Exogenous peptide and protein expression levels using retroviral vectors in human cells.

Pseudotyped retroviral vectors combine the advantages of broad host range, high expression, stable chromosomal integration, and ease of preparation. These vectors greatly facilitate delivery into mammalian cells of sequences encoding individual peptide inhibitors-including those with therapeutic utility-and inhibitor libraries. However, retroviral vectors vary in behavior, particularly with respect to expression levels in different cell lines. Expression level is especially important in transdominant experiments because the concentration of an inhibitor (for example, an expressed peptide) is one of the key determinants in the degree of complex formation between the inhibitor and its target. Thus, inhibitor concentration should have an impact on the expressivity and/or penetrance of an induced phenotype. Here, we compare several retroviral vectors and human cell lines for relative expression levels using a green fluorescent protein reporter. We show for a subset of these lines that cellular protein concentrations produced by single-copy vectors range up to about 2 microM. We also examine other variables that contribute to expression level, such as the nature of the expressed protein's carboxy terminus. Finally, we test the effect of increased concentration on phenotype with a nine-amino-acid peptide derived from the human papilloma virus protein E7 which overcomes E7-mediated cell growth.

Transcriptional transactivation by selected short random peptides attached to lexA-GFP fusion proteins.

BACKGROUND: Transcriptional transactivation is a process with remarkable tolerance for sequence diversity and structural geometry. In studies of the features that constitute transactivating functions, acidity has remained one of the most common characteristics observed among native activation domains and activator peptides. RESULTS: We performed a deliberate search of random peptide libraries for peptides capable of conferring transcriptional transactivation on the lexA DNA binding domain. Two libraries, one composed of C-terminal fusions, the other of peptide insertions within the green fluorescent protein structure, were used. We show that (i) peptide sequences other than C-terminal fusions can confer transactivation; (ii) though acidic activator peptides are more common, charge neutral and basic peptides can function as activators; and (iii) peptides as short as 11 amino acids behave in a modular fashion. CONCLUSIONS: These results support the recruitment model of transcriptional activation and, combined with other studies, suggest the possibility of using activator peptides in a variety of applications, including drug development work.

Suppressor analysis of fimbrin (Sac6p) overexpression in yeast.

Yeast fimbrin (Sac6p) is an actin filament-bundling protein that is lethal when overexpressed. To identify the basis for this lethality, we sought mutations that can suppress it. A total of 1326 suppressor mutations were isolated and analyzed. As the vast majority of mutations were expected to simply decrease the expression of Sac6p to tolerable levels, a rapid screen was devised to eliminate these mutations. A total of 1324 mutations were found to suppress by reducing levels of Sac6p in the cell. The remaining 2 mutations were both found to be in the actin gene and to make the novel changes G48V (act1-20) and K50E (act1-21). These mutations suppress the defect in cytoskeletal organization and cell morphology seen in ACT1 cells that overexpress SAC6. These findings indicate that the lethal phenotype caused by Sac6p overexpression is mediated through interaction with actin. Moreover, the altered residues lie in the region of actin previously implicated in the binding of Sac6p, and they result in a reduced affinity of actin for Sac6p. These results indicate that the two mutations most likely suppress by reducing the affinity of actin for Sac6p in vivo. This study suggests it should be possible to use this type of suppressor analysis to identify other pairs of physically interacting proteins and suggests that it may be possible to identify sites where such proteins interact with each other.

A G protein alpha subunit from Cochliobolus heterostrophus involved in mating and appressorium formation.

A Galpha subunit-encoding gene (CGA1) was cloned from Cochliobolus heterostrophus, a heterothallic foliar pathogen of corn. The deduced amino acid sequence showed similarity to Galpha proteins from other filamentous fungi and suggested that CGA1 is a member of the Galphai class. cga1 mutants had reduced ability to form appressoria on glass surfaces and on corn leaves; mutants nevertheless caused lesions on corn plants like those of wild type. cga1 mutants were female sterile; sexual development was completely abolished when the mutant allele was homozygous in a cross. Ascospores produced in crosses heterozygous at Cga1 were all wild type. The signal transduction pathway represented by CGA1 appears to be involved in developmental pathways leading to either appressorium formation or mating; in sexual development CGA1 is required for both fertility and ascospore viability.

Laser emission of erbium-doped fluoride bulk glasses in the spectral range from 2.7 to 2.8 mum.

