Showing papers in "Nucleic Acids Research in 1995"
TL;DR: The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity that allows the specific co-amplification of high numbers of restriction fragments.
Abstract: A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.
12,960 citations
TL;DR: A set of tools for molecular biologists that allow generation of new matrix and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices is presented in this article.
Abstract: The identification of potential regulatory motifs in new sequence data is increasingly important for experimental design. Those motifs are commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. However, sufficiently large libraries of pre-compiled matrices are a prerequisite for practical application of any matrix-based approach and are just beginning to emerge. Here we present a set of tools for molecular biologists that allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices. We also supply a large library (> 200) of transcription factor binding site matrices that has been compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. Our search method includes position weighting of the matrices based on the information content of individual positions and calculates a relative matrix similarity. We show several examples suggesting that this matrix similarity is useful in estimating the functional potential of matrix matches and thus provides a valuable basis for designing appropriate experiments.
2,596 citations
1,287 citations
TL;DR: Improvements are improved upon the adaptor ligation method by combining 'vectorette PCR' with a newly developed method termed 'suppression PCR' (12), which is based upon adaptors ligated to the ends of DNA fragments generated by digestion of human genomicDNA.
Abstract: Several PCR-based methods are available for walking from a known region to an unknown region in cloned or uncloned genomic DNA. The methods are of three types: inverse PCR (1), randomly primed PCR (2) and adaptor ligation PCR (3-6). However, these methods have not been generally applied to walking in uncloned genomic DNA because they are either complicated or inefficient. Recent improvements to these methods have been applied to uncloned genomic DNA (7,8), however, walks have been limited to distances of <1 kb. We have investigated the application of 'long and accurate PCR' (9,10) to walking in uncloned genomic DNA. We initially examined the use of 'unpredictably primed PCR' (3), a new method based on randomly primed PCR and 'vectorette PCR' (5), which is based upon adaptor ligation. As a model system we attempted to walk upstream from exon 1 of the human tissue-type plasminogen activator (TPA) gene which has been previously characterized (11). In our hands both 'unpredictably primed PCR' and 'vectorette PCR' generated multiple PCR products even after nested PCR was perforned. Although upon analysis some of the PCR products were found to be derived from successful walks, the presence of multiple PCR products would complicate further characterization without Southern blot hybridization or extensive cloning. We have improved upon the adaptor ligation method by combining 'vectorette PCR' with a newly developed method termed 'suppression PCR' (12). A special adaptor is ligated to the ends of DNA fragments generated by digestion of human genomicDNA with EcoRV, Scal, Dral, PvuII and SspI separately. Following adaptor ligation, a small amount of the DNA is used
1,116 citations
TL;DR: The Genome Assembly Program (GAP), a new program for DNA sequence assembly, is described, which retains the useful components of the previous work, but includes many novel ideas and methods.
Abstract: We describe the Genome Assembly Program (GAP), a new program for DNA sequence assembly. The program is suitable for large and small projects, a variety of strategies and can handle data from a range of sequencing instruments. It retains the useful components of our previous work, but includes many novel ideas and methods. Many of these methods have been made possible by the program's completely new, and highly interactive, graphical user interface. The program provides many visual clues to the current state of a sequencing project and allows users to interact in intuitive and graphical ways with their data. The program has tools to display and manipulate the various types of data that help to solve and check difficult assemblies, particularly those in repetitive genomes. We have introduced the following new displays: the Contig Selector, the Contig Comparator, the Template Display, the Restriction Enzyme Map and the Stop Codon Map. We have also made it possible to have any number of Contig Editors and Contig Joining Editors running simultaneously even on the same contig. The program also includes a new 'Directed Assembly' algorithm and routines for automatically detecting unfinished segments of sequence, to which it suggests experimental solutions.
951 citations
TL;DR: A transcriptionally inactive mutant of the murine oestrogen receptor is used which is unable to bind oestrogens yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen.
Abstract: A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use in cells in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum in order to avoid constitutive activation. Secondly, control of oestrogen receptor fusion proteins in vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors problematical. In order to overcome these drawbacks we have used a transcriptionally inactive mutant of the murine oestrogen receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen. When the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced proliferation and apoptosis in fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17 beta-oestradiol.
