RESUMO
Six genes, CYC1, UTR1, UTR3, OSM1, tRNAGly, and RAD7, have been localized within an 8-kilobase region on chromosome X of the yeast Saccharomyces cerevisiae. The physical structures and the transcripts of these genes were identified by analyzing a normal strain and six deletion mutants by genomic blotting, transcriptional analysis, and gene disruption procedures. The well-studied CYC1 gene encodes iso-1-cytochrome c; the tRNAGly gene encodes a tRNA; deletion of OSM1 and RAD7 causes sensitivity to hypertonic medium and UV irradiation, respectively. There were no observable phenotypes in strains having deletions of the UTR1, UTR3, and tRNAGly gene. The high density of transcripts, with little or almost no intragenic regions, indicates that the chromosomal organization of S. cerevisiae resembles the chromosomal organization of procaryotes rather than higher eucaryotes.
Assuntos
DNA Fúngico/genética , Genes Fúngicos , Saccharomyces cerevisiae/genética , Mapeamento Cromossômico , Grupo dos Citocromos c/genética , Reparo do DNA , Genes , Ligação Genética , Genótipo , Fenótipo , RNA Fúngico/genética , RNA Mensageiro/genética , RNA de Transferência/genética , Transcrição Gênica , Raios Ultravioleta , Equilíbrio HidroeletrolíticoRESUMO
A vector was constructed that directs the expression of foreign genes in the yeast Saccharomyces cerevisiae. This vector contains an expression site that was constructed by in vitro modification of the iso-1-cytochrome c (CYC1) gene of S. cerevisiae. The expression of heterologous sequences can be experimentally controlled by catabolite control sequences, promoter and transcription initiation sequences and termination sequence derived from the CYC1 gene. A portion of a genomic wheat alpha-gliadin gene consisting of the entire 861 bp of protein-coding sequence, 18 bp of 5' leader sequence and 54 bp of 3'-noncoding sequence was inserted into the expression site. A CYC1::alpha-gliadin transcript of approx. 1050 nucleotides was synthesized in transformed yeast under the control of the CYC1 regulatory region. The transcripts terminated within the alpha-gliadin 3'-noncoding region, near a nucleotide sequence similar to the yeast transcription termination consensus sequence. The alpha-gliadin was immunochemically detected in total protein extracts from transformed cells and accounted for approx. 0.1% of the total cellular protein. The size of alpha-gliadin synthesized in yeast is the same as that of mature wheat alpha-gliadin. This is consistent with recognition and cleavage of the signal peptide by yeast. Due to the amino acid composition of alpha-gliadin, the availability of glutamine tRNA is a potential translational limitation to high-level synthesis in yeast.
Assuntos
Gliadina/genética , Proteínas de Plantas/genética , Triticum/genética , Códon , Vetores Genéticos , Gliadina/biossíntese , Regiões Promotoras Genéticas , Aminoacil-RNA de Transferência/fisiologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/genética , Transcrição GênicaAssuntos
Clonagem Molecular/métodos , DNA Recombinante/isolamento & purificação , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Oligonucleotídeos/genética , Bacteriófago lambda/genética , Sequência de Bases , Escherichia coli/genética , Proteínas Fúngicas/genética , Genes Virais , Óperon Lac , Saccharomyces cerevisiae/genéticaRESUMO
A genetic linkage map of papaya (Carica papaya L.) was constructed using randomly amplified polymorphic DNA (RAPD) markers and a F2 population derived from a University of Hawaii UH breeding line 356 x 'Sunrise' cross. A total of 596 10-mer primers were screened, and 96 polymorphisms were detected. At LOD 4.0, 62 of these markers mapped to 11 linkage groups comprising 999.3 cM. About 80% of the markers conformed to expected Mendelian segregation ratios. We have mapped the locus that determines sex to a 14-cM region flanked by RAPD markers. The results demonstrate the usefulness of RAPD markers for developing a basic genetic linkage map in papaya.
RESUMO
We have applied the recently developed technique of random amplification of polymorphic DNA (RAPD) to the analysis of the relationships among ten cultivars of papaya (Carica papaya L.). Eleven ten-base synthetic oligonucleotides were chosen that gave multiple PCR amplification products using papaya DNA as template. These 11 primers amplified a total of 102 distinct fragments. Cultivars were scored for presence or absence of RAPD fragments and grouped by cluster analysis using simple matching coefficients of similarity. A dendrogram of the ten cultivars was constructed. Of the ten cultivars seven were of the Hawaiian type, and all of these grouped to one branch of the tree. Divisions within the Hawaiian, branch were mostly consistent with the known genetic background of these cultivars. Three non-Hawaiian, cultivars were also analyzed. The minimum similarity detected was 0.7 suggesting that the domesticated papaya germ plasm is quite narrow. Our results show that RAPD technology is a rapid, precise and sensitive technique for genomic analysis.
RESUMO
We have constructed a plasmid that selectively integrates adjacent to the CYC1 locus, which determines iso-1-cytochrome c in the yeast Saccharomyces cerevisiae. Different CYC1 alleles can be conveniently recovered by digestion of total DNA from transformed strains with BgI II, a restriction endonuclease that does not cut the vector or the CYC1 gene, followed by transformation of Escherichia coli, selecting the ampicillin resistance gene carried on the original vector. This procedure was used to clone the cyc1-362 gene, which contains an alteration in front of the AUG initiation codon. The cyc1-362 mutational causes a deficiency of the iso-1-cytochrome c protein but still allows transcription of the iso-1-cytochrome c mRNA. DNA sequence analysis showed that the cyc1-362 mutation consisted of two single-base-pair substitutions, producing an A leads to G change 18 nucleotides and a G leads to A change 30 nucleotides in front of the AUG initiation codon in the mRNA. The A leads to G change at position -18 resulted in the creation of an AUG triplet, which is proximal to the normal initiation site and out of phase with the normal reading frame. The deficiency of iso-1-cytochrome c is most simply explained by assuming that translation initiates at the more proximal abnormal AUG site but not at the normal AUG site.
Assuntos
Grupo dos Citocromos c/análogos & derivados , Citocromos c , DNA Fúngico/genética , Mutação , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Bases , Clonagem Molecular , Códon , Grupo dos Citocromos c/genética , Biossíntese de Proteínas , RNA Fúngico/genética , RNA Mensageiro/genéticaRESUMO
We have developed molecular markers tightly linked to Sex1, the gene that determines plant sex in papaya ( Carica papaya L.). Three RAPD products have been cloned and a portion of their DNA sequenced. Based on these sequences SCAR primers were synthesized. SCAR T12 and SCAR W11 produce products in hermaphrodite and male plants and only rarely in females. SCAR T1 produces a product in all papayas regardless of plant sex. SCAR T12 and SCAR W11 showed no recombination in a population of 182 F2 plants from a 'SunUp' by 'Kapoho' cross. Based on these results a PCR-based technique for rapidly and accurately determining the sex of papaya plants was developed using either W11 or T12 to detect the hermaphrodite or male allele and T1, which amplifies a product regardless of sex type, as a positive control. The sexing technique, using SCAR T12 and SCAR T1 as a positive control, was used to correctly predict hermaphrodite papaya plants in a population of seedlings with an overall accuracy of 99.2%.