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Biomedical subjects

E Jay

Publications and source records attributed to E Jay.

At least 37 records · Page 2Linked to original sources
PubMed1982

Gene expression: chemical synthesis of E. coli ribosome binding sites and their use in directing the expression of mammalian proteins in bacteria.

Mammalian genes, when inserted into bacterial plasmid or phage DNAs, will not be expressed into the corresponding specific proteins in E. coli unless proper initiations signals required for recognition by E. coli ribosomes are provided. We have studied these signals and chemically synthesized two DNA duplexes each containing different initiation signals. These have been inserted in front of the Simian virus 40 (SV40) small tumor antigen gene (SV40 t gene) at varying distances from the ATG initiation codon prior to its cloning into pBR322 plasmid DNA. Plasmid containing clones carrying either of these two synthetic ribosome binding sites (RBS) at varying distances from the SV40 t gene all produced a 17K protein identical to authentic t antigen by immunologic, electrophoretic and proteolytic digestion analyses. This provides a novel method to ensure the specific expression of any contiguous mammalian gene to be cloned to bacteria, and also a unique in vivo method for studying the structure-function (efficiency) relationship of RBS with specific base changes.

Animals↗
PubMed1981

Construction of a general vector for efficient expression of mammalian proteins in bacteria: use of a synthetic ribosome binding site.

With the premise that mRNAs transcribed in Escherichia coli from cloned eukaryotic DNA inserts do not possess the necessary regulatory signals for recognition by prokaryotic ribosomes, we have constructed a general plasmid vector carrying a chemically synthesized prokaryotic ribosome binding site that will ensure the efficient expression of eukaryotic proteins in E. coli. In addition to the regulatory signals necessary for ribosome recognition, the synthetic segment contains, at one end, a Pst I cleavage site which will direct its insertion to pBR322 DNA and, at the other end, a HindIII site to facilitate attachment of the passenger eukaryotic gene. Using simian virus 40 (SV40) tumor (t) antigen as a model system, we have ligated the SV40 DNA fragment containing the entire t antigen gene in tandem with the synthetic ribosome binding site to pBR322 DNA at the Pst I site, which lies within the coding sequence of the beta-lactamase gene. Initiation of transcription at the beta-lactamase promoter would produce a chimeric mRNA with the synthetic ribosome binding signals and the SV40 sequence flanked by beta-lactamase coding sequences. Utilization of the synthetic regulatory signals for initiation of translation is demonstrated by the efficient synthesis, in bacterial transformants, of authentic SV40 t antigen. Excision of the entire SV40 insert by HindIII from those clones that have retained intact HindIII sites at the junction between the ribosome binding site and the SV40 sequence would allow insertion of other heterologous DNAs by using HindIII linkers. The efficient expression of any DNA insert would require that the entire coding sequence be contiguous and that its termini be randomized by treatment with exonuclease III and nuclease S1 to vary the distance between the translational initiation codon and the synthetic ribosome binding site.

Antigens, Neoplasm↗
PubMed1981

Tissue and species-specific effects of small molecular weight nuclear RNA's on transcription in isolated mammalian nuclei.

Small molecular weight nuclear RNA's (SnRNA's) purified from the 0.35 M NaCl extract of chromatin from human and monkey tissues have been found to stimulate transcription of chromatin in isolated nuclei in a tissue- and species-specific manner. While SnRNA from normal human cells (WI38 fibroblasts and placenta) stimulates homologous transcription to some extent, it has a greater activity on transcription of heterologous tissue of the same species and no activity on heterologous tissue of a different species (monkey kidney cells). Likewise SnRNA from monkey cells stimulates transcription of homologous chromatin but has no effect on human cells. Fractionation of the RNA's in polyacrylamide gradient slab gels revealed that in all cases the active RNA was 160-175 nucleotides in length. Our results are compatible with the hypothesis that the active RNA's are involved in the determination and maintenance of tissue differentiation by recognizing promoter or regulator sequences in the DNA and act at the level of the nucleosome to induce tissue-specific genes.

Animals↗
PubMed1980

A study of the efficiency and the problem of sulfonation of several condensing reagents and their mechanisms for the chemical synthesis of deoxyoligoribonucleotides.

