Identification of a Novel Gene Involved in Stable Maintenance of Plasmid pGA1 from Corynebacterium glutamicum

The cryptic plasmid pGA1 (4.8 kb) from Corynebacterium glutamicum, replicating in the rolling-circle mode, has been reported to contain four open reading frames longer than 200 bp (ORFA/per, ORFA2, ORFB, ORFC/rep). Here we present another pGA1 gene, ORFE (174 bp), located in the region downstream of the per-ORFA2 gene cluster. The ORFE is transcribed into two RNA species in a direction opposite to that of the per-ORFA2 RNA. Introduction of ORFE in trans into the cells harboring the pGA1 derivatives carrying the main stability determinant, the per gene coding for a product that positively influences the pGA1 copy number and maintenance, increased their segregational stability. Mutation of the putative translational start of the ORFE abolished this observed positive effect in trans. ORFE thus codes for a protein acting as an accessory element involved in stable maintenance of plasmid pGA1 and was hence designated the aes gene (accessory effector of stable maintenance).     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

Molecular and functional analyses of COPT/Ctr-type copper transporter-like gene family in rice

Background  

The copper (Cu) transporter (COPT/Ctr) gene family has an important role in the maintenance of Cu homeostasis in different species. The rice COPT-type gene family consists of seven members (COPT1 to COPT7). However, only two, COPT1 and COPT5, have been characterized for their functions in Cu transport.     

Strong linkage disequilibrium near the selected Yr17 resistance gene in a wheat experimental population

Dynamic management (DM) is a method of genetic resources conservation that aims at maintaining evolutionary process in subdivided populations cultivated in contrasted environments. Such populations are often submitted to strong natural selection as it was the case for experimental wheat populations maintained under DM. Understanding impacts of selection on genetic diversity around selected genes is necessary for the middle-term maintenance of genetic variability in DM populations. Evolution of diversity at six neutral markers located near the yellow rust resistance gene Yr17 has been studied for the parental lines and for generations 1, 5, 10 and 17 in one of the DM populations. Yr17 provided complete resistance to yellow rust in France until 1997 and thus was suspected to be under strong selection. The gene is located on a fragment introgressed in winter wheat from a wild species. The presence of the gene was estimated using a marker closely related to the gene. We showed that the Yr17 gene has been selected between generations 5 and 10. Generally, selection tends to reduce diversity around selected genes, generating linkage disequilibrium (LD) between a gene and adjacent markers. Here, the major effect of the Yr17 gene selection was a reduction of multilocus diversity and the maintenance of strong pre-existing LD in the zone surrounding the gene for a distance of 20 cM. As expected, the presence of the exogenous introgression was responsible for restrictions to recombination which contributed to the maintenance of strong correlations between loci. However, we found a noticeable number of recombinations around the gene indicating a progressive incorporation of the fragment into the wheat genome.     

rpsL+: a dominant selectable marker for gene replacement in mycobacteria

Molecular genetic manipulations in mycobacteria would benefit from procedures which efficiently select for double-crossover events by homologous recombination. Here we describe a vector-host system for gene replacement in mycobacteria, the utility of which was investigated using functional inactivation of the pyrF gene in Mycobacterium smegmatis as a model. This system is based on the expression of the wild-type rpsL gene coding for ribosomal protein S12 in a streptomycin-resistant host. Owing to the absence of a mycobacterial origin the plasmids are unable to replicate autonomously in mycobacteria. The first selection for maintenance of cloned sequences is conferred by the kanamycin-resistance gene. The second simultaneous selection by streptomycin is against maintenance of cloned sequences which contain the gene encoding the streptomycin-sensitive allele of the rpsL gene. By placing the gene for positive selection and that used for negative selection within and outside the target gene of interest, respectively, gene replacement is obtained. A one-step double selection procedure provides a means to distinguish strictly between gene replacement by double crossover versus homologous recombination by single crossover events. The system should have considerable potential for genera or species where single-crossover events or even illegitimate recombination are the predominant recombination mechanisms. It should also be of wide use for the construction of mutants without a selectable phenotype.     

Regions associated with the stable maintenance of plasmid pSC101 and its tetracycline resistance

Summary Two regions tentatively called unsA and unsR were identified on pSC101. One, unsA, corresponds to less than 650 bp of the N-terminal in the tetracycline resistance structural gene and seems to inhibit stable maintenance of pSC101. The other, unsR, is defined within the 1 kb XhoI-EcoRI region located upstream of the tetracycline resistance structural gene and is a regulatory gene clearly distinct from tetR (Unger et al. 1984); it serves as a suppressor of the unsA function.     

