Caesium fluoride-induced changes in the c.d. spectra of synthetic DNA fragments.

Ten DNA fragments containing self-complementary alternating sequences of adenine and thymine differing in length and the starting nucleotide were studied by c.d. spectroscopy. It was found that d(TATATATA) but not d(ATATATAT), d(TATATA), d(CTATATAG) or (dT-dA)20 isomerized into the unusual X-DNA double helix at molar concentrations of CsF in solution. But in contrast to poly(dA-dT), the octamer (dT-dA)4, isomerized very slowly, at relatively low CsF concentrations and the isomerization was strongly dependent on the octamer concentration. A model is proposed to account for the observed properties of the B-to-X isomerization on the oligomer level.

Synthesis and immunogenicity of hepatitis B virus envelope antigen expressed by recombinant vaccinia virus. Finding of retention signal in the C-terminal portion of the preS1 domain of subtype adyw.

Five different recombinant vaccinia viruses expressing the envelope antigen of hepatitis B virus (HBsAg) under the control of the P7.5 promoter were constructed. Cell cultures infected with some of the recombinant viruses synthesized both middle (M) and major surface (S) protein of HBsAg. It was shown that the length of the nontranslated sequence preceding preS2-ATG influenced the extracellular or intracellular HBV antigen distribution and the preS2:S antigen ratio. Some recombinants synthesized an M protein that was enlarged by additional 35 amino acids of preS1 domain and was entirely retained within the infected cells. Antibody responses to the S and preS2 antigens in mice revealed significant differences in the immunogenicity of individual recombinants.

[Sex determination using the polymerase chain reaction method for amplification of DNA segments on the X and Y chromosomes]. / Stanovení pohlaví metodou polymerázové retezové reakce (PCR) amplifikací úseku DNA chromosomu X a Y.

The authors tested the rapid and accurate prenatal and postnatal diagnosis of sex by the method of amplification of specific portions of DNA of sex chromosomes. To ensure a maximum reliability of the diagnosis the authors recommend combined examination of specific sequences from the heterochromatin region of the long arm of the Y chromosome (Yq) and from alphoid satellite sequences of DNA from the pericentromeric portion of chromosome X and Y (Xc and Yc). For further improvement of the reliability the authors recommend to digest the Yc amplification product obtained by means of restriction endonucleases Msp I, Eco Ri and Hini I. Correct assessment of the diagnosis was confirmed in all examined subjects (12) from DNA and chorium.

[Experience with rapid molecular genetic diagnosis using the polymerase chain reaction]. / Zkusenosti s metodou rychlé molekulárne genetické diagnostiky polymerázovou retezovou reakcí.

The authors give an account of their experience with 1000 amplifications of DNA by the polymerase chain reaction for the prenatal diagnosis of cystic fibrosis. The method is demonstrated on examples of examinations of the informativity value and prenatal diagnosis in the first trimester of pregnancy in families with a 25% risk of cystic fibrosis, using J 3.11 (Msp I), met H (Msp I), KM 19 (Pst I), CS 7 (Hha I), Mp6d9 (Msp I), XV 2c (Taq I) probes. The authors summarize methodical check-up and safety measures to ensure the reliability of diagnoses made by the PCR method.

A recombinant vaccinia virus expressing hepatitis B virus middle surface protein. Restricted expression of HBV antigens in human diploid cells.

Several vaccinia virus recombinants inducing the synthesis of the middle surface (M) protein of hepatitis B virus (HBV) were constructed. One of them, denoted v137, was examined in some detail. The virus replicated nearly to the same extent in various cell lines, viz. human embryo diploid fibroblast LEP and MRC-5 cells, rabbit embryo fibroblast REF cells, TK- rat RAT-2 cells, and green monkey CV-1 cells. However, the production of M protein was found considerably lower in the human LEP and MRC-5 than in the other cells examined. In addition, the kinetics of M formation were different in these two cell systems, LEP cells lagging significantly behind CV-1 cells. The low-level production of M protein in LEP cells was not increased by repeated v137 passages in LEP cells, nor by a passage in a laboratory worker accidentally infected with the v137 virus, nor by shortening the leader sequence preceding the translation initiation codon. The greater part of the M antigen was found to be cell associated, more so in the cells of human than monkey origin. From the major HBV S antigen (HBsAg) isolated from the plasma of chronically infected subjects, the antigen released by cell destruction differed by binding to polymerized human albumin. This property was utilized in ELISA to detect anti-preS2 antibody. Rabbits inoculated intradermally with the v137 virus developed antibodies reactive in this assay as well as with a synthetic peptide corresponding in the amino acids 14-34 of the NH2 terminus of the HBsAg preS2 region.

Cystic fibrosis marker testing in Bohemia with polymerase chain reaction.