874 citations
TL;DR: A series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication that achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components.
Abstract: We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.
746 citations
TL;DR: This work has divided the SNF2 family into multiple subfamilies, each of which represents what it proposes to be a functionally and evolutionarily distinct group, and used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNf2 family.
Abstract: The SNF2 family of proteins includes representatives from a variety of species with roles in cellular processes such as transcriptional regulation (e.g. MOT1, SNF2 and BRM), maintenance of chromosome stability during mitosis (e.g. lodestar) and various aspects of processing of DNA damage, including nucleotide excision repair (e.g. RAD16 and ERCC6), recombinational pathways (e.g. RAD54) and post-replication daughter strand gap repair (e.g. RAD5). This family also includes many proteins with no known function. To better characterize this family of proteins we have used molecular phylogenetic techniques to infer evolutionary relationships among the family members. We have divided the SNF2 family into multiple subfamilies, each of which represents what we propose to be a functionally and evolutionarily distinct group. We have then used the subfamily structure to predict the functions of some of the uncharacterized proteins in the SNF2 family. We discuss possible implications of this evolutionary analysis on the general properties and evolution of the SNF2 family.
734 citations
TL;DR: Results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals.
Abstract: A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.
652 citations
498 citations
TL;DR: Sequence analysis of 236 promoters recognized by the Bacillus subtilis sigma A-RNA polymerase reveals an extended promoter structure that indicates that the alpha subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase.
Abstract: Sequence analysis of 236 promoters recognized by the Bacillus subtilis cARNA polymerase reveals an extended promoter structure The most highly conserved bases include the -35 and -10 hexanucleotide core elements and a TG dlnucleotide at position -15,-14 In addition, several weakly conserved A and T residues are present upstream of the -35 region Analysis of dinucleotide composition reveals Ar and T2-rich sequences in the upstream promoter region (-36 to -70) which are phased with the DNA helix: A,, tracts are common near -43, -54 and -65; Tn tracts predominate at the intervening positions When compared with larger regions of the genome, upstream promoter regions have an excess of A,, and Tn sequences for n > 4 These data indicate that an RNA polymerase binding site affects DNA sequence as far upstream as -70 This sequence conservation is discussed in light of recent evidence that the a subunits of the polymerase core bind DNA and that the promoter may wrap around RNA polymerase
TL;DR: An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoriberibon nucleotides and not for 2'-deoxy oligorIBonucleotsides.
Abstract: The nuclease stability and melting temperatures (Tm) were compared for fully modified oligoribonucleotide sequences containing 2'-fluoro, 2'-O-methyl, 2'-O-propyl and 2'-O-pentyl nucleotides. Duplexes formed between 2' modified oligoribonucleotides and RNA have typical A-form geometry as observed by circular dichroism spectroscopy. Modifications, with the exception of 2'-O-pentyl, were observed to increase the Tm of duplexes formed with complementary RNA. Modified homoduplexes showed significantly higher Tms, with the following Tm order: 2'-fluoro:2'fluoro > 2'-O-propyl:2'-O-propyl > 2'-O-methyl:2'-O- methyl > RNA:RNA > DNA:DNA. The nuclease stability of 2'-modified oligoribonucleotides was examined using snake venom phosphodiesterase (SVPD) and nuclease S1. The stability imparted by 2' modifications was observed to correlate with the size of the modification. An additional level of nuclease stability was present in oligoribonucleotides having the potential for forming secondary structure, but only for 2' modified oligoribonucleotides and not for 2'-deoxy oligoribonucleotides.
TL;DR: The design of a bidirectional promoter which would allow the simultaneous regulation of two transcriptional units from centrally located multiple tetO sequences is suggested, which may allow the co-regulation of the synthesis of two gene products in stoichiometric amounts.
