Showing papers in "Nucleic Acids Research in 1992"

The Ribosomal Database project

Abstract: The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services, and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous ftp (rdp.life.uiuc.edu), electronic mail (server/rdp.life.uiuc.edu) and gopher (rdpgopher.life.uiuc.edu). The electronic mail server also provides ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for chimeric nature of newly sequenced rRNAs, and automated alignment.

A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues

Abstract: Molecular approaches have become an important tool for mycologists studying fungal systematics, molecular evolution, population genetics or plant-fungus interactions. For this purpose the analysis of restriction fragment length polymorphisms (RFLPs), PCR-based techniques like amplification of polymorphic sequences by one random primer (RAPDs) or amplification of defined genomic regions are applied. Prerequisite for such studies is the rapid isolation of pure genomic DNA of high molecular weight not only allowing simultaneous processing of large numbers of samples but also providing reproducibility with regard to restriction, ligation, and PCR. Most common problems encountered in fulfilling these conditions are the rather high production of polysaccharides by some fungal cultures, and extraction of insufficient amounts of DNA from fruit bodies or from lignified and/or infected plant tissues. Few published protocols include steps for efficient removal of polysaccharides (Do and Adams 1991). Moreover, most procedures contain phenol—chloroform treatments (e.g. Garber and Yoder 1983, Blaiseau et al. 1992) and/or more than one precipitation (e.g. Rodriguez and Yoder 1991: four precipitations) — both latter steps are possible causes of shearing. The short protocol given below combines inactivation of proteins by SDS/Proteinase K with precipitation of acidic polysaccharides by hot CTAB in the presence of SDS and high salts (Kim et al. 1990) and only a single selective precipitation of DNA with isopropanol (Marmur 1961, Cryer et al. 1975). The method is appropriate for simultanous processing of many samples because all steps can be done in Eppendorf tubes, but it can also be scaled up easily for bulk preparations. A treatment with methanol containing 0.1 % mercaptoethanol preceding DNA extraction was performed when basidiocarps and plant tissues contained large amounts of reactive compounds (resins, phenolics) interfering with solubilization of DNA. Methanol extraction was found to be superior over other solvents such as acetone (Schneiderbauer et al. 1991). In this way we isolated DNAs from a wide variety of fungi including Fusarium, Pseudocercosporella, Phytophthora, Setosphaeria, Armillaria, Heterobasidion, Boletus, Russula, Lactarius, Suillus, Cortinarius, and from conifer roots and needles (Picea abies, Pinus sylvestris). DNAs were subjected to Pulsed Field Gel Electrophoresis to verify their high molecular weight: the majority of DNA molecules was in the size range of 50-150 kbp and only insignificant amounts of DNA <50 kbp were present (Figure 1, lane 1). DNAs are suited as substrate for restriction, ligation and PCR (Figure 1, lanes 4 6 and 8-10). Yields depend on the species/isolate. For example, of Fusarium spp. yield averaged 100 ng from 30 mg dried mycelium with an average purity measured as A26o/28O °f 1 -78 ± 0.05. Needles and basidiocarps yielded less (10-30 /*g).

Strand displacement amplification—an isothermal, in vitro DNA amplification technique

Abstract: Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

Slippage synthesis of simple sequence DNA

Abstract: The analysis of slippage synthesis of simple sequence DNA in vitro sheds some light on the question of how simple sequences arise in vivo. We show that it is possible to synthesize all types of repetitious di- and trinucleotide motifs starting from short primers and a polymerase in vitro. The rate of this synthesis depends on a sequence specific slippage rate, but is independent of the length of the fragments being synthesized. This indicates that only the ends of the DNA fragments are involved in determining this rate and that slippage is accordingly a short range effect. Slippage synthesis occurs also on a fixed template where only one strand is free to move, a situation which resembles chromosome replication in vivo. It seems therefore likely that slippage during replication is the cause of the observed length polymorphism of simple sequence stretches between individuals of a population.

Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications.

Abstract: A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).

Thioredoxin regulates the DNA binding activity of NF-kappa B by reduction of a disulphide bond involving cysteine 62.

Abstract: A role for redox regulation in activation of the NF-kappa B transcription factor was suggested by the observation that DNA binding activity of free protein, but not preformed DNA-protein complex, is inhibited by -SH modifying agents but enhanced by reducing agents. Mutagenesis of conserved cysteine residues in the p50 subunit identified amino acid 62 as being important for DNA binding, as a serine substitution at this position reduces DNA binding affinity, but renders the protein insensitive to -SH modifying agents. DNA binding activity of the wild type protein but not the amino acid 62 mutant was also stimulated by thioredoxin while detection of disulphide cross linked dimers in p50 but not the amino acid 62 mutant suggests that thioredoxin stimulates DNA binding by reduction of a disulphide bond involving cysteine 62. The physiological relevance of these findings was supported by the observation that cotransfection of a plasmid expressing human thioredoxin and an HIV LTR driven reporter construct resulted in an NF-kappa B dependent increase in expression of the reporter gene. Thus modification of p50 by thioredoxin, a gene induced by stimulation of T-lymphocytes in parallel with NF-kappa B translocation, is a likely step in the cascade of events leading to full NF-kappa B activation.

DNA diversity among clinical isolates of Helicobacter pylori detected by PCR-based RAPD fingerprinting

Abstract: The RAPD (or AP-PCR) DNA fingerprinting method was used to distinguish among clinical isolates of Helicobacter pylori, a bacterium whose long term carriage is associated with gastritis, peptic ulcers and gastric carcinomas. This method uses arbitrarily chosen oligonucleotides to prime DNA synthesis from genomic sites to which they are fortuitously matched, or almost matched. Most 10-nt primers with > or = 60% G + C yielded strain-specific arrays of up to 15 prominent fragments, as did most longer (> or = 17-nt) primers, whereas most 10-nt primers with 50% G+C did not. Each of 64 independent H. pylori isolates, 60 of which were from patients in the same hospital, was distinguishable with a single RAPD primer, which suggests a high level of DNA sequence diversity within this species. In contrast, isolates from initial and followup biopsies were indistinguishable in each of three cases tested.

Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications

Abstract: The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.

