylation and the initiation of DNA replication on oriC plasmids.

Plasmid DNA containing the replication origin of the Escherichia coli chromosome (oriC) has been shown to be inefficient as a template for DNA synthesis in vitro when isolated from dam mutants. Here, we extend this study to hemimethylated oriC plasmids and to replication in dam-3 mutant enzyme extracts. The results show that: (1) hemimethylated oriC plasmids replicate with the same low efficiency as nonmethylated DNA; (2) DNA synthesis starts at oriC regardless of the methylated state of the template; (3) replication in dam-3 enzyme extracts is inefficient because this strain is deficient in DnaA protein; and (4) consistent with this observation, the copy number of the oriC plasmid pFH271 is reduced in the dam-3 mutant. However, we have found that low DnaA protein levels in dam-3 mutants are not sufficient to explain the reduced transformation efficiency of oriC plasmids. We suggest that there must exist in vivo inhibitory factors not present or present in low quantities in vitro which specifically recognize the hemimethylated or nonmethylated forms of the oriC region.

Bacterial Proteins↗
PubMed2003

Flexibility of the endogenous progesterone lactonisation pathway in Aspergillus tamarii KITA: transformation of a series of cortical steroid analogues.

A range of cortical steroids have been transformed by the fungus Aspergillus tamarii, which has the ability to convert progesterone to testololactone in high yield through a four step enzymatic pathway. 16alpha,17alpha-Epoxyprogesterone underwent a rare epoxide opening resulting in a unique inversion of stereochemistry to give 16beta-hydroxy-17alpha-oxa-D-homo-androst-4-en-3,17-dione. The metabolism of deoxycorticosterone resulted in relatively efficient transformation to testololactone with no other products isolated. Transformation of 17alpha-hydroxyprogesterone yielded 17alpha-oxa-D-homo-androst-1,4-dien-3,17-dione, a lactone not previously isolated from A. tamarii. Cortexolone was transformed to the 20(R)-alcohol with no further transformation observed. Evidence is also presented for the presence of a highly flexible but stereospecific keto-reductase. All metabolites were isolated by column chromatography and were identified by 1H, 13C NMR, DEPT analysis and other spectroscopic data.

Aspergillus↗
PubMed2003

Development of cloning vectors and transformation methods for Amycolatopsis.

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA- rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.

Actinomycetales↗
PubMed2000

Native plasmids of Fusobacterium nucleatum: characterization and use in development of genetic systems.

Three native plasmids of Fusobacterium nucleatum were characterized, including DNA sequence analysis of one plasmid, pFN1. A shuttle plasmid, pHS17, capable of transforming Escherichia coli and F. nucleatum ATCC 10953 was constructed with pFN1. pHS17 was stably maintained in the F. nucleatum transformants, and differences in the transformation efficiencies suggested the presence of a restriction-modification system in F. nucleatum.

Amino Acid Sequence↗