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Methods and compositions are provided for high level production of foreign proteins in yeast. The system is exemplified by production of α1 -antitrypsin in yeast strain AB110, derived from yeast strains 2150-2-3 and AB103.1.
S. carlsbergensis/S. cerevisiae hybrid strain AB110 containing plasmid pCl/1GAPATi9 was deposited at the A.T.C.C. on May 9, 1984, and given Accession No. 20709.
1. An expression system comprising an S. carlsbergensis/S. cerevisiae hybrid strain which is pep- and has an extrachromosomal element having a marker for selection, a truncated promoter region from a yeast glycolytic enzyme structural gene, wherein said promoter region includes the RNA polymerase binding site and transcription initiation site and is less than about 600 bp, and a gene foreign to yeast under the transcriptional control of said promoter region.
2. A system according to claim 1, wherein said strain is auxotrophic and said marker complements said auxotrophy.
3. A system according to claim 1, wherein said hybrid strain is obtained by mating an S. carlsbergensis strain with an S. cerevisiae strain containing said extrachromosomal element.
4. A system according to claim 1, wherein said hybrid strain is AB110, having A.T.C.C. Accession No. 20709.
5. A system according to claim 4, wherein said foreign gene codes for a mammalian α1 -antitrypsin.
6. A system according to claim 1, wherein said foreign gene codes for a mammalian α1 -antitrypsin.
7. A system according to claim 1, which is pep4-.
8. An expression system comprising an S. carlsbergensis/S. cerevisiae hybrid strain which is pep4-, has at least one auxotrophic mutation in at least one gene and has a extrachromosomal element having a gene complementing at least one auxotrophic mutation, a truncated promoter region from a yeast glyceraldehyde-6-phosphate dehydrogenase of less than about 500 bp and extending downstream to at least about nucleotide -20, wherein said promoter region includes the RNA polymerase binding site and transcription initiation site, and a gene foreign to yeast under the transcriptional control of said region.
9. An expression system according to claim 8, wherein upstream from said promoter region and contiguous therewith is a prokaryotic sequence of at least about 500 bp.
10. An expression system according to claim 8, wherein upstream from said promoter region and contiguous therewith is a yeast transcriptional regulatory sequence.
11. An expression system according to claim 10, wherein said regulatory sequence is associated with the regulation of GAL1, GAL10 or PHO5.
12. An expression system according to claim 8, wherein said foreign gene is α1 -antitrypsin.
13. A method for producing the expression product of a foreign gene in yeast comprising growing the expression system according to claim 1 in an appropriate nutrient medium.
14. A method for producing the expression product of a foreign gene in yeast, comprising growing the expression system according to claim 8 in an appropriate nutrient medium.
15. A method according to claim 14, wherein said foreign gene is α1 -antitrypsin.
16. An S. carlsbergensis/S. cerevisiae hybrid strain obtained by crossing auxotrophic leu- S. carlsbergensis yeast cells with non-complementary leu- pep4- S. cerevisiae yeast cells and selecting for an auxotrophic leu- S. carlsbergensis/S. cerevisiae hybrid strain having higher levels of expression of a foreign gene relative to said S. cerevisiae cells.
17. A hybrid strain according to claim 19 which strain is AB110, A.T.C.C. Accession No. 20709.
18. A hybrid strain resulting from crossing a hybrid strain according to claim 19 with a leu- S. carlsbergensis strain.
1. Field of the Invention
Hybrid DNA technology provides the opportunity to produce any amino acid sequence from naturally occurring amino acids. However, to use this ability in a useful manner has involved the overcoming of numerous obstacles, many of them associated with unknown factors involved with replication and expression in unicellular microorganism hosts. While the basic concepts of transcription of DNA to messenger RNA, splicing, and translation have seen enormous expansions in understanding of the processes involved, nevertheless the stability of messenger RNA, the rapidity with which it is translated, the stability of foreign proteins in the microorganisms, and the efficiency of processing and secretion, are still understood only at a rudimentary level.
For many polypeptides, the economies of manufacture will determine their utility. It will therefore be important that systems be developed, where the vectors, markers, regulatory signals, and host cooperate to provide for the production of the desired polypeptide in a form and at a level of total protein in which it may be readily isolated and purified. Desirably, where the natural product is processed to a mature protein, the host may afford partial or complete processing.
It is thus of vital importance to the development of useful proteins for industrial and therapeutic purposes to efficiently produce in high yields polypeptides of interest employing systems which can be readily developed, can be stably maintained and employ a host which can be grown in industrially efficient ways.
2. Description of the Prior Art
Carrell et al., Nature (1982) 298: 329-334 provide the amino acid sequence of human α1 -antitrypsin, with discussion of various known antitrypsin mutants. Kurachi et al., Proc. Natl. Acad. Sci. USA (1981) 78: 6826-6830 present the entire cDNA sequence of baboon α1 -antitrypsin. A partial human cDNA clone sequence is also shown. Bollen et al., DNA (1983) 2: 255-264 describe the cloning and expression in E. coli of a full cDNA coding for human α1 -antitrypsin. Courtney et al., Proc. Natl. Acad. Sci. USA (1984) 81: 669-673 describe the expression of human cDNA for α1 -antitrypsin in E. coli. Kawasaki et al., "The Production of Human α1 -antitrypsin in Yeast and Fission Yeast" in The Molecular Biology of Yeast, Cold Spring Harbor Laboratory, 1983, describe the expression of human α1 -antitrypsin in S. cerevisiae and S. pombe employing the yeast triosephosphate isomerase promoter and terminator sequences. The protein is reported to be unglycosylated, but biologically active against trypsin and elastase.
Methods, compositions and yeast systems are provided for expression of heterologous proteins in high yield in yeast. Particularly, yeast DNA sequences associated with transcriptional and translational control of glycolytic enzymes are employed in conjunction with structural genes encoding proteins foreign to yeast, where the construction is employed in a yeast host providing for high yield of the expression product. Particularly, human α1 -antitrypsin is produced in excess of 5% of total soluble protein.
FIG. 1 shows the nucleotide and predicted amino acid sequences of alpha-antitrypsin.
Novel yeast systems are provided for the enhanced production of proteins foreign to yeast. The system involves the selection of a yeast host obtained by crossing different strains of Saccharomyces having the desired genotype, wherein one of the strains contains an expression plasmid which includes a structural gene of interest. The expression plasmid has an efficient transcriptional initiation system for transcriptional regulation of the structural gene. The resulting crosses are selected for enhanced production of the polypeptide of interest. In this manner, novel strains are produced which are specifically adapted to the efficient and enhanced expression of a desired foreign protein.
Of particular interest as the host is a cross between two strains, employing the common fermenting strain S. carlsbergensis having the appropriate auxotrophy, which may be a laboratory or industrial strain, mated with an S. cerevisiae strain having the appropriate genotype, so as to provide for a strain which can be selected having the desired properties. The two strains will usually have at least one common genetic lesion, so that the progeny will also have the same lesion. Particularly, the resulting strain should be pep- particularly pep4- and auxotrophic in a convenient metabolite, which can be complemented with an extrachromosomal marker, for example a cross between two different Saccharomyces strains, the carlsbergensis leu- strain 2150-2-3 (available from Lee Hartwell, University of Washington) and the cerevisiae leu- strain AB103.1 (ATCC Accession No. 20711), where one of the cell lines, particularly the AB103.1 cell line bears the pep4- mutation and carries the expression plasmid. A strain was selected, "cured" of the plasmid and designated AB110, which has the following genotype: MAT α, ura3-52, leu2-04 or both leu2-3 and leu2-112, pep4-3, his4-580 [cir�].
