Molecular mechanisms of spider silk.

Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers.

Isolation and characterization of the activated B-cell factor 1 homolog in Caenorhabditis elegans.

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate a wide array of developmental processes in many cell types, including cell fate specification, differentiation and morphogenesis. Our studies describe the cloning of a gene from the nematode Caenorhabditis elegans that is closely related to the vertebrate-activated B-cell factor (ABF) gene. The nematode gene product CeABF-1 was detected by northern blot analysis from RNA isolated from pooled nematodes representing different developmental stages. The developmental expression profile of CeABF-1 was shown by RT-PCR analysis to be predominantly expressed in the larval stages L3 and L4, with lower levels observed in the L2 larval stage and adult. We also show that CeABF-1 is capable of forming heterodimers with E2A proteins and binding E-box target sites. Mammalian cells transfected with CeABF-1 expression plasmids were capable of blocking E2A-mediated gene transcription, but full repression activity required the presence of two conserved amino acid residues found within the first helix of the CeABF-1 bHLH domain. These results suggest a conserved mechanism of gene repression between certain class II bHLH and class I bHLH proteins found in vertebrates and invertebrates.

Characterization of a basic helix-loop-helix protein, ABF-1: nuclear localization, transcriptional properties, and interaction with Id-2.

The activated B-cell factor (ABF)-1 cDNA was initially isolated from Epstein-Barr virus (EBV)-infected B cells and codes for a DNA-binding protein belonging to the basic helix-loop-helix (bHLH) family of transcription factors. In this study, we characterized the nuclear localization signal of ABF-1, mapped two distinct transcriptional repression domains, and identified one ABF-1-interacting protein, Id-2. By examining the subcellular location of deletion mutants of ABF-1 fused to green fluorescent protein (GFP), critical regions involved in nuclear localization were determined. Analysis of GFP-tagged ABF-1 deletion mutants revealed two separate regions capable of directing nuclear localization. One region mapped to the N-terminal amino acids 71 to 103, whereas the second region localized to the C-terminal bHLH domain. Transient transfection of ABF-1 deletion mutants demonstrated that the N-terminal amino acids 1 to 40 and the bHLH domain function together to achieve maximum repression of E2A activity. Taken together, these results indicate that ABF-1 is a nuclear transcriptional repressor with two distinct regions that function in a synergistic fashion to attenuate E2A-mediated gene activation.

The genomic structure and promoter analysis of the human ABF-1 gene.

The human ABF-1 gene is expressed in activated B-cells and Epstein-Barr virus-immortalized lymphoblastoid cell lines. ABF-1 represents the only member belonging to the basic helix-loop-helix (bHLH) family of transcription factors whose expression pattern is restricted to B-cells. ABF-1 forms heterodimeric complexes with E2A to modulate gene transcription. We report the cloning and characterization of the human ABF-1 gene and the promoter region. The gene spans more than 3 kb and contains two exons. Exon 1 contains 274 bp of a 5'-untranslated sequence (UTR) while exon 2 contains 1097 bp of 3'-UTR. Promoter analysis of the 5'-flanking region revealed no apparent B-cell-restricted control elements within approximately 700 bp, but clearly demonstrated the presence of a functional minimal promoter residing immediately upstream of the transcription start site. Analysis of the region containing the minimal promoter activity identified no CCAAT or TATA sequence. Lastly, we have assigned the ABF-1 gene to human chromosome 8q21.1 using fluorescence in situ hybridization (FISH). The cloning of the human ABF-1 gene will facilitate further biochemical and genetic studies of its function in the regulation of B-cell differentiation.

The Pan basic helix-loop-helix proteins are required for insulin gene expression.

The rat I insulin enhancer contains two principal regulatory elements, the Nir and Far motifs of an identical 9-base pair sequence, which function both as positive and negative cis-acting elements. The Nir and Far elements are targets for DNA-binding proteins, which play a predominant role in the selective transcription of the insulin gene in endocrine beta-cells. In vitro DNA-binding studies have demonstrated the ability of several helix-loop-helix (HLH) proteins, including the Pan/E2A proteins, upstream stimulating factor, human beta-HLH factor (rat beta-HLH factor), and E2-2/ITF-2, to bind the Nir and Far enhancer motifs. The presence of the aforementioned different HLH proteins in endocrine beta-cells, all of which display similar binding affinities for the Nir and Far elements in vitro, raises the question of which HLH proteins actively participate in the transcriptional regulation of the rat insulin I gene in pancreatic endocrine beta-cells. To investigate the specific role that Pan proteins play in regulating insulin gene expression, we have created endocrine beta-cell stable integrants that constitutively express Pan antisense transcripts that selectively inhibit endogenous Pan protein synthesis in differentiated beta-cells. We demonstrate that diminished Pan protein levels in beta-cells, caused by Pan antisense transcripts, accompanies a dramatic attenuation of rat insulin gene transcription. We also show that the decrease in Pan protein expression correlates with a specific reduction of the beta-endocrine-specific Nir and Far element-binding activity, insulin enhancer factor 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of E. coli-synthesized Pan proteins and development of a Pan-specific monoclonal antibody.

