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K Nam

Publications and source records attributed to K Nam.

34 records · Page 2Linked to original sources
PubMed1999

A novel glutamic acid to aspartic acid mutation near the end of the 2B rod domain in the keratin 1 chain in epidermolytic hyperkeratosis.

We report a mutation in a mild case of epidermolytic hyperkeratosis that results in a glutamic acid to aspartic acid substitution in a novel location, codon 477 or position 106 of the 2B rod domain of the keratin 1 chain. This residue has been conserved in all intermediate filament chains and lies near the beginning of the highly conserved helix termination sequence and just prior to the predicted molecular overlap region. Keratin filaments assembled in vitro from chains bearing this substitution are abnormal, indicating that the glutamic acid residue is critically involved in ionic interactions in intermediate levels of filament structure.

Amino Acid Substitution↗
PubMed1997

An alanine to proline mutation in the 1A rod domain of the keratin 10 chain in epidermolytic hyperkeratosis.

We report a mutation in a case of epidermolytic hyperkeratosis that results in a proline for alanine substitution in the residue position 12 of the 1A subdomain of the keratin 10 chain (codon 158). The disease phenotype is consistent with the inappropriate substitution of a proline near the beginning of the rod domain, because it is likely to seriously disrupt the structural organization of coiled-coil molecules within keratin intermediate filaments. Mutations/substitutions in this position have not been reported in any keratin disease. Position 12 is an alanine in all intermediate filament chains, and lies in the outer b heptad position of the coiled-coil. In vitro peptide interference assembly assays revealed that substitutions that alter residue size or charge at this position primarily interfere with keratin filament elongation.

Adolescent↗
PubMed1997

Severe growth defect in a Schizosaccharomyces pombe mutant defective in intron lariat degradation.

The cDNAs and genes encoding the intron lariat-debranching enzyme were isolated from the nematode Caenorhabditis elegans and the fission yeast Schizosaccharomyces pombe based on their homology with the Saccharomyces cerevisiae gene. The cDNAs were shown to be functional in an interspecific complementation experiment; they can complement an S. cerevisiae dbr1 null mutant. About 2.5% of budding yeast S. cerevisiae genes have introns, and the accumulation of excised introns in a dbr1 null mutant has little effect on cell growth. In contrast, many S. pombe genes contain introns, and often multiple introns per gene, so that S. pombe is estimated to contain approximately 40 times as many introns as S. cerevisiae. The S. pombe dbr1 gene was disrupted and shown to be nonessential. Like the S. cerevisiae mutant, the S. pombe null mutant accumulated introns to high levels, indicating that intron lariat debranching represents a rate-limiting step in intron degradation in both species. Unlike the S. cerevisiae mutant, the S. pombe dbr1::leu1+ mutant had a severe growth defect and exhibited an aberrant elongated cell shape in addition to an intron accumulation phenotype. The growth defect of the S. pombe dbr1::leu1+ strain suggests that debranching activity is critical for efficient intron RNA degradation and that blocking this pathway interferes with cell growth.

Amino Acid Sequence↗
PubMed1996

A novel H1 mutation in the keratin 1 chain in epidermolytic hyperkeratosis.

We report a novel mutation in a case of epidermolytic hyperkeratosis that results in a proline for arginine substitution in the penultimate residue position of the H1 subdomain of the keratin 1 chain, which is near the beginning of the rod domain. This causes a severe clinical disease classified as PS-2. Therefore, the H1 subdomain is probably equally important for the maintenance of keratin intermediate filament integrity as the rod domain. Since earlier concepts had implied that mutations in the H1 subdomain produce milder disease, this case suggests that attempts to correlate mutations with disease presentation remain problematic.

Amino Acid Sequence↗
PubMed1995

S gene expression and the timing of lysis by bacteriophage lambda.

The S gene of bacteriophage lambda encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single lambda late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2... N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.

Amino Acid Sequence↗
PubMed1995

Plus-strand strong-stop DNA synthesis in retrotransposon Ty1.

Reverse transcription in the yeast retrotransposon Ty1 follows the general "rules" of retroviral replication overall. However, some details of the retroviral and Ty1 reverse transcription processes are different. We have identified and determined the structure of plus-strand strong-stop DNA and examined the effect of polypurine tract deletion mutations on its synthesis. Furthermore, we have defined the stop signal for plus-strand strong-stop DNA synthesis as an unusual 2'-O-ribosylated nucleotide in the primer tRNA. Full-length plus-strand strong-stop DNA, following strand transfer, would have a terminal 2-base mismatch with minus-strand DNA. These findings indicate that the mechanism of plus-strand strong-stop DNA transfer in Ty1 differs from that of the retroviral transfer and suggest that full-length plus-strand strong-stop DNA is not a direct intermediate in Ty1 retrotransposition.