Abstract: Recently, we have described a regulatory system that allows the stringent control of individual gene activities in higher eukaryotic cell lines (1), in plants (2) and in animals (3,4). The essential components of this system are (i) an RNA polymerase II minimal promoter placed downstream of multiple operator sequences (tetO) of the Escherichia coli TnlO tetracycline resistance operon and (ii) a fusion between the Tet repressor (TetR) and the herpes simplex virus protein 16 (VP16), named tTA (1). In the absence of tetracycline (Tc), tTA binds to the tet operators to activate transcription from the minimal promoter, whereas in the presence of Tc its association and consequently its transcription activation is prevented. After the binding of tTA, minimal promoters derived from the cytomegalovirus IE promoter (PhCMv; 5) and fused to seven tetO sequences reach the remarkable strength of the parent promoter in HeLa cells when compared in transient expression assays (6). This high activation potential of tTA and the arrangement of its binding sites within PhCMV*-i [(!)'»see Fig. 1A] suggested the design of a bidirectional promoter which would allow the simultaneous regulation of two transcriptional units from centrally located multiple tetO sequences (Fig. 1A). Such a promoter should be useful for a number of experimental approaches. First, it may allow the co-regulation of the synthesis of two gene products in stoichiometric amounts, frequently a prerequisite for the production of heterodimeric (or hetero-oligomeric) proteins. Second, by fusing minimal promoters of differing efficiencies to the centrally located tetO sequences, two gene products may be co-regulated at different but defined levels. Third, by integrating an appropriate reporter gene at one side of the bidirectional promoter, the regulation of a not-readily-assayable gene of interest may be monitored via the reporter function. This latter possibility may also facilitate—at the cellular as well as at the organismal level—the screening for properly integrated expression units controlling the gene of interest.
TL;DR: NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides.
Abstract: The effect of the modified nucleoside pseudouridine (psi) on RNA structure was compared with uridine. The extent of base stacking in model RNA oligonucleotides was measured by 1H NMR, UV, and CD spectroscopy. The UV and CD results indicate that the model single-stranded oligoribonucleotides AAUA and AA psi A form stacked structures in solution and the CD results for AA psi A are consistent with a general A-form helical conformation. The AA psi A oligomer exhibits a greater degree of UV hypochromicity over the temperature range 5-55 degrees C, consistent with a better stacked, more A-form structure compared with AAUA. The extent of stacking for each nucleotide residue was inferred from the percent 3'-endo sugar conformation as indicated by the H1'-H2' NMR scalar coupling. This indirect indication of stacking was confirmed by sequential NOE experiments. NMR measurements as a function of temperature indicate that pseudouridine forms a more stable base stacking arrangement than uridine, an effect that is propagated throughout the helix to stabilize stacking of neighboring purine nucleosides. The N1-H imino proton in AA psi A exchanges slowly with solvent, suggesting a role for the extra imino proton in stabilizing the conformation of pseudouridine. These results show that the conformational stabilization is an intrinsic property of pseudouridine occurring at the nucleotide level. The characteristics of pseudouridine in these models are consistent with earlier studies on intact rRNA, indicating that pseudouridine probably performs the same stabilizing function in most structural contexts.
TL;DR: Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function.
Abstract: The abundance of Alu RNA is transiently increased by heat shock in human cell lines. This effect is specific to Alu repeats among Pol III transcribed genes, since the abundance of 7SL, 7SK, 5S and U6 RNAs is essentially unaffected by heat shock. The rapid induction of Alu expression precedes the heat shock induction of mRNAs for the ubiquitin and HSP 70 heat shock genes. Heat shock mimetics also transiently induce Alu expression indicating that increased Alu expression is a general cell-stress response. Cycloheximide treatment rapidly and transiently increases the abundance of Alu RNA. Again, compared with other genes transcribed by Pol III, this increase is specific to Alu. However, as distinguished from the cell stress response, cycloheximide does not induce expression of HSP 70 and ubiquitin mRNAs. Puromycin also increases Alu expression, suggesting that this response is generally caused by translational inhibition. The response of mammalian SINEs to cell stress and translational inhibition is not limited to SINEs which are Alu homologues. Heat shock and cycloheximide each transiently induce Pol III directed expression of B1 and B2 RNAs in mouse cells and C-element RNA in rabbit cells. Together, these three species exemplify the known SINE composition of placental mammals, suggesting that mammalian SINEs are similarly regulated and may serve a common function.
