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At least 451 records · Page 25Linked to original sources
PubMed1984

Tandem arrangement of the albumin and alpha-fetoprotein genes in the human genome.

A genomic clone containing human albumin mRNA sequences was isolated from a lambda phage gene library derived from a human hepatoma cell line that produces alpha-fetoprotein (AFP) and albumin. This clone was also found to hybridize with the 5'-flanking region of the human AFP gene. Restriction mapping and nucleotide sequencing revealed that the albumin and AFP genes are present in tandem, in the same transcriptional orientation, with the albumin gene 14.5 kb upstream of the AFP gene. We have also isolated a genomic clone carrying both the albumin and AFP gene sequences from human fibroblasts, which produce neither AFP nor albumin. This DNA showed a restriction map that was indistinguishable from that of the clone obtained from the hepatoma described above, demonstrating that no gross rearrangements of the intergenic DNA sequence are involved in control of expression of the AFP and albumin genes.

Bacteriophage lambda↗
PubMed1978

A hybridization procedure for the isolation of specific RNA segments applied to the analysis of bacteriophage Qbeta RNA.

A method for the isolation of RNA fragments originating from defined regions of bacteriophage Qbeta RNA minus strands is described. Large RNase T1 oligonucleotides were isolated on a preparative scale from Qbeta RNA. The nucleotide sequences (13 to 26 nucleotides) and map positions of these oligonucleotides were known from previous work (Billeter, M. A. (1978) J. Biol. Chem. 253, 8381-8389). After addition of AMP residues (50 in the average) using terminal adenylate transferase, these pure oligonucleotides were hybridized to 32P-labeled Qbeta RNA minus strands synthesized in vitro. Fragments in the size range of 100 to 500 nucleotides were then generated by partial digestion with RNase T1. Fragments hybridized to such oligonucleotides were recovered by chromatography on poly(U)-Sephadex and then resolved according to their size by polyacrylamide gel electrophoresis. The specificity and reproducibility of the method as well as its suitability for the sequence analysis of Qbeta RNA was verified by using in particular a linker oligonucleotide derived from a Qbeta RNA region near the 3' end. The sequence catalogues of the RNase T1 and RNase A oligonucleotides of two fragments isolated in this way, 202 and 310 nucleotides in length, were established and all fragments isolated were shown to contain a sequence complementary to the linker oligonucleotide.

Base Sequence↗
PubMed1985

Nucleotide sequence and transcription of a bacteriophage 29 early promoter.

We have studied the in vitro and in vivo transcription of a promoter for Bacillus subtilis RNA polymerase on bacteriophage phi 29 DNA. The promoter is identified as an early promoter as it is transcribed in vitro by uninfected B. subtilis sigma 55-containing RNA polymerase; is transcribed in vivo at both 7 min after infection and in the presence of chloramphenicol; and is transcribed right to left on the standard phi 29 map. The nucleotide sequence of the promoter and the initiation site for RNA synthesis are reported. We have also examined the kinetics of RNA synthesis initiation using a single round run-off transcription assay. The overall rate of initiation was found to be 1.6 X 10(6) M-1 s-1 while the rate of conversion of unstable to stable polymerase-promoter complexes was 0.049 s-1. These values are comparable to those for similar promoters for Escherichia coli RNA polymerase.

Bacteriophages↗
PubMed1988

The ethanol regulon in Aspergillus nidulans: characterization and sequence of the positive regulatory gene alcR.

The regulatory gene, alcR, of Aspergillus nidulans, encodes a protein that induces the expression of the alcA and aldA genes. The alcR gene is inducible, autoregulated, and subject to carbon catabolite repression. We report the complete nucleotide sequence of the alcR gene and its 5' and 3' non-coding regions. In the 5' flanking region of the alcR gene, several repeats and inverted repeats were found, and small sequence similarities were also found with the 5' flanking regions of the alcA and aldA genes. One intron of small size interrupts the open reading frame. The start point of transcription was mapped 50 nucleotides upstream from the putative start codon, and a sequence CAATG was found 5' to the polyadenylation site of the transcript that could play a role in selection of the polyadenylation site. The putative alcR-encoded protein was identified in vivo as an inducible polypeptide of 96 kDa in a transformant carrying multiple copies of the alcR gene.

Alcohol Dehydrogenase↗
PubMed1993

Molecular characterization and hrp dependence of the avirulence gene avrPto from Pseudomonas syringae pv. tomato [corrected].

