Simultaneous on/off regulation of transgenes located on a mammalian chromosome with Cre-expressing adenovirus and a mutant loxP

The site-specific recombinase Cre has often been used for on/off regulation of expression of transgenes introduced into the mammalian chromosome. However, this method is only applicable to the regulation of a single gene and cannot be used to simultaneously regulate two genes, because site-specific recombination occurs from the target loxP sequence of one regulating unit to the loxP sequence of any other unit and would eventually disrupt the structure of both regulating units. We previously reported a mutant loxP sequence with a two base substitution called loxP V (previously called loxP 2272), with which wild-type loxP cannot recombine but with which the identical mutant loxP recombines efficiently. In the present study we isolated cell lines bearing two regulating units on a chromosome containing a pair of wild-type loxP sequences or mutant loxP V sequences. After infection with Cre-expressing recombinant adenovirus AxCANCre, expression of a gene in each regulating unit was simultaneously turned on and off. Southern analyses showed that both regulating units were processed simultaneously and independently, even after infection with a limited amount of AxCANCre. The results showed that simultaneous regulation of gene expression on a mammalian chromosome mediated by Cre can be achieved by using mutant loxP V and wild-type loxP. This method may be a useful approach for conditional transgenic/knockout animals and investigation of gene function involving two genes simultaneously. Another possible application is for preparation of a new packaging cell line of viral vectors through simultaneous production of toxic viral genes.     

Reconstitution of the mammalian DNA double-strand break end-joining reaction reveals a requirement for an Mre11/Rad50/NBS1-containing fraction

The non-homologous end-joining pathway promotes direct enzymatic rejoining of DNA double-strand breaks (DSBs) and is an important determinant of genome stability in eukaryotic cells. Although previous work has shown that this pathway requires Ku, DNA-PKcs and the DNA ligase IV/XRCC4 complex, we found that these proteins alone did not promote efficient joining of cohesive-ended DNA fragments in a cell-free assay. To identify factors that were missing from the reaction, we screened fractions from HeLa cell extracts for the ability to stimulate the joining of cohesive DNA ends in a complementation assay containing other known proteins required for DNA DSB repair. We identified a factor that restored end-joining activity to the level observed in crude nuclear extracts. Factor activity copurified with Rad50, Mre11 and NBS1, three proteins that have previously been implicated in DSB repair by genetic and cytologic evidence. Factor activity was inhibited by anti-Mre11 antibody. The reconstituted system remained fully dependent on DNL IV/XRCC4 and at least partially dependent on Ku, but the requirement for DNA-PKcs was progressively lost as other components were purified. Results support a model where DNA-PKcs acts early in the DSB repair pathway to regulate progression of the reaction, and where Mre11, Rad50 and NBS1 play a key role in aligning DNA ends in a synaptic complex immediately prior to ligation.     

Solution structure of an arabinonucleic acid (ANA)/RNA duplex in a chimeric hairpin: comparison with 2′-fluoro-ANA/RNA and DNA/RNA hybrids

Hybrids of RNA and arabinonucleic acid (ANA) as well as the 2′-fluoro-ANA analog (2′F-ANA) were recently shown to be substrates of the enzyme RNase H. Although RNase H binds to double-stranded RNA, no cleavage occurs with such duplexes. Therefore, knowledge of the structure of ANA/RNA hybrids may prove helpful in the design of future antisense oligonucleotide analogs. In this study, we have determined the NMR solution structures of ANA/RNA and DNA/RNA hairpin duplexes and compared them to the recently published structure of a 2′F-ANA/RNA hairpin duplex. We demonstrate here that the sugars of RNA nucleotides of the ANA/RNA hairpin stem adopt the C3′-endo (north, A-form) conformation, whereas those of the ANA strand adopt a ‘rigid’ O4′-endo (east) sugar pucker. The DNA strand of the DNA/RNA hairpin stem is flexible, but the average DNA/RNA hairpin structural parameters are close to the ANA/RNA and 2′F-ANA/RNA hairpin parameters. The minor groove width of ANA/RNA, 2′F-ANA/RNA and DNA/RNA helices is 9.0 ± 0.5 Å, a value that is intermediate between that of A- and B-form duplexes. These results rationalize the ability of ANA/RNA and 2′F-ANA/RNA hybrids to elicit RNase H activity.     

