PubMed Health⌕ Search

Biomedical subjects

V L Murtif

Publications and source records attributed to V L Murtif.

7 recordsLinked to original sources
PubMed1988

Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase.

The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.

Amino Acid Sequence↗
PubMed1987

Mutagenesis affecting the carboxyl terminus of the biotinyl subunit of transcarboxylase. Effects on biotination.

Biotin is added to biotin-containing enzymes as a post-translational modification catalyzed by holoenzyme synthetase. This reaction is fairly general in that synthetase from one organism will modify enzymes from heterologous sources. This suggests that the polypeptides share some structural characteristic(s) that define(s) them as biotin enzymes. We have reported previously that when the gene coding for the 1.3 S biotinyl subunit of transcarboxylase is expressed in Escherichia coli, the polypeptide produced is biotinated by the cellular synthetase. Using in vitro mutagenesis of this gene, we have begun to define the primary structure involved in the enzymatic addition of biotin to a lysine residue. We show here that the carboxyl terminus of the 1.3 S subunit is critical in biotination. Mutations affecting the COOH-terminal residue do not influence the modification, but elimination of the hydrophobic side chain of the penultimate residue abolishes biotin addition.

Amino Acid Sequence↗
PubMed1985

In vivo transcription of rDNA spacers in Drosophila.

The sequence-nonhomologous rDNA spacers of D. melanogaster and D. virilis exhibit remarkably similar organizations and transcription capabilities. In spacers of both species, tandemly repeated sequences are present upstream of the beginning of the pre-rRNA coding region, and the repeats are capable of transcriptional activity. Short, nuclear transcripts homologous to the spacer repeats have been identified by S1 nuclease protection experiments and by northern blot analyses. Although spacer transcripts are rare in steady state RNA populations, the presence of multiple promoter elements may suggest a regulatory role for rDNA spacers.

Animals↗
PubMed1985

Cloning and expression of the 1.3S biotin-containing subunit of transcarboxylase.

We have cloned the gene coding for the 1.3S biotin-containing subunit of transcarboxylase (EC 2.1.3.1) from Propionibacterium shermanii. Transcarboxylase is a well-characterized enzyme composed of 30 polypeptides of three different types: twelve 1.3S biotinyl subunits, six 5S dimeric outer subunits, and one 12S hexameric central subunit. In propionic acid fermentation, the enzyme catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. The 1.3S subunit binds the outer and central subunits of the enzyme together, and its biotin serves as carboxyl carrier between subsites on the central and outer subunits where each partial reaction occurs. The cloned gene has been expressed in Escherichia coli, and the 1.3S subunit accumulates to 7% of total cellular protein. The foreign protein is recognized and biotinated by biotin holoenzyme synthetase of E. coli. The identifications of the gene and its product were confirmed by four independent approaches: DNA sequence analysis, immunoprecipitation, incorporation of labeled biotin, and measurement of enzymatic activity in the first partial reaction.

Amino Acid Sequence↗
PubMed1981

Nontranscribed spacers in Drosophila ribosomal DNA.

Ribosomal DNA nontranscribed spacers in Drosophila virilis DNA have been examined in some detail by restriction site analysis of cloned segments of rDNA, nucleic acid hybridizations involving unfractionated rDNA, and base composition estimates. The overall G + C content of the spacer is 27--28%; this compares with 39% for rDNA as a whole, 40% for main band DNA, and 26% for the D. virilis satellites. Much of the spacer is comprised of 0.25 kb repeats revealed by digestion with Msp I, Fnu DII or Rsd I, which terminate very near the beginning of the template for the ribosomal RNA precursor. The spacers are heterogeneous in length among rDNA repeats, and this is largely accounted for by variation among rDNA units in the number of 0.25 kb elements per spacer. Despite its high A + T content and the repetitive nature of much of the spacer, and the proximity of rDNA and heterochromatin in Drosophila, pyrimidine tract analysis gave no indication of relatedness between the spacer and satellite DNA sequences. Species of Drosophila closely related to D. virilis have rDNA spacers that are homologous with those in D. virilis to the extent that hybridization of a cloned spacer segment of D. virilis rDNA to various DNA is comparable with hybridization to homologous DNA, and distributions of restriction enzyme cleavage sites are very similar (but not identical) among spacers of the various species. There is spacer length heterogeneity in the rDNA of all species, and each species has a unique major rDNA spacer length. Judging from Southern blot hybridization, D. hydei rDNA spacers have 20--30% sequence homology with D. virilis rDNA spacers, and a repetitive component is similarly sensitive to Msp I and Fnu DII digestion. D. melanogaster rDNA spacers have little or no homology with counterparts in D. virilis rDNA, despite a similar content of 0.25 kb repetitive elements. In contrast, sequences in rDNA that encode 18S and 28S ribosomal RNA have been highly conserved during the divergence of Drosophila species; this is inferred from interspecific hybridizations involving ribosomal RNA and a comparison of distributions of restriction enzyme cleavage sites in rDNA.

Animals↗