The avrPto avirulence gene from Pseudomonas syringae pv. tomato (Pst) race 0 governs race-specific resistance to bacterial speck disease in tomato cultivars containing the Pto resistance gene. The avrPto gene encodes 0.7 and 0.75 kb mRNAs whose predicted translation product is a mostly hydrophilic 164 amino acid protein of 18.3 kD a that reveals no homology to protein sequences in GenBank or EMBL databases. Highest expression of avrPto in cell culture is observed in minimal media containing sugars and sugar alcohols as carbon sources and lowest expression in minimal media containing tricarboxylic acid intermediates and in complex media. Expression of avrPto in planta is induced within 1 h following infection of both resistant and susceptible tomato plants by Pst, and increases over the first 6 h. Transcription of avrPto requires the hrpSR pathogenicity functions, but is independent of other Pst hrp genes. A region of the avrPto promoter shows homology to hrp box sequences upstream of other P. syringae genes that require the hrpSR locus for expression, and both avirulence activity and avrPto mRNA accumulation are abolished by deletions extending into this region. The avrPto transcription start site maps 31 nucleotides downstream of the hrp box motif.

Amino Acid Sequence↗
PubMed1988

Isolation and restriction endonuclease analysis of a mycoplasma plasmid.

A 1.7-kb plasmid was isolated from a large colony type strain of Mycoplasma mycoides subsp. mycoides. It was cloned into the Escherichia coli vector M13mp19, and DNA from the resultant recombinant was used to construct a restriction map of the plasmid. Because mycoplasmas show deviation from normal coding patterns, the availability of a mycoplasma plasmid could be useful in the development of a cloning vector for them.

DNA Restriction Enzymes↗
PubMed1997

Mapping replication fork direction by leading strand analysis.

Replication fork polarity methods measure the direction of DNA synthesis by taking advantage of the asymmetric nature of DNA replication. One procedure that has been used on a variety of cell lines from different metazoans relies on the isolation of newly replicated DNA strands in the presence of the protein synthesis inhibitor emetine. Since Okazaki fragments are not synthesized under such conditions, DNA strands produced during continuous exposure to emetine consist mainly of leading strands. In the protocol described, leading strands isolated from emetine treated cells are detected with single-stranded probes representing each strand of the DNA duplex in the region of interest. Hybridization of leading strands to one strand of a cloned genomic template identifies the direction of replication fork movement. If initiation of DNA synthesis occurs from a preferred site, leading strands should diverge from the corresponding initiation region. The leading strand method is particularly useful for mapping initiation in chromosomal loci that do not replicate immediately on entry into S phase and in mapping the replication fork patterns in which candidate initiation regions have not been identified. Cautious interpretation of the results is needed because the method relies heavily on quantitative hybridization. Leading strand data can be difficult to interpret when the genomic targets are very close to initiation regions and when the targets vary in their hybridization efficiency or in the efficiency of incorporation of nucleotide analogs. The experimental details of the method are reviewed, controls to avoid common pitfalls are suggested, protocols to facilitate the accurate interpretation of the results are provided.

Animals↗
PubMed1988

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus.

A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5' termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.

DNA Restriction Enzymes↗
PubMedDate not supplied

Applications of pharmacogenetics to drug development: the Glaxo Wellcome experience.

The Genetics Directorate was established at Glaxo Wellcome (GW) in 1997. The goals of the Directorate are to identify susceptibility genes for common diseases with large unmet therapeutic need, apply genetic methods for the targeted development of medicines so that the right medicine is developed for the right patient, assist in translating gene discoveries into target selection, and represent genetics accurately internally within GW and externally (to laypersons, the medical community, the business community, government representatives, and regulatory agencies). As part of the goal of developing the right medication for the right patient, GW has added genetic research to its clinical drug studies in every major therapeutic area. GW worked closely with international ethicists experienced in the area of genetic research to develop the process and documents that are currently in use; these undergo frequent, rigorous review in light of the changing regulatory and legal environment and the needs of clinical investigators and patients. The addition of genetic research to clinical studies was accompanied by significant education efforts within GW and for study-site personnel, ethics committees, and regulatory authorities. Feedback from all those involved is an integral part of implementing GW's genetic research and is used to fine-tune the processes and protocol and consent document templates. A recently completed review of the approval rate from ethics committees in several countries has revealed trends in EC/IRB (Ethics Committees/Institutional Review Boards) questions and concerns about genetic research. This article will focus on the lessons learned from incorporating genetic research into clinical studies at over 1,500 international sites and will include summaries of the feedback from investigators, EC/IRBs, and regulatory authorities. It also will include a discussion of the potential applications of SNP (single nucleotide polymorphism) map technologies to pharmacogenetics by increasing the ability to correlate patients' genetic information with their response to medicines.

Drug Design↗
PubMed1989

Mapping initiation sites of DNA replication in vivo using polymerase chain reaction amplification of nascent strand segments.

