PubMed Health⌕ Search

Biomedical subjects

F Maley

Publications and source records attributed to F Maley.

At least 73 records · Page 4Linked to original sources
PubMed1990

Plastic adaptation toward mutations in proteins: structural comparison of thymidylate synthases.

The structure of thymidylate synthase (TS) from Escherichia coli was solved from cubic crystals with a = 133 A grown under reducing conditions at pH 7.0, and refined to R = 22% at 2.1 A resolution. The structure is compared with that from Lactobacillus casei solved to R = 21% at 2.3 A resolution. The structures are compared using a difference distance matrix, which identifies a common core of residues that retains the same relationship to one another in both species. After subtraction of the effects of a 50 amino acid insert present in Lactobacillus casei, differences in position of atoms correlate with temperature factors and with distance from the nearest substituted residue. The dependence of structural difference on thermal factor is parameterized and reflects both errors in coordinates that correlate with thermal factor, and the increased width of the energy well in which atoms of high thermal factor lie. The dependence of structural difference on distance from the nearest substitution also depends on thermal factors and shows an exponential dependence with half maximal effect at 3.0 A from the substitution. This represents the plastic accommodation of the protein which is parameterized in terms of thermal B factor and distance from a mutational change.

Amino Acid Sequence↗
PubMed1990

Characterization of the restriction site of a prokaryotic intron-encoded endonuclease.

The 1016-base-pair (bp) intron in the T4 bacteriophage thymidylate synthase gene (td) contains a 735-bp open reading frame that encodes a protein product with endonucleolytic activity. The endonuclease shows specificity for the intronless form of the td gene. Highly purified endonuclease cleaves the DNA of the intronless form of the td gene in vitro at 24 bp upstream of the exon 1-exon 2 junction, generating a 2-base staggered cut with 3'-hydroxyl overhangs. Although the endonuclease cleaves in exon 1, it requires some exon 2 sequence for recognition. The maximum recognition sequence lies in an 87-bp stretch, from 52 bp upstream to 35 bp downstream of the cleavage site, ending at 11 bp into exon 2. The td intron endonuclease appears involved in the conversion of the intronless form of td to intron-containing td gene in the T-even phages. A role for intron mobility is discussed.

Base Sequence↗
PubMed1990

Intron-associated splicing reactions in bacteriophage T4.

Group I introns are present in at least three bacteriophage T4 genes: td, nrdB and sunY. The transcription products of these three genes have similar intron consensus regions and secondary structures, which render them capable of guanosine-mediated in vitro autocatalytic splicing reactions. Moreover, it has been shown that the 245-amino-acid protein encoded in the td intron expresses an endonuclease that cleaves near the joining site for the two exons in the intron-deleted thymidylate synthase gene. The intron-containing td gene is resistant to the enzyme. As in the case of other group I intron-containing genes that have been described in eukaryotes, which also encode site-specific endonucleases, the td intron is highly mobile and can insert into the intron-less td gene by a process initiated by endonuclease cleavage near the insertion site. Whether intron transposition reactions have any physiological significance to the phage, or represent an early imprint on the evolution of introns, remains to be determined.

Base Sequence↗
PubMed1989

Evidence that the intron open reading frame of the phage T4 td gene encodes a specific endonuclease.

The phage T4 thymidylate synthase (td) gene contains an intron open reading frame that encodes a 245-amino acid-long basic protein (Chu, F. K., Maley, G. F., West, D. K., Belfort, M., and Maley, F. (1986) Cell 45, 157-166). The open reading frame (Irf) has been cloned as a fusion protein behind a phage T7 promoter and overexpressed in Escherichia coli. The amplified Irf protein is associated with insoluble inclusion bodies and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 7 kDa smaller than expected. Data obtained from DNA sequencing, amino acid sequencing of the fusion protein, and carboxypeptidase Y digestion suggest that although the cloned gene is not altered and the protein is made from the expected start codon, it appears to terminate about 90 amino acids before the encoded stop codon. Proteolytic cleavage during or soon after synthesis appears to be responsible for the truncated Irf. The expressed protein is solubilized in guanidine HCl and renatured by dialysis against high salt. This partially purified preparation has been found to contain a DNA endonuclease activity specific for the td delta I gene, which contains a precise deletion of the intron.