The host is used with an expression vector, which includes an efficient promoter system. Particularly, the promoter regions from yeast glycolytic enzyme genes are employed, where fewer than 600 bp, usually from about 300 to 550 bp of the promoter region upstream from the initiation codon of the structural gene is employed. This sequence from the promoter region extends downstream to at least about the nucleotide -20, more usually to at least about the nucleotide -10, and generally not beyond the nucleotide +3. (The numbering is based upon the first base of the initiation codon as +1, while the adjacent upstream base is -1 and numbering in opposite directions therefrom accordingly.) The sequence includes the RNA polymerase binding site (the sequence including nucleotides upstream from the transcription initiation site which provides for initiation of formation of messenger RNA), as well as the transcription initiation site, and may also include the capping sequence, and other sequences associated with transcriptional regulation, as well as with translational regulation.
The promoter region of yeast glycolytic enzyme structural genes appears to be divided into two domains, one domain proximal to the structural gene, which involves the binding of the RNA polymerase and initiation of transcription and a second domain contiguous to the first domain and distal to the structural gene, which provides for enhanced efficiency and/or regulation of transcription. However, the presence of this distal region in recombinant constructs is found to reduce the efficiency of transformation, as well as the viability of the host. Therefore, the RNA polymerase binding region employed in the subject expression system will usually involve solely the first domain, with the second domain being substituted with various other sequences.
These sequences may include various sequences involved with the regulation of transcription of structural genes, such as sequences which result in regulation of transcription in response to a carbon source, nutrient, metabolite, ligand, or the like; or responsive to changes in the physical environment, e.g., temperature. That is, sequences where transcription can be induced by a change in the chemical or physical environment of the host. Alternatively, sequences other than the native sequence may be employed to provide for enhanced efficiency of constitutive transcription.
The proximal domain may be obtained from the promoter region providing for transcription of such glycolytic enzyme structural genes as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, triosephosphate isomerase, phosphoglucoisomerase, phosphofructokinase, glucose-6-phosphate dehydrogenase, or the like.
The distal domain when involving regulatory regions will generally be of at least about 100 base pairs and may be 500 bp or more, usually not exceeding about 2 kbp, more usually not exceeding about 1 kbp. These regions may be derived from such regulatory regions as GAL1, PHO5, GAL10, etc., or may be any of a number of temperature-sensitive sequences, which have been extensively described in the literature. Alternatively, prokaryotic sequences of at least 500 bp, preferably at least about 1000 bp, and usually not exceeding 6 kbp, more usually not exceeding about 5 kbp may be employed to enhance efficiency.
A transcriptional-translational termination region will also be employed 3' to the structural gene, which may be associated with the same or different structural gene from the structural gene with which the promoter domain is associated. Conveniently, it may be a transcriptional termination region associated with the same structural gene. The termination region will usually be at least about 300 bp, and may be 500 bp or more and will include the necessary regulatory sequences for transcriptional and translational termination.
Intermediate the transcriptional and translational regulatory initiation and termination sequences will be a structural gene of interest usually including associated translational initiation and termination codons, the gene usually being foreign to the yeast host. Of particular interest will be genes encoding mammalian proteins, proteins of pathogens, such as viruses or unicellular microorganisms, e.g., bacteria and protozoa, or plant proteins. Of particular interest are proteins which function as enzymes, hormones, structural proteins, lymphokines, plasma proteins, enzyme inhibitors, or the like. Illustrative proteins include α1 -antitrypsin, which will be used as exemplary of the subject invention, α- , β- or γ-interferons, interleukin-2, tissue plasminogen activator, blood factors V to XI, and portions and precursors thereof, e.g., Factor VIIIR and Factor VIIIC, growth hormone, epidermal growth factor, insulin, platelet derived growth factor, superoxide dismutase, thrombin, plasmin, albumin, etc.
The expression vector will have a stable replication system, unless integration is desired. Usually, it will be preferable to maintain an independent extrachromosomal element of either high or low copy number, depending upon the particular construction and the effect of the construct on the viability of the host. Conveniently, the 2 μm plasmid replication system may be employed. Alternatively a combination may be employed of a centromere sequence and an autonomously replicating segment, e.g., CEN3 and ars1 or 3.
In addition to the other functional sequences, one or more genetic markers may be employed for selection and maintenance. These markers may provide for protection from a biocide, e.g., antibiotic, toxin, or heavy metal; provide prototrophy to an auxotrophic host, e.g., the amino acid his, leu, or trp or other metabolite, e.g., ura, etc., gene; immunity; etc. Preferably the host cell phenotype will be caused by a mutation or lesion resulting in a metabolic block, i.e., in auxotrophy, which can then be complemented by the wild-type, functional, plasmid DNA which was introduced.
Frequently, it will be desirable to have a replication system recognized by bacteria, e.g., E. coli, included in the construct, so that during construction of the expression plasmid or afterwards, the construct may be cloned in a bacterial host at intermediate and final stages, isolated and purified. There are numerous replication systems described in the literature derived from such plasmids or viruses as ColE1, lambda, R6, or the like.
The various sequences may be isolated and joined in conventional ways. Gene libraries may be employed, either genomic or cDNA libraries, which are searched employing probes which may be obtained in different ways, depending upon the information which is available. Where an amino acid sequence is known, mixtures of probes can be prepared which encode for the particular sequence. Alternatively, where a fragmentary DNA or RNA sequence is known, a probe can be prepared having the particular sequence. These sequences may be used to identify regions associated with the fragment of interest, for example, a yeast glycolytic enzyme structural gene or desired foreign, e.g., human, gene.
Once the fragment(s) containing the gene(s) of interest is(are) identified by means of the probe, the fragment(s), which will usually be substantially larger than the gene and the regulatory regions associated with the gene, may be manipulated in a variety of ways. Particularly, restriction mapping and sequencing may be employed to identify the specific regions concerned with transcriptional and translational regulation and the coding region. As appropriate, various manipulative changes may be made. One or more bases may be modified, or fragments truncated or extended, by using such techniques as primer repair, in vitro mutagenesis, ligating of adapters or linkers, resection, restriction, or the like. These techniques are well established and may be found in the text, Maniatis et al., Molecular Cloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982, or other publications.
Frequently, vectors will be available which can be used in preparing the expression plasmid. Cassettes may be prepared, where the transcriptional and translational initiation and termination regions are separated by a relatively short sequence containing one or more unique restriction sites. In this way, the gene may be inserted at the unique restriction site or may be used as a substitution for a segment between two restriction sites. Once the expression plasmid has been prepared, it may be introduced into the host by conventional techniques, such as polyethylene glycol precipitated DNA with spheroplasts, conjugation, transfection or the like. See, for example, Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929-1933.
