Biomedical subjects

J Zabielski

Publications and source records attributed to J Zabielski.

At least 19 recordsLinked to original sources

PubMed1997

Inactivation of the replication-termination system affects the replication mode and causes unstable maintenance of plasmid R1.

Two so-called Ter sites, which bind the Escherichia coli Tus protein, are located near the replication origin of plasmid R1. Inactivation of the tus gene caused a large decrease in the stability of maintenance of the R1 mini-derivative pOU47 despite the presence of a functional partition system on the plasmid. Deletion of the right Ter site caused a drop in stability similar to that observed after inactivation of the tus gene. Substitution of 2bp required for Tus binding also caused unstable plasmid maintenance, whereas no effects on stability were observed when the left Ter site was deleted. Inactivation of the tus gene was coupled to an increased occurrence of multimeric plasmid forms as shown by gel electrophoresis of pOU47 DNA. Inactivation of the recA gene did not increase plasmid stability, suggesting that the multimerization was not mediated by RecA. Plasmid DNA was isolated from the tus strain carrying plasmid pOU47 and from a wild-type strain carrying pOU47 in which the right Ter site had been inactivated; in both cases, electron microscopy revealed the presence of multimers as well as rolling-circle structures with double-stranded tails. Thus, the right Ter site in plasmid R1 appears to stabilize the plasmid by preventing multimerization and shifts from theta to rolling-circle replication.

Bacterial Proteins↗

PubMed1992

Rolling-circle replication of a high-copy BPV-1 plasmid.

We investigated the replicating form of a bovine papillomavirus type 1 (BPV-1) deletion mutant by direct electron-microscopic analysis of low molecular weight cellular DNA fractions. The detection of viral plasmid DNA replication intermediates was facilitated by the isolation of a spontaneously transformed mouse cell subclone containing an unusually high viral genome copy number (approx. 1000 per cell), and by employing a slight modification of the Hirt fractionation procedure to reduce the level of contaminating linear chromosomal DNA fragments. We observed exclusively rolling-circle-type viral DNA replication intermediates, at a frequency of detection of approximately one replication intermediate per 200 monomeric circular viral DNA molecules. The demonstration of rolling-circles with longer-than-genome-length tails indicated that this high-copy viral plasmid was not subject to a strict once-per-cell-cycle mode of DNA replication. Our observations provide further evidence in favour of an alternative replication mode of the BPV-1 genome, and may help to explain earlier conflicting findings concerning the mechanism of stable BPV-1 plasmid copy-number-control.

Animals↗

PubMed1989

Evidence for multiple vegetative DNA replication origins and alternative replication mechanisms of bovine papillomavirus type 1.

By following up the chance detection in the electron microscope of a DNA replication intermediate within a preparation of bovine papillomavirus (BPV-1) DNA isolated from purified virus particles, information was obtained about the mechanism of BPV-1 genome replication during the final stages of virus multiplication in naturally infected bovine wart tissue. The structure of viral replication intermediates was investigated by electron microscopic analysis of viral DNA linearized by digestion with restriction endonucleases which cleave the circular BPV-1 chromosome at defined sites. Both Cairns and rolling circle-type molecules were identified. Furthermore, replication eyes were widely distributed within the viral genome, indicating that vegetative BPV-1 DNA replication origins are largely uncoupled from previously described plasmid maintenance sequence elements.

Bovine papillomavirus 1↗

PubMed1987

Properties of a distal regulatory element controlling transcription of the U2 small nuclear RNA.

The upstream region of human U2 genes contains a distal transcriptional control element, previously mapped between nucleotide (nt) positions -198 and -258 (Westin et al., 1984b). In the present study we show that it resembles transcriptional enhancers in being active even from a distance of 1.4 kb. However, in contrast to most other enhancers it functions unidirectionally in Xenopus laevis oocytes. The distal control element was further mapped by construction of truncated templates for U2 RNA transcription. The results showed that templates, which extended to either of nt positions -214 and -218, were inactive. Templates comprising sequences to nt positions -225 or -226 displayed an intermediate level of activity whereas templates which extend to nt -258 were fully active. It has previously been shown that the human U2 enhancer contains binding sites for the so-called octamer binding protein and for transcription factor Sp1 [Janson et al., Nucl. Acids Res. 15 (1987) 4997-5016]. The partially active templates included one binding site for the octamer binding protein, whereas the fully active template included, in addition, two Sp1 binding sites, thus indicating that these transcription factors are of importance for U2 RNA transcription. The structure of the enhancer was also probed by inserting a pair of complementary synthetic oligodeoxynucleotides which represented the region between nt positions -235 and -215 into a truncated template which lacked the enhancer. The oligodeoxynucleotide enhanced transcription to approximately 50% of the level obtained with templates extending to position -258.

