RESUMO
The precursor of the ribulose-1,5-bisphosphate carboxylase small subunit and other proteins from Chlamydomonas reinhardtii are efficiently transported into chloroplasts isolated from spinach and pea. Thus, similar determinants specify precursor-chloroplast interactions in the alga and vascular plants. Removal of all or part of its transit sequence was found to block import of the algal small subunit into isolated chloroplasts. Comparison of available sequences revealed a nine amino acid segment conserved in the transit sequences of all small subunit precursors. A protease in the vascular plant chloroplasts recognized this region in the Chlamydomonas precursor and produced an intermediate form of the small subunit. We propose that processing of the small subunit precursor involves at least two proteolytic events; only one of these has been evolutionarily conserved.
Assuntos
Chlamydomonas/enzimologia , Cloroplastos/enzimologia , Precursores Enzimáticos/metabolismo , Plantas/enzimologia , Ribulose-Bifosfato Carboxilase/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Cinética , Substâncias Macromoleculares , Biossíntese de Proteínas , Processamento de Proteína Pós-Traducional , Especificidade da EspécieRESUMO
Mutations caused by the insertion of members of the Ac or Spm family of transposable elements result in a great diversity of phenotypes. With the cloning of the mutant genes and the characterization of their products, the mechanisms underlying phenotypic diversity are being deciphered. These mechanisms include (i) imprecise excision of transposable elements, which can result in the addition of amino acids to proteins; (ii) DNA methylation, which has been correlated with the activity of the element; (iii) transposase-mediated deletions within elements, which can inactivate an element or lead to a new unstable phenotype; and (iv) removal of transcribed elements from RNA, which can facilitate gene expression despite the insertion of elements into exons. An understanding of the behavior of the maize elements has provided clues to the function of cryptic elements in all maize genomes.
Assuntos
Elementos de DNA Transponíveis , Plantas/genética , Alelos , Mutação , Fenótipo , Zea mays/genéticaRESUMO
In some instances, insertion of maize transposable elements into exons does not result in the total loss of enzymatic activity. In other instances, messenger RNAs of wild-type size are encoded by genes known to contain the maize transposable element Dissociation (Ds) in exons. To understand how Ds is processed from RNA, a study was made of transcripts encoded by two alleles of the maize waxy (wx) gene containing Ds insertions in exon sequences. The analysis was carried out in strains where the Ds element could not excise from the wx gene. Despite insertions of 4.3- and 1.5-Ds elements, the predominant transcripts encoded by these two genes were wild type in size. For both alleles, DNA sequencing of complementary DNAs revealed that the Ds elements had been spliced in a similar manner. Splicing was accomplished by the utilization of multiple 5' donor splice sites in the Ds termini and a 3' acceptor site within the wx gene adjacent to the Ds element. The net effect in both cases was the removal of most of the Ds element from the messenger RNA.
Assuntos
Elementos de DNA Transponíveis , Splicing de RNA , RNA Mensageiro/genética , Zea mays/genética , Sequência de Bases , Clonagem Molecular , DNA/genética , Éxons , Transcrição GênicaRESUMO
The temporal and spatial patterns of anthocyanin pigmentation in the maize plant are determined by the presence or absence of the R protein product, a presumed transcriptional activator. At least 50 unique patterns of pigmentation, conditioned by members of the R gene family, have been described. In this study, microprojectiles were used to introduce into maize cells a vector containing the transcription unit from one of these genes (Lc) fused to a constitutive promoter. This chimeric gene induces cell autonomous pigmentation in tissues that are not normally pigmented by the Lc gene. As a reporter for gene expression studies in maize, R is unique because it can be quantified in living tissue simply by counting the number of pigmented cells following bombardment. R may also be useful as a visible marker for selecting stably transformed cell lineages that can give rise to transgenic plants.