The modified yeast host is then grown in an appropriate nutrient medium to provide for expression of the desired polypeptide. Conventional rich nutrient media may be used or any other medium in which yeast can be satisfactorily grown, lacking the appropriate metabolite(s). For induced expression, the host will be grown to high density under conditions where expression of the polypeptide is not induced and when the desired cell density is achieved, the conditions will be changed to induce expression. For constitutive expression, the host will be grown in a rich medium to ensure the efficient production of the desired polypeptide.
After sufficient time for the polypeptide to be obtained, the cells may be isolated, lysed by any convenient means, e.g., mechanical shearing or detergent, and the polypeptide of interest isolated and purified by conventional techniques, e.g., chromatography, such as affinity chromatography, electrophoresis, density gradient, crystallization, ultrafiltration, or the like. Where the polypeptide is secreted, the cells may be separated from the supernatant and the supernatant treated as described above.
Of particular interest is the use of the glyceraldehyde-3-phosphate dehydrogenase or pyruvate kinase promoter regions having the RNA polymerase binding site and the transcription initiation site. In conjunction with these promoter regions or proximal domains are desirably used for regulated expression, the GAL4 regulator region or the PHO5 regulator region or a temperature sensitive regulator. The terminator region may be derived from any convenient structural gene, particularly the glycolytic enzyme genes, which have been described previously.
By employing the constructions of the subject invention, polypeptides may be produced in amounts usually of at least 1% of total soluble protein, more usually at least 2% of total soluble protein, and frequently at least 5% or higher, where amounts of about 25% or higher of total soluble protein are attainable, the total soluble protein being the sum of total host and expression product soluble protein.
The following examples are offered by way of illustration and not by way of limitation.
All DNA manipulations were done according to standard procedures. See Molecular Cloning, T. Maniatis et al., Cold Spring Harbor Lab., 1982. Enzymes used in cloning were obtained either from New England Biolabs or Bethesda Research Laboratories and employed according to the supplier's directions. Yeast were transformed and grown using a variety of media including selective medium (yeast nitrogen base supplemented with amino acids, etc. as appropriate but without leucine; YEPD medium, containing 1% (w/v) yeast extract, 2% (w/v) peptone and 2% (w/v) glucose; and in the case of plating medium contained 2% (w/v) agar and for transformation 3% top agar).
A cDNA library was made from 10 μg of polyA+ RNA isolated from part of a human liver. This library was prepared by oligo-dT priming of the first cDNA strand and self-priming of the second cDNA strand. The ds cDNA was size fractionated on a Sepharose CL4B column and those molecules greater than 300 bp isolated. This fraction was treated with nuclease S1 and tailed with dCTP, using terminal transferase. The tailed cDNA was annealed to pBR322 which had been digested with PstI and tailed with dGTP. Transformation of E. coli HB101 yielded 60,000 colonies, where greater than 90% of the clones were recombinant.
Two synthetic oligonucleotide probes were used to isolate the alpha-1-antitrypsin (α1 -AT) cDNA, the first probe corresponding to amino acid residues 344-350 near the C-terminus of the protein was used to probe 5,000 colonies and the second probe, corresponding to amino acid residues -23 to -17 (+1 being the first nucleotide of the first codon of the mature α1 -AT, with the negative numbers being upstream therefrom) of the signal peptide, was used to probe 25,000 colonies. The probe sequences were taken from the partial nucleotide sequence described by Kurachi et al., Proc. Natl. Acad. Sci. USA (1981) 78: 6826; Leicht et al., Nature (1982) 297: 655). Approximately 3% of the colonies hybridized to the C-terminal probe and four hybridized to the N-terminal probe. The four N-terminal clones and 12 C-terminal clones were isolated and subjected to restriction analysis. From these, three overlapping clones which cover the entire cDNA were subjected to further study and were used to construct the full-length cDNA clone.
The entire sequence of a composite full-length cDNA derived from the three plasmids is shown in FIG. 1. The reactive center met-ser at positions 358-359 is boxed. The subscripts to the amino acids in parentheses identify differences between the subject protein sequence and those derived from (a) protein sequencing [Carrell et al., 1982], (b) the cDNA of Woo et al. [see Carrell et al., 1982], and (c) the cDNA of Bollen et al., 1983. The synthetic DNA molecules used in the construction of the BamHI to SalI fragment encoding the mature protein are shown as are the cDNA restriction sites used in this construction.
The above sequence was determined using the dideoxy sequencing method of Sanger et al., Proc. Natl. Acad. Sci. USA (1977) 74: 5463, in the M13 vectors of Messing et al., Nucleic Acids Res. (1981) 9: 309. The differences at the nucleotide and amino acid level from the published cDNA sequences are shown.
Construction of the full length clone for expression of yeast began with three fragments isolated from cDNA clones: (1) a 630 bp BamHI-BstEII fragment; (2) a 450 bp BstEII-AvaI fragment; and (3) an 85 bp AvaI-HinfI fragment. A synthetic adapter was employed having the following sequence: ##STR1## Approximately two pmoles of fragments 1 and 2 were ligated together and after removal of the ligase, digested with BamHI and AvaI. Fragment 3 and the synthetic adapter were ligated and digested with AvaI and SalI and the two resulting fragment mixtures were ligated followed by digestion with BamHI and SalI. Fragments migrating upon electrophoresis in the region of about 1000-1400 bp were isolated and cloned by substitution into BamHI and SalI digested and alkaline phosphatase treated pBR322. The resulting plasmid is referred to as pATi.
A yeast expression vector was prepared called pPGAP having a polyrestriction site linker between the GAPDH terminator and a truncated GAPDH promoter region. Plasmids pGAP2 and pGAP1 were obtained as follows: A yeast gene library was prepared by inserting fragments obtained after partial digestion of total yeast DNA with restriction endonuclease Sau3A in lambda-phage Charon 28 (Blattner et al., Science (1977) 196: 161-169). The phage library was screened with DNA complementary to the yeast GAPDH mRNA and the yeast GAPDH gene GAP49 (Holland and Holland, J. Biol. Chem. (1979) 254: 5466-5474) from one of these clones was subcloned as either an about 3.3 kb BamHI fragment in the BamHI site of pBR322 (pGAP-2) or as an about 2.1 kb HindIII fragment in the HindIII site of pBR322 (pGAP-1). After digestion of the plasmid pGAP1 with HinfI, a 500 bp fragment was gel isolated, the fragment resected with Bal31 to remove about 50 bp, ligated with HindIII linkers, followed by digestion with HindIII and the resulting about 450 bp fragment inserted into the HindIII site of pBR322 after treatment of the plasmid with HindIII followed by alkaline phosphatase. The resulting plasmid pGAP128 was digested with HindIII, the fragment made blunt-ended with the Klenow fragment of DNA polymerase I and nucleotide triphosphates and the resulting blunt-ended about 450 bp fragment gel isolated and inserted into the SmaI site of plot5 after SmaI digestion and alkaline phosphatase treatment to provide the plasmid plot5pGAP128. plot5 was prepared by inserting the 40 bp polylinker fragment of the following sequence ##STR2## into pBR322 as an EcoRI-PvuII substitution followed by insertion of the trp-lac promoter (Russell and Bennett, Gene (1982) 20: 231-245) into the PvuII site with transcription oriented toward the polylinker sequence. Plasmid plot5pGAP128 was then digested with BamHI and TaqI to yield an approximately 390 bp BamHI-TaqI fragment having the -400 to -26 bp of the GAPDH promoter. The BamHI-TaqI fragment was ligated to a synthetic fragment having the following sequence: ##STR3## and containing -26 to -1 bp of the GAPDH promoter and an NcoI site. The resulting BamHI-SalI fragment, which was digested with BamHI and SalI and used to replace the BamHI-SalI fragment of BamHI-SalI digested pBR322 treated with alkaline phosphatase. After ligation, the plasmid pGAPNRS was obtained which was digested with BamHI and SALI to provide a 400 bp BamHI-SalI fragment which was gel isolated. This fragment was ligated to an about 1 kb SalI-BamHI fragment containing the GAPDH terminator region and a short segment of 3' coding region and the resulting 1.4 kb BamHI-BamHI fragment digested with BamHI. The about 1 kb SalI-BamHI GAPDH terminator fragment was obtained by gel electrophoresis after SalI and BamHI digestion of pGAP2, prepared as described above.
