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J D Alfonzo

Publications and source records attributed to J D Alfonzo.

7 recordsLinked to original sources
PubMed1999

APT1, but not APT2, codes for a functional adenine phosphoribosyltransferase in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae has two separate genes (APT1 and APT2) that encode two potentially different forms of adenine phosphoribosyltransferase (APRT). However, genetic analysis indicated that only APT1 could code for a complementing activity. Cloning and expression of both the APT1 and APT2 genes in Escherichia coli showed that although discrete proteins (APRT1 and APRT2) were made by these genes, only APRT1 had detectable APRT activity. Northern and Western blot analyses demonstrated that only APT1 was transcribed and translated under normal physiological conditions in yeast. Phylogenetic analysis revealed that APRT1 and APRT2 are evolutionary closely related and that they arise from a gene duplication event. We conclude that APT1 is the functional gene in S. cerevisiae and that APT2 is a pseudogene.

Adenine Phosphoribosyltransferase↗
PubMed1998

Purification and characterization of MAR1. A mitochondrial associated ribonuclease from Leishmania tarentolae.

A relatively thermostable 22-kDa endoribonuclease (MAR1) was purified more than 10,000-fold from a mitochondrial extract of Leishmania tarentolae and the gene cloned. The purified nuclease has a Km of 100-145 +/- 33 nM and a Vmax of 1.8-2.9 +/- 2 nmol/min, depending on the RNA substrate, and yields a 3'-OH and a 5'-phosphate. Cleavage was limited to several specific sites in the substrate RNAs tested, but cleavage of pre-edited RNAs was generally independent of the addition of cognate guide RNA. The MAR1 gene was expressed in Escherichia coli or in L. tarentolae cells, and the recombinant protein was affinity-purified. The cleavage specificity of the recombinant enzyme from L. tarentolae was identical to that of the native enzyme. The single copy MAR1 gene maps to an 820-kilobase pair chromosome and contains an open reading frame of 579 nucleotides. The 18-amino acid N-terminal sequence shows characteristics of an uncleaved mitochondrial targeting sequence. Data base searching revealed two homologues of MAR1 corresponding to unidentified open reading frames in Caenorhabditis elegans (GenBankTM accession number Z69637) and Archaeoglobus fulgidus (GenBankTM accession number AE000943). The function of MAR1 in mitochondrial RNA metabolism in L. tarentolae remains to be determined.

Amino Acid Sequence↗
PubMed1998

U-insertion/deletion Edited Sequence Database.

Uridine insertion/deletion RNA editing is a post-transcriptional RNA modification occurring in the mitochondria of kinetoplastid protozoa. The U-insertion/deletion Edited Sequence Database is a compilation of mitochondrial genes and edited mRNAs from five kinetoplastid species. It contains separate files with the DNA, mRNA (both unedited and edited) and predicted protein sequences, as well as alignments of the Leishmania tarentolae and Trypanosoma brucei protein sequences from edited and unedited genes. The sequence files are in GCG format. A 'map' sequence file showing the location of U-deletions, U-insertions and the translated amino acid sequences is also provided for each gene. Genomic maps for each species are also provided with clickable genes, including maxicircle-encoded gRNAs. Sets of aligned nuclear rRNA sequences from kinetoplastid protozoa are also provided, which were used for phylogenetic reconstructions in an analysis of the origin of RNA editing. The database is available through the World Wide Web as an HTML document at the URLhttp://www.lifesci.ucla.edu/RNA/trypanosome/ database.html

Animals↗
PubMed1997

The mechanism of U insertion/deletion RNA editing in kinetoplastid mitochondria.

Recent advances in in vitrosystems and identification of putative enzymatic activities have led to the acceptance of a modified 'enzyme cascade' model for U insertion/deletion RNA editing in kinetoplastid mitochondria. Models involving the transfer of uridines (Us) from the 3'-end of gRNA to the editing site appear to be untenable. Two types of in vitrosystems have been reported: (i) a gRNA-independent U insertion activity that is dependent on the secondary structure of the mRNA; (ii) a gRNA-dependent U insertion activity that requires addition of a gRNA that can form an anchor duplex with the pre-edited mRNA and which contains guiding A and G nucleotides to base pair with the added Us. In the case of the gRNA-mediated reaction, the precise site of cleavage is at the end of the gRNA-mRNA anchor duplex, as predicted by the original model. The model has been modified to include the addition of multiple Us to the 3'-end of the 5'-cleavage fragment, followed by the formation of base pairs with the guiding nucleotides and trimming back of the single-stranded oligo(U) 3'-overhang. The two fragments, which are held together by the gRNA 'splint', are then ligated. Circumstantial in vitroevidence for involvement of an RNA ligase and an endoribonuclease, which are components of a 20S complex, was obtained. Efforts are underway in several laboratories to isolate and characterize specific components of the editing machinery.

Animals↗
PubMed1997

Purification and characterization of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.

Adenine phosphoribosyltransferase (APRT) from Saccharomyces cerevisiae was purified approximately 1500-fold. The enzyme catalyzes the Mg-dependent condensation of adenine and 5-phosphoribosylpyrophosphate (PRPP) to yield AMP. The purification procedure included anion exchange chromatography, chromatofocusing and gel filtration. Elution of the enzyme from the chromatofocusing column indicated a pI value of 4.7. The molecular mass for the native enzyme was 50 kDa; however, upon electrophoresis under denaturing conditions two bands of apparent molecular mass of 29 and 20 kDa were observed. We have previously reported the presence of two separate coding sequences for APRT, APT1 and APT2 in S. cerevisiae. The appearance of two bands under denaturing conditions suggests that, unlike other APRTs, this enzyme could form heterodimers. This may be the basis for substrate specificity differences between this enzyme and other APRTs. Substrate kinetics and product inhibition patterns are consistent with a ping-pong mechanism. The Km for adenine and PRPP were 6 microM and 15 microM, respectively and the Vmax was 15 micromol/min. These kinetic constants are comparable to the constants of APRT from other organisms.

Adenine↗
PubMed1995

Cloning and characterization of the adenine phosphoribosyltransferase-encoding gene (APT1) from Saccharomyces cerevisiae.

We have cloned, sequenced and characterized the APT1 (adenine phosphoribosyltransferase) gene from Saccharomyces cerevisiae. The APT1 sequence includes an open reading frame encoding 221 amino acids and is contained within a 1322-bp insert that complements APRT-deficient mutants to wild-type levels of enzyme activity. Analysis by primer extension revealed multiple transcription start points (tsp) and a major tsp 21-bp upstream from the ATG start codon. A transcript initiated at the major tsp would yield a 700-nt mRNA which is in agreement with the size observed by Northern analysis. Sequence comparison indicates that the yeast enzyme shares strong similarities with other known APRT of bacterial, invertebrate, plant and mammalian origins.

Adenine Phosphoribosyltransferase↗