RESUMO
The Drosophila protein Chip potentiates activation by several enhancers and is required for embryonic segmentation. Chip and its mammalian homologs interact with and promote dimerization of nuclear LIM proteins. No known Drosophila LIM proteins, however, are required for segmentation, nor for expression of most genes known to be regulated by Chip. Here we show that Chip also interacts with diverse homeodomain proteins using residues distinct from those that interact with LIM proteins, and that Chip potentiates activity of one of these homeodomain proteins in Drosophila embryos and in yeast. These and other observations help explain the roles of Chip in segmentation and suggest a model to explain how Chip potentiates activation by diverse enhancers.
Assuntos
Proteínas de Drosophila , Proteínas de Homeodomínio/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Cromatografia de Afinidade , Proteínas de Ligação a DNA/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Modelos Biológicos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras , Transativadores/química , Transativadores/genética , Transcrição Gênica , Ativação TranscricionalRESUMO
The induction of cellulases by cellulose, an insoluble polymer, in the filamentous fungus Trichoderma reesei is puzzling. We previously proposed a mechanism that is based on the presence of low levels of cellulase in the uninduced fungus; this basal cellulase activity would digest cellulose-releasing oligosaccharides that could enter the cell and trigger expression of cellulases. We now present experiments that lend further support to this model. We show here that transcripts of two members of the cellulase system, cbh1 and egl1, are present in uninduced T. reesei cells. These transcripts are induced at least 1100-fold in the presence of cellulose. We also show that a construct containing the hygromycin B resistance-encoding gene driven by the cbh1 promoter confers hygromycin B resistance to T. reesei cells grown in the absence of cellulose. Moreover, cellulose-induced production of the cbh1 transcript was suppressed when antisense RNA against three members of the cellulase system was expressed in vivo. Experiments are presented indicating that extracellular cellulase activity is the rate-limiting event in induction of synthesis of the cellulase transcripts by cellulose. The results reveal a critical requirement for basal expression of the cellulase system for induction of synthesis of its own transcripts by cellulose.
Assuntos
Celulase/biossíntese , Celulose/metabolismo , Trichoderma/enzimologia , Autorradiografia , Sequência de Bases , Northern Blotting , Celulase/genética , Celulase/metabolismo , Celulose 1,4-beta-Celobiosidase , DNA Complementar/química , Indução Enzimática , Regulação Enzimológica da Expressão Gênica , Glucanos/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , RNA Antissenso/metabolismo , RNA Mensageiro/metabolismoRESUMO
Four mutants of Trichoderma reesei defective in cellulose utilization were characterized at the molecular level. Genomic analysis of the cellulase-encoding genes (cel) and transcript induction using two well-established inducers of the cel system--the insoluble polymer, cellulose and the soluble inducer, sophorose,--revealed that these mutants are defective in the transcription of cel genes. The results also indicate that the cel genes are coordinately expressed and most probably are regulated by the same mechanism. Using a heterologous gene construct, in which the hygromycin-B-resistance-encoding gene was placed under the control of the promoter of the major cel gene, cbh1, we showed that the mutants synthesize basic levels of cellulase, but are defective in the cel induction.
Assuntos
Celulase/genética , Celulose/biossíntese , Mutação , Trichoderma/genética , Celulase/biossíntese , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Trichoderma/enzimologia , Trichoderma/metabolismoRESUMO
The first enzyme of the lysine degradation pathway in maize (Zea mays L.), lysine-ketoglutarate reductase, condenses lysine and [alpha]-ketoglutarate into saccharopine using NADPH as a cofactor, whereas the second, saccharopine dehydrogenase, converts saccharopine to [alpha]-aminoadipic-[delta]-semialdehyde and glutamic acid using NAD+ or NADP+ as a cofactor. The reductase and dehydrogenase activities are optimal at pH 7.0 and 9.0, respectively. Both enzyme activities, co-purified on diethylaminoethyl-cellulose and gel filtration columns, were detected on nondenaturing polyacrylamide gels as single bands with identical electrophoretic mobilities and share tissue specificity for the endosperm. The highly purified preparation containing the reductase and dehydrogenase activities showed a single polypeptide band of 125 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native form of the enzyme is a dimer of 260 kD. Limited proteolysis with elastase indicated that lysine-ketoglutarate reductase and saccharopine dehydrogenase from maize endosperm are located in two functionally independent domains of a bifunctional polypeptide.