pBR322 was digested with EcoRI and SalI, the termini blunt-ended and ligated to BamHI linkers, followed by BamHI digestion and the BamHI-BamHI 3.8 kb fragment gel isolated, recircularized by self-ligation, cloned and designated pBRΔR1-Sal. The 1.4 kb BamHI-BamHI fragment was inserted into the BamHI digested, alkaline phosphatase treated pBRΔR1-Sal vector to provide the plasmid pPGAP of about 5.3 kb with the orientation in the opposite direction of the ampr.
Plasmid pPGAP was digested with NcoI, followed by blunt-ending, followed by SalI digestion and treatment with alkaline phosphatase. The NcoI-SalI fragment was substituted with an approximately 1250 bp blunt-ended (BamHI)-SalI fragment obtained from plasmid pATi, by BamHI digestion, blunt ending, and SalI digestion. This was inserted into the pPGAP vector to produce the plasmid pGAPATi, a 6.6 kb plasmid, which was digested with NcoI and BamHI and a 2.3 kb NcoI-BamHI fragment obtained having the α1 -AT gene and the GAPDH terminator and approximately 400 bp BamHI-NcoI fragment obtained having the GAPDH promoter. These fragments were ligated together and inserted into the BamHI site of pC1/1. (Plasmid pC1/1 is a derivative of pJDB219 (Beggs, Nature (1978) 275: 104) in which the region corresponding to bacterial plasmid pMB9 in pJDB219 has been replaced by pBR322 pC1/1.)
The plasmids pC1/1GAPATi8 and pC1/1GAPATi9 were obtained with the orientation of expression clockwise in the former and counterclockwise in the latter, with ampr being in the counterclockwise direction. These plasmids were transformed in S. cerevisiae AB103.1 (MATα, pep4-3, leu2-3, leu2-112, ura3-52, his4-580 [cir�]) (A.T.C.C. No. 20711, deposited June 8, 1984) by standard methods (Hinnen et al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929-1933), selecting for leucine prototrophy and grown as described above. Yeast extracts were prepared by lysis with glass beads followed by centrifugation at 12,000�g for 10 min and the α1 -AT activity determined by inhibition of human leukocyte elastase.
Assays contained in 1 ml: 0.1-0.2 human leukocyte elastase (HLE); 0.1 mM MeO-Suc-Ala-Ala-Pro-Val-p-nitroanilide (Beatty et al., J. Biol. Chem. (1980) 255: 3931); 50 mM Tris, pH 8, 0.5M NaCl, and the indicated amounts of yeast extract or human α1 -AT. Assays were initiated by the addition of elastase, incubated at 28� C. for 15 min, terminated by the addition of 100 μl of 8N acetic acid and the absorbance at 410 nm determined. Typical results are shown in the following Table 1.
TABLE 1__________________________________________________________________________ Amt. Extract Amt. HLE Amt. Protein % ElastasePlasmid Strain (μl) (μg) (μg) Activity % α1 -AT*__________________________________________________________________________pC1/1GAPATi8 AB103.1 5.0 0.1 50.0 40 0.17 10.0 0.1 100.0 26 0.11pC1/1GAPATi9 AB103.1 0.25 0.1 2.3 89 0.7 1.0 0.1 9.1 26 1.2pC1/1GAPATi9 AB110** 0.2 0.2 2.9 39 6.1 0.4 0.2 5.8 14 4.3__________________________________________________________________________ *Calculation based upon the Mol. wt. of HLE (29kD), the amount of protein added and the degree of inhibition. **See below for preparation.
The above data demonstrate that plasmids having the orientation of the expression cassette in the counterclockwise orientation, the promoter proximal to the long sequence of pBR322, make 10-20 times more α1 -AT than the same cassette in the other orientation.
Yeast strain S. carlsbergensis 2150-2-3 (available from Lee Hartwell, University of Washington) was crossed with a yeast S. cerevisiae strain AB103.1 transformant containing pC1/1GAPATi9. The diploids were sporulated and the tetrads dissected. Strains were maintained on leucine selective plates in order to ensure maintenance of the plasmid, since the parents are auxotrophs. A series of colonies were screened for their genotype with respect to a number of markers. The most vigorous strains were selected and cultures grown on leucine selective media. The best strain was designated AB110 (pC1/1GAPATi9), gave 6-7.5% of the total cell protein as α1 -AT as shown in the above Table 1. The strain AB110 has the following genotype: MATα, ura3-52, leu2-04 or both leu2-3 and leu2-112, pep4-3, his4-580 [cir�] and is obtained by curing the above strain AB110(pC1/1GAPATi9) of its resident plasmid by growth in the presence of leucine (absence of selective pressure) and then selection for leu- colonies by replica plating.
It is evident from the above results, that by employing a robust brewer's yeast S. carlsbergensis-baker's yeast S. cerevisiae hybrid strain as a host, auxotrophic as to a particular amino acid, for example, leucine, histidine, or tryptophan, and having a pep- mutation particularly in the pep4 gene, greatly enhanced yields of foreign proteins may be obtained. Desirably, after the original mating of the strains, the resulting pep4- strains may be selected and crossed against the parent S. carlsbergensis strain with continuous selection for enhanced yields of the desired product.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.
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Ok, I pretty much hate my new The Laundress products
I was pretty sure yesterday that I did not think The Laundress products were so great (the Classic scent in the bottle is so great though). After trying again today, I know they are total sh*t! Once items are dried, they do not smell clean at all (and are not very soft).
Even my spouse asked yesterday why we don't have clean sheets (Wednesday is usually clean sheets day). They were washed with The Laundress Classic detergent and softener!! Nothing smells clean after washing and drying! What a waste of like $35.00!
Also, I did a load of cottons today. Mainly darks and work out t-shirts and shorts. Seriously, after drying...the clothes did not even smell laundered. I had to re-wash.
SO...I really like and highly recommend:
Persil Color Gel
Vaska Herbatergent
Clorox Green Works
Mrs. Meyer's Geranium
Seventh Generation 4x
Avoid The Laundress. In my opionion the brand is nothing more than pretty packaging, clever marketing and hype. So disappointed!!!
That's funny. For the most part it gets good reviews.
I only have the wool & cashmere one and it is great. It gets really good reviews (9 reviews on Amazon a perfect 5/5).