Rapid extraction of fungal DNA for PCR amplification

Abstract: Polymerase chain reaction (PCR) is a technique widely used in fungal research. One of its advantages is the ability to amplify very small amounts of DNA, in the picograms range, even in the presence of diverse contaminants. In spite of this, most of the extraction protocols of fungal DNA are designed for the obtention of microgram amounts of very pure DNA, requiring the establishment of relatively large fungal cultures and long extraction procedures. These protocols are needlessly complicated for PCR experiments. On the other hand, some authors have pointed out the feasibility of using single spores (1) or boiled mycelium (2) as a source of DNA in PCR experiments. This is advantageous for detection purposes, but when working with hundreds of strains in population studies, obtaining the material from the culture plate can be cumbersome and favor contaminations. Two widely cited methods (1,3) allow the DNA extraction in Eppendorf tubes, simplifying and reducing the scale of the extraction. Both methods start from lyophilized or fresh mycelium, ground by hand with a mortar and pestle. However, this procedure is cumbersome, and implies careful sterilization of the material and the use of liquid nitrogen. We have developed a faster and less prone to contamination procedure by culturing the fungus directly in an Eppendorf tube, and suppressing the phenolization step, as suggested in other recent protocol for plant DNA extraction (4). The proposed method is as follows: A 1.5 ml Eppendorf tube is filled with 500 ftl of liquid Potato—dextrose medium. The culture is started by inoculating some hyphal threads and allowed to grow for 72 hours at 25 °C. The mycelial mat is pelleted by centrifugation for 5 minutes at 13,000 rpm in a microfuge, washed with 500 p\\ of TE buffer and pelleted again. The TE is decanted and 300 y\\ of extraction buffer are added. This buffer is the same as that described by Raeder and Broda (3) (200 mM Tris HC1 pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS). The mycelium is crushed with a conical grinder (Treff AG, Degersheim, Switzerland), fitting exactly the tube and actioned by hand or electric potter at 200 rpm for some minutes. After that, 150 pA of 3 M sodium acetate, ph 5.2 are added, and tubes are placed at —20°C for about ten minutes. Tubes are then centrifuged in a microfuge and the supernatant transferred to another tube. Then, an equal volume of isopropanol is added, and after at least 5 minutes at room temperature, the precipitated DNA is pelleted by centrifugation in a microfuge. After a wash with 70% ethanol, the pellet is vacuum dried for some minutes and resuspended in 50 fil of TE. The amount of DNA obtained in this way, is around 3 6 /ig. This is enough for at least 50 PCR reactions, as determined by titration of DNA concentration in several reactions. Alternatively, in case more material is needed, the fungal culture can be made in a 15 ml centrifuge tube. After the mycelium is pelleted and washed in a benchtop centrifuge, it is transferred to an Eppendorf tube and crushed in the way described above. This culture yields about 25 — 35 /ig of DNA, enough for at least 500 PCR reactions. Fungal DNA obtained in this way, has been used in two PCR experiments. The first one was intended for detection of polymorphisms by random amplified polymorphic DNA (RAPDPCR) (5) in the phytopathogenic fungi Fusarium oxysporum and Rhizoctonia solani. This experiment was performed according to the conditions and cycling parameters described by Williams et al. (5). The primer used was OPA-2 (Operon Technologies, Alameda, CA), with sequence 5'-CAGGCCCTTC-3'. The second experiment was designed for amplification of a rDNA fragment in the same fungi. In this case, the reaction buffer was composed as before, but primers were 5'-ACCCGCTGAACTTAAGC-3' and 5'-TACTACCACCAAGATCT-3'. Cycling parameters were: 94°C for 1 minute, 50°C for 1 minute and 68°C for 2 minutes, for 35 cycles and a final autoextension step of 72°C for 5 minutes. In all cases, 1 p\\ of the described fungal miniprep was used as DNA template. Fifteen /il of reaction product were analysed in a 1.4% agarose gel, and visualized by ethidium bromide staining. (Figure 1).

Arbitrarily primed PCR fingerprinting of RNA.

Abstract: Fingerprinting of RNA populations was achieved using an arbitrarily selected primer at low stringency for first and second strand cDNA synthesis. PCR amplification was then used to amplify the products. The method required only a few nanograms of total RNA and was unaffected by low levels of genomic double stranded DNA contamination. A reproducible pattern of ten to twenty clearly visible PCR products was obtained from any one tissue. Differences in PCR fingerprints were detected for RNAs from the same tissue isolated from different mouse strains and for RNAs from different tissues from the same mouse. The strain-specific differences revealed are probably due to sequence polymorphisms and should be useful for genetic mapping of genes. The tissue-specific differences revealed may be useful for studying differential gene expression. Examples of tissue-specific differences were cloned. Differential expression was confirmed for these products by Northern analysis and DNA sequencing uncovered two new tissue-specific messages. The method should be applicable to the detection of differences between RNA populations in a wide variety of situations.

Oligonucleotide hybridizations on glass supports: a novel linker for oligonucleotide synthesis and hybridization properties of oligonucleotides synthesised in situ.

Abstract: A novel linker for the synthesis of oligonucleotides on a glass support is described. Oligonucleotides synthesised on the support remain tethered to the support after ammonia treatment and are shown to take part in sequence specific hybridisation reactions. These hybridizations were carried out with oligonucleotides synthesised on 'ballotini' solid sphere glass beads and microscope slides. The linker has a hexaethylene glycol spacer, bound to the glass via a glycidoxypropyl silane, terminating in a primary hydroxyl group that serves as starting point for automated or manual oligonucleotide synthesis.

