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At least 487 records · Page 27Linked to original sources
PubMed1994

A photo-cross-linkable tertiary structure motif found in functionally distinct RNA molecules is essential for catalytic function of the hairpin ribozyme.

We have identified an essential UV-sensitive tertiary structure domain within the hairpin ribozyme. Irradiation at 254 nm produces two cross-linked RNA species that are resolved from the unmodified structure by denaturing gel electrophoresis. One cross-link forms at high efficiency and maps between nucleotides G21 and/or A22 and U41, all essential bases located within an internal loop joining helices 3 and 4. A second cross-link forms between nucleotides A20 and U42 as a result of ribozyme dimerization at concentrations greater than 0.5 microM. Both cross-linked species retain cleavage activity and so presumably reflect catalytically proficient structures of the ribozyme. Formation of the intramolecular cross-link is independent of Mg2+ and substrate and is blocked by base substitutions within the reactive domain that inhibit catalysis. A 36-nt RNA fragment containing the photoreactive domain but lacking the substrate binding domain also cross-links with high efficiency and maps between G21 and U41, as observed with the intact molecule. The sequence and cross-linking sites of the UV-sensitive internal loop are strikingly similar to those found in several other RNA molecules, including loop E of 5S rRNA. These results suggest that the loop E-like structure may be a common RNA folding domain that is utilized in a variety of functionally important RNA molecules.

Base Sequence↗
PubMed1988

Molecular cloning of three structurally related genes for chicken avidin.

The chicken genomic library was screened using the 32P-labelled 3'-end of avidin cDNA as a hybridization probe. A positive clone, lambda gAV12201, containing a 15-16 kb insert, was detected. The EcoRI subclones, pgAV0.4, pgAV1.8, pgAV3.3 and pgAV3.7 from the genomic clone were subjected to hybridization and restriction enzyme mapping analysis. The preliminary results suggest the existence of three structurally related genes for chicken avidin. Whether the natural gene is within the subclones can only be established when sequencing analyses of the subclones have been completed.

Animals↗
PubMed1989

A cosmid clone map derived from a small region of human chromosome 11.

We have used cosmid "fingerprinting" to construct an overlapping DNA clone "map" of the human DNA in a mouse/human hybrid cell line, E65-9, that contains about 4 x 10(6) bp, including the H-Ras gene, as its human component. We have additionally used 32P-labeled RNA probes to establish linkage of particular sets of clones, and the final map comprises about 300,000 bp and is contained in three nonoverlapping segments. The reasons for failure to close the gaps by direct probing are discussed. We have developed techniques to search for rare cutting restriction enzyme cleavage sites in large numbers of cloned DNAs and have positioned sites for EagI and BssHII on our clone map. The methods we used are capable of considerable scale-up and are currently being applied to the short arm of human chromosome 11.

Animals↗
PubMed1989

Chromosomal localization of DBL oncogene sequences.

The DBL oncogene was generated by rearrangements involving three discontinuous regions of the human genome. Analyses of panels of human X rodent somatic cell hybrids demonstrated that the DBL proto-oncogene located on the X chromosome (just proximal or distal to bands q26-27.2) underwent recombination at its 5' and 3' ends with sequences derived from chromosomes 3 (p13q-ter) and 16 (p13-q22), respectively. DBL was localized to chromosome Xq27-q28 by in situ hybridization. Another oncogene, MCF2, was previously shown to contain sequences derived from Xq27 as well. Comparison of the restriction maps and nucleotide sequences of genomic and cDNA clones representing the chromosome X-specific sequences of the DBL oncogene and MCF2, taken together with their chromosomal localization, indicates that these oncogenes were derived from the same genetic locus.

Animals↗
PubMed1988

FRAGMAP--a program for restriction site map comparison.

A program is described which aligns pairs of linear restriction site maps using an algorithm which maximizes the number of coincident sites through the addition of extra 'padding' fragments; and makes maps of the non-conserved sites between pairs and triplets of input maps. A range of display facilities, hardcopy functions and database management routines are also included. The program runs on a stand-alone microcomputer, and is stored along with a database of several hundred files on a single floppy disc.