Lovely cedar scent and leaves wool soft and clean.
I thought the others were WAY too perfumey for me so I've never tried them.
They even make some items for J Crew.
This is the background of the founders of The Laundress. You would think these two might know something about fabric care.
Lindsey Wieber Boyd and Gwen Whiting, co-founders of The Laundress
Lindsey and Gwen were both educated in Fiber Science, Textile and Apparel Management and Design at Cornell University. Their extensive professional experience in retail management and product development in the luxury-brand business merged with their educational backgrounds has created a unique knowledge and passion in fine fabrics and garment care.
Here is a link that might be useful: J. Crew - The Laundress
Ford, GM, BMW, Volvo, etc, etc know a thing or two about building great cars...but they have made disasters in the past!
Kraft and Nestle know a thing or two about making tasty foods, but they have had their share of disasters as well...
I think The Laundress is just really slick (NYC) advertising and marketing. With or without J.Crew...the items I got suck!! Maybe using some of their specialties for Cashmere or silk is fine, but their "basic" detergents left my close feeling worse than Sun detergent from the Dollar Tree Store!!! :)
I'm surprised to see another laundress non lover. I junked mine for the exact same reasons. A few peeps I know, who have to deal with textiles, swear by their gentle washes. I keep seeing their stuff in local shops (Village Apothecary) here in NYC that I usually love. Maybe it's the fabric or washing method b/c the everyday stuff from the machine approached uncomfortable. Blech.
I'm not often persuaded by Amazon's reviews either; too mass and some complain about packaging rather than product. When I was a publicist we placed a lot of stories that were about building personal or corporate brand equity. It's my own fault but I'm jaded about marketing, lol.
westvillager, I worked in advertising in the "prestige" fragrance industry for more than 10 years. I know all about "fluff" ... lol.
I do like the cashmere and wool wash. I was told by a local "upscale" shop that it's the best selling item from The Laundress. Maybe it's the one that works the best ... who knows.
I am officially done with these pricey "boutique" brands that do nothing. Look at Consumer Reports review of Martha Stewart's laundry detergent... they said it's just as effective as water! I've tried Le Blanc, another boutique brand, and it doesn't live up to a fraction of what is said about the product. In fact, I wrote to the manufacturer about the strong perfume scent and they basically said that nobody else is complaining so too bad.
These companies just want your money and will say anything to get it. Stay away from these "luxury" detergents - they don't work. Say what you will about P&G and other "mainstream" manufacturers, but at least they put their resources into formulations and research. How much research and product testing do companies like The Laundress, Le Blanc, and other similar companies do?
Products which I've tried and dislike:
- Charlie's Soap - doesn't clean
- All Small & Mighty - too many suds
- Everything Le Blanc and L'Amour - strong perfume scent and doesn't clean
- Country Save - doesn't clean
- Method Squeaky Green - too many suds
- Persil Universal Powder - not concentrated and must use ridiculous amounts
- EcoVantage - cleans OK but "eco" claims are suspect
Products which I've tried and like:
- Persil Megaperls
- Vaska Herbatergent - my current fave
- Tide HE Liquid w/ Bleach
- Bi-O-Kleen Premium Powder
- LanoSoft
- Clorox Green Works
I agree 100% with Sstava! Designer/boutique detergents are just a bunch of hyped up fragrances and claims of greatness, and almost always yield crappy results.
I love, love, love Persil Color Gel, Vaska Herbatergent and Clorox Greenworks.
I really like Seventh Generation 4x and Method Peony Blossom.
I have been Downy free now for 2 complete wash cycles...and so far, so good. I really, really like Mrs. Meyer's Geranium Softener and Ecover Sunny Day softener. Both soften as well as (or close to) Downy. And NO animal fat!!! :)
Do you (Larsi/Sstava) not think Vaska was a "boutique" brand?
A very small company started by a single person who worked in the fashion industry. Sounds a lot like The Laundress. She created a very different product and I do like it a lot.
Also, BioKleen was started by one guy who worked in the chemical industry. It too would have been a small brand before gaining larger distribution.
For anyone interested, here is BioKleen's story:
Jim Rimer founded Biokleen in 1989. As a previous sales representative of cleaning chemicals in the commercial and industrial industry he became more and more aware of the dangers of the products he was selling and the ingredients they contained.
He saw first hand his chemicals were creating health problems for his customers, causing negative effects - including cancer - on families and pets in the homes and businesses where these supplies were being used. Jim was selling poison and this realization shook him to the core. Jim's drive to take a stand and make a difference helped motivate him to study chemistry. With his newfound knowledge and driven passion he formulated his first cleaner using natural, non-toxic ingredients. Today Biokleen continues to develop and create innovative products, which still measure up to Jim's beliefs and standards.
If you're not using a product produced by one of the "biggie" companies, chances are it is or started as a small independent brand. I don't think you can make a blanket statement that all small brands are crap. I'm sure some are but some go on to become pretty large companies.
Method and Seventh Generation were once small guys trying to get attention too.
I think the Mrs. Meyer's Geranium detergent is very good actually. No obas and it has enzymes! Of course using it with the Geranium Softener leaves my clothes fragranced with a spicy Geranium/Rose scent that is really soft, yet there & I LOVE it! I tried it with a load of my 4 years old's preschool clothes (ketcup, paint, grass, dirt, pudding, cheetos, juice, markers....). The clothes were stain free, soft and fluffy!! I was surprised actually!
@livebetter... I think there are brands out there like Le Blanc/L'Amour, the Laundress and others who are specifically trying to capitalize on this notion of a "boutique" detergent without really having a superior product. Seventh Generation, Bi-O-Kleen, Method, etc. are all brands that not only have a statement to make, but they also work. The same can't be said for some of these other brands that I feel are trying to cash-in on the high priced detergent bandwagon.
I can make my own detergent using readily available products, call it the best thing since sliced bread, put it in a fancy bottle or slap a trendy label on it, and charge ridiculous amounts for it without delivering any performance at all. Oh wait, Martha Stewart already did that.
As far as Vaska is concerned, I don't consider it a boutique detergent and here's why. It's most recently been available at Albertson's, Target and Sam's Club. It's also not priced in the stratosphere like some of these other brands. A 52-load bottle of Tide HE Liquid w/ Bleach Alternative costs $13.64 after discount at SOAP.COM. That's 26 cents per load. A 64-load bottle of Vaska Herbatergent costs $13.48 after discount at SOAP.COM. That's 21 cents per load. Vaska is cheaper than Tide, and as such does not meet my basic criteria for a "boutique" detergent - high price.
Wow, you have serious laundry challenges :)
I think Mrs. Meyer's works well too. I just don't love the lavender version. I'm tempted to try the geranium. Maybe I'll start with the FS and use it with unscented detergent.
FWIW, Mrs. Meyer's was also a small "boutique" brand once. Started by Monica Nassif (who started Caldrea first and then introducted Mrs. Meyer's). Mrs. Meyer's is named after her mother (who raised 9 kids!).
"MRS. MEYER'S CLEAN DAY household products were inspired by Mrs. Thelma A. Meyer, an Iowa homemaker and mother of nine. It all started when one of her daughters (Monica Nassif) was walking down a cleaning aisle, eyeing all the products with their harsh, stinky chemicals. Right then and there, she had an idea: "Let's make cleaners that smell nice, like a garden, but still work like the dickens on daily dirt and grime." She named the product line after her very practical mom and the common sense values she passed along to her kids."