Amino Acid Sequence↗
PubMed1989

An anomaly in the active site region of thymidylate synthase.

A putative thyA gene from Escherichia coli was cloned into a high expression vector and the thymidylate synthase produced was purified to homogeneity. Comparison of the monomer molecular weight of this protein with that of authentic E. coli thymidylate synthase revealed the two to differ, suggesting that they were derived from different sources. This was confirmed by Ochterlony immunodiffusion analysis, which revealed that while the unknown thymidylate synthase formed a precipitin band with guinea pig antibody to the putative E. coli synthase, pure E. coli TS did not. In addition, the specific enzyme activity of the purified unknown thymidylate synthase was about 4-fold higher than that of the pure authentic enzyme. Sequence analysis of the active site peptide revealed that the amino acid linked to the carboxyl end of the active site cysteine was valine. The only instance where this has been found in the 11 thymidylate sequences reported so far is in the thyP3 sequence of the Bacillus subtilis phage-3T. In all the other cases, a histidine has been found in this position. Amino end group sequence analysis of the unknown synthase for about 30 residues confirmed the close identity of this protein to that of the B. subtilis phage thymidylate synthase. To determine whether the replacement of the active site histidine with a valine enhances the activity of the resulting thymidylate synthase, we affected this change in T4-phage thymidylate synthase by site-directed mutagenesis and found that instead of an increase in activity there was an 80 percent decrease.

Amino Acid Sequence↗
PubMed1989

Folate analogues. 32. Synthesis and biological evaluation of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid and related compounds.

The chemical synthesis of three close analogues (2-4) of N10-propargyl-5,8-dideazafolate (PDDF) is described. The quinazoline ring of 2 and 4 was constructed from the pivotal intermediate 9 in a novel and unambiguous manner during the final step of the synthesis under very mild conditions. 2-Desamino-2-methyl-N10-propargyl-5,8-dideazafolate (DMPDDF) (2) was a strong inhibitor of human and Lactobacillus casei thymidylate synthases, whereas 2-desamino-2-(trifluoromethyl)-N10-propargyl-5,8-didezafolate (3) and 2-desamino-2,3-dimethyl-N10-propargyl-5,8-dideazafolate (4) were only weak inhibitors of this enzyme. DMPDDF exhibited excellent growth inhibition of Manca human lymphoid leukemia and H35 hepatoma cells in culture. The inhibitor activities of 2 were 43- and 65-fold greater than that of PDDF, respectively, in these cell lines. H35R cells that are resistant to methotrexate by virtue of a transport defect were cross resistant to DMPDDF but not to PDDF. H35FF cells which have 70-fold greater amounts of thymidylate synthase compared to H35N cells were 130-fold resistant to DMPDDF. Furthermore, the toxicity of DMPDDF to H35 hepatoma cells could be completely reversed by thymidine, establishing its locus of action as thymidylate synthase. Transport studies in vitro established that DMPDDF effectively inhibits MTX influx into H35 hepatoma cells, whereas PDDF has no effect on MTX transport in this cell line. These data suggest that the greater activity of DMPDDF relative to PDDF is partly due to the ability of the former compound to enter cells via the MTX/reduced folate transport system. Enzyme inhibition data of 4 suggest that the presence of N3H in DMPDDF is essential for binding to thymidylate synthase.

Breast Neoplasms↗
PubMed1988

Properties of a defined mutant of Escherichia coli thymidylate synthase.