We describe a sensitive method for mapping replication initiation sites near regions of sequenced genomic DNA in vivo. It is based on selective amplification of sets of segments in purified nascent DNA strands and subsequent determination of the lengths of these strands required to include each member of the set. We demonstrate the ability of this method to accurately map a well-defined origin, that of replicating SV40 DNA. Pulse-labeled DNA from infected CV-1 cells was size-fractionated on an alkaline sucrose gradient and newly-synthesized strands purified by immunoprecipitation using anti-BrdU antibodies. Three pairs of synthetic oligonucleotide primers were used to amplify three SV40 segments, using the polymerase chain reaction (PCR), at known distances from the origin. Lengths of the nascent DNA strands that allow amplification were determined by hybridization to probes homologous to the amplified segments and used to calculate position of the origin. Experiments with a mix of SV40 and human HeLa cell DNA demonstrate the applicability of the method to mapping origins present at the level of single-copy genomic sequences in mammalian cells.

Animals↗
PubMed1991

Efficient cloning of hepatitis B virus DNA from single-stranded replicative intermediates and its application to S1 mapping of viral RNA in human liver tissue.

An efficient method for cloning subgenomic fragments of the hepatitis B virus (HBV) was developed, utilizing its abundant single-stranded replicative intermediates. The total genomic DNA obtained from the liver tissue of patients with chronic HBV infection was treated by using the Klenow fragment of E. coli DNA polymerase I without adding any exogenous primers. Single-stranded replicative intermediates were efficiently converted to double-stranded linear DNA, one end of which terminated at (or near to) the direct repeat 1 (DR1) sequence of the HBV genome. By screening less than 1,000 recombinants from a DNA library after this treatment, we obtained a subgenomic HBV fragment of 2.0 kilobases. We then analysed HBV RNA in human liver tissue by S1 mapping. It was possible to map HBV RNA only when a DNA probe from the same tissue was used.

Cloning, Molecular↗
PubMed1995

Identification of a coat protein binding site on southern bean mosaic virus RNA.

The cowpea strain of Southern bean mosaic virus (SBMV-C), a T = 3 icosahedral RNA virus, was dissociated to yield a ribonucleoprotein complex (RNPC) composed of the viral RNA and coat protein subunits. To determine if the coat protein subunits were bound to a specific site on the viral RNA, the RNPC was treated with ribonuclease, and the remaining coat protein--RNA complexes were recovered by filter binding. A single species of RNA was isolated by this procedure and further characterized by sequencing. The RNA was mapped to nucleotides 1410-1436 of the SBMV-C genome. This region of the viral RNA was predicted to fold into a hairpin with a 4-base loop and a duplex stem of 24 nucleotides. The stability and specificity of the coat protein-RNA complex isolated from dissociated virus suggest a possible role for this interaction in the selective encapsidation of the viral RNA.

Amino Acid Sequence↗
PubMed1987

Isolation of the yeast gene encoding elongation factor 3 for protein synthesis.

The gene YEF-3 encoding the elongation factor 3 (EF-3) for peptide chain elongation in Saccharomyces cerevisiae has been isolated by immunoscreening of a yeast genomic library in the phage lambda gt11. The identity of the EF-3 gene was confirmed by several methods. First, a clone-encoded protein could affinity purify the antibody that specifically reacted with EF-3. Second, a recombinant fusion protein, which reacted with anti-beta-galactosidase antibody as well as with anti-EF-3 antibody, was found in the lysate of a positive clone lysogen. Third, the function of EF-3 in a yeast mutant in which the EF-3 activity is temperature-sensitive in an in vitro assay could be complemented by transformations. EF-3 protein was overproduced in a transformant which contained the EF-3 gene on a multicopy plasmid YEp-13. Southern blot analysis shows that YEF-3 is a single copy gene. The transcript unit as mapped by S1 nuclease mapping, is consistent with the size of the message determined by Northern blot analysis and shows no evidence of introns.

Cloning, Molecular↗
PubMed1989

Detection and mapping of spliced RNA from a human hepatoma cell line transfected with the hepatitis B virus genome.

HepG2 cells, known to support the replication and virion formation of hepatitis B virus (HBV), were transfected with a cosmid constructed to contain 12 tandem head-to-tail repeats of the HBV genome for effective HBV genome expression. We detected previously identified RNAs of 3.3, 2.3, and 2.0 kilobases (kb) that code for core antigen, large surface antigen, and middle/major surface antigen, respectively. We also detected four additional RNAs of 2.1, 1.7, 1.1, and 0.7 kb [the lengths exclude the poly(A) tail]. S1 mapping and nucleotide sequencing data showed that the 2.1-kb RNA is a spliced RNA whose 5' and 3' ends are identical to those of the 3.3-kb RNA. The results suggest that the 2.1-kb RNA codes for an altered core antigen lacking the last amino acid, cysteine, and that expression of the 3.3-kb pregenomic RNA is regulated, at least in part, by splicing. The map positions of the 1.7- and 1.1-kb RNAs suggest that they code for the carboxyl-terminal portions of the putative polymerase, whereas the 0.7-kb RNA codes for the X protein.

