Cloning and expression of full-length Trichoderma reesei cellobiohydrolase I cDNAs in Escherichia coli.

The process of converting lignocellulosic biomass to ethanol via fermentation depends on developing economic sources of cellulases. Trichoderma reesei cellobiohydrolase (CBH) I is a key enzyme in the fungal cellulase system; however, specific process application requirements make modification of the enzyme by site-directed mutagenesis (SDM) an attractive goal. To undertake SDM investigations, an efficient, cellulase-free host is required. To test the potential of Escherichia coli as a host, T. reesei CBH I cDNA was expressed in E. coli strain GI 724 as a C-terminal fusion to thermostable thioredoxin protein. Full-length expression of CBH I was subsequently verified by molecular weight, Western blot analysis, and activity on soluble substrates.

Analysis of the primary sequence and microtubule-binding region of the Drosophila 205K MAP.

We have sequenced cDNA clones encoding the Drosophila 205K microtubule-associated protein (MAP), a protein that may be the species specific homologue of mammalian MAP4. The peptide sequence deduced from the longest open-reading frame reveals a hydrophilic protein, which has basic and acidic regions that are similar in organization to mammalian MAP2. Using truncated forms of the 205K MAP, a 232-amino acid region could be defined that is necessary for microtubule binding. The amino acid sequence of this region shares no similarity with the binding motif of MAP2 or tau. We also analyzed several embryonic cDNA clones, which show the existence of differentially spliced mRNAs. Finally, we identified several potential protein kinase target sequences. One of these is distal to the microtubule-binding site and fits the phosphorylation consensus sequence of proteins phosphorylated by the mitosis specific protein kinase cdc2. Our data suggest that the 205K MAP uses a microtubule-binding motif unlike that found in other MAPs, and also raise the possibility that the activities of the 205K MAP may be regulated by alternative splicing and phosphorylation.

A three-domain structure of kinesin heavy chain revealed by DNA sequence and microtubule binding analyses.

The structure and function of kinesin heavy chain from D. melanogaster have been studied using DNA sequence analysis and analysis of the properties of truncated kinesin heavy chain synthesized in vitro. Analysis of the sequence suggests the existence of a 50 kd globular amino-terminal domain that contains an ATP binding consensus sequence, followed by another 50-60 kd domain that has sequence characteristics consistent with the ability to fold into an alpha helical coiled coil. The properties of amino- and carboxy-terminally truncated kinesin heavy chains synthesized in vitro reveal that a 60 kd amino-terminal fragment has the nucleotide-dependent microtubule binding activities of the intact kinesin heavy chain, and hence is likely to be a "motor" domain. Finally, the sequence data indicate the presence of a small carboxy-terminal domain. Because it is located at the end of the molecule away from the putative "motor" domain, we propose that this domain is responsible for interactions with other proteins, vesicles, or organelles. These data suggest that kinesin has an organization very similar to that of myosin even though there are no obvious sequence similarities between the two molecules.

Identification of the gene for fly non-muscle myosin heavy chain: Drosophila myosin heavy chains are encoded by a gene family.

In contrast to vertebrate species Drosophila has a single myosin heavy chain gene that apparently encodes all sarcomeric heavy chain polypeptides. Flies also contain a cytoplasmic myosin heavy chain polypeptide that by immunological and peptide mapping criteria is clearly different from the major thoracic muscle isoform. Here, we identify the gene that encodes this cytoplasmic isoform and demonstrate that it is distinct from the muscle myosin heavy chain gene. Thus, fly myosin heavy chains are the products of a gene family. Our data suggest that the contractile function required to power myosin based movement in non-muscle cells requires myosin diversity beyond that available in a single heavy chain gene. In addition, we show, that accumulation of cytoplasmic myosin transcripts is regulated in a developmental stage specific fashion, consistent with a key role for this protein in the movements of early embryogenesis.

Structure of transcripts from the homeotic Antennapedia gene of Drosophila melanogaster: two promoters control the major protein-coding region.

The Antennapedia (Antp) homeotic gene of Drosophila melanogaster regulates segmental identity in the thorax. Loss of Antp function results in altered development of the embryonic thoracic segments or can cause legs to be transformed into antennae. Certain combinations of Antp recessive lethal alleles complement to permit normal development. The structure of the Antp gene, analyzed by sequencing cDNA clones and exons and by transcript mapping, revealed some of the basis for its genetic complexity. It has two promoters governing two nested transcription units, one unit 36 and one 103 kilobase pairs (kb) long. Both units incorporated the same protein-coding exons, all of which are located in the 3'-most 13 kb of the gene. The two promoters resulted in the attachment of either of two long noncoding leader sequences (1.5 and 1.7 kb) to a 1.1-kb open reading frame. Both transcription units used the same pair of alternative polyadenylation sites 1.4 kb apart; the choice of sites was developmentally regulated. Some of the mutations that disrupt the larger transcription unit complemented a mutation affecting the smaller one. Dominant mutations that transform antennae into legs split the gene but left the coding exons intact. The encoded protein has unusually long runs of glutamine and a homeodomain near the C terminus.

The localization and regulation of Antennapedia protein expression in Drosophila embryos.

The homeotic Antennapedia (Antp) gene of Drosophila is required for the normal differentiation of the thoracic segments during embryonic development and metamorphosis. Antibodies to a recombinant Antp protein were used to localize the protein in whole mount embryos. Antp is expressed in the nuclei of cells of the thoracic embryonic epidermis and several segments of the ventral and peripheral nervous systems. Analysis of Antp expression in mutant embryos revealed three levels of Antp regulation by genes of the bithorax complex, pleiotropic homeotic loci, and Antp itself. The distributions of the Antp and the Ultrabithorax (Ubx) proteins in doubly-labeled embryos suggest that the Ubx protein may be one direct negative regulator of Antp gene expression.

A microtubule-associated protein in Drosophila melanogaster: identification, characterization, and isolation of coding sequences.

Microtubules and microtubule-associated proteins (MAPs) have been isolated from cultured cells of Drosophila melanogaster by a taxol-dependent polymerization procedure. The principal MAPs are a group of four polypeptides with similar electrophoretic mobilities corresponding to approximately Mr 205,000 (the 205K MAP). These proteins are resistant to precipitation by boiling. One mouse monoclonal antibody and one polyclonal rabbit antiserum specific for the Mr 205,000 MAP were produced and characterized by immunoblotting and indirect immunofluorescence. Both antibody preparations stain the Mr 205,000 molecules and an Mr 255,000 molecule in immunoblots of Drosophila cell homogenates; the rabbit antiserum also stains an Mr 150,000 triplet. Both preparations stain the microtubules of the mitotic spindle, and the rabbit antiserum stains the cytoplasmic microtubules as well. Experiments using affinity-purified rabbit antiserum demonstrate that it is the Mr 205,000 species that is located in the mitotic apparatus and on cytoplasmic microtubules. A random shear genomic library was produced in the expressing vector lambda gt11 and screened with the rabbit antiserum to isolate the DNA sequences encoding these polypeptides. Several cross-hybridizing clones were recovered, shown to encode antigenic determinants in the Mr 205,000 MAP, and characterized by hybridization to Northern blots of mRNA and Southern blots of genomic DNA. Analysis by in situ hybridization reveals that the gene encoding the 205K MAP is located in polytene region 100EF.