Improving the PCR protocol to amplify a repetitive DNA sequence.

Although PCR-based techniques have become an essential tool in the field of molecular and genetic research, the amplification of repetitive DNA sequences is limited. This is due to the truncated nature of the amplified sequences, which are also prone to errors during DNA polymerase-based amplification. The complex structure of repetitive DNA can form hairpin loops, which promote dissociation of the polymerase from the template, impairing complete amplification, and leading to the formation of incomplete fragments that serve as megaprimers. These megaprimers anneal with other sequences, generating unexpected fragments in each PCR cycle. Our gene model, MaSp1, is 1037-bp long, with 68% GC content, and its amino acid sequence is characterized by poly-alanine-glycine motifs, which represent the repetitive codon consensus. We describe the amplification of the MaSp1 gene through minor changes in the PCR program. The results show that a denaturation temperature of 98°C is the key determinant in the amplification of the MaSp1 partial gene sequence.

The absence of detectable fetal microchimerism in nontransgenic goats (Capra aegagrus hircus) bearing transgenic offspring.

Regulations for the disposal of genetically engineered animals are strict due to concern for their inappropriate introduction into the food chain, and of the possible public health and environmental impacts of these organisms. Nontransgenic animals that give birth to transgenic offspring are treated as if they are transgenic due to concern of fetal cells crossing the placental barrier and residing in the mother (fetal-maternal microchimerism). Determining whether or not fetal-fetal or fetal-maternal transfer of DNA or cells occurs during caprine gestation is critical to effectively protect the public without culling animals that pose no risk. Additionally, fetal-maternal transfer, should it exist in the goat, could contraindicate the rebreeding of nontransgenic dams due to the possible transfer of fetal cells from 1 pregnancy to the fetus of subsequent pregnancies. Fetal-maternal transfer in Capra hircus has not been reported in the literature, although it has been reported in another ruminant, Bos taurus. We examined blood from nontransgenic dams that carried transgenic offspring using a PCR method sensitive enough to detect the presence of a spider silk transgene to a 1:100,000 dilution. At this sensitivity, we did not detect the occurrence of fetal-maternal transfer in 5 nontransgenic dams. Likewise, fetal-fetal transfer was not observed from a transgenic to a nontransgenic twin in utero. To test tissue-specific expression of the silk transgene, proteins purified from standard necropsy tissue from a lactating transgenic dam were examined by Western blot analysis. Silk protein expression was only observed in mammary tissue consistent with the tissue specificity of the ß-casein promoter used in the transgenic construct. We report evidence collected from a limited caprine breeding pool against transfer of transgenes in utero from fetus to dam and fetus to fetus. In addition, we show evidence that the ß-casein promoter in our expression construct is not expressed ectopically as previously suggested. These results suggest that transgene transfer in utero does not occur, but further study is warranted with a larger sample group to confirm these results.

Spidroins from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae).

Spiders produce up to six different kinds of silk, each one for a specific biological function. Spider silks are also known for their unique mechanical properties. The possibility of producing new materials with similar properties motivated research on these silk proteins (spidroins). Using expression sequence tags, we identified four spidroins produced by major ampullate, minor ampullate, flagelliform and tubuliform silk glands from the Brazilian spider Nephilengys cruentata (Araneae: Nephilidae). The new protein sequences showed substantial similarity to other spidroins previously described, with high content of alanine and glycine due to the presence of the highly repetitive motifs (polyAla, (GA)n, (GGX)n, (GPGGX)n). Similarities among sequences were also observed between the different spidroins with the exception of tubuliform spidroin, which presents a unique complex amino acid sequence with high amounts of serine and low amounts of glycine.

A novel methodology to explore the viscoelasticity of spider major ampullate silk.

Even before material science was a recognized discipline, the amazing mechanical properties of spider silk were documented and became the object of much study. In addition to the exceptional material properties of spider silk and the reported low immunogenicity, its concatenated amino acid motif arrangement facilitates a distinct possibility of manipulating the silk to create a designer biomaterial for medical applications. Crystalline protein regions imbedded in a mobile protein matrix give it a distinct set of viscoelastic abilities. Consequently, elasticity cannot be simply quantified by only measuring extensibility. To understand how the sequence of the major ampullate proteins affects elasticity, the hysteresis of single fibers from two different species, Argiope aurantia and Nephila clavipes, were examined using cyclic loading and unloading. The yield point that discriminates a transition from elastic extension to a plastic extension was analyzed by examining three different properties: Young's modulus, energy recovery and slack in the fiber after recovery. Young's modulus remained relatively constant regardless of the cycle. However, the energy recovered decreased as the slack and cycle number increased. Large standard deviations masked any quantitative differences between species and substantiated the necessity of developing synthetic silk to harness the amazing mechanical properties of spider silk.