Animals↗

PubMed1986

Subtypes of bovine adenovirus type 2 exhibit major differences in region E3.

The genomes of two adenovirus type 2 strains which were isolated from different hosts have been investigated. One of these strains designated ORT-111 was originally isolated from a lamb in Hungary during an outbreak of pneumoenteritis. This isolate was typed as bovine adenovirus type 2 (Ad bos 2) in a neutralization assay. The genome of ORT-111 was compared to that of the prototype strain of Ad bos 2, a virus which exclusively has been isolated from cattle. Electron microscopic heteroduplex analysis showed that 95% of the genomes were well matched, forming stable duplexes at Tm -6 degrees. Two distinct substitution loops were, however, seen which were approximately 0.5 and 1.0 kbp long. The centers of the two loops were located 5.3 and 7.7 kbp from one end of the Ad bos 2 genome. In order to map these regions relative to the gene map of human adenovirus type 2 (Ad2), restriction enzyme cleavage fragments of the two bovine viruses were cloned and hybridized to different sets of restriction fragments of human Ad2. From these results it was apparent that the centers of the two substitution loops were located at coordinates 76 and 83, respectively; thus at positions which fall within region E3 and the adjacent gene for polypeptide VIII of human Ad2. The observed differences between the genomes of the two Ad bos 2 strains are in sharp contrast to those previously observed when the genomes of different human adenovirus serotypes were compared. In the latter case the hexon and the fiber genes showed the most pronounced variation.

Adenoviridae↗

PubMed1986

Genes for human U4 small nuclear RNA.

A study of human genes coding for U4 small nuclear RNA is presented. It is known from previous studies that mammalian cells contain three major U4 RNA species, designated U4A, U4B, and U4C (Krol and Branlant, 1981). A clone was isolated from a human DNA library which contained two transcriptionally active genes for U4 RNA. U4 transcription was sensitive to low concentrations of alpha-amanitin, inferring that U4 RNA is a product of RNA polymerase II or RNA polymerase II-like activity. One of the two genes contains a coding region which matches the sequence of U4C RNA perfectly. The coding region of the second gene resembles U4B RNA although there are two differences between the sequence of this gene and the U4B RNA sequence, suggesting that it may encode a minor, hitherto undetected U4 RNA species. The 5'-flanking regions of the two U4 genes contain several almost perfectly conserved sequence motifs. One is located between positions -50 and -60. This motif is present in equivalent positions in the two U4 genes as well as in human U1 and U2 genes. A second motif, which is 19 nucleotides (nt) long and centered around nt position -140, is present in the two U4 genes but absent from U2 RNA genes. A third highly conserved region, located between nt positions -210 and -250, is a putative enhancer element. It includes one copy of the so-called octanucleotide motif, previously identified as adjacent to the early SV40 promoter and immunoglobulin promoters. Another highly conserved sequence motif, CTCTGTGA, is located approximately one helical turn upstream from the octanucleotide motif in both U2 and U4 genes. The human genome appears to contain a family of U4 RNA genes comprising at least 100 copies.

Animals↗

PubMed1985

Messenger RNAs from the transforming region of bovine papilloma virus type I.

Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.

Animals↗

PubMed1984

Genes and pseudogenes for human U2 RNA. Implications for the mechanism of pseudogene formation.