TL;DR: It is reported here that the hmdm2 gene also contains an intronic, p53-dependent promoter, and the structural features of this promoter are highly conserved between mouse and man, as opposed to the lack of conservation of the first exon.
Abstract: Themdm2gene isatarget fortranscriptional activation bythep53tumorsuppressor geneproduct. Previous workhasrevealed that themousemdm2genecontains twopromoters: oneislocated upstream tothegeneand isactive intheabsence ofp53, theother resides within thefirst intron andrequires p53fortranscriptional activity. Todetermine whether thisunique promoter activation pattem isbiologically important, weinvestigated thestructure andfunction ofthecorresponding region ofthehumanmdm2(hmdm2) gene. Wereport herethatthehmdm2genealsocontains anintronic, p53-dependent promoter. Thestructural features ofthis promoter arehighly conserved between mouseand man,asopposed tothelack ofconservation ofthefirst exon. Thispromoter istriggered invivo inthepresence ofactivated wild typep53, leading totheproduction of novelmRNA species. Theintronic hmdm2promoter contains twotandemp53binding elements. Deletion analysis suggests thatoptimal promoter activity requires thesimultaneous binding ofp53tobothelements; this mayserve toprevent premature triggering ofthepromoter byp53.
TL;DR: The synthesis and DNA binding properties of bis-PNA (peptide nucleic acid) are reported, which form triple-stranded complexes of somewhat higher thermal stability than monomeric PNA with complementary oligonucleotides and the thermal melting transition shows very little hysteresis.
Abstract: The synthesis and DNA binding properties of bis-PNA (peptide nucleic acid) are reported. Two PNA segments each of seven nucleobases in length were connected in a continuous synthesis via a flexible linker composed of three 8-amino-3,6-dioxaoctanoic acid units. The sequence of the first strand was TCTCTTT (C- to N-terminal), while the second strand was TTTCTCT or TTTJTJT, where J is pseudoisocytosine. These bis-PNAs form triple-stranded complexes of somewhat higher thermal stability than monomeric PNA with complementary oligonucleotides and the thermal melting transition shows very little hysteresis. When the J base is placed in the strand parallel to the DNA complement ('Hoogsteen strand'), the DNA binding was pH independent. The bis-PNAs were also superior to monomeric PNAs for targeting double-stranded DNA by strand invasion.
TL;DR: Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11, indicating that the XR CC1-DNA ligase III complex is much reduced in the two CHO mutants.
Abstract: The human DNA repair protein XRCC1 was overexpressed as a histidine-tagged polypeptide (denoted XRCC1-His) in Escherichia coli and purified in milligram quantities by affinity chromatography. XRCC1-His complemented the mutant Chinese hamster ovary cell line EM9 when constitutively expressed from a plasmid or when introduced by electroporation. XRCC1-His directly interacted with human DNA ligase III in vitro to form a complex that was resistant to 2 M NaCl. XRCC1-His interacted equally well with DNA ligase III from Bloom syndrome, HeLa and MRC5 cells, indicating that Bloom syndrome DNA ligase III is normal in this respect. Detection of DNA ligase III on far Western blots by radiolabelled XRCC1-His indicated that the level of the DNA ligase polypeptide was reduced approximately 4-fold in the mutant EM9 and also in EM-C11, a second member of the XRCC1 complementation group. Decreased levels of polypeptide thus account for most of the approximately 6-fold reduced DNA ligase III activity observed previously in EM9. Immunodetection of XRCC1 on Western blots revealed that the level of this polypeptide was also decreased in EM9 and EM-C11 (> 10-fold), indicating that the XRCC1-DNA ligase III complex is much reduced in the two CHO mutants.
TL;DR: A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR and a growth factor/cytokine-specific induction of TIEG has been shown.