Compartmentalised expression of <Emphasis Type="Italic">Delta-like 1</Emphasis> in epithelial somites is required for the formation of intervertebral joints

Background  

Expression of the mouse Delta-like 1 (Dll1) gene in the presomitic mesoderm and in the caudal halves of somites of the developing embryo is required for the formation of epithelial somites and for the maintenance of caudal somite identity, respectively. The rostro-caudal polarity of somites is initiated early on within the presomitic mesoderm in nascent somites. Here we have investigated the requirement of restricted Dll1 expression in caudal somite compartments for the maintenance of rostro-caudal somite polarity and the morphogenesis of the axial skeleton. We did this by overexpressing a functional copy of the Dll1 gene throughout the paraxial mesoderm, in particular in anterior somite compartments, during somitogenesis in transgenic mice.     

Minimal regions in the Arabidopsis PISTILLATA promoter responsive to the APETALA3/PISTILLATA feedback control do not contain a CArG box

PISTILLATA (PI) is a floral homeotic B function gene in Arabidopsis and together with the other B function gene, APETALA3 (AP3), is involved in specifying petal and stamen identities. The expression of PI and AP3 is under similar developmental control. The initiation of AP3 and PI expression is at least partly caused by the floral meristem identity gene LEAFY, but the maintenance of AP3 and PI expression involves an autoregulatory loop requiring the activity of both genes. PI and AP3 are MADS domain proteins that form, and appear to function as, a heterodimer. AP3/PI binds in vitro to a sequence motif, CC(A/T)₆GG, a MADS domain protein consensus binding site also known as the CArG box. We identified a 481-bp PI promoter region that confers both the initiation and the maintenance of PI expression patterns. We further dissected the promoter and identified minimal regions responsible for the AP3/PI-dependent expression. No CArG box is present in these minimal regions, suggesting that either AP3/PI does not bind directly to the PI promoter for the maintenance control, or that it requires additional factors to bind to the PI promoter. Our results suggest that the mechanisms of regulation of the two B function genes, AP3 and PI, are different, because CArG boxes are present in the AP3 promoter and are necessary for the AP3 feedback control. Received: 1 March 2000 / Revision accepted: 15 June 2000     

Epigenetic regulation of vertebrate Hox genes: A dynamic equilibrium

Temporal and spatial control of Hox gene expression is essential for correct patterning of many animals. In both Drosophila and vertebrates, Polycomb and Trithorax group complexes control the maintenance of Hox gene expression in appropriate domains. In vertebrates, dynamic changes in chromatin modifications are also observed during the sequential activation of Hox genes in the embryo, suggesting that progressive epigenetic modifications could regulate collinear gene activation.     

Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene: understanding the role of Enhancers of trithorax and Polycomb

Background  

Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements (ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos. To understand better the role of ETP, we addressed genetic and molecular interactions between corto and dsp1.     

Rhythmic gene expression in somite formation and neural development

In mouse embryos, somite formation occurs every two hours, and this periodic event is regulated by a biological clock called the segmentation clock, which involves cyclic expression of the basic helix-loop-helix gene Hes7. Hes7 expression oscillates by negative feedback and is cooperatively regulated by Fgf and Notch signaling. Both loss of expression and sustained expression of Hes7 result in severe somite fusion, suggesting that Hes7 oscillation is required for proper somite segmentation. Expression of a related gene, Hes1, also oscillates by negative feedback with a period of about two hours in many cell types such as neural progenitor cells. Hes1 is required for maintenance of neural progenitor cells, but persistent Hes1 expression inhibits proliferation and differentiation of these cells, suggesting that Hes1 oscillation is required for their proper activities. Hes1 oscillation regulates cyclic expression of the proneural gene Neurogenin2 (Ngn2) and the Notch ligand Delta1, which in turn lead to maintenance of neural progenitor cells by mutual activation of Notch signaling. Taken together, these results suggest that oscillatory expression with short periods (ultradian oscillation) plays an important role in many biological events.     