The helix-loop-helix (HLH) transcription factors, Pan-1 (E47) and Pan-2 (E12), are produced by the mechanism of alternative transcript splicing. Pan-1 and Pan-2 were expressed in Escherichia coli, and a purification scheme was developed. Purified Pan-2 was used to immunize Smith-Webster mice and a hybridoma was generated that produced a monoclonal antibody (Yae) that specifically recognized both native and denatured Pan-1 and Pan-2. Deletion mapping and sequence transfer studies have localized the determinant recognized by the Yae antibody to the region 195-208 of Pan-2. This region is conserved in Pan-1 and Pan-2. The Yae antibody recognized in vitro-synthesized ITF-1, a third E2A (Pan) gene product also produced by the mechanism of alternative RNA splicing, but did not recognize the related HLH proteins, ITF-2, REB alpha, or REB beta. By Western blot assay of pancreatic acinar cells, the Yae antibody detected a single protein species of 72 kD that comigrated with in vitro-synthesized Pan-1 and Pan-2.

Patterns of Pan expression and role of Pan proteins in endocrine cell type-specific complex formation.

The Pan gene encodes at least two distinct transcripts, Pan-1 and Pan-2 (also known as E47 and E12, respectively), by the mechanism of alternative RNA splicing. Northern blot analyses performed on rat and mouse tissues have detected ubiquitously expressed Pan transcripts, but the abundance, distribution, and form of Pan proteins have not been clearly defined. Studies of cell lines representing endocrine, fibroblast, and lymphoid lineages using polyclonal antisera to detect E2A proteins have suggested that significant E2A protein expression is restricted to B-lymphocytes. We have developed a monoclonal antibody, Yae, which is specific for Pan/E2A proteins, and have used the Yae antibody to examine a variety of endocrine and nonendocrine cell lineages for differences in Pan/E2A protein expression, subcellular localization, and heteromeric complex formation. In contrast to previous results obtained using polyclonal antiseras to detect Pan/E2A proteins, we report comparable levels of Pan proteins in GH/PRL- and insulin-producing, B- and T-lymphocyte cells. IEF-1, a pancreatic beta-cell type-specific complex believed to regulate insulin expression, is demonstrated to consist of at least two distinct species, one of which does not contain Pan molecules. Although it has been postulated that pituitary endocrine cells and pancreatic endocrine beta-cells share identical Pan/E2A complexes, native-Western analyses of pituitary and endocrine beta-cells detect Pan proteins in distinct cell type-specific complexes.

E2A expression, nuclear localization, and in vivo formation of DNA- and non-DNA-binding species during B-cell development.

A monoclonal antibody (Yae) was characterized and shown to specifically recognize E2A proteins in vivo, including the E2A-Pbx1 fusion gene products, p77E2A-Pbx1 and p85E2A-Pbx1. E2A proteins of a predominant molecular mass of 72 kDa, which comigrated with in vitro-produced rat E12 and and rat E47, were detected in human pro-B, pre-B, mature B, and plasma cell lines. The Yae antibody detected an E2A-containing microE2 enhancer element-binding complex (BCF-1) in pre-B- and mature B-cell lines in electrophoretic mobility shift assays which displayed a migration rate similar to that of in vitro-produced rat E12 and rat E47. A new E2A-containing microE2-binding species (P-E2A) was identified in plasma cells by using electrophoretic mobility shift assays. E2A proteins were detected in pro-B cells but were unable to bind the microE2 site. These observations suggest that the microE2 site is the target of stage-specific E2A regulatory complexes during B-cell development. Immunostaining analyses demonstrated the predominant nuclear localization of E2A proteins. Finally, we have identified an E2A form, designated I-E2A, which is unable to bind DNA. Our observations demonstrate novel in vivo mechanisms for the regulation of transcription by E2A proteins during B-cell development.