Abstract: The TGF-beta family of growth factors has been extensively studied and found to play major roles in bone physiology and disease. A novel, TGF-beta-inducible early gene (TIEG) in normal human fetal osteoblasts (hFOB) has been identified using differential-display PCR. Using this differentially expressed cDNA fragment of TIEG to screen a hOB cDNA library, a near full-length cDNA for this gene was isolated. Northern analyses indicated that the steady-state levels of the 3.5 kb TIEG mRNA increased within 30 min of TGF-beta treatment of human osteoblasts and reached a maximum of 10-fold above control levels at 120 min post-treatment. This regulation was independent of new protein synthesis. Computer sequence analyses indicates that TIEG mRNA encodes for a 480 amino-acid protein. The TIEG protein contains three zinc finger motifs, several proline-rich src homology-3 (SH3) binding domains at the C-terminal end, and is homologous in this region to the zinc finger-containing transcription factor family of genes. A growth factor/cytokine-specific induction of TIEG has been shown. TIEG expression in hFOB cells was highly induced by TGF-beta and bone morphogenetic protein-2 (BMP-2), with a moderate induction by epidermal growth factor (EGF), but no induction by other growth factors/cytokines was observed. In addition to osteoblastic cells, high levels of TIEG expression were detected in skeletal muscle tissue, while low or no detectable levels were found in brain, lung, liver or kidney. Because TIEG is an early induced putative transcription factor gene, and shows a growth factor induction and tissue specificity, its protein product might play an important role as a signalling molecule in osteoblastic cells.
TL;DR: Characterization of the genomic structure revealed that the AML1 gene consists of nine exons and spans > 150 kb of genomic DNA, which can all be explained by the existence of two promoters, alternative splicing and differential usage of three polyadenylation sites.
Abstract: We previously isolated the AML1 gene, which is rearranged by the t(8;21) translocation in acute myeloid leukemia. The AML1 gene is highly homologous to the Drosophila segmentation gene runt and the mouse transcription factor PEBP2 alpha subunit gene. This region of homology, called the Runt domain, is responsible for DNA-binding and protein--protein interaction. In this study, we isolated and characterized various forms of AML1 cDNAs which reflect a complex pattern of mRNA species. Analysis of these cDNAs has led to the identification of two distinct AML1 proteins, designated AML1b (453 amino acids) and AML1c (480 amino acids), which differ markedly from the previously reported AML1a (250 amino acids) with regard to their C-terminal regions, although all three contain the Runt domain. The large C-terminal region common to AML1b and AML1c is suggested to be a transcriptional activation domain. AML1c differs from AML1b by only 32 amino acids in the N-terminal. Characterization of the genomic structure revealed that the AML1 gene consists of nine exons and spans > 150 kb of genomic DNA. Northern blot analysis demonstrated the presence of six major transcripts, encoding AML1b or AML1c, which can all be explained by the existence of two promoters, alternative splicing and differential usage of three polyadenylation sites. A minor transcript encoding AML1a which results from alternative splicing of a separate exon can be detected only by reverse transcription-polymerase chain reaction amplification. The distinct proteins encoded by the AML1 gene may have different functions, which could contribute to regulating cell growth and/or differentiation through transcriptional regulation of a specific subset of target genes.
TL;DR: A method of separating polynucleotides from a solution containing polyn DNA, RNA and PNA by reversibly and non-specifically binding the polyn nucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface is disclosed.
Abstract: A method of separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface is disclosed. The salt and polyalkylene glycol concentration of the solution is adjusted to levels which result in polynucleotide binding to the magnetic microparticles. The magnetic microparticles with bound polynucleotides are separated from the solution and the polynucleotides are eluted from the magnetic microparticles.
TL;DR: It is found that RZR alpha and ROR alpha 1 vary in their constitutive transactivational activity and are activated to a different extent by melatonin, and a synthetic RzR-ligand, the thiazolidine dione CGP 52608, is a functional analogue of melatonin at its nuclear receptor, but does not bind to the high affinity membrane receptor for melatonin.