A mutant of Escherichia coli thymidylate synthase (F3-TS), resulting from the replacement of a tyrosine for a cysteine 50 amino acids from the amino-terminal end, has been purified to homogeneity and found to contain less than 0.2% of the activity of the native enzyme (thyA-TS). Although this protein formed a ternary complex with 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) and 5,10-methylenetetrahydrofolate, like the native enzyme, the extent of complex formation was significantly impaired as determined by equilibrium dialysis and circular dichroism. Thus, unlike the native enzyme, where 2 mol of FdUMP were present in each mole of ternary complex, F3-TS contained less than 1 mol of FdUMP/mol of ternary complex. Similarly, the binding of dUMP by F3-TS was greatly diminished relative to thyA-TS, but its binding as well as that of FdUMP could be improved by the presence of either the folate substrate or a tight binding folate analogue, 10-propargyl-5,8-dideazafolate (PDDF). However, despite the fact that PDDF enhanced the binding of FdUMP and dUMP to F3-TS, the binding of PDDF to the mutant enzyme was also greatly impaired. This contrasts with the native enzyme, which, under the same conditions, bound about 2 mol of PDDF/mol of enzyme in the presence or absence of either FdUMP or dUMP. Circular dichroism analyses with PDDF in the presence of dUMP or FdUMP yielded analogous results, but the effects were less dramatic than those obtained by equilibrium dialysis. Evidence in support of a structural difference between thyA-TS and F3-TS was obtained by demonstrating that the latter protein was 15-fold slower in forming a ternary complex with dUMP and PDDF than the former and that the mutant enzyme was less stable than the native enzyme.

Amino Acids↗
PubMed1988

Characterization of the glycosylation sites in yeast external invertase. II. Location of the endo-beta-N-acetylglucosaminidase H-resistant sequons.

There are 14 potential Asn-X-Thr/Ser glycosylation sites, or sequons, in the yeast external invertase sequence. Of these, 13 are wholly or partially glycosylated to give an average of 9-10 oligosaccharides/subunit (Reddy, V. A., Johnson, R. S., Biemann, K., Williams, R. S., Ziegler, F. D., Trimble, R. B., and Maley, F. (1988) J. Biol. Chem. 263, 6978-6985). On digestion of native holoenzyme by endo-beta-N-acetylglucosaminidase H (Endo H) an average of about seven oligosaccharides per subunit are released without affecting enzyme activity (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine whether the remaining Endo H-resistant chains were restricted to a limited number of unique sequons or were randomly distributed on all 13, Endo H-treated native invertase was digested with either thermolysin or trypsin and the resultant glycopeptides isolated by reversed-phase high pressure liquid chromatography and gel filtration. It was found that the oligosaccharides attached to Asn92, Asn247, and Asn350 were partially resistant to Endo H, while those at Asn45 and Asn337) were completely resistant. Bio-Gel P-4 analysis revealed the Endo H-resistant oligosaccharides at Asn45, Asn92, Asn247, and Asn337 to be Man8-14GlcNAc, while the minor residual carbohydrate at Asn350 was Man greater than 50GlcNAc. The Endo H-susceptible oligosaccharides at Asn4, Asn146, and Asn256 were Man greater than 50GlcNAc while all other glycosylation sites contained Man8-14GlcNAc. Based on a hydropathic analysis of invertase, the two most Endo H-resistant oligosaccharides at Asn45 and Asn337 were located in the more hydrophobic regions of the molecule. These may form part of the folded protein structure or interacting subunit surfaces, thus restricting their accessibility to Endo H.

Acetylglucosaminidase↗
PubMed1988

Characterization of the glycosylation sites in yeast external invertase. I. N-linked oligosaccharide content of the individual sequons.

External invertase is the product of the SUC2 gene of Saccharomyces cerevisiae. The deduced sequence of this enzyme (Taussig, R., and Carlson, M. (1983) Nucleic Acid Res. 11, 1943-1954) reveals it to contain 14 potential N-linked glycosylation sites, or sequons, although only 9-10 appear to be glycosylated (Trimble, R. B., and Maley, F. (1977) J. Biol. Chem. 252, 4409-4412). To determine the location of the glycosylated sequons, external invertase was deglycosylated with endo-beta-acetylglucosaminidase H and its component peptides analyzed by both fast atom bombardment mass spectrometry (FABMS) and classical peptide isolation procedures. By use of the former technique most of the glucosamine-containing sequons could be located and by the latter sufficient amounts of small glucosamine-containing peptides were isolated to enable their quantitation. From the combined FABMS and glucosamine analyses, it was established that eight of the sequons in a subunit of invertase are either completely or almost completely glycosylated, while five others are glycosylated to the extent of about 50% or less. In the case of two overlapping sequons (4 and 5), which include Asn92-Asn93-Thr-Ser, only the first Asn was glycosylated. Thus, all but one of the sequons of external invertase are glycosylated to some extent, giving an appearance of only 9-10 N-linked oligosaccharides/subunit. The sequence identity of both external and internal invertase was verified by FABMS and by peptide sequence analysis. In only one site was an amino acid found to differ from that deduced from the DNA sequence of the SUC2 gene. This occurred at position 390 where a proline was found in place of alanine, which could result from a single base change in the triplet specifying the latter amino acid.