The efficiencies and problems of sulfonation of several condensing reagents for deoxyoligoribonucleotide synthesis have been studied. While 2,4,6-triisopropylbenzenesulfonyl chloride (TPSC1) gave very low yield and slow rate of coupling, the new 1:3 mixture of TPSC1 and tetrazole afforded excellent yield and extremely fast rate of reaction with the lowest rate of sulfonation. The difference and advantages of this mixture over triisopropylbenzenesulfonyl tetrazole (TPSTe) and mesitylenesulfonyl tetrazole (MSTe) are discussed. Mechanisms for the coupling reactions with these condensing reagents to account for the difference in their rates and yields of coupling are discussed.

Benzene Derivatives↗
PubMed1980

Specific binding of a chemically synthesized prokaryotic ribosome recognition site. Prospect for molecular cloning and expression of eukaryotic genes.

An icosadeoxyribonucleotide containing the several features found in prokaryotic mRNA ribosome binding sites has been synthesized. This sequence can stimulate the binding of initiator fMet-tRNAf to the ribosome to form a stable 71 S initiation complex identical with those induced by natural messengers. The binding of this synthetic ribosome binding site is absolutely dependent upon initiation factor IF3, and the bound fMet-tRNAf is sensitive to puromycin indicating the formation of a functional initiation complex. A heptadecadeoxyribonucleotide, identical with the icosanucleotide but lacking the terminal A-T-G codon, can also stimulate the stable binding of fMet-tRNAf to the ribosome, suggesting that the selection of the proper A-U-G initiation codon by fMet-tRNAf is subsequent to and a result of the recognition and binding of the fMet-tRNAf . 30 S ribosome complex to the initiation site. The prospect of ligating a similar synthetic ribosome binding site in front of a eukaryotic gene for cloning in an appropriate prokaryotic vector to assure the expresion of the protein is discussed.

Base Sequence↗
PubMed1980

Stimulation of transcription of chromatin by specific small nuclear RNAs.

Small molecular weight nuclear RNAs (SnRNA) purified from the chromatin of SV40-transformed W138 human fibroblasts have been found to stimulate transcription of chromatin in homologous isolated nuclei as well as in nuclei of untransformed human and monkey cells. Stimulation in normal cell nuclei involves an increase in both initiation sites and rate of elongation of RNA chains. Fractionation of the SnRNA in polyacrylamide gradient slab gels revealed that the "active" RNA was 160-175 nucleotides in length.

Cell Line↗
PubMed1977

Relaxed circular SV40 DNA as cleavage intermediate of two restriction endonucleases.

We have determined the mode of cleavage of superhelical SV40 DNA (Form I) by restriction endonucleases EcoRI and HpaII at 37 degrees C. By analysis with agarose gel electrophoresis and direct examination with dark field electron microscopy, we found that a large amount of the single-nicked circular DNA (Form II) was produced before the linear SV40 DNA (Form III) appeared. Thus, both restriction enzymes cleave only one strand of the superhelical DNA first. The second cleavage on the complementary strand occurred after a lag period. The first order rate constant for the second cleavage by EcoRI endonuclease was determined and a kinetic reaction scheme for both enzymes is proposed.

DNA Restriction Enzymes↗
PubMed1977

Chemical synthesis of the hexanucleotide d(A-C-C-A-G-C) required to isolate fibroin mRNA on an affinity column.

The synthesis of the hexanucleotide d)A-C-C-A-G-C), complementary to the 2 major triplets of fibroin mRNA, using the phosphotriester methodology is described. The protected dinucleotides ((MeO)2Tr)dbzA.anC, ((MeO)2Tr)danC.bzA and ((meO)2Tr)dacG.anC were synthesized; the latter two were detritylated and joined in stepwize fashion to the 1st to form the protected hexanucleotide ((MeO)2Tr)dbzA.anC.anC.bzA.acG.anC. The latter was deblocked with NH3 and acid to form the hexanucleotide d(A-C-C-A-G-C). In view of the ability of a prototype affinity column, oligo dC-cellulose, to isolate fibroin mRNA, prospects appear excellent for the d(A-C-C-A-G-C)-cellulose affinity column isolation of fibroin mRNA.

Chromatography, Affinity↗
PubMed1976

Arthrobacter luteus restriction endonuclease recognition sequence and its cleavage map of SV40 DNA.

The nucleotide sequence at the cleavage site of the restriction endonuclease isolated from Arthrobacter luteus (Alu) has been determined. The endonuclease cleaves at the center of a palindromic tetranucleotide sequence to give even-ended duplex DNA fragments phosphorylated at the 5'-end. The endonuclease cleaves SV40 form I DNA into 32 fragments. The order and sizes of these fragments have been determined to provide an Alu cleavage map of the SV40 genome.

Arthrobacter↗
PubMed1976

Synchronous digestion of SV40 DNA by exonuclease III.

