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S R Jaskunas

Publications and source records attributed to S R Jaskunas.

At least 37 records · Page 2Linked to original sources
PubMed1990

Characterization of a modified human tissue plasminogen activator comprising a kringle-2 and a protease domain.

To study structure/function relationships of tissue plasminogen activator (t-PA) activity, one of the simplest modified t-PA structures to activate plasminogen in a fibrin-dependent manner was obtained by constructing an expression vector that deleted amino acid residues 4-175 from the full-length sequence of t-PA. The expression plasmid was introduced into a Syrian hamster cell line, and stable recombinant transformants, producing high levels of the modified plasminogen activator, were isolated. The resulting molecule, mt-PA-6, comprising the second kringle and serine protease domains of t-PA, produced a doublet of plasminogen activator activity having molecular masses of 40 and 42 kDa. The one-chain mt-PA-6 produced by cultured Syrian hamster cells was purified in high yield by affinity and size exclusion chromatography. The purified mt-PA-6 displayed the same two types of microheterogeneity observed for t-PA. NH2-terminal amino acid sequencing demonstrated that one-chain mt-PA-6 existed in both a GAR and a des-GAR form. Purified mt-PA-6 also existed in two glycosylation forms that accounted for the 40- and 42-kDa doublet of activity produced by the cultured Syrian hamster cells. Separation of these two forms by hydrophobic interaction chromatography and subsequent tryptic peptide mapping demonstrated that both forms contained N-linked glycosylation at Asn448; in addition, some mt-PA-6 molecules were also glycosylated at Asn184. Plasmin treatment of one-chain mt-PA-6 converted it to a two-chain molecule by cleavage of the Arg275-Ile276 bond. This two-chain mt-PA-6, like t-PA, had increased amidolytic activity. The fibrinolytic specific activities of the one- and two-chain forms of mt-PA-6 were similar and twice that of t-PA. The plasminogen activator activity of one-chain mt-PA-6 was enhanced greater than 80-fold by CNBr fragments of fibrinogen, and the one-chain enzyme lysed human clots in vitro in a dose-dependent manner. The ability to produce and purify a structurally simple plasminogen activator with desirable fibrinolytic properties may aid in the development of a superior thrombolytic agent for the treatment of acute myocardial infarction.

Amino Acid Sequence↗
PubMed1990

Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin.

Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by alpha-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Asp167 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Arg169-Leu170 which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium. At 3 mM calcium, alpha-thrombin activated the derivatives 5- to 8-fold faster compared with the wild-type, an effect which was shown to be caused by a decreased inhibitory effect of calcium on the reaction. These same single amino acid substitutions enhanced the affinity of the thrombomodulin-thrombin complex for the substrate at 3 mM calcium 3-(Gly-substitution) to 6-(Phe-substitution) fold, either without influencing kcat (Gly-substitution) or with a 2.5-fold decrease of kcat. For both derivatives, the calcium concentrations resulting in half maximal inhibition of activation by alpha-thrombin and in half maximal stimulation of activation by the thrombomodulin-thrombin complex increased from 0.3 mM to 0.6 mM. It is concluded that Asp167 is involved in the calcium induced inhibition of protein C activation by thrombin. Moreover, our studies demonstrate that it is feasible to enhance the efficiency of enzymatic reactions by introducing point mutations in the substrate.

Amino Acid Sequence↗
PubMed1989

Direct expression of recombinant activated human protein C, a serine protease.

Human protein C, like other serine proteases, is normally secreted as an inactive zymogen. It is converted to its active form extracellularly by limited proteolysis with the thrombin-thrombomodulin complex. This activation results from the removal of a 12-residue activation peptide from the NH2 terminus of the heavy (COOH-terminal) chain. We report here a successful strategy for the activation of human protein C during post-translational cellular processing, resulting in the secretion of activated protein C from transfected mammalian cells. Deletion of the nucleotides encoding the activation peptide resulted in the expression of a protease with less than 5% of the expected activity. However, the replacement of the activation peptide with an 8-residue sequence (Pro-Arg-Pro-Ser-Arg-Lys-Arg-Arg) involved in the proteolytic processing of the human insulin receptor precursor resulted in the direct expression of fully activated protein C. The mutant protein was shown to be correctly processed by NH2-terminal sequence analysis. This strategy for successful expression of an activated form of protein C may apply to the expression of active forms of other proteases which are naturally expressed as zymogens.

Amino Acid Sequence↗
PubMed1983

RecA-independent recombination between direct repeats of IS50.

Recombination between the IS50 sequences that flank Tn5 has been found to occur readily after the transformation of E. coli recA strains with a plasmid that contains direct repeats of these sequences. Analysis of mutants of IS50 indicates that the polypeptide encoded by IS50 that is required for transposition is also required for the recombination. Surprisingly, the mechanism of recombination appears to be similar to general homologous recombination rather than site-specific recombination. This IS50 recombination system may be responsible for resolving transient cointegrate structures containing direct repeats of IS50 or Tn5 during the transposition of IS50 and Tn5.

