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PubMed2000

Two SecG molecules present in a single protein translocation machinery are functional even after crosslinking.

SecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA. Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant. All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro. Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent. The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane. Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery. The crosslinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.

Amino Acid Sequence↗
PubMed2006

Gene targeting of ErbB3 using a Cre-mediated unidirectional DNA inversion strategy.

Recombinase-mediated unidirectional DNA inversion and transcriptional arrest is a promising strategy for high throughput conditional mutagenesis in the mouse. Banks of mouse embryonic stem cells with defined, transcriptionally silent insertions that can be activated by Cre recombinase would take advantage of existing transgenic Cre lines to rapidly produce hundreds of lineage specific and temporally controlled knockout mice for each gene, thereby introducing significant parallelism to functional gene annotation. However, the extent to which this strategy results in effective gene knockout has not been established. To test the feasibility of this strategy we targeted ErbB3, a member of the ErbB family of tyrosine kinase receptors, using this strategy. Insertion of a reversed "flipflox" vector consisting of a gene inactivation cassette (GI) and an internal ribosome entry site (IRES)-GFP reporter into intron 1 of ErbB3 was transcriptionally silent and did not affect ErbB3 expression. Crosses with ubiquitous and lineage specific Cre recombinase expressing lines permanently inverted the inserted GI cassette and blocked ErbB3 expression. Unidirectional DNA inversion by in vivo recombination is an effective strategy for targeted or ubiquitous gene knockout.

Animals↗
PubMed2006

Identification of the lacZ insertion site and beta-galactosidase expression in transgenic chickens.

The quail:chick chimera system is a classical research model in developmental biology. An improvement over the quail:chick chimera system would be a line of transgenic chickens expressing a reporter gene. Transgenic chickens carrying lacZ and expressing bacterial beta-galactosidase have been generated, but complete characterization of the insertion event and characterization of beta-galactosidase expression have not previously been available. The genomic sequences flanking the retroviral insertion site have now been identified by using inverse polymerase chain reaction (PCR), homozygous individuals have been identified by using PCR-based genotyping, and beta-galactosidase expression has been evaluated by using Western analysis and histochemistry. Based upon the current draft of the chicken genome, the viral insertion carrying the lacZ gene has been located on chromosome 11 within the predicted gene for neurotactin/fractalkine (CX3CL1); neurotactin mRNA expression appears to be missing from the brain of homozygous individuals. When Generation 2 (G2) lacZ-positive individuals were inter-mated, they generated 361 G3 progeny; 82 were homozyous for lacZ (22.7%), 97 were wild-type non-transgenic (26.9%), and 182 (50.4%) were hemizygous for lacZ. Western analysis revealed the highest expression in the muscle and liver. With the identification of homozygous birds, the line of chickens is now designated NCSU-Blue1.

Animals↗
PubMed1987

Unusual conformational effect exerted by Z-DNA upon its neighboring sequences.

Supercoiled plasmid DNA harboring an insert of (dG-dC)16, a sequence known to form Z-DNA upon negative supercoiling, was reacted with chloroacetaldehyde. Chloroacetaldehyde, like bromoacetaldehyde, was found to be a specific probe for detecting unpaired DNA bases in supercoiled plasmid DNA. Under torsional stress (at bacterial superhelical density), chloroacetaldehyde reacted at multiple discrete regions within the neighboring sequences of the (dG-dC)16 insert. When the plasmid population was considered as a whole, the distribution of the chemically reactive bases exhibited a pattern of inversion symmetry with the center of inversion in the middle of the (dG-dC)16 insert. However, when a single supercoiled plasmid molecule was considered, chloroacetaldehyde reacted with only one of the neighboring sequences, either 5' or 3' of the (dG-dC)16 insert, but not with both. The possibility that the supercoiled plasmid DNA is in equilibrium with these two structural forms is discussed.

Acetaldehyde↗
PubMed2000

A simple and efficient method to determine the terminal sequences of restriction fragments containing known sequences.