Conditions for assessing KM-19 probe detected by Pst-1 restriction fragment length polymorphism (RFLP) by means of polymerase chain reaction were provided. Computer controlled mechanical arm with waterbaths and cloned heat-stable DNA polymerase was used. Results of KM-19 allelic frequencies on 90 cystic fibrosis chromosomes are presented. Allele two frequency was -0.833.

The rapid molecular genetic diagnosis of cystic fibrosis by polymerase chain reaction: an experience report.

The authors report their experience with about two thousand DNA amplifications by polymerase chain reaction (PCR) in prenatal diagnosis of cystic fibrosis. The method is demonstrated on examples of diagnostic informativity and prenatal diagnosis examination in a family at 1 in 4 risk of the disease using closely CF-linked diagnostic polymorphisms: J3.11/MspI, MetH/MspI, CS7/HhaI, KM19/PstI, Mp6-d9/MspI and XV2c/TaqI, PCR methodology and safety precautions are discussed.

Nucleotide sequence of the right early region of Bacillus phage phi 15 and comparison with related phages: reorganization of gene 17 during evolution.

The rightmost 2016 bp of the Bacillus subtilis phage phi 15 genome were sequenced. The nucleotide sequence was compared with the homologous regions of the related phages PZA and phi 29. There are six open reading frames (ORFs) in this region of the phi 15 genome; all of them are present in the PZA and phi 29 genomes. One of the ORFs was assigned to gene 17, which is involved in the replication of the phage DNA. Gene 17 has undergone reorganization during the evolution of this phage family. Comparison of the nucleotide sequence of its mRNA-like strand in phi 15, PZA and phi 29 showed that deletions in its central and 3'-end-proximal parts are tolerated and do not interfere with the gene 17 product function. It seems that the only portion of gene 17 that has to be conserved to encode the functional product is its 5'-end-proximal part.

Solid-phase assembly of cow colostrum trypsin inhibitor gene.

A gene for cow colostrum trypsin inhibitor (CTI) was constructed from synthetic oligonucleotides using a novel method of solid-phase gene assembly. In the first step an anchor oligonucleotide was covalently bound to the CNBr-activated Sephacryl S-500 support. Next, triads or tetrads of separately annealed oligonucleotides were stepwise hybridized to the immobilized complementary sequence, with washing after each step. In the last step a linearized vector molecule was ligated to the assembled gene. The whole construct was released from the solid support with a restriction enzyme, circularized, and used for transformation, with a high yield of recombinant clones being obtained. The method represents a generally applicable approach to rapid and efficient assembly of extended DNA duplexes.

Lipid derivatives of (2'-5')oligo A.

The synthesis of adenylyl-(2'-5')-adenylyl-(2'-5')-2', 3'-O-(1-methoxyhexadecylidene)-adenosine (III) and its 5'-phosphorylated analogue (V) is described. Phosphorylation was achieved by (2-cyanoethyl)-phosphodichloridite agent followed by iodine oxidation.

Chloridite and amidite automated synthesis of oligodeoxyribonucleotides using amidine protected nucleosides.

Automated synthesis of oligodeoxyribonucleotides using in situ prepared methylchlorophosphite intermediates from amidine protected purine nucleosides is described. Phosphoramidite method using 1-methylimidazole trifluoromethanesulfonate as mediator is described.

Automated 3'-H-phosphonate synthesis of oligodeoxyribonucleotides using amidine protected purine synthons.

Side reaction during phosphitylation of 5'-O-dimethoxytrityl-N2-isobutyryl-2'-deoxyguanosine was observed. As a suitable dG synthon, 5'-O-dimethoxytrityl-N2-dimethylaminomethyl-ene-3'-H-phosphonate was developed and used, along with 3'-H-phosphonates of 5'-O-dimethoxytrityl derivatives of dT, N4--benzoyl-dC and N6-dimethylaminoethylidene-dA, in automated synthesis of several oligodeoxyribonucleotides containing 30-40 bases.

Solid-phase assembly of DNA duplexes from synthetic oligonucleotides.

A new method of rapid and efficient assembly of extended DNA duplexes in solid phase was developed. Subassemblies of separately annealed oligonucleotides were stepwise hybridized to each other on a solid support. Two types of supports with anchor oligonucleotide were tested: Fractosil-1000 with oligo-dT sequence and Sephacryl S-500 with an oligonucleotide bound via CNBr-activation procedure. Sephacryl S-500 turned out to be the support of choice since all enzymatic reactions of the assembly procedure (phosphorylation, ligation, restriction enzyme digestion) could be efficiently performed with DNA immobilized on Sephacryl S-500 particles.

Design of synthetic oligonucleotide duplexes for site-directed mutagenesis with recombinant plasmids.

Screening for site-mutated plasmids may be greatly facilitated by the different occurrence of the restriction sites distinguishing the altered sequence from the original one. Synthetic oligonucleotides can be so designed that they, apart from defining the mutation, also create a new restriction site recognizable in the restriction pattern of the mutant plasmid.