Copying Processes↗
PubMed1988

Differentiation-induced gene expression in 3T3-L1 preadipocytes. Characterization of a differentially expressed gene encoding stearoyl-CoA desaturase.

Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the activation of transcription of an unidentified gene which encodes a 4.9-kilobase (kb) mRNA. Several cDNAs that include the complete sequence of this mRNA were obtained and used to isolate and characterize the gene. Analysis of the nucleotide and amino acid sequences of both cDNA and genomic clones revealed that the gene encodes the mouse stearoyl-CoA desaturase (SCD), an enzyme known to be expressed upon differentiation of 3T3-L1 preadipocytes. The predicted amino acid sequence (355 residues) of the mouse 3T3-L1 adipocyte SCD exhibits 92% identity to that of the rat liver SCD. There is also a high degree of nucleotide sequence identity between the mouse and rat mRNAs in their unusually long approximately 3.5-kb 3'-untranslated regions. Mice fed a diet containing unsaturated triacylglycerides express SCD mRNA only in adipose tissue, whereas mice starved and refed a fat-free diet, express SCD mRNA in both liver and adipose tissue. The mouse gene for the desaturase spans approximately 15 kb and contains 6 exons and 5 introns with all intron-exon junctions conforming to the GT/AG splicing rule. As determined by S1 nuclease mapping and primer extension analysis, the transcriptional initiation site maps 152 nucleotides upstream from the initiation methionine codon. A canonical promoter "TATA" box is located 30 base pairs upstream of the Cap site. A typical "CCAAT" box sequence is not present in the adjacent 5'-flanking region; however, there is a GC-rich sequence (at nucleotide -215) similar to the binding site for the nuclear transcription factor Sp1. Upstream from the transcriptional initiation site are elements with homology (approximately 75%) to the putative fat-specific transcriptional element FSE2 and core consensus sequences for cAMP and glucocorticoid regulatory elements. A chimeric construct, containing 363 base pairs of 5'-flanking sequence and 30 nucleotides of 5'-untranslated sequence of the mouse SCD gene ligated to the bacterial chloramphenicol acetyltransferase gene, was transfected into 3T3-L1 cells. When cells were induced to differentiate into adipocytes, expression of the SCD chloramphenicol acetyltransferase gene increased approximately 63-fold, suggesting that the SCD promoter region contains elements that mediate the response to adipogenic agents which induce differentiation.

Adipose Tissue↗
PubMedDate not supplied

Characterization of the regulatory region of Adra2c, the gene encoding the murine alpha2C adrenoceptor subtype.

The 5' flanking sequence (3,227 base pairs, bp) of the mouse Adra2c subtype gene was determined and characterized. The transcription start site was mapped to nucleotide 'A' of two initiator motifs in tandem array, i.e. 1,159 and 1,153 bp upstream from the initiation codon of the open reading frame (ORF) of Adra2c, respectively. One structural feature salient to the 5' regulatory region of Adra2c is present in the sequence 1 kb immediately upstream from the receptor ORF, which is highly enriched in GC content (76%) and CpG island counts (i.e. CpG/GpC, 146:177), and thus rich in Sp1-binding motifs. At the 3' flanking region, the polyadenylation signal was mapped to 481 bp downstream from the termination codon. The transcript defined by sequence data thereby is consistent with a size of 3 kb (brain form) determined by Northern blot analysis. The transgene, Adra2c-NN- lacZ, which links the promoter region of Adra2c to the lacZ reporter gene, was constructed in order to evaluate the functional capacity of the promoter and the putative motifs residing within the defined regulatory region (1.9 kb upstream from the ORF) in directing the reporter gene expression in vitro in transiently transfected cells and in vivo in transgenic (Tg) mice. Permissive cell types to Adra2c-NN include those derived from neural and kidney lineages. Significant Adra2c-NN-driven reporter expression in Tg mice established suggests that alpha2C adrenoceptor expression is permissive under Adra2c-NN in central (cerebral cortex, hippocampus, subthalamus, hypothalamus, superior colliculus, cerebellum, and brain stem) and peripheral (pancreatic beta-islets) tissues.

Amino Acid Motifs↗
PubMed1987

RNA recombination of coronaviruses: localization of neutralizing epitopes and neuropathogenic determinants on the carboxyl terminus of peplomers.

