Biomedical subjects

N P Fiil

Publications and source records attributed to N P Fiil.

At least 19 recordsLinked to original sources

PubMed1988

Competitive expression of two heterologous genes inserted into one plasmid in Saccharomyces cerevisiae.

Plasmids were constructed which contained two expression units encoding single-chain insulin precursors. Surprisingly, the total amount of insulin precursor produced was similar to that produced from plasmids containing a single expression unit. In this system, therefore, two expression cassettes can be brought to compete for the limited ability of the yeast cell for synthesis and secretion. Using genes encoding B(1-29)-A(1-21) and B(1-29)-Ala-Ala-Lys-A-(1-21), the slightly different precursors could be quantified individually after separation by high-performance liquid chromatography from the culture supernatant. The two-cassette system allowed a sensitive and well controlled comparison of parameters important for optimal expression of a heterologous gene in Saccharomyces cerevisiae. The system was used to compare two promoter constructions and also to evaluate the position of expression cassettes in the plasmid. Finally the codon usage in the gene to be expressed was found to influence its ability to compete for expression.

Amino Acid Sequence↗

PubMed1988

Rhizomucor miehei triglyceride lipase is synthesized as a precursor.

A Rhizomucor miehei cDNA library constructed in Escherichia coli was screened with synthetic oligonucleotides designed from knowledge of a partial amino acid sequence of the secreted triglyceride lipase (triacylglycerol acylhydrolase EC 3.1.1.3) from this fungus. Lipase-specific recombinants were isolated and their insert sequenced. Unlike characterized bacterial and mammalian triglyceride lipases, the fungal enzyme is synthesized as a precursor, including a 70 amino acid residue propeptide between the 24 amino acid residues of the signal peptide and the 269 residues of the mature enzyme. The precursor processing mechanism, which involves cleavage between a methionine and a serine residue, is unknown. By sequence comparison with other lipases, a serine residue involved in substrate binding was identified in the fungal lipase. The sequence around this residue is well-conserved among characterized lipases. Conservation of an intron in an isolated cDNA recombinant and immunoprecipitation of in vitro synthesized R. miehei translation products indicates that the expression of the lipase gene might involve inefficient mRNA splicing.

Amino Acid Sequence↗

PubMed1986

Primary structure of a precursor to the aspartic proteinase from Rhizomucor miehei shows that the enzyme is synthesized as a zymogen.

In order to characterize the zymogen of the milk-clotting enzyme from Rhizomucor miehei, we constructed a cDNA library on pBR327 in Escherichia coli. Aspartic proteinase-specific recombinants were isolated by colony hybridization to a specific oligonucleotide mixture, and the cDNA sequence corresponding to a precursor form of the enzyme was determined. The deduced amino acid sequence shows that this secreted fungal proteinase is synthesized as a precursor. The first 22 amino acid residues in this precursor constitute a typical signal peptide. The amino acid sequence of the following 47-amino-acid-long prosegment shows homology to the prosegments from both the extracellular and intracellular vertebrate aspartic proteinases, and to the prosegments from the yeast and Mucor pusillus aspartic proteinases as well. These observations suggest that all aspartic proteinases are synthesized with a prosegment and that this prosegment is essential for the correct folding of all the mature enzymes. The active Rhizomucor miehei enzyme consists of 361 amino acid residues with a total molecular weight of 38,701. Clusters of identities around the active site cleft support the assumption that these proteinases have a common folding of their peptide chains. The disulphide bridges were localized in the fungal enzyme, and 2 N-glycosylation sites were identified.

Amino Acid Sequence↗

PubMed1986

Secretion and processing of insulin precursors in yeast.

A series of dibasic insulin precursors including proinsulin was expressed and secreted from Saccharomyces cerevisiae. Recombinant plasmids were constructed to encode fusion proteins consisting of a modified mating factor alpha 1 leader sequence and an insulin precursor. The leader sequence serves to direct the fusion protein into the secretory pathway of the cell and to expose it to the Lys-Arg processing enzyme system. The secreted peptides were purified from the fermentation broth and characterized by sequencing and amino acid analysis. Processing at one or both dibasic sequences was shown in proinsulin and in other insulin precursors containing a short spacer peptide in place of the C peptide. In contrast, no processing was observed in the absence of a spacer peptide in the insulin precursor molecule, e.g., B-Lys-Arg-A (where A and B are the A and B chain of human proinsulin, respectively). This type of single-chain insulin precursors isolated from such constructions could be enzymatically converted into insulin by treatment with trypsin and carboxypeptidase B. The above results suggest that the C-peptide region of proinsulin serves to direct the trypsin-like converting enzyme to process at the two dibasic sequences. We propose that in hormone precursors in general the spacer peptides serve to expose dibasic sequences for processing.