RESUMO
Retrotransposons are an abundant and ancient component of plant genomes, yet recent evidence indicates that element activity in many modern plants is restricted to times of stress. Stress activation of plant retrotransposons may be a significant factor in somaclonal variation, in addition to providing an important means to isolate new active elements. Long terminal repeat retrotransposons and a second class of elements we have called miniature inverted-repeat transposable elements (MITEs) have recently been found to be associated with the genes of diverse plants where some contribute regulatory sequences. Because of their sequence diversity and small size, MITEs may be a valuable evolutionary tool for altering patterns of gene expression.
Assuntos
Evolução Biológica , Elementos de DNA Transponíveis , Genoma de Planta , Plantas/genética , Sequências Repetitivas de Ácido Nucleico , Retroelementos , Animais , Sequência de Bases , Variação Genética , Mamíferos/genética , Transcrição GênicaRESUMO
All known active plant retrotransposons are largely quiescent during development but activated by stresses, including wounding, pathogen attack and cell culture. This may reflect a survival strategy based on plant biology, or retrotransposons could be the stress-induced generators of genomic diversity proposed by McClintock.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Plantas/genética , Retroelementos , Fenômenos Fisiológicos VegetaisRESUMO
The null wx-ml allele contains a 409-bp Dissociation 1 (Ds1) element in exon 9 of the maize waxy (Wx) gene. In the absence of the autonomous Activator (Ac) element, the Ds1 element cannot transpose, and this allele encodes several Wx transcripts that arise following alternative splicing of Ds1 sequences from Wx pre-mRNA. Splicing involves the utilization of three 5' splice sites and three 3' splice sites. All but one of these splice sites are in Ds1 sequences near the ends of the element. The presence of 5' and 3' splice sites near the Ds1 termini and the element's small size and AT richness are features that distinguish Ds1 elements from all other known Ds elements. It is suggested that these features may enhance the ability of Ds1 to function as a mobile intron.
Assuntos
Elementos de DNA Transponíveis , Éxons , Splicing de RNA , Zea mays/genética , Alelos , Sequência de Bases , Northern Blotting , Southern Blotting , DNA , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Three mutations, each causing constitutive expression of the Salmonella typhimurium leu operon, were cloned into phage vector lambda gt4 on EcoRI DNA fragments carrying all of that operon except for part of the promoter-distal last gene. Sequence analysis of DNA from these phage demonstrated that each contains a single base change in the leu attenuator. Transcription of mutant DNA in vitro resulted in transcription beyond the usual site of termination. The level of beta-IPM dehydrogenase, the leuB enzyme, was elevated 40-fold in a strain carrying one of these mutations, and starvation of this strain for leucine had little effect on the amount of activity expressed. Using a strain with a wild-type promoter-leader region of the leu operon, the rates of synthesis and degradation of leu leader RNA and readthrough RNA (leu mRNA) were measured by DNA-RNA hybridizations with specific DNA probes. The rate of synthesis of the leu leader was about the same in cells grown with excess or with limiting leucine. On the other hand, the rate of synthesis of leu mRNA was 12-fold higher for cells grown in limiting leucine as opposed to excess leucine. The rate of degradation of these RNA species was the same under both conditions of growth. Thus, the variation in expression of the leu operon observed for cells grown in minimal medium is, for the most part, not caused by control over the frequency of initiation or by the differential stability of these RNA species. Rather, the variation is a direct result of the frequency of transcription termination at an attenuator site. These results taken together suggest that transcription attenuation is the major mechanism by which leucine regulates expression of the leu operon of S. typhimurium for cells growing in a minimal medium.
