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PubMed1989

Mapping of heteroplasmic mitochondrial DNA deletions in Kearns-Sayre syndrome.

Kearns-Sayre syndrome (KSS) is a progressive neuromuscular disease characterised by ophtalmoplegia, cardiac bloc branch, pigmentary retinopathy associated with abnormal mitochondrial function. We have studied the mitochondrial DNA organization of patients presenting KSS and have found large deletions ranging from 3 to 8.5 kilobase pairs. DNA molecules containing deletion are accompanied by the presence of the normal sized mtDNA molecule forming heteroplasmic genomes. The deletions always map in the region which is potentially single stranded during mitochondrial DNA replication. The deletions differ in length and position between individuals but are similar within the different tissues of an individual suggesting that they arise during or before embryogenesis.

Biopsy↗

PubMed1989

Functional mapping of the activity of the R region in the human T-cell leukemia virus type I long terminal repeat to increase gene expression.

We previously demonstrated the activity of the R fragment in the long terminal repeat of human T-cell leukemia virus type I for elevation of the level of gene expression. In this study, the fragment was deleted with BAL31 nuclease to determine its functional domain. Series of the shortened R fragments were linked to the simian virus 40 promoter unit, which regulated expression of a reporter gene. Examination with the R fragments deleted from the 5' and 3' ends showed that borders of the functional domain were mapped within nucleotide positions 458 to 473 for the 5' end and nucleotide positions 559 to 594 for the 3' end, respectively. Thus we conclude that a 136-base-pair fragment corresponding to the second half of the R region was sufficient to allow elevation of the level of gene expression.

Base Sequence↗

PubMed1988

Evidence for a role of topoisomerase II in the Ca2+-dependent basal level expression of the rat prolactin gene.

The PRL gene is expressed at a high basal level in rat pituitary tumor GH3 cells, and this basal level enhancement of PRL gene expression is maintained through a Ca2+-calmodulin-dependent mechanism. We have now examined whether the enzyme, DNA topoisomerase II, which has been shown to be phosphorylated by a Ca2+-calmodulin-dependent protein kinase, plays a role in the Ca2+-calmodulin-dependent basal level enhancement of PRL gene expression. The topoisomerase II inhibitor, novobiocin, at concentrations in the range of 35-140 microM, effectively blocked the ability of Ca2+ to increase PRL mRNA levels. Examination of the effects of novobiocin on the levels of protein synthesis, glucose-regulated protein (GRP) 78 mRNA, histone 3 mRNA, and 18S ribosomal RNA indicated that the drug selectivity inhibited PRL gene expression. Two other topoisomerase II inhibitors, m-AMSA and VM26, also diminished the Ca2+-induced levels of PRL mRNA at concentrations (100-400 nM) that did not lower total mRNA levels. We then examined whether topoisomerase II interacted nonrandomly with DNA from the 5' transcribed and 5'-flanking region of the rat PRL gene by in vitro mapping of topoisomerase II DNA cleavage sites. In initial assays with a 10.5 kilobase (kb) PRL genomic DNA fragment containing 3.5 kb of 5'-transcribed DNA and 7 kb of 5'-flanking DNA, we detected 4 major cleavage sites in the following regions: site 1, +1500 to +1600; site 2, +1 to -100; site 3, -1200 to -1300; and site 4, -2900 to -3000.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

In vivo and in vitro stimulation by antitumor drugs of the topoisomerase II-induced cleavage sites in c-myc proto-oncogene.

Stimulation of topoisomerase II cleavage activity by antitumor drugs with or without DNA intercalative ability has been tested in vivo on the c-myc proto-oncogene. Two human tumor cell lines (N417 and HL60 cells) were treated with mAMSA, OH-9-ellipticine, VM26, and BD-40 (an aza-ellipticine analog), and DNA breaks were mapped in the gene locus by Southern blot hybridization with c-myc probes. Most of the major cleavage sites induced in vivo by drugs in the 5' end of c-myc were also observed in vitro using purified topoisomerase II and a c-myc gene DNA insert. Moreover, they closely mapped to some DNAse I hypersensitive sites, the presence of which reflects gene activity. DNA from drug treated cells probed with a human beta 1 globin pseudogene, and the c-mos proto-oncogene did not reveal topoisomerase II cleavage bands, suggesting that topoisomerase II-mediated drug activity may correlate with gene activity.

Amsacrine↗

PubMed1984

Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site.

The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenylation at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40-associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed.

Animals↗

PubMed1990

Characterization of the breakpoint of a t(14;14)(q11.2;q32) from the leukemic cells of a patient with T-cell acute lymphoblastic leukemia.

The leukemic cells and derivative cell line from a 74-year-old male with T-cell acute lymphoblastic leukemia showed chromosomal abnormalities including a t(14;14)(q11.2;q32). This translocation is characteristic of a variety of T-cell malignancies, particularly T-cell prolymphocytic leukemia and the clonal proliferations of peripheral T cells in patients with ataxia-telangiectasia. Using DNA probes that spanned the T-cell receptor alpha chain (TCRA) joining (J) locus, the DNA rearrangement caused by the translocation was identified, cloned, and sequenced. The breakpoint shows site-specific juxtaposition of a TCRA joining segment and DNA from a region of 14q32 centromeric to the immunoglobulin heavy chain locus. Comparison of restriction map and nucleotide sequence from this translocation with other related chromosomal breakpoints suggests a dispersion of breakpoints throughout the 14q32 region.