Linkage disequilibrium between haplotypes and allelic incompatibilities detected with p49f,a probe located on the Y chromosome

We have analyzed linkage disequilibrium between haplotypes and allelic incompatibilities betweenTaqI RELPs concerning the probe 49f, located on the Y chromosome. For most (but one) observed haplotypes, discrepancies between observed and expected frequencies can be easily explained allowing a small number of incompatibilities beetween alleles.     

Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype

The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by ~50% and the poorly hydrolyzed GTP analog, GTPγS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP_GTP and TB-RBP_GTP no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP_GTP will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP_GTP into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP_GTP in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.     

The “switching on” of mammalian spermatozoa: Molecular events involved in promotion and regulation of capacitation

Following the discovery of mammalian sperm capacitation and its fundamental importance for the acquisition of fertilizing potential, it has gradually become possible to identify some specific molecules and molecular events that play pivotal roles in the “switching on” of spermatozoa. These are discussed in the context of the promotion and regulation of capacitation, emphasizing differences between commonly used conditions in vitro and the environment in vivo where spermatozoa normally undergo capacitation. Although typical culture media used in vitro do support capacitation, they do not prevent capacitated cells from undergoing spontaneous acrosome reactions and so losing fertilizing potential. This is not a problem in vitro, but could be in vivo where few spermatozoa reach the site of fertilization. Several small molecules, known to be present in vivo, have been shown in vitro to bind to spermatozoa and to regulate capacitation, first accelerating capacitation and then inhibiting spontaneous acrosome reactions, by regulating cAMP production. Since spermatozoa would contact these molecules during and after ejaculation, it is plausible that they serve a similar function in vivo. The mechanisms whereby the presence or absence of decapacitation factors might alter plasma membrane architecture and so alter functionality of a number of membrane‐associated enzymes involved in capacitation are also considered. Finally, several unresolved issues relating to events during capacitation are discussed. Mol. Reprod. Dev. 77: 197–208, 2010. © 2009 Wiley‐Liss, Inc.     

Recovery and potential utility of YACs as circular YACs/BACs

A method has been established to convert pYAC4-based linear yeast artificial chromosomes (YACs) into circular chromosomes that can also be propagated in Escherichia coli cells as bacterial artificial chromosomes (BACs). The circularization is based on use of a vector that contains a yeast dominant selectable marker (G418R), a BAC cassette and short targeting sequences adjacent to the edges of the insert in the pYAC4 vector. When it is introduced into yeast, the vector recombines with the YAC target sequences to form a circular molecule, retaining the insert but discarding most of the sequences of the YAC telomeric arms. YACs up to 670 kb can be efficiently circularized using this vector. Re-isolation of megabase-size YAC inserts as a set of overlapping circular YAC/BACs, based on the use of an Alu-containing targeting vector, is also described. We have shown that circular DNA molecules up to 250 kb can be efficiently and accurately transferred into E.coli cells by electroporation. Larger circular DNAs cannot be moved into bacterial cells, but can be purified away from linear yeast chromosomes. We propose that the described system for generation of circular YAC derivatives can facilitate sequencing as well as functional analysis of genomic regions.     

A Y639F/H784A T7 RNA polymerase double mutant displays superior properties for synthesizing RNAs with non-canonical NTPs

A T7 RNA polymerase in which Tyr639 is mutated to Phe readily utilizes 2′-deoxy, 2′-NH₂ and 2′-F NTPs as substrates and has been widely used to synthesize modified RNAs for a variety of applications. This mutant does not readily utilize NTPs with bulkier 2′-substituents, nor does it facilitate incorporation of NTPs with modifications at other positions. Introduction of a second mutation (H784A) into the Y639F background markedly enhances utilization of NTPs with bulky 2′-substituents (2′-OMe and 2′-N₃), and may also enhance use of NTPs with modifications at other than the 2′-position. The Y639F/H784A double mutant may therefore be exceptionally useful for incorporation of a variety of non-canonical NMPs into RNA.     