Another interesting "boutique" brand is the Good Home Co. The founder, Christine Dimmick was only 25 when she started the Good Home Co. in the kitchen of her New York City apartment in 1995. Today (2004) the company's 25 products are sold in high-end hardware stores and specialty shops like Restoration Hardware and Gracious Home, and its annual sales exceed $4 million.
You can read about her and Monica Nassif in this article from Time, "The Smell of Clean".
Read more: http://www.time.com/time/magazine/article/0,9171,993833-1,00.html#ixzz1J95Wn5ZR
Here is a link that might be useful: Mrs. Meyers - Meet Thelma
sshrivastava, I was just pointing out the fact that all the brands that are not part of the big boys (P&G, Henkel, etc) started out small.
Vaska didn't start out with distribution in these large chains. They had to work themselves up there. Her pricing is (I'm sure) part of their "stategy". Her earliest distribution was as a diaper wash so high cost would have been an issue (I think).
Same with Method. "it's hard to believe that 10 years ago, this week, two guys walked into a grocery store and sold the first bottle of method, launching our little revolution into the world." (see link)
They didn't start with huge distribution or a large product offering. Two guys had an idea and went for it.
I was arguing against that point that all boutique/designer brands are crap. Obviously the ones with legs go on to become large brands. They clearly have to have a product that works or people wouldn't keep buying it.
The only way to know is to try. None of us would be using and enjoying Vaska if we didn't give it a try.
Sometimes we try it and hate it (Laundress) - oh well.
I think each brand can be given the benefit of the doubt until you try it. That's all ;)
Here is a link that might be useful: Method 10 year Anniversary
Yes, you are right. If we don't try some "off" brands, we will never know if they are good or not. I do agree with Sstava that many, many designer/boutique brands are sadly just fancy labels on un-tested or un-proven detergents.
If it wasn't for you guys (especially you)...I would never have tried Vaska, and you might never have tried Green Works.
It is all basically trial and error. And while it annoys me when I feel I wasted $40 as with The Laundress...for a laundry junkie like me, waiting for FedEx or UPS to deliver a new box of holy grail laundry products is sometimes worth "wasting" $40!! LOL
Larsi, based on your glowing reviews I have also tried Green Works. I like it a lot! I did a few loads in it over the weekend and I agree that it cleans and rinses very well.
But as to cleaning as well as Persil, I don't think it does. I performed (completely by accident) a very unscientific test;-) I meant to wash a pair of grimey (formerly) white socks with the rest of my extra hot white wash but they got separated by accident. So one sock got washed on Sanitary, Extra Rinse, with Dixan Classic (Italian Persil) and the other on Perm Press (Warm), Extra Rinse, with Green Works. The sock washed in Persil was visibly cleaner. As I said, the experiment was unscientific and I suspect the length of the wash and the water temp played a significant role in the final results, but the sock washed in Persil was much whiter. Having said that, I am very happy with Green Works and will continue using it. Clothes and towels came out clean, soft and smelling fresh.


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Recent Stories
- Science Day at Heysham Power Station
- New Head of Physics
- Keswick School rockets to success in STEM Challenge day
- Major feat of engineering opens doors to future
- New Business Partnerships and Enterprise Deputy Director in post
- New guidelines to support research fellowships
- Strategic partnership with Danish university to boost research collaboration
- A decade of 'Innovation, Interaction and Impact': InfoLab21 turns Ten Years Old
- The scientific method and the Higgs Boson
- Physics student wins blogging award
Don't worry if you think you've seen ghosts running around campus, researchers from the Informatics Group within the Department of Communication Systems have been testing their new mobile location based game inspired by the arcade classic PACMAN (which they have named PAC-LAN) but with human players.
The story has gained a lot of media coverage and a TV production company has also shown some interest, so get your autographs early!
Tue 20 December 2005
Associated Links
- Computer Games of the Future - LU News article
- PAC-LAN - RFID-enabled mixed reality mobile phone game
Latest News
Science Day at Heysham Power Station
A Faculty team representing science, technology, engineering and maths took part in EDF Energy's 'Science Day' on Saturday 21st March at Heysham Power Station.
Wed 25 March 2015
New Head of Physics
Professor Roger Jones has replaced Professor Peter Ratoff as Head of the Physics Department. Roger gained a PhD studying neutrino interactions at CERN and Fermilab before starting his career at CERN working at the Large Electron-Positron (LEP) Collider.
Tue 24 March 2015
Keswick School rockets to success in STEM Challenge day
As part of British Science week, 170 students from 14 schools across the region came to Lancaster University on Wednesday 18th March to compete in science, technology, engineering and mathematics challenges.
Mon 23 March 2015
Major feat of engineering opens doors to future
Lancaster University's new flagship Engineering Building will provide a beacon of excellence for research and recruitment for the UK's buoyant and significant engineering industry.
Mon 23 March 2015


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Gujarat elections 2012: Cong files model code violation plaint against CM Narendra Modi
Top Stories
- Patna High Court stays Nitish Kumar's election as JD(U) legislature party chief
- Arvind Kejriwal gets down to business, calls for full statehood for Delhi
- President Pranab Mukherjee warns against deviation from constitutional principles
- Sunanda Pushkar murder case: SIT to quiz Shashi Tharoor tomorrow
- Shanti Bhushan accuses Arvind Kejriwal of accepting 'tainted' money
Congress state unit today filed a complaint against Chief Minister Narendra Modi for allegedly violating the Gujarat elections 2012 model code of conduct by 'campaigning' on election day at the polling station where he cast his vote, turning it virtually into a victory parade surrounded by a thick crowd of supporters.
Watch Video: Narendra Modi casts his vote
"After coming out from the polling booth, the Chief Minister addressed the crowd outside and by showing the 'V' sign he appealed the voters to make victorious the BJP candidate," alleged advocate and Congress unit's legal cell member Vijay Kanara, who filed the complaint with the EC authorities.
At about 9.45 am when Modi exercised his franchise by casting vote at Nishan High School in Ranip area in the city, scores of supporters and party workers were present there.
Congress has also alleged that Modi had come there with a big convoy and scores of party workers and also addressed them after he came out of the polling booth.
"He is not a candidate from the area where he cast his ballot and appealing for victory of BJP candidate is a clear violation of poll model code of conduct and EC should immediately take action against him," said Kanara.
In Ranip, Modi expressed confidence of winning the Assembly elections and forming the next BJP government.
"I thank all the voters. In this election, people of Gujarat will make a hat-trick by giving a third term to us. The people of the state will once again vote BJP to power... the sentiments of people here is giving you a clear indication of that," Modi told reporters there.


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Goldman Sachs's War On Plants PAYS OFF
Goldman Sachs said profit dropped 58 percent, beating analysts' estimates as the company cut compensation in response to falling revenue. Fourth-quarter net income dropped to $1.01 billion, or $1.84 a share, from $2.39 billion, or $3.79, in the same period a year earlier, the New York-based company said today in a statement. Per-share earnings exceeded […]
Wall Street Protests North: Boston Police Officers Imply They'll Take A Bullet For City's Shrubs
Should it come to that.