Cloning by recognition site screening of two novel GT box binding proteins: a family of Sp1 related genes

Abstract: Previous analyses of the uteroglobin gene promoter revealed a GT1 box which is also found in the SV40 enhancer. The GT1 element in the context of the uteroglobin promoter is active in Ishikawa cells, a human endometrial cell line, but not in HeLa cells. Here we report the cloning by recognition site screening of two factors (SPR-1 and SPR-2) which bind to this GT1 motif. SPR-1 and SPR-2 are homologues of the transcription factor Sp1. All three proteins are closely related members of a gene family encoding proteins with very similar structural features. Like Sp1, SPR-1 and SPR-2 contain glutamine and serine/threonine rich amino acid stretches. Most significantly, the DNA binding domains of all three proteins are highly conserved and they recognize GT as well as GC boxes identically. SPR-2 mRNA is expressed ubiquitously, whereas SPR-1 transcripts are abundant in the brain but barely detectable in other organs. The possible function of these factors for the activity of the uteroglobin promoter is discussed.

Conserved sequence motifs in the initiator proteins for rolling circle DNA replication encoded by diverse replicons from eubacteria, eucaryotes and archaebacteria

Abstract: An amino acid motif was identified that consists of the sequence HisHydrHisHydrHydrHydr (Hydr--bulky hydrophobic residue) and is conserved in two vast classes of proteins, one of which is involved in initiation and termination of rolling circle DNA replication, or RCR (Rep proteins), and the other in mobilization (conjugal transfer) of plasmid DNA (Mob proteins). Based on analogies with metalloenzymes, it is hypothesized that the two conserved His residues in this motif may be involved in metal ion coordination required for the activity of the Rep and Mob proteins. Rep proteins contained two additional conserved motifs, one of which was located upstream, and the other downstream from the 'two His' motif. The C-terminal motif encompassed the Tyr residue(s) forming the covalent link with nicked DNA. Mob proteins were characterized by the opposite orientation of the conserved motifs, with the (putative) DNA-linking Tyr being located near their N-termini. Both Rep and Mob protein classes further split into several distinct families. Although it was not possible to find a motif or pattern that would be unique for the entire Rep or Mob class, unique patterns were derived for large subsets of the proteins of each class. These observations allowed the prediction of the amino acid residues involved in DNA nicking, which is required for the initiation of RCR or conjugal transfer of single-stranded (ss) DNA, in Rep and Mob proteins encoded by a number of replicons of highly diverse size, structure and origin. It is conjectured that recombination has played a major part in the dissemination of genes encoding related Rep or Mob proteins among the replicons exploiting RCR. It is speculated that the eucaryotic small ssDNA replicons encoding proteins with the conserved RCR motifs and replicating via RCR-related mechanisms, such as geminiviruses and parvoviruses, may have evolved from eubacterial replicons.

Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA

Abstract: Methylated DNA in vertebrates is associated with transcriptional repression and inactive chromatin. Two activities have been identified, MeCP1 and MeCP2, which bind specifically to DNA containing methyl-CpG pairs. In this report we characterize MeCP2. We show that it is more abundant than MeCP1, is more tightly bound in the nucleus, and is distinguishable chromatographically. The two proteins share widespread expression in somatic mammalian cells, and barely detectable expression in early embryonic cells. DNAs containing thymidine which has a methyl group at position 5 are not ligands for the MeCPs. The possible role of MeCP2 in methylation-associated gene inactivation was tested in in vitro transcription extracts. Purified MeCP2 inhibited transcription from both methylated and nonmethylated DNA templates in vitro, probably due to the presence of nonspecific DNA binding domains within the protein. We hypothesise that MeCP2 normally binds methylated DNA in the context of chromatin, contributing to the long-term repression and nuclease-resistance of methyl-CpGs.

