RESUMO
In addition to the bZIP protein Opaque2 (O2), there are other maize endosperm nuclear proteins that recognize the O2 box in 22 kDa zein gene promoters. In an effort to understand the effect of these factors on 22 kDa zein expression, we have cloned one of these and identified it as the putative maize (Zea mays L.) orthologue of the wheat bZIP protein EmBP-1 (mEmBP-1). The mEmBP-1 protein exhibits 52% sequence identity and 68% similarity with the wheat protein and recognizes a similar spectrum of DNA sequences, albeit with slightly altered specificity. The mEmBP-1 gene exists as duplicate loci in maize on chromosomes 7S (mEmBP-1a) and 2L (mEmBP-1b). The mEmBP-1 genes are expressed in endosperm, embryo, immature ears, tassel, roots, and seedling shoots at low levels. Although mEmBP-1 binds to the O2 box from the 22 kDa zein gene promoter as a homodimer, it is unable to heterodimerize with O2. The mEmBP-1 protein can activate transcription from a truncated promoter containing a pentamer of the O2 site in yeast cells; however, it inhibited regulated transcription of a 22 kDa zein promoter in a transient expression assay using cultured maize endosperm cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/genética , Sequência de Aminoácidos , Fatores de Transcrição de Zíper de Leucina Básica , Células Cultivadas , Proteínas de Ligação a DNA/biossíntese , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Sinais de Localização Nuclear , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico , Sementes/citologia , Sementes/genética , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/biossíntese , Zea mays/citologia , Zeína/genéticaRESUMO
Site-directed mutations in the Escherichia coli ssb gene were tested for the ability to complement a chromosomal ssb deletion for viability, and only the ssb W54-->G mutation failed to do so at the pSC101 copy level. Non-aromatic amino acid substitutions for SSB Trp-54 (ssb W54-->L and ssb W54-->S) produced the greatest effects on in vivo protein function including altered marker linkage subsequent to generalized transduction, extreme UV sensitivity, and a lack of ability to support SOS induction. Additionally, the ssb-113 (ssb P176-->S) mutation demonstrated the existence of both uvrA-dependent and uvrA-independent components of SOS induction. Although nucleotide excision repair appeared unaffected by alterations in the SSB protein, the mutational analysis suggests a direct role for SSB in recombinational repair.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Resposta SOS em Genética , Bacteriófago P1/metabolismo , Clonagem Molecular , Conjugação Genética , Teste de Complementação Genética , Óperon Lac , Mutagênese Sítio-Dirigida , Recombinases Rec A/genética , Recombinação Genética , Temperatura , Transdução Genética , Raios UltravioletaRESUMO
Site-directed mutations involving selected amino acids of Escherichia coli single-stranded DNA-binding protein (SSB) were tested for their in vivo functionality when introduced into a chromosomal ssb deletion strain on a plasmid. All mutants complemented the ssb deletion for viability when present on a pSC101 derivative. The generation time with ssbW54S doubled in comparison to the ssb+ control, and both the ssbW54S- and ssbH55K-containing strains exhibited temperature sensitivity. ssbH55K, ssbW54S, ssbW88T, and ssbH55Y (ssb-1) strains displayed reduced survival to ultraviolet irradiation, while ssbW40T and ssbF60L strains were comparable to the ssb+ control strain. This study represents the first investigation of the in vivo properties of ssb mutations constructed for in vitro analysis of DNA binding by SSB.