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PubMed1989

Isolation and characterization of the promoter and flanking regions of the gene encoding the human protein-synthesis-initiation factor 2 alpha.

The promoter region of the gene (eIF-2 alpha) for eukaryotic initiation factor 2 alpha (eIF-2 alpha) was isolated from a human genomic library and its structure was determined by restriction mapping and nucleotide (nt) sequence analysis. The promoter region and twelve in vivo transcriptional start points (tsp) have been identified by endonuclease S1 mapping and their location confirmed by primer-extension analysis, using RNA isolated from human cells. The untranslated leader is 102 to 140 nt long depending upon the tsp, and the 5' region of the mRNA has the potential for forming stable stem-loop structures. The nt sequence of the regions upstream and downstream from the tsp contains neither a 'TATA box' nor a 'CAAT box', but does contain several direct and inverted repeats, as well as palindromic sequences near the tsp. In addition, multiple consensus binding sites for a wide variety of regulatory proteins are present throughout upstream and downstream tsp-flanking regions.

Base Sequence↗
PubMed1993

PCAP: probe choice and analysis package--a set of programs to aid in choosing synthetic oligomers for contig mapping.

In the program, PCAP, we provide a methodology for choosing synthetic oligonucleotide probes to be used in contig mapping experiments. The package serves the purpose of presenting a series of short oligonucleotides (8-12mers) that are chosen based on constraints with respect to frequency of occurrence within a particular genome and the G+C content of the oligonucleotides. The four programs contained within the package: (i) convert GenBank files to a format useable by the package; (ii) calculate trinucleotide and tetranucleotide frequencies in available sequence data on a particular species; (iii) present the user with upper and lower bounds on the frequencies of hybridization sites for oligonucleotide probes of length 8-12, (iv) allow the user to place constraints on site frequency and G+C content and provides a list of short probe sequences that fit these criteria. These sequences can then be synthetically produced and used in hybridization experiments to carry out contig mapping.

Base Sequence↗
PubMed1987

Isolation and characterization of Streptococcus cremoris Wg2-specific promoters.

By cloning MboI fragments in the promoter selection vector pGKV210, which replicates in Streptococcus lactis, Bacillus subtilis, and Escherichia coli and carries a promoterless chloramphenicol acetyltransferase gene, we obtained a number of fragments endowed with promoter activity, partly by direct selection for chloramphenicol resistance in S. lactis IL1403 and partly by selection in B. subtilis. Five fragments were sequenced, and the promoters were mapped with S1 nuclease. The promoters agreed with the E. coli promoter consensus and the B. subtilis vegetative sigma 43 promoter consensus. The promoters were preceded by an A + T-rich region (ranging from 64 to 78% A + T). S1 nuclease mapping data showed that the transcriptional start point in three of the fragments was at a TAG sequence 5 to 9 nucleotides downstream from the promoter. Three fragments carried an open reading frame preceded by a ribosome-binding site which can be recognized by E. coli, B. subtilis, and S. lactis ribosomes.

Bacillus subtilis↗
PubMed1998

Mapping oxidative DNA damage at nucleotide level.

DNA damage induced by reactive oxygen species (ROS) is considered an important intermediate in the pathogenesis of human conditions such as cancer and aging. By developing an oxidative-induced DNA damage mapping version of the Ligation-mediated polymerase chain reaction (LMPCR) technique, we investigated the il vivo and in vitro frequencies of DNA base modifications caused by ROS in the human p53 and PGK1 gene. Intact human male fibroblasts were exposed to 50mM H2O2, or purified genomic DNA was treated with 5 mM H2O2, 100 microM Ascorbate, and 50 microM, 100 microM, or 100 microM of Cu(II), Fe(II), or Cr(VI) respectively. The damage pattern generated in vivo was nearly identical to the in vitro Cu(II) or Fe(III) damage patterns; damage was non-random with guanine bases heavily damaged. Cr(VI) generated an in vitro damage pattern similar to the other metal ions, although several unique thymine positions were damaged. Also, extra nuclear sites are a major contributor of metal ions (or metal-like ligands). These data show that the local probability of H2O2-mediated DNA damage is determined by the primary DNA sequence, with chromatin structure having a limited effect. The data suggest a model in which DNA-metal ion binding domains can accommodate different metalions. LMPCR's unique aspect is a blunt-end ligation of an asymmetric double-stranded linker, permitting exponential PCR amplification. An important factor limiting the sensitivity of LMPCR is the representation of target gene DNA relative to non-targeted genes; therefore, we recently developed a method to eliminate excess non-targeted genomic DNA. Restriction enzyme-digested genomic DNA is size fractionated by Continuous Elution Electrophoresis (CEE), capturing the target sequence of interest. The amount of target DNA in the starting material for LMPCR is enriched, resulting in a stronger amplification signal. CEE provided a 24-fold increase in the signal strength attributable to strand breaks plus modified bases created by ROS in the human p53 and PGK1 genes, detected by LMPCR. We are currently taking advantage of the enhanced sensitivity of target gene-enriched LMPCR to map DNA damage induced in human breast epithelial cells exposed to non-cytotoxic concentrations of H2O2.