We present an improvement of the inverse PCR method for the determination of end sequences of restriction fragments containing unknown DNA sequences flanked by known segments. In this approach, a short "bridge" DNA is inserted during the self-ligation step of the inverse PCR technique. This bridge DNA acts as primer annealing sites for amplification and subsequent direct sequencing. Successive PCR amplifications enable selective amplification of the unknown sequences from a complex mixture. Unlike previously described methods, our method does not require special materials, such as synthetic adapters or biotinylated primers that must be prepared each time to adapt the target. Furthermore, no complex steps such as dephosphorylation or purification are needed. Our method can save time and reduce the cost of cloning unknown sequences; it is ideal for routine, rapid gene walking. We applied this method to a GC-rich bacterial genome and succeeded in determining the end sequences of a 4.5-kb fragment.

Chromosome Walking↗
PubMed1987

Mismatch repair during pneumococcal transformation of small deletions produced by site-directed mutagenesis.

The genetic behaviour of short non-homologous regions has been studied during transformation of Streptococcus pneumoniae. Amethopterin-resistant mutants belonging to the amiA locus were used for these investigations. Five mutants deleted for 1-5 bp were obtained by oligonucleotide-directed mutagenesis. Their efficiency of transformation was measured using recipient strains either able to excise and repair mismatched bases (Hex+) or Hex- derivatives. Deletions or insertions of 1 and 2 bp are fully recognized by the Hex system, and are efficiently repaired whereas 3-bp deletions or insertions are only partially excised and repaired. The efficiency of repair is inversely related to the size of the non-homology. Markers with 5-bp deletions or insertions are poorly repaired and thus transform at very high frequency: similar results are obtained in reciprocal crosses. It is proposed that 1- or 2-bp deletions or insertions are included in the heteroduplex structure as transition mutations. The Hex system would detect only small deviations from the normal DNA structure.

Chromosome Deletion↗
PubMed1993

Genetic instability in Drosophila melanogaster mediated by hobo transposable elements.

Eight independent recessive lethal mutations that occurred on derivatives of an unstable X chromosome (Uc) in Drosophila melanogaster were analyzed by a combination of genetic and molecular techniques. Seven of the mutations were localized to complementation groups in polytene chromosome bands 6E; 7A. In situ hybridization and genomic Southern analysis established that hobo transposable elements were associated with all seven of the mutations. Six mutations involved deletions of DNA, some of which were large enough to be seen cytologically, and in each case, a hobo element was inserted at the junction of the deletion's breakpoints. A seventh mutation was associated with a small inversion between 6F and 7A-B and a hobo element was inserted at one of its breakpoints. One of the mutant chromosomes had an active hobo-mediated instability, manifested by the recurrent production of mutations of the carmine (cm) locus in bands 6E5-6. This instability persisted for many generations in several sublines of an inbred stock. Two levels of instability, high and basal, were distinguished. Sublines with high instability had two hobo elements in the 6E-F region and produced cm mutations by deleting the segment between the two hobos; a single hobo element remained at the junction of the deletion breakpoints. Sublines with low instability had only one hobo element in the 6E-F region, but they also produced deletion mutations of cm. Both types of sublines also acquired hobo-mediated inversions on the X chromosome. Collectively, these results suggest that interactions between hobo elements are responsible for the instability of Uc. It is proposed that interactions between widely separated elements produce gross rearrangements that restructure the chromosome and that interactions between nearby elements cause regional instabilities manifested by the recurrence of specific mutations. These regional instabilities may arise when a copy of hobo transposes a short distance, creating a pair of hobos that can interact to produce small rearrangements.

Animals↗
PubMed2005

Populations of a Connecticut River midge structured by geological history and downstream gene flow.

Connecticut River midges in the genus Axarus are constrained to semi-isolated populations associated with submerged clay exposures, and gene flow between populations is restricted. I sampled larval midges from fifteen spatially separated sites in the Connecticut River and determined karyotypes for 3111 larvae in two closely related species. In one of these species, an undescribed species referred to as Axarus species varvestris, 5 chromosomal rearrangements were present as polymorphisms. Four of these are paracentric inversions, and the fifth is a terminal insertion or deletion. I used chromosomal rearrangement frequencies to calculate Nei's genetic distance measures for all pairwise population comparisons and I constructed a neighbor-joining phenetic tree for the populations using these distances. Genetic distance between populations of A. sp. varvestris increases with geographic separation, and tree structure indicates that restricted gene flow only occurs in a downstream direction. The effect of unidirectional (downstream) gene flow on population genetic structure in riverine midges is described here for the first time. A genetic break is apparent between populations associated with 12 000-year-old sediments originating in different ancient lakes. The geological break between lakes also coincides with a break in the species composition of the Axarus populations.