Three loci, designated U2/4, U2/6 and U2/7, which contain sequences related to human U2 RNA, have been studied. The U2/6 locus contains a tandem array of bona fide U2 genes. U2/4 and U2/7, in contrast, contain pseudogenes of whose sequences deviate significantly from that of mammalian U2 RNA. The two pseudogenes appear to have been created by different mechanisms. The sequences that flank the pseudogene in the U2/4 locus lack homology to the corresponding sequences in functional human U2 genes, except for 10 base-pairs immediately following the 3' end. The conserved 3'-flanking segment is homologous to those nucleotides that are present in a U2 RNA precursor. No direct repeats flank the pseudogene in the U2/4 locus. The observations thus suggest that a complementary DNA copy of the U2 RNA precursor was inserted into a blunt-ended chromosomal break to generate the U2/4 locus. The U2/7 locus, in contrast, revealed flanking sequence homology when compared to functional U2 genes, both on the 5' and 3' sides of the pseudogene. The homology was interrupted on both sides by repetitive sequences belonging to the Alu family. On the 5' side the homology continues beyond the Alu repeats whereas on the 3' side it ends precisely at the Alu repeat. This Alu repeat is inserted in a region where a homocopolymeric region of alternating C and T residues is located in functional U2 loci. The observed organization of the U2/7 locus suggests that a previously functional U2 locus was invaded by Alu repeats and subsequently accumulated base substitutions to become a pseudogene.

Base Sequence↗

PubMed1984

Clustered genes for human U2 RNA.

Genes for the human small nuclear RNA U2 are present within 6.2-kilobase-pair-long tandem repeats. The haploid human genome contains approximately 20 such repeats, organized in one or a few very large clusters.

Base Sequence↗

PubMed1983

Loci for human U1 RNA: structural and evolutionary implications.

Three clones U1-1, U1-6, and U1-8 containing sequences related to human U1 RNA have been studied by sequence analysis. The results show that each of the three clones represents a distinct locus. The U1-6 locus is closely related to the HU1-1 locus, which is believed to represent a functional U1 gene. The U1-1 and U1-8 loci are pseudogenes by definition, since they contain sequences that are closely related to but not identical with the human U1 RNA sequence. The U1-6 locus contains the sequence T-A-T-A-T close to the 5'-end of the U1 sequence but it is unclear if this represents the promoter. When the U1-8 locus was compared to the U1-6 locus, it was observed that the 5'-flanking sequences, except in the immediate vicinity of the pseudogene, are as well-conserved as the U1-related sequence itself, at least up to position -220. The high degree of homology in the 5'-flanking region suggests that U1 genes have a much more strict sequence requirement with regard to 5'-flanking sequences than most other eukaryotic genes. The U1-6 and U1-8 loci contain the sequence T-A-T-G-T-A-G-A-T-G-A between positions -211 and -221. An identical sequence is present in the equivalent position in the HU1-1 locus, and may represent the promoter. The high degree of conservation in the postulated promoter region indicates that pseudogenes like U1-8 possibly could be expressed. A truncated U1-related sequence is present between 106 to 150 nucleotides upstream from the U1 gene/pseudogene in the U1-6, the U1-8 and the HU1-1 loci, suggesting that the U1 genes may have been clustered early in evolution. The U1-1 locus has a strikingly different structure from the U1-8 locus; the pseudogene itself is as closely related to the U1 RNA sequence as is the U1-8 pseudogene but the flanking sequences, both on the 5' and the 3' side, share no detectable homology with the corresponding regions in the U1-6 or U1-8 loci. It may therefore be postulated that small nuclear RNA pseudogenes are created by several different mechanisms.

Base Sequence↗

PubMed1983

DNA homology between the arsenate resistance plasmid pSX267 from Staphylococcus xylosus and the penicillinase plasmid pI258 from Staphylococcus aureus.

