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J D Friesen

Publications and source records attributed to J D Friesen.

At least 73 records · Page 4Linked to original sources
PubMed1984

RNA secondary structure and translation inhibition: analysis of mutants in the rplJ leader.

We have carried out measurements of the stable binding of the ribosomal protein (r-protein) complex L10-L7/L12 to mutant forms of the mRNA leader of the rplJ operon of Escherichia coli. One of the point mutations, base 1548, which lies within the L10-L7/L12-protected region, almost completely abolishes in vitro formation of a stable complex of L10-L7/L12 with rplJ mRNA leader, and a second point mutation, base 1634, strongly reduces it. These observations constitute strong support for the proposition that L10-L7/L12 binds to the rplJ leader in bringing about translational feedback. To account for the action of these and other mutations, and to explain the mechanism of translation feedback inhibition, we suggest a secondary structure model involving alternate forms of the rplJ mRNA leader.

DNA-Directed RNA Polymerases↗
PubMed1984

Molecular cloning and biosynthetic regulation of cry1 gene of Saccharomyces cerevisiae.

The cryptopleurine resistance gene, cry1, of Saccharomyces cerevisiae has been molecularly cloned using genetic complementation of cryptopleurine sensitivity by the cryptopleurine resistance gene contained in a clone library prepared from DNA of a cryptopleurine resistant strain. Analysis of RNA transcripts indicated that the cry1 gene is the template for a transcript of approximately 900 bases and that the primary transcript contains an intron of approximately 300 bases. In vitro hybrid selection translation experiments indicated that this transcript encodes a protein of molecular weight 17 kilodaltons which on two-dimensional SDS polyacrylamide gels exactly coincides with ribosomal protein rp59. Further analysis showed that when the gene was present on a plasmid of about five copies per cell the amount of messenger RNA was elevated approximately five-fold compared to a cell that had only a single chromosomal copy. The rate of synthesis of ribosomal protein rp59 was not detectably elevated. These data suggest that the cry1 gene is regulated, at least in part, post-transcriptionally.

Alkaloids↗
PubMed1984

Identification, molecular cloning, and mutagenesis of Saccharomyces cerevisiae RNA polymerase genes.

Three different regions of Saccharomyces cerevisiae DNA were identified by using as hybridization probe a fragment of Drosophila melanogaster DNA that encodes an RNA polymerase II (EC 2.7.7.6) polypeptide. Two of these regions have been molecularly cloned. Each contains a sequence related not only to the D. melanogaster DNA fragment that was used as a probe in its isolation but also to the immediately adjacent DNA fragment of the D. melanogaster RNA polymerase II gene. The two cloned S. cerevisiae DNA sequences are each the template for single transcripts in vivo, one of 5.9 kilobases and the other of 4.6 kilobases. In vitro translation of hybrid-selected cellular RNA indicated that the former locus encodes a protein of Mr 220,000, equal in size to the largest polypeptide subunit of S. cerevisiae RNA polymerase II. Disruption of either gene by targeted integration of URA3+ DNA demonstrated that each is single-copy and essential in a haploid genome. We suggest that these S. cerevisiae loci are members of a family of related genes encoding the largest subunit polypeptides of RNA polymerases I, II, and III.

Animals↗
PubMed1984

Isolation and characterization of the RNA2+, RNA4+, and RNA11+ genes of Saccharomyces cerevisiae.

We used genetic complementation to isolate DNA fragments that encode the Saccharomyces cerevisiae genes RNA2+, RNA4+, and RNA11+ and to localize the genes on the cloned DNA fragments. RNA blot-hybridization analyses coupled with genetic analyses indicated the RNA2+ is coded by a 3.0-kilobase (kb) transcript, RNA4+ is coded by a 1.6-kb transcript, and RNA11+ is coded by a 1.3-kb or a 1.7-kb transcript or both; none of the cloned genes contains detectable introns. All three genes were transcribed into messages of very low abundance (approximately 20 times lower than a ribosomal protein message). DNA blot-hybridization revealed that all cloned genes are represented only once in the yeast chromosome. mRNA for RNA2+ and RNA4+ is produced in approximate proportion to gene dosage, whereas RNA11+ transcription appears to be not nearly so dependent on gene dosage. On a medium-copy plasmid (5 to 10 copies per cell), each cloned gene complemented mutations only in its own gene, indicating that each gene encodes a unique function. Genetic analysis by integrative transformation indicated that we cloned the RNA2+, RNA4+, and RNA11+ structural genes and not second-site suppressors.