Bacterial Proteins↗
PubMed1983

Role of short regions of homology in intermolecular illegitimate recombination events.

The structures of three recombinants between bacteriophage lambda DNA and plasmid pBR322 that were generated in a recA derivative of Escherichia coli are described. Each resulted from two illegitimate recombination events that resulted in the substitution of part of the lambda genome by part of the plasmid genome. The nucleotide sequences at the six lambda-plasmid junctions were determined and compared with the sequences of the lambda and plasmid genomes before recombination. Each recombination occurred at a short region of homology in the two genomes, and other short regions of homology were found near some of the junctions. The structures of these junctions are similar to those resulting from illegitimate recombination in animal cells. A model to explain how these multiple illegitimate recombination events could result from a cascade of DNA gyrase-catalyzed recombinations is discussed.

Bacteriophage lambda↗
PubMed1982

Nucleotide sequence analysis of in vivo recombinants between bacteriophage lambda DNA and pBR322.

The nucleotide sequences involved in the illegitimate recombination of four recombinants between bacteriophage lambda DNA and pBR322 in E. coli (lambda TA6, lambda KA3, lambda TA1R, and lambda KA7) were determined. Each resulted from recombination between regions of homology of 10 to 13 base pairs. The presence of a recA+ allele was found to stimulate recombination between lambda DNA and pBR322 approximately 10-fold. Lambda TA6, lambda KA3, and lambda KA7 were isolated in the presence of a recA+ allele and therefore, may have been generated by the recA recombination system. However, lambda TA1R was isolated in a recA mutant, and was presumably generated by a different recombination system. The possibility that it was generated by DNA gyrase is discussed. Two recombination events were required to form lambda KA7, which may indicate that it also was generated by DNA gyrase.

Alleles↗
PubMed1980

Isolation of E. coli mutants containing multiple transpositions of IS sequences.

The characterization of three E. coli mutants that appeared to have unselected IS1 insertions on the chromosome are described. One had a single new IS1 sequence. The second had three new IS1 sequences. The third had two new IS1 sequences and one of the IS1 sequences in the parent was missing. These mutants were found in a collection on strains that contained IS insertions in the spc operon. The frequency of finding mutants with unselected IS1 transpositions was at least 100 times greater than expected. The results suggest several transposition events may frequently occur in the same cell.

Base Sequence↗
PubMed1980

Fate of donor insertion sequence IS1 during transposition.

The number of insertion sequence IS1 segments in Escherichia coli K-12 and 20 mutants in which an ISI had been inserted at a new site was measured by Southern blot hybridization analysis. The parent strain appeared to contain seven IS1 segments. Each of the mutants contained these seven IS1 and one additional IS1 corresponding to the new IS1 insertion. These results suggest that a copy of a donor ISI is inserted at the new site. A model for transposition is prevented that postulates that a reactive intermediate is formed by a tandem duplication of a transposable sequence.

Base Sequence↗
PubMed1980

Recombination between bacteriophage lambda and plasmid pBR322 in Escherichia coli.

Recombinant lambda phages were isolated that resulted from recombination between the lambda genome and plasmid pBR322 in Escherichia coli, even though these deoxyribonucleic acids (DNAs) did not share extensive regions of homology. The characterization of these recombinant DNAs by heteroduplex analysis and restriction endonucleases is described. All but one of the recombinants appeared to have resulted from reciprocal recombination between a site on lambda DNA and a site on the plasmid. In general, there were two classes of recombinants. One class appeared to have resulted from recombination at the phage attachment site that probably resulted from lambda integration into secondary attachment sites on the plasmid. Seven different secondary attachment sites on pBR322 were found. The other class resulted from plasmid integration at other sites that were widely scattered on the lambda genome. For this second class of recombinants, more than one site on the plasmid could recombine with lambda DNA. Thus, the recombination did not appear to be site specific with respect to lambda or the plasmid. Possible mechanisms for generating these recombinants are discussed.

Bacteriophage lambda↗
PubMed1980

Identification of a second tetracycline-inducible polypeptide encoded by Tn10.

Three Tn10 polypeptides were detected by analyzing the proteins synthesized in ultraviolet light-irradiated Escherichia coli cells after infection with lambda::Tn10. One of these polypeptides was the previously identified 36,000-dalton TET polypeptide. The other two had approximate sizes of 25,000 and 13,000 daltons. The syntheses of both the TET polypeptide and the 25,000-dalton polypeptide were inducible by tetracycline in lambda-immune hosts. Similarly, the synthesis of the TET polypeptide was inducible in nonimmune hosts. However, the synthesis of the 25,000-dalton polypeptide was constitutive in nonimmune hosts. An amber mutation in a gene required for tetracycline resistance on lambda::Tn10 was isolated that eliminated the synthesis of the TET polypeptide in sup+ hosts but not the synthesis of the 25,000-dalton or the 13,000-dalton polypeptides. The expression of tetracycline resistance from wild-type Tn10 was found to be anomalous in E. coli strains carrying the amber suppressors supD, supE, and supF. In general, strains containing these nonsense suppressors were less resistant to tetracycline.

