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F K Chu

Publications and source records attributed to F K Chu.

34 records · Page 2Linked to original sources
PubMed1985

Diverse properties of external and internal forms of yeast invertase derived from the same gene.

It has been shown by genetic analysis that the external and internal invertases from Saccharomyces cerevisiae share a common structural gene [Taussig, R., & Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954]. However, the only amino acid composition of these two forms of invertase reported to date has revealed extensive differences [Gascon, S., Neumann, N.P., & Lampen, J.O. (1968) J. Biol. Chem. 243, 1573-1577]. We have found from amino acid analyses of both enzymes and sodium dodecyl sulfate-polyacrylamide gel analysis of their cyanogen bromide peptides that they are most likely identical in their amino acid sequence. However, the invertases exhibit dramatically different physical properties, particularly in their stability. The most striking difference was in their renaturation following guanidine treatment where it was shown that inactivated external invertase could be renatured completely. Endo-beta-N-acetylglucosaminidase H treated external invertase was restored to 40% of its original activity while internal invertase remained completely inactive. The observed differences may be attributed to the presence and absence of the oligosaccharide moiety in the external and internal invertases, respectively.

Amino Acids↗
PubMed1985

In vitro expression of the intron-containing gene for T4 phage thymidylate synthase.

The mechanism of expression of the structural gene (td) of T4 phage thymidylate synthase, which contains a 1,017-base pair intron, was studied by employing a coupled transcription-translation system with a td containing recombinant plasmid (pKTd2) as template. The [3H]leucine-labeled protein products synthesized in this system were treated with antibody to the synthase and the resulting immunoprecipitate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two labeled polypeptides were obtained, one with an Mr of 32,000 and the other with an Mr of 25,000. The former corresponds in molecular weight to a subunit of T4-thymidylate synthase and the other to the 183-amino acid peptide encoded by exon I, the 5'-end of the interrupted td gene. When pKTd2 restricted in exon I was used as a template, labeled immunopeptides were not detected but, when restricted in the intron region or in exon II, only the 25,000 Mr exon I product was obtained. Both peptides (Mr = 25,000 and 32,000) were synthesized when the gene was restricted downstream to exon II. Active enzyme, as measured by the tritium release assay, was shown to form about 6 min after the td gene was added to the in vitro protein synthesizing system, and followed the appearance of mature mRNA, as evidenced by S1 nuclease protection studies. The enzyme increased linearly for another 14 min in conjunction with the appearance of the Mr = 32,000 immunopeptide. The exon I product, however, preceded the Mr = 32,000 peptide, indicating that a post-transcriptional processing event may be required for mature mRNA to be formed. Measurement of the RNA products from the td gene in a transcriptional system, with labeled probes from specific regions of the td gene, provided evidence in support of an RNA processing mechanism involving intron excision and exon splicing.

Base Sequence↗
PubMed1984

Intervening sequence in the thymidylate synthase gene of bacteriophage T4.

The continuous sequence of 2.3 kilobases in a 3-kilobase DNA fragment encoding the structural gene for coliphage T4 thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) was determined by using the M13 dideoxy chain-termination method. From the coding information within this gene and that provided by sequence analysis of selected CNBr peptides from the protein product, the primary structure of T4 thymidylate synthase was determined. The most significant finding of these studies is the presence of a 1017-base-pair interruption two-thirds of the way through the nucleotide sequence of the structural gene. The 5'- and 3'-terminal ends of this intron are demarcated by an apparent stop and start codon, respectively. The corresponding methionine preceding the second coding region of the synthase is not incorporated into the final protein product. Structural evidence confirming the presence of the intervening sequence in the phage genome was obtained by restriction and hybridization analysis. Support for the presence of the intron was also obtained at the functional level by enzyme expression studies using selected td gene fragments. This work also confirms the findings of Purohit and Mathews [ Purohit , S. & Mathews , C. K. (1983) Fed. Proc. Fed. Am. Soc. Exp. Biol. 42, 1759], which reveal that the termination codon for the dihydrofolate reductase gene and the triplet initiating thymidylate synthase overlap by a four-base stretch, A-T-G-A. The implications of this unusual gene arrangement are discussed.