Identification of a human heart FABP pseudogene located on chromosome 13

The fatty acid-binding proteins (FABPs) constitute a conserved group of cytosolic low molecular mass proteins, which consists of several types: liver, heart, myelin, epidermal, adipocyte, brain, intestinal and ileal type. The FABP gene structure is well conserved during evolution and exhibits a four-exon/three-intron structure. In the past, multiple hybridizing fragments were detected upon Southern blot analysis using heart FABP (H-FABP) cDNA as a probe. The origin of these fragments was not clear. We screened a human genomic library and isolated an intronless gene (FABP3-ps) with 85% similarity to the human H-FABP cDNA and high similarity (76 and 79%) to the H-FABP cDNAs of mouse and bovine, respectively. By means of fluorescence in situ hybridization this processed pseudogene could be assigned chromosome 13q13-q14, whereas the gene for human H-FABP (FABP3) resides on chromosome 1p32-p33. No expression of the processed pseudogene could be detected in skeletal muscle or fetal brain.     

Induction of a human carbonyl reductase gene located on chromosome 21

Carbonyl reductase (EC 1.1.1.184) belongs to the group of enzymes called aldo-keto reductases. It is a NADPH-dependent cytosolic protein with specificity for many carbonyl compounds including the antitumor anthracycline antibiotics, daunorubicin and doxorubicin. Human carbonyl reductase was cloned from a breast cancer cell line (MCF-7). The cDNA clone contained 1219 base paires with an open reading frame corresponding to 277 amino acids encoding a protein of Mr 30,375. Southern analysis of genomic DNA digested with several restriction enzymes and analyzed by hybridization with a labeled cDNA probe indicated that carbonyl reductase is probably coded by a single gene and does not belong to a family of structurally similar enzymes. Southern analysis of 17 mouse/human somatic cell hybrids showed that carbonyl reductase is located on chromosome 21. Carbonyl reductase mRNA could be induced 3-4-fold in 24 h with 10 microM 2,(3)-t-butyl-4-hydroxyanisole (BHA), beta-naphthoflavone or Sudan 1.     

ADAM-TS8, a novel metalloprotease of the ADAM-TS family located on mouse chromosome 9 and human chromosome 11

A disintegrin-like and metalloprotease domain with thrombospondin type I modules (ADAM-TS) describes a novel family of zinc metalloendopeptidases. Its members have a common domain organization, which includes, typically, a pre-pro-metalloprotease domain, a disintegrin-like domain, and one or more thrombospondin-like (TS) modules. We describe here the complete primary structure of mouse ADAM-TS8, through cloning of Adamts8 cDNA. This novel member of the family contains two TS modules and is highly similar in sequence and domain organization to three other recently described gene products, ADAM-TS5, ADAM-TS6, and ADAM-TS7. Adamts8 is expressed at low levels throughout development and in adult mouse lung and heart. Through analysis of an interspecific backcross panel, we place the Adamts8 locus on mouse chromosome 9 at a consensus position of 11 cM and its human ortholog, recently reported as the METH2 gene, on human chromosome 11q25.     

The alteration in the architecture of a T‐DNA insertion rice mutant osmtd1 is caused by up‐regulation of MicroRNA156f

Plant architecture is an important factor for crop production. Some members of microRNA156 (miR156) and their target genes SQUAMOSA Promoter‐Binding Protein‐Like (SPL) were identified to play essential roles in the establishment of plant architecture. However, the roles and regulation of miR156 is not well understood yet. Here, we identified a T‐DNA insertion mutant Osmtd1 (Oryza sativa multi‐tillering and dwarf mutant). Osmtd1 produced more tillers and displayed short stature phenotype. We determined that the dramatic morphological changes were caused by a single T‐DNA insertion in Osmtd1. Further analysis revealed that the T‐DNA insertion was located in the gene Os08g34258 encoding a putative inhibitor I family protein. Os08g34258 was knocked out and OsmiR156f was significantly upregulated in Osmtd1. Overexpression of Os08g34258 in Osmtd1 complemented the defects of the mutant architecture, while overexpression of OsmiR156f in wild‐type rice phenocopied Osmtd1. We showed that the expression of OsSPL3, OsSPL12, and OsSPL14 were significantly downregulated in Osmtd1 or OsmiR156f overexpressed lines, indicating that OsSPL3, OsSPL12, and OsSPL14 were possibly direct target genes of OsmiR156f. Our results suggested that OsmiR156f controlled plant architecture by mediating plant stature and tiller outgrowth and may be regulated by an unknown protease inhibitor I family protein.     

The mouse Cebp gene encoding a DNA-binding protein is polymorphic and is located on chromosome 7

The mouse gene Cebp, encoding the DNA-binding protein C/EBP, has been localized to the proximal region of chromosome 7 by determining the strain distribution patterns of a restriction fragment length polymorphism among the BXD and AKXL recombinant inbred mouse lines.     