Amino Acid Sequence↗
PubMed1988

RNA splicing in the T-even bacteriophage.

Group 1 introns, first demonstrated in the nuclear large rRNA of Tetrahymena thermophila and subsequently in many yeast, fungal mitochondrial, and chloroplast precursor RNAs, are capable of intron excision and exon ligation in vitro, although this process occurs much more rapidly in vivo. The discovery and characterization of a similar intron in the T4 phage thymidylate synthase gene (td) led to the finding of additional group 1 introns in other T4 genes and in genes of the related T2 and T6 phages. Because protein factors are not required in the splicing of group 1 introns in vitro, it has been postulated that the precursor RNA can assume a critical conformation enabling it to undergo site-specific autocatalytic cleavage and ligation (self-splicing). By means of site-directed mutation, it has been shown unequivocally that several sequence elements in the Tetrahymena rRNA intron are involved in the formation of base-paired stem structures that are essential for the self-splicing process. These sequence elements have been demonstrated in other eukaryotic group 1 introns, as well as in the td intron. In this brief review we shall describe the biochemical and structural properties of the td intron in relation to other newly found phage introns. The interesting implications arising from these revelations will also be discussed.

Base Sequence↗
PubMed1987

Mechanism and requirements of in vitro RNA splicing of the primary transcript from the T4 bacteriophage thymidylate synthase gene.

The splicing of a procaryotic precursor RNA transcribed from the T4 phage thymidylate synthase (td) gene with SP6 RNA polymerase was investigated in vitro. The intron excision-cyclization reaction increased progressively to 60 degrees C. Exon ligation, though barely detectable at the lower temperatures, was greatly enhanced at 60 degrees C. Both reactions required Mg2+. The addition of guanosine to the 5' end of an intron-exon II intermediate via a 3',5'-phosphodiester bond was essential for the ligation of exon I to exon II. The added guanosine and the first intron-encoded uridine are subsequently lost as a dinucleotide from the 5' end during cyclization of the linear form of the excised intron RNA. Exon ligation is intramolecular and occurs more readily in the nascent RNA molecule (cotranscriptionally) than in the finished transcript (posttranscriptionally). These data and the identification of various structural elements (P, Q, R, S, E, E') in the td intron that are found typically in eucaryotic class I introns firmly establish the td intron as the first example of class I intron of procaryotic origin.

DNA Restriction Enzymes↗
PubMed1987

Localization of the T4 phage ribonucleotide reductase B1 subunit gene and the nucleotide sequence of its upstream and 5' coding regions.

The nucleotide (nt) sequence in a 757-bp [corrected] segment downstream from the intron-containing T4 phage thymidylate synthase gene (td) has been determined. This region was found to contain two open reading frames (ORFs). The first ORF(ORF2) [corrected] 261 bp [corrected] in length, is 24 [corrected] nt downstream from the td gene. The second ORF(ORF3) [corrected]) is 200 bp long at 558 [corrected] nt from the td gene and extends to the end of the Eco RI fragment. The amino acid (aa) sequence (66 aa residues) deduced from the second truncated ORF shows 59% homology to the sequence of the N-terminal portion of the ribonucleotide reductase large subunit of either Escherichia coli (B1 subunit) or mouse (M1 subunit). This tentatively identifies the truncated gene to be the 5' end of the T4 phage ribonucleotide reductase subunit B1 (nrdA) gene and pinpoints its exact location on the T4 phage genomic map. Southern hybridization analysis suggests good sequence homology among the nrdA genes of various T-even phages.

Amino Acid Sequence↗
PubMed1987

Interrupted thymidylate synthase gene of bacteriophages T2 and T6 and other potential self-splicing introns in the T-even bacteriophages.