Compilation and analysis of DNA sequences associated with apparent streptomycete promoters

Abstract: The DNA sequences associated with 139 apparent streptomycete transcriptional start sites are compiled and compared. Of these, 29 promoters appeared to belong to a group which are similar to those recognized by eubacterial RNA polymerases containing sigma 70-like subunits. The other 110 putative promoter regions contain a wide diversity of sequences; several of these promoters have obvious sequence similarities in the -10 and/or -35 regions. The apparent Shine-Dalgarno regions of 44 streptomycete genes are also examined and compared. These were found to have a wide range of degree of complementarity to the 3' end of streptomycete 16S rRNA. Eleven streptomycete genes are described and compared in which transcription and translation are proposed to be initiated from the same or nearby nucleotide. An updated consensus sequence for the E sigma 70-like promoters is proposed and a potential group of promoter sequences containing guanine-rich -35 regions also is identified.

Secondary structure of the 5' nontranslated regions of hepatitis C virus and pestivirus genomic RNAs.

Abstract: The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5' nontranslated regions (5'NTRs) sharing short segments of conserved primary nucleotide sequence. The functions of these 5'NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5'NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5'NTRs are highly conserved structurally, despite substantial differences in their primary nucleotide sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5'NTR of HCV resulted in a model of the secondary structure of the HCV 5'NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3' 200 bases of the 5'NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.

A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae.

Abstract: We report the discovery of a group of highly conserved DNA sequences located, in those cases studied, within intergenic regions of the chromosome of the Gram positive Streptococcus pneumoniae. The S. pneumoniae genome contains about 25 of these elements called BOX. From 5' to 3', BOX elements are composed of three subunits (boxA, boxB, and boxC) which are 59, 45 and 50 nucleotides long, respectively. BOX elements containing one, two and four copies of boxB have been observed; boxB alone was also detected in one instance. These elements are unrelated to the two most thoroughly documented families of repetitive DNA sequences present in the genomes of enterobacteria. BOX sequences have the potential to form stable stem-loop structures and one of these, at least, is transcribed. Most of these elements are located in the immediate vicinity of genes whose product has been implicated at some stage in the process of genetic transformation or in virulence of S. pneumoniae. This location raises the intriguing possibility that BOX sequences are regulatory elements shared by several coordinately controlled genes, including competence-specific and virulence-related genes.

Assessment of protein coding measures

Abstract: A number of methods for recognizing protein coding genes in DNA sequence have been published over the last 13 years, and new, more comprehensive algorithms, drawing on the repertoire of existing techniques, continue to be developed. To optimize continued development, it is valuable to systematically review and evaluate published techniques. At the core of most gene recognition algorithms is one or more coding measures--functions which produce, given any sample window of sequence, a number or vector intended to measure the degree to which a sample sequence resembles a window of 'typical' exonic DNA. In this paper we review and synthesize the underlying coding measures from published algorithms. A standardized benchmark is described, and each of the measures is evaluated according to this benchmark. Our main conclusion is that a very simple and obvious measure--counting oligomers--is more effective than any of the more sophisticated measures. Different measures contain different information. However there is a great deal of redundancy in the current suite of measures. We show that in future development of gene recognition algorithms, attention can probably be limited to six of the twenty or so measures proposed to date.

The bromodomain: a conserved sequence found in human, Drosophila and yeast proteins

