Small RNA molecules related to the Alu family of repetitive DNA sequences.

A rodent 4.5S RNA molecule with extensive homology to the Alu family of interspersed repetitive DNA sequences has been found physically associated with polyadenylated nuclear and cytoplasmic RNAs (W. Jelinek and L. Leinwand, Cell 15:205-214, 1978; S. Haynes et al., Mol. Cell. Biol. 1:573-583, 1981). In this report, we describe a 4.5S RNA molecule in rat cells whose RNase fingerprints are identical to those of the equivalent mouse molecule. We show that the rat 4.5S RNA is part of a small family of RNA molecules, all sharing sequence homology to the Alu family of DNA sequences. These RNAs are synthesized by RNA polymerase III and are developmentally regulated and short-lived in the cytoplasm. Of this family of small RNAs, only the 4.5S RNA is found associated with polyadenylated RNA.

Targeted mutagenesis of DNA with alkylating RecA assisted oligonucleotides.

Site-specific mutation was demonstrated in a shuttle vector system using nitrogen mustard-conjugated oligodeoxyribonucleotides (ODNs). Plasmid DNA was modified in vitro by ODNs containing all four DNA bases in the presence of Escherichia coli RecA protein. Up to 50% of plasmid molecules were alkylated in the targeted region of the supF gene and mutations resulted upon replication in mammalian cells. ODNs conjugated with either two chlorambucil moieties or a novel tetrafunctional mustard caused interstrand crosslinks in the target DNA and were more mutagenic than ODNs that caused only monoadducts.

The N-terminal sequence of the ribosomal 'A' protein from two moderate halophiles, Vibrio costicola and an unidentified moderate (NRCC 11227).

The 'A' protein, equivalent to ribosomal protein EL7/L12 from Escherichia coli, has been isolated and purified from two moderate halophiles Vibrio costicola and NRCC 11227. The 'A' protein from V. costicola contained an N-terminal serine and separated into two forms on DEAE-cellulose and two-dimensional electrophoresis while the equivalent protein in NRCC 11227 contained an N-terminal alanine residue and was present in only one form. The amino acid composition and mobility on two-dimensional gels indicated these proteins were very similar to EL7/L12. The first 38 residues of the 'A' proteins were sequenced and compared to the equivalent protein from E. coli and the extreme halophile Halobacterium cutirubrum. The N-terminal region of the 'A' protein from both moderate halophiles showed substantial homology to EL 12 (75--80%) but no evidence of any homology to the equivalent protein from the extreme halophile. The ribosomal proteins equivalent to ES1A in E. coli were also isolated and their amino acid compositions determined.

Expression efficiency of the human thrombomodulin-encoding gene in various vector and host systems.

Expression systems were developed for evaluating recombinant human thrombomodulin (TM) production in different host cell lines by investigating the performance of five mammalian expression vectors. The expression vectors were constructed so that they contain multiple monocistronic gene cassettes which include a gene encoding a dominant selectable marker, HyR (hygromycin B phosphotransferase), under the regulation of the thymidine kinase promoter, the target gene which encodes a truncated human re-TM under the regulation of various promoters, an amplifiable gene (Dhfr) encoding murine dihydrofolate reductase under the regulation of either the SV40 early or late promoter along with the SV40 enhancer and the SV40 ori. We tested the performance of the five expression vectors in human embryonic kidney cells (HEK293), baby hamster kidney cells (BHK), human melanoma cells (CHL-1) and Dhfr- Chinese hamster ovary cells (CHO/Dhfr-). We found that the efficiency of DNA uptake, transient expression and stable expression of the different expression vectors were all cell-line dependent. However, the myeloproliferative sarcoma virus (MPSV) LTR promoter consistently showed higher expression levels in all cell lines, particularly in HEK293 cells. These results were confirmed by the distribution curves of the level of expression of individual clones. Furthermore, by amplifying Dhfr in transfected CHO/Dhfr- cells with 100 nM methotrexate, we achieved a 20-fold increase in re-TM production using the SV40 late promoter to control murine Dhfr expression. Our data from DNA and mRNA analysis reveal that pMPSV-TM has a high transcription efficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant human protein C: comparative functional studies with human plasma protein C.