Murine coronaviruses undergo RNA recombination at a very high frequency. We have obtained a series of recombinant viruses using neutralizing monoclonal antibodies in conjunction with temperature-sensitive markers. All of the recombinants obtained have a crossover within gene C, which encodes the peplomer protein of the virus. The genetic structure of these recombinants suggests that the antigenic regions recognized by these neutralizing monoclonal antibodies are localized on the carboxyl-terminal one-third of the peplomer protein. Since the two monoclonal antibodies used are also associated with the critical determinants of virus neuropathogenicity, we conclude that both the neutralizing antibody binding sites and determinants of pathogenicity are localized at the carboxyl-terminal one-third of the peplomer. The variation of crossover sites in different recombinant viruses also allowed precise mapping of additional antigenic sites. RNA recombination thus presents a powerful genetic tool, and the carboxyl-terminal localization of the biological functions of peplomers suggests a distinct conformation of these viral membrane proteins.

Animals↗
PubMed2001

An oligonucleotide fingerprint normalized and expressed sequence tag characterized zebrafish cDNA library.

The zebrafish is a powerful system for understanding the vertebrate genome, allowing the combination of genetic, molecular, and embryological analysis. Expressed sequence tags (ESTs) provide a rapid means of identifying an organism's genes for further analysis, but any EST project is limited by the availability of suitable libraries. Such cDNA libraries must be of high quality and provide a high rate of gene discovery. However, commonly used normalization and subtraction procedures tend to select for shorter, truncated, and internally primed inserts, seriously affecting library quality. An alternative procedure is to use oligonucleotide fingerprinting (OFP) to precluster clones before EST sequencing, thereby reducing the re-sequencing of common transcripts. Here, we describe the use of OFP to normalize and subtract 75,000 clones from two cDNA libraries, to a minimal set of 25,102 clones. We generated 25,788 ESTs (11,380 3' and 14,408 5') from over 16,000 of these clones. Clustering of 10,654 high-quality 3' ESTs from this set identified 7232 clusters (likely genes), corresponding to a 68% gene diversity rate, comparable to what has been reported for the best normalized human cDNA libraries, and indicating that the complete set of 25,102 clones contains as many as 17,000 genes. Yet, the library quality remains high. The complete set of 25,102 clones is available for researchers as glycerol stocks, filters sets, and as individual EST clones. These resources have been used for radiation hybrid, genetic, and physical mapping of the zebrafish genome, as well as positional cloning and candidate gene identification, molecular marker, and microarray development.

Animals↗
PubMed1994

Site-directed mutagenesis of adeno-associated virus type 2 structural protein initiation codons: effects on regulation of synthesis and biological activity.

It has been shown that two of the three adeno-associated virus type 2 capsid proteins, B and C, are synthesized from a single spliced transcript. Protein C arises from an AUG codon at nucleotide 2810, whereas protein B is initiated by a unique eucaryotic initiation codon (ACG) that lies 65 triplets upstream from the C origin. The third capsid component, protein A, is synthesized from a second spliced transcript which uses an alternative 3' acceptor site. In this study we used oligonucleotide-directed mutagenesis to confirm the positions of the B initiation codon and the 3' acceptor sites for the alternatively spliced B/C and A protein messages. We also located definitively the protein A initiation codon, an AUG triplet mapping to nucleotide 2203. Mutagenesis of the B initiator permitted a direct test of the effect of increased B initiator strength on the translational efficiencies of the B and C proteins. It was found that conversion of the relatively inefficient protein B initiator (ACG) to an AUG enhanced the level of B synthesis while abolishing the synthesis of C from its downstream AUG initiator. Protein C synthesis thus depends on the strength of the B initiator, i.e., the relatively higher levels of C (approximately 20-fold greater than B) must result from frequent readthrough of the weak B initiator. Finally, we examined the abilities of mutants deficient in the synthesis of A, B, or C to produce infectious virions. We found that at least two of the structural proteins, B and C, are required for the production of infectious virions and that sequestration of single-stranded adeno-associated virus genomes from the pool of replicating DNA molecules does not occur in the absence of either of these proteins.