Abstract: Identification of conserved domains or motifs in proteins may aid in the localization and analysis of important structural and functional regions. We report here a protein sequence motif, called the bromodomain (1), that has been found in six genes from humans (CCG1 and RING3), Drosophila (fsh and brm), and yeast (SPT7 and SNF2). The fsh and brm genes are required maternally for proper expression of certain homeotic genes (1,2). The SPT7 and SNF2 genes of Saccharomyces cerevisiae encode transcriptional activators (3,4). The SNF2 and brm proteins have extensive sequence homology (1). It is not clear whether the two human genes are involved in processes. CCG1 is a DNA-binding protein that complements temperature sensitive mutations that cause cell cycle arrest in Gl (5). The RING3 gene is a newly identified human gene of unknown function, mapping to the class II region of the human major histocompatibility locus, that has substantial homology to the fsh gene (6). Each of these proteins has one or two copies of the 61 —63 amino acid bromodomain (Figure 1). The sequence identity is highest (~ 80%) between the corresponding fsh and RING3 repeats. More typically, any two motifs show 25-40% identity, with 50-60% identity between two repeats within the same protein. There are seven invariant residues, four of which are aromatic amino acids, and numerous conservative substitutions. The location of the motif(s) within the individual proteins is variable, and two motifs may be present in tandem or separated by sequences unrelated to the motif. Secondary structure prediction methods were applied to the bromodomain and revealed two strongly predicted amphipathic a helices followed by reverse turns. Four of the invariant residues are located within the turns, and the other three are located among four highly conserved proline residues at the N terminus. The functional significance of the bromodomain is unknown, and experiments with the two yeast proteins indicate that it may be dispensable in some cases (6; P.Tan and F.W., unpublished data). However, the widespread conservation of the bromodomain suggests that it is important for some aspects of protein function. We speculate that the hydrophobic surfaces of the helices and the invariant hydrophobic residues could serve as sites of intramolecular or intermolecular protein-protein interaction. Such interactions could influence the assembly or activity of multicomponent complexes involved in transcription activation or other cellular processes.

Hairpin and parallel quartet structures for telomeric sequences

Abstract: The role of thymine residues in the formation of G-quartet structures for telomeric sequences has been investigated using model oligonucleotides of the type d(G4TnG4), with n = 1-4. Sequences d(G4T3G4) and d(G4T4G4) adopt a G-quartet structure formed by hairpin dimerization in 70 mM NaCl as judged by a characteristic circular dichroism signature with a 295 nm positive and 265 nm negative bands while d(G4TG4) adopts a parallel G-quartet structure like d(G12) which exhibits a strong positive band at 260 nm and a negative band at 240 nm. The sequence d(G4T2G4) exhibits a mixture of both conformations. The stability of hairpin G-quartet structures decreases with decrease in the number of intervening thymine residues. Potassium permanganate, a single strand specific probe has been used to establish the presence of loops composed of T residues in the hairpin G quartet structures formed by the oligonucleotides d(G4TnG4) with n = 2-4 in 70 mM NaCl. The formation of hairpin G quartet structure for the above sequences is further supported by the enhanced electrophoretic mobility observed on non-denaturing polyacrylamide gels. Human telomeric sequence d(TTAGGG)4 which showed enhanced electrophoretic mobility like Tetrahymena telomeric sequence d(T2G4)4 also exhibited a characteristic CD spectrum for a folded-back G-quartet structure. A detailed model for G-quartet structure involving hairpin dimer with alternating syn-anti-syn-anti conformation for the guanine residues both along the chain as well as around the G tetrad with at least two thymine residues in the loop is proposed. Intermolecular association of short telomeric sequences reported here provides a possible model for chromosomal pairing.

Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR

Abstract: Thermus aquaticus (Taq) DNA polymerase was used to measure the extension efficiency for all configurations of matched and mismatched base pairs at template-primer 3'-termini. The transition mispairs, A(primer).C, C.A, G.T, and T.G were extended 10(-3) to 10(-4)-fold less efficiently than their correctly paired counterparts. Relative efficiencies for extending transversion mispairs were 10(-4) to 10(-5) for T.C and T.T, about 10(-6) for A.A, and less than 10(-6) for G.A, A.G, G.G and C.C. The transversion mispair C(primer).T was extended with high efficiency, about 10(-2) compared to a correct A.T basepair. The unexpected ease of extending the C.T mismatch was not likely to have been caused by primer-template misalignment. Taq polymerase was observed to bind with similar affinities to each of the correctly paired and mispaired primer-template 3'-ends. Thus, the failure of Taq polymerase to extend mismatches efficiently appears to be an intrinsic property of the enzyme and not due to an inability to bind to 3'-terminal mispairs. For almost all of the mispairs, C.T being the exception, Taq polymerase exhibits about 100 to 1000-fold greater discrimination against mismatch extension compared to avian myeloblastosis reverse transcriptase and HIV-1 reverse transcriptase which extend most mismatched basepairs permissively. Relative mismatch extension efficiencies for Taq polymerase were measured at 45 degrees C, 55 degrees C and 70 degrees C and found to be independent of temperature. The mispair extension data should be important in designing experiments using PCR to distinguish between sequences that vary by a single nucleotide.