Animals↗
PubMed1983

R46 encodes a site-specific recombination system interchangeable with the resolution function of TnA.

Transposition of Tn4 onto the IncN plasmic R46 generates unstable DNA molecules. The R46::TnA recombinant plasmids undergo further DNA rearrangements which depend on the orientation in which the TnA element is inserted into the plasmid, and deletions and inversions of R46 and TnA sequences have been observed. Both types of rearrangement have the same specific endpoints, one within TnA and one located between the R46 coordinates, 36.0 and 37.0. The results are consistent with the operation of a recA-independent, site-specific recombination system utilizing, at least in part, the transposon cointegrate resolution system of TnA, together with R46-encoded functions. Data are presented that indicate that R46 encodes analogs of both the res site of TnA and its tnpR gene, although little homology between this element and the plasmid is apparent. Models for the TnA-induced generation of site-specific deletions and inversions upon transposition of TnA to R46 are presented.

Chromosome Deletion↗
PubMed2000

Apparent coactivation due to interference of expression constructs with nuclear receptor expression.

Transient cotransfection in COS-7 cells, a standard approach to demonstrate coactivation, was used to study the coactivation properties of NuRIP183, a new nuclear receptor interacting protein of 183 kDa, isolated by a yeast two-hybrid screening. Transfection with a NuRIP183 expression construct strongly increased the ligand-dependent response of reporter constructs for several nuclear receptors when compared to transfection with the empty expression vector. A more detailed study, however, revealed major changes in the expression level of the nuclear receptors in cotransfection experiments, indicating that the observed changes in receptor activity were not due to coactivation but to differences in receptor concentration caused by interference from the cotransfected expression constructs with the expression of the receptor. Such interference, which is inversely related to the length of the insert, was observed, not only in COS-7 cells but also in CV-1 and MCF-7 cells, using different transfection techniques (FuGENE-6 and calcium phosphate) and different expression vectors (pSG5, pcDNA1.1 and pIRESneo). These data cast some doubt on coactivation of nuclear receptors based on similar cotransfection experiments without measurement of receptor concentration. Moreover, it is recommended to limit the amounts of (co)transfected expression plasmid and to avoid the use of empty expression plasmid as a control. Finally, one should be aware of similar misleading results in other experimental set-ups based on cotransfection.

Animals↗
PubMed2005

Transcriptional activation by bidirectional RNA polymerase II elongation over a silent promoter.

Transcriptional interference denotes negative cis effects between promoters. Here, we show that promoters can also interact positively. Bidirectional RNA polymerase II (Pol II) elongation over the silent human endogenous retrovirus (HERV)-K 18 promoter (representative of 2.5 x 10(3) similar promoters genomewide) activates transcription. In tandem constructs, an upstream promoter activates HERV-K 18 transcription. This is abolished by inversion of the upstream promoter, or by insertion of a poly(A) signal between the promoters; transcription is restored by poly(A) signal mutants. TATA-box mutants in the upstream promoter reduce HERV-K 18 transcription. Experiments with the same promoters in a convergent orientation produce similar effects. A small promoter deletion partially restores HERV-K 18 activity, consistent with activation resulting from repressor repulsion by the elongating Pol II. Transcriptional elongation over this class of intragenic promoters will generate co-regulated sense-antisense transcripts, or, alternatively initiating transcripts, thus expanding the diversity and complexity of the human transcriptome.

Animals↗
PubMed1997

Identification of Pseudomonas aeruginosa genes required for epithelial cell injury.

We have developed a simple, reproducible and rapid genetic screen for Pseudomonas aeruginosa-induced epithelial cell cytotoxicity in cultures of MDCK cells. This screen was used to isolate isogenic transposon-tagged non-cytotoxic mutants of a cytotoxic and lung-virulent strain of P. aeruginosa (PA103). The transposon-insertion site was determined by using an inverse polymerase chain reaction followed by DNA-sequence analysis. On the basis of phenotype and sequence analysis, these mutants fell into four classes. One class had absent or defective pill, based on their resistance to phage PO4 and/or loss of twitching motility (twt-). A second class exhibited decreased adherence. A third class of mutants exhibited probable defects in the machinery or targets of type III protein secretion. A final class of mutants exhibited decreased but not absent cytotoxicity. This class included members of the first three classes as well as other mutants. These results suggest that localized cytotoxicity is likely to require several steps and several components, including pili and other (unidentified) extracellular proteins. The type III protein-secretion apparatus appears to be involved in this process.

