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PubMed1987

Protein-binding sites within the 5' DNase I-hypersensitive region of the chicken alpha D-globin gene.

We mapped at high resolution and as a function of development the hypersensitive domain in the 5'-flanking region of the chicken alpha D-globin gene and determined the specific protein-binding sites within the domain. The domain extends from -130 to +80 nucleotides (nt) relative to the cap site. DNase I footprinting within intact embryonic erythrocyte nuclei revealed a strongly protected area from -71 to -52 nt. The same area was weakly protected in adult nuclei. A factor was present in extracts of erythrocyte nuclei from both embryos and adults that protected the sequence AAGATAAGG (-63 to -55 nt) in DNase I footprinting experiments; at higher concentrations of extract, sequences immediately adjacent (-73 to -64 and -53 to -38) were also protected. The same pattern of binding was revealed by gel mobility shift assays. The identical AAGATAAGG sequence is found in the 5'-flanking region of the beta rho gene; it competed for binding of the alpha D-specific factor, suggesting that regulatory elements are shared.

Animals↗
PubMed2006

Mapping phenotypic plasticity and genotype-environment interactions affecting life-history traits in Caenorhabditis elegans.

Phenotypic plasticity and genotype-environment interactions (GEI) play an important role in the evolution of life histories. Knowledge of the molecular genetic basis of plasticity and GEI provides insight into the underlying mechanisms of life-history changes in different environments. We used a genomewide single-nucleotide polymorphism map in a recombinant N2 x CB4856 inbred panel of the nematode Caenorhabditis elegans to study the genetic control of phenotypic plasticity to temperature in four fitness-related traits, that is, age at maturity, fertility, egg size and growth rate. We mapped quantitative trait loci (QTL) for the respective traits at 12 and 24 degrees C, as well as their plasticities. We found genetic variation and GEI for age at maturity, fertility, egg size and growth rate. GEI in fertility and egg size was attributed to changes in rank order of reaction norms. In case of age at maturity and growth rate, GEI was caused mainly by differences in the among-line variance. In total, 11 QTLs were detected, five QTL at 12 degrees C and six QTL at 24 degrees C, which were associated with life-history traits. Five QTL associated with age at maturity, fertility and growth rate showed QTL x environment interaction. These colocalized with plasticity QTL for the respective traits suggesting allelic sensitivity to temperature. Further fine mapping, complementation analyses and gene silencing are planned to identify candidate genes underlying phenotypic plasticity for age at maturity, fertility and growth.

Animals↗
PubMed2000

Mapping oxidative DNA damage using ligation-mediated polymerase chain reaction technology.

Reactive oxygen species induce a pharmacopoeia of oxidized bases in DNA. DNA can be cleaved at most of the sites of these modified bases by digestion with a combination of two base excision repair glycosylases from Escherichia coli, Fpg glycosylase, and endonuclease III. The frequency of the resulting glycosylase-dependent 5'-phosphoryl ends can be mapped at nucleotide resolution along a sequencing gel autoradiogram by a genomic sequencing technique, ligation-mediated polymerase chain reaction (LMPCR). In cultured rat cells, the frequency of endogenous oxidized bases in mitochondrial DNA is sufficiently high, about one oxidized base per 100 kb, to be directly mapped from 0.1 microg of total cellular DNA preparations by LMPCR. Nuclear DNA has a lower frequency of endogenous oxidative base damage which cannot be mapped from 1-microg preparations of total cellular DNA. Preparative gel electrophoresis of the PGK1 and p53 genes from 300 microg of restriction endonuclease-digested genomic DNA showed a 25-fold enrichment for the genes and, after endonuclease digestion followed by LMPCR, gave sufficient signal to map the frequency of oxidized bases from human cells treated with 50 microM H2O2.

Animals↗
PubMed1990

Specific neuronal expression of human NGF receptors in the basal forebrain and cerebellum of transgenic mice.

