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E Jay

Publications and source records attributed to E Jay.

16 recordsLinked to original sources
PubMed1990

Construction of a Co1E1 plasmid bearing inducible high-copy-number phenotype.

In order to construct plasmids bearing inducible high-copy-number phenotype, the cloning plasmid pBR322 was modified as follows: a DNA fragment containing a strong synthetic promoter (P1), synthetic lac operator (O1), DNA sequence corresponding to the RNAI/RNAII region of the Co1E1 replicon and the CAT gene transcription terminator was substituted for the 29 bp EcoRI/HindIII DNA fragment. Two types of plasmids were constructed in this way, differing in the orientation of the RNAI/RNAII fragment. Depending on the orientation these plasmids coded for RNA molecules representing either RNAI or RNAII domains. It was found that when RNAII molecules were overproduced the plasmid copy number was about 4 times higher than that of pBR322 and only negligible change in the plasmid copy-number value was observed upon overproduction of RNAI molecules.

Bacteriocin Plasmids↗
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↗
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 Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold 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↗
PubMed1975

Influence of modified atmosphere storage on aflatoxin production in high moisture corn.

Samples of freshly harvested corn and remoistened corn were inoculated with Asphergillus flavus and stored for 4 weeks at about 27 C in air and three modified atmospheres. Aflatoxins and fat acidity were determined weekly. Corn stored in the modified atmospheres did not accumulate over 15 mug of total aflatoxins per kg. Corn from the high CO2 treatment (61.7 per cent CO2, 8.7 per cent O2, and 29.6 per cent N2) was visibly molded at 4 weeks and had a higher fat acidity than the other treatments. In the N2 (99.7 per cent N2 and 0.3 per cent O2) and controlled atmosphere (13.5 per cent CO2, 0.5 per cent O2, 84.5 per cent N2) treatments, a fermentation-like odor was detected. When the corn was removed from the modified atmospheres it deteriorated rapidly and was soon contaminated with aflatoxins.

Aflatoxins↗
PubMed1975

Survival of Aspergillus flavus and Fusarium moniliforme in high-moisture corn stored under modified atmospheres.

Freshly harvested high-moisture corn with 29.4% moisture and corn remoistened to 19.6% moisture were inoculated with Aspergillus flavus Link ex Fr. and stored for 4 weeks at about 27 C in air (0.03% CO2, 21% O2, and 78% N2) and three modified atmospheres: (i) 99.7% N2 and 0.3% O2; (ii) 61.7% CO2, 8.7% O2, and 29.6% N2; and (iii) 13.5% CO2, 0.5% O2, and 84.8% N2. Kernel infections by A. flavus, Fusarium moniliforme (Sheld.) Snyd. et Hans., and other fungi were monitored weekly. The modified-atmosphere treatments delayed deterioration by A. flavus and F. moniliforme, but their growth was not completely stopped. A. flavus survived better in the remoistened than in the freshly harvested corn. F. moniliforme survived in both. A. flavus and F. moniliforme were the dominant fungi in corn removed from the modified atmospheres and exposed to normal air for 1 week.

Air↗