Association between polymorphisms of VKORC1 and CYP2C9 genes with warfarin maintenance dose in a group of warfarin users in Birjand city,Iran

Warfarin is the cardinal anticoagulant drug prescribed around the world. Due to stochastic bleeding in patients, it is essential to adjust the dose for every individual. The aim of the present study was to evaluate the frequency of CYP2C9 and VKORC1 gene polymorphisms and their association with warfarin maintenance dose in a sample of cardiovascular patients in Birjand, South-Khorasan province of Iran. Patients with a history of cardiovascular disorders who take warfarin daily were selected. CYP2C9 and VKORC1 gene polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism in all participants. A total of 114 patients (mean age: 52.7 ± 14.9 years, M/F ratio: 0.76) participated in this study. Regarding CYP2C9 gene polymorphisms, the most frequent genotype was 1*/1* (80.4% in females and 62.5% in males). The frequency of 1*/2* and 2*/2* variants was 13% and 6.5% in females and 25% and 12.5% in males, respectively. The frequency of VKORC1 gene (1639 G > A), was 31.5%, 39.5%, and 29% for GG, GA, and AA in males, respectively. Besides, the mentioned genotype frequencies for females were 50%, 40.5%, and 9.5%, respectively. Moreover, there was a statistically significant correlation between VKORC1 gene −1639 G > A variant and warfarin maintenance dose (P < 0.001) but not for CYP2C9 variants. The results of the current study confirmed that the mutant variants of CYP2C9 are not frequent and do not have any impact on warfarin dose. In the case of VKORC1, the mutant allele (A) showed a positive correlation with warfarin dose adjustment.     

Improved binary vectors for Agrobacterium-mediated plant transformation

Improved plant transformation vectors were constructed which utilize the pRiHRI origin of replication for highly stable maintenance in Agrobacterium tumefaciens, the ColE1 origin of replication for high copy maintenance in Escherichia coli, and a gentamycin resistance gene as a strong selectable marker for bacteria. Concise T-DNA elements were engineered with border sequences from the TL-DNA of pTiA6, the Tn5 neomycin phosphotransferase gene (npt II) expressed from either CaMV 35S or mannopine synthase (mas) promoters, and the lac Z gene segment from pUC18 as a source of unique restriction sites as well as an insertional inactivation marker for cloned DNA. The order of T-DNA components in all vectors is left border, plant marker cassette, lac Z, and right border, respectively. The prototype vector, pCGN1547, was shown to be very stable in A. tumefaciens strain LBA4404 and to act as an efficient donor of T-DNA in tomato transformation experiments. Use of the other vectors is also described.     

Nitrite toxicity inAspergillus nidulans: Effect of mutation at thenihB gene

ThenihB gene ofAspergillus nidulans was found to confer sensitivity to elevated concentrations of nitrite, compact morphology and absence of conidiation. ThenihB locus was allocated to linkage group II and was recessive in heterozygous diploids. When thenihB1 mutant was grown on a mixture of nitrite plus NH ₄ ⁺ its sensitivity to nitrite was unchanged. A possible role for this gene in nitrite transport and/or the maintenance of membrane integrity is discussed.     

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

Neurofibromatosis type 1 is a disorder of dysplasia: The importance of distinguishing features,consequences, and complications

BACKGROUND : The disorder neurofibromatosis type 1 (NF1) is caused by mutations in the NF1 gene, which influences the availability of activated Ras and the latter's control of cellular proliferation. Emphasis on this aspect of NF1 has focused attention on the tumor suppression function of NF1 and thereby displaced attention from the gene's role in initial normal tissue formation, maintenance, and repair. METHODS : Clinical and neuroimaging data systematically compiled over more than 30 years are analyzed to document the involvement of multiple organs and tissues, often with an embryonic origin. In addition, recent literature based on selective knockout mouse experiments is cited to corroborate embryonic dysplasia as an element of NF1 pathogenesis. RESULTS : Tissue dysplasia, both ab initio and as part of tissue maintenance and wound healing, is a key clinical and pathogenetic aspect of NF1 and thereby provides a rationale for differentiating the elements of NF1 into features, consequences, and complications. CONCLUSIONS : NF1 is a histogenesis control gene that also has properties that overlap with those of a tumor suppressor gene. Both its neoplastic and dysplastic manifestations become more amenable to understanding and treatment if they are differentiated at three levels—specifically, features, consequences and complications. Birth Defects Research (Part A), 2010. © 2009 Wiley‐Liss, Inc.