A 29.5-kb plasmid, pSX267, from Staphylococcus xylosus DSM 20267 was found to code for arsenate, arsenite, and antimony (III) resistance. The isolated plasmid was transformed into S. aureus, where the same resistances were expressed. It was of special interest to see whether pSX267 showed any DNA sequence homology with the well-studied penicillinase plasmid from S. aureus pI258, also conferring arsenate, arsenite, and antimony III resistance. By the use of the Southern blotting technique, it was found that DNA sequence homology exists in the region of arsenate, arsenite, and antimony resistance, in addition to the region where the origin of replication, the incompatibility, and the replication A function were mapped on pI258. This finding was confirmed by electron microscope heteroduplex analysis, which allowed a correlation between the genetic and physical maps of pI258 and pSX267. Duplex DNA was formed at the arsenate operon of pI258, with a length of 2.6 kb, and at the incompatibility and replication A region, comprising a length of 2.5 kb. Adjacent to this latter region, two small regions of DNA homology were present, with lengths of 0.2 and 0.27 kb. Both plasmids share approximately 20% DNA sequence homology. The DNA homology of the arsenate, arsenite, and antimony III resistance coding regions between pI258 and pSX267 indicate that these plasmid-determined resistance markers are highly conserved and distributed among different staphylococcal species.

Antimony↗

PubMed1983

Avian acute leukemia virus OK 10: analysis of its myc oncogene by molecular cloning.

Several DNAs representing the genome of the avian acute leukemia virus OK 10 were isolated by molecular cloning from a transformed quail cell line, 9C, which contained at least six OK 10 proviruses. Recombinant lambda phages harboring the OK 10 genome and additional flanking cellular DNA sequences were studied by restriction endonuclease mapping and hybridization to viral cDNA probes. Six of the clones represented complete proviruses with similar, if not identical, viral sequences integrated at different positions in the host DNA. The organization of the OK 10 genome was determined by electron-microscopic analysis of heteroduplexes formed between the cloned OK 10 DNA and DNAs representing the c-myc gene and the genomes of two other avian retroviruses, Rous-associated virus-1 and MC29. The results indicated that the OK 10 proviral DNA is about 7.5 kilobases in size with the following structure: 5'-LTR-gag-delta polmyc-delta env-LTR-3', where LTR indicates a long terminal repeat. The oncogene of OK 10, v-mycOK 10, forms a continuous DNA segment of around 1.7 kilobases between pol and env. It is similar in structure and length to the v-myc gene of MC29, as demonstrated by restriction endonuclease and heteroduplex analyses. Two of the OK 10 proviruses were tested in transfection experiments: both DNAs gave rise to virus with the transforming capacities of OK 10 when Rous-associated virus-1 was used to provide helper virus functions.

Animals↗

PubMed1982

Sequence organization of a viral DNA insertion present in the adenovirus-type-5-transformed hamster line BHK268-C31.

The hamster cell line BHK268-C31 contains two large viral inserts which both include sequences from the left-hand end of adenovirus type 5 (Ad5) DNA. One of these viral inserts has been cloned in the lambda vector Charon 4A. Electron microscopic analysis and restriction enzyme mapping shows that the recombinant carries a 4.4-kb-long colinear segment of viral DNA, which is located between map positions 1.5 and 14.2 in the Ad5 genome. The junctions between viral DNA and flanking sequences have been sequenced and found not to show any specific features. One of the junctions is located in the E1a coding region, 573 bp from the left-hand end of the Ad5 genome, whereas the other junction is situated in the coding region for polypeptide IVa2. The promoter region as well as the cap site for the mRNAs from region E1a are thus missing from this insert and its role in viral transformation is unclear.

Animals↗

PubMed1982

Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29.

The chicken genome contains nucleotide sequences homologous to transforming genes (oncogenes) of a number of avian retroviruses. We have isolated chicken DNA (c-myc) that is homologous to the oncogene (v-myc) of the avian myelocytomatosis virus MC29 and have compared the structures of the cellular and viral genes. Results from restriction endonuclease mapping of c-myc and from analysis of heteroduplexes between the DNAs of the cellular and viral genes show that c-myc is homologous to 1,500 nucleotides in v-myc DNA. This homologous region is interrupted in c-myc by an intron-like sequence of 1,100 nucleotides which is absent from v-myc. Nuclear RNA from normal chicken cells contains at least five species of transcripts from c-myc ranging from 2.5 to 6.5 kilobases in length. By contrast, cytoplasm contains only the 2.5-kilobase c-myc RNA. These features of the c-myc gene and its nuclear transcripts are characteristic of normal cellular genes and suggest that the myc gene is of cellular rather than viral origin. The exons in c-myc may define two functional domains in the gene and may therefore facilitate the dissection of the different oncogenic potentials of the MC29 virus.