Splicing signals in Drosophila: intron size, information content, and consensus sequences

Abstract: A database of 209 Drosophila introns was extracted from Genbank (release number 64.0) and examined by a number of methods in order to characterize features that might serve as signals for messenger RNA splicing. A tight distribution of sizes was observed: while the smallest introns in the database are 51 nucleotides, more than half are less than 80 nucleotides in length, and most of these have lengths in the range of 59-67 nucleotides. Drosophila splice sites found in large and small introns differ in only minor ways from each other and from those found in vertebrate introns. However, larger introns have greater pyrimidine-richness in the region between 11 and 21 nucleotides upstream of 3' splice sites. The Drosophila branchpoint consensus matrix resembles C T A A T (in which branch formation occurs at the underlined A), and differs from the corresponding mammalian signal in the absence of G at the position immediately preceding the branchpoint. The distribution of occurrences of this sequence suggests a minimum distance between 5' splice sites and branchpoints of about 38 nucleotides, and a minimum distance between 3' splice sites and branchpoints of 15 nucleotides. The methods we have used detect no information in exon sequences other than in the few nucleotides immediately adjacent to the splice sites. However, Drosophila resembles many other species in that there is a discontinuity in A + T content between exons and introns, which are A + T rich.

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins

Abstract: We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.

Effective incorporation of 2'-O-methyl-oligoribonucleotides into liposomes and enhanced cell association through modification with thiocholesterol.

Abstract: Cholesterol was linked to 2'-O-methyl-oligoribonucleotides (2'-OMe-RNA) via a disulfide bond by reacting the 3'-(pyridyldithio)-modified 2'-OMe-RNA with thiocholesterol in dichloromethane-methanol solution. This ligation reaction was made possible by a novel strategy in which the highly charged oligonucleotide was rendered soluble in nonaqueous solvent through conversion to a lipophilic amidinium salt. The biodegradable lipophilic modification of 2'-OMe-RNA resulted in a large increase in incorporation of such oligonucleotides into liposomes prepared by reversephase evaporation. Furthermore, association of these modified oligonucleotides with cultured TIB 73 cells was 100-fold higher than that seen with unmodified 2'-OMe-RNA in serum-free medium and about 10 to 30-fold higher in the presence of 10% calf serum. During incubation with cells, release of the internalized oligonucleotide from the thiocholesteryl moiety can be demonstrated.

Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants.

Abstract: We have used the polymerase chain reaction to isolate fragments of Ty1-copia group retrotransposons from a wide variety of members of the higher plant kingdom 56 out of 57 species tested generate an amplified fragment of the size expected for reverse transcriptase fragments of Ty1-copia group retrotransposons Sequence analysis of subclones shows that the PCR fragments display varying degrees of sequence heterogeneity Sequence heterogeneity therefore seems a general property of Ty1-copia group retrotransposons of higher plants, in contrast to the limited diversity seen in retrotransposons of Saccharomyces cerevisiae and Drosophila melanogaster Phylogenetic analysis of all these sequences shows, with some significant exceptions, that the degree of sequence divergence in the retrotransposon populations between any pair of species is proportional to the evolutionary distance between those species This implies that sequence divergence during vertical transmission of Ty1-copia group retrotransposons within plant lineages has been a major factor in the evolution of Ty1-copia group retrotransposons in higher plants Additionally, we suggest that horizontal transmission of this transposon group between different species has also played a role in this process