Morgan Stanley Joins Goldman Sachs In FirmScaping
Back in August, it was revealed that Goldman Sachs had added a disturbing element to its cost-cutting efforts: plant murder. The lobby philodendrons? Gone. Boston Ferns by the elevator? To the dumpster. The third floor Geraniums that lined the windows? Left for dead. The While every bank on Wall Street is bracing for serious reductions […]
Layoffs Watch '11: Goldman Sachs' Philodendrons In The Line Of Fire
As you may have heard, Wall Street is set to layoff a whole lot of financial services employees before the year is over. Last month the figure was pegged at around 100,000, although it got cranked up a notch on Friday when Bank of America said in September, it plans to tell 10,000 employees to […]


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Quality Resort & Spa Stromstad
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Phone: (46) 526 303 00
Fax: (46) 526 303 20
Outdoor Activities
Stadsmuseet (Göteborg City Museum)
Norra Hamngatan 12
Gothenburg, 411 14
+46 31 368 36 00
Tu to Su from 10:00 AM to 05:00 PM,We from 10:00 AM to 08:00 PMStadsmuseet is housed in a building commissioned by the Swedish East India Company in the eighteenth century. This museum of cultural history has collections of items gathered from Gothenburg as well as the rest of the world, from a wide variety of eras. On the last Sunday in every month between January and May, the museum organizes special family displays at 12:15p and 2p. In the spring, many of the Vetenskapsfestivalen (International Science Festival) events take place here.
Earn valuable points toward free nights.


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Library Branch
Book Clubs & Writers' Groups
Goldhawk Park
Join us to discuss great Chinese books once a month. Registration required. To register please call 416-396-8964.
Join us Tuesday evenings to chat about selected books. Copies of the books are available at the branch.
Tue Mar 31
Tue Apr 28
Tue May 26
Tue Jun 30
Friendly, informal discussion group. We read mostly literary fiction, some non-fiction, new books and old ones too. The books are provided and the book club meets the first Thursday of the month....
Thu Apr 02
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Mimico Centennial
Join the book club for an informal book discussion, with lively and stimulating conversation. New members welcome. March's title is "Quiet: The Power of Introverts in a World That Can't Stop Talking" by Susan Cain. April's ti...
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Join us for lively conversation about selected books.
Thu Apr 30
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If you like mysteries, consider joining a group of mystery lovers for informal discussions. Tell us about your favourite books and learn about new authors. The Mystery Book Club is currently reading classic tales of mystery a...
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Discuss new books and old favourites with library staff. Call branch at 416-394-5330 for more information.
Thu Apr 30
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Discuss new books and old favourites with library staff. Light refreshments provided. Call the library for more information.
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Do you write fiction, non-fiction, or poetry? Share and develop your skills in a supportive environment. Bring a piece of your writing to read aloud. Maximum length 500 words, approximately 2 pages. Come prepared to offer ...
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What's under and/or around your Japanese Maple? Pictures please!
I'll bet this has been asked, but when i did a search i came up with a million threads that didn't help. I also posted in the "Maples" forum, but didn't get much response.
Hope ya'll can help me.
I have decided that a Japanese maple (variety to be decided) would look good in a corner of my yard. What shorter plants would look good and thrive under or around it? I am going for a natural, but manicured look that will be easy to maintain. Already walked miles around my neigborhood looking for examples, but all the yards are covered with old junipers of every size, shape, and condition!!! These northern Californinas LOVE their junipers! :)
I live in the Oakland hills, and I have bearded iris, vinca major (a major mistake, I wouldn't recommend it), bacopa, a very pretty yellow-leaved plectranthus you can't see in this photo, and a variegated alstroemeria that isn't doing well in this much shade. The hydrangea does well as it's far enough way from the canopy of this 'Emperor 1' JMaple to do well. This is from last year, the maple is about 5' taller now.
The thread linked below might give you a few ideas. You are both in South Carolina; though zone 7 versus zone 9. You don't mention how much sun your area gets...I would hope you plan on giving that maple a little break from the hot afternoon sun.
Somewhere around here is a picture of a Japanese maple (maybe Crimson Queen) in my front yard. It and the pink flowering Evening Primrose that surrounds it were planted by the previous owner and the combo is rather ugly.
- Brent
Here is a link that might be useful: I am NOT happy with this bed!
Classic companion plants that would also be found in a japanese garden would include dwarf Nandina cultivars, Baby Tears, Carex morrowii, Acorus gramineus 'Ogon', Azaleas, Aucuba, Bletilla striata, etc. If you have room for them, Daphne odora marginata or Edgeworthia chrysantha are beautiful accent shrubs as companions to Japanese Maples.
Don't give people the wrong impression of northern California, you certainly don't see mass quantities of Junipers for landscaping here in Berkeley!
per bahia's "you certainly don't see mass quantities of Junipers for landscaping here in Berkeley!" -- actually, we live just a few miles away in the Laurel district of the Oakland hills. And here we DO see tons of junipers, both old and new plantings, by many homeowners. Not my favorite either, but they are tough, pest-free and drought resistant, so it's understandable.
And I will say that the only planting I've seen of Strelitzia (bird of paradise) that hasn't gotten away from the owner, is completely surrounded by mature junipers. The perfect containment strategy, LOL!!
Brent beat me to it! But here are some imbedded pics--just to make your life easier.
From this angle you can see: Rhodedendron 'Yaku Prince', Heuchera 'Obsidian', Japanese Painted Fern, Autumn fern, Hakonechloa 'aurea', Ophiopogon planiscapus 'Nigrescens' (black mondo grass),and hosta 'Gold Standard.' Oh, and some ripening daffodil foliage--ignore THAT.
Same bed--different angle:
Mostly what you see here is the heuchera...and some sedum whose name I've forgotten.
The JM is a 'Waterfall'--btw.
I have low-growing catmint on one side under my Japanese maple. Plant association-wise, it makes no sense, but it works in my garden. On the other side of the catmint there are some toad lilies, but I don't think those would do quite what you want.
On the other side, I have a low growing thyme. (Also makes no sense.) I like to think that it looks like a lake next to the tree. It is dying out - not sure if it is water competition or just a harsh summer.
Sorry I don't have a picture.
I think that a lot of people use the term "look natural" to describe something not looking out of place - like "act natural". Others use it or hear it more literally. I'll assume the latter.
What makes a plant not look out of place? Very often it is when the plant is within expected surroundings or appears the way that we are familiar with it looking. What is a familiar scene for a Japanese Maple? Is it in a corner planting in someone's yard surrounded by lower plants that are easy to maintain?
I believe that a Japanese Maple, at least many cultivars, don't really lend themselves well to a flat bed with other plants in them when it is to be viewed from a distance. When it is going to be viewed up close the detail of the plant dominates and captures our attention. When the plant is at a greater distance, even twenty feet away, its form is what captures our attention. Many of the cultivars have weeping forms and/or interesting branch structure. Those forms are very unexpected in an orderly garden and are very hard to make "natural looking" because they are unfamiliar.
What is a familiar look of a Japanese Maple that is seen by us from a distance? Can we envision a cutleaf weeping Japanese maple in the middle of a lawn with a mulch ring cut around it? ... in a border perennial bed? .... a foundation planting? What about in a Japanese Garden? (ding!)