FLAGdb/FST: a database of mapped flanking insertion sites (FSTs) of Arabidopsis thaliana T-DNA transformants

A large collection of T-DNA insertion transformants of Arabidopsis thaliana has been generated at the Institute of Agronomic Research, Versailles, France. The molecular characterisation of the insertion sites is currently performed by sequencing genomic regions flanking the inserted T-DNA (FST). The almost complete sequence of the nuclear genome of A.thaliana provides the framework for organising FSTs in a genome oriented database, FLAGdb/FST (http://genoplante-info.infobiogen.fr). The main scope of FLAGdb/FST is to help biologists to find the FSTs that interrupt the genes in which they are interested. FSTs are anchored to the genome sequences of A.thaliana and positions of both predicted genes and FSTs are shown graphically on sequences. Requests to locate the genomic position of a query sequence are made using BLAST programs. The response delivered by FLAGdb/FST is a graphical representation of the putative FSTs and of predicted genes in a 20 kb region.     

Effects of Saxitoxin (STX) and Veratridine on Bacterial Na+-K+ Fluxes: a Prokaryote-Based STX Bioassay

Saxitoxin (STX) is a potent natural sodium channel blocker and represents a significant health concern worldwide. We describe here the antagonistic effects of STX and veratridine (VTD), an Na⁺ channel activator, on three gram-negative bacteria and their application to an STX bioassay. STX reduced the total cellular levels of both Na⁺ and K⁺, as measured by flame photometry, whereas VTD increased the cellular concentrations relative to control ion fluxes in the cyanobacterium Cylindrospermopsis raciborskii AWT205. Endogenous STX production in toxic cyanobacterial strains of C. raciborskii and Anabaena circinalis prevented cell lysis induced by VTD stress. Microscopic cell counts showed that non-STX producing cyanobacteria displayed complete cell lysis and trichome fragmentation 5 to 8 h after addition of VTD and vanadate (VAN), an inhibitor of sodium pumps. The addition of STX, or its analogue neoSTX, prior to treatment with VTD plus VAN prevented complete lysis in non-STX-producing cyanobacteria. VTD also affected cyanobacterial metabolism, and the presence of exogenous STX in the sample also ameliorated this decrease in metabolic activity, as measured by the cellular conversion of tetrazolium into formazan. Reduced primary metabolism was also recorded as a decrease in the light emissions of Vibrio fischeri exposed to VTD. Addition of STX prior to VTD resulted in a rapid and dose-dependent response to the presence of the channel blocker, with samples exhibiting resistance to the VTD effect. Our findings demonstrate that STX and VTD influence bacterial Na⁺ and K⁺ fluxes in opposite ways, and these principles can be applied to the development of a prokaryote-based STX bioassay.     

Biochemical and genetical characterization of a fiber-defective temperature-sensitive mutant of type 2 adenovirus.

The adenovirus type 2 fiber mutant H2 ts 125 synthesized an unstable, temperature-sensitive fiber polypeptide with an apparent mol. wt. smaller by 2500 than the wild-type (62 K). The polypeptide of 59.5 K was found to be stable at the permissive temperature (33 degrees C). H2 ts 125 fiber synthesized in reticulocyte lysates had the same apparent mol. wt. of 59.5 K as the mutant fiber produced in vivo. Neither structural nor functional differences between wild-type and mutant fibers were detected in the N-terminal and C-terminal sequences, excluding the occurrence of a new initiation or termination codon. Restriction analysis of H2 ts 125 DNA also ruled out the hypothesis of a deletion mutant. The 59.5 K mutant fiber unit was normally glycosyated, N-acetylated, assembled into 6S oligomeric fiber and incorporated into virions. DNA sequencing of the H2 ts 125 fiber gene revealed two point mutations at nucleotides 3970 (C*TT leads to T*TT) and 4958 (GC*T leads to GT*T), corresponding to two amino acid changes at positions 105 and 434, respectively. The 105 mutation consisted of a conservative change Leu leads to Phe; the 434 interchange was Ala leads to Val, usually considered as nonconservative. The possibility of a donor site for splicing created by the mutation at codon GTT was eliminated on the basis of S1 nuclease analysis data. All these results suggested that either one or both mutations concerned highly organized domain(s) of the fiber polypeptide chain, resulting in aberrant mobility in SDS-polyacrylamide gels and temperature-sensitivity.