Southern hybridization analyses of procaryotic DNA from Escherichia coli, lambda bacteriophage, and T1 to T7 phages were carried out. The hybridization probes used consisted of DNA restriction fragments derived from the T4 phage intron-containing thymidylate synthase gene (td) and short synthetic oligodeoxynucleotides defining specific exon and intron regions of the gene. It was shown that intact as well as restricted DNA from the T-even phages hybridized not only to both T4 phage td intron- and exon-specific probes but also to probes defining the td 5' (exon I-intron) and 3' (intron-exon II) presplice junctions. These data strongly suggest that, analogous to the T4 phage, only the T2 and T6 phages among the procaryotes tested contain interrupted td genes. The td intervening sequence in each phage is roughly 1 kilobase pair (kb) in size and interrupts the td gene at a site analogous to that in the T4 phage. This was confirmed by data from Northern (RNA) hybridization analysis of td-specific in vitro transcripts of these phage DNAs. [alpha-32P]GTP in vitro labeling of total RNA from T4 phage-infected cells produced five species of labeled RNAs that were 1, 0.9, 0.83, 0.75, and 0.6 kb in size. Only the 1-, 0.9-, and 0.75-kb species were labeled in RNA from T2- or T6-infected cells. The commonly present 1-kb RNA is the excised td intron, which exists in both linear and circular forms in the respective T-even-phage-infected cells, while the 0.6-kb RNA unique to T4 may be the excised intron derived from the ribonucleotide reductase small subunit gene (nrdB) of the phage. The remaining labeled RNA species are likely candidates for other self-splicing introns.

DNA, Bacterial↗
PubMed1986

Cloning and expression of an intron-deleted phage T4 td gene.

The 1017-bp intron within the cloned phage T4 td gene was deleted by oligonucleotide-directed mutagenesis. Induction of thymidylate synthase activity and mature td mRNA from this intronless construct (pKTd delta I) was compared both in vivo and in vitro with expression from plasmids bearing td genes in which the introns contain either no change (pKTd2), an XbaI linker inserted about 200 nucleotides from the 3'-end (pKTdX-1), or a deletion of two-thirds of the central portion (pKTd delta 1-3). Slightly more synthase accumulated in cells carrying pKTd delta I as compared to the other td genes when induction was performed at 30, 37, or 42 degrees C. Dramatically different results were observed in vitro, where enzyme activity synthesized from pKTd delta I DNA appeared earlier and reached severalfold higher levels than with pKTd2 DNA. In addition, thymidylate synthase expression from pKTdX-1 was impaired relative to pKTd2, while pKTd delta 1-3 accumulated enzyme at levels intermediate between those of pKTd2 and pKTd delta I. Under both in vivo and in vitro conditions, increasing levels of mature td mRNA preceded and paralleled those in enzyme activity for all four plasmids, demonstrating comparable translation of the mRNAs produced. From these results it would appear that the splicing of td RNA is much more efficient in vivo than in vitro, suggesting that other cellular components may facilitate in vivo processing of this intron-containing transcript.

Chromosome Deletion↗
PubMed1986

Transfer of glycerol by Endo-beta-N-acetylglucosaminidase F to oligosaccharides during chitobiose core cleavage.

N-Linked oligosaccharides, when hydrolyzed by glycerol-containing preparations of endo-beta-N-acetylglucosaminidase (Endo) F from Flavobacterium meningosepticum were found to have glycerol attached to their reducing ends. The absence of a reducing end was confirmed by high-field 1H NMR spectroscopy, and the incorporated glycerol was verified through mass spectrometry and collisionally activated decomposition fast atom bombardment/mass spectrometry/mass spectrometry techniques. Periodate oxidation of [1(3)-14C]glycerol-labeled oligosaccharides indicated glycerol was glycosidically linked via its 1(3) carbon to the C1 of the reducing end N-acetylglucosamine. In a second, less favored reaction, the glycerol glycoside was hydrolyzed by Endo F using water as the terminal nucleophile, thus regenerating the N-acetylglucosamine reducing end. Glycerol could be removed from Endo F preparations without affecting enzyme stability, and chitobiosyl core hydrolysis in its absence provided intact oligosaccharides with normal N-acetylglucosamine reducing ends. The incorporation of labeled glycerol may provide a useful method for monitoring of Endo F release of oligosaccharides.

Acetylglucosamine↗