Nonsense mutation in factor VIII gene of a severe haemophiliac patient with anti-factor VIII antibody.
Explore the source record for details and available documents.
Adolescent↗
Biomedical subjects
T Hashimoto-Gotoh
Publications and source records attributed to T Hashimoto-Gotoh.
High-copy-number and low-copy-number plasmid vectors for lacZ alpha-complementation and chloramphenicol- or kanamycin-resistance selection.
Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'. These vectors are essentially based on high- or low-copy-number replicons. The low-copy-number vectors, 3.61 kb in size, confer CmR and contain the pSC101 replicon and pUC8-/pUC9-type polylinker. On the other hand, the high-copy-number vectors, 2.21 to 2.68 kb in size, confer either CmR or KmR, and contain the pBR322 replicon and pUC18-/pUC19-type or other modified polylinkers. All cloning sites except HindIII and SmaI sites in the KmR vectors are unique in each plasmid. Since almost all frequently used plasmid vectors confer ampicillin resistance, these vectors may be useful to simplify the subcloning/DNA joining experiments due to unnecessity of radioisotope labelling, size fractionation and purification of foreign DNA segments.
Base Sequence↗
Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence.
We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.
Amino Acid Sequence↗
Identification of the principal promoter sequence of the c-H-ras transforming oncogene: deletion analysis of the 5'-flanking region by focus formation assay.
A number of deletion mutants were isolated, including 5', 3', and internal deletions in the 5'-flanking region of the human cellular oncogene related to the Harvey sarcoma virus (c-H-ras), and their transforming activities were examined in NIH 3T3 cells. DNA sequences which could not be detected without losing transforming activity were localized to a relatively short stretch upstream of the region which showed homology to the 5'-flanking region of v-H-ras oncogene. S1 nuclease analysis indicated that there were two clusters of mRNA start sites at positions that were about 1,371 and 1,298 base pairs upstream of the first coding ATG. The minimum region required for promoter function was estimated to be a 51-base-pair-long (or less) DNA segment. The promoter was GC rich (78%) and did not contain the consensus sequences that are usually observed in PolII-directed promoters but contained a GC box within which one of the mRNA start sites was included. In addition, two sets of positive and negative elements seemed to be located between the promoter and the protein-coding region, which appeared to influence positively and negatively, respectively, the efficiency of transformation with the c-H-ras oncogene.
Animals↗
Improved vector, pHSG664, for direct streptomycin-resistance selection: cDNA cloning with G:C-tailing procedure and subcloning of double-digest DNA fragments.
A plasmid cloning vector, pHSG664, has been constructed which is suitable for the direct-selection of transformed cells with recombinant DNAs. The plasmid contains the replicon and ApR-gene region of pUC9 ligated to the strA+ (rpsL+) gene derived from pNO1523 [Dean, Gene 15 (1981) 99-102]. The vector contains ten unique restriction sites: EcoRI, HpaI, PvuII, SphI, PstI, SmaI(XmaI), BamHI, SalI(AccI, HincII), XbaI and HindIII. Five sites (bold-face lettering) are located within the coding region of the strA+ gene. Any insertion at the five bold-faced sites or any nucleotide replacement involving the strA+ gene region and the other unique sites can be selected by Ap and Sm double selection in a strA- (SmR) strain such as E. coli HB101. Thus, this vector is useful for cDNA cloning at either the SphI or the PstI site with the G:C-tailing procedure, as well as for the cloning of double-digest DNA segments.
Base Sequence↗
New cosmid vectors developed for eukaryotic DNA cloning.
A series of ColE1 and pSC101 cosmid vectors have been constructed suitable for cloning large stretches of DNA. All contain a single BamHI site allowing cloning of Sau3A, MboI, BglII, BclI , and BamHI-generated fragments. These vectors have the following characteristics: (i) they are relatively small (1.7-3.4 kb); (ii) the BamHI cloning site is flanked by restriction enzyme sites enabling direct cloning of unfractionated insert DNA without generating multiple insert or vector ligation products [ Ish - Horowitz and Burke, Nucl . Acids Res. 9 (1981) 2989-2998]; (iii) two vectors ( pHSG272 and pHSG274 ) contain a hybrid Tn5 KmR/ G418R gene which is selectable in both prokaryotic and eukaryotic cells, making them suitable for transferring DNA into eukaryotic cells, and (iv) the different prokaryotic selectable markers available in the other vectors described facilitate cosmid rescue of the transferred DNA sequences from the eukaryotic cell: CmR, ApR, KmR, ( pHSG429 ), CmR, ( pHSG439 ), colicin E1 immunity ( pHSG250 ), (v) the cosmid pHSG272 was used successfully to construct a shuttle vector based on the BPVI replicon [ Matthias et al., EMBO J. 2 (1983) 1487-1492].
Animals↗
A bovine papilloma virus vector with a dominant resistance marker replicates extrachromosomally in mouse and E. coli cells.