Animals↗

PubMed1981

Human DNA sequences complementary to the small nuclear RNA U2.

Clones containing sequences complementary to the small nuclear RNA U2 were isolated from a human DNA library (1). Three clones, designated U2/4, U2/6 and U2/7 were purified and characterized by restriction enzyme cleavage, hybridization and heteroduplex analysis. Hybridization showed that the three clones each contained one single region which is complementary to U2 RNA. Restriction enzyme cleavage revealed furthermore that the inserted fragments in the three recombinants are different. Heteroduplex analysis identified a 240-380 bp long duplex region in each heteroduplex which includes sequences complementary to U2 RNA. Heteroduplexes between clones U2/4 and U2/7 as well as between U2/4 and U2/6 revealed two additional approximately 200 bp long homologies. The remainder of the inserts were found to lack measurable sequence homology. Two fragments from clone U2/4 were subcloned in the pBR322 vector and the subclones were used to determine the nucleotide sequence of a region in clone U2/4 which is complementary to U2 RNA. A comparison between the established sequence and the sequence for rat U2 RNA (2) reveals several discrepancies.

Base Sequence↗

PubMed1981

The sequence of the 3' non-coding region of the hexon mRNA discloses a novel adenovirus gene.

We report the sequence of a 1164 nucleotide long DNA segment, located between map positions 59.5 and 62.8 on the adenovirus type 2 genome. The sequence comprises the 701 nucleotides long 3' non-coding region of the hexon mRNA as well as several important processing signals. The sequence revealed unexpectedly that the 3' non-coding region of the hexon mRNA contains a 609 nucleotide long uninterrupted translational reading frame following a potential initiator AUG. A late 14S mRNA, corresponding to the open reading frame, could be identified by S1 nuclease mapping and electronmicroscopy. The mRNA shares a poly(A) addition site with the hexon and pVI mRNAs, and carries a leader sequence which is related, and probably identical, to the tripartite leader, found in late adenovirus mRNAs. The junction between the leader and the body of this novel mRNA is located within the coding part of the hexon gene.

Adenoviruses, Human↗

PubMed1981

Structure and restriction enzyme maps of the circularly permuted DNA of staphylococcal bacteriophage phi 11.

One restriction enzyme map of Staphylococcus aureus bacteriophage phi 11 DNA was established by reciprocal double digestions with the enzymes EcoRI, HaeII, and KpnI. The sequential order of the EcoRI fragments was thereafter established by a novel approach involving blotting of DNA partially cleaved with EcoRI and the probing the blots with nick-translated terminal fragments. A circular map of the phi 11 DNA was established, and the phage genome was circularly permuted based on the failure to end label mature viral DNA, restriction maps of replicating DNA, and finally, homoduplex analysis in the electron microscope. A restriction enzyme map of the prophage form of phi 11 DNA was obtained by analysis of chromosomal DNA from a lysogenic strain.

Chromosome Mapping↗

PubMed1980

Multiple mRNA species for the precursor to an adenovirus-encoded glycoprotein: identification and structure of the signal sequence.

Early region 3 of the adenovirus type 2 genome encodes three proteins with molecular weights of 16,000, 14,500, and 14,000 (E2/16, E3/14.5, and E3/14). The E3/16 protein is the precursor to the E3/19 glycoprotein and is around 1500 daltons larger than the unglycosylated E3/19O protein. The E3/14.5 and E3/14 proteins are structurally related to each other but different from E3/16. Three mRNA species were identified for E3/16; all have common 5' ends with the same spliced region but with different 3' ends. E3/14 was translated from a 13S mRNA with the same 5' structure as the E3/16 mRNA but followed by a second spliced region with a different 3' end. A partial amino acid sequence was determined for E3/16 after radioactive labeling in vitro and this sequence can be aligned with a known DNA sequence. It contains a hydrophobic signal sequence, two presumptive glycosylation sites, and a hydrophobic region close to the COOH terminus.

Adenoviruses, Human↗