Amino Acid Sequence↗

PubMed1984

A cDNA encoding a small common precursor for human pancreatic polypeptide and pancreatic icosapeptide.

A cDNA for the hormone, human pancreatic polypeptide (PP), was isolated by oligodeoxynucleotide screening from a cDNA library constructed from normal human pancreatic mRNA. The primary structure of the precursor protein as deduced from the cDNA sequence is 95 amino acids long and is composed of a typical, but rather long signal peptide of 29 residues, followed by the sequence of the 36 amino acid human pancreatic polypeptide, which again is separated from the human pancreatic icosapeptide sequence by a classic cleavage and amidation site, Gly-Lys-Arg. The precursor terminates in a heptapeptide which is cleaved from the icosapeptide at a monobasic processing site. Both the size and the structure of the PP precursor was supported by the results of peptide analysis of biosynthetically labeled pro-PP isolated from canine PP cells in which processing was prevented by the arginine analogue canavanine. It is concluded that the precursor for mammalian PP gives rise to two peptide products, the well preserved, carboxyamidated PP and an icosapeptide which is preserved only in its COOH-terminal end, plus a small highly variable COOH-terminal oligopeptide.

Amino Acid Sequence↗

PubMed1984

Glucoamylases G1 and G2 from Aspergillus niger are synthesized from two different but closely related mRNAs.

By the use of glucoamylase-specific synthetic oligodeoxyribonucleotides and molecular cloning of cDNA synthesized from Aspergillus niger total poly(A) + RNA, the primary structure of the glucoamylase G1 mRNA was determined. Glucoamylase G1 is synthesized as a precursor of 640 amino acid residues containing a putative signal peptide of 18 residues, a short propeptide of six residues and the 616 residues long mature enzyme. In vitro translations of mRNA and immunoprecipitations with glucoamylase-specific antisera showed that two glucoamylase polypeptides are synthesized. The larger form with an apparent mol. wt. of 71 000 corresponds to the precursor of glucoamylase G1, and the shorter form with an apparent mol. wt. of 61 000 corresponds to the precursor of glucoamylase G2. From the nucleotide sequencing data of several glucoamylase-specific cDNA recombinants it is shown that the G1 mRNA contains a 169 bp long intervening sequence that can be spliced out to generate a G2 mRNA. Only the 3' part of the G1 mRNA is modified by this splicing event. This kind of differential mRNA processing to give different protein products from one primary transcript has previously only been demonstrated in higher eukaryotes.

Amino Acid Sequence↗

PubMed1984

Two different types of intervening sequences in the glucoamylase gene from Aspergillus niger.

One single glucoamylase gene could be identified in the chromosomal DNA of Aspergillus niger by Southern blot analysis. This glucoamylase gene was isolated from a genomic library of A. niger DNA. The glucoamylase gene is situated on a 2.5-kb EcoRI-EcoRV fragment and contains five intervening sequences in the coding region. One 169-bp intron is involved in differential mRNA processing leading to the two different glucoamylase enzymes G1 and G2; the other four introns are all very small ranging from 55 to 75 bp in length. One intron has a significant homology to the coding region which immediately follows, and it contains the internal conserved sequence TACTAAC, which is also found in yeast chromosomal gene introns, and is thought to participate in mRNA splicing. Two transcription initiation sites and a typical eukaryotic promoter region with TATAAT and CAAT boxes are located upstream from the gene.

Amino Acid Sequence↗

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↗

PubMed1982

Autogenous control: ribosomal protein L10-L12 complex binds to the leader sequence of its mRNA.