We report on the laser emission of erbium-doped fluoride glasses in the spectral range from 2.7 to 2.8 mum . The pump source was a fiber-coupled InGaAs semiconductor laser emitting near lambda(p)~970 nm . The laser performance of bulk samples with various ErF(3) contents was investigated with respect to the optimum doping concentration, which is estimated to be higher than 8 mol. %. A maximum output power of P(out) > 160 mW and a slope efficiency of eta = 10% were achieved. Results are compared with previously published data on erbium-doped ZBLAN fiber and LiYF(4) crystal lasers.

Diode-pumped 1-W Er-doped fluoride glass M-profile fiber laser emitting at 2.8 mum.

We report a novel short-length diode-pumped cw fiber laser with high output power at a wavelength of 2.8mum . The system combines continuous-wave diode pumping at 970 nm, a high doping concentration (5-mol. % ErF(3)) of the active fiber, and an annular geometry of the laser medium, an M-profile fiber. Output powers in excess of 1 W with a slope efficiency of 25% were obtained.

Self-starting mode locking of a Nd:glass fiber laser by use of the third-order nonlinearity of low-temperature-grown GaAs.

We present a diode-pumped Nd:glass fiber laser, emitting at 1060 nm, that is passively mode locked by fast nonlinear loss in low-temperature-grown GaAs (LT-GaAs). This new mode-locking mechanism is based on intensity-dependent defocusing in LT-GaAs that occurs after nonresonant generation of free carriers by two-photon absorption. Mode locking is self-starting and produces pulses as short as 4.1 ps.

A fungal kinesin required for organelle motility, hyphal growth, and morphogenesis.

A gene (NhKIN1) encoding a kinesin was cloned from Nectria haematococca genomic DNA by polymerase chain reaction amplification, using primers corresponding to conserved regions of known kinesin-encoding genes. Sequence analysis showed that NhKIN1 belongs to the subfamily of conventional kinesins and is distinct from any of the currently designated kinesin-related protein subfamilies. Deletion of NhKIN1 by transformation-mediated homologous recombination caused several dramatic phenotypes: a 50% reduction in colony growth rate, helical or wavy hyphae with reduced diameter, and subcellular abnormalities including withdrawal of mitochondria from the growing hyphal apex and reduction in the size of the Spitzenkörper, an apical aggregate of secretory vesicles. The effects on mitochondria and Spitzenkörper were not due to altered microtubule distribution, as microtubules were abundant throughout the length of hyphal tip cells of the mutant. The rate of spindle elongation during anaphase B of mitosis was reduced 11%, but the rate was not significantly different from that of wild type. This lack of a substantial mitotic phenotype is consistent with the primary role of the conventional kinesins in organelle motility rather than mitosis. Our results provide further evidence that the microtubule-based motility mechanism has a direct role in apical transport of secretory vesicles and the first evidence for its role in apical transport of mitochondria in a filamentous fungus. They also include a unique demonstration that a microtubule-based motor protein is essential for normal positioning of the Spitzenkörper, thus providing a new insight into the cellular basis for the aberrant hyphal morphology.

Allele-specific suppression by formation of new protein-protein interactions in yeast.

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have conducted a biochemical analysis of the interaction between various combinations of wild-type and mutant actin and Sac6 proteins. Previously, we showed that actin mutations that are suppressed by sac6 mutations encode proteins with a reduced affinity for wild-type Sac6p. In the present study, we have found that mutant Sac6 proteins bind more tightly to mutant actin than does wild-type Sac6p, and thus compensate for weakened interactions caused by the mutant actin. Remarkably, we have also found that mutant Sac6 proteins bind more tightly to wild-type actin than does wild-type Sac6p. This result indicates that suppression does not occur through the restoration of the original contact site, but rather through the formation of a novel contact site. This finding argues against suppression occurring through a "lock-and-key" mechanism and suggests a mechanism involving more global increases in affinity between the two proteins. We propose that the most common kind of suppressors involving interacting proteins will likely occur through this less specific mechanism.

High-power continuous-wave upconversion fiber laser at room temperature.

We report cw laser emission of a Pr, Yb-doped ZrF(4)-BaF(2)-LaF(3)-AlF(3)-NaF fiber in the red spectral region. Laser emission was achieved on the transition P(0)(3)?F(2)(3)(lambda(L)=635 nm) with a Ti:sapphire pump laser tuned to lambda(p)=850 nm . A maximum output power of P(out)=675 mW was obtained at an incident pump power of P(in)=3.37 W . The output power was increased to P(out)=1020 mW when pumping with P(in)=5.51 W was provided by two Ti:sapphire lasers. A photon avalanche process was found to contribute strongly to the population of the upper laser level.

Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin.

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.