Amino acid sequences and double-stranded messages--a means of directing the site of mutation?
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Amino Acid Sequence↗
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Results for “FIBROIN”
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Processing of nuclear heterogeneous RNA: recent developments.
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Animals↗
Transcription signals and factors of the silk genes.
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Animals↗
Full length and discrete partial reverse transcripts of globin and chorion mRNAs.
Rabbit globin mRNA was copied by AMV reverse transcriptase in the presence of various concentrations of deoxyribonucleotides (dNTPs). The cDNAs were analyzed by electrophoresis under denaturing conditions in formamide-polyacrylamide gels. Discrete size products were detected, ranging from 65 to 650 nucleotides-that is, up to the full length of the mRNA template. Increasing the concentrations of all four dNTPs stimulated formation of full-length transcripts and made the incomplete copies less abundant. Hybridization and nuclease digestion experiments indicated that the full-size product is indeed a complete transcript of globin mRNA. Similar results were obtained with chorion mRNAs. The possible usefulness of the discrete partial transcripts is discussed.
Animals↗
Transcription of a cloned Bombyx mori tRNA2Ala gene: nucleotide sequence of the tRNA precursor and its processing in vitro.
We have analyzed the transcription of a cloned silkworm tRNA2Ala gene in germinal vesicle extracts of X. laevis oocytes. The primary transcript was sequenced; it is 98 nucleotides long, beginning with a 5' triphosphate nucleotide and ending in a 3' oligouridine stretch. After transcription for long periods of time, enzymes in the frog extract also process the tRNA2Ala precursor to remove extra 5' and 3' nucleotides and to add a CCA end. The twenty-two extra nucleotides at the 3' end of this precursor are recovered as an intact fragment, implicating a new site of endoribonuclease cleavage in eucaryotic tRNA processing. This enzyme activity has also been demonstrated by reincubation of isolated pre-tRNA2Ala with a germinal vesicle extract. The products of in vitro cleavage are the same as those seen in the transcription reactions. The tRNA2Ala precursor molecules are made faithfully in the system with as few as 6 bp of Bombyx morti DNA upstream of the transcription initiation site of the tRNA2Ala gene. This result narrows down the minimal amount of DNA adjacent to the 5' end of a eucaryotic tRNA gene needed to support proper initiation by RNA polymerase III.
Animals↗
The amino acid composition of the silk of Calpodes ethlius (Hesperiidae, Lepidoptera).
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Amino Acids↗
Evidence for an immune function of lepidopteran silk proteins.
Hemolymph coagulation stops bleeding and protects against infection. Clotting factors include both proteins that are conserved during evolution as well as more divergent proteins in different species. Here we show that several silk proteins also appear in the clot of the greater wax moth Galleria mellonella. RT-PCR analysis reveals that silk proteins are expressed in immune tissues and induced upon wounding in both Galleria and Ephestia kuehniella, a second pyralid moth. Our results support the idea that silk proteins were co-opted for immunity and coagulation during evolution.
Animals↗
Novel assembly properties of recombinant spider dragline silk proteins.
Spider dragline silk, which exhibits extraordinary strength and toughness, is primarily composed of two related proteins that largely consist of repetitive sequences. In most spiders, the repetitive region of one of these proteins is rich in prolines, which are not present in the repetitive region of the other. The absence of prolines in one component was previously speculated to be essential for the thread structure. Here, we analyzed dragline proteins of the garden spider Araneus diadematus, ADF-3 and ADF-4, which are both proline rich, by employing the baculovirus expression system. Whereas ADF-3 represented an intrinsically soluble protein, ADF-4 was insoluble in vitro and self-assembled into filaments in the cytosol of the host insect cells. These ADF-4 filaments displayed the exceptional chemical stability of authentic silk threads. We provide evidence that the observed properties of ADF-3 and ADF-4 strongly depend on intrinsic characteristics such as hydropathicity, which differs dramatically between the two proteins, as in most other pairs of dragline silk proteins from other Araneoidea species, but not on their proline content. Our findings shed new light on the structural components of spider dragline silk, allowing further elucidation of their assembly properties, which may open the door for commercial applications.
Animals↗
Inter-specific sequence conservation and intra-individual sequence variation in a spider silk gene.
