RESUMEN
Expression of the vestigial plastid genome of the nonphotosynthetic, parasitic flowering plant Epifagus virginiana was examined by northern analysis and by characterization of cDNAs. Probes for each of 12 plastid genes tested hybridized to all lanes of northern blots containing total RNA prepared from stems and fruits of Epifagus and from leaves of tobacco. Certain transcript patterns in Epifagus plastids are highly complex and similar to those of tobacco operons. In contrast, genes such as rps2, which have become orphaned in Epifagus as a result of evolutionary loss of formerly cotranscribed genes, show simpler transcript patterns in Epifagus than in tobacco. Sizing and sequencing of cDNAs generated by reverse transcriptase-PCR for three genes, rps12, rpl2, and clpP, show that their transcripts are properly cis- and/or trans-spliced at the same five group II intron insertion sites used in photosynthetic plants. A single, conventional C-->U edit in rps12 was found among the total of 1401 nucleotides of cDNA sequence that was determined for the three genes. An octanucleotide sequence identical to a putative guide RNA of plant organelles and perfectly complementary to the rps12 edit site itself was identified just 200 bp upstream of the edit site. These data, together with previous results from the complete sequencing of the Epifagus plastid genome, provide compelling evidence that this degenerate genome is nonetheless expressed and functional. Analysis of the putative maturase MatK, encoded by the group II intron of trnK in photosynthetic land plants but by a freestanding gene in Epifagus, leads us to hypothesize that it acts 'in trans' to assist the splicing of group II introns other than the one in which it is normally encoded.
Asunto(s)
Adenosina Trifosfatasas , Plantas/genética , Plastidios/genética , Procesamiento Postranscripcional del ARN , ARN de Planta/genética , Secuencia de Aminoácidos , Secuencia de Bases , Endopeptidasa Clp , Expresión Génica , Genes de Plantas , Intrones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Tirosina Quinasas/genética , Edición de ARN , Precursores del ARN/genética , Empalme del ARN , ARN Mensajero/genética , Proteínas Ribosómicas/genética , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/genética , Especificidad de la EspecieRESUMEN
pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases. The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 degrees. The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursors forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.
Asunto(s)
Proteínas Fúngicas/genética , Genes Fúngicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Codón , Proteínas Fúngicas/análisis , Prueba de Complementación Genética , Hidrolasas/metabolismo , Immunoblotting , Microscopía Electrónica , Datos de Secuencia Molecular , Mutación , Plásmidos , Mapeo Restrictivo , Saccharomyces cerevisiae/ultraestructura , Transcripción Genética , Vacuolas/enzimología , Proteínas de Transporte VesicularRESUMEN
The vacuolar protease B of Saccharomyces cerevisiae is a subtilisin-like protease encoded by the PRB1 gene. Antibodies raised against a synthetic peptide and an Escherichia coli-derived PRB1 open reading frame (ORF) protein cross-react with authentic protease B from yeast. By using these antibodies, the posttranslational biosynthetic pathway of protease B has been elucidated. Preproprotease B is a 76-kD unglycosylated precursor that enters the endoplasmic reticulum (ER), where it receives one asparagine-linked (Asn-linked) and an undetermined number of non-Asn-linked carbohydrate side chains. The large glycosylated intermediate is proteolytically processed to a 39-kD form before exiting the ER. In the Golgi complex, the 39-kD form becomes 40 kD, due to elaboration of the Asn-linked side chain. The carboxyterminal end of the 40-kD proprotease B undergoes protease A-mediated processing to a 37-kD intermediate, which in turn is quickly processed to 31-kD mature protease B. The ultimate processing step removes a peptide containing the Asn-linked chain; mature PrB has only non-Asn-linked carbohydrate.