Protein C (PC) is the central protein in a major antithrombotic regulatory mechanism. Hereditary deficiencies of PC are associated with thrombosis. Therapeutic PC replacement may be an important treatment if pure functional human protein C is available in sufficient quantity. Human PC has been produced on a commercial scale using recombinant techniques. To study the functional properties of recombinant protein C (r-PC), we undertook a comparative investigation of the basic properties of r-PC and plasma protein C (n-PC). Both were isolated by immunopurification methods. Protac C activation proceeded at the same rate and kinetics for both forms. With thrombin-thrombomodulin (T-TM) activation, r-PC is significantly better than the activation of n-PC (for r-PC: Kcat/Km = 378 vs. n-PC: Kcat/Km = 35). No difference in the anticoagulant (aPTT prolongation) or profibrinolytic activities (inactivation of PAI-1 and PAI-3) were observed between activated r-PC and n-PC. Based on these functional studies, recombinant protein C has similar properties to the plasma form of protein C. However, T-TM activation of r-PC occurs faster than the n-PC. The mechanism is unknown, but may be due to the presence of larger amounts of single chain protein C which exists in a conformation more rapidly activated by the T-TM complex.

Recombinant soluble human thrombomodulin: a randomized, blinded assessment of prevention of venous thrombosis and effects on hemostatic parameters in a rat model.

UNLABELLED: Thrombomodulin is an endothelial surface receptor that binds thrombin and accelerates the activation of protein C. We compared the effects of a recombinant thrombomodulin analog (TME), recombinant hirudin (r-HIR), heparin sodium (HEP), and normal saline (Control) on thrombus formation, activated partial thromboplastin time (APTT), thrombin time (TT), platelet aggregation and tail transection bleeding time (BT) in a rat model of vena cava thrombosis. RESULTS: TME, r-HIR and HEP prevented venous thrombosis in this model in a dose-dependent manner. At the dose required to reduce vena cava thrombosis by 50% (ED50), TME did not prolong the APTT or TT as did HEP and r-HIR. Platelet aggregation in response to thrombin was not effected by TME but was inhibited by both r-HIR and HEP. BT did not differentiate the agents tested. CONCLUSION: TME inhibited venous thrombosis in a rat vena cava model with less effect on hemostatic variables than HEP or r-HIR.

A new animal model of venous thrombosis in rats with low flow conditions in the venous blood stream.

The aims of the present investigation were to develop a new venous thrombosis animal model with low flow conditions in the venous blood stream and then evaluate this model for testing new anticoagulants. In this model, the vena cava of rats was narrowed with a Doppler flow probe, blood flow velocity continuously recorded and thrombus formation initiated by thromboplastin infusion. Sixty-five minutes following thromboplastin infusion the animals were sacrificed and the following parameters measured: thrombus wet weight, fibrinopeptide A (FpA), activated partial thromboplastin time and platelet number. The new model was evaluated with aspirin, a PGI2 mimetic, heparin and a soluble thrombomodulin analogue. Without thromboplastin infusion no thrombus formation or reduction of blood flow was observed. Controls receiving thromboplastin infusion developed a thrombus, blood flow was arrested, platelet number decreased and FpA was elevated. In contrast, animals pretreated with anticoagulants maintained a residual flow, while thrombus weight, thrombocytopenia and FpA elevation were reduced. The antiplatelet agents were not effective. This study demonstrates that, under low flow conditions, only a combination of blood flow reduction with a hypercoagulable state results in venous thrombus formation. This improved model of venous thrombosis more closely resembles the clinical situation and is applicable for testing anticoagulants.

An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis.

HFE-linked hereditary hemochromatosis (HH) is one of the most common inherited diseases among individuals of Northern European ancestry. Two sites of point mutations in the HFE gene--C282Y and H63D--are associated with greater than 90% of HH cases. We have developed a sensitive real time PCR (TaqMan) 5'-nuclease assay for single nucleotide polymorphism (SNP) detection using novel DNA chemistry, and successfully applied this method to detect these mutations. Fluorogenic PCR probes, chemically modified with a minor groove binding agent to increase duplex stability, were used in single and multiplex probe closed tube formats. The probes were tested in two commercially available thermocycling fluorimeters (the Light Cycler and the ABI Prism 7700). Comparison of the results obtained from the analysis of 43 samples showed no discrepancies between our 5' nuclease assay and the restriction length polymorphism analysis, which is routinely used in hospitals. The reported real time PCR technology is ideal for the clinical setting as it is sensitive, eliminates the labor and supply costs of post-PCR steps, reduces the risk of crossover contamination, minimizes sources of error, and can be fully automated.

Synthesis, chemical and pharmacological properties of some 2,4-dioxo-1,2,3,4,5,6,7,8-octahydropyrimido [4,5-d]pyrimidines.

Synthesis of 6-substituted 2,4-dioxo-1,2,3,4,5,6,7,8-octahydropyrimido[4,5-d]pyrimidines [III-VI] obtained by cyclocondensation of 1-phenyl-6-aminouracil with formaline and the primary amines is described. Compounds III, V, VI in the Mannich reaction with secondary cyclic amines yield the corresponding 3-substituted N-aminomethyl derivatives VII-X. Some of them were active pharmacologically.