Transgenic mice carrying multiple copies of the human NGF receptor gene have been generated. Using a monoclonal antibody specific for the human receptor, we have detected specific expression in cholinergic neurons in the basal forebrain and Purkinje cells in the cerebellum during the postnatal period. Expression in the PNS was exemplified by immunostaining of sympathetic and sensory neurons during an early embryonic age. Transection of the sciatic nerve in transgenic animals resulted in induction of human NGF receptors, indicating that the inserted gene can be appropriately regulated. These transgenic mice will provide an opportunity to study the elements regulating the NGF receptor. Furthermore, the ability to obtain specific expression in transgenic mice will permit directed expression of heterologous genes in discrete cells important in the cholinergic septal-hippocampal pathway and the PNS.

Animals↗
PubMed1987

Isolation of a human genomic fragment, co-amplified with c-Ki-ras, that affects plasmid supercoiling in E. coli.

Amplification of cellular proto-oncogenes has been implicated in the development of human malignancies. A library was constructed from genomic DNA extracted from a lung tumour, previously shown to carry an amplified c-Ki-ras 2 gene. Using a v-Ki-ras probe, a fragment with ras homology was isolated and shown to be amplified in the original tumour DNA to the same level as c-Ki-ras. Studies with human hamster hybrids demonstrated that it is normally located on human chromosome 12 (as is c-Ki-ras). The restriction map of the fragment is different from that of the known Ha, Ki or N-ras genes and its sequence shows evolutionary conservation, as demonstrated by hybridisation to the genomic DNA of several mammalian species. A pUC19 subclone (pK42), carrying a 1.3kb insert, shows supercoil heterogeneity in plasmid preparations, as does a second compatible plasmid introduced into the same bacterial host with pK42. It appears therefore that the subclone is encoding a product that affects DNA topoisomerase activity in E. coli.

Animals↗
PubMed1996

Biochemical and molecular characterization of the extracellular esterase from Streptomyces diastatochromogenes.

An esterase of Streptomyces diastatochromogenes was purified to homogeneity from culture filtrate. The purified enzyme had a molecular mass of 30,862 +/- 5.8 Da, as determined by electrospray mass spectrometry. The esterase-encoding gene was cloned on a 5.1-kb MboI fragment from S. diastatochromogenes genomic DNA into Streptomyces lividans TK23 by using plasmid vector pIJ702. Nucleotide sequence analysis predicted a 978-bp open reading frame, estA, encoding a protein of 326 amino acids, a potential ribosome binding site, and a putative 35- or 36-residue signal peptide for secretion in S. lividans or S. diastatochromogenes, respectively. The transcriptional initiation site was mapped 29 nucleotides upstream from the predicted translational start codon of estA in S. diastatochromogenes. The protein sequence deduced from the estA gene was similar to that of the esterase from the plant pathogen Streptomyces scabies. Both enzymes lacked the conserved motif GXSXG carrying the active-site serine of hydrolytic enzymes. A serine modified by [1,3-3H]diisopropyl fluorophosphate was located at position 11 of the mature enzyme in the sequence GDSYT. This finding and results obtained by site-directed mutagenesis studies indicate that serine 11 may be the active-site nucleophile.

Amino Acid Sequence↗
PubMed1988

Structure, chromosome location, and expression of the human gamma-actin gene: differential evolution, location, and expression of the cytoskeletal beta- and gamma-actin genes.