Hydrolysis of adenylyl(2'-5')adenosine in chick embryo sera.

Similar to adult chick sera in chick embryo sera the enzyme activity hydrolyzing the core of the dimer of A-5A - adenylyl(2' -5')adenosine is present. The enzyme activity possesses the same properties in both sera. Heating of the serum to 60 degrees C for 30 minutes does not affect the enzyme activity. A marked decrease in activity occurs after heating of the serum to above 65 degrees C. Hydrolysis of A2' p5' A in the serum is more efficient under alkaline conditions (pH 9-10) than at physiological pH 7.2-7.4.

Chemical synthesis of octadeoxyribonucleotides containing sequence-specific 3'-deoxy-3'-chlorouridine residues and 2'-5' internucleotide linkages by the phosphite chloridite approach.

3'-Deoxy-3'-chlorouridine has been chemically incorporated into two octanucleotides at various positions using in situ prepared phosphite chloridite intermediates. The behaviour of these modified selfcomplementary DNA fragments containing 2'-5' internucleotide bonds in the neighbourhood of 3'-deoxy-3'-chlororibose in respect to chemical sequencing, polynucleotide kinase and T4 DNA ligase has been studied.

Molecular cloning and identification of cDNA recombinants coding for bovine prochymosin in E. coli.

Poly(A) RNA was isolated from the gastric mucosa of the bovine fourth stomach (the abomasum) using and analysing several calves not older than 12 days. The amount of the preprochymosin mRNA in the mucosa of those animals at best reaches about 5-10% of the poly(A) RNA as estimated by in vitro translation and immunoprecipitation. Starting from that material double-stranded complementary DNA was synthesized, inserted by dG dC tailing into the PstI site of the vector plasmid pBR322 and used for transformation of E. coli. Tetracycline resistant clones containing DNA sequences coding for the full length of prochymosin were recognized by colony hybridization with five specific d-oligonucleotides corresponding either to the N-terminal, the middle or the C-terminal part of prochymosin. Six recombinants were detected by screening of 1 500 recombinants with an oligonucleotide which corresponds to positions 649 to 663 of the nucleotide sequence published by Harris et al. (1982). Two of them were found to cover together the complete prochymosin sequence as evidenced by both positive colony hybridization with either the N-terminal or the C-terminal oligonucleotide probe, as well as by the restriction pattern of the selected plasmids.

Cloning and expression in Escherichia coli of the synthetic proenkephalin analogue gene.

Enkephalins are pentapeptides with opioid activity that have been found in brain and other neural tissues. They are released by proteolytic processing of the proenkephalin, which contains several enkephalin sequences each flanked by pairs of basic amino acid (aa) residues. We have constructed an artificial variant of the proenkephalin gene by concatenation of synthetic oligodeoxynucleotides (oligo) coding for Met-enkephalin preceded by two arginines. One of the resulting structures, containing eleven enkephalin sequences separated by pairs of arginine codons, was cloned in the expression vector pRE31. The biological activity of enkephalin was detected after the digestion of the isolated plasmid-coded protein with trypsin and carboxypeptidase B. The product of the synthetic gene may thus serve as a defined simplified substrate for the study of the not yet fully understood enzymatic mechanisms of proenkephalin processing.

Stability of (2'-5')oligoriboadenylates in various sera.

Avian and mammalian sera were found to contain an enzyme activity degrading 2-5A oligonucleotides. The most extensive degradation of the A2' p5' A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56 degrees C for 30 min. The 5-mU3' p5' A has also been degraded in chicken serum.

Antiviral and anticellular effects of synthetic (2'-5')-oligoadenylate (A 2' p 5' A 2' p 5' A) in Rauscher murine leukaemia.

Antiviral and antileukaemic effects of the synthetic (2'-5')-oligoadenylate trimer [(2'-5')-ApApA] were demonstrated in BALB/c mice infected with Rauscher murine leukaemia virus (RMLV) by intraperitoneal (i.p.) treatment for 5-20 days (100 micrograms--1 mg daily doses) as evidenced by 72% suppression of viraemia and by decreased activity of serum reverse transcriptase levels. Electron microscopy revealed more than 95% inhibition of RMLV replication as compared to controls in transformed spleen cells from mice treated 5 times with 1 mg dose of (2'-5') ApApA. A significant and dose-dependent reduction of spleen weights of the RMLV-infected mice treated with (2'-5') ApApA was also observed. The antileukaemic effect of (2'-5-') ApApA was enhanced by simultaneous i.p. injection of amphotericin B (20 micrograms/mouse). In comparison to the effect of interferon (IFN) on RNA tumour viruses, our results suggest a higher antiviral activity of the synthetic (2'-5') ApApA oligonucleotide in suppressing RMLV replication in vivo.