Amino Acid Sequence↗
PubMed2005

Rates of intron loss and gain: implications for early eukaryotic evolution.

We study the intron-exon structures of 684 groups of orthologs from seven diverse eukaryotic genomes and provide maximum likelihood estimates for rates and numbers of intron losses and gains in these same genes for a variety of lineages. Rates of intron loss vary from approximately 2 x 10(-9) to 2 x 10(-10) per year. Rates of gain vary from 6 x 10(-13) to 4 x 10(-12) per possible intron insertion site per year. There is an inverse correspondence between rates of intron loss and gain, leading to a 20-fold variation among lineages in the ratio of the rates of the two processes. The observed rates of intron gain are insufficient to explain the large number of introns estimated to have been present in the plant-animal ancestor, suggesting that introns present in early eukaryotes may have been created by a fundamentally different process than more recently gained introns.

Animals↗
PubMed1986

Location of the site-specific recombination system of R46: a function necessary for plasmid maintenance.

The R46 site-specific recombination system comprises a per (plasmid-encoded recombinase) gene and a site at which the gene product acts, the per site. The two functions have been cloned into pACYC184. They are encoded by sequences within a region of approximately 2 kb on the R46 genome. These R46 sequences are closely related to the site-specific recombination systems of the ampicillin resistance transposons collectively designated TnA. The R46 per function is interchangeable with the tnpR gene product of TnA. Both enzymes can mediate recombination between the related res and per sites in R46::TnA recombinant plasmids to generate site-specific deletions and inversions. Similar DNA rearrangements occur when TnA inserts into pACYC184 derivatives carrying the cloned R46 per functions. Carriage of this site-specific recombination system contributes to the stable maintenance of R46. By converting plasmid dimers to monomers the R46 per functions help to ensure equal partitioning at cell division.

Cloning, Molecular↗
PubMed2006

Transcribing paramyxovirus RNA polymerase engages the template at its 3' extremity.

For the non-segmented, negative-stranded RNA viruses, the mechanism controlling transcription or replication is still a matter of debate. To gain information about this mechanism and about the nature of the RNA polymerase involved, the length of an intervening sequence separating the 3' end of Sendai virus minigenomes and a downstream transcription-initiation signal was increased progressively. It was found that transcription, as measured by green fluorescent protein (GFP) expression, decreased progressively in proportion to the increase in length of the intervening sequence. GFP expression correlated well with the levels of GFP mRNA in the cells, as measured by quantitative primer extension and by RNase protection. Thus, mRNA transcription was inversely proportional to the length of the inserted sequence. These data are evidence that the RNA polymerase initiating transcription at the downstream transcription signal somehow sees the distance separating this signal and the template 3' extremity. Implication of this observation for the nature of the Sendai virus RNA polymerase and for the mechanism by which it synthesizes mRNAs or replication products is presented.

3' Flanking Region↗
PubMed1996

The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family.

To transposon tag the tomato Dwarf (D) gene, a tomato line that carries a T-DNA containing a maize Activator (Ac) transposable element closely linked to D was pollinated with a stock homozygous for the d mutation. Hybrid seedlings were screened for dwarf progeny, and three independent dwarf lines were obtained. Two of these lines showed inheritance of a recessive phenotype similar to that conferred by the extreme dwarf (dx) allele. Variegation for the dwarf phenotype in one of these lines suggested that D had been tagged by Ac. Genomic DNA adjacent to Ac in these two lines was isolated by use of the inverse polymerase chain reaction, and the two insertions mapped approximately 2 kb apart. Partial complementation of d was observed when the corresponding wild-type sequence was used in transformation experiments. A cDNA clone of D was sequenced, and the predicted amino acid sequence has homology to cytochrome P450 enzymes.

Alleles↗
PubMed2003

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association.

Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.

Bacterial Proteins↗