Conformational changes in pediocin AcH upon vesicle binding and approximation of the membrane-bound structure in detergent micelles.

Pediocin AcH is a 44-residue antimicrobial peptide with bactericidal potency against Gram-positive bacteria such as Listeria. It belongs to a family of bacteriocins that, when membrane-associated, is predicted to contain beta-sheet and alpha-helical regions. All bacteriocins in this family have a conserved N-terminal disulfide bond. An additional C-terminal disulfide bond in pediocin AcH is thought to confer enhanced potency and broader specificity range against sensitive bacteria. The C-terminal disulfide bond may also affect the conformation of the C-terminus. The secondary structures of pediocin AcH in aqueous solution and vesicles from susceptible cells, as well as the ability of trifluoroethanol (TFE) and detergent systems to induce secondary structures like those induced in vesicles, were studied by circular dichroism (CD) spectroscopy. Like related peptides, pediocin AcH was highly unordered in aqueous solution, 56%. However, it also contained 20% beta-strand and 15% beta-turn structures. Upon complete binding to vesicles, 32% alpha-helical structure formed, the unordered structure decreased to 32%, and the beta-strand and beta-turn structures remained largely unchanged. Thus, a betaalpha domain structure formed in vesicles. The helical structure likely forces the C-terminal tail to loop back on the helix so that the C24-C44 disulfide bond can form. Detergent micelles were superior to TFE in their ability to induce secondary structural fractions in pediocin AcH comparable to those observed in vesicles. This demonstrates the importance of a hydrocarbon-water interface to pediocin AcH structure induction and suggests that it is preferable to use detergent micelles as solvents in NMR studies of pediocin AcH structure.

Spider flagelliform silk: lessons in protein design, gene structure, and molecular evolution.

Spiders spin multiple types of silks that are renowned for their superb mechanical properties. Flagelliform silk, used in the capture spiral of an orb-web, is one of the few silks characterized by both cDNA and genomic DNA data. This fibroin is composed of repeating ensembles of three types of amino acid sequence motifs. The predominant subrepeat, GPGGX, likely forms a beta-turn, and tandem arrays of these turns are thought to create beta-spirals. These spring-like helices may be critical for the exceptional ability of capture silk to stretch and recoil. Each ensemble of motifs was found to correspond to a different exon within the flagelliform gene. The pattern of sequence similarity among exons indicates intragenic concerted evolution. Surprisingly, the introns between the iterated exons are also homogenized with each other. This unusual molecular architecture in the flagelliform silk gene has implications for the evolution and maintenance of spider silk proteins.

Synthetic spider silk: a modular fiber.

Spiders make their webs and perform a wide range of tasks with up to seven different types of silk fiber. These different fibers allow a comparison of structure with function, because each silk has distinct mechanical properties and is composed of peptide modules that confer those properties. By using genetic engineering to mix the modules in specific proportions, proteins with defined strength and elasticity can be designed, which have many potential medical and engineering uses.

Molecular architecture and evolution of a modular spider silk protein gene.

Spider flagelliform silk is one of the most elastic natural materials known. Extensive sequencing of spider silk genes has shown that the exons and introns of the flagelliform gene underwent intragenic concerted evolution. The intron sequences are more homogenized within a species than are the exons. This pattern can be explained by extreme mutation and recombination pressures on the internally repetitive exons. The iterated sequences within exons encode protein structures that are critical to the function of silks. Therefore, attributes that make silks exceptional biomaterials may also hinder the fixation of optimally adapted protein sequences.

Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins.

Several types of silks and silk protein coding genes have been characterized from orb-web weaving spiders. When the protein sequences of major ampullate, minor ampullate, and flagelliform silks from Nephila clavipes are compared, they can be summarized as sets of shared amino acid motifs. Four of these motifs and their likely secondary structures are described. Each structural element, termed a module, is then associated with its impact on the mechanical properties of a silk fiber. In particular, correlations are drawn between an alanine-rich 'crystalline module' and tensile strength and between a proline-containing 'elasticity module' and extensibility.