The accumulation of the cytoskeletal beta- and gamma-actin mRNAs was determined in a variety of mouse tissues and organs. The beta-isoform is always expressed in excess of the gamma-isoform. However, the molar ratio of beta- to gamma-actin mRNA varies from 1.7 in kidney and testis to 12 in sarcomeric muscle to 114 in liver. We conclude that, whereas the cytoskeletal beta- and gamma-actins are truly coexpressed, their mRNA levels are subject to differential regulation between different cell types. The human gamma-actin gene has been cloned and sequenced, and its chromosome location has been determined. The gene is located on human chromosome 17, unlike beta-actin which is on chromosome 7. Thus, if these genes are also unlinked in the mouse, the coexpression of the beta- and gamma-actin genes in rodent tissues cannot be determined by gene linkage. Comparison of the human beta- and gamma-actin genes reveals that noncoding sequences in the 5'-flanking region and in intron III have been conserved since the duplication that gave rise to these two genes. In contrast, there are sequences in intron III and the 3'-untranslated region which are not present in the beta-actin gene but are conserved between the human gamma-actin and the Xenopus borealis type 1 actin genes. Such conserved noncoding sequences may contribute to the coexpression of beta- and gamma-actin or to the unique regulation and function of the gamma-actin gene. Finally, we demonstrate that the human gamma-actin gene is expressed after introduction into mouse L cells and C2 myoblasts and that, upon fusion of C2 cells to form myotubes, the human gamma-actin gene is appropriately regulated.

Actins↗
PubMed1987

Genetic characterization of an Escherichia coli mutant deficient in organophosphonate biodegradation.

An E. coli mutant deficient in organophosphonate biodegradation has been complemented with a cosmid library prepared from a BamHI partial digest of wild-type E. coli W3110. Mutant E. coli SL724, when transformed with cosmid pSL163 and plasmid pSL263, regained the ability to exploit ethylphosphonate as a sole source of phosphorus during growth. In route to complementation, the Tn5 insert of SL724 was subcloned and restriction enzyme mapped. Complementing pSL163 and pSL263 were also characterized via restriction enzyme digests.

Cosmids↗
PubMed1988

Molecular cloning and restriction enzyme mapping of an African swine fever virus isolate from Malawi.

DNA prepared from a field isolate of African swine fever virus, which causes high mortality and severe disease in domestic pigs, was cloned in bacteriophage lambda and plasmid vectors. Clones containing DNA inserts overlapping with each other and together covering the complete genome, apart from short fragments close to the cross-linked termini of the genome, were obtained. A complete restriction enzyme site map of the genome for three enzymes was deduced.

African Swine Fever↗
PubMed1988

Polymorphisms in the umuDC region of Escherichia species.

The umuDC operon of Escherichia coli encodes mutagenic DNA repair. The umuDC regions of multiple isolates of E. coli, E. alkalescens, and E. dispar and a single stock of E. aurescens were mapped by nucleotide hybridization. umuDC is located at one end of a conserved tract of restriction endonuclease sites either 12.5 or 14 kilobase pairs long. Rearrangements, including possible deletions, were seen in the polymorphic DNA flanking the conserved tract. Restriction site polymorphisms were not found around the DNA repair gene recA or polA. The junctions of the conserved region contain direct repeats of nucleotide sequences resembling the termini of the Tn3 group of transposons. Possible mechanisms for the generation of these variants are discussed.

Base Sequence↗
PubMedDate not supplied

[Structural-functional organization of the SV40 virus chromosome. II. Characteristics of viral genome containing deletion in the regulatory region].

Structure of SV40 wild type virus genome differing from the well known 776 strain has been characterized. This particular strain (SV40) was used previously for viral chromatin analysis. We show here that SV40 strain differs from 776 strain by deletion in the beginning of the "late" region (enhancer). Restriction nucleases mapping and nucleotide sequencing through this region reveal the absence of one full-length copy of 72 bp repeat.

Base Sequence↗
PubMed1983

Vaccinia virus thymidine kinase and neighboring genes: mRNAs and polypeptides of wild-type virus and putative nonsense mutants.