Base Sequence↗
PubMed1994

Yeast lariat debranching enzyme. Substrate and sequence specificity.

Yeast RNA lariat debranching enzyme has been purified to near homogeneity using a bacterial overproducer of the enzyme. The enzyme is capable of digesting a variety of branched nucleic acid substrates, including group II intron lariats, multicopy single-stranded DNAs (msDNAs), and a variety of synthetic branched RNAs. A trinucleotide release assay using radiolabeled msDNA substrates was developed and used to determine the basic biochemical parameters for the enzyme. The debranching enzyme shows a strong preference for purines at the 2'-position in both msDNA and synthetic branched RNA substrates, in accord with the structure of its native substrate, which always has a 2'-G residue. The use of small synthetic branched RNA substrates will allow systematic mechanistic and structural studies of this unique enzyme.

Base Sequence↗
PubMed1994

Mapping of DBR1 and YPK1 suggests a major revision of the genetic map of the left arm of Saccharomyces cerevisiae Chromosome XI.

The Saccharomyces cerevisiae dbr1 mutation has been mapped on the left arm of chromosome XI. XIL is a chromosome arm that was until now rather sparsely populated with accurately mapped markers. On the basis of physical data, the overall order of markers is inverted relative to the existing genetic map of XI. We present tetrad analyses using a variety of markers on XI that indicate that the existing genetic map of XIL should be inverted, at least for the strains in which our mapping was carried out, and probably for other S. cerevisiae strains.

Chromosome Mapping↗
PubMed1993

Synthesis of two bacteriophage lambda S proteins in an in vivo system.

Bacteriophage lambda has two genes which are essential for lysis: R, a gene encoding a 158-amino-acid (aa) transglycosylase that attacks the peptidoglycan, and S, a gene encoding two inner-membrane-associating proteins, designated S105 and S107 for their predicted lengths in aa residues. S105 and S107 are thought to have opposing roles in lysis, with the former acting as the lethal lysis effector and the latter as a lysis inhibitor. Here, we used a T7-polymerase-mediated expression system to show that S105 and S107 are synthesized at a constant ratio of about 2.5:1 throughout the period leading up to lysis, indicating that lysis scheduling does not require a translationally controlled switch from inhibitor (S107) to effector (S105) synthesis. However, evidence is presented that the mRNA sequences immediately 5' to the ribosome-binding site (RBS) of the S gene are required for the rather limited translation, but not the stability, of the S mRNA. No difference could be found in the pattern of ternary complex formation over the two S start codons in in vitro toe-printing assays with the wild-type mRNA and with mRNA deleted of the upstream sequences. Nevertheless, these results may suggest a role for translational control in S gene expression, if not in its temporal regulation or in the partition between S105 and S107 production.

Amino Acid Sequence↗
PubMed1990

Conservation of a dual-start motif in P22 lysis gene regulation.

Gene 13 of bacteriophage P22 is functionally equivalent to lambda lysis gene S. Gene S codes for two products, the polypeptides S105 and S107, produced from translational initiation events at the third and first codon, respectively. We have shown that the two polypeptides have opposing functions in lysis: S105 is the lethal lysis effector, and S107 acts as an inhibitor of lysis (U. Bläsi, K. Nam, D. Hartz, L. Gold, and R. Young, EMBO J. 11:3501-3510, 1989). Gene 13 has a 108-codon reading frame and its product begins with a similar motif: Met-1-Lys-2-Lys-3-Met-4. Here, we present in vivo and in vitro evidence for the expression of a 13(108) and a 13(105) product and show that the lambda lysis control mechanisms is evolutionarily conserved in phage P22. In this case 13(108), like S107 in lambda, functions as the inhibitor of the lysis effector 13(105). Although the DNA sequences upstream of the S and 13 gene starts showed less homology, the same structural characteristics, i.e., stem-loop structures immediately upstream and about 10 codons downstream of the start region, were present in both reading frames. Using in vitro mutagenesis and toeprinting, we show that the upstream stem-loop structures of genes 13 and S, containing the Shine-Dalgarno sequence for initiations at Met-1, are interchangeable. Moreover, our data indicate that the stability of the secondary structures present in the translational initiation regions of genes S and 13 is set to create a particular ratio of initiation events at Met-1 and Met-3 or Met-4. The ratio of effector to inhibitor was much higher in P22 than in lambda. We propose that this reflects less transcriptional readthrough at the late terminator t(R) and suggests that the dual-start motif in genes 13 and S may be important for establishment of maintenance of the lysogenic state.

