Biomedical subjects

J D Friesen

Publications and source records attributed to J D Friesen.

At least 91 records · Page 5Linked to original sources

PubMed1981

Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene.

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. beta-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH26 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in minimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine.

3-Isopropylmalate Dehydrogenase↗

PubMed1980

"Two out of three" codon reading leading to mistranslation in vivo.

Strains of Escherichia coli were starved for asparagine or lysine in order to increase the in vivo level of mistranslation. In a relA strain, asparagine starvation increased the error frequency in elongation factor Tu to 0.12 mistake per asparagine codon, while with lysine starvation in the same strain the error frequency per lysine codon was 0.008. The pattern of isoelectric point changes in the altered protein produced is consistent with third position misreading in the AAN codon group. This high level of mistranslation is not seen in streptomycin resistant (rpsL) strains or in most relA+ strains.

Asparagine↗

PubMed1980

Post-transcriptional regulatory mutants in a ribosomal protein-RNA polymerase operon of E. coli.

A high copy number plasmid that carries the promoter PJ, intact rplJ and a deletion of the 3' terminal portion of rplL is detrimental to the growth of the host bacterium. Six independent point mutations on the plasmid that overcome this detriment have been isolated. Nucleotide sequence analysis demonstrates that all six mutants are single base pair alterations, occur within the leader region of the rplJ operon and are well removed from the presumed position of the primary promoter, PJ. These mutant plasmids exhibit normal transcription of rplJ-rplL but do not translate rplJ messenger RNA to yield plasmid-specified L10 ribosomal protein. We suggest that these mutations define a regulatory region within the leader sequence of the RNA transcript that serves to modulate the translational efficiency of rplJ messenger RNA.

Bacterial Proteins↗

PubMed1980

The nucleotide sequence of tufB and four nearby tRNA structural genes of Escherichia coli.

In order to approach some of the problems suggested by the presence of two genes for EF-Tu in Escherichia coli we have determined the nucleotide sequence of tufB and its genetic surroundings. Comparison with the known amino acid sequence of EF-Tu (Arai et al., 1980) and with the tufA nucleotide sequence (Yokota et al., 1980) allows some conclusions to be made regarding possible relationships between tufA and tufB. We also report the sequence of four tRNA structural genes (thrU, tyrU, glyT and thrT) that lie immediately to the 5' side of tufB.

Bacterial Proteins↗

PubMed1980

Bacteriophage lambda vehicle for the direct cloning of Escherichia coli promoter DNA sequences: feedback regulation of the rplJL-rpoBC operon.

A derivative of bacteriophage lambda, lambda 21, has been constructed and used for the cloning of Escherichia coli DNA fragments carrying promoters. Phage lambda 21 lacks the lac promoter operator and can accept DNA fragments up to 9.8 kilobases in size at a unique HindIII restriction endonuclease site adjacent to lacZ. Recombinant phage that carry promoters are readily identified by their expression of lacZ. Lysogens of these phages in strains harboring a deletion of the chromosomal lac operon are capable of growth on lactose as sole carbon source and can be used to study some of the regulatory signals that act upon the cloned promoter. In principle, lambda 21 can be used to clone any promoter DNA sequence with HindIII termini. PJ, the primary promoter for the rplJL-rpoBC operon, and P beta, a weak promoter for rpoBC, have been cloned in lambda 21. Transcription of lacZ from PJ was found to be subjected to feedback control by ribosomal protein L10 and to a lesser extent by ribosomal protein L7/L12. This suggests a possible L10-binding site near PJ that regulates transcription from that promoter. Lysogens of the phage that carries P beta responded to two regulatory signals: a rho-sensitive termination site preceding rpoBC and induction of beta-galactosidase synthesis by rifampicin. This suggests that P beta is a bona fide promoter for rpoBC.

Bacterial Proteins↗

PubMed1980

Characterization of promoter-cloning plasmids: analysis of operon structure in the rif region of Escherichia coli and isolation of an enhanced internal promoter mutant.