We describe the construction of a bovine papilloma virus-based vector (pCGBPV9) which contains a dominant selectable marker and replicates autonomously in both mouse and Escherichia coli cells. This vector contains the complete bovine papilloma virus genome, a ColE1 replication origin and a dominant selectable marker conferring resistance to kanamycin in bacteria and G418 in eukaryotic cells. A high number of G418R colonies are obtained after transfer of pCGBPV9 into mouse C127 cells. These G418R colonies contain vector DNA which replicates autonomously at approximately 10-30 copies per cell. The molecules are in most cases unrearranged and can be rescued into E. coli cells by bacterial transformation.
Animals↗
Temperature sensitive replication plasmids are passively distributed during cell division at non-permissive temperature: a new model for replicon duplication and partitioning.
To see whether plasmid molecules in bacteria are equally partitioned or randomly distributed at cell division, the segregation properties of temperature sensitive replication mutants of the E. coli plasmid pSC101 were tested at non-permissive temperature. The results support the idea that at least unreplicated molecules are passively distributed and thus the Equipartition Model is unlikely even under physiological conditions if plasmids replicate randomly. Therefore, we developed a new model which involves the Random Replication Hypothesis and assumes that only the two products of the last plasmid replication event are actively partitioned into two daughter cells and the others are randomly distributed. Mathematical studies revealed that the incompatibility segregation rate predicted by this model fits the experimental data.
DNA Replication↗
Incompatibility properties of Col E1 and pMB1 derivative plasmids: random replication of multicopy replicons.
The incompatibility properties of Col E1-like plasmids have been examined in Rec+ and RecA- bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.
Bacterial Proteins↗
Specific-purpose plasmid cloning vectors. I. Low copy number, temperature-sensitive, mobilization-defective pSC101-derived containment vectors.
Two cloning vector plasmids, pHSG415 (7100 bp) and a lambda phage cos site-containing derivative (cosmid) thereof, pHSG422 (8760 bp), were constructed from a low copy number plasmid (pSC101) replicon to permit the propagation of cloned DNA segments at low gene dosage levels. Two features of the vectors, namely temperature sensitivity of replication and inability to be mobilized by conjugative plasmids, cause them to exhibit a high level of "biological containment". The essential characteristics of pHSG415 and pHSG422 may be summarized as follows: (1) their genome copy number is low (4--6 copies/chromosome); (2) their replication ceases at high temperature and they are rapidly lost from host cells grown at temperatures of 37 degrees C and above; (3) the relaxation nick site of pSC101, which is thought to be synonymous with its origin of transfer replication, is absent from the vectors; as a consequence, they are not mobilized to a significant extent by co-existing conjugative plasmids that are able to mobilize wild-type pSC101; (4) they contain unique insertion sites for DNA fragments generated by the following restriction endonucleases: EcoRI, XhoI, XmaI, HindIII and PstI; pHSG415 additionally contains single BamHI, BstEII and HincII sites and may also be used to clone PvuI-generated fragments; (5) the plasmids confer upon their host cells resistance to chloramphenicol, kanamycin and ampicillin, and every unique cloning site, except those of BamHI and BstEII, is located within one of these antibiotic-resistance genes.
Anti-Bacterial Agents↗
Location of two relaxation nick sites in R6K and single sites in pSC101 and RSF1010 close to origins of vegetative replication: implication for conjugal transfer of plasmid deoxyribonucleic acid.
A nick-labeling method has been used to localize the relaxation complex nick sites in three plasmids (pSC101, RSF1010, and R6K) that differ markedly in their host range, deoxyribonucleic acid replication, and conjugal transfer properties. Single specific relaxation sites were located in pSC101 and RSF1010, but surprisingly two distinct sites could be identified in the bi-origin plasmid R6K. In all cases, relaxation nick sites, which are thought to be origins of plasmid conjugal transfer, were shown to be located near origins of vegetative replication. This result suggests a functional interaction between these two types of deoxyribonucleic acid loci, and we speculate here that application events initiated at origins of replication may constitute an integral part of the process of conjugal transfer of small plasmids among bacteria. Consistent with this proposal is the finding that inhibition of vegetative replication of the pSC101 and ColE1 plasmids results in a severe inhibition of their conjugal transfer ability.
Conjugation, Genetic↗
Isolation and characterization of replication-deficient mutants of ColE1 plasmids.
Replication-defective mutants of plasmid ColE1 were isolated from a chimeric plasmid formed by ligating a temperature-sensitive replication derivative of pSC101, pHSG1, with a ColE1-Tn3-containing plasmid. The replication-defective ColE1 mutants isolated were all spontaneous deletion mutants that had lost the ColE1 replication origin and regions adjacent to it. The extent of a deletion was determined by analyzing restriction endonuclease-generated deoxyribonucleic acid fragments of the ColE1 plasmid component of the chimeras by both agarose and polyacrylamide gel electrophoresis. None of the chimeras containing the replication-defective ColE1 mutants was able to replicate in the presence of chloramphenicol. The expression of ColE1 incompatibility was either markedly reduced or not detectable in the replication mutants isolated.