Ascorbic Acid↗
PubMed2003

Hypervariability of the membrane-associated mucin and cancer marker MUC1.

The highly heterogeneous epithelial mucins show considerable inter-individual variability attributable to allelic variations in their tandem repeat (TR) peptide domains. Most mucins are known to show variations in repeat number but variation in the sequence of the individual TRs is not as well characterised. Here, we have studied variation in the immunodominant PDTR motif in the TR domain of the membrane-associated "cancer" mucin MUC1 by using the Minisatellite Variant Repeat-Polymerase chain reaction (MVR-PCR) technique. We have fully or partially mapped two nucleotide changes that encode two amino-acid changes, PDTR to PESR, across the arrays of 149 alleles. A total of 103 different maps was obtained when these changes alone were considered and additional variations were also observed. Most maps showed blocks of PDTR repeats interspersed with PESR repeats, although these were possibly more irregular in the longer alleles that also tended to have more PESR repeats. This variability has potential functional consequences and possible implications for some individuals with respect to the efficacy of immune targetting and immune therapy.

Amino Acid Sequence↗
PubMed1990

Physical mapping of a low-copy DNA sequence in rye (Secale cereale L.).

A 900-base-pair (bp) sequence from a cDNA clone of the rye endosperm-storage-protein gene Sec-1 was labeled with biotin and hybridized to Secale cereale 'Blanco' chromosomes. Hybridization was seen on the satellite part of the short arm of chromosome 1R, where the Sec-1 gene has been genetically mapped, in approximately 8.5% of the cells analyzed. The clone cross-hybridized at a lower frequency to rye chromosome arms 1R (long) (4.4%) and 2R (short) (2.6%), where two additional rye storage-protein loci, Sec-3 and Sec-2, respectively, have been mapped. A fourth hybridization site was observed on the short arm of chromosome 6R at a frequency of 3.0%. The cross-hybridization is attributed to a combination of residual sequence homology between the protein loci and the low-stringency conditions used in in situ hybridization. In situ hybridization mapping, in combination with chromosome walking by using molecular techniques, is suggested as an excellent approach to physical mapping of chromosomes.

Base Sequence↗
PubMed1992

Interaction of murine ets-1 with GGA-binding sites establishes the ETS domain as a new DNA-binding motif.

The proto-oncogene ets-1 is the founding member of a new family of eukaryotic transcriptional regulators. Using deletion mutants of murine ets-1 cDNA expressed in Escherichia coli, we show that the DNA-binding domain corresponds closely to the ETS domain, an 85-amino-acid region that is conserved among ets family members. To investigate the specificity of DNA binding of the ETS domain, we mapped the DNA contacts of a monomeric Ets-1 fragment by chemical protection and interference assays. DNA backbone interactions span a 20-nucleotide region and are localized on one face of the helix. Close phosphate and base contacts are restricted to 10 central nucleotides. Contacts map to the major groove in the center of the site. Flanking minor groove interactions also are predicted. To determine the sequence preference in the close contact zone, we selected a pool of high-affinity binding sites using a purified Ets-1 carboxy-terminal fragment. Our Ets-1-selected consensus, 5'-A/GCCGGAA/TGT/C-3', differs from the binding consensus for the Drosophila ETS domain protein E74A, suggesting that specificity of action of ets family members is mediated by the ETS domain. Compared to other well-characterized classes of DNA-binding proteins, Ets-1 produces a unique pattern of DNA contacts. These studies demonstrate that the ETS domain proteins bind DNA in a novel manner.

Animals↗
PubMed1991

Gene encoding two alkali-soluble components of the spore coat from Bacillus subtilis.

We report the cloning and characterization of a gene called cotF from Bacillus subtilis that encodes alkali-soluble polypeptides of 5 and 8 kDa that are components of the spore coat. The 5- and 8-kDa polypeptides are generated by proteolytic cleavage of the primary product of the cotF gene, which is 160 codons in length and is capable of encoding a polypeptide of 19 kDa. Amino acid sequence analysis indicates that the 5-kDa species is derived from the NH2-terminal portion of the primary gene product and that the 8-kDa species is derived from the COOH-terminal portion. A mutant bearing an in vitro-constructed cotF null mutation produced normal-looking spores that contained an apparently complete set of coat proteins except for the absence of the 5- and 8-kDa polypeptides. The map position of cotF is 349 degrees. Transcription of cotF commenced coincidently (during h 6 of sporulation) with genes known to be under the control of sporulation transcription factor sigma kappa.