Using the promotor-cloning vehicle described by An and Friesen (J. Bacteriol. 140:400-410, 1979), Escherichia coli chromosomal deoxyribonucleic acid fragments derived from the lambda drifd18 transducing phage were cloned in one of several unique restriction endonuclease sites adjacent to tetracycline(tet) genes that lack their own promotor. One of these plasmids has been used to isolate nine variants having mutations that lie in a putative internal promoter which is located between rplL and rpoB. Deoxyribonucleic acid sequence analysis revealed that, in all nine mutants, a single base change, C to T, in the ribonucleic acid polymerase recognition site led to a large increase in promoter activity. Analysis of a variety of plasmids in which tet is fused to various promoters yielded the following results: (i) rplK and rplA, genes for ribosomal protein L11 and L1, respectively, were cotranscribed from a common promoter located upstream from rplK; (ii) there was a strong promoter in the region between the rplKA operon and rplJ, the gene for ribosomal protein L10; (iii) an attenuator region was located between rplL, the gene for ribosomal protein L12, and rpoB, the gene for ribonucleic acid polymerase subunit beta; (iv) transcription terminated immediately after rpoC, the gene for ribonucleic acid polymerase subunit beta'; (v) a gene coding for unknown protein U, which is located between tufB and the rplKA operon, had its own promoter; (vi) the tufB gene was separated from all of the genes described above and had its own promoter.

Bacterial Proteins↗

PubMed1980

Identification and quantitation of elongation factor EF-P in Escherichia coli cell-free extracts.

EF-P, an elongation factor that stimulates peptide bond synthesis in vitro with some aminoacyl-tRNAs, has been identified by two-dimensional gel electrophoresis and the cellular content at three points in the growth curve has been measured. The molecular weight of EF-P is estimated to be 21 000. EF-P is a slightly acidic protein whose isoelectric point is close to RNA polymerase subunit alpha. The amount of EF-P present in Escherichia coli is about 1/10th that of EF-G and the level is independent of the stage of cell growth; there is about one EF-P per 10 ribosomes. It is also shown that a highly purified preparation of EF-P is free of all known protein synthesis factors and ribosomal proteins.

Escherichia coli↗

PubMed1979

Expression of Escherichia coli ribosomal protein and RNA polymerase genes cloned on plasmids.

Fragments of lambda drifd 18 DNA with different end-points within the set of structural genes of ribosomal proteins L11 (RPLK), Li (rplA), L10 (rplJ) and L12 (rplL) as well as the beta (rpoB) ANd beta' (rpoC) subunits of RNA polymerase have been cloned on plasmids. These plasmids were transformed in host cells which were mutant for each of the genes, enabling expression of both wild-type (plasmid-borne) and mutant (chromosomal) genes to be differentiated. On the basis of these results we propose the following genetic structure for the region: rplK and rplA are in one operon; rplL, rpoB and rpoC are in a second. Our data suggest the possibility that rplJ is by itself in an operon situated between the other two.

DNA, Recombinant↗

PubMed1979

Individual messenger RNA half lives in Saccharomyces cerevisiae.

We have measured the decay half-life of functional messenger RNA (mRNA) for some thirty different proteins in the yeast Saccharomyces cerevisiae. Production of newly synthesized mRNA was halted by raising the temperature of a culture of a temperature-sensitive mutant, ts 136. Aliquots of this culture were pulsed-labelled with [35S]-methionine at various times after the temperature shift and the radioactive proteins separated on the two-dimensional gel electrophoresis system of O'Farrell. We find a range in the decay half lives of individual mRNA species which varies from 3.5 min to greater than 70 min. We find three general classes of decay curves, (a) simple exponential (first order); some of these showed a shoulder before onset of exponential decay; (b) bi-component or multi-component concave upward; (c) initial stimulation of rate of mRNA synthesis, followed by virtually undetectable decay.

Electrophoresis↗

PubMed1979

Cloning the spoT gene of Escherichia coli: identification of the spoT gene product.

We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.

Alleles↗

PubMed1979

Two-dimensional protein patterns during growth and sporulation in Saccharomyces cerevisiae.

Proteins synthesized by Saccharomyces cerevisiae in presporulation and sporulation media were compared by using sporulating (a/alpha) and nonsporulating (a/a and alpha/alpha) yeast strains. Total cellular proteins were labeled with [35S]methionine and analyzed by two-dimensional polyacrylamide gel electrophoresis. Autoradiograms and/or fluorograms showed some 700 spots per gel. Nine proteins were synthesized by a/alpha cells which were specific to vegetative, log-phase conditions. During incubation in sporulation medium, sporulating (a/alpha) cells synthesized 11 proteins not present in vegetatively growing cell. These same 11 proteins, however, were synthesized by nonsporulating (a/a and alpha/alpha) cells on sporulation medium as well. Nonsporulating diploids (a/a and alpha/alpha) were also examined with the electron microscope at various times during their incubation in sporulation medium. Certain cellular responses found to be unique to meiotic yeast cells in previous studies were exhibited by the nonsporulating controls. The degree to which all cell types (a/alpha, a/a, and alpha/alpha) were committed to sporulation was also determined by shifting cells from sporulation medium to vegetative medium. Some commitment to the meiotic pathway was observed in both the a/alpha and the a/a, alpha/alpha cells.