Chloramphenicol↗
ColE1 plasmid incompatibility: localization and analysis of mutations affecting incompatibility.
Deletion mutants of plasmid ColE1 that involve the replication origin and adjacent regions of the plasmid have been studied to determine the mechanism by which those mutations affect the expression of plasmid incompatibility. It was observed that (i) a region of ColE1 that is involved in the expression of plasmid incompatibility lies between base pairs -185 and -684; (ii) the integrity of at least part of the region of ColE1 DNA between base pairs -185 and -572 is essential for the expression of ColE1 incompatibility; (iii) the expression of incompatibility is independent of the ability of the ColE1 genome to replicate autonomously; (iv) plasmid incompatibility is affected by plasmid copy number; and (v) ColE1 plasmid-mediated DNA replication of the lambda phage-ColE1 chimera lambda imm434 Oam29 Pam3 ColE1 is inhibited by ColE1-incompatible but not by ColE1-compatible plasmids.
Colicins↗
Random replication and random assortment model for plasmid incompatibility in bacteria.
Explore the source record for details and available documents.
DNA Replication↗
Mutations of temperature sensitivity in R plasmid pSC101.
Temperature-sensitive (Ts) mutant plasmids isolated from tetracycline resistance R plasmid pSC101 were investigated for their segregation kinetics and deoxyribonucleic acid (DNA) replication. The results fit well with the hypothesis that multiple copies of a plasmid are distributed to daughter cells in a random fashion and are thus diluted out when a new round of plasmid DNA replication is blocked. When cells harboring type I mutant plasmids were grown at 43 degrees C in the absence of tetracycline, antibiotic-sensitive cells were segregated after a certain lag time. This lag most likely corresponds to a dilution of plasmids existing prior to the temperature shift. The synthesis of plasmid DNA in cells harboring type I mutant plasmids was almost completely blocked at 43 degrees C. It seems that these plasmids have mutations in the gene(s) necessary for plasmid DNA replication. Cells haboring a type II mutant plasmid exhibited neither segregation due to antibiotic sensitivity nor inhibition of plasmid DNA replication throughout cultivation at high temperature. It is likely that the type II mutant plasmid has a temperature-sensitive mutation in the tetracycline resistance gene. Antibiotic-sensitive cells haboring type III mutant plasmids appeared at high frequency after a certain lag time, and the plasmid DNA synthesis was partially suppressed at the nonpermissive temperature. They exhibited also a pleiotrophic phenotype, such as an increase of drug resistance level at 30 degrees C and a decrease in the number of plasmid genomes in a cell.
DNA, Bacterial↗
A novel monitoring of immunosuppression in recipients with DNA-binding activity of nuclear factor in activated T cells.
Explore the source record for details and available documents.
Binding Sites↗
Possible role of the first intron of c-H-ras in gene expression: anti-cancer elements in oncogenes.
When members of ras gene family, which encode structurally related proteins of approximately 21,000 daltons (p21), are activated by either structural mutations or enhanced promoter activities, immortalized mammalian cells can be transformed into malignant forms. The ras genes are classified as "housekeeping genes" which express relatively constantly at low levels in all tissues and throughout all developmental stages. It is therefore important to understand how regulatory, cis-acting in particular, elements of the genes control their expression. We have previously reported that the principal promoter, 51 bp long or less, located around 1370 bp upstream from the first coding ATG, plays the most important role for expression, though there seem to be residual promoter activities further downstream as well. The most upstream mRNA start site is located at the 3'-end of the principal promoter, which lies directly adjacent to the homologous sequence between human and rat c-H-ras 5'-flanking regions. It is therefore proposed that the c-H-ras gene may have a dispersed promoter in which the principal promoter and subpromoters may be distributed. On the other hand, the first intron region seems to contain two sets of positive and negative elements which appear to have, respectively, a positive and negative influence on the efficiency of focus formation with the activated c-H-ras oncogene. From testing done in our laboratory, it was further revealed that these cis-acting elements in the intron affect c-H-ras gene expression at the post-transcriptional level. The middle part of the first intron was in fact shown unusually conserved between human and rat DNA sequences. Moreover, computer analyses revealed that the intron sequences of various oncogenes (both human and rodent sequences available from GenBank DNA sequence data-bank) such as ras, fos, c-myc, N-myc and int-1, are in general significantly more highly conserved when compared with sequences of non-oncogene introns. The significance of this oncogene-related conserved sequence is discussed and the proposal is made that it may function as an "anti-cancer element" or "safety valve" against gene truncation/translocational activation of oncogenes.
Base Sequence↗