Antigens, Viral↗
PubMed1991

Fluorescence resonance energy transfer mapping of the fourth of six nucleotide-binding sites of chloroplast coupling factor 1.

Equilibrium dialysis measurements of adenine nucleotide binding to chloroplast coupling factor 1 suggest that the enzyme has six binding sites for ADP, adenylyl-beta,gamma-imidodiphosphate (AMP-PNP), and 2'(3')-O-2,4,6-trinitrophenyl-ATP (TNP-ATP). High affinity binding at all six sites requires the divalent cation, Mg2+. Three of the nucleotide-binding sites, sites 1, 2, and 3, have been mapped by fluorescence resonance energy transfer distance measurements (see McCarty, R. E., and Hammes, G. G. (1987) Trends Biochem. Sci. 12, 234-237). Using the same technique, we mapped the location of nucleotide-binding site 4, a tight, exchangeable site (Shapiro, A. B., Huber, A. H., and McCarty, R. E. (1991) J. Biol. Chem. 266, 4194-4200). Two arrangements of the energy transfer map of coupling factor 1 were found which are compatible with the available data. The two arrangements make different predictions about which sites interact in cooperative catalysis.

Adenosine Diphosphate↗
PubMed1987

Restriction map of a capsule plasmid of Bacillus anthracis.

The capsule plasmid pTE702 of Bacillus anthracis has been physically mapped with the restriction endonucleases HindIII, PstI, BamHI, SalI, and XhoI. A HindIII fragment map of pTE702 (96.5 kb) was obtained by analysis of the recombinant plasmids and cosmids containing overlapping fragments partially digested with HindIII. The physical map for PstI, BamHI, SalI, and XhoI was obtained by double digestion mapping of these sites in relation to the HindIII sites. The replication region of pTE702 was determined by in vitro genetic replicon labeling in B. subtilis.

Bacillus anthracis↗
PubMed1999

Single nucleotide polymorphism (SNP) discovery and linkage mapping of bovine cytokine genes.

Polymorphic markers at bovine gene loci facilitate the integration of cattle genetic maps with those of humans and mice. To this end, 31 single nucleotide polymorphism (SNP) markers were developed for seven bovine chemokine genes. Loci were amplified from bovine genomic DNA by the polymerase chain reaction, and candidate amplicons were sequenced to determine their identity. Amplified loci from 24 founding parents and select progeny from a beef cattle reference population were sequenced and analyzed for SNPs. SNP haplotype alleles were determined by examining segregation patterns and used to establish the locus position on the bovine linkage map. Loci for growth-related proteins (GRO3, GRO1, and GROX) were clustered with the related CXC chemokine genes, interleukin (IL) 8, and epithelial cell inflammatory protein 1, at 84 cM from the centromeric end of the bovine chromosome (BTA) 6 linkage group. Bovine loci for a cluster of IL8 receptors, a stromal cell-derived factor 1, interferon-gamma, and tumor necrosis factor-alpha were mapped at 90, 55, 59, and 34 cM, respectively, from the centromeric ends of the BTA 2, 28, 5, and 23 linkage groups. The positions of these bovine loci were compared with those of orthologous loci on the human map to refine the boundaries of conserved synteny. These seven loci provide examples of SNP development in which the efficiency was largely dependent on the availability of bovine genomic or cDNA sequence. The polymorphic nature of these SNP haplotype markers suggests that they will be useful for mapping complex traits in cattle, such as resistance to infectious disease.

Animals↗
PubMed1988

Difference between two major size classes of carrot rDNA repeating units is due to reiteration of sequences of about 460 bp in the large spacer.

Based on the previous observation that the different regions between two major size classes of carrot rRNA genes are located in their large spacer, detailed physical maps of the spacer region were constructed by cloning, followed by restriction analysis. As a result, the different regions were restricted to BamHI segments of 1.3 kb and 1.8 kb. Sequence analysis of these segments revealed that the shorter one carried one truncated and two complete copies of about 460 bp of repetitious sequences, while the longer one contained one truncated and three full copies of the repetitious sequences. S1 nuclease mapping data suggest that either transcription initiation sites or processing sites of precursor rRNA are located in the repetitious sequence closest to the 18 S rRNA coding region.

Base Sequence↗