Bacterial Proteins↗
PubMed1978

Isolation and characterization of a promoter mutant in the str ribosomal protein operon in E. coli.

The lambda fus3 transducing phage carries several operons for ribosomal proteins of E. coli, including the str operon. A mutant transducing phage with a promoter mutation in this operon has been isolated. This mutant shows reduced stimulation of synthesis of proteins encoded by the operon, S12, S7, and elongation factors G and Tu, in ultraviolet-irradiated cells. This mutation also abolishes in vitro transcription from the str promoter. The DNA sequence of the mutant promoter shows that it is a point mutation 6 bases upstream from the in vitro transcription start site, changing the "Pribnow box" sequence from TAAAATT to TAAAACT. These results indicate that the site altered by the mutation, which is in the region just preceding the transcription start site, is important for the expression of the str operon.

Bacterial Proteins↗
PubMed1977

Identification and organization of ribosomal protein genes of Escherichia coli carried by lambdafus2 transducing phage.

We describe the isolation of lambdafus2, which carries bacterial DNA from the str-spc region of the Escherichia coli chromosome. Genes for 27 ribosomal proteins were found on the genome of this transducing phage by identifying the ribosomal proteins whose synthesis was stimulated after infection of UV-irradiated bacteria. These were genes for S3, S4, S5, S7, S8, S10, S11, S12, S13, S14, S17, S19, L2, L3, L4, L5, L6, L14, L15, L16, L17, L18, L22 L23, L24, L29, and L30. Subsets of these genes were identified on the genomes of the phages lambdaspc1, lambdaspc2-delta 9, and lambdaspc2-delta16, all of which carry parts of the bacterial DNA present on lambdafus2. From the known structures of these phage genomes, it has been possible to determine the relative order of many of these genes on the lambdafus2 genome and thus on the E. coli chromosome. Our evidence also suggests that the genes for S3, S17, S19, L2, L4, L16, L22, L23, and L29 are part of a single transcription unit. These results, along with the observations described in the previous and accompanying papers, indicate that the ribosomal protein genes on lambdafus2 are organized into at least four transcription units.

Coliphages↗
PubMed1977

Organization of ribosomal protein genes of Escherichia coli as analyzed by polar insertion mutations.

Several mutants of lambdaspc1 and lambdafus3 have been isolated carrying DNA insertion elements that were selected for their ability to reduce the expression of the spc gene. The sizes and locations of the insertions on the phage genomes were determined by heteroduplex analysis. They were found to be located at different positions in the Spc transcription unit. The effect of these insertions on the expression of the ribosomal protein genes carried by these phages in ultraviolet light-irradiated bacteria was investigated. The insertions at intermediate positions in the transcription unit reduced the expression of some of the genes in the unit but not others. Assuming that the genes whose expressions were reduced are distal to the insertion, it was possible to determine the relative position of most of the genes in the unit. The results indicate the order of genes in the Spc transcription unit is: promoter, L14, L24, L5, S14, S8, L6, L18, (S5, L15, L30).

Coliphages↗
PubMed1977

Insertion of DNA carrying ribosomal protein genes of Escherichia coli into Charon vector phages.

DNA fragments from lambdaspc1 and lambdafus2, carrying ribosomal protein genes from Escherichia coli, were inserted into lambda phage vectors Charon 3 and Charon 4. Eight of the resulting clones were characterized by agarose gel electrophoresis of EcoRI digests, analytical CsCl equilibrium centrifugation, and electron micrographic analysis of heteroduplexes. In each case, the identity, order, and orientation of each cloned fragment was determined. In all, 8 of the 12 EcoRI fragments of lambdafus2 were cloned in various arrangements. In the accompanying paper, genes for 15 ribosomal and related proteins and three bacterial promoters were detected in these phages. In addition, four of the hybrid phages carried fragments of lambda-DNA including the phage origin of replication (ori), the late promoter, PR', and the cohesive ends (cos site) in both orientations. The latter phages yield a circularly permuted collection of DNA molecules.

Coliphages↗
PubMed1976

Transfer RNA genes between 16S and 23S rRNA genes in rRNA transcription units of E. coli.

We have identified genes for tRNAGLU/2 on the transducing phages o80d3ilvsu7+ (see Ohtsubo et al., 1974) and lambdarifd18 (Kirschbaum and Konrad, 1973), and a gene for tRNAlle/1 on the transducing phage o80rifr (Konrad, Kirschbaum, and Austin, 1973). All these phages have previously been shown to carry genes for rRNA (Ohtsubo et al., 1974; Lindahl et al., 1975; Jaskunas et al., 1975a). We have analyzed the position of these tRNA genes by hybridizing purified RNAs to restriction fragments of the phage DNA. The tRNA genes are located inside the rRNA transcription unit in the spacer region between the 16S and 23S rRNA genes.

Chromosome Mapping↗