Structure of functional A salina -- E coli hybrid ribosome by electron microscopy.

Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination -- small 30S E coli and large 60S A salina -- fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A slina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.

Cell-bound cations of the moderately halophilic bacterium Vibrio costicola.

Over most of the range of salt concentrations in which the moderately halophilic bacterium Vibrio costicola could grow, the sum of the cell-associated Na+ + K+ ions was at least as high as in the external medium. This is in contrast to other moderate halophiles, which have substantially lower internal than external salt concentrations for most of their growth range. The relative amounts of Na+ and K+ in V. costicola varied with environmental conditions. The K+/Na+ ratio fell during anaerobic incubation or when cells were poisoned. As Na+ ions left the cells, K+ ions entered. However, movement of these ions was not tightly coupled, since K+ content of cells could increase without a corresponding decrease in Na+ content. The Mg2+ contents of cells varied little with environmental conditions.

Molecular analysis of the gene for vitamin K dependent protein S and its pseudogene. Cloning and partial gene organization.

Protein S is a vitamin K dependent plasma protein and a cofactor to activated protein C, a serine protease that regulates blood coagulation. The haploid genome contains two protein S genes (alpha and beta) with the protein S alpha-gene corresponding to the cloned cDNA. We have now isolated and mapped overlapping genomic clones that cover an area of 50 kilobases of the protein S alpha-gene which code for the 3' part of the gene, i.e., the thrombin-sensitive region, the four domains that are homologous to the epidermal growth factor (EGF) precursor, the COOH-terminal part of protein S that is homologous to a plasma sex hormone binding globulin (SHBG), and, finally, the 3' untranslated region. The thrombin-sensitive region and the EGF-like domains are each coded on a separate exon. The sizes of the exons coding for the COOH-terminal half of protein S and the location of the introns are nearly identical with those in the homologous SHBG gene. Furthermore, the phase class of the splice junctions is the same in these two genes. We have also isolated and mapped genomic clones that cover 25 kilobases of the protein S beta-gene, which was found to contain stop codons and a 2 bp deletion which introduces a frame shift, suggesting that it is a pseudogene. The structure of the two protein S genes and a comparison with the vitamin K dependent clotting factors support a model for their origin by exon shuffling and recruitment of the 3' part of the gene from an ancestor shared with the sex hormone binding globulin.

Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity.

Protein C is a vitamin K-dependent regulator of blood coagulation. It has beta-hydroxyaspartic acid in position 71 which is in the first of its two domains that are homologous to epidermal growth factor (EGF). This region has recently been demonstrated to have a Ca2+ binding site with a Kd of approximately 100 microM. Recombinant human protein C, expressed in mammalian tissue culture, had full biological activity and contained beta-hydroxyaspartic acid. Furthermore, it had a Ca2+-dependent epitope in the EGF-like domain, recognized by a monoclonal antibody. In contrast, a mutant recombinant human protein C in which beta-hydroxyaspartic acid had been replaced with glutamic acid in position 71 did not have the Ca2+-dependent epitope, and its biological activity was reduced to about 10% of normal. Fab' fragments of this antibody inhibited the anticoagulant activity of plasma-derived activated protein C, apparently by interfering with the interaction between activated protein C and its cofactor, protein S. The latter contains four tandemly arranged EGF homology domains. We propose that beta-hydroxyaspartic acid is directly involved in Ca2+ binding in protein C and in related proteins and that protein C interacts with protein S by means of its EGF homology regions.

Moloney murine sarcoma proviral DNA is a transcriptional unit.

A portion of Moloney murine sarcoma virus DNA which is repeated at both ends of the provirus has been sequences. The nucleotide sequence, together with hybridization data obtained with in vitro pulse-labelled nascent viral RNA, indicate that initiation and termination of RNA synthesis occur within that region of the proviral DNA. A model for transcriptional readthrough of termination signals during RNA synthesis in this system is suggested.

Cytoplasmic processing of myosin heavy chain messenger RNA: evidence provided by using a recombinant DNA plasmid.