Blotting, Northern↗
PubMed1990

Structure and function of the Epstein-Barr virus BZLF1 protein.

Five DNA-binding sites for the Epstein-Barr virus BZLF1 protein have been identified within three of the early viral promoters, and four of these binding sites contain a consensus AP-1 site. The part of the BZLF1 protein required for sequence-specific DNA binding to one of these AP-1-like sites was identified by deletion mapping. Site-directed mutagenesis of this DNA target suggests that BZLF1 may work partly by overcoming a cellular repressor of viral transcription.

Amino Acid Sequence↗
PubMed1980

Expression of Ti-plasmid DNA in E. coli: comparison of homologous fragments cloned from Ti plasmids of Agrobacterium strains C58 and Ach5.

Fragments of two Ti plasmids from Agrobacterium tumefaciens were cloned and studied for cross-hybridization. Homologous fragments were further investigated for areas of strong homology and mapped with various restriction enzymes. The fragments were inserted in two directions and the recombinant plasmid was brought into E. coli strains producing minicells. About five proteins were found to be coded by those fragments of the Ti plasmids.

Bacterial Proteins↗
PubMed1989

Recognition and cleavage signals for mRNA processing lie within local domains of the phage f1 RNA precursors.

In Escherichia coli infected with the filamentous phage f1, a number of the abundant phage mRNAs, species C-G, are products of post-transcriptional processing. The approach of cloning the phage sequences likely to include the processing signals in a plasmid under transcriptional control of the lambda PL promoter (Blumer, K. J., and Steege, D. A. (1984) Nucleic Acids Res. 12, 1847-1861) was extended to additional sites to show that processing at all five major sites is mediated by nuclease activity encoded by the bacterial genome. Primer extension methods were used to map more accurately in the f1 DNA sequence the 5' end points of the processed RNAs. The DNA segments that encode the mRNA processing signals were delimited from parallel series of 5'-3' and 3'-5' deletions made into the regions in which the RNA 5' ends map. For each deletion variant, in vivo f1 mRNA processing activity was assessed by primer extension and S1 nuclease mapping methods. The data indicate that the processing signals are comprised of relatively local regions near the point of RNA cleavage. Whereas cleavage occurs at the 5' border of the sequences that comprise the D, E, and F processing sites and thereby places most of the recognition information in the mature or product portion of the precursor, it occurs more centrally within the region comprising the C site. The C and D sites function independently as substrates for cleavage, with the necessary information contained in regions of 70 and 90 nucleotides, respectively. Cleavage at the E site appears to require a region of 130 nucleotides which completely contains the F site. From the effects on processing activity of deleting sequences in this region, the overlapping E and F processing sites appear to consist functionally of two subdomains. Each has a cleavage site at its immediate 5' end which can be substituted by foreign sequences, but both utilize a common recognition domain downstream from the point of strand scission.

Base Sequence↗
PubMed1988

A new murine Ig VH gene family.

A novel murine VH gene family, termed VH10, has been found and characterized. Based on RFLP analysis, this family exhibits extensive polymorphism among inbred strains of mice and encompasses two to five members, depending on the Igh haplotype. Analyses of recombinant inbred strains suggest a map position of this family 5' to the 7183 and Q52 VH gene families. A VH10 gene has been found to encode anti-DNA autoantibodies from lupus mice; another one may be a pseudogene.

Amino Acid Sequence↗
PubMed2001

Transcript map and comparative analysis of the 1.5-Mb commonly deleted segment of human 5q31 in malignant myeloid diseases with a del(5q).

Loss of a whole chromosome 5, or a del(5q), are recurring abnormalities in malignant myeloid diseases. In previous studies, we defined a commonly deleted segment (CDS) of 1.5 Mb between D5S479 and D5S500 in patients with a del(5q), and we established a P1 artificial chromosome-based contig encompassing this interval. To identify candidate tumor suppressor genes (TSGs), we developed a transcript map of the CDS. The map contains 18 genes and 12 expressed sequence tags/UniGenes. Among the 18 genes are 10 genes that were previously cloned and 8 novel genes. The newly identified genes include CDC23, which encodes a component of the anaphase-promoting complex; RAB6KIFL, which encodes a kinesin-like protein involved in organelle transport; and KLHL3, which encodes a human homologue of the Drosophila ring canal protein, kelch. We determined the intron/exon organization of 14 genes and eliminated each gene as a classical TSG by mutation analysis. In addition, we established a single-nucleotide polymorphism map as well as a map of the mouse genome that is syntenic to the CDS of human 5q31. The development of a transcription map will facilitate the molecular cloning of a myeloid leukemia suppressor gene on 5q.