Assuntos
Leucina/genética , Óperon , Salmonella typhimurium/genética , Transcrição Gênica , Bacteriófago lambda/genética , Sequência de Bases , DNA Bacteriano , DNA Recombinante , DNA Viral , Mutação , Hibridização de Ácido Nucleico , RNA Bacteriano , RNA Mensageiro/metabolismo , Moldes GenéticosRESUMO
The waxy gene, which is responsible for the synthesis of amylose in endosperm and pollen, is genetically well characterized in many grasses including maize and rice. Homology between the previously cloned maize waxy gene and the rice gene has facilitated our cloning of a 15-kb HindIII fragment that contains the entire rice gene. A comparison of the restriction maps of the maize and rice genes indicates that many restriction sites within translated exons are conserved. In addition, the rice gene encodes a 2.4-kb transcript that programs the in vitro synthesis of a 64-kD pre-protein which is efficiently precipitated with maize waxy antisera. We demonstrate that these gene products are altered in rice strains containing mutant waxy genes. Southern blot analysis of 16 rice strains, ten containing waxy mutations, reveals that the waxy gene and flanking restriction fragments are virtually identical. These results contrast dramatically with the high level of insertions and deletions associated with restriction fragment length polymorphism and spontaneous mutations among the waxy alleles of maize.
Assuntos
Amilose/genética , Genes , Mutação , Oryza/genética , Zea mays/genética , Alelos , Clonagem Molecular , Immunoblotting , Peso Molecular , Polimorfismo de Fragmento de Restrição , Testes de Precipitina , Precursores de Proteínas/genética , Mapeamento por RestriçãoRESUMO
Anthocyanin pigmentation patterns in different plant species are controlled in part by members of the myc-like R regulatory gene family. We have examined the molecular evolution of this gene family in seven plant species. Three regions of the R protein show sequence conservation between monocot and dicot R genes. These regions encode the basic helix-loop-helix domain, as well as conserved N-terminal and C-terminal domains; mean replacement rates for these conserved regions are 1.02 x 10(-9) nonsynonymous nucleotide substitutions per site per year. More than one-half of the protein, however, is diverging rapidly, with nonsynonymous substitution rates of 4.08 x 10(-9) substitutions per site per year. Detailed analysis of R homologs within the grasses (Poaceae) confirm that these variable regions are indeed evolving faster than the flanking conserved domains. Both nucleotide substitutions and small insertion/deletions contribute to the diversification of the variable regions within these regulatory genes. These results demonstrate that large tracts of sequence in these regulatory loci are evolving at a fairly rapid rate.
Assuntos
Evolução Biológica , Genes de Plantas , Genes Reguladores , Família Multigênica , Poaceae/genética , Sequência de Aminoácidos , Sequência de Bases , Primers do DNA , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de AminoácidosRESUMO
A few families of retrotransposons characterized by the presence of long terminal repeats (LTRs) have amplified relatively recently in maize and account for >50% of the genome. Surprisingly, none of these elements have been shown to cause a single mutation. In contrast, most of the retrotransposon-induced mutations isolated in maize are caused by the insertion of elements that are present in the genome at 2-50 copies. To begin to understand what limits the amplification of this mutagenic class of LTR-retrotransposons, we are focusing on five elements previously identified among 17 mutations of the maize waxy gene. One of these elements, Stonor, has sustained a deletion of the entire gag region and part of the protease domain. Missing sequences were recovered from larger members of the Stonor family and indicate that the deletion probably occurred during retrotransposition. These large elements have an exceptionally long leader of 2 kb that includes a highly variable region of approximately 1 kb that has not been seen in previously characterized retrotransposons. This region serves to distinguish each member of the Stonor family and indicates that no single element has yet evolved that can attain the very high copy numbers characteristic of other element families in maize.
Assuntos
Variação Genética , Genoma de Planta , Retroelementos/genética , Zea mays/genética , Sequência de Aminoácidos , Dados de Sequência Molecular , Mutação , Alinhamento de Sequência , Especificidade da EspécieRESUMO
R and B genes and their homologues encode basic helix-loop-helix (bHLH) transcriptional activators that regulate the anthocyanin biosynthetic pathway in flowering plants. In maize, R/B genes comprise a very small gene family whose organization reflects the unique evolutionary history and genome architecture of maize. To know whether the organization of the R gene family could provide information about the origins of the distantly related grass rice, we characterized members of the R gene family from rice Oryza saliva. Despite being a true diploid, O. sativa has at least two R genes. An active homologue (Ra) with extensive homology with other R genes is located at a position on chromosome 4 previously shown to be in synteny with regions of maize chromosomes 2 and 10 that contain the B and R loci, respectively. A second rice R gene (Rb) of undetermined function was identified on chromosome 1 and found to be present only in rice species with AA genomes. All non-AA species have but one R gene that is Ra-like. These data suggest that the common ancestor shared by maize and rice had a single R gene and that the small R gene families of grasses have arisen recently and independently.