Amino Acid Sequence↗

PubMed1993

Regulation of the expression of bacterial iron transport genes: possible role of an antisense RNA as a repressor.

Expression of the iron transport gene, fatB and fatA, in the bacterium Vibrio anguillarum 775 is negatively regulated by the iron concentration in the medium. Here, we show that iron represses fatB and fatA mRNA levels and concomitantly induces the synthesis of an antisense RNA (RNA alpha). The presence of RNA alpha correlates with the inhibition of FatA protein synthesis and thus may play a role in the iron repression of fatA expression. Since the 5' end of RNA alpha maps 125 nucleotides upstream from the start codon of fatA and this RNA also extends into the coding region of fatB, it may also be involved in the iron regulation of fatB expression. RNA alpha may thus constitute a novel component of the bacterial iron regulatory circuit.

Bacterial Outer Membrane Proteins↗

PubMed1991

The mouse transition protein 1 gene contains a B1 repetitive element and is located on chromosome 1.

The gene for mouse transition protein 1 (mTP1) was isolated, sequenced, and chromosomally mapped. The nucleotide sequence of 1895 bp of a 6.4-kb mTP1 genomic subclone was determined to include 788 bp of 5' flanking region, 564 bp of coding region including a 396-bp intron and a TAA stop codon, and 543 bp of 3' flanking region. The mTP1 gene contains a B1 repeat sequence within the only intron of the gene. The transcriptional start site of the mTP1 mRNA was determined to be located 31 bases upstream of the ATG translational start codon. Southern blot analysis demonstrated the presence of sequences homologous to the mTP1 cDNA in the genomes of the rat, hamster, bull, boar, dog, horse, ram, human, and two marsupials (the American opossum and Monodelphis), suggesting that the mTP1 gene sequence is widely conserved. The TP1 gene has been mapped by analysis of restriction fragment length variants (RFLV) in an interspecific backcross to a position 0.7 +/- 0.4 cM telomeric of Mylf and 1.2 +/- 0.5 cM centromeric of Vil on mouse chromosome 1.

Amino Acid Sequence↗

PubMed1988

The design of synthetic genes.

Computer programs are described that aid in the design of synthetic genes coding for proteins that are targets of a research program in site directed mutagenesis. These programs "reverse-translate" protein sequences into general nucleic acid sequences (those where codons have not yet been selected), map restriction sites into general DNA sequences, identify points in the synthetic gene where unique restriction sites can be introduced, and assist in the design of genes coding for hybrids and evolutionary intermediates between homologous proteins. Application of these programs therefore facilitates the use of modular mutagenesis to create variants of proteins, and the implementation of evolutionary guidance as a strategy for selecting mutants.

Algorithms↗

PubMed1988

[Exon-intron structure and organization of the 5'-terminal region of the human p53 gene].

S1 mapping and nucleotide sequencing were used for localization of exon-intron junctions of the human p53 gene. The 3'-end of the gene was localized 12 nucleotides downstream AATAAA sequence, though an mRNA, 94 nucleotides shorter, was observed in one tumor. No typical promoter sequences were found at the 5'-end of the gene. In cell-free HeLa extracts transcription of RNA initiates within the sequence of possible hairpin-loop structure. Organization of the promoter region of the human p53 gene is discussed.

Endonucleases↗

PubMed1997

Mapping replication origins by quantifying relative abundance of nascent DNA strands using competitive polymerase chain reaction.

A procedure was developed for mapping origins of DNA replication in mammalian cell chromosomes based on determining the relative abundance of nascent DNA strands throughout a specific genomic region. The method entails purification of short strands of nascent DNA derived from recently activated origins and the quantification, within this sample, of the relative abundances of different adjacent DNA segments by a competitive polymerase chain reaction technique. It is expected that the abundance of defined markers within the origin region is greatest at the site where DNA replication begins. This origin mapping procedure (i) allows analysis of single-copy genomic regions, (ii) can be performed on cultured and primary cells in the absence of any chemical treatment, (iii) does not require cell synchronization, and (iv) allows mapping origins to within a few hundred base pairs. This high degree of resolution permits a study of the cis- and trans-acting elements required for origin function. Application of this method to single-copy sequences in mammalian cells has identified replication origins within an approximately 500-bp segment in the human lamin B2 gene domain and within an approximately 800-bp segment in the hamster dihydrofolate reductase gene locus.

Animals↗

PubMed1986

Organization of the human genes for insulin-like growth factors I and II.

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb.

Amino Acid Sequence↗

PubMed1984

Characterization of the expressed gene and several processed pseudogenes for the mouse ribosomal protein L30 gene family.