A recombinant DNA plasmid, designated pMHC25, has been constructed that contains structural gene sequences for rat skeletal muscle myosin heavy chain (MHC). The identity of the MHC sequence insert in pMHC25 was determined by muscle-tissue specificity, inhibition of MHC protein synthesis in vitro by hybrid-arrested translation, purification of mRNA that directs the synthesis of MHC protein in vitro, and hybridization to a 33S cytoplasmic mRNA found only in differentiated muscle cells. pMHC25-DNA-excess filter hybridizations were used to show that more than 90% of the newly synthesized MHC mRNA that appears in the cytoplasm of differentiated L6E9 myotubes contains a long 3' poly(A) tail. In contrast, 90% of the MHC mRNA that accumulates in the cytoplasm of these same cells during myogenic differentiation lacks this long 3' poly(A) tail. These results suggest the occurrence of a posttranscriptional event in differentiated L6E9 myotubes that involves the cytoplasmic processing of poly(A)+ MHC mRNA to poly(A)- or poly(A)-short MHC mRNA.

Characterization of sarcomeric myosin heavy chain genes.

Myosin heavy chain is encoded by a large multigene family. Using pMHC-25, a recombinant cDNA clone isolated from the rat myogenic cell line L6E9, four members of this family in the rat have been isolated and shown to be tissue-specific and developmentally regulated. The coding regions of these genes share regions of homology interspaced with regions of non-homology. Detailed analysis of one embryonic and one adult myosin heavy chain gene shows that the coding sequences are interrupted by numerous intervening sequences whose number, size, and distribution do not appear to be conserved in the same organism or between species.

Sarcomeric myosin heavy chain is coded by a highly conserved multigene family.

pMHC25, a recombinant plasmid containing myosin heavy chain (MHC) cDNA sequences from differentiated myotubes of the L6E9 rat cell line, has been shown to hybridize to all sarcomeric MHC mRNAs so far tested but not to nonsarcomeric MHC mRNAs. In addition, pMHC25 hybridizes to multiple restriction endonuclease-digested fragments of rat genomic DNA corresponding to different MHC genomic sequences. Thus, the MHC gene represented by pMHC25 is a member of a sarcomeric MHC multigene family that has regions of sequence homology shared among its members. This sarcomeric MHC multigene family has been estimated to be composed of a minimum of seven genes, some of which are polymorphic in the rat. We have also determined that pMHC25 hybridizes to MHC gene sequences in genomic DNA of all species that have striated muscle, ranging from nematodes to man. Sarcomeric MHC genes, therefore, have been horizontally and vertically conserved in evolution. Additionally, we have used the pMHC25 plasmid to demonstrate that MHC genes do not undergo rearrangement or amplification during muscle cell differentiation.

Expression of completely gamma-carboxylated and beta-hydroxylated recombinant human vitamin-K-dependent protein S with full biological activity.

Human anticoagulant vitamin-K-dependent protein S was expressed in mouse C127 cells using a bovine papilloma virus vector system. A full-length cDNA construct was introduced into the vector in the 5' untranslated region of the mouse metallothionein-I gene. Transfected cells expressed approximately 10 micrograms/ml of the recombinant protein which was purified by ion-exchange chromatography followed by affinity chromatography using Ca2(+)-dependent monoclonal antibodies against the region of protein S containing 4-carboxyglutamic acid. Recombinant protein S was structurally and functionally similar to protein S purified from plasma. On SDS/polyacrylamide-gel electrophoresis recombinant protein S had a slightly higher molecular mass than plasma protein S. After treatment with endoglycosidase F, the proteins comigrated suggesting the observed molecular mass difference to be due to alterations in the N-linked carbohydrate side chains. Recombinant and plasma protein S demonstrated identical amino-terminal sequences, similar amino acid composition and number of 4-carboxyglutamyl and 3-hydroxyaspartyl/asparaginyl residues. Recombinant protein S had the same affinity for Ca2+ as protein S from plasma and the two proteins had the same activated protein C cofactor activity in a functional assay. In addition, both forms of protein S formed complexes with C4b-binding protein with the same apparent Kd. Protein S is the most extensively post-translationally modified vitamin-K-dependent protein, and all the modifications were carried out in the recombinant DNA system yielding a recombinant protein S with full biological activity.

Isolation and sequence of the cDNA for human protein S, a regulator of blood coagulation.

Protein S is a cofactor of activated protein C; together they function as a regulator of blood coagulation. A human liver cDNA library constructed in bacteriophage lambda gt11 was screened with DNA fragments from a full-length bovine cDNA clone encoding protein S. Several cDNA clones were isolated and sequenced. The combined cDNA sequences encoded the mature protein and 15 residues of the leader sequence when compared to bovine protein S. Human protein S is a single-chain protein consisting of 635 amino acids with 82% homology to bovine protein S. After an NH2-terminal gamma-carboxyglutamic acid-containing region, there is a short region with thrombin-sensitive bond(s), followed by a region with four repeat sequences that are homologous to the precursor of mouse epidermal growth factor. In contrast to the other vitamin K-dependent plasma proteins, the COOH-terminal portion of human protein S does not show any resemblance to serine proteases.