Assuntos
Mapeamento Cromossômico , DNA de Plantas , Genes de Plantas , Genoma de Planta , Oryza/genética , Zea mays/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Dosagem de Genes , Dados de Sequência Molecular , Filogenia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
The molecular basis for the unusual phenotype conditioned by the waxy(Wx)-m5 Ds allele has been elucidated. Unlike most Ds alleles, Wx-m5 is phenotypically wild-type in the absence of Ac. We find that the Wx-m5 gene contains a 2-kb Ds element at -470 relative to the start of Wx transcription, representing the most 5' insertion of any transposable element allele characterized to date in plants. Despite its wild type phenotype, Wx-m5 has reduced levels of Wx enzymatic activity indicating that Ds insertion influences Wx gene expression. In the presence of Ac, Wx-m5 kernels have sectors of null expression on a wild-type background and give rise to stable wx and unstable wx-m germinal derivatives. Seventeen of 20 derivatives examined are wx-m alleles and at least 15 of these appear to result from intragenic transposition of Ds from -470 to new sites within the Wx gene. Three wx-m alleles contain two Ds elements, one at -470 and a second in Wx coding sequences. Surprisingly, only 3 out of 20 derivatives are stable wx mutants and these have sustained gross rearrangements of Wx and flanking sequences. For most other maize transposable element alleles somatic sectors and germinal derivatives usually arise following element excision or deletions of element sequences. In contrast, element insertion following intragenic transposition is apparently responsible for most of the somatic sectors and germinal derivatives of Wx-m5.
Assuntos
Elementos de DNA Transponíveis/genética , Proteínas de Plantas/genética , Zea mays/genética , Alelos , Sequência de Bases , Southern Blotting , Regulação da Expressão Gênica/genética , Dados de Sequência Molecular , FenótipoRESUMO
A mutation at the maize waxy locus, wx1240, was isolated following treatment of pollen with EMS and self-pollinating ears on M1 plants. This allele was cloned and found to contain a 30-bp deletion within the gene and additional lesions upstream of the transcription start site. Using fine structure genetic mapping, we determined that the deletion is responsible for the mutant phenotype. In addition, the position of wx1240 on the genetic map coincided with the previously determined positions of two other waxy mutations, the spontaneous wx-C, which is reference allele, and the putative ethyl methanesulfonate (EMS)-induced wx-BL2. Molecular analysis of these alleles revealed that both contain the same deletion as wx1240, and that the wx-BL2 allele is similar to wx-C and possibly resulted from wx-C contamination. The deleted sequence responsible for these mutations is flanked by a short, 4-bp, direct repeat. Similar structures are favored sites for spontaneous deletions in other organisms. The data suggests that EMS is capable of inducing structural alterations in plant genes in addition to the point mutations normally ascribed to EMS-induced mutations.