Spider minor ampullate silk proteins contain new repetitive sequences and highly conserved non-silk-like "spacer regions".

Spider minor ampullate silk is a strong non-elastic deformably stretchable silk used in web formation. This silk from Nephila clavipes is composed of two proteins, MiSp 1 and 2, whose transcripts are 9.5 and 7.5 kb, respectively, as determined by Northern blots. Both MiSp proteins are organized into a predominantly repetitive region and a small nonrepetitive carboxy terminal region. These highly repetitive regions are composed mainly of glycine and alanine, but also contain tyrosine, glutamine, and arginine. The sequences are mainly GGX and GA repeats. The repetitive regions are interrupted by nonrepetitive serine-rich spacer regions. Although the sequences of the spacer regions differ from the repetitive regions, sequences of the spacers from different regions of the proteins are nearly identical. The sequence differences between major and minor ampullate silks may explain the differing mechanical properties of the fibers.

Evidence from flagelliform silk cDNA for the structural basis of elasticity and modular nature of spider silks.

Orb-web weaving spiders rely on their aerial nets to entrap flying prey. A key mechanical feature of orb-web design is the high elasticity of the capture spiral. We report the cloning of substantial cDNA for flagelliform gland silk protein, which forms the core fiber of the catching spiral. Like all silks, the flagelliform protein is composed largely of iterated sequences. The dominant repeat of this protein is Gly-Pro-Gly-Gly-X, which can appear up to 63 times in tandem arrays. This motif likely forms Pro2-Gly3 type II beta-turns and the resulting series of concatenated beta-turns are thought to form a beta-spiral. We propose that this spring-like helix is the basis for the elasticity of silk. The variable fifth position of the motif (X) is occupied by a small subset of residues (Ala, Ser, Tyr, Val). Moreover, these X amino acids occur in specific patterns throughout the repeats. This ordered variation strongly suggests that with hydration, the beta-spirals form hydrogen-bonded networks that increase the elasticity of flagelliform silk. The self-assembly of flagelliform protein monomers into silk fibers may be promoted by beta-spiral/beta-spiral interactions. Additionally, the other two motifs in the flagelliform protein, Gly-Gly-X and a spacer that disrupts the glycine-rich regions, may contribute to the alignment of monomers into fibers. The flagelliform protein cDNA was compared to the other members of the spider silk gene family. We show that all spider silk proteins can be characterized as sets of shared structural modules. The occurrence of these modules among the proteins is inconsistent with the phylogenetic relationships inferred from the C-terminal regions. This observation, along with the high level of variation among individual flagelliform protein repeats, but striking lack of such variation in the other silk proteins, suggests that unusual homogenization processes are involved in silk protein evolution.

Structural studies of spider silk proteins in the fiber.

Although spider silk has been studied for a number of years the structures of the proteins involved have yet to be definitely determined. X-ray diffraction and solid-state nuclear magnetic resonance (NMR) were used to study major ampullate (dragline) silk from Nephila clavipes. The silk was studied in its natural state, in the supercontracted state and in the restretched state following supercontraction. The natural silk structure is dominated by beta-sheets aligned parallel to the fiber axis. Supercontraction is characterized by randomizing of the orientation of the beta-sheet. When the fiber is restretched alignment is regained. However, the same reorientation was observed for wetting of minor ampullate silk which does not supercontract. Thus, the reorientation of beta-sheets alone cannot explain the supercontraction in dragline silk. Cocoon silk showed very little beta-sheet orientation in the natural state and there were no changes upon wetting. NMR and X-ray diffraction data are consistent with the beta-sheets arising from the poly-alanine sequences known to be present in the proteins of major ampullate silk as has been proposed previously.

Expression and purification of a spider silk protein: a new strategy for producing repetitive proteins.