Enzymatically active thymidine kinase (TK) was made in reticulocyte lysates programmed with early vaccinia mRNA that hybridized to plasmid recombinants containing either of two adjacent small DNA subsegments of the viral HindIII-J fragment. The map position of an early polypeptide, with a molecular weight of 19,000 (19K), coincided precisely with that of the TK. The absence of the 19K polypeptide in cell-free translation products of hybridization-selected mRNAs from several TK-negative mutants provided an independent identification of the TK polypeptide. The small size of the TK polypeptide of vaccinia virus distinguishes it from that of procaryotes, eucaryotes, and herpesvirus. Five early mRNAs of 3,840, 2,390, 1,790, 1,070, and 590 nucleotides were mapped within the HindIII-J fragment by RNA blotting and nuclease S1 digestion of RNA-DNA hybrids. The RNAs of 590 and 2,380 nucleotides were found to have 5' coterminal ends and represent major and minor forms, respectively, of the TK message. The 3' end of the minor TK mRNA appeared to be coterminal with the 3' end of the 1,790-nucleotide transcript which encodes a 41K polypeptide. The 1,070-nucleotide RNA was identified as the message for a 21K polypeptide. All of these RNAs, including the two forms of the TK message, were made by the putative TK-negative nonsense mutants.

DNA Restriction Enzymes↗
PubMed1986

The gene for Spirodela oligorhiza chloroplast ribosomal protein homologous to E. coli ribosomal protein L16 is split by a large intron near its 5' end: structure and expression.

The nucleotide sequence of a Spirodela chloroplast DNA fragment, which directs the synthesis of a approximately 15 kD chloroplast ribosomal protein in an E. coli cell free system, has been determined. The deduced aminoacid sequence of the open reading frame shows extensive homology with E. coli ribosomal protein L16. Primer extension analysis, S1 nuclease mapping and nucleotide sequence analysis indicate that the chloroplast L16 gene (rpl16) is interrupted by a 1411 bp intron, which separates a short 5' exon from a large 3' exon. The shorter in vitro synthesized ribosomal protein results from an artificial initiation event at an internal ATG codon in the 3' exon. The sequences at the 5' and 3' splice sites of the intron are similar to consensus sequences described for other, class II intron containing, protein coding chloroplast genes. Northern hybridization experiments reveal 6 stable transcripts of rpl16 ranging from 500 b to greater than 4000 b. As determined by S1 nuclease mapping, the 3'-end of the smallest transcript maps exactly after the stem of a proposed termination signal. Finally, the implications of the finding of a cluster of several chloroplast ribosomal protein genes and possible polycistronic transcription of this chloroplast DNA region, are discussed in relation to the organization and expression of ribosomal protein genes found in the S10 operon of E. coli.

Amino Acid Sequence↗
PubMed1983

Transduction of c-myb into avian myeloblastosis virus: locating points of recombination within the cellular gene.

The oncogene (v-myb) of avian myeloblastosis virus apparently arose by transduction of nucleotide sequences from a cellular gene (c-myb). In c-myb the nucleotide sequences that formed v-myb exist at seven distinct regions separated by nontransduced stretches of sequence that are flanked by eucaryotic splice signals. By contrast, the sequences at the outside boundaries of the transduced region of c-myb do not resemble splice sites. We mapped the nucleotide sequences that are homologous to the ends of v-myb with respect to the exons and introns of c-myb. The results indicate that the leftward recombination between c-myb and the transducing retrovirus occurred within an intron of the cellular gene, whereas the rightward recombination took place in an exon of c-myb. Transduction of c-myb sequences may therefore have involved a DNA rearrangement.

Animals↗
PubMed2002

Molecular cytogenetic analysis of the breakpoint region at 6q21-22 in T-cell lymphoma/leukemia cell lines.