Base Sequence↗
PubMed1990

Translational efficiency of phi X174 lysis gene E is unaffected by upstream translation of the overlapping gene D reading frame.

The lysis gene E of bacteriophage phi X174 is entirely embedded in gene D. Expression studies of genes D and E in Escherichia coli minicells and lysis times obtained in the presence or absence of D translation showed that the simultaneous expression of gene D does not affect protein E production. Thus, unlike other overlapping gene pairs, gene E expression is independent from the upstream translation of gene D. lacZ fusion studies and primer extension inhibition analysis (toeprinting) revealed an intrinsically weak E ribosome-binding site, which seems to be the major factor determining the low expression rate of the gene and thus proper scheduling of cell lysis.

Amino Acid Sequence↗
PubMed1989

Effects of phorbol ester and teleocidin on Ca2+-induced fusion of liposomes.

The effects of different types of lipid membrane defects on Ca2+-induced fusion of liposomes containing phosphatidylserine (PS) were investigated using fluorescent probes. Teleocidin enhanced the fusion of phospholipid vesicles in an assay system using terbium/dipicolinic acid during mixing of internal aqueous phases of vesicles upon fusion. 12-O-Tetradecanoylphorbol-13-acetate (TPA) suppressed the fusion. This latter phenomenon was also observed by measuring the excitation energy transfer. The promotion of membrane fusion by teleocidin was ascribed to dehydration of the membrane surface, the suppressive effect of TPA to desorption of Ca2+ from the membrane surface. Thus, Ca2+-induced fusion of PS vesicles was shown to be sensitive to defects of the membrane surface, but insensitive to defects of the hydrophobic core of the lipid membrane.

Calcium↗
PubMed1989

Dual translational initiation sites control function of the lambda S gene.

Lysis gene S of phage lambda has a 107 codon reading frame beginning with the codons Met1-Lys2-Met3. Genetic data have suggested that translational initiation occurs at both Met1 and Met3, generating two polypeptides, S107 and S105 respectively. We have proposed a model in which the proper scheduling of lysis depends on the partition of translational initiations between the two start codons. Here, using in vitro methods, we show that two stem-loop structures, one immediately upstream of the reading frame and a second approximately 10 codons within the gene, control the partitioning event. Utilizing primer-extension inhibition or 'toeprinting', we show that the two S start codons are served by two adjacent Shine-Dalgarno sequences. Moreover, the timing of lysis supported by the wild-type and a number of mutant alleles in vivo can be correlated with the ratio of ternary complex formation over Met1 and Met3 in vitro. Thus the regulation of the S gene is unique in that the products of two adjacent in-frame initiation events have opposing function.

Bacteriolysis↗
PubMed1981

Atmospheric pressure ionization mass spectrometry: the routine determination of 2,4,5-trichlorophenoxyacetic acid and its metabolites.

This study demonstrated the utility of negative ion atmospheric pressure ionization mass spectrometry for the routine quantitation of 2,4,5-trichlorophenoxyacetic acid and its glycine and taurine amide metabolites in mouse blood, urine and feces samples. The quantitation of 2,4,5-trichlorophenoxyacetic acid in blood follows a short cleanup procedure and used 2,4,5-trichlorophenoxy-13C6-acetic acid as the stable label isotope diluent. A more extensive cleanup procedure utilizing high-pressure liquid chromatography was required for the determination of 2,4,5-trichlorophenoxyacetic acid and its two metabolites in urine and feces. The glycine amide metabolite was quantitated by the 13C stable isotope diluent method. The taurine amide relied on an initial separation step followed by using a second 2,4,5-trichlorophenoxy-13C6-acetic acid spike fot its isotope diluent. Alkaline hydrolysis of the metabolites, followed by methylation, allowed the methyl ester of 2,4,5-trichlorophenoxyacetic acid to be solely used as the analyte in the negative ion atmospheric pressure ionization mass spectrometry quantitation step.

2,4,5-Trichlorophenoxyacetic Acid↗