Synthetic genes were constructed based on the known sequence of the spider dragline silk protein MaSp 2. The genes had 8, 16, or 32 contiguous units of the consensus repeat sequence of the protein. These artificial genes were constructed using a strategy involving compatible but nonregenerable restriction sites, which allowed construction of very large inserts in a precisely controlled manner. This strategy should have general utility in the controlled construction of repetitive proteins composed of identical or different repeat units. The protein from the 16-unit repeat was produced in Escherichia coli at levels up to 10 mg/g wet wt of cells although yields of 1-2 mg/g were more typical. The protein was easily purified with high recovery using an affinity column. The purified protein had the predicted amino acid composition and N-terminal sequence after cleavage of a leader sequence. The methodology described will allow production of sufficient quantities of protein for basic structure/function studies including production of synthetic fibers.

ScreenMax plasmid mini-prep: super rapid plasmid DNA extraction method.

An ultra-quick method for plasmid DNA extraction using the ScreenMax Plasmid Mini-prep Kit was optimized. Since the method has fewer steps than current methods, the entire process takes only 12 min for two sets of plasmid DNA extractions. DNA quality was excellent for further analyses, including DNA sequencing. The kit's new medium, MMB, provided 5 times the viable cell count and 7 times the dry cell weight compared with the conventional LB medium. The yield of plasmid DNA was 5 times better than from LB medium. From 200 microL of culture, the maximum yield was 7.5 micrograms. The ScreenMax Plasmid Mini-Prep is not only simple and economical but also safe because of its mild reagents. This new procedure is both reliable and reproducible.

Enkephalin-containing peptides processed from proenkephalin significantly enhance the antibody-forming cell responses to antigens.

Highly conserved enkephalin containing peptides (ECPs) are selectively processed from proenkephalin, which is synthesized in both the neuroendocrine and immune systems. The reported regulatory effects within the central nervous system and the biologic release patterns from both activated lymphocytes and stimulated adrenal chromaffin cells suggest the ECPs may act as regulatory factors of the immune system. We tested the effects of three of the ECPs, Peptides F, E, and B, on the in vitro Ab-forming cell (AFC) response murine splenocytes to antigenic challenge. In contrast to the immunosuppressive effects of the pentapeptide enkephalins, physiologic concentrations of the ECPs significantly enhanced the AFC response to both T cell-dependent and T cell-independent Ags. The effects are not sensitive to competition by the opiate receptor antagonist, naloxone, suggesting cell surface interactions that do not involve classical opiate receptors. These studies provide evidence that the effects are mediated through T cells rather than B cells. Peptide F-treated splenocytes also showed a significant enhancement of the AFC response to suboptimal Ag concentrations, suggesting a mechanism of action in which the ECPs may act to lower the threshold of activation of the effector cell. These results suggest that the ECPs are physiologically important modifiers of the humoral immune response. Given their release patterns and demonstrated action on the in vitro immune response, the proenkephalin-derived ECPs have the potential to be involved in both paracrine and autocrine regulatory networks within the immune system and as a positive immunoregulatory effect from the neuroendocrine system.

Adverse reactions with beta-adrenoceptor blocking drugs. An update.

beta-Adrenoceptor blocking drugs are widely used throughout the world, and serious adverse reactions are relatively uncommon. Many of those that do occur, including bronchospasm and peripheral ischaemia, are due primarily to blockade of beta 2-adrenoceptors. Recently developed beta-blockers with enhanced beta 1-selectivity and partial beta 2-agonist activity appear, in general, to have lesser effects upon airways function and vascular resistance, but none are regarded as being entirely 'safe' in patients with asthma. In the treatment of hypertensive patients with co-existing airways disease there are now effective alternatives to the beta-blockers, including calcium antagonists, alpha-adrenoceptor antagonists and angiotensin converting enzyme (ACE) inhibitors. However, in the presence of ischaemic heart disease, beta-blockers have specific advantages and may still be considered necessary in patients with airways disease. In this situation, agents with beta 2-agonist activity are preferable to 'conventional' beta-blockers. However, there is still some risk that bronchospasm may occur in certain individuals, and the bronchodilator response to inhaled beta 2-agonists might be impaired. In patients with peripheral vascular disease, beta-blockers with beta 2-agonist activity are less likely to worsen the symptoms and signs of peripheral ischaemia, and may reduce the prevalence of peripheral coldness, a common adverse effect of beta-blockers. There is concern that beta-blockers may have significant central effects, including impairment of memory and concentration, although these are difficult to quantify. A number of pharmacologically unpredictable adverse reactions may occur rarely, including skin reactions, alopecia and arthropathy.