Abstract: Many important physiological functions are controlled by hormones via binding and activating members of the nuclear receptor superfamily. This group of structurally related transcription factors also includes a still growing number of orphan receptors for which no ligand is known so far. The identification of ligands for orphan receptors is a key to understanding their physiological role, as has been successfully shown for retinoid X receptors and the discovery of 9-cis retinoic acid as a specific ligand. We have discovered very recently that the pineal gland hormone melatonin is a specific ligand for the brain-specific nuclear receptor RZR beta. Here we report that the alpha-subtype of RZR, RZR alpha and its splicing variant ROR alpha 1, is also a nuclear receptor for melatonin with binding specificities in the low nanomolar range. In contrast to RZR beta, RZR/ROR alpha is expressed in many tissues and cells outside the brain. We found that RZR alpha and ROR alpha 1 vary in their constitutive transactivational activity and are activated to a different extent by melatonin. Furthermore, we identified a synthetic RZR-ligand, the thiazolidine dione CGP 52608. This compound is a functional analogue of melatonin at its nuclear receptor, but does not bind to the high affinity membrane receptor for melatonin. Therefore, this specific RZR-ligand may help to differentiate between nuclear and membrane signalling of melatonin.
TL;DR: The data imply that a wide spectrum of mtDNA rearrangements accumulate in old individuals, which correlates with the marked age related decrease in OXPHOS capacity observed in post-mitotic tissues.
Abstract: Several reports have shown that individual mitochondrial DNA (mtDNA) deletions accumulate with age. However, the overall extent of somatic mtDNA damage with age remains unclear. We have utilized full-length PCR to concurrently screen for multiple mtDNA rearrangements in total DNA extracted from skeletal muscle derived from physiologically normal individuals (n = 35). This revealed that both the number and variety of mtDNA rearrangements increases dramatically between young and old individuals (P < 0.0001). We further examined the mtDNA from both the younger and older subjects by Southern blot analysis and observed an age-related increase in mtDNA(s) comparable in size to mtDNA products unique to patients with known mtDNA deletions. These data imply that a wide spectrum of mtDNA rearrangements accumulate in old individuals, which correlates with the marked age related decrease in OXPHOS capacity observed in post-mitotic tissues.
TL;DR: It is argued here that this unique genetic code change in cellular mRNAs cannot be explained by the 'Codon Reassignment Theory', and the amino acid specified by the CUG codon has been reassigned within the mRN as C. albicans.
Abstract: Previous studies have shown that the yeast Candida albicans encodes a unique seryl-tRNA(CAG) that should decode the leucine codon CUG as serine. However, in vitro translation of several different CUG-containing mRNAs in the presence of this unusual seryl-tRNA(CAG) result in an apparent increase in the molecular weight of the encoded polypeptides as judged by SDS-PAGE even though the molecular weight of serine is lower than that of leucine. A possible explanation for this altered electrophoretic mobility is that the CUG codon is decoded as modified serine in vitro. To elucidate the nature of CUG decoding in vivo, a reporter system based on the C. albicans gene (RBP1) encoding rapamycin-binding protein (RBP), coupled to the promoter of the C. albicans TEF3 gene, was utilized. Sequencing and mass-spectrometry analysis of the recombinant RBP expressed in C. albicans demonstrated that the CUG codon was decoded exclusively as serine while the related CUU codon was translated as leucine. A database search revealed that 32 out of the 65 C. albicans gene sequences available have CUG codons in their open reading frames. The CUG-containing genes do not belong to any particular gene family. Thus the amino acid specified by the CUG codon has been reassigned within the mRNAs of C. albicans. We argue here that this unique genetic code change in cellular mRNAs cannot be explained by the 'Codon Reassignment Theory'.
TL;DR: It is shown here that CGG repeats readily form a series of barriers to DNA synthesis in vitro, which are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines.
Abstract: A large increase in the length of a CGG tandem array is associated with a number of triplet expansion diseases, including fragile X syndrome, the most common cause of heritable mental retardation in humans. Expansion results in the appearance of a fragile site on the X chromosome in the region of the CGG array. We show here that CGG repeats readily form a series of barriers to DNA synthesis in vitro. There barriers form only when the (CGG)n strand is used as the template, are K(+)-dependent, template concentration-independent, and involve hydrogen bonding between guanines. Chemical modification experiments suggest these blocks to DNA synthesis result from the formation of a series of intrastrand tetraplexes. A number of lines of evidence suggest that both triplet expansion and chromosome fragility are the result of replication defects. Our data are discussed in the light of such evidence.