Currently, studies on major ampullate spidroin 1 (MaSp1) genes of non-orb weaving spiders are few, and it is not clear whether genes of these organisms exhibit the same characteristics as those of orb-weavers. In addition, many studies have proposed that MaSp1 might be a single gene with allelic variants, but supporting evidence is still lacking. In this study, we compared partial DNA and amino acid sequences of MaSp1 cloned from different spider guilds. We also cloned partial MaSp1 sequences from genomic DNA and cDNA of the same individuals of spiders using the same primer combination to see if different molecular forms existed. In the repetitive region of partial MaSp1 sequences obtained, GGX, GA and poly-A motifs were present in all Araneomorphae and Mygalomorpae species examined. An extreme similarity in MaSp1 non-repetitive portions was found in sequences of ecribellate, cribellate and Mygalomorphae web-builders and such a result suggested that this sequence might exhibit an important function. A comparison of sequences amplified from the same individual showed that substitutions in amino acids occurred in both repetitive and non-repetitive regions, with a much higher variation in the former. These results suggest that the MaSp1 of Araneomorphae spiders exhibits several forms in an individual spider and it might be either a multiple gene or a single gene with a multiple exon/intron organization.
Amino Acid Sequence↗
Conformational polymorphism, stability and aggregation in spider dragline silks proteins.
Spider silk is spun in a complex and unique process, thought to depend on a hydrophobic conversion of a predominantly disordered to a beta-sheet rich protein structures. To test this hypothesis we monitored the effect of cationic (DOTAC) and anionic (alkyl sulfate) detergents and of (ii) solvent polarity using a series of alcohols on the secondary structure transition in dilute solutions of native spidroin. Our results showed that the detergents hydrophilic head charge and hydrophobic tail length cooperatively induced either a transition to the beta-sheet rich form or a stable helical state. Changing the solvent polarity showed that HFIP and TFE induced formation of stable helical forms whereas MeOH, EtOH and IsoP induced a kinetically driven formation of beta-sheet rich structure.
2-Propanol↗
Comparative analysis of the development of the mandibular salivary glands and the labial silk glands in the mulberry silkworm, Bombyx mori.
The mulberry silkworm, Bombyx mori has a pair of salivary glands arising from the mandibular segment, in addition to the labial silk glands which are generally considered as modified salivary glands. Here we report the characterization of salivary glands and the comparative gene expression profiling of the silk and salivary glands. The two independent salivary glands made up by 330 cells, grow about 1000 fold during larval development. These individual glands extend up to the T(1) thoracic segment unlike silk glands with fused anterior ends and extending up to the caudal region. The salivary glands also undergo endomitosis resembling the silk glands. The B. mori homologue of the homeotic gene Deformed (BmDfd) was expressed in the mandibular and maxillary segments in stage 17 embryo and got localized to the centre of the mandibular segment at stage 18 to form the salivary gland placodes. The expression was also seen in the distal ends of the leg appendages after blastokinesis (stage 22). Only low variations in BmDfd expression ranging from 1.6 to 2.1 fold were apparent during embryonic development. BmDfd expression was observed in the salivary glands all through the larval instars but not in the silk glands. The transcription factor, Forkhead and the segment polarity gene, Wingless were expressed throughout the salivary glands, the latter confirming the absence of physiological compartmentation within these glands unlike the silk glands. The expression of Amylase and Fibrohexamerin was restricted to the salivary and silk glands, respectively and therefore, served as molecular markers for these tissues.
Amylases↗
Mechanochemical mechanism for peptidyl free radical generation by amyloid fibrils.
beta-Amyloid peptides (A beta) form the core of Alzheimer's disease (AD) senile plaques, and are implicated in AD neurotoxicity. A beta and some derivatives generate free radicals upon fibrilogenesis. A mechanism for free radical generation is proposed, based upon fibril cross beta-sheet structure: (1) During fibrilogenesis there is a small probability of mispacking of A beta monomers, resulting in abnormal fibril packing. (2) Continued fibrilogenesis traps a packing defect within the beta-sheet. Surrounding beta-sheet resists distortion, and the abnormally packed polypeptide(s) is strained. (3) Thermal processes cause homolytic bond scission and radical production from strained polypeptide through mechanically activated thermal decomposition. (4) Reaction with oxygen produces peroxy radicals, prevents unproductive radical recombination, and promotes observed cross-linking, production of reactive oxygen species and peptide fragmentation. Adiabatic mapping suggested significant strain would be generated by beta-sheet misalignment. The mechanism relates the common structure of fibrils to radical production, and may be relevant to cytotoxicity in prion and other amyloidoses.