Acute Disease↗
PubMed1988

The effects of HPFH mutations in the human gamma-globin promoter on binding of ubiquitous and erythroid specific nuclear factors.

Genetic evidence indicates that single point mutations in the gamma-globin promoter may be the cause of high expression of the mutated gene in the adult period (Hereditary Persistence of Fetal Hemoglobin, HPFH). Here we show that one of these mutations characterized by a T----C substitution at position -175 in a conserved octamer (ATGCAAAT) sequence, abolishes the ability of a ubiquitous octamer binding nuclear protein to bind a gamma-globin promoter fragment containing the mutated sequence; however, the ability of two erythroid specific proteins to bind the same fragment is increased three to five fold. DMS interference and binding experiments with mutated fragments indicate that the ubiquitous protein recognizes the octamer sequence, while the erythroid specific proteins B2, B3 recognize flanking nucleotides. Competition experiments indicate that protein B2 corresponds to an erythroid-specific protein known to bind to a consensus GATAG sequence present at several locations in alpha, beta and gamma-globin genes. Although the distal CCAAT box region of the gamma-globin gene shows a related sequence, an oligonucleotide including this sequence does not show any ability to bind the above mentioned erythroid protein; instead, it binds a different erythroid specific protein, in addition to a ubiquitous protein. The -117 G----A mutation also known to cause HPFH, and mapping two nucleotides upstream from the CCAAT box, greatly decreases the binding of the erythroid-specific, but not that of the ubiquitous protein, to the CCAAT box region fragment.

Base Sequence↗
PubMed1988

Transcript mapping using [35S]DNA probes, trichloroacetate solvent and dideoxy sequencing ladders: a rapid method for identification of transcriptional start points.

A simple method for RNA transcript mapping has been developed that combines the use of 35S-labeled M13 DNA probes and the presence of high concentrations of sodium trichloroacetate in the hybridization buffer. These hybridization conditions permit the use of M13 probes without purification from the template. The dideoxy sequencing ladders used for sizing the protected DNA fragments are obtained from the same M13 templates utilized to synthesize the DNA probes. The method was tested by analyzing the transcripts controlled by lac, ptr and trxA promoters. Comparison of the results with previously published data obtained with the conventional S1 nuclease mapping technique indicated that the present method is just as precise and at least 50 times more sensitive. Clones constructed for sequencing a gene of interest can be used directly to identify transcriptional start points.

Base Sequence↗
PubMed1987

Cloning and mapping of infA, the gene for protein synthesis initiation factor IF1.

The gene for translation initiation factor IF1, infA, has been identified by using two synthetic oligonucleotides to screen a Charon 30 library of Escherichia coli DNA. A recombinant lambda phage, C1921, was purified from a plaque positive for both probes. A 2.8 kb BglII fragment and a 2.0 kb HindIII fragment isolated from C1921 were subcloned into the BamHI and HindIII sites of pBR322 to yield pTB7 and pTH2 respectively. Synthesis of IF1 in maxicells transformed with pTB7 or pTH2 indicates the presence of inf A in both inserts. This was confirmed by DNA sequencing: a region was found that codes for a 8,119 dalton protein with an amino acid sequence corresponding to IF1. The chromosomal location of inf A was determined by mapping the closely linked beta-lactamase gene (Ampr) in pTB7 and pTH2. pTB7 and pTH2 were transformed into polA Hfr hosts, and integration of the plasmid by homologous recombination near inf A was selected on the basis of ampicillin resistance. The site of integration was confirmed by Southern blot analysis of restriction nuclease digested wild type and transformed genomic DNA. The Ampr marker (and therefore inf A) was mapped to about 20 minutes by Hfr interrupted matings and P1 transduction experiments. The structure and regulation of the inf A operon currently are being investigated.

Amino Acid Sequence↗