Amino Acid Sequence↗
PubMed1985

Characterization of a new simian virus 40 mutant, tsA3900, isolated from deletion mutant tsA1499.

The simian virus 40 (SV40) mutant tsA1499 contains an 81-base-pair deletion in the region of A gene encoding the C-terminal portion of the large T antigen. This mutant is particularly interesting, since it is a temperature-sensitive mutant that is apparently able to separate the lytic growth and transforming functions of the SV40 large T antigen at 38.5 degrees C. We report the isolation of a tsA1499 revertant (tsA1499-Rev) which is no longer temperature sensitive for lytic growth but still contains the 81-base-pair deletion of tsA1499. Marker rescue experiments with tsA1499-Rev or wild-type strain 830 (wt830) DNAs revealed that the original tsA1499 mutant contained a second mutation within the HindIII-Fnu4HI restriction fragment between 0.425 and 0.484 map units. Sequencing of this DNA fragment from the tsA1499, tsA1499-Rev, and wt830 viruses revealed that tsA1499 contained a single-base transversion (C to G) at 0.455 map units (nucleotide 4261). This transversion resulted in the creation of a new RsaI cleavage site in the tsA1499 DNA and predicts an arginine-to-threonine substitution at amino acid position 186 in the mutant large T antigen. The DNA sequence of the tsA1499-Rev HindIII-Fnu4HI fragment was identical to that of wt830. To determine whether tsA1499 was temperature sensitive for lytic growth solely as a result of the newly discovered point mutation or because of a combination of the point and deletion mutations, a series of viruses were constructed which contained the point mutation, the deletion mutation, both mutations, or neither. This was done by ligating the PstI A and B DNA fragments from either tsA1499 or wt830 and transfecting the ligated DNA into BSC-1H monkey kidney cells. This experiment revealed that all viruses containing the point mutation (the tsA1499 PstI A DNA fragment) were temperature sensitive for lytic growth, regardless of the presence of the 81-base-pair deletion (the tsA1499 PstI B DNA fragment). This newly discovered point mutation, at nucleotide 4261, is therefore unique, since to our knowledge it is the first tsA mutation to be described in the 0.455-map-unit region of the SV40 genome. We then tested the effect of this unique mutation on the ability of the SV40 virus to transform cultured rat cells to anchorage independence.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.

Aging↗
PubMed1998

Mapping of complex traits by single-nucleotide polymorphisms.

Molecular geneticists are developing the third-generation human genome map with single-nucleotide polymorphisms (SNPs), which can be assayed via chip-based microarrays. One use of these SNP markers is the ability to locate loci that may be responsible for complex traits, via linkage/linkage-disequilibrium analysis. In this communication, we describe a semiparametric method for combined linkage/linkage-disequilibrium analysis using SNP markers. Asymptotic results are obtained for the estimated parameters, and the finite-sample properties are evaluated via a simulation study. We also applied this technique to a simulated genome-scan experiment for mapping a complex trait with two major genes. This experiment shows that separate linkage and linkage-disequilibrium analyses correctly detected the signals of both major genes; but the rates of false-positive signals seem high. When linkage and linkage-disequilibrium signals were combined, the analysis yielded much stronger and clearer signals for the presence of two major genes than did two separate analyses.

Biosensing Techniques↗
PubMed1988

Mapping the genome.

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Base Composition↗
PubMed1990

cis-acting sequences involved in protein binding and in vitro transcription of the human alcohol dehydrogenase gene ADH2.

The human alcohol dehydrogenase gene ADH2 is expressed at high levels in the liver. At least five different complexes between nuclear proteins from mouse liver or rat hepatoma cells and the proximal promoter region extending from nucleotides -188 to +31 are detected by gel retardation assays. Using oligonucleotides to compete for the binding, and also as the probes, allowed localization of sequences within this region that bind nuclear proteins. Mutated oligonucleotides did not bind or compete. Nucleotides which are contacted by the proteins have been localized by methylation interference assays to two regions homologous to the related mouse Adh-1 gene. One maps between nucleotides -94 and -84; the other is from nucleotides -72 through -64. The 5' region of the human ADH2 gene is capable of directing accurate in vitro transcription in extracts from hepatoma cells. Deletion analysis indicates that the smallest portion of the proximal promoter region capable of directing significant transcription extends to nucleotide -93, which just contains both of the identified contact regions. The longer promoter fragments do not work as well, suggesting the presence of negative elements. In vitro transcription assays using oligonucleotides and mutated oligonucleotides as competitors demonstrate that both of the cis-acting sequences identified here are important to efficient initiation of transcription from the ADH2 promoter.

Alcohol Dehydrogenase↗