Amyloid beta-Peptides↗
Comparative studies of the organic matrices of invertebrate mineralized tissues.
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Amino Acids↗
Swelling behavior of a genetically engineered silk-elastinlike protein polymer hydrogel.
The influence of environmental conditions such as pH, temperature, and ionic strength on the equilibrium swelling ratio of physically crosslinked networks of a genetically engineered silk-elastinlike protein-based copolymer (SELP) with an amino acid repeat sequence of [(GVGVP)4GKGVP(GVGVP)3(GAGAGS)4]12 was investigated. The effects of gelation cure time and initial polymer concentration on the equilibrium swelling ratio and soluble fraction of the hydrogels were also studied. It was found that the soluble fraction linearly correlated with the initial polymer concentration at higher gelation times. Soluble fraction results suggest that final hydrogel water content may be controlled by both initial polymer concentration and gelation time. Equilibrium swelling studies demonstrated that these hydrogels are relatively insensitive to environmental changes such as pH, temperature, and ionic strength. Over the concentration range studied, it was found that an increase in gelation time at 37 degrees C resulted in lower hydrogel weight equilibrium swelling ratios, which corresponds to less soluble polymer released post-gelation. Together, these results have implications for the controlled delivery of bioactive agents from silk-elastinlike hydrogels.
Amino Acid Sequence↗
Solute diffusion in genetically engineered silk-elastinlike protein polymer hydrogels.
The partitioning and diffusion behavior of theophylline, vitamin B(12), and cytochrome c in physically crosslinked networks of a genetically engineered silk-elastinlike protein-based (SELP) copolymer with an amino acid sequence of [(GVGVP)(4)GKGVP(GVGVP)(3)(GAGAGS)(4)](12) was investigated. The effect of gelation kinetics on the equilibrium swelling ratio and normalized dimensions of loaded SELP hydrogel disks before and after release studies was also examined. Size dependent release behavior was quantified by diffusion studies with equilibrium loaded SELP hydrogels. Direct loading diffusion studies confirmed that hydrogels produced by direct incorporation of cytochrome c with the aqueous SELP solution did not significantly influence the release behavior compared to equilibrium loaded hydrogels. An overall increase in the equilibrium swelling ratio after the release studies was observed. Analysis of the hydrogel disk dimensions after the release studies revealed no expansion of the disk dimensions. The apparent increase in the equilibrium swelling ratio was most likely due to a decrease in the hydrogel crosslinking density following the removal of the polymer soluble fraction over the course of the release study.
Amino Acid Sequence↗
Genetic engineering of fibrous proteins: spider dragline silk and collagen.
Various strategies have been employed to genetically engineer fibrous proteins. Two examples, the subject of this review, include spider dragline silk from Nephila clavipes and collagen. These proteins are highlighted because of their unique mechanical and biological properties related to controlled release, biomaterials and tissue engineering. Cloning and expression of native genes and synthetic artificial variants of the consensus sequence repeats from the native genes has been accomplished. Expression of recombinant silk and collagen proteins has been reported in a variety of host systems, including bacteria, yeast, insect cells, plants and mammalian cells. Future utility for these proteins for biomedical materials is expected to increase as needs expand for designer materials with tailored mechanical properties and biological interactions to elicit specific responses in vitro and in vivo.
Amino Acid Sequence↗
Genetically engineered silk-elastinlike protein polymers for controlled drug delivery.
The silk-elastinlike class of genetically engineered protein polymers is composed of tandemly repeated silk-like (Gly-Ala-Gly-Ala-Gly-Ser) and elastin-like (Gly-Val-Gly-Val-Pro) amino acid blocks. The precision with which these polymers can be synthesized, as well as the ability to incorporate motifs that allow for gel-formation, stimuli-sensitivity, biodegradation, and biorecognition have stimulated interest in their use for controlled drug and gene delivery. This review will focus on the synthesis and characterization of silk-elastinlike polymers as related to controlled drug delivery. The design and biological synthesis of the copolymers, by recombinant DNA techniques, are reviewed. The characterization of the polymers is discussed. Finally, biocompatibility of the polymers and recent studies to determine their potential utility for controlled drug and gene delivery are reviewed.
Bacterial Proteins↗