Amino Acid Sequence↗
PubMed1984

Cell cycle-dependent expression of thymidylate synthase in Saccharomyces cerevisiae.

Synchronous populations of Saccharomyces cerevisiae cells, generated by two independent methods, have been used to show that thymidylate synthase, in contrast to the vast majority of cellular proteins thus far examined, fluctuates periodically during the S. cerevisiae cell cycle. The enzyme, as assayed by two different methods, accumulated during S period and peaked in mid to late S phase, and then its level dropped. These observations suggest that both periodic synthesis and the instability of the enzyme contribute to the activity profile seen during the cell cycle. Accumulation of thymidylate synthase is determined at the level of its transcript, with synthase-specific mRNA levels increasing at least 10-fold to peak near the beginning of S period and then falling dramatically to basal levels after the onset of DNA synthesis. This mRNA peak coincided with the time during the cell cycle when thymidylate synthase levels were increasing maximally and immediately preceded the peak of DNA synthesis, for which the enzyme provides precursor dTMP.

Cell Cycle↗
PubMed1983

Factors affecting the oligomeric structure of yeast external invertase.

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.

Extracellular Space↗
PubMed1980

Growth phase dependence of invertase mRNA levels in yeast.

Poly(A)-containing RNA from Saccharomyces cerevisiae was translated in the wheat germ protein-synthesizing system and yielded as one of its products a polypeptide which was identified as invertase. This characterization was based on the presence of a protein band at 60,000 daltons following immunoprecipitation with a specific invertase antibody, and the formation from this protein of staphylococcal V8 protease and cyanogen bromide peptides, which were similar in size to those from carbohydrate-depleted invertase. The observed increase in invertase activity from the early to mid logarithmic stage of cell growth was synchronous with the appearance of translatable invertase mRN in these cells. No translatable invertase mRNA, however, was detected in late logarithmic cells, although a constant level of invertase activity was present in the culture medium.

Cell-Free System↗
PubMed1976

Protein binding and subunit association activity in particles reconstituted from Escherichia coli MRE600 50S ribosomal components.

Reconstituted 37S and 48S ribonucleoprotein particles were constructed by incubating Escherichia coli ribosomal RNA with total 50S ribosomal proteins by a sequential incubation method. By comparing the protein compositions of the two types of particles, the proteins that bind to 37S complexes to form 48S particles have been determined. Although only 48S particles could associate with 30S subunits, isolated 37S reconstituted particles could do so if incubated with exogenous 50S proteins. The proteins that bind under these conditions and confer upon particles the ability to associate are L2, L11, L15, L18, and L25. The involvement of these proteins in 5S RNA binding is discussed.

Binding Sites↗
PubMed1975

Reconstitution of 48 S particles with subunit association activity from Escherichia coli 50 S ribosomal components.

Previous studies have shown that 50 S ribosomal proteins bind sequentially to RNA's of the 50 S subunit via temperature-dependent steps (0 leads to 37 leads to 55 degrees C) to form 28, 32, (43) and 48 S particles (Chu, F.K., and Maeba, P.Y. (1973) Can. J. Biochem. 51, 129-139). When three equivalents of 50 S ribosomal proteins are used per equivalent of 23 S RNA, the yield of reconstituted 48 S particles is greatly increased. The addition of 10 mM spermidine increases the rate of reconstitution and abolishes the need for prior incubation at lower temperatures when 48 S particles are formed at 55 degrees C. In the presence of excess 50 S ribosomal proteins and 10 mM spermidine, 48 S particles formed at 50 degrees C are able to associate with 30 S subunits in a stoichiometric manner to form 70 S complexes. These 48 S particles compete effectively with 50 S subunits for 30 S subunits and have an association activity equal to that of 50 S subunits. As compared to the 34 proteins in 50 S subunits, 4 are either absent or present in marginal amounts, 11 in reduced amounts, and 19 in approximately normal amounts in the 48 S particles.

Binding Sites↗