Assuntos
Amilose/biossíntese , Mutação , Zea mays/genética , Alelos , Sequência de Bases , Southern Blotting , Cruzamentos Genéticos , DNA , Metanossulfonato de Etila , Dados de Sequência Molecular , Mutagênese , Fenótipo , Mapeamento por RestriçãoRESUMO
The R and B proteins of maize are required to activate the transcription of several genes in the anthocyanin biosynthetic pathway. To determine the structural requirements for R function in vivo, we are exploiting its sensitive mutant phenotype to identify transposon (Ds) insertions that disrupt critical domains. Here we report that the ability of the r-m1 allele to activate transcription of at least three structural genes is reduced to only 2% of wild-type activity because of a 396-bp Ds element in helix 2 of the basic helix-loop-helix (bHLH) motif. Residual activity likely results from the synthesis of a mutant protein that contains seven additional amino acids in helix 2. This protein is encoded by a transcript where most of the Ds sequence has been spliced from pre-mRNA. Two phenotypic classes of stable derivative alleles, very pale and extremely pale, condition <1% of wild-type activity as a result of the presence of two- and three-amino-acid insertions, respectively, at the site of Ds excision. Localization of these mutant proteins to the nucleus indicates a requirement for an intact bHLH domain after nuclear import. The fact that deletion of the entire bHLH domain has only a minor effect on R protein activity while these small insertions virtually abolish activity suggests that deletion of the bHLH domain may bypass a requirement for bHLH-mediated protein-protein interactions in the activation of the structural genes in the anthocyanin biosynthetic pathway.
Assuntos
Elementos de DNA Transponíveis , Genes de Plantas , Sequências Hélice-Alça-Hélice , Mutagênese , Proteínas Nucleares/genética , Proteínas de Plantas/genética , Zea mays/genética , Alelos , Sequência de Aminoácidos , Sítios de Ligação , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fenótipo , Proteínas de Plantas/metabolismo , RNA Mensageiro , RNA de Plantas , Frações Subcelulares , Transformação GenéticaRESUMO
There are six examples of maize transposable elements that are spliced from pre-mRNA. These represent the first introns that have been added to nuclear genes in recent years. All six are members of the Ac/Ds or Spm/dSpm family of elements and all have been inserted into exons of active genes within the last twenty-five years. The structure of these element-introns and the sequences involved in their splicing are presented. These examples illustrate how active transposable elements can also function as introns and how they may evolve into stable introns.
Assuntos
Elementos de DNA Transponíveis , Íntrons , Precursores de RNA/metabolismo , Splicing de RNA , Zea mays/genética , Alelos , Mutação , Precursores de RNA/genética , Homologia de Sequência do Ácido NucleicoRESUMO
Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker ( Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (=0.20). Only two markers (3%) were monomorphic in all samples. Although DNA sequencing indicated that 5.5% of same-sized DNA fragments were non-homologous, this result did not affect the cluster analyses (i.e., relationships obtained from the Hbr data were congruent with those derived from pedigree information). Distance matrices generated from Hbr markers were significantly correlated ( p<0.001) with those obtained from pedigree ( r=0.782), RFLPs ( r=0.747), and SSRs ( r=0.719). Overall, these results indicated that Hbr markers could be used in conjunction with other molecular markers for genotyping and relationship studies of related maize inbred lines.
RESUMO
Most plant genomes are populated with enormous quantities of transposable elements (TEs) or sequences derived from TEs. The impact of TEs on their host has been addressed by characterizing mutations of the maize waxy and R genes caused by TE insertions. Association between a new class of TEs (called MITEs) and normal plant genes is also reviewed. The notion that different classes of TEs have found their respective niches in the maize genome is discussed.
Assuntos
Elementos de DNA Transponíveis , Evolução Molecular , Genes de Plantas , Expressão Gênica , Zea mays/genéticaRESUMO
The nucleotide sequence of the 1494 bp wxB4 Ds element is presented. A comparison with previously characterized Ds elements reveals several novel features. This element has less Ac terminal sequence than other Ac-like Ds elements. The left terminus contains 398 bp of Ac sequence interrupted by a transposon-like DNA insertion, leaving only 317 bp of contiguous Ac sequence. The right terminus has 259 bp of Ac terminal sequence. The interior of the element contains sequences not found in other cloned members of the Ac/Ds family. We suggest that the role of this non-Ac DNA is to separate the Ac termini by a minimum distance and may be a cis requirement for Ds transposition in maize.