TL;DR: The Schizosaccharomyces pombe SWI6 protein, involved in repression of the silent mating-type loci, is a member of the chromo shadow group, which supports the model that the specificity of action of chromatin proteins is generated by combinations of protein modules.
Abstract: The chromo domain was originally identified as a protein sequence motif common to the Drosophila chromatin proteins, Polycomb (Pc) and heterochromatin protein 1 [HP1; Paro and Hogness (1991) Proc. Natl. Acad. Sci. USA, 88, 263-267; Paro (1990) Trends Genet., 6, 416-421]. Here we describe a second chromo domain-like motif in HP1. Subsequent refined searches identified further examples of this chromo domain variant which all occur in proteins that also have an N-terminally located chromo domain. Due to its relatedness to the chromo domain, and its occurrence in proteins that also have a classical chromo domain, we call the variant the 'chromo shadow domain'. Chromo domain-containing proteins can therefore be divided into two classes depending on the presence, for example in HP1, or absence, for example in Pc, of the chromo shadow domain. We have also found examples of proteins which have two classical chromo domains. The Schizosaccharomyces pombe SWI6 protein, involved in repression of the silent mating-type loci, is a member of the chromo shadow group. The similar modular architecture of SpSW16, HP1 and HP1-like proteins supports the model that the specificity of action of chromatin proteins is generated by combinations of protein modules.
TL;DR: The characterization of LSIRF, a new member of the IRF gene family cloned from mouse spleen by the polymerase chain reaction using degenerate primers, was found to encode a 51 kDa protein that shares a high degree of amino acid sequence homology in the DNA-binding domain with other IRF family members.
Abstract: Interferon regulatory factor (IRF) genes encode a family of DNA-binding proteins that are involved in the transcriptional regulation of type-I interferon and/or interferon-inducible genes. We report here the characterization of LSIRF, a new member of the IRF gene family cloned from mouse spleen by the polymerase chain reaction using degenerate primers. LSIRF was found to encode a 51 kDa protein that shares a high degree of amino acid sequence homology in the DNA-binding domain with other IRF family members. LSIRF expression was detectable only in lymphoid cells. In contrast to other IRF genes, LSIRF expression was not induced by interferons, but rather by antigen-receptor mediated stimuli such as plant lectins, CD3 or IgM crosslinking. In in vitro DNA binding studies, LSIRF was able to bind to the interferon-stimulated response element (ISRE) of the MHC class I promoter. The expression pattern and DNA binding activities suggest that LSIRF plays a role in ISRE-targeted signal transduction mechanisms specific to lymphoid cells.
TL;DR: Improved version of differential display, which is modified to enhance reproducibility and to facilitate sequencing and cloning, is reported here and generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts.
Abstract: Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.
TL;DR: The atomic force microscope (AFM) is used to explore the structural characteristics of long, linear polymers formed by the telomeric oligonucleotide d(GGGGTTGGGG) in the presence of specific metal cations, which could provide a basis for developing advanced biomaterials.
Abstract: G-DNA is a polymorphic family of quadruple helical nucleic acid structures containing guanine tetrad motifs [G-quartets; Williamson, J.R., Raghuraman, M.K. and Cech, T.R. (1989) Cell 59, 871-880; Williamson, J.R. (1993) Proc. Natl. Acad. Sci. USA 90, 3124-3124]. Guanine rich oligonucleotides that are self-complimentary, as found in many telomeric G-strand repeat sequences, form G-DNA in the presence of monovalent and/or divalent metal cations. In this report we use the atomic force microscope (AFM) to explore the structural characteristics of long, linear polymers formed by the telomeric oligonucleotide d(GGGGTTGGGG) in the presence of specific metal cations. In the AFM these polymers, termed G-wires, appear as filaments whose height and length are determined by the metal ions present during the self-assembly process. The highly ordered, controllable self-assembly of G-wires could provide a basis for developing advanced biomaterials.