Base Sequence↗
PubMed1983

The lethal effect of a plasmid resulting from transcriptional readthrough of rplJ from the rplKA operon in Escherichia coli.

A high-copy plasmid, pGA217, which carries a deletion (lacking the carboxy-terminal 20 amino acids) of the structural gene for ribosomal protein L10 (rplJ) is lethal to the cell in the absence of the gene (rplL) for r-proteins L7/L12, but only if the upstream operon for r-proteins L11 (rplK) and L1 (rplA) is present on the same plasmid. Measurements of beta-galactosidase activity of a hybrid protein expressed by a rplL-lacZ fusion indicated that the L10 fragment peptide which lacks the carboxy-terminal 20 amino acids is capable of exerting feedback regulation. Double transformation experiments with two compatible plasmids showed that the detrimental effect of the rplJ deletion on pGA217 can be reversed by the addition of a second plasmid which carries a functional gene for L7/L12. These two pieces of evidence suggest that the lethal effect of pGA217 is due to its property of feeding back on L7/L12 production from the chromosomal rplK gene. The upstream rplKA operon was inferred to have a cis-acting, stimulating effect on rplJ expression from the following evidence: (1) donor plasmids carrying the genes for L11 and/or L1 fail to exert a trans-acting effect, (2) deletion mutants which removed portions of rplK and/or rplA, but maintained the rplKA promoter, rplKp, still retained a severe growth-inhibiting effect. We suggest that these results can be explained by assuming that there is transcription from the rplKA promoter through rplJ and perhaps beyond.

Chromosome Deletion↗
PubMed1983

Mutations in the rpIJ leader of Escherichia coli that abolish feedback regulation.

We have isolated mutants that fail to exhibit biosynthetic feedback regulation of a rpIJ-lacZ fusion. Analysis of these mutants and of others that were isolated earlier indicates that crucial sequences for both translational feedback regulation and efficient translation lie closely intermingled in the central region of the rpIJ mRNA leader 70-195 bases upstream from the translation start of rpIJ. We suggest that our point mutations define a region of the rpIJ leader mRNA to which L10 binds in effecting autogenous translational regulation.

Bacterial Proteins↗
PubMed1983

Expression vehicles used in recombinant DNA technology.

We survey cloning vehicles whose function is to carry and express a gene in host cells including Escherichia coli, Saccharomyces cerevisiae and mammalian cells. In E. coli these include vehicles based on the lac operon, the trp operon, the rho leftward operon, and the recA gone; open reading frame cloning vehicles are also discussed, as are steps that can be taken to extrude a gene product from the cell and the use of plasmids with runaway replication. In S. cerevisiae we discuss vehicles based on the PGK gene, the ADH1 gene, the acid phosphatase gene and the GAL1-GAL10 gene cluster. In mammalian cells we discuss vehicles based on SV40 promoters, the metallothionein gene, retroviral LTR promoters, bovine papilloma virus and vaccinia virus.

Journal Article↗
PubMed1983

Nucleotide sequence of the tcml gene (ribosomal protein L3) of Saccharomyces cerevisiae.

The yeast tcml gene, which codes for ribosomal protein L3, has been isolated by using recombinant DNA and genetic complementation. The DNA fragment carrying this gene has been subcloned and we have determined its DNA sequence. The 20 amino acid residues at the amino terminus as inferred from the nucleotide sequence agreed exactly with the amino acid sequence data. The amino acid composition of the encoded protein agreed with that determined for purified ribosomal protein L3. Codon usage in the tcml gene was strongly biased in the direction found for several other abundant Saccharomyces cerevisiae proteins. The tcml gene has no introns, which appears to be atypical of ribosomal protein structural genes.

Amino Acid Sequence↗
PubMed1982

Isolation of a recombinant lambda phage carrying nusA and surrounding region of the Escherichia coli K-12 chromosome.

A recombinant bacteriophage lambda, lambda argG-6, has been isolated which carries the argG gene and neighbouring loci on an EcoRI-generated 15.5 Kb DNA fragment from the Escherichia coli chromosome. The locations of the argG, nusA and pnp genes on the 15.5 Kb DNA fragment have been determined. In the case of nusA, a Tn5 insertion and sub-cloning of restriction fragments were used to locate the gene. The gene products of nusA and pnp have been identified on one- and two-dimensional polyacrylamide gels. The clockwise gene order was found to be argG-nusA-pnp.

Bacterial Proteins↗
PubMed1982

Evidence for an internal promoter preceding tufA in the str operon of Escherichia coli.

We constructed plasmids carrying tufA from which the major promoter for the rpsL-rpsG-fus-tufA operon (also called the str operon) had been removed. These plasmids continued to express tufA, as judged by the ability to complement mocimycin resistance and by electrophoretic analysis of synthesized proteins. Tn5 transpositions into fus, the gene for elongation factor G, which lies immediately on the 5' side of tufA, failed to obstruct the expression of tufA. The subcloning of a 2,000-base-pair PstI-SmaI DNA fragment (containing the intercistronic region between tufA and fus, the distal portion of fus, and the proximal portion of tufA) next to promoterless tetracycline resistance genes (tet) yielded a plasmid that was capable of bestowing resistance to 12 microgram of tetracycline per ml. The removal of an EcoRI fragment that lies within fus destroyed the ability of the 2,000-base-pair PstI-SmaI fragment to promote the transcription of tet. These data indicate that, in addition to the operon's major promoter rpsLp, there is an internal promoter, tufAp, which can be used for the transcription of tufA, tufAp probably lies within fus, about 50 base pairs upstream from its 3' end and 120 base pairs from the start codon of tufA. The relative activities of tufB and of tufA-from-tufAp were estimated by a comparison of beta-galactosidase activities of almost identical EF-Tu-beta-galactosidase protein fusions; they were approximately equal.

Cloning, Molecular↗
PubMed1982

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.

DNA Restriction Enzymes↗
PubMed1981

Organization and nucleotide sequence of a new ribosomal operon in Escherichia coli containing the genes for ribosomal protein S2 and elongation factor Ts.

We report the nucleotide sequence of the four min region of the Escherichia coli genetic map that includes the genes for ribosomal protein S2 (rpsB) and translation elongation factor EF-Ts (tsf), and the possible location of regulatory sites within this two gene operon. The data indicate that the gene order is: rpsBp-rpsB-tsf-tsft. One potential regulatory site is a 16 nucleotide sequence in the rpsB leader region encompassing the ribosome binding site and the translation initiation codon. This has a high degree of homology with nucleotides 8 through 23 on the 5' end of 165 ribosomal RNA, and might signify a sequence that is necessary for post-transcriptional control of rpsB expression. The data allow one to infer the amino acid sequences of S2 and EF-Ts.

Amino Acid Sequence↗
PubMed1981

Cloning and the nucleotide sequence of the genes for Escherichia coli ribosomal proteins L28 (rpmB) and L33 (rpmG).

The specialized transducing bacteriophage lambda dpyrE DNA was used as a source of DNA to clone two ribosomal protein genes rpmB (L28) and rpmG (L33) on the cloning vehicle pACYC184. Using one of these plasmids, the nucleotide sequence of these two genes and their flanking regions were determined. The amino acid sequences of both proteins deduced from the nucleotide sequences match with the amino acid sequences previously determined, with one exception. The nucleotide sequences suggest that these two ribosomal protein genes are cotranstribed. There was no expression of the second gene of the operon, rpmG, in the absence of the 5' sequences adjacent to the first gene, rpmB. Observation of the structure of mRNA also strongly supports the idea that rpmB and rpmG are in a single transcription unit whose order is: rpmBp-rpmB-rpmG-rpmGt.

Amino Acid Sequence↗
PubMed1981

Expression of the Herpes simplex virus thymidine kinase gene in Saccharomyces cerevisiae.

Yeast plasmids have been constructed that carry the Herpes simplex Virus type 1 (HSV-1) thymidine kinase (TK) gene which is functionally expressed in Saccharomyces cerevisiae. The expression of the TK gene appears to be due to transcriptional read-through from a yeast promoter that lies on the 3' side of the HIS3 gene. The TK+ yeast possesses in vitro thymidine kinase activity which is absent in the original yeast strain. Yeast strains auxotrophic for thymidine monophosphate (dTMP) (tmp1) can grown on thymidine-containing medium after transformation with these plasmids. Tmp+, TK+ S. cerevisiae whose de novo synthesis of dTMP is inhibited with amethopterin plus sufanilamide is also capable of growth in thymidine. S. cerevisiae transformed with such plasmids is capable of incorporating thymidine and bromodeoxyuridine into DNA.

Bromodeoxyuridine↗
PubMed1981

Organization of genes in the four minute region of the Escherichia coli chromosome: evidence that rpsB and tsf are co-transcribed.

Restriction endonuclease-generated DNA fragments of lambda polC-9 (Friesen et al. 1976) have been cloned on plasmid vehicles. Expression of genes carried by these plasmids was determined either by genetic complementation of the appropriate mutants, or in ultraviolet-irradiated cells. On the basis of these experiments we have inferred the following gene order in the four minute region of the Escherichia coli chromosome: tonA-dapD4-dapD2-rpsB-tsf-22 kilodalton protein - fir 27,000-firA-dnaE. We suggest that rpsB and tsf are in one transcriptional unit, with rpsB being promoter-proximal. We also suggest the possible position of the promoter for dnaE.

Chromosomes, Bacterial↗
PubMed1981

Location of the tufB promoter of E. coli: cotranscription of tufB with four transfer RNA genes.

Previous nucleotide sequence studies have demonstrated that the structural genes for four transfer RNA species (thrU, tyrU, glyT and thrT) are positioned close to one another and near tufB, one of the structural genes for elongation factor Tu. We have carried out experiments to determine the position of the tufB promoter and thus to infer whether these five genes are in a single transcription unit. tufB cloned on plasmids was fused to Tc (in operon fusion) or to lacZ (in gene fusion). These plasmids were then subjected to in vitro deletion to locate the promoter responsible for tufB transcription. In addition, the ability of wild-type tufB to complement kirromycin resistance was determined with deletion plasmids. The results indicate that the major promoter for tufB lies upstream from the four transfer RNA genes, and that there might be at least one weak internal promotor, possibly adjacent to tufB. Assuming that the four tRNA genes that lie between the major promoter and tufB are also transcribed from that promoter, we suggest that all five genes lie in a single transcription unit (thrUp-thrU-tyrU-glyT-thrT-tufB-tufBt) whose primary transcript is thus both a transfer RNA precursor and messenger RNA.

Bacterial Proteins↗
PubMed1981

The identification and partial characterization of a plasmid containing the gene for the membrane-associated hydrogenase from E. coli.

Escherichia coli DNA was digested with restriction endonuclease PstI and ligated into the PstI site of plasmid pBR322. Recombinant plasmids that were constructed in this manner were used to transform E. coli H61, a mutant with a decreased level of hydrogenase activity. Complementation of this hydrogenase mutation identified a bacterial clone carrying the gene for the membrane-associated E. coli hydrogenase in plasmid pBL101. In E. coli minicells, the pBL101 DNA directed the synthesis of a protein of a size corresponding to that of the precursor of the E. coli membrane-associated hydrogenase, which appears to contain an uncleaved leader peptide. A restriction map of the cloned DNA was determined for 14 endonucleases.

Cell Membrane↗