Chromosome band 6q21 is reported to be one of the most frequent target regions in T-cell lymphoma for both translocations and deletions. To explore whether the breakpoint clustering in T-cell malignancy indicates the presence of a common breakpoint region in 6q, we employed fluorescence in situ hybridization analysis using various YAC, BAC, and PAC clones aligned at 6q21-22. We identified two T-cell lymphoma/leukemia cell lines with different differentiation stages that had breakpoints within the same novel gene, TCBA1 (T-cell lymphoma breakpoint associated target 1). In a T-cell lymphoblastic lymphoma cell line, HT-1, the TCBA1 fused to SUSP1 (SUMO-1-specific protease), creating a SUSP1-TCBA1 chimeric gene. However, in an adult T-cell leukemia cell line, ATN-1, no chimeric gene was detected, although aberrant TCBA1 transcripts were produced. We conclude that TCBA1 is a possible target gene for T-cell lineage-specific chromosome aberrations at 6q21.

Amino Acid Sequence↗
PubMed1999

Structural organization and expression of the mouse gene for Pur-1, a highly conserved homolog of the human MAZ gene.

We have characterized the genomic structure and expression of the mouse gene for Pur-1. The cloned Pur-1 gene spans a 5-kb region encompassing the promoter, five exons, four introns and the 3'-untranslated region. All exon-intron junction sequences conform to the GT/AG rule. The promoter region has typical features of a housekeeping gene: a high G + C content (77.5%); a high frequency of CpG dinucleotides, in particular within the region 0.5 kb upstream of the site of initiation of translation; and the absence of canonical TATA and CAAT boxes. S1 nuclease protection assay demonstrated the presence of multiple sites for initiation of transcription around a site 108 nucleotides upstream of the ATG codon. Comparison of Pur-1 with the human gene for MAZ (Myc-associated zinc finger protein) revealed a striking homology of both their nucleotide and deduced protein sequences, an identical genomic organization and high similarity in promoter architecture and mRNA expression pattern. Sequence analysis of the 5'-flanking region of Pur-1 revealed numerous potential binding sites for transcription factors Sp1, AP-2 and Pur-1/MAZ itself. An element required for basal Pur-1 expression was mapped from nucleotide -258 to +43. This region also mediated stimulation of basal transcription by ectopically expressed MAZ protein. We conclude that the Pur-1 gene is the murine homolog of human MAZ and, like it, belongs to the family of housekeeping genes.

Animals↗
PubMed1987

Phylogenetic implication of heterogeneity of the nontranscribed spacer of rDNA repeating unit in various Neurospora and related fungal species.

Nontranscribed spacer (NTS) regions of ribosomal (r)RNA genes are non-conserved and are shown to be useful for phylogenetic studies. 32P-labelled N. crassa NTS pCC3400 DNA, was used as a molecular probe to hybridize Southern blots of genomic DNAs obtained from Neurospora, Gelacinospora, Sordaria, bacteria, plants, and animals. Our studies conclude that: (a) the homotahllic species of Neurospora should not belong to genus Gelacinospora (a historical question) and that Neurospora homothallic species are closer to Gelacinospora than to Sordaria; and that (b) all of the filamentous fungal species tested are indeed closer to the higher plant genome than to higher primate animal genome based on shared restriction sites of 12 enzymes. Our studies also demonstrate the usefulness of nontranscribed rRNA gene probes in resolving questions regarding phylogenetic relatedness between widely separated organisms using the parsimony principle based on mutation sites from DNA restriction maps; it has not been possible to do this using DNA: DNA hybridization procedures that involved the total genome.

DNA Restriction Enzymes↗
PubMed1989

Transcriptional analysis of the repressor gene of the temperate Streptomyces phage phi C31.

A 397-bp fragment that contained the 5' end of the coding region of the repressor gene of the temperature Streptomyces phage phi C31 was shown by in vivo promoter-probing to possess bidirectional promoter activity. In vitro runoff transcription experiments, and high resolution transcript mapping of mRNA species produced in vivo using both nuclease S1 and mung bean nuclease, indicated the probable presence of two promoters for the repressor gene with two further promoters oriented in the opposite direction. An inverted repeat sequence is situated 20 bp downstream from the translational stop codon of the repressor gene; high resolution transcript mapping revealed an mRNA endpoint close to this sequence, indicating its likely role as a transcriptional terminator.

Bacteriophages↗