Five cloned genes encoding the mouse ribosomal protein L30 were isolated from a recombinant DNA library and characterized by restriction mapping and nucleotide sequence analysis. Only one of these genes has introns and is expressed; the others are inactive processed pseudogenes. The expressed gene consists of five exons and four introns spanning 2,723 nucleotides. Transcripts of this gene are processed into the mature L30 mRNA by pathways that exhibit both constraints and flexibility with regard to the order of intron excision. The L30 mRNA which is 457 to 468 nucleotides in length excluding the polyadenylic acid tail, exhibits some microheterogeneity at its 3' end and encodes a basic protein of 115 amino acids. The 5' portion of the rpL30 gene has some novel features which are remarkably similar to the previously characterized mouse rpL32 gene. These include homologous sequences in the -60 to -340 region, the absence of a good TATA consensus sequence, and the presence of a palindromic pyrimidine sequence that spans the cap site.

Animals↗

PubMed1989

Linkage of the mouse LDL receptor gene on chromosome 9.

We identified restriction fragment length variants of the mouse low-density lipoprotein receptor gene and used these to map the gene, designated Ldlr, to the proximal region of chromosome 9. An interspecific backcross between strains MEV and CAST/Ei yielded the following gene order and distances in centimorgans: Ldlr-18.8 +/- 5.6-Apoa-4-7.3 +/- 3.5-Mpi-1-10.2 +/- 3.9-Emv-3 or dilute. Analysis of recombinant inbred strains also indicated that Ldlr is tightly linked to a previously unmapped retroviral marker, Xmmv-67.

Animals↗

PubMed1985

[Characteristics of the genome of poliovirus type 3 isolated from patients with poliomyelitis in Moldavia].

Twelve poliovirus isolates of serotype 3 from patients with paralytic poliomyelitis have been analyzed by oligonucleotide mapping of the viral genomes. All the studied strains were isolated from patients in different regions of the Moldavian SSR in 1982. The maps of all isolates are similar but they do not practically possess any large oligonucleotides characteristic of the vaccine strain of type 3 poliovirus. It is concluded that a wild neurovirulent strain of type 3 poliovirus, that circulated in 1982 in the Moldavian SSR was the cause of paralytic poliomyelitis cases. All the studied isolates are suggested to have been derived relatively recently from the common ancestor.

DNA, Viral↗

PubMed1997

Mapping replication origins, pause sites, and termini by neutral/alkaline two-dimensional gel electrophoresis.

Neutral/alkaline two-dimensional gel electrophoresis is a robust, easily interpretable, sensitive technique that has yielded insights about the in vivo replication of many types of DNA, from multicopy yeast plasmids to single-copy chromosome regions in unsynchronized mammalian cells. It can provide information about directions of replication fork movement and locations of replication origins, termini, and pause sites. Especially when combined with its partner technique, neutral/neutral two-dimensional gel electrophoresis, it is a method of choice for investigation of unknown situations.

Animals↗

PubMed1997

Mapping the regions carrying the three contiguous antibiotic resistance genes aadE, sat4, and aphA-3 in the genomes of staphylococci.

Tn5405 (12 kb) is a staphylococcal composite transposon delimited by two inverted copies of IS1182, one of which contains IS1181. The internal part of this transposon carries three antibiotic resistance genes, aphA-3, aadE, and sat4, and three open reading frames (ORFs), orfx, orfy, and orfz, of unknown function. The dispersion of Tn5405 and the genes and ORFs included in this transposon were investigated in 50 epidemiologically unrelated staphylococci carrying aphA-3. Twenty-three maps, distinguishable by the presence or absence of the investigated genes or ORFs and/or by the sizes of the restriction fragments carrying them, were identified. Four isolates carried Tn5405, and 15 other isolates contained a Tn5405-related element. IS1182 was not detected in the aphA-3 regions mapped in 31 isolates which carried the following combinations: orfx, orfy, aadE, sat4, and aphA-3 +/- orfz; orfy, aadE, sat4, and aphA-3 +/- orfz; and aadE, sat4, aphA-3, and orfz. In all isolates, the genes and ORFs investigated were in relative positions similar to those in Tn5405. Thus, the internal part of Tn5405 appeared to be partially conserved with the maintenance, in all of the isolates, of at least the three antibiotic resistance genes.

DNA, Bacterial↗

PubMed1988

Genetic variants of influenza A/Taiwan/1/86 cocirculating in Canada during the winter of 1986 to 1987.

The first isolate of influenza virus in Canada during the winter of 1986 to 1987 was a genetic variant of A/Taiwan/1/86. This genetic variant type was the predominant strain obtained from several of the western provinces. The variant strains were antigenically indistinguishable from A/Taiwan/1/86 but were remarkably distinct by T1 oligonucleotide mapping. T1 mapping of individual genome segments indicated that the variants evolved from an A/Taiwan/1/86-like virus through the accumulation of point mutation or deletion or insertion events and probably do not contain foreign genes. The relative distribution of genetic variation was approximately equal among the individual genes, with the possible exception of segments 1 or 2 that were analyzed in combination and thus could not be individually associated with the observed variation.

Canada↗