Ribosomal proteins L10 and L12 are encoded in the L10 operon, situated at position 89.5 min on the Escherichia coli genetic map, and are able to regulate their own translation. The two proteins form a L10-L12 complex that is able to bind specifically to the leader sequence of the L10 operon mRNA and prevent translation. We show that the leader sequence: (i) is required for the translation of mRNA into L10 and L12 proteins; and (ii) contains a unique binding site for the inhibitory L10-L12 complex. We suggest that a specific secondary structure of the leader RNA is required for translation. When this structure is perturbed by L10-L12 binding, by deletion, or point mutations, translation is inhibited. The block on the synthesis of L10 and L12 can presumably be removed by the incorporation of the inhibitory L10-L12 complex into assembling 50S ribosome subunits. We observed that rRNA prevents the binding of L10-L12 to the mRNA. Furthermore, we have identified extended sequence homologies within the 23S rRNA and L10 leader region RNA. The L10-L12 binding site on the mRNA includes part of the homologous sequences.

Bacterial Proteins↗

PubMed1981

Transcriptional and post-transcriptional control of ribosomal protein and ribonucleic acid polymerase genes.

A partial restriction of ribonucleic acid (RNA) polymerase activity has been used to dissociate the coordinate synthesis of ribosomal proteins and subunits of RNA polymerase and to identify transcriptional and post-transcriptional control signals which regulate the expression of these component genes. Within the beta operon [which has the genetic organization: promoter (p beta), rplJ (L10), r;lL (L7/L12), attenuator, rpoB (beta), rpoC (beta'), terminator], the restriction caused a disproportionate increase between proximal and distal gene transcriptions; the transcriptional intensities of the proximal ribosomal protein genes and the distal RNA polymerase genes were elevated about two- and fourfold, respectively. Transcription within the operon containing four ribosomal protein genes and the RNA polymerase alpha gene was also enhanced, whereas transcription within operons containing only ribosomal protein genes was virtually unaffected by the restriction. It was thus concluded that the mechanisms controlling transcription initiation or attenuation or both in operons containing RNA polymerase subunit genes are coupled to the global rate of RNA synthesis. By introducing the composite ColE1 plasmid pJC701 carrying the proximal portion of the L10 operon, including the beta subunit gene, it was possible to achieve a 10- and a 30-fold range in the transcriptional intensities of the genes specifying L10 and L7/L12 and beta, respectively. Under these conditions, the relative synthesis rates of L7/L12 and beta protein varied by less than 2-fold and by about 15-fold, respectively. These observations corroborate the existence of a post-transcriptional mechanism which severely restricts translation of excess L7/L12 and L10 ribosomal protein messenger RNA; this mechanism is probably important in maintaining the balanced synthesis of ribosome components under conditions in which their messenger RNA levels are dissociated. Furthermore, the observed reduction in the translation efficiency of beta subunit messenger RNA may be related to an inhibitory effect caused by accumulation of RNA polymerase assembly intermediates.

Bacterial Proteins↗

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

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↗

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↗

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↗

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

Genetics of the relB locus in Escherichia coli.

A mutant of Escherichia coli with a delayed relaxed phenotype very similar to that of a previously described relB mutant has been obtained using a new selection procedure. The mutation giving rise to this phenotype has been shown to map at 34.5 min and to be 12% cotransducible with man. It is recessive, revertible, and most likely an allele of the relB gene.

Chromosome Mapping↗

PubMed1976

A transducing bacteriophage lambda carrying the structural gene for elongation factor Ts.

A specialized transducing bacteriophage lambdadpolCdap D-9 has been isolated that carries the structural gene for EF-Ts1 (tsf). The presence of EF-Ts among the proteins synthesized under the direction of this phage in UVL-inactivated cells has been detected by two-dimensional gel electrophoresis and has been verified by antibody precipitation. In an induced lysogen of this phage the relative rate of synthesis of EF-Ts is increased 4-fold. Evidence is presented which suggest that the structural genes for ribosomal protein S2 (rpsB) and RNA polymerase sigma factor (sit) also lies on lambdadpolCdap D-9.

Coliphages↗

PubMed1976

Isolation of transducing phage carrying rps T, the structural gene for ribosomal protein S20.

Lambda transducing phages carrying segments of the Escherichia coli chromosome in the dapB region have been isolated in their in vivo gene products analyzed by two-dimensional gel electrophoresis. One of these phages, lambdaddapB-2, carries the structural genes for ribosomal protein S20 (rps T) and isoleucyltransfer RNA synthetase (ileS.) The most likely gene order is thr-rpsT-ileS-dapB-pyrA.

Bacterial Proteins↗