We have established an optimal condition for the synchronous digestion of SV40 DNA with Escherichia coli exonuclease III. Electron microscopy and polyacrylamide gel electrophoresis were used to obtain accurate measurements on the lengths of DNA before and after exonuclease III digestion. Based on this finding, a new method for determining the sequence of long duplex DNA can be realized. It involves (a) the synchronous digestion of the DNA from the 3' ends with exonuclease III, followed by (b) repair synthesis with labeled nucleotides and DNA polymerase, and (c) sequence analysis of the repaired DNA.

Base Sequence↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 11. Enzymatic joining to form the total DNA duplex.

The DNA duplex corresponding to the entire length (126 nucleotides) of the precursor for an Escherichia coli tyrosine tRNA has been synthesized. Duplex [I] (Sekiya, T., Besmer, P., Takeya, T., and Khorana, H. G.(1976) J. Biol. Chem. 251, 634-641), corresponding to the nucleotide sequence 1-26, containing single-stranded ends and carrying one appropriately labeled 5'-phosphate group, was joined to duplex [II] (Loewen, P. C., Miller, R. C., Panet, A., Sekiya, T., and Khorana, H. G. (1976) J. Biol. Chem. 251, 642-650) (nucleotide sequence 23-66 or 23-60) was phosphorylated with [gamma-33P]ATP at the 5'-OH ends. Duplex [III] (Panet, A., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 651-657) (nucleotide sequence 57-94 (Fig. 2)) was also phosphorylated at 5'-ends with [gamma-33P]ATP and was joined to duplex [IV] (Caruthers, M. H., Kleppe, R., Kleppe, K., and Khorana, H. G. (1976) J. Biol. Chem. 251, 658-666) (nucleotide sequence 90-126) which carried a 33P-labeled phosphate group on nucleotide 90. The joined product, duplex [III + IV] (nucleotide sequence 57-126) was characterized. The latter duplex was joined to the duplex [I + II] to give the total duplex. The latter contains singlestranded ends (nucleotides 1 to 6 and 121 to 126) which can either be "filled in" to produce the completely base-paired duplex or may be used to add the promoter and terminator regions at the appropriate ends.

Base Sequence↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction.

With the ultimate objective of the total synthesis of a tRNA gene including its transcriptional signals, an Escherichia coli tyrosine suppressor tRNA gene was chosen. The arguments in favor of this choice are presented. A plan for the total synthesis of the 126-nucleotide-long DNA duplex corresponding to a precursor (Altman S., and Smith, J. D. (1971) Nature New Biol. 233, 35) to the above tRNA is formulated. The plan involves: (a) the chemical synthesis of 26 deoxyribooligonucleotide segments, (b) polynucleotide ligase-catalyzed joining of several segments at a time to form a total of four DNA duplexes with appropriate comlementary single-stranded ends, and (c) the joining of the duplexes to form the entire DNA duplex. Ten accompanying papers describe the experimental realization of this objective.

Base Sequence↗
PubMed1976

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 5. Synthesis of the deoxyribopolynucleotide segments representing the nucleotide sequence 71-103.

Chemical syntheses of the pentadecanucleotide, d(G-G-T-G-G-G-G-T-T-C-C-C-G-A-G), the undecanucleotides, d(G-G-T-G-G-G-G-T-T-C-C) and d(C-C-C-C-A-C-C-A-C-G-G), the decanucleotide, d(G-T-A-A-T-G-C-T-T-T), and the nonanucleotides, d(A-T-T-A-C-C-C-G-T) and d(A-G-T-A-A-A-A-G-C) are described. The deoxyribopolynucleotides together represent the DNA duplex corresponding to the nucleotide sequence 71-103 (from the 3'-end) of the gene for the tyrosine suppressor tRNA. Synthesis of the guanine-rich undecanucleotide d(G-G-T-G-G-G-G-T-T-C-C) was performed by the use of a new protecting group for the guanine ring, the methylbutyryl group. The heptanucleotide d[(MeOTr)mbG-mbG-T-mbG-mbG-mbG-mbG], prepared by the new method, was condensed with the tetranucleotide d[panC-anC-T-T(Ac)]. All of the condensations described followed previously developed chemical principles and started with the N- and 5'-protected deoxyribonucleosides. Successive condensations at the 3'-end with protected mononucleotides, preformed di-, tri-, or tetranucleotides gave products which were separated by anion exchange chromatography and characterized by chemical and enzymatic methods.

Base Sequence↗
PubMed1976

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Cobalt↗