How are they handled in a Japanese Garden? Are they on flat terrain surrounded by several of the same smaller shrubs? More likely there is some topography to the garden and there is a wide variety of individual plants of contrasting form, texture, and foliage color. There is usually stone involved to unify the composition. The form is what you have to make viewable because the detail is too far away. Red leafed varieties are easily viewed as strong color up close, but at a distance the greens of surrounding plants absorb the red of the maple making it far weaker than you might expect. Elevation isolates the form as well.
Here is an example of elevating the form:
Japanese maple can be pruned into a lollipop or meatball just like any other shrub or small tree and if it is used that way its purpose is to add contrasting leaf colour, when surrounded but green leaved plants pruned similarly. Trust me, this is a very common technique here and it negates every thing laag says about looking out of place. I am certainly NOT suggesting anyone does this, I only wanted us to be clear about what Andrew is therorizing about 'natural'.
I have candytuft planted around my japanese maple. It's a Crimson Queen with a contorted trunk planted on a slight slope. It has holly behind it, and the green mounds of candytuft in front of it have a nice texture. When the candytuft is covered by white blooms, it creates a nice contrast with the red leaves of the maple.
Improve with age, well, it depends if and who puts a set of pruners to it. It may well be a mushroom that touches the ground 360 degrees all around with no hint of the rocks and under planting. It is in a nice area of a rough city that I do not go to or go through. It would be interesting to see that house again, though. My father originally landscaped it in 1966. I'm afraid that all of his work is fading away. That bums me out, but time marches on.
Funny you should ask this. I was outside for a stroll today and was noting the things that had to be done now that Jack Frost has kissed so many things.
Under our good ol' stand-by "Bloodgood" (I think?) I've been patiently cultivating Asarum europaeum. I plunked a clump in a couple of years ago and it likes the site very much... little babies here and there. I have to be careful not to "weed" them. As more established clumps of it attain dividable size I add to the planting. It's a handsome, carefree groundcover, providing all season interest when we have a "brown" winter. There are also a couple of Hostas in the immediate vicinity. And bulbs.


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1. This is Tom Brady:
Elsa / Getty
2. This is the cowardly lion:
Via imdb.com
3. This is Tom Brady dressed as the cowardly lion:
4. Or maybe shag carpet?
5. Or possibly this mop?
6. I don't know. I just know this wasn't his idea and it makes me laugh.
7. Wait. Nope. He's an alpaca. Definitely an alpaca.
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At Esxence, Etat Libre d'Orange were showing their recent fragrance True Lust and their latest scent Remarkable People.… [Read More]
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Fetish (Parfum) opens with an orange-green citric petitgrain with significant ashy green oakmoss support before quickly moving to its heart. As the composition moves to its early heart the ashy green oakmoss takes the fore for an hour or so, gradual...More
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Acharya Institute of Technology, Bangalore
Year Founded: 2000
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Acharya Institute of Technology (AIT), Bangalore was established in 2000 and is a part of Acharya Institutes. The college is approved by AICTE and affiliated to Visvesvaraya Technological University. The college is NBA accredited, and offers a wide range of BE programs, M. Tech Programs and PG Programs in Computer Application and Business Management and also offers a Doctoral Study Program in Mathematics, leading to Ph.D. The Vision of Acharya is committed to the cause of value based education in all disciplines, envisions itself as a fountainhead of innovative human enterprise, with inspiration initiatives for academic excellence.
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Steigen lane 2012 at Acharya Institute of Technology, Bangalore
Acharya Institute of Technology, Bangalore will conduct Steigen lane 2012, a national level technical fest which is to be held from May 4 to May 5, 2012. Steigen lane 2012 is a national level technical fest conducted by the department of Information Science and Engineering, AIT, Bangalore.... Read more
Other Colleges in Karnataka
- A) Vogue Institute of Fashion Technology, Bangalore
- A. J. Institute of Management
- A.B.P. Systems, Bangalore
- A.J Institute of Management
- A.S.Patil College of Commerce
- A.V. Kanthamma College for Women
- Aakash Institute of Business Management (AIBM)
- Aakson Academy For Technology & Management
- Aakson Institute for Management Studies, Bangalore
- Abhyudaya Degree College (ADC)
- Academy of Aviation & Engineering, Bangalore
* This information has been collected from www.acharya.ac.in/ait.php or by contacting the institute on phone. Trade Marks belong to the respective owners. If you are an official representative of the institute and wish to modify this information, please write to us at firstname.lastname@example.com The listing was last modified on 29/03/2015


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BARRE A Granite City man accused of having sex with a 15-year-old is now facing charges involving sexual contact with another 15-year-old girl.
Jonathan Amell, 20, pleaded not guilty Thursday in Washington County criminal court to felonious lewd and lascivious conduct with a child. If convicted, Amell faces a maximum of 15 years in prison and a $5,000 fine.
According to the police affidavit for the most recent charges, the girl told police in November 2011 that Amell forced her to the ground and had intercourse with her, saying repeatedly that he loved her. She told police she said no to Amell a few times during the incident and said the encounter hurt.
Police said that when they questioned Amell, he admitted there was sexual contact between him and the girl but that she had initiated it and it only involved her hand.
Police said a DNA sample from the girls underwear and a sample given voluntarily by Amell matched. Amell told police before the DNA results came back that his DNA probably would be on the girls clothing from her touching him; he said she tried to take off her pants and have sex with him but that he refused.
Amell is already facing a rape charge from an incident in December. According to the police affidavit in that case, Amell and a girl were drinking at Bert Wendels house in Middlesex and the girl got extremely intoxicated. The girl said she and Amell went outside for a cigarette and that Amell asked her if she wanted to have sex. The girl said she turned him down, according to police, but said the next thing she remembered was walking into the woods and having sex with Amell.
In that case, Amell faces a maximum of 20 years in prison and a $10,000 fine.
Wendel, 47, is facing charges of having a sexual relationship with the girl who had been at his house.MORE IN Central VermontLeahy joins Marshfield woman in calling for stricter labeling requirements for generic drugs Full StoryMaj. Gen. Patrick A. Full StoryThe City Council has approved a parking ordinance change that paves the way for CarShare Vermont... Full Story
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openSUSE 11.4 Released
Published on 10 March 2011 09:41 AM EST
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The openSUSE developers in Nuremberg and around the world have a reason to celebrate today: openSUSE 11.4 was just released. openSUSE 11.4 has been developed over the past eight months and features a number of improvements and package updates.
Included with openSUSE 11.4 is the Linux 2.6.37 kernel, GRUB2, systemd, ZYpp, KDE Software Compilation 4.6, GNOME 2.32, Mozilla Firefox 4.0, Xen 4.0.2, VirtualBox 4.0.4, KVM 0.14, and various other features.
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Michael Larabel is the principal author of Phoronix.com and founded the web-site in 2004 with a focus on enriching the Linux hardware experience and being the largest web-site devoted to Linux hardware reviews, particularly for products relevant to Linux gamers and enthusiasts but also commonly reviewing servers/workstations and embedded Linux devices. Michael has written more than 10,000 articles covering the state of Linux hardware support, Linux performance, graphics hardware drivers, and other topics. Michael is also the lead developer of the
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