Culture Media↗

PubMed1979

Chimeric plasmids for cloning of deoxyribonucleic acid sequences in Saccharomyces cerevisiae.

Two sets of plasmids, each carrying a Saccharomyces cerevisiae gene and a portion or all of the yeast 2-micron circle linked to the Escherichia coli plasmid pBR322, have been constructed. One of these sets contains a BamHI fragment of S. cerevisiae deoxyribonucleic acid that includes the yeast his3 gene, whereas the other set contains a BamHI fragment of S. cerevisiae that includes the yeast leu2 gene. All plasmids transform S. cerevisiae and E. coli with a high frequency, possess unique restriction endonuclease sites, and are retrievable from both host organisms. Plasmids carrying the 2.4-megadalton EcoRI fragment of the 2-micron circle transform yeast with 2- to 10-fold greater frequency than those carrying the 1.5-megadalton EcoRI fragment of the 2-micron circle. Restriction endonuclease analysis of plasmics retrieved from S. cerevisiae transformed with plasmics carrying the 2.4-megadalton EcoRI fragment showed that in 13 of 96 cases the original plasmic has acquired an additional copy of the 2-mcron circle. These altered plasmids appear to have arisen by means of an interplasmid recombination event while in S. cerevisiae. A clone bank of S. cerevisiae genes based upon one of these composite plasmids has been constructed. By using this bank and selecting directly in S. cerevisiae, the ura3, tyr1, and met2 genes have been cloned.

Cloning, Molecular↗

PubMed1979

Plasmid vehicles for direct cloning of Escherichia coli promoters.

A multicopy plasmid cloning vehicle, pGA22, which carries genes for ampicillin resistance (Apr), tetracycline resistance (Tcr), chloramphenicol resistance (Cmr), and kanamycin resistance (Kmr) has been constructed. This plasmid has five unique sites for restriction endonucleases EcoRI, PstI, XhoI, SmaI, and SalI within antibiotic resistance genes. pGA22, which is 5.1 megadaltons in size, has a low copy number (probably fewer than 10 per genome), is capable of relaxed replication, and is mobilized by F-factor at a frequency of 10(-5). A series of promoter-cloning vehicles, pGA24, pGA39, and pGA46, has been developed from pGA22. In these plasmids the natural promoter for Tcr has been removed and has been replaced by small deoxyribonucleic acid fragments carrying unique sites for several restriction endonucleases. Cells carrying these vectors are sensitive to tetracycline unless insertional activation of the Tcr occurs by cloning a promoter-carrying deoxyribonucleic acid fragment in one of the unique sites adjacent to the 5' end of Tcr. In this way, promoters carried on a HindIII-generated deoxyribonucleic acid fragment can be inserted at the HindIII site of plasmid pGA24, pGA39, or pGA46. A promoter in fragments generated by digestion with restriction endonuclease XmaI or PstI or by any restriction endonucleases which generate flush ends, such as SmaI, PvuII, HpaI, HincII, or HaeIII, can be clones in plasmid pGA39. Plasmid pGA46 can be used to detect a promoter fragment carried on a BglII, BamHI, MboI, or PstI fragment. We also describe a plasmid, pGA44, with a unique KpnI site in the rifampin resistance gene rpoB.

Bacterial Proteins↗

PubMed1978

Nonsense and insertion mutants in the relA gene of E. coli: cloning relA.

We have made use of lysogens of a specialized transducing bacteriophage, lambdapyrG+ relA+, to select nonsense (relAnon) and insertion (relAins) mutations in the relA gene. Three independent relAnon mutants were isolated on the phage. In all three, the relaxed phenotype was suppressed by supD, supE, supF or sup6. Three independent relAins mutants were isolated, all containing an insertion element (probably IS2) in an apparently identical location in the relA gene. Polyacrylamide gel electrophoretic analysis of peptides synthesized by the phages in ultraviolet lightkilled host cells revealed that no stringent factor was coded for by either the relAins or relAnon phages (the latter in a sup+ cell); stringent factor was detected when the relAnon phages were used in a similar experiment with supD or supE host cells. The relAnon and relAins mutations could be crossed in haploid form in the E. coli chromosome. These recombinants grew with a normal doubling time, had a ppGpp pool which was between 70 and 100% compared with the classical relA strain, and underwent a normal carbon source shift-down. A restriction endonuclease map of the pyrG relA region of the specialized transducing phage is presented in which the position of the insertion element (recognized by a novel Hind III-cut site) defines the position of the relA gene. This position was verified by an analysis of the structure of five plasmids formed by cloning portions of the region in the pBR322 cloning vehicle. Our results indicate that the relA gene is not an essential cellular function, that there might be a second mechanism for the synthesis of basal level ppGpp in the cell and that the sole function of the relA gene is apparently the high level ppGpp synthesis triggered in response to deacylated tRNA.

Bacterial Proteins↗

PubMed1978

Stuttering: high-level mistranslation in animal and bacterial cells.

In both bacterial and mammalian cells, extreme starvation for certain amino acids resulted in translational errors that could be easily detected by two-dimensional polyacrylamide gel electrophoresis. On two-dimensional gels, the faulty proteins were shown as a trail of spots with molecular weights similar to those of the authentic proteins but separated in the isoelectric focusing dimension, a phenomenon we call "stuttering." The direction of charge shift depended on the amino acid substituted and could be predicted from misreading of pyrimidines for purines at the third position of the codon. It is expected that this phenomenon will provide a rapid means of measuring the fidelity of the translational machinery from cell type to cell type.

Amino Acids↗

PubMed1977

Interaction of alleles of the relA, relC and spoT genes in Escherichia coli: analysis of the interconversion of GTP, ppGpp and pppGpp.

Mutants in the spo T gene have been isolated as stringent second site revertants of the relC mutation. These show varying degrees of the characteristics associated with the spoT1 gene, viz relative amount and absolute levels of both pppGpp and ppGpp and the decay rate of the latter. The entry of 3H-guanosine into GTP and ppGpr pools in spoT+ and spoT1 cells either growing exponentially or during amino acid starvation was determined, and the rate of ppGpp synthesis and its decay constant calculated. During exponential growth the ppGpp pool is 2-fold higher, its decay constant 10-fold lower, and its synthesis rate 5-fold lower in spoT- than in spoT+ cells; during amino acid starvation the ppGpp pool is 2-fold higher, its decay constant 20-fold lower, and its synthesis rate 10-fold lower in spoT than in spoT+ cells. In one of the "intermediate" spoT mutants the rate of entry of 3H-guanosine into GTP, ppGpp and pppGpp was measured during amino acid starvation. The data form the basis of a model for the interconversion of the guanosine nucleotides in which the flow is: GDP leads to GTP leads to pppGpp leads to ppGpp leads to Y. Calculations of the rates of synthesis and conversion of pppGpp and ppGpp under various conditions in various spoT+ and spoT- strains indicate that the ppGpp concentration indirectly controls the rate of pppGpp synthesis.

Alleles↗

PubMed1977

Fine-structure mapping of the rts, rplK, rplL, and rpoB genes of Escherichia coli.

The specialized transducing phage lambdacI857S7drifd18 was used as a donor in a transductional mapping of four genes in the rif region of the Escherichia coli genome. The gene order was rts-2.9-rplL-0.8-rpoB, where the numbers indicate intermarker distances in kilobases. The possible orientation of these genes with respect to each other and to neighboring genes is discussed.

Bacterial Proteins↗

PubMed1976

Biosynthetic regulation of individual proteins in relA+ and relA strains of Escherichia coli during amino acid starvation.

An isogenic pair of Escherichia coli mutants (relA+ tufB valSts and relA1 tufB valSts) has been cultured at several temperatures to establish various degrees of limitation for valyl-tRNA synthetase. The biosynthetic rate of 16 identifiable proteins, most of which are components of the transcription and translation apparatus, was measured by pulse-labelling with [35S]-methionine, followed by protein separation using two-dimensional gel electrophoresis (O'Farrell, 1975). No single pattern of response to amino acid starvation of biosynthetic rate was observed. EF-Ts, L12 and S6 were found to be under strong stringent and relaxed regulation; EF-G, EF-Tu-A and S1 are under strong stringent, but weak relaxed regulation; EF-Tu-B, alpha, VRS, IRS and ARS are under week stringent and weak relaxed regulation; beta is under weak stringent regulation and does not respond at all to relaxed conditions; the biosynthetic rate of a protein called stringent starvation protein is strongly stimulated